Investigating cell
dynamics and death
by conventional and
confocal microscopy
International Symposium
Pavia, May 3-6, 1999
University of Pavia - Italy
Dipartimento di Biologia Animale
Piazza Botta 10, 27100 Pavia, Italy
EUROPROGRAM LEONARDO DA VINCI
Investigating cell dynamics and death
by conventional and confocal
microscopy
International Symposium
Department of Animal Biology, University of Pavia
Pavia, May 3-6, 1999
Internet WEB page:
www.unipv.it/webbio/anatcomp/leonardo/sympo99/progsy99.htm
Scientific and Organizing Committee
Carlo E. Pellicciari (Pavia)
2
Isabel Freitas (Pavia)
Marie Christine Dabauvalle (Wurzburg)
Heinz Gundlach (Oberkochen)
Luciana Dini (Lecce)
Giuseppe Gerzeli (Pavia)
Sergio Barni (Pavia)
3
Organizers
Prof. Carlo Pellicciari
Laboratorio di Biologia Cellulare
Departimento di Biologia Animale
Università di Pavia
Piazza Botta 10, 27100 Pavia, Italy
phone: + 39 - 0382 - 506420
FAX: + 39 - 0382 - 506325
e-mail: pelli@unipv.it
Prof. Isabel Freitas
Laboratorio di Anatomia Comparata e Citologia
Dipartimento di Biologia Animale
Università di Pavia
Piazza Botta 10, 27100 Pavia, Italy
phone: + 39 - 0382 - 506317
FAX: + 39 - 0382 - 506406
e-mail: freitas@unipv.it
Dr. Heinz Gundlach
Carl Zeiss GmbH
Carl Zeiss Str. 1
D-7347 Oberkochen, Germany
phone: + 49 - (0)7364 - 202071
FAX: + 49 - (0)7364 - 204013
e-mail: gundlach@zeiss.de
Layout, Artwork and Web Design by Dr.Vittorio Bertone
With the collaboration of
A CN Biosciences Company
by
4
PROGRAM
MAY 3RD, 1999 "PHAGOCYTOSIS AND APOPTOSIS"
 Luciana Dini (Dipartimento di Biologia, University of Lecce): "Apoptosis: the
importance of being phagocytosed"
 Paola Ramoino, Francesco Beltrame, Alberto Diaspro and Mario Fato
(DIP.TE.RIS, Dipartimento di Fisica e INFM, University of Genova): "Membrane
flow during phagocytosis and exocytosis of Paramecium primaurelia, as
studied by confocal laser scanning microscopy"
 Patrizia Rovere, Attilio Bondanza, Valerié Zimmermann, Cristina Vallinoto and
Angelo A. Manfredi (H.S. Raffaele, Milano and University of Marseille):
"Functional outcomes of apoptotic clearance by dendritic cells"
 Carlo Pellicciari, Maria Grazia Bottone, Debora Duò, Marco Biggiogera, Patrizia
Rovere and Angelo A. Manfredi (Dipartimento di Biologia Animale, University of
Pavia and H.S. Raffaele, Milano): "Phagocytosis of apoptotic bodies by
microglial cells in culture: fate of ribonucleoproteins"
MAY 4TH, 1999 "CYTOSKELETON"
 Jürgen Wehland (Gesellschaft für Biotechnologische Forschung, Braunschweig):
"Bacterial manipulation of the actin cytoskeleton"
 Luigi Sciola, Alessandra Spano and Sergio Barni (Universities of Sassari and
Pavia): "Cell membrane and cytoskeleton modifications during apoptosis in
neoplastic cell lines"
 Heinz Gundlach (Carl Zeiss, Oberkochen): "Laser scanning microscopy:
principle, instrumentation and application in the study of cytoskeleton"
5
MAY 5TH, 1999 "NUCLEAR
FUNCTION,
RNA
SYNTHESIS AND PROCESSING, AND NUCLEUS-
TO-CYTOPLASM TRANSPORT"
 Marie Christine Dabauvalle (University of Wurzburg): "Structure and functional
aspects of the nuclear envelope"
 Giuseppe Biamonti, Ilaria Chiodi, Fabio Cobianchi, Florian Weighardt and Silvano
Riva (IGBE CNR,
Pavia) "Dynamic distribution of hnRNP proteins in
mammalian cells: identification of new nuclear compartments "
 Marco Biggiogera, Andrea Trentani, Maria Grazia Bottone and Terence E. Martin
(Universities of Pavia and Chicago) “In vivo incorporation of anti-RNP
antibodies in cultured cells"
 Peter J.S Hutzler (GSF Research Center, Neuherberg ): "Analysis of interphase
FISH on histological sections using confocal laser scanning microscopy"
 Christian Castelli and Gabriele A. Losa (Laboratorio di Biologia Cellulare, Istituto di
Patologia, Locarno) "Apoptotic cell death in human breast cancer cells and
tissues: from light microscopy to quantitative electron microscopy"
 Pier Carlo Marchisio (DiBit, H.S. Raffaele, Milano) “New molecules controlling
basic cell functions”
MAY 6TH, 1999 "STUDYING
LIVING CELLS IN SITU "
 Rosario Rizzuto (University of Ferrara): "Green Fluorescent Protein mutants in
the study of calcium signalling"
 Mike White, C.D. Wood, D.G. Spiller, K. Bateman, D.G. Fernig, S. Hawley, S.
Pennington, N. Takasuka, A. Stirland , A. Loudon, J. Davis (Universities of
Liverpool and Manchester): "Real time imaging of transcriptionin mammalian
cells using luciferase genes "
 Sergio Barni, Luigi Sciola, Alessandra Spano, Vittorio Bertone (Universities of
Pavia and Sassari): “Fluorescent probes for supravital cell staining"
6
Index
Pag.
7
13
16
20
23
24
28
30
33
38
41
43
47
49
51
53
"PHAGOCYTOSIS AND APOPTOSIS"
Luciana Dini (Dipartimento di Biologia, University of Lecce): "Apoptosis: the
importance of being phagocytosed"
Paola Ramoino, Francesco Beltrame, Alberto Diaspro and Mario Fato
(DIP.TE.RIS, Dipartimento di Fisica e INFM, University of Genova): "Membrane
flow during phagocytosis and exocytosis of Paramecium primaurelia, as
studied by confocal laser scanning microscopy"
Patrizia Rovere, Attilio Bondanza, Valerié Zimmermann, Cristina Vallinoto and
Angelo A. Manfredi (University of Marseille and H.S. Raffaele, Milano):
"Functional outcomes of apoptotic clearance by dendritic cells"
Carlo Pellicciari, Maria Grazia Bottone, Debora Duò, Marco Biggiogera, Patrizia
Rovere, Angelo A. Manfredi (Dipartimento di Biologia Animale, University of Pavia
and H.S. Raffaele, Milano): "Phagocytosis of apoptotic bodies by microglial
cells in culture: fate of ribonucleoproteins"
"CYTOSKELETON"
Jürgen Wehland (Gesellschaft f. Biotechnologische Forschung, Braunschweig):
"Bacterial manipulation of the actin cytoskeleton"
Luigi Sciola, Alessandra Spano, Sergio Barni (Universities of Sassari and Pavia):
"Cell membrane and cytoskeleton modifications during apoptosis in
neoplastic cell lines"
Heinz Gundlach (Carl Zeiss, Oberkochen): "Laser scanning microscopy:
principle, instrumentation and application in the study of cytoskeleton"
"NUCLEAR FUNCTION, RNA SYNTHESIS AND PROCESSING, AND NUCLEUS-TOCYTOPLASM TRANSPORT"
Marie Christine Dabauvalle (University of Wurzburg): "Structure and functional
aspects of the nuclear envelope"
Giuseppe Biamonti, Ilaria Chiodi, Fabio Cobianchi, Florian Weighardt and Silvano
Riva (IGBE CNR, Pavia) "Dynamic distribution of hnRNP proteins in
mammalian cells: identification of new nuclear compartments "
Marco Biggiogera, Andrea Trentani, Maria Grazia Bottone, Terence E. Martin
(Univ. Pavia and Chicago) “In vivo incorporation of anti-RNP antibodies in
culture cells”
Peter J.S Hutzler (GSF Research Center, Neuherberg ): "Analysis of interphase
FISH on histological sections using confocal laser scanning microscopy"
Christian Castelli and Gabriele A. Losa (Laboratorio di Biologia cellulare, Istituto di
Patologia, Locarno) "Apoptotic cell death in human breast cancer cells and
tissues: from light microscopy to quantitative electron microscopy"
Pier Carlo Marchisio (DiBit, H.S. Raffaele, Milano) “New molecules controlling
basic cell functions”
"STUDYING LIVING CELLS IN SITU "
Rosario Rizzuto (University of Ferrara): "Green Fluorescent Protein mutants in
the study of calcium signalling"
Mike White, C.D. Wood, D.G. Spiller, K. Bateman, D.G. Fernig, S. Hawley, S.
Pennington, N. Takasuka, A. Stirland , A. Loudon, J. Davis (University of
Liverpool): "Real time imaging of transcription in mammalian cells using
luciferase genes "
Sergio Barni, Luigi Sciola, Alessandra Spano (Universities of Pavia and Sassari):
"Fluorescent probes for supravital cell staining"
7
57
Speakers' addresses
APOPTOSIS: THE IMPORTANCE OF BEING PHAGOCYTOSED
Luciana Dini
Dipartimento di Biologia, University of Lecce, Via per Monteroni 73100 Lecce
Tel.: 0832320614, FAX: 0832320626, email: ldini@ilenic.unile.it
Apoptosis in vivo is followed almost inevitably by rapid uptake into adjacent
phagocytic cells, a critical process in tissue remodelling, regulation of the immune
response or resolution of inflammation (Savill 1997). Condemned cells are swiftly
identified and engulfed by phagocytes. The fact that 'free' or 'non-phagocytosed'
dying cells are rarely observed in vivo because of their swift removal partly explains
why we have only recently identified apoptosis as a frequent physiological event.
Phagocyte recognition of 'apoptotic self' is also essential in protecting tissues from
inflammatory injury due to leakage of noxious contents from dying cells and possibly
limiting the development of auto-immune responses (Ren and Savill 1998). In fact,
unlike other receptor-mediated phagocytic responses of macrophages, ingestion of
apoptotic neutrophils does not lead to release of pro-inflammatory mediators
(Meagher et al. 1992).
The phagocytosis of apoptotic neutrophils, in contrast to immunoglobulin Gopsonized apoptotic cells, actively inhibits the production of interleukin (IL)-1, IL-8,
IL-10, granulocyte macrophage colony-stimulating factor (G-MCSF) and tumor
necrosis factor-, as well as leukotriene C4 and tromboxane B2, by human
monocyte-derived macrophages (Fadok et al. 1998).
In contrast, production of transforming growth factor (TGF)-beta1, prostaglandin E2
or PAF resultes in inhibition of lipopolysaccharide-stimulated cytokine production
(Fadok et al. 1998). The final intracellular fate of intact ingested cells undergoing
apoptosis is the lysosomal enzyme destruction. However, little is still known about
signalling events downstream of apoptotic cell binding to specific receptors. Recently,
Liu and Hengartner (1998) cloned the ced-6 gene from C. elegans that is required for
engulfment of apoptotic cells. It encodes a protein with a phosphotyrosine-binding
domain and appears to be an adaptor molecule that functions within a specific signaltransduction pathway.
But what are the mechanisms underlining the phagocytosis of apoptotic cells?
8
Recent data indicate that apoptotic cells are marked for disposal by mechanisms
which remain poorly understood. Available data have identified candidate phagocyte
molecules for restraining apoptotic cells (i.e. lectins, thrombospondin (TPS); CD14;
scavenger receptors), transmembrane signalling for phagocytosis (avb3, CD36,
ABC1, an ATP binding Cassette transporter, CED-6) and cytoskeletal reorganization
(CED-5) (Dini et al. 1996; Savill 1998; Wu & Horvitz 1998; Devitt et al. 1998; Luciani
and Chimini1996; Liu and Hengartner 1998).
Therefore, individual phagocytes might employ parallel or redundant phagocytic
receptor systems. It is conceivable that the several systems of recognition on the
surface of the phagocyte proposed to trigger or execute the apoptotic engulfment
may act sequentially, each recognising cells at different stages of the death program.
Nevertheless, a full understanding of this complexity will require definition of
recognition mechanisms which operate in vivo in higher organisms. These aberrant
exposures, as well as several independent mechanisms, allow for the recognition of
apoptotic cells by different phagocyte populations and by non phagocytic cells such
as fibroblasts and epithelial cells.
Recognizing death: phagocytosis of apoptotic cells in the liver.
Phagocytosis, that is one of the peculiar functions of the liver, is beautifully operated
by the sinusoidal cells (i.e. endothelial and Kupffer cells) (Smedsrod et al. 1990; Toth
and Thomas 1992). Endothelial and Kupffer cells have many specific functions that
are essential for the preservation of homeostasis in liver under several conditions
and the endocytosis (including also particulate material from the blood such as
apoptotic cells) is pivotal for this role.
Although apoptosis occurs at a negligible rate in the normal liver, a variety of
physiological conditions, diseases, and xenobiotic treatments can cause this form of
cell death, like stimulation with mitogens or hyperplasia-inducing treatments
(Columbano et al. 1985; Bursch et al. 1986; Dini et al., 1993).
The location of sinusoidal cells in the sinusoids makes these cells the first cells of the
mononuclear phagocyte system to come into contact with particulate and
immunoreactive materials coming from the blood, potentially noxious like apoptotic
cells (Reske et al. 1981).
Among the several alternative mechanisms reported for removal of apoptotic cells, in
the liver, the recognition and phagocytosis of apoptotic cells is operated by means of
hepatic lectin-like receptors (Dini et al. 1996).
The first demonstration that the asialoglycoprotein receptor (ASGPR) (likely in
cooperation with other carbohydrate receptors) is involved in the phagocytosis of
9
apoptotic hepatocytes by healthy ones, was performed on newborn hepatocyte
cultures induced to undergo apoptosis by hormonal treatments (Dini et al 1992).
The presence of galactose/N-acetyl-galactosamine, mannose/N-acetyl-glucosamine
on the surface of apoptotic hepatocytes was observed on cells derived both from the
supernatant of the cultures as well as isolated from livers of rat treated to induce
apoptosis in vivo (Dini et al. 1993).
In the liver the clearance of galactose-terminated particles from the circulation is
performed by a galactose-specific uptake mechanism on Kupffer and endothelial
cells. This receptor shows a high affinity for particulate ligands that expose galactose
group, like (desialylated) erythrocytes. Moreover, the liver endothelial and Kupffer
cells take up a wide range of molecules with a net negative charge by the so-called
scavenger receptor (Van Berker et al.1992) and with mannose- and Nacetylglucosamine residues by lectin-like receptors.
All the main three liver cell types possess receptors that can potentially recognize
apoptotic cells and therefore, play a role in the recognition and subsequent
engulfment of apoptotic cells.
Modification of cell surface molecules has been reported for cells undergoing the
process of apoptosis in different experimental conditions (Dini et al. 1992; Fadok et
al. 1992; Flora and Gregory 1994; Emoto et al. 1997) but very little is known about
receptorial molecules on the dying cells or on the neigbhouring healthy ones.
On the cell surface of non-apoptotic liver cells (i.e. hepatocytes, Kupffer cells,
endothelial cells), the expression of the ASGP-R, the galactose-specific receptor and
the mannose-specific receptor is modulated (enhanced or decreased) during the
entire process of apoptosis, induced in vivo by administration of a potent liver
mitogen, lead nitrate (Dini et al. 1995).
The number and distribution of binding sites is receptor and cell-type dependent
during the days following the metal injection, thus indicating different (time and
modality) involvement during the process of apoptosis for hepatocytes, Kupffer cells
and endothelial cells.
Data also suggest that the galactose and the mannose receptors are acting in
cooperation for the removal of apoptotic cells: decrement of galactose binding sites
are paralleled by mannose binding sites overexpression. In this way, carboydrate
binding sites are always expressed in a great amount on the cell surface, irrespective
of the type of cells and receptors.
The meaning of the all above described changes has to be better understood. To this
purpose we are currently studying the modification of hepatic membrane composition
10
in relation to apoptosis, whose composition may be under the control of
mitochondria.
It is emerging the central role of mitochondria in apoptosis. In fact, a single
intravenous injection of lead nitrate into rats was able to lower the activity of the
mitochondrial tricarboxylate carrier and the lipogenic enzymes as well as to modify
the lipid mitochondrial composition.
However, unlike these biochemical modifications, the ultrastructure of the
mitochondria was not altered (manuscript in preparation). In particular the reduced
activities of cytosolic lipogenic enzymes could suggest a putative mitochondrial
function of apoptotic membrane alterations through tricarboxylate transport activity
decrement (personal communication). Therefore, the mitochondrial control of the
lipogenic enzymes could in turn promote the rapid removal of apoptotic hepatocytes
by neigbhouring and sinusoidal liver cells, by modifications of their cell surface.
With the use of in vivo experiments the role of galactose- and mannose-specific
receptors in the recognition of dead cells was highlighted. In particular, Kupffer cells
at five and fifteen days from the lead nitrate injection are very active in internalizing
apoptotic cells (two-three fold the control), but phagosomes containing apoptotic
hepatocytes are often seen inside the cytoplasm of parenchymal cells and
endothelial cells. Moreover, liver endothelial cells were able to recognize apoptotic
lymphocytes even after isolation and cultivation. Interestingly, apoptotic peripheral
blood lymphocytes are retained by the sinusoids in a heterogeneic distribution:
apoptotic cells in the periportal tract are double those in the perivenous region.
The reason should be find in the differences existing between periportal and
centrilobular endothelial cells regarding fenestration pattern and to the uneven
expression of galactose and mannose-specific receptors.
Mannose receptor expression on the liver endothelium is up-regulated by IL-1, and
was associated with increased removal of apoptotic cells and tumor cell adhesion
(Vidal-Vanaclocha et al. 1994; Dini et al. 1995).
Of note, IL-1b, in addition to the upregulation of mannose receptor activity,
contributes to the sublobular compartmentalization of this endothelial cell function. In
fact, endothelial cells showed a significant heterogeneity of mannose receptormediated endocytosis in response to this cytokine treatment (Asumendi et al. 1996
Summarizing, the entire carbohydrate recognition system (mainly galactose and
mannose specific receptors) of the liver is involved in the homeostatic function of the
regulation of the cell numbers of liver tissue. Multiple data are in favour of the
involvement of hepatic carbohydrate receptors in the apoptotic cell and/or body
clearance: i) the cell surface of dead hepatocytes express great amounts of
11
galactose/ N-acetyl-galactosamine/ mannose residues; ii) hepatocytes, Kupffer and
endothelial cells express on their cell surfaces the carbohydrate receptor systems; iii)
these receptors are modulated differently during the in vivo onset of apoptosis; iv)
during in vivo onset of apoptosis hepatocytes, Kupffer and endothelial cells show
large phagosome containing apoptotic bodies; v) LPS and IL1b stimulation of
endothelial cells markedly enhances the phagocytosis of apoptotic lymphocytes
probably by increasing the carbohydrate receptors expressed on the cell surfaces; vi)
the increase in the rate of apoptosis is paralleled by a modulation of the receptorial
expression of the carbohydrate recognition systems and the removal is reduced of
about 70% by addition of specific saccharide.
REFERENCES
Asumendi A, Alvarez, A, Martinez I, Smedsrod B and Vidal-Vanaclocha F. (1996) Hepatic
sinusoidal endothelium heterogeneity with respect to mannose receptor activity is
interleukin 1 dependent. Hepatology 23: 1521-1529
Bursch W, Dusterberg B and Schulte-Hermann R (1986) Growth, regression and cell death
in rat liver as related to tissue levels of the hepatomitogen cytoproterone acetate. Arch
Toxicol 59: 221-227
Columbano A, Ledda-Columbano GM, Coni P, Faa G, Liguori C, Santacruz G and Pani G
(1985) Occurrence of cell death (apoptosis) during the involution of liver hyperplasia.
Lab Invest 52: 670-677
Devitt A, Moffatt OD, Raykundalia C, Capra JD, Simmons DL and Gregory CD (1998)
Human CD 14 mediates recognition and phagocytosis of apoptotic cells. Nature 392:
505-508
Dini L, Autuori F, Lentini A, Oliverio S and Piacentini M (1992) The clearance of apoptotic
cells in the liver is mediated by the asialoglycoprotein receptor. FEBS Lett 296:174-178
Dini L, Falasca L, Lentini A, Mattioli P, Piacentini M, Piredda L and Autuori F (1993)
Galactose-specific receptor modulation related to the onset of apoptosis in rat liver. Eur
J Cell Biol 61: 329-337
Dini L, Lentini A, Diez Diez G, Rocha M, Falasca L, Serafino L and Vidal-Vanaclocha F
(1995) Phagocytosis of apoptotic bodies by liver endothelial cells. J Cell Sci 108: 967973
Dini L, Ruzittu M and Falasca L (1996a) Recognition and phagocytosis of apoptotic cells.
Scanning Microsc. 10:239-252
Dini L and Carlà EC (1998) Hepatic sinusoidal endothelium heterogeneity with respect to the
recognition of apoptotic cells. Exp Cell Res 240: 388-393
Dini L (1998) Endothelial liver cell recognition of apoptotic peripheral blood lymphocytes.
Biochem Soc Trans 26: 636-639
12
Emoto K, Toyama-Sorimachi N, Karasuyama H, Inoue K and Umeda M (1997) Exposure of
phosphatidylethanolamine on the surface of apoptotic cells. Exp Cell Res 232:430-434
Fadok VA, Savill JS, Haslett C, Bratton DL, Doherty DE, Campbell Paa and Henson PM
(1992a) Different populations of macrophages use either the vitronectin receptor or the
phosphatidylserine receptor to recognize and remove apoptotic cells. J Immunol 149:
4029-4035
Fadok VA, Bratton DL, Frasch SC, Warner ML and Henson PM (1998a) The role of
phosphatidylserine in recognition of apoptotic cells by phagocytes. Cell Death Differ 5:
551-562
Flora PK and Gregory CD (1994) Recognition of apoptotic cells by human macrophages:
inhibition by a monocyte/macrophage-specific monoclonal antibody. Eur J Immunol 24:
2625-2632
Liu QA and Hengartner MO (1998) Candidate adaptor protein CED-6 promotes the
engulfment of apoptotic cells in C.elegans. Cell 93: 961-972
Luciani MF and Chimini G (1996) The ATP binding cassette transporter ABCD1, is required
for the engulfment of corpses generated by apoptotic cell death. EMBO J 15: 226-235
Meagher LC, Savill JS, Baker A, Fuller R and Haslett C (1992) Phagocytosis of apoptotic
neutrophils does not induce macrophage release of thromboxane B2. J Leuk Biol 52:
269-273
Ren V and Savill J (1998) Apoptosis: the importance of being eaten. Cell Death Differ 5:
563-568
Savill JS (1997) Recognition and phagocytosis of cells undergoing apoptosis. Br Med Bull
53: 491-508
Savill JS (1998) Phagocytic docking without shocking. Nature 392: 442-443
Smedsrod B, Pertoft H, Gustafson S and Laurent TC (1990) Scavanger functions of the liver
endothelial cell. Biochem J 266: 313-327
Toth CA and Thomas P (1992) Liver endocytosis and Kupffer cells. Hepatology 16: 255-266
Van Berkel TJC, De Rijke Jb and Kruijt JK (1992) Recognition of modified lipoprotein by
various scavenger receptors on Kupffer and endothelial liver cells. In: (Windler E and
Greten H, eds.) Hepatic Endocytosis of Lipids and Proteins. Munchen, FRG:
Zuckschwerdt Verlag p 443
Wu YC and Horvitz HR (1998) C.elegans phagocytosis and cell-migration protein CED-5 is
similar to human DOCK 180. Nature 392: 501-504
13
MEMBRANE FLOW DURING PHAGOCYTOSIS AND EXOCYTOSIS
IN Paramecium primaurelia, AS STUDIED BY
CONFOCAL LASER SCANNING MICROSCOPY
Paola Ramoino1, Francesco Beltrame2, Alberto Diaspro3 and Marco Fato2
1Dip.
per lo Studio del Territorio e delle sue Risorse (DIP.TE.RIS.), Via Balbi 5, 16126
Genova, 2Dip. di Informatica, Sistemistica e Telematica (DIST), Viale Causa 13, 16145
Genova, and 3INFM & Dip. di Fisica, Via Dodecaneso 33, 16146 Genova.
1Tel.:
+39-0102099459; FAX: +39-0102099323; e-mail: zoologia@unige.it
An elaborate membrane flow system occurs in Paramecium during the
phagocytosis and exocytosis processes. Phagocytosis begins with food vacuole
formation at the posterior end of the oral cavity. Fluid and suspended food particles are
collected in the buccal cavity and directed, by oral membranelle beating, downward to
the cytopharynx and cytostome to form a new food vacuole. Food vacuoles undergo a
series of sequential changes from their formation at the cytostome to their defecation at
the cytoproct (Fok and Allen, 1993). Several types of vesicles enter and leave the food
vacuole membrane during the vacuole’s progression throughout the cell. As the
vacuolar content is digested by lysosomal enzymes, the breakdown substances pass
into the cytoplasm through the vacuolar membrane or by way of small pinocytic
vesicles. The vesicles evaginated from the membrane, go away from the food vacuole
and move in the cytoplasm toward the cytopharynx where they enlarge the membrane
of the nascent food vacuole or can fuse with other food vacuoles (Allen and Fok, 1980).
Trichocyst excretion is similar to other exocytic processes: the trichocyst membrane
fuses with the plasma membrane, the content is released outside the cell and the
membrane is retrieved back into the cell, fragmented and recycled (Plattner, 1989).
In paramecia fed with indigestible particles, the duration of the digestive cycle is
relatively short (20 to 60 minutes), and the phagocytic processes are so ordered with
respect to both timing and intracellular position as to allow the food vacuole selection in
a specific digestion stage using a pulse-chase protocol. By immobilizing living cells
(Sikora and Wasik, 1978) pulsed with a food vacuole marker at succeeding times after
a chase of unlabeled medium, we have visualized in vivo vesicle formation, movement
and fusion during phagocytosis of P. primaurelia using the confocal laser scanning
microscopy (CLSM). The vesicle movement was shown by means of the multimodal
image analysis in fluorescence and the use of the false-color technique (Beltrame et al.,
14
1995; Diaspro et al, 1996). By associating three different colors (red, green or blue)
with three images of the same cell, taken at succeeding time points, a composite image
showing the different positions versus time of the vesicle inside the cytoplasm was
generated.
When the cells previously fed with BSA-FITC are immobilized between 15 to 20
min after chase in sterile medium, the food vacuoles are in the digestion stage and
small pinocytic fluorescent vesicles pinch off. The multimodal image analysis utilizing
the false-color technique shows changes on the direction of movement of the vesicles
going away from the vacuole (Ramoino et al., 1996). Conversely, vesicles which fuse
with food vacuoles move apparently in unidirectional manner. The reutilization of FITClabeled vesicles formed from condensing food vacuoles and from egested food
vacuoles at the cytoproct is evidenced by feeding the cells with a second fluorescent
probe. The nascent and the new-formed food vacuoles are double labeled.
As to the membrane reutilization after the exocytosis process, when synchronous
and massive extrusion of trichocysts is induced (Plattner et al., 1985) in the presence of
WGA-FITC, the lectin in the medium is trapped in the ghosts of the fragmented
trichocyst membrane. The ghost number decreases with
increased time,
and
contemporaneously some vacuoles become fluorescent. We have evidenced the
relation between trichocyst ghosts and the phagosome-lysosme vesicle system by a
double labeling (WGA-FITC for trichocyst membrane retrieval and BSA-Texas Red for
food vacuoles) and by the multimodal analysis utilizing the false-color technique. A
fundamental (red, green or blue) was associated with each of the two fluorescent
images and with a third image taken in phase-contrast mode. This allowed for the
display of a composite image showing the relationship between the markers and their
location inside the cell. It was shown that immediately after trichocyst exocytosis,
ghosts are found in the cortex and are green labeled, while pinocytic vesicles and food
vacuoles are red labeled. As trichocyst ghosts fuse with food vacuoles, the vacuoles
become both Texas Red and FITC labeled (Ramoino et al., 1997).
Hence, the optical CLSM used for in vivo and in situ acquisition of images and the
multimodal analysis utilizing the false-color technique can be considered as powerful
tools to characterize the spatial and temporal features of cellular organelle motion as
well as vesicle fusion.
15
REFERENCES
Allen, R. D., A. K. Fok: Membrane recycling and endocytosis in Paramecium confirmed
by horseradish peroxidase pulse-chase studies. J. Cell Sci., 45: 131-145 (1980).
Beltrame, F., A. Diaspro, M. Fato, I. Martin, P. Ramoino, I. Sobel: Use of stereo vision
and 24-bit false colour imagering to enhance visualisation of multimodal confocal
images. Proc. SPIE, 2412: 222-229 (1995).
Diaspro, A., F. Beltrame, M. Fato, A. Palmeri, P. Ramoino: CLSM image fusion to study
dynamics and networking processing in biological systems through the use of a
bioimage-oriented device. J. Comp. Assisted Microscopy, 8: 119-129 (1996).
Fok, A. K., R. D. Allen: Membrane flow in the digestive cycle of Paramecium. In: H.
Plattner (ed.): Advances in Cell and Molecular Biology of Membranes, Membrane
Traffic in Protozoa. pp. 311-337. JAI Press Inc., Greenwich, CT (1993).
Plattner, H.: Regulation of membrane fusion during exocytosis. Int. Rev. Cytol., 119:
197-286 (1989).
Plattner, H., R. Stürzl, H. Matt: Synchronous exocytosis in Paramecium
cells. IV.
Polyamino compounds as potent trigger agents for repeatable trigger-redocking
cycles. Eur. J. Cell Biol., 36: 32-37 (1985).
Ramoino, P., F. Beltrame, A. Diaspro, M. Fato: Time-variant analysis of organelle and
vesicle movement during phagocytosis in Paramecium primaurelia by means of
fluorescence confocal laser scanning microscopy. Microsc. Res. Tech., 35: 377384 (1996).
Ramoino, P., F. Beltrame, A. Diaspro, M. Fato: Trichocyst membrane fate in living
Paramecium primaurelia visualized by multimodal confocal imaging. Eur. J. Cell
Biol., 74: 79-84 (1997).
Sikora, J., A. Wasik: Cytoplasmic streaming with Ni2+ immobilized Paramecium aurelia.
Acta Protozool., 17: 389-397 (1978).
16
FUNCTIONAL OUTCOMES OF APOPTOTIC CLEARANCE
BY DENDRITIC CELLS
Patrizia Rovere, Attilio Bondanza, Valerié Zimmermann,
Cristina Vallinoto and Angelo A. Manfredi
Laboratory of Tumor Immunology, H.S. Raffaele
Via Olgettina 60, 20132, Milano - Italy.
Tel. 02.2643.2612; FAX: 02.2643.2611; E-mail: Rovere.Patrizia@hsr.it
Single cells are deleted from the midst of living tissue during normal turnover
and embryogenesis. This event is not associated with inflammation or
autoimmunity. However it has been recently pointed out as that apoptotic
cells contain relevant antigens targeted in autoimmune diseases, which are
selectively cleaved, phosphorylated and clustered in membrane blebs of cells
undergoing apoptosis (1,2). Little is known of the clearance of apoptotic cells
during "dangerous" situations, accompanied by extensive cell death and
tissue damage. In these situations macrophages are overwhelmed by
apoptotic cells and other phagocytes, including immature dendritic cells
(DCs), may become involved.
DCs are professional antigen presenting cells involved in the initiation of
immune and autoimmune reactions (3,4). Immature DCs express low levels
of MHC class I, class II and co-stimulatory molecules and are poor at
initiating immune responses (3). They satisfy the requirements for tolerance
induction via cross-presentation of self antigens, since they capture antigens
at the periphery, process them into the class I and class II pathways and
traffic to draining lymph nodes (3,5). Infectious agents induce proinflammatory cytokine secretion and DCs maturation: DCs lose the ability to
take up antigens while up-regulate MHC molecules, adhesion and costimulatory signals, thus becoming able to trigger productive activation of
antigen-specific T cells (4,5).
The outcome of the interaction of apoptotic cells with dendritic cells
(DCs) is still under debate. To analyze the interaction between DCs and
bystander apoptotic cells, we relayed on the possibility to generate in vitro
high numbers of immature DCs by culturing bone marrow precursors in the
presence of recombinant mouse GM-CSF (20 ng/ml) and IL-4 (50 ng/ml). As
a source of apoptotic cells we used a murine lymphoma, RMA, which
17
express a leaderless non-secreted mutant of the OVA antigen (OVA-RMA
cells). OVA-RMA cells were committed to apoptosis by U.V. irradiation (6).
Immature DCs internalize apoptotic biotin-labelled OVA-RMA cells, as
assessed by flow cytometry after addition of FITC-labelled streptavidin. The
phagocytosis was inhibited at 4°C or by CCD (that disrupts actin-based
cytoskeleton) and depended on the number of apoptotic cells.
Antigens from internalized apoptotic cells are also processed and
presented to T lymphocytes (7). Interestingly, DCs which internalized either
low or high numbers of OVA-RMA apoptotic cells activated OVA-specific
class I- (B3Z) and class II- (BO97.10) restricted hybridoma T cells, as
assessed by measuring specific IL-2 secretion. Intracellular processing by
DCs of the internalized apoptotic OVA-RMA cells was necessary for T cell
activation. CCD substantially inhibited T cell activation in vitro, suggesting
that active phagocytosis and integrity of the actin based cytoskeleton are
required. BFA, which inhibits newly synthesized protein egress from the
endoplasmic reticulum, interfered with both class I- and class II-restricted T
hybridoma cell activation, implicating nascent MHC molecules in antigen
presentation. DCs pulsed with living OVA-RMA cells or with OVA-RMA cells
killed by necrosis did not activate T cell hybridomas, suggesting that the
mode of cell death, and possibly the re-organisation of the plasma membrane
typical of apoptosis, is relevant for correct internalization, processing and
presentation of dying cells. Both early (i.e. cells irradiated and allowed to
undergo apoptosis in the presence of DCs) and late apoptotic cells (i.e. cells
allowed to proceed into the apoptotic pathway until they all were permeable
before incubation with DCs) cross-activated T cells. Therefore apoptotic cells
that escape immediate clearance by professional scavenger phagocytes in
vivo and reach a late apoptotic stage may retain the ability to sustain immune
(and possibly autoimmune) responses. Supernatants of apoptotic OVA-RMA
cells did not endow D1 cells with the ability to activate T cell, ruling out a role
of soluble antigen release from apoptotic cells.
Antigen presentation may result either in T cell activation or in their
functional blockade. The outcome is influenced by pro-inflammatory
maturative signals: efficient T cell cross-priming requires fully mature DCs
(discussed in 8,9). To determine the release of IL-1, TNF-, IL-10, and IFN we challenged DCs with increasing amounts of apoptotic OVA-RMA cells.
DCs cells secreted substantial amounts of IL-1 and TNF-, but not of INF-,
when challenged with high numbers of apoptotic cells. Of interest, the
18
recognition of high numbers of apoptotic cells induced the release of minute
amounts of IL-10 (around 10 pg/ml). Minimal cytokine production was
detected when DCs were challenged with low number of apoptotic cells
(0.5:1 apoptotic cells:DCs ratio). The molecular basis of the different
response to the same stimulus are currently under investigation.
We then verified whether the secretion of proinflammatory cytokines
induced by high numbers of apoptotic cells was accompanied by DC
maturation: it induced the consistent up-regulation of the membrane
expression of MHC class II (I-A), CD86 and CD40 molecules. A similar effect
was obtained by adding exogenous recombinant TNF-. Low numbers of
apoptotic cells, that induced the secretion of minimal amounts of TNF- and
of no IL-1 did not influence the maturation of DCs. Phagocytosis of an
excess of inert substrates was not per se sufficient to trigger DC maturation.
All together these results support the hypothesis that DCs control their own
maturation by selectively releasing maturative factors when challenged with a
relative excess of apoptotic cells.
These data suggest that the number of apoptotic cells that die at a given
time influences DCs maturation and therefore their ability to cross-activate T
lymphocytes. High numbers of apoptotic cells, mimicking a failure of their in
vivo clearance, are therefore sufficient to trigger DCs maturation and
presentation of intracellular antigens from apoptotic cells, even in the
absence of exogenous "danger" signals.
REFERENCES
1. Casciola-Rosen L. A., G. Anhalt, and A. Rosen. 1994. Autoantigens
targeted in systemic lupus erythematosus are clustered in two populations
of surface structures on apoptotic keratinocytes. J. Exp. Med. 179:1317.
2. Rosen, A., L. Casciola-Rosen. 1999. Autoantigens as substrates for
apoptotic proteases: implications for the pathogenesis of systemic
autoimmune disease. Cell Death Diff. 6, 6-12.
3. Cella, M., F. Sallusto, and A. Lanzavecchia. 1997. Origin, maturation and
antigen presenting function of dendritic cells. Curr. Opin. Immunol. 9:10.
4. Banchereau J., and R. M. Steinman. 1998. Dendritic cells and the control
of immunity. Nature 392:245.
5. Winzler, C., P. Rovere, M. Rescigno, F. Granucci, G. Penna, L. Adorini,
V. S. Zimmermann, J. Davoust, and P. Ricciardi-Castagnoli. 1997.
19
Maturation stages of mouse dendritic cells in growth-factor-dependent
long-term cultures. J. Exp. Med. 185:317
6. Bellone, M., G. Iezzi, P. Rovere, G. Galati, A. Ronchetti, M. P. Protti, J.
Davoust, C. Rugarli, and A. A. Manfredi. 1997. Processing of engulfed
apoptotic bodies yields T cell epitopes. J. Immunol. 159:5391.
7. Rovere, P., C. Vallinoto, A. Bondanza, M. C. Crosti, M. Rescigno, P.
Ricciardi-Castagnoli, C. Rugarli, and A.A. Manfredi. 1998. Cutting edge:
Bystander apoptosis triggers dendritic cell maturation and antigen
presentation function. J. Immunol. 161:4467.
8. Rovere, P., A. A. Manfredi, C. Vallinoto, V. S. Zimmermann, U. Fascio, G.
Balestrieri, P.
Ricciardi-Castagnoli, C. Rugarli, A. Tincani, M.G.
Sabbadini. 1998. Dendritic cells preferentially internalize apoptotic cells
opsonized by anti-
-glycoprotein I antibodies. J. Autoimmun., 11, 403-
411.
9. Rovere, P., C. Vallinoto, U. Fascio, M. Rescigno, M.C. Crosti, P.
Ricciardi-Castagnoli, P., G. Balestrieri, A. Tincani, M.G. Sabbadini, and A.
A. Manfredi. 1999. Dendritic cells present antigens from opsonized
apoptotic cells in a proinflammatory context. Arthritis Rheum. In press.
20
PHAGOCYTOSIS OF APOPTOTIC BODIES BY MICROGLIAL CELLS
IN CULTURE: FATE OF RIBONUCLEOPROTEINS
Carlo Pellicciari1, Maria Grazia Bottone1, Debora Duò1, Marco Biggiogera1,
Patrizia Rovere2 and Angelo A. Manfredi2
1Dipartimento
di Biologia Animale, Piazza Botta 10, University of Pavia and
2H.S.
1Tel.
Raffaele, Via Olgettina 60, 20132, Milano
+39.0382.506420; FAX: +39.0382.506325; E-mail: pelli@unipv.it
Phagocytosis is the final common event in vivo for most of apoptotic cells, which are
recognized and removed by phagocytes. Several receptors have been described
which mediate the recognition and binding of apoptotic cells by macrophages
(reviewed by Luciana Dini, page 7 of this volume).
It is generally accepted that phagocytosis of apoptotic cells prevents the onset of
secondary necrotic events, that would make the cell potentially noxious and injurious
for the surrounding tissue. In particular, one may assume that degradation of
macromolecular complexes would take place in phagolysosomes, whether or not
apoptosis-activated cleavage has occurred beforehand (Gregory, 1998).
However, there is growing consensus on the fact that some pathologies like
autoimmune diseases may be the consequence of alterations in the mechanism of
clearance of apoptotic cells by phagocytes (see Piacentini, 1999).
In fact, it has been proposed that during apoptosis proteolytic enzymes such as
caspases may generate protein fragments which represent neoantigens, capable of
eliciting an autoimmune response in susceptible individuals; furthermore, potential
autoantigens may generate novel protein-protein or protein-nucleic-acid complexes,
as a consequence of apoptosis-induced molecular rearrangements (Casciola-Rosen
et al., 1995). In addition, failed apoptotic clearance and release of autoantigens from
disintegrating apoptotic cells and bodies could potentiate presentation of constituent
autoantigen peptides (Ren and Savill, 1998). In particular, specific groups of
intracellular autoantigens (e.g., ribosomal 52kDa Ro) were found to cluster in the
small blebs at the cell surface of apoptotic cells, whereas others (such as
deoxyribonucleoproteins
of
nucleosomes
and
U1-70kDa
small
nuclear
ribonucleoproteins [snRNPs]) are found in apoptotic bodies, often within the nuclear
fragments resulting from karyorrhexis. It is interesting to observe that in systemic
lupus erythematosus (SLE), autoantigens may be divided into two groups, depending
on their sensitivity to proteolytic cleavage by apoptotic caspases: many of the
frequent targets of high-titer autoantibody response (in more than 15% of SLE
21
patients) are not cleaved during apoptosis (among which, deoxyribonucleoprotein
and RNP antigens, Ro and Sm: reviewed by Rosen and Casciola-Rosen, 1999).
We have recently demonstrated (Biggiogera et al., 1997a,b; 1998; Pellicciari et al.,
1999) that during spontaneous and drug-induced apoptosis RNP-containing
structures
undergo
severe
rearrangement.
In
fact,
along
with
chromatin
condensation, perichromatin fibrils, interchromatin and perichromatin granules, and
nucleolar components (which usually occupy distinct nuclear domains in non
apoptotic cells) segregate in the areas of non-condensed chromatin, giving rise to
heterogeneous
fibrogranular
aggregates
which
we
called
HERDS
(for
Heterogeneous Ectopic Ribonucleoprotein Derived Structures: Biggiogera et al.,
1998).
HERDS are extruded from the nucleus into the cytoplasm, and are finally released at
the cell surface inside apoptotic blebs and bodies (Biggiogera et al., 1997a).
Cytochemical investigations at light and electron microscopy demonstrated that
during this process RNP protein moieties are always recognized by specific
antibodies (Biggiogera et al., 1997b), and that RNA is also present in sufficient
amount to be detected by cytochemical techniques (Biggiogera et al., 1998). This
demonstrates that in general RNP aggregates, even when present ectopically, are
very resistant to degradation by apoptosis-activated proteases.
In the attempt to elucidate the fate of apoptotic nuclear RNPs after they had been
phagocytosed, we set up a simple cell system in vitro. Microglial N9 cells (derived
from fetal mouse brain, as described in Righi et al., 1989) were used, which
constitutively exhibit the capability of phagocytosing apoptotic cells and bodies. As
apoptotic cell model, mouse thymocytes were selected in which apoptosis was
induced by etoposide treatment (10 M for 1 hr followed by 4 hr recovery in
etoposide-free medium: described in Pellicciari et al., 1996).
Apoptotic thymocytes were supravitally stained with PKH2-GL (Sigma Chem. Co.: a
fluorescent dye with an aliphatic tail that interact with the membrane phospholipids,
thus allowing an effective superficial cell labeling, without affecting membrane
integrity and cell-to-cell interaction).
To assess the occurrence of phagocytosis, N9 were "fed" with PKH2-stained
apoptotic thymocytes and observed in fluorescence microscopy: it was found that
after 15 min, 10-15% N9 cells had already phagocytosed apoptotic thymocytes, while
after 90 min more that 70% of N9 cells did it. A previous treatment with chloroquine
(a weak diffusible base, largely used in autoimmune patients, which is known to
impair lysosome activity) allowed to confirm that apoptotic cells and bodies do follow
the phagolysosome pathway.
22
Immunolabeling with antibodies directed against snRNPs demonstrated that nuclear
RNPs of apoptotic origin may be detected in the cytoplasm of N9 cells at both
fluorescence and electron microscopy, up to 60 min after exposure to apoptotic
thymocytes.
This evidence suggests that degradation of RNP clusters from apoptotic cells in the
lysosome compartment of phagocytes may be a slow (and possibly incomplete)
process, from which potentially harmful polypeptide molecules may result, whenever
usually cryptic determinant are processed by antigen presenting cells, thus becoming
visible to the immune system.
REFERENCES
Biggiogera M., Bottone M.G., Martin T.E., Uchiumi T., Pellicciari C. (1997): Still immunodetectable nuclear RNPs are extruded from the cytoplasm of spontaneously apoptotic
thymocytes: Exp. Cell Res., 234: 512-520
Biggiogera M., Bottone M.G., Pellicciari C. (1997): Nuclear ribonucleoprotein-containing
structures undergo severe rearrangement during spontaneous thymocyte apoptosis. A
morphological study at electron microscopy. Histochemistry Cell Biol., 107: 331-336.
Dini L. (1999): Apoptosis: the importance of being phagocytosed. In: Investigating cell
dynamics and death by conventional and confocal microscopy. Pavia, May 3-6, 1999,
pp: 7-12
Gregory C.D. (1998): Phagocytotic clearance of apoptotic cells: food for thought. Cell Death
Differ. 5: 549-550,.
Pellicciari C., Bottone M.G., Biggiogera M. (1999) Restructuring and extrusion of nuclear
ribonucleoproteins (RNPs) during apoptosis. Gen. Physiol. Biophys (in press)
Pellicciari C., Bottone M.G., Schaack V., Barni S., Manfredi A.A. (1996): Spontaneous
apoptosis of thymocytes is uncoupled with progression through the cell cycle. Exp. Cell
Res., 229: 370-377.
Pellicciari C., Bottone M.G., Schaack V., Manfredi A.A., Barni S. (1996): Etoposide at
different concentrations may open different apoptotic pathways in thymocytes. Eur. J.
Histochem., 40, 289-298.
Piacentini M. (1999): Apoptosis and autoimmunity: two sides of the coin. Cell Death Differ. 6:
1-2
Ren Y. And Savill J. (1998): Apoptosis: the importance of being eaten. Cell Death Differ. 5:
563-568.
Righi M., Mori L., De Libero G., Sironi M., Biondi A., Mantovani A., Denis-Donini S., RicciardiCastagnoli P. (1989). Monokine production by microglial cells. Eur. J. Immunol. 19:
1443-1451.
Rosen A. and Casciola-Rosen L. (1999): Autoantigens as substrates for apoptotic proteases:
implications for the pathogenesis of systemic autoimmune disease. Cell Death Differ. 6:
6-12.
23
BACTERIAL MANIPULATION OF THE ACTIN CYTOSKELETON
Jürgen Wehland
Department of Cell Biology, Gesellschaft für Biotechnologische Forschung
D-38124 Braunschweig, Germany
Tel: +49.531.6181.415; FAX: +49.531.6181.444; E-mail: jwe@gbf.de
The
actin-based
motility
of
the
intracellular
bacterial
pathogen
Listeria
monocytogenes has recently attracted much attention as a model system to analyse
actin dynamics, e.g. the process of actin filament nucleation. When Listeria enter the
cytoplasm of host cells, they induce the polymerizationof actin filaments at their
surface. The actin cloud is then rearranged into a "comet tail" at one bacterial pole
and the assembly of new actin filaments provides the driving force for intra- and
intercellular motility. The bacterial surface protein ActA is both necessary and
sufficient for the recruitment of actin. Thus far only two host cell proteins have been
shown to bind directly to ActA: the vasodilator-stimulated phosphoprotein (VASP)
and the related murine protein Mena. Both proteins are ligands for profilin. In
addition, the Arp 2/3 complex, initially identified in Acanthamoeba, is thought to be
involved in actin-based motility of Listeria since this protein complex stimulates the
assembly of actin filaments on the bacterial surface in cell free extracts, and also
promotes the nucleation of actin filaments in vitro.
Using a mouse brain extract
reconstituted system, combined with transfection studies based on myc- and GFPtagged proteins, as well as Listeria actA mutants, we are able to dissect the process
of actin filament formation induced by Listeria. VASP and Mena are required for
efficient intracellular movement and cell to cell spreading of Listeria, whereas the Arp
2/3 complex is essential for Listeria motility.
24
CELL MEMBRANE AND CYTOSKELETON MODIFICATIONS DURING
APOPTOSIS IN NEOPLASTIC CELL LINES
Sciola L., °Spano A. and °Barni S.
Dipartimento di Scienze Fisiologiche, Biochimiche e Cellulari, Università di Sassari;
°Dipartimento di Biologia Animale, Università di Pavia.
Tel.
+39-079-228651;
FAX +39-079-228615; e-mail: sciola@ssmain.uniss.it
Several subcellular parameters can be used to evaluate the cellular changes
that lead to apoptosis. In the early stages of apoptosis, dynamic modifications occur
in plasma membrane structure and on the cell surface, such as the expression of
specific antigens (e.g. receptors) or alterations in carbohydrate structure. These
modifications have a great significance because represent signals for recognition and
removal of apoptotic cells by phagocytes (1).
The loss of plasma membrane asymmetry is an early event in apoptosis,
independent of the cell type, resulting in the exposure of phosphatidylserine (PS)
residues on the outer phospholipid leaflet, while the membrane integrity remains
unchanged (2).
The phospholipid asymmetric distribution is maintained partially in cells by an
enzyme, aminophospholipid translocase, which catalyzes the translocation of lipid
molecules from the outer to the inner leaflet of membrane. It is proposed that the
surface exposure of PS in the apoptotic cells is due to both the failure of this enzyme
and modification of actin cytoskeleton (3).
Furthermore, during apoptosis, cytoskeleton could play an important role even
in the modifications that appear at level of other surface molecules by contributing to
a general rearrangement (e.g. clustering of membrane proteins). The actin
microfilaments can have a particular role in the dynamics of cytoplasmic
modifications related to the formation and the release of blebs, and during cytoplasm
cleavage with formation of apoptotic bodies (4).
Our previous ultrastructural analysis, performed by means of scanning
electron microscopy, evidentiated that the progression of apoptotic event (besides
the early phase) reflects on dramatic modifications of cell surface, which mainly
consist on loss of blebs, with consequent persistence of cell residues showing
depressions or scars in correspondence of bleb release areas. These situations
represent the probable expression of advanced stages of apoptotic degeneration. To
clarify these aspects we have reproduced experimental conditions that could
originate situations with different incidence of cells in early and late stages of
25
apoptosis. We have stimulated a promyelocytic cell line (HL-60) with etoposide
(VP16) at different concentrations and times; the assessment of the apoptotic death
has been performed by flow cytometry.
In this work we have therefore analysed, at plasma membrane level, the
progression of the apoptotic phenomenon by different approaches. The role of PS
was evaluated by means of flow cytometry and fluorescence microscopy after a
double staining with Annexin V-FITC, to label PS and PI to label DNA.
In the same experimental conditions, this evaluation has been integrated with
phospholipid pattern determination by thin layer chromatography (TLC), after
labelling the aminophospholipids with fluorescamine; the aim was to evidence the
possible changes of phospholipid distribution.
Furthermore the reorganisation of the microfilament network, during apoptosis,
was evaluated by fluorescence microscopy in the hepatoma adherent Chang cell line
treated with VP-16. This aspect has been related to the determination of the lectin
ConA-FITC binding quantitatively analyzed by flow cytometry also in the
promyelocytic cell line HL-60.
The results obtained by means of both ultrastructural and fluorescence
microscopy seem to indicate that blebbing phenomenon is the result of a deep
rearrangement of apoptotic cell membrane that, if it could reflect signal organization
for their removal, on the other hand could represent the onset of dramatic structural
and functional modifications.
Our data indicate that bleb membrane release, as the presence of depressions
and scars testifies at cellular residue level, could coincide with the loss of some
typical signals of the early apoptotic phase, as the data obtained after evaluation of
both Annexin V-FITC and ConA-FITC binding seem to demonstrate (Figg. 2a, 2d).
The morphological data related to these fluorochromizations are partially confirmed
by flow cytometry (Fig. 1).
The analysis of microfilament staining (phalloidin-TRITC) allowed us to
evidence specific sequential changes and reorganisation of actin: i) retraction from
the adhesion substrate; ii) loss of stress fibers; iii) accumulation in the perinuclear
region in a ring-like structure, that could play a role during nuclear fragmentation. In
general it is often possible to evidence a less remarkable expression of actin,
especially in the advanced stages of apoptosis (Fig. 2b).
1) The simultaneous fluorochromization with ConA-FITC showed that the progression
of apoptosis is related to a decrease of intensity of lectin binding which loses its
uniform distribution in the cell periphery and appears as typical patching
26
Cell number
a
d
g
e
h
f
i
G1
G1
Ap
S G2/M
S



bb
Ap

c
DNA content
Fig. 1
Fig.2
27
features, until its disappearance in the late stages of the phenomenon (Fig. 2d).
Migration, polarisation and loss of ConA binding seems to be related to formation and
release of surface protrusions, as a consequence of the actin network
rearrangement.
However, the absence of these signals does not seem to influence the
recognition and the removal of apoptotic cells by scavenger cells. In fact preliminary
SEM analysis performed on HL-60/THP-1 co-cultures also indicates that the loss of
typical early apoptosis signals does not seem to influence the phagocytosis of cells
during the late apoptosis.
References
1. Savill J., Fadock V.A.., Henson P. and Haslett C.: Phagocyte recognition of cells
undergoing apoptosis. Immunol. Today 14, 131-136, 1993.
2. van Engeland M., Nieland L.J.W, Ramaekers F.C.S., Schutte B. and Reutelingsperger
C.P.M.: Annexin V-affinity assay: a review on an apoptosis detection system based on
phosphatidylserine exposure. Cytometry 31, 1-9, 1998.
3. Fadock V.A., Voelker D.R., Campbell P.A., Cohen J.J., Bratton D.L. and Henson P.M.:
Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific
recognition and removal by macrophages. .J. Immunol. 148, 2207-2216, 1992.
4. Cotter T.G., Lennon S.V., Glynn J.M. and Green D.R.: Microfilament-disrupting agents
prevent the formation of apoptotic bodies in tumor cells undergoing apoptosis. Cancer
Res.. 52, 997-1005, 1992.
Fig. 1 - Flow cytometry of cell DNA content, bivariate Annexin-FITC/PI analysis and
profiles of ConA-FITC binding in untreated (a, d, g) and VP-16-treated cells
for 12 h (b, e, h) and 24 h (c, f, i).
Fig. 2 - Fluorescence microscopy: a) Annexin V-FITC/PI double labelling of VP16treated HL-60 cells (arrow: early apoptosis; arrowhead: middle apoptosis;
asterisk: late apoptosis). b) Staining with phalloidin-TRITC/HO342 in VP16treated Chang cells (arrows: lamellipodia with evident persistence of actin in
an apoptotic cell in karyorrhexis): c, d) Same microscopic field of VP16treated Chang cells: c) fluorochromization with phalloidin-TRITC/HO342; d)
fluorochromization with ConA-FITC/HO342.
28
LASER SCANNING MICROSCOPY: PRINCIPLE, INSTRUMENTATION
AND APPLICATIONS IN THE STUDY OF CYTOSKELETON
Heinz Gundlach
Research and Technology Division, Carl Zeiss,Oberkochen
Tel. +49 - 7364-20-2071;
FAX +49 - 7364-20-4013; e-mail: gundlach@zeiss.de
The last 20 years have seen an unexpected great renaissance and partially
revolution in light microscopy. One of these reasons are the recent progress in
fluorescence microscopy, achieved by multiparameter fluorescence techniques, by
improvement of conventional photomicrography as well as by optoelectronic imaging
using CCD techniques, confocal laser scanning microscopy, image processing and
image analysis. In multiple fluorescence microscopy digital imaging techniques are
more and more being used to supplement conventional fluorescence documentation
and analysis. The potential of image processing by gain = contrast and offset =
brightness facilitates the optimized image and analysis of a labeled specimen and
quantitative data on signal intensities or measurements can be easily assessed.
A laser scanning microscope consists of a light microscope ( upright or inverted),
laser light sources instead of halogen or HBO/Xenon sources. Since only a small
specimen point is recorded, the entire image of a specimen is generated by either
moving the specimen (stage scanning) or moving the beam of light across the
stationary specimen (beam scanning). The final image is performed by means of
photomultiplier or diode sensors. One of the major advantages is the possibilty of
programmable zooming to enlarge the final magnification without loss of contrast and
resolution.
The confocal laser scanning microscope is a powerful tool for the study of the threedimensional structure of microscopic biological specimen revealed with fluorescent
labels. The digital images of the optical sections provided by the CLSM can be
transferred into the memory of a peripheral computer where they can be organized
and combined to reconstruct the original volume. The confocal laser scanning
microscope combines two different principles. The first element is scanning laser
technology. Instead of illuminating the whole field of view, only a single specimen
point is illuminated at a time with a flying spot (laser beam) and the light emitted or
reflected from this single point is recorded by a sensor (photomultiplier, CCD or
diode). A deflection device generally based on galvanometric mirrors permits to scan
the specimen point by point. The advantage of this method is that the stray light and
flare are reduced and the image becomes a closer rendition of the specimen. Mainly
29
in the transmitted light DIC Nomarski and phase contrast the final image are more
crisp and of higher contrast compared to conventional microscopy.
The second element is the confocal filtering. This is obtained by placing a pinhole
aperture in the intermediate image plane. Only the emitted or reflected light coming
from the focal plane passes completely through the pinhole aperture since it
converges exactly at this level. Light coming from out of focus planes is mostly
rejected. This results in a reduction of the depth of focus, therefore an increase of the
axial resolution by rejection of out of focus flare.
In the fluorescence mode, the light source is generated by various lasers at different
excitation wavelength: a current setting is made of a helium-neon internal laser with a
543 nm ray and an external argon laser with two rays (488 nm and 514 nm )
The different fluorescences of a specimen stained by more than one fluorochrome
may be recorded simultaneously by means of multiple photodetectors associated to
beam splitter systems (dichroic mirror) that will seperate the different emission
wavelangth.
Confocal microscopic equipment is available as an add-on to a standard microscope
or in the form of more fully integrated and stable devices, including computer control
equipment. The inverted confocal microscope allows the application of living
specimens in chambers and offers easy handling of micromanipulating or injecting
devices.
The latest generation of confocal laser scanning microscopes consists of highly
integrated scanning modul and fully motorized microscopes, upright as well as
inverted. Up to four confocal detection channels are available for fluorescence or
reflected-light applications. Each channel has a photomultiplier of high sensitivity
across the entire spectrum and a pinhole with individual diameter and xy
adjustments. To achieve perfect results, the electronics must match the quality of the
optics. Up to 2048 x 2048 pixels brings out finest details, even with subsequent
zooming. Some applications will be shown in the fielt of cell biology and cytoskeleton
research.
REFERENCES (Selection of reviews only):
Arndt-Jovin, D.J. et al.: Fluorescence Digital Imaging Microscopy in Cell Biology. Science
230, 247 - 256, 1985
Pawley, J.B. editor : Handbook of Biological Confocal Microscopy. Plenum Press New York
and London, 1990
Shotton, D.M.: Confocal Scanning Optical Microscopy and its Applications for Biological
Specimen (a review). J. Cell Sciences 94, 175 – 206, 1989
Wilson, T. : Confocal Microscopy. Academic Press, London and New York, 1984
30
STRUCTURE AND FUNCTIONAL ASPECTS
OF THE NUCLEAR ENVELOPE.
Marie-Christine Dabauvalle
Department of Cell and Developmental Biology
Biocenter of the University of Würzburg,
Am Hubland, D-97074, Germany.
Tel.: +49.931.8884273; FAX: +49.931.8884252
E-mail: dabauvalle@biozentrum.uni-wuerzburg.de
The nuclear envelope of eukaryotic cells is composed of three separate membrane
domains: (i) the outer nuclear membrane is in continuity with the endoplasmic
reticulum; (ii) the membranous wall of the nuclear pore complex is located where the
outer and inner nuclear membrane fuse to form the pore channel; (iii) the inner
nuclear membrane is associated with a meshwork of proteins, the nuclear lamina.
The inner nuclear membrane is structurally and functionally distinct from the other
two membrane domains. It contains specific integral membrane proteins that link the
membrane to the underlying lamina and the peripheral chromatin (1). Integral
membrane proteins of the inner nuclear membrane are implicated in the structural
organization of the nucleus by their binding to lamins and chromatin (1). These are
the lamin B receptor/p58 (2), the lamina-associated polypeptides 1A-C (3) and 2 (4),
otefin (5) and emerin (6).
We have analyzed the distribution of emerin during interphase and mitosis, especially
in relation to the nuclear lamins, to LAP2 proteins and to the spindle apparatus during
nuclear envelope assembly. Confocal laser scanning microscopy after doublelabelling experiments with emerin and tubulin suggests a close interaction of these
proteins and indicates that emerin participates in the re-organisation of the nuclear
envelope after mitosis.
Another field of interest are the nuclear pore complexes (NPC). The nuclear
envelope is interrupted at numerous sites by NPCs, which provide plasmatic
channels through the double-layered nuclear membrane barrier. Although several
protein constituents, collectively termed “ nucleoporins ”, have been described and
molecularly characterized (7), only limited information is presently available on the
biochemical nature of a minority of the various gross morphological components of
the NPCs. By using indirect immunofluorescence and immunogold electron
microscopy we have analyzed the structure of the nuclear pore complex. The pore
channel is flanked by two coplanar rings or annuli, one attached to the cytoplasmic
31
and one to the nucleoplasmic pore margin (8). The central plug or “ transporter
assembly ” is located within the pore channel through which active nucleocytoplasmic
transport takes place (9). In addition, short fibrils emanate from the cytoplasmic face
of the NPCs and significantly longer NPC-associated fibrils extend from the inner
annulus into the nucleoplasm. It is conceivable that both of these fibrils provide
docking sites in the receptor-mediated nucleoplasmic transport of proteins prior to
their translocation through the NPC (10,11).
Numerous studies from different laboratories have shown that most proteins
translocate through the NPC in an energy and signal-dependent manner (12).
Nuclear import is mediated by short stretches of basic amino acids, called nuclear
localization signals (NLS), which interact with the importin /complex or directly with
alternative import receptors (12). Nuclear export of proteins, on the other hand, is
mediated by so called nuclear export signals (NES), characterized by clusters of
leucine residues as identified in a variety of cellular and viral proteins (12). The
prototypic NES was originally identified in the activation domain of the HIV Rev
protein (13,14). It has been shown that the importin- like factor CRM1 is essential
for NES mediated nuclear export, suggesting that CRM1 is a general export receptor
for leucine-rich NESs in eukaryotic cells (15).
In our study we were able to show by immunogold electron microscopy that the
eukaryotic initiation factor 5A (eIF-5A) accumulates at the nucleoplasmic face of the
NPC and in particular at the NPC-associated filaments. Furthermore, microinjection
studies revealed that eIF-5A interact with the general nuclear export receptor
CRM1and that eIF-5A is transported from the nucleus to the cytoplasm. Our data
demonstrate that eIF-5A is a factor which constantly shuttles between the nucleus
and the cytoplasm of eukaryotic cells.
REFERENCES
(1) Ye, Q., Barton, R. M. and Worman, H.J. (1998) In Subcellular Biochemistry:
Intermediate filaments (ed. H. Hermann and J. Robin Harris), pp. 587-610.
(2) Worman, H.J., Evans, C.D. and Blobel, G. (1990) J. Cell Biol. 117, 245-258.
(3) Martin, L., Crimaudo, C, and Gerace, L. (1995) J. Biol. Chem. 270, 8822-8828.
(4) Dechat, T., Gotzmann, J., Stockinger, A., Harris, C.A., Talle, M.A., Siekierka, J.J.
and Foisner, R. (1998) EMBO J. 17, 4887-4902.
(5) Ashery-Padan, R., Ulitzur, N., Arbel, A., Goldberg, M., Weiss, A., Maus, N.,
Fisher and Gruenbaum, Y. (1997) Mol. Cell Biol. 17, 4114-4123.
(6) Manilal, S., Nguyen, T. M., Sewry, C. A. and Morris, G. E (1996). Hum. Molec.
Genet. 5, 801-806.
32
(7) Bastos, R., Pante, N. and Burke, B. (1995) Int. Rev. Cytol. 162B, 257-302.
(8) Akey, C.W. and Rademacher, M. (1993) J. Cell Biol. 122, 1-19.
(9) Feldherr, C.M. and Akin, D., (1997) J. Cell Sci. 110, 3065-3070.
(10) Wilken, N., Senécal, J.L., Scheer, U. and Dabauvalle, M.C. (1995) Eur. J. Cell
Biol. 68, 211-219.
(11) Bastos, R., Lin, A., Enarson, M. and Burke, B. (1996) J. Cell Biol. 134, 1141-1156.
(12) Görlich, D. (1998) EMBO J. 17, 2721-2727.
(13) Fischer, U., Huber, J., Boelens, W.C., Mattaj. I.W. and Lührmann, R. (1995) Cell
82, 475-483.
(14) Wen, W., Meinkoth, J.L., Tsien, R.Y. and Taylor, S.S. (1995) Cell 82, 463-473.
(15) Stade, K., Ford, C.S., Guthrie, C. and Weis, K. (1997) Cell 90, 1041-1050.
33
DYNAMIC DISTRIBUTION OF hnRNP PROTEINS IN MAMMALIAN
CELLS: IDENTIFICATION OF NEW NUCLEAR COMPARTMENTS
Giuseppe Biamonti, Ilaria Chiodi, Claudia Ghigna, Fabio Cobianchi,
Florian Weighardt and Silvano Riva
IGBE CNR Via Abbiategrasso 207, 27100 Pavia.
Tel: +39-0382-546322
FAX: +39-0382-422286
E-mail: biamonti@igbe.pv.cnr.it
The hnRNP protein family: an overview.
Structural Properties
Origin of complexity
RNA binding domains
Binding to Nucleic Acids
Auxiliary domains
Protein-Protein interactions
Functions
Role in splicing
Subcellular localization and role in mRNA export.
Role in translation
HAP: a new hnRNP protein relocates in few nuclear granules upon heat shock.
---------------------------The hnRNP protein family: an overview
In eukaryotic cells RNA polymerase II transcripts (pre-mRNA or hnRNA) are
complexed with a set of polypeptides called heterogeneous ribonucleoproteins or
hnRNPs. In human cells the hnRNP family consists of about twenty major proteins or
group of proteins, as abundant as histones, named A1 (34 kDa) to U (120 kDa)
according to their molecular mass. Until recently, hnRNPs were regarded as mere
structural proteins that, upon packaging nascent hnRNAs in a nucleosome like
structure (hnRNP complex), protect RNA from unspecific degradation. In the last few
years a direct and specific role of these proteins in different aspect of mRNA life,
namely pre-mRNA splicing and export to the cytoplasm of mature mRNA, has started
to emerge.
34
Protein composition of hnRNP complexes as
revealed by 2xD gel analysis.
Structural Properties
Origin of complexity.
The hnRNP family consists of 20 major proteins identifiable by 2xD gel
electrophoresis. The complexity of this pattern is increased both by the differential
expression of the hnRNP genes and by post-translational modifications.
Some hnRNPs are subjected to cell-type and growth-dependent regulation of
gene expression that takes place at both transcriptional and translational levels. Most
of the satellite spots detectable by 2-D gel originate either by alternative splicing or
by post-translational modifications. Three types of post-translational modification of
hnRNPs have been described: phosphorylation, methylation of Arg residues,
glycosylation. Although their role is still matter of discussion, they are likely to affect
both the RNA binding capacity and the nucleo-cytoplasmic distribution of the
modified proteins.
RNA binding domains.
The cDNAs for most of the hnRNP proteins have been isolated. All hnRNP
proteins have a modular structure in which RNA binding domains are associated with
“auxiliary” or “effector” domains. Three types of RNA binding domains have been so
far identified in hnRNP proteins.
1) The RBD (RNA binding domain) / RRM (RNA Recognition Motif) / RNP-CS
motif (RNP -consensus) is the best characterized RNA binding domain and its tertiary
structure has been determined. It consists of a 90 amino acid sequence and contains
two short highly conserved motifs: RNP1 and RNP2. The four-stranded anti-parallel
ß-sheet RNA binding platform leaves the bound RNA accessible to interactions with
other RNAs or proteins during splicing.
35
2) The RGG box is formed by closely spaced Arg-Gly-Gly repeats often
interspersed with aromatic residues.
3) The K-homology motif, initially identified in hnRNP K, has been found in
several proteins such as the product of the X-fragile gene involved in mental
retardation. The functional and structural characterization of this 45 amino acid
domain is still lacking.
The structures and functions of the auxiliary domains are still poorly defined,
but they are thought to determine the functional properties of the hnRNP proteins,
namely RNA binding, protein-protein interactions, nucleo-cytoplasmic trafficking and
subnuclear localization.
Binding to Nucleic Acids.
In addition to a general affinity for RNA, many hnRNPs display a certain
sequence specificity of binding which supports the concept of a transcript-specific
assembly of the complexes. High affinity binding sites or “winner sequences for
hnRNP A1, C, I/PTB and L have been identified by means of different in vitro and in
vivo assays.
Protein-Protein interactions
Although RNA bridging is highly probable, direct protein-protein interactions between
hnRNPs were suggested to occur. The determinants for these interactions have been
identified in hnRNP A1, A2, B1, and C1/C2. The ability of hnRNP A1 to enter homo
and heterocomplexes relies on the regularly spaced aromatic residues interspersed
between glycines and positively charged amino acids and distributed over the entire
Gly-rich auxiliary domain. The same domain mediates the interaction of A1 with a
subset of splicing factors of the SR family.
Role in splicing
An involvement in splicing was shown both in vitro and in vivo for hnRNP A, B,
C. I/PTB and F. The relative ratio between hnRNP A1 and the splicing factor
ASF/SF2 determines the choice between alternative 5’ splice sites, one target being
the A1 pre-mRNA itself. Binding of hnRNP I to the poly-pyrimidine tract of certain
introns governs the choice between the skeletal and muscle-specific splicing of the ?
and ß tropomyosin. Recently hnRNP F has been implicated in a neuronal specific
splicing of the src pre-mRNA.
36
Subcellular localization and role in mRNA export.
In general, hnRNP proteins are homogeneously distributed throughout the
nucleoplasm with the exclusion of nucleoli. On the other hand, some distinctive
patterns can be observed for hnRNP A1 (small nuclear dots), hnRNP F and H
(granules distributed throughout the nucleoplasm) and hnRNP I and L (accumulation
in perinucleolar compartments or PNC).
As expected for factors involved in mRNA export, a subset of hnRNPs (A1,
A2, B1, B2, D, I, and K) continuously shuttle between the nucleus and the cytoplasm
and their nuclear re-import requires active transcription. Both nuclear import and
export of hnRNP A1 depend on a 30 amino acid sequence in the Gly rich domain
termed M9, which mediates the interaction with a protein of the “importin ß” family,
called “transportin”. A different sequence mediates the nucleo-cytoplasmic shuttling
of hnRNP K.
Role in translation.
The observation that the hrp36 protein of Chironomous tentans, homologous
to hnRNP A1, remains associated with mRNA on polysomes suggests a role of
hnRNPs in translation. A direct role in the translation of specific mRNAs has been
recently shown for hnRNP I, K and E1.
HAP: a new hnRNP protein relocates in few nuclear granules upon heat shock.
Through a two-hybrid screening in yeast for proteins interacting with the
human hnRNP A1, we have isolated a nuclear protein of 917 amino acids called
hnRNP A1 associated protein (HAP). HAP contains an RNA Binding Domain (RBD)
flanked by a negatively charged domain and by an S/K-R/E-rich region which
mediates the interaction with hnRNP A1. HAP was found to be identical to the
previously described Scaffold Attachment Factor B (SAF-B) and to HET, a
transcriptional regulator of the Hsp27 gene. We show that HAP is a bona fide hnRNP
protein, since anti-HAP antibodies immunoprecipitate from HeLa cell nucleoplasm
the complete set of hnRNP proteins. Thus these findings reinforce the idea of a tight
relationship between components of the nuclear matrix and those involved in the
maturation of pre-mRNA molecules.
Unlike most hnRNP proteins, the subnuclear distribution of HAP is profoundly
modified in heat shocked cells. In fact, heat shock at 42°C causes a transcription
dependent recruitment of HAP to a few large nuclear granules that exactly coincide
with sites of accumulation of Heat Shock Factor 1 (HSF1). The recruitment of HAP to
37
the granules is temporally delayed with respect to HSF1 and persists for a longer
time during recovery at 37°C.
We have observed that a similar phenomenon occurs also in the case of hnRNP M,
that is recruited to a large number of nuclear granules, a subset of which seems to
correspond to the sites of HAP accumulation.
The observation that hnRNP complexes immunoprecipitated from nucleoplasm of
heat shocked cells with anti-HAP antibodies have an altered protein composition with
respect to canonical complexes suggests an involvement of HAP in the cellular
response to heat shock, possibly at the RNA metabolism level.
38
IN VIVO INCORPORATION OF ANTI-RNP ANTIBODIES
IN CULTURE CELLS.
Marco Biggiogera1, Andrea Trentani2, Maria Grazia Bottone1 and Terence E.
Martin3,
1Dip.
di Biologia Animale (Laboratorio di Biologia Cellulare) and Centro di Studio per
l'Istochimica del CNR, University of Pavia, Piazza Botta 10, 27100 Pavia, Italy.
2Dept.
of Biological Psychiatry, Academic Hospital, Groningen, The Netherlands
3Department
1Tel.:
of Molecular Genetics and Cell Biology, University of Chicago, U.S.A.
+39.0382.506322
FAX: +39.0382.506325
E-mail: marcobig@unipv.it
Transcription and splicing of RNA are complex mechanisms involving several groups
of proteins such as RNA polymerases, hnRNPs, snRNPs and splicing factors
(Lamond, 1995; Will and Lührmann, 1997) . The role of both hnRNPs and snRNPs
has ben studied for several years also with specific antibodies recognizing these
proteins (Lerner et al., 1981; Steitz et al., 1988) and their subcellular location has
been extensively elucidated (see Fakan, 1994 for a review). In particular, the nature
and the role played by perichromatin fibrils in transcription and co-transcriptional
splicing has been almost unanimously recognized (Misteli and Spector, 1997). An
efficient way to probe the role played by a specific protein has been utilized by
Benavente et al. (1988) by microinjecting antibodies anti-RNA polymerase I into
nucleoli of living cells. This treatment can give rise to nucleolar segregation due to
the block of the transcriptional activity of the RNA polymerase I. However,
microinjection treatment limits seriously the number of cells which can be used for a
futher ultrastructural analysis. On the other hand, an interesting approach has been
proposed
by
Shea
and
Beermann
(1991).
These
authors
use
L--
lysophosphatidylcholine (LPC) to create in the cell membrane transitory gaps which
allow the penetration of antibodies in vivo. Thereafter, if LPC is removed from the
culture medium, the gaps close and the integrity and functionality of the cell
membrane is restored. Ultrastuctural studies (Shea and Beermann, 1991) have
demonstrated that in these conditions the cell membrane is indistinguishable from
that of an untreated cell. We have therefore used LPC to incorporate in vivo specific
anti-hnRNP or snRNP antibodies into two different cell lines, normal human
fibroblasts and rat glioma C6 cells. The rate of transcription has then been monitored
by flow cytometry, immunofluorescence and electron microscopy following the
incorporation of bromo-uridine (BrU) after the penetration of the antibodies, as well
39
as with a series of antibodies detecting splicing factors and other proteins involved in
this process.
Our results indicate that:
1) the permeabilization with LPC allows in vivo incorporation of antibodies into the
cell nucleus;
2) LPC does not damage the ability of cell to incorporate and phosphorilate BrU;
3) after the incorporation of anti-RNP antibodies, pre-mRNA transcription decreases
both in fibroblasts and in C6 cells;
4) after anti-RNP incorporation, the presence of RNA polymerase I and of the
ribosomal proteins P0P1P2 increase in C6 cells. In fibroblasts the nucleolar
markers are unchanged.
The use of LPC, to create transitory gaps in the cell membrane, allows in vivo
incorporation of antibodies not only in the cytoplasm (Shea and Beermann, 1994) but
also in the nucleus. This permeabilization can be used to introduce IgG class
antibodies (like anti-snRNP Y12 antibody; Lerner et al., 1981) but also IgM class
antibodies (like anti-hnRNP ID2 antibody; Leser et al., 1984) that have a higher steric
hindrance, being a pentamer of immunoglobulins. In our conditions, this treatment
did not damage the cell: in fact fibroblasts and C6 cells were able to utilize BrU as a
precursor of RNA. BrU is hence incorporated at the level of perichromatin fibrils,
which represent the sites of active gene transcription (Fakan, 1994) and probably are
constituted by newly synthesized hnRNA (Jackson et al., 1993; Wansink et al.,
1993), and in the nucleolus at the fibrillar dense component level, where pre-rRNA
transcription occurs. Since BrU must be phosphorylated to be incorporated, it follows
that the functionality of the transcription apparatus was not damaged. However, we
do not know the modality by which the antibodies enter the nucleus. It seems likely
that LPC could cause also gaps in the nuclear envelope, since the nuclear pores
should, in principle, not allow IgG entering.
The in vivo incorporation of antibodies directed against hnRNPs or snRNPs is able to
modify the rate of the transcription. Our flow cytometry data show a decrease of
incorporation of BrU in fibroblasts which, at the ultrastructural level, corresponds to a
decrease of immunolabeling both at level of the perichromatin fibrils and of the
nucleolus. In C6 cells, where flow cytometry did not show a marked decrease of
incorporation of BrU, it was evident at ultrastructural level that the incorporation of
BrU occurred exclusively on the nucleolus while there was very weak incorporation at
perichromatin fibrils level.
40
The consequences of an induced decrease of the transcription are different in
fibroblasts and in C6 cells. In the first case the incorporation of anti-hnRNP or antisnRNP antibodies had effect on pre-mRNA transcription, while nucleolar activity (in
terms of BrU incorporation or of presence of P0P1P2 proteins) was not affected. In
C6 cells, instead, the presence of P0P1P2 proteins, in the nucleolus, increases: these
proteins are also detectable in the nucleoplasm, where, in normal conditions, they
should not be present in large amounts. Moreover, the immunolabeling for RNA
polimerase I increases. This seems to point out that, in this cell line, the block of the
transcription of pre-mRNA is accompanied by a marked increase of rRNA synthesis
and of its transport toward the cytoplasm. An analogous situation can be observed
after a-amanitin treatment: this drug blocks the RNA polymerase II (Petrov and
Sekeris, 1971). Also in this case, RNA polymerase I increases its activity (Hozak et
al., 1994).
Finally, we would like to point out that the use of LPC to incorporate antibodies into
living cells seems to be a useful tool which can give interesting information on
nuclear factors. By this method, in fact, the effect of blocking a specific protein in vivo
can be studied on a large population of cells.
REFERENCES
Benavente R, Reimer G, Rose KM, Hugle-Doerr B, Scheer U: Chromosoma 97: 115-123,
1988.
Fakan S: Trends Cell Biol. 4: 86-90, 1994.
Hozak P, Cook PR, Schofer C, Mosgoller W, Wachtler F: J Cell Sci 107: 639-648, 1994.
Jackson DA, Hassan AB, Errington RJ, Cook PR: EMBO J 12: 1059-1065, 1993.
Lamond AI: Pre-mRNA processing. Heidelberg: Springer-Verlag, 1995.
Lerner EA, Lerner MR, Janeway CA, Steitz J: Proc Natl Acad Sci USA 78: 2737-2741, 1981 .
Leser GP, Escara-Wilke J, Martin TE: J Biol Chem 259: 1827-1833, 1984.
Misteli T, Spector DL: Trends Cell Biol 7: 135-138, 1997.
Petrov P, Sekeris CE: Exp Cell Res 69: 393-401, 1971.
Shea TB, Beermann ML: Biotechniques 10: 288-294, 1991.
Shea TB, Beermann ML: Mol Biol Cell 5: 863-875, 1994.
Steitz JA, Black DL, Gerke V, Parker KA, Kramer A, Frendewey D, Keller W. Structure and
Function of Major and Minor Small Nuclear Ribonucleoprotein Particles. Berlin: Springer.
pp 115-154, 1988.
Wansink DG, Schul W, Van Der Kraan I, Van Steensel B, Van Driel R, De Jong L: J Cell Biol
98: 358-363, 1993.
Will CL, Lührmann R: Curr Op Cell Biol 9: 320-328, 1997.
41
ANALYSIS OF INTERPHASE FISH ON HISTOLOGICAL SECTIONS
USING CONFOCAL LASER SCANNING MICROSCOPY
Peter J.S. Hutzler
GSF - Research Center, Postfach 1129, D-85758 Neuherberg
Tel.: +49-89.3187-2410; FAX: +49-89.3187-3349; E-mail: hutzler@gsf.de
Fluorescence in situ hybridization (FISH) using chromosome specific DNA probes on
histological sections has emerged as a powerful and reliable instrument for the
discrimination of chromosomal aberrations in interphase cell nuclei of solid tumors. It
allows the combination of cytogenetic and histological analysis (1). FISH on paraffinembedded tissue sections was applied in studies on various tumors like prostatic
carcinoma (2), primary cutaneous melanomas (3), or mamma carcinoma.
The essential steps in preparation after deparaffinization of the sections mounted on
a microscopic slide are
 pretreatment, e.g. digestion, to make the section permeable,
 denaturation, to produce single stranded DNA
 hybridization with fluorescently labelled DNA probe
 Counterstain of the nuclei with a fluorescent DNA stain.
Whereas hybridization
became reliable by the availability of tested commercial
probes, the steps of pretreatment and denaturation are crucial because the influence
the morphological quality. This results in a competition of the parameters: acceptable
thickness of the section, morphological quality, and quality of pretreatment for
hybridization. On the other side reliable signal counting requires complete, uncut
nuclei (2), resulting in section thickness of 15 µm or more.
Best image quality for a spatial correlation of FISH signals and nuclei is obtained by
confocal imaging. From a field of view a sequence of optical sections is taken at
distances of 0.5 µm or less. Acquisition of the different fluorescence channels should
be done sequentially to avoid cross talk. Dot counting normally is done interactively,
starting with an extended focus image, obtained by projection of the optical sections,
or a pair of them and using stereoscopic view on them. In critical situations where
there is doubt on the edge of a nucleus and the location of a signal with respect to it
an animation of the image sequence or a representation in orthogonal cuts shows
more details.
Software for automated dot counting was developed at different sites. However at its
application the quality of the results strongly depends on the image quality. One
crucial step is the 3-D segmentation of the nuclei based on the counterstain image
42
data. Segmentation needs distinct edges of the nuclei. But ‘over-digestion’ results in
smeared diffuse edges. The problem becomes crucial at certain tumor regions with
densely packed nuclei. A second problem is the identification of the FISH signals and
there separation from artifacts. E.g. certain histological sections like those from
prostate contain brightly autofluorescent particles with size down to that of the
signals. Future success in automated analysis of interphase FISH on histological
sections needs concerted effort in optimization of the involved steps as there are
molecular pathological preparation, confocal data acquisition, and computerized
analysis.
REFERENCES
1. Van Dekken, H., et al., J. Pathol., 171: 161-171 (1993)
2. Aubele, M., et al., Histochem. Cell Biol., 107: 121-126 (1997)
3. D’Alessandro, I., et al., J. Cutan. Pathol. 24: 70-75 (1997)
43
APOPTOTIC CELL DEATH IN HUMAN BREAST CANCER CELLS
AND TISSUES: FROM LIGHT MICROSCOPY TO QUANTITATIVE
ELECTRON MICROSCOPY
Christian Castelli and Gabriele A. Losa
Laboratorio di Patologia Cellulare, Istituto di Patologia
Via In Selva 24, CH-6601 Locarno Switzerland
Tel : +41 091 756 2680; FAX : +41 091 756 2691; e-mail: glosa@guest.cscs.ch
Apoptosis is a physiological form of cell death that occurs in all tissues and in which
cells committed to die undergo a series of characteristic functional and morphological
changes which can be examined by conventional or electron microscopy. In contrast
to thymocytes and other lymphoid tissues, it is more difficult to recognize and
quantify apoptosis in solid tissues under physiological and pathological conditions for
several reasons. First, apoptosis is a relatively rapid phenomenon in vivo and
second, apoptotic cells are efficiently removed from a tissue by phagocytic
elements.On the other hand, the methods currently available do not adequately
identify the early phases of apoptosis which probably occur at the cell surface while
the cells continue to appear overtly normal and healthy. Finally, the frequency of
measurable apoptosis may be low in living tissues because this is a natural residual
process.
Several
reports
based
on
morphological,
cytometric,immunologic and genetic examination, have indeed
biochemical,
flow
documented the
paucity of the apoptotic process in various types of tissue either normal, abnornal or
neoplastic (1,2,3,4,5). Even in human breast tissues with ductal invasive carcinoma
the measurable apoptotic process was found to occur at very low frequency (less
than 2%) when examined in histological sections by conventional quantitative
microscopy (6) and TUNEL immunohistochemical staining or in cell tissue
preparations by flow cytometry of propidium iodide uptake and biochemical
determination of DNA fragmentation (7).There are only few cases where the
mechanism of apoptosis activation is known and concerns mostly the lymphocytes:
the perforin / granzyme B pathway and the Fas ligand binding to the Fas/Apo-1
receptor, both leading to the activation of caspases responsable of the proteolytic
death cascade (8,9). It has been reported that distinct biochemical pathways
occuring at the cell surface of lymphocytes could be activated in the early phases of
apoptosis induced by various stimuli, such as the asymmetrical exposure of
phosphatidylserine on the outer leaflet of plasma membrane, the activation of signal
transduction (phosphatydlinositol phospholipase C) and transport membrane (44
glutamyltransferase) enzymes, the activation of the sphingomyeline cycle and
breakdown of sphingolipids (10,11,12). Crosslinking of Fas/Apo-1 receptor by mAbs
in human T78 lymphoma cells induced early signals of apoptosis which resulted in
the activation of a phosphatidylcholine specific phospholipase C and of both neutral
and acidic sphyngomyelinases leading to the hydrolysis of membrane sphyngomyelin
and generation of ceramide which in turn contributed to the propagation of the
Fas/Apo-1-generated apoptotic signal (13). Recently it has been shown that
proliferating Hela cells arrested in the G2/M phase of cell cycle by nocodazole
treatement expressed an high level of survivin, a new inhibitor of the apoptosis,
whose dissociation from microtubules at the beginning of mitosis increased the
caspase-3 activity which propagates the apoptotic process. (14). All these
biochemical events, however, occur rarely in living tissues and may not necessarily
express in correspondence with initial apoptotic phases. They could eventually be
more efficiently examined in cultured breast cancer cells together with morphological
ultrastructural changes which may be assessed by means of a morphometric
approach based on the principle of the fractal geometry, developed for the
description of irregular structures including cell and tissue components (15). Its
reliability has been ascertained by studying the early effects of steroid hormones on
the nuclear heterochromatin organization of breast cancer cells (16). Hence, finest
modifications in the heterochromatin structure or in plasma and nuclear membrane
outlines which may intervene at the beginning of apoptotic cell death, even if not
detectable by visual inspection could be numerically assessed. To achieve this goal,
non-estrogen sensitive SKBR-3 breast cancer cells were cultured in presence of 1M
calcimycine (A23187), a potent Ca++ ionophore able to arrest cell cycle in the G0/G1
phase and to induce active apoptosis, while the apoptotic pathways were assessed
by measuring functional and ultrastructural changes of plasma membrane and
nuclear components. Propidium iodide permeability, phosphatidylserine expression,
activity of -glutamyltransferase (-GT) involved in the antioxidant glutathion cycle,
antiproliferative acidic sphingomyelinase, and surface gangliosides were found
decreased only after 48 hr of ionophore treatement whereas the activity of
mitochondrial
dehydrogenases
was
dramatically
reduced
still
after
4
hr.
Characteristic features of ultimate apoptotic stages, frankly apparent at 96 hr in most
treated cells, were monitored by DNA fragmentation gel electrophoresis, sub-diploid
DNA peak emergence and DNA strand breaks labelled by deoxynucleotidyl
transferase (TUNEL). However, changes in the ultrastructural complexity of plasma
membrane, nuclear envelope and nuclear heterochromatin outlines were measured
at 12-24 hr of treatement with calcimycin. They resulted in a significant smoothing of
45
all the examined structures, as documented by the reduced fractal capacity
dimension [Df]. A similar trend was evidenced by measuring the ultrastructural
feature of the intranuclear scattered heterochromatin. These findings documented
that
calcimycine-induced
apoptosis
enhanced
the
compactness
of
the
heterochromatin while reducing the nuclear complexity. This ionophore is a strong
apoptogen in SKBR-3 cancer cells, that probably activates the late steps of the
apoptotic machinery by bypassing the caspase cascade pathway. No apparent
alterations of cell surface constituents could be appreciated with conventional
biochemical and morphologic approaches within 24 h of treatment. In contrast, the
fractal descriptor has enabled to evaluate subtle morpho-structural modifications of
membranes and nuclear components still in the early phases of the apoptotic
process.
REFERENCES:
1.Drachenbery C., Papadimitriou JC. Significance of apoptosis in prostatic
neoplasias. Arch.Pathol Lab Med 1997, 121, 54-59.
2.Gaffney EF. The extent of apoptosis in different types of high grade prostatic
carcinomas. Histopathology 1994, 25, 269-273.
3. Allan DJ, Howell A, Roberts SA, Williams TG, et al. Reduction in apoptosis relative
to mitosis in histologically normal epithelium accompanies fibrocystic change
and carcinoma of the premenopausal human breast. J Pathology 1992,167,2532.
4. Battersby S, Anderson TJ. Proliferative and secretory activity in the pregnant and
lactating human breast. Virchows Archiv A Pathol. 1988, 413, 189-196.
5. Lipponen P. et al. Apoptosis in breast cancers as related to histopathological
characteristics and prognosis.Eur. J. Cancer 1994, 30, 2068.
6. Van de Shepop H.A.M.et al. Counting of apoptotic cells: a methodological study in
invasive breast cancers. J.Clin.Pathol.Mol.Pathol. 1996, 49, 214-217.
7. Losa GA, Graber R. Apoptotic cell death and the proliferative capacity of human
breast cancers. Anal. Cell. Pathol. 1998, 16, 1-10.
8. M.Raff. Cell suicide for beginners. Nature 1998, 396, 119-122.
9. G.M.Cohen. Caspases: the executioners of apoptosis. Biochem. J. 1997, 326, 116.
10. R.Graber, G.A.Losa. Changes in the activities of signal transduction and
transport membrane enzymes in CEM lymphoblastoid cells by glucocorticoidinduced apoptosis. Anal.Cell.Pathol. 1995, 8, 159-176.
46
11. W.D.Jarvis, S.Grant,R.N.Kolesnick. Ceramide and the induction of apoptosis.
Clin.Cancer Res. 1996,2,1-6.
12. S.J.Martin et al. Early redistribution of plasma membrane phosphatidylserine is a
general feature of apoptosis regardless of the initiating stimulus. Inhibition by
overexpression of Bcl-2 and Abl. J.Exp.Med. 19995,182,1545-1557.
13. M.G.Cifone et al..Multiple pathways originate at the Fas/Apo-1 (CD95) receptor:
sequential involvement of phosphatidylcholine-specific phospholipase C and
acidic sphingomyelinase in the propagation of the apoptotic signal. The EMBO
Journal 1995,14, 5859-68.
14. Fengzhi Li et al. Cell cycle control of apoptosis and mitotic spindle checkpoint by
survivin. Nature 1998,396, 580-84.
15. G.A.Losa, T.F.Nonnenmacher. Self-similarity and fractal irregularity in pathologic
tissues. Modern Pathology 1996,9,174-182.
16. G.A.Losa et al. Steroid hormones modify nuclear heterochromatin structure and
palsma membrane enzyme of MCF-7 cells. A combined fractal, electron
microscopical and enzymatic analysis. Eur.J.Histochem. 1998,42,21-29.
47
NEW MOLECULES CONTROLLING BASIC CELL FUNCTIONS
Pier Carlo Marchisio
DIBIT, Department of Biological and Technological Research
San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milano.
Tel.: +39-02.26434834; FAX: +39-02.26434855; E-mail: marchisio.piercarlo@hsr.it
The integrin has a long cytodomain necessary for hemidesmosome formation. A
yeast two-hybrid screen using 4 cytodomain uncovered a protein called p27BBP
that represents a 4 interactor (1). This protein has been cloned and sequenced and
proved to be highly conserved. Both in yeast and in vitro, p27BBP binds the two Nterminal fibronectin type III modules of 4, a region required for signaling and
hemidesmosome formation. Sequence analysis of p27BBP revealed that p27BBP
was not previously known and has no homology with any isolated mammalian
protein, but 85% identical to a yeast gene product of unknown function. Expression
studies by Northern analysis and in situ hybridization showed that, in vivo, p27BBP
mRNA is highly expressed in epithelia and proliferating embryonic epithelial cells. An
antibody raised against p27BBP C-terminal domain showed that all 4-containing
epithelial cell lines expressed p27BBP. A fraction of the p27BBP protein is insoluble
and present in the intermediate filament pool. Furthermore, subcellular fractionation
indicated the presence of p27BBP both in the cytoplasm and in the nucleus.
Confocal analysis of cultured cells showed that part of p27BBP immunoreactivity was
both nuclear, and notably in the nucleolar shell, and in hemidesmosomes closely
apposed to 4. These results suggest that the p27BBP is an in vivo interactor of 4,
possibly linking to the intermediate filament cytoskeleton and also provided with
previously unknown functions in protein biosynthetic pathways (2). Finally, p27BBP
has been been found to be overexpressed in carcinomas both in vitro and in situ, and
we are currently studying whether p27BBP is involved in apoptosis protection.
In apoptosis protection is certainly involved the newly discovered protein survivin. In
a collaborative effort with Yale University we have found (3) that survivin represents a
checkpoint acting at mitosis by directly binding the mictrotubules of the spindle. Cell
cycle progression and control of apoptosis (programmed cell death) are thought to be
intimately
linked
morphogenesis.
processes,
preserving
homeostasis
and
developmental
Although apoptosis regulatory proteins have been implicated in
restraining cell cycle entry, and controlling ploidy, the effector molecules at the
interface between cell proliferation and cell survival have remained elusive. We have
48
shown that a novel IAP inhibitor of apoptosis, survivin, is expressed in G2/M in a cellcycle-regulated manner. At the beginning of mitosis, survivin associates with spindle
microtubules in a specific and saturable reaction, regulated by microtubule dynamics.
Disruption of survivin-microtubule interaction results in loss of anti-apoptosis function
and increased caspase-3 activity in mitosis. These findings suggest that survivin
counteracts a default induction of apoptosis in G2/M. The over-expression of survivin
in cancer may overcome this apoptotic checkpoint and favor aberrant progression of
transformed cells through mitosis.
REFERENCES
1) S. Biffo, F. Sanvito, S. Costa, L. Preve, R. Pignatelli, L. Spinardi and P.C.
Marchisio: Isolation of a novel β4 integrin binding protein (p27BBP) highly
expressed in epithelial cells. J. Biol. Chem. 272: 0314-30321, 1997.
2) F. Sanvito, S. Piatti, A. Villa, M. Bossi, G. Lucchini, P. C. Marchisio and S. Biffo:
The β4 integrin interaction of P27BBP/eIF6 is an essential nuclear matrix
protein involved in 60S ribosomal subunit assembly. J. Cell Biol., in press, 1999.
3) F. Li, G. Ambrosini, E. Y.Chu, J. Plescia, S. Tognin, P.C. Marchisio and D.C.
Altieri: Cell cycle control of apoptosis and mitotic spindle check-points by
survivin. Nature 396: 580-584, 1998.
49
GREEN FLUORESCENT PROTEIN MUTANTS IN THE STUDY OF
CALCIUM SIGNALLING
Rosario Rizzuto
Dept. of Experimental and Diagnostic Medicine, Section of General Pathology
Via Borsari 46, 44100 Ferrara, Italy
Tel. +39-0532-291361; E-mail: rzr@unife.it
Green fluorescent protein has emerged as a novel, versatile tool for
investigating in vivo events as diverse as protein sorting, gene expression or
embryonic development1. In these tasks, the spectral variants of GFP significantly
widen the possible applications, by allowing to label simultaneously different
molecular components, subcellular structures or cell lineages.
In this presentation, I will review our recent work, employing specifically
targeted GFP mutants2 and high-resolution imaging systems3 to investigate signal
transduction events in living cells.
In the past years, by using a specifically targeted Ca2+-sensitive photoprotein, we
demonstrated that, in the process of calcium signalling, mitochondria undergo major
changes in the matrix [Ca2+]. This observation, which implies the activation of
mitochondrial Ca2+-sensitive dehydrogenases and thus the close coupling of
metabolism to the increased cell needs, was largely unexpected, given the low
affinity of mitochondrial Ca2+ uptake systems. The apparent discrepancy was solved
by showing, with appropriately targeted GFP chimeras, that mitochondria are in close
contacts with the endoplasmic reticulum, the main intracellular Ca 2+ store, and thus
may sense, upon opening of IP3-gated channels microdomains of high [Ca2+], well in
the range of their uptake systems. The high-resolution 3D imaging also showed that
mitochondria form, in living cells, a continuous, highly interconnected network in rapid
motion, with fusions and fissions4. Finally, we studied mitochondrial morphology in
the course of fas-mediated apoptosis, demonstrating that the release of pro-apoptotic
factors early in the process of cell death does not occur via major changes (e.g.
swelling) of mitochondrial morphology.
More recently, we have developed novel tools for investigating the signalling
events downstream of the [Ca2+] change, namely GFP-labelled PKCs, allowing to
monitor the translocation to the plasma membrane of the various isoforms of PKC.
Thus in combination with fluorescent indicators, these tools allow to correlate the
spatio-temporal complexity of calcium signalling to the recruitment of specific
effectors. Finally, I will also discuss how energy transfer between GFP chimeras is a
50
successful, albeit difficult route for generating probes for important physiological
parameters and processes, such as, for example, Ca 2+ or ATP concentration,
enzyme activity, etc.
REFERENCES
1. Cubitt et al., Trends Biochem. Sci. 20, 448-455, 1995;
2. Rizzuto et al. Curr. Biol. 6, 183-188, 1996;
3. Rizzuto et al., Trends Cell Biol. 8, 288-292, 1998;
4. Rizzuto et al. Science 280, 1763-1766, 1998.
51
REAL TIME IMAGING OF TRANSCRIPTION IN MAMMALIAN CELLS USING
LUCIFERASE GENES.
Michael RH White, C.D. Wood, D.G. Spiller, K. Bateman, D.G. Fernig,
S. Hawley*, S. Pennington*, N. Takasuka^, A. Stirland§ , A. Loudon§, J. Davis^
School of Biological Sciences, University of Liverpool, Crown Street, Liverpool, L69
3BX, UK.. *Department of Cell Biology and Human Anatomy, University of Liverpool.
^Department of Medicine and §School of Biological Sciences, Manchester University.
Tel. +44.0151.7944371; FAX. +44.0151.7944349; E-mail: mwhite@liv.ac.uk
Firefly luciferase has become a widely used reporter for the analysis of transcription
in mammalian cells. Commonly, luciferase activity is measured in cell lysates.
Luciferase activity can also be measured in intact living cells following the addition of
the specific substrate luciferin to the cell medium. Highly sensitive imaging cameras
such as intensified photon-counting cameras can quantify luciferase activity in single
living cells.
Recently, we have developed an improved microscopy system for the long term
measurement of gene expression in single living cells. This involves the use of a
CO2 environmental chamber placed over a heated microscope stage. This approach
has allowed the long term imaging of living cells for more than 1 week after addition
of the substrate luciferin to the culture media at the start of the experiment. A
particular advantage of the firefly luciferase enzyme as a real-time reporter of gene
expression lies in the short half life of the enzyme activity. This means that
luminescence may be used to track dynamic changes in gene expression rather than
just measuring integrated expression.
We have coupled this technology with mammalian cell microinjection. This approach
allows multiple DNAs (or other molecules) to be injected simultaneously into cells.
The use of a second luciferase from the sea pansy Renilla reniformis has allowed
dual luciferase imaging following addition of the respective substrates, firefly luciferin
and coelenterazine to the cells. This allows the expression from one promoter to be
measured relative to that from a second control promoter. In addition, microinjection
is a useful tool for single cell experiments since it may be used to introduce activators
or inhibitors of specific gene expression pathways. These approaches allow single
cells to be used as test-tubes for the analysis of gene expression.
I will describe recent work using this technology, including the analysis of the
mechanism of the timing of transcription in the mammalian cell cycle and the analysis
of the molecular basis by which restricted cell spreading creates a block in cell cycle
52
progression. We are also studying the regulation of the prolactin promoter in stably
transfected pituitary (GH3) cells where there is considerable dynamic heterogeneity
in the basal level of gene expression. We have recently found that a serum shock
synchronises long term oscillations in the prolactin promoter directed gene
expression.
This work is supported by MRC, BBSRC, The Wellcome Trust, Society for
Endocrinology, North West Cancer Research Fund, Department of Trade and
Industry, Hamamatsu Photonics, Carl Zeiss Ltd. Kinetic Imaging Ltd, Astra
Pharmaceuticals
53
FLUORESCENT PROBES FOR SUPRAVITAL CELL STAINING
Barni S°., Sciola L.*, Spano A. and Bertone V.
Dipartimento di Biologia Animale, Università di Pavia
*Dipartimento di Scienze Fisiologiche, Biochimiche e Cellulari, Università di Sassari
°Tel. +39-0382-506.316; FAX +39-0382-506.406; E-mail: anatcomp@unipv.it
The last years of cytological research have demonstrated the importance of
fluorescent dyes and fluorogenic compounds in the study of living cells, providing
useful knowledge about the dynamic and functional behaviour of cell organelles.
At the present time the supravital fluorescence cytochemistry coupled to the use of
technologically sophisticated instrumental techniques (e.g. video lapse recording,
confocal microscopy with digital image processing, multiparametric flow cytometry)
can provide important information in different fields of the cell biology: normal
physiology, experimental stimulations, pathological alterations, etc.
Detectability of fluorescent markers specific for labelling different macromolecule
classes, employed at nontoxic concentrations, allows the study of living cells for
several hours. Therefore, different aspects of the structure and function of live cell
organelles may be investigated by monitoring uptake and fate of fluorescent trackers.
Generally, a fluorescent dye comprises two domains, namely a fluorophore and a
targeting group. Sometimes, the interaction with the target induces deep specific
changes in the fluorescence efficiency and/or in the spectral features of the probe
(e.g. Acridine orange, JC-1, Nile red) producing differential bicolor fluorescence
(fluorochromasia).
For the interaction of fluorescent dyes with living cells, some basic physiological and
biological properties include electric charge, hydrophilicity, lipophilicity, pinocytosis,
phagocytosis, etc. In addition to spontaneous capacity of fluorescent dyes to enter
the live cell, the delivery of more polar molecules requires different strategies such as
hypo-osmotic shock, cationic liposomes, microinjection, etc. In some cases the cell
membrane integrity is detectable by lipophilic precursors of fluorescent derivatives
(e.g. Fluorescein diacetate, Calcein AM) that are activated by intracellular esterases,
producing, in living cells, a stable cytoplasmic labelling. The fluorochromes that are
not membrane permeant may also be used as an indicator of the cell viability (dye
exclusion test). In particular, complementary DNA-markers (e.g. Propidium
iodide/Hoechst 33342), as regard the capacity to cross the membrane hydrophobic
barrier, are frequently used to distinguish intact cells from injured cells in different
54
degenerative phase (necrosis, early apoptosis, late apoptosis, secondary necrosis
etc.)(Fig.1).
At the present a large and heterogeneous group of fluorescent dyes is available to
label supravitally the different chemical (ions, lipids, enzymes, nucleic acids, etc.) and
structural (nucleus, endoplasmic reticulum, Golgi complex, lysosomes, mitochondria,
etc.) components of the cell. In this contest a particular attention is focused on the
membrane potential probes for studying the mitochondrial activity in single living
cells. For example, the membrane permeant cationic fluorochromes (e.g. DiOC 6, JC1) are significantly accumulated in the metabolically active mitochondria as a
consequence of the negative inner membrane potential. The distribution of the
mitochondrial fluorescent marker can be monitored in time, obtaining information on
the changes in the morphology and distribution of the chondriome during the normal
cell activity (Fig.2), after perturbation of cells with chemical and pharmacological
substances or in consequence of various pathological situations.
Fig. 1 Fluorescence microscopy assessment of cell degeneration in culture after
double supravital fluorochromisation with the membrane-permeable DNA dye
Hoechst 33342 (blue fluorescence) followed by the DNA dye Propidium iodide (red
fluorescence), which is impermeable to the intact cell membrane. A) normal nucleus,
B) pre-pyknotic nucleus, C) pyknotic nucleus, D) nucleus of cell with damaged
membrane, E) pyknotic nucleus of cell with damaged membrane, F) fragmented
nucleus of cell with damaged membrane.
Possible biological condition: A = normal cell; B = pre-apoptosis; C = early apoptosis;
D = necrosis; E, F = advanced apoptosis. (Photo contributed by A.F. Castoldi)
Fig. 2 Changes in morphology and distribution of chondriome during the cell cycle of
cultured cells submitted to a double supravital fluorochromisation with the DNA dye
Hoechst 33342 (blue fluorescence) and the mitochondrial dye DiOC 6 (green
fluorescence): a) interphasic cell, b) metaphasic cell, c) telophasic cell, d) just divided
cells. During the cell cycle phases the evident changes of the cell size and shape are
in part the consequence of the different spreading capacity. The arrows point to the
zone of cytoplasmic cleavage.
55
Fig.1
Fig.2
56
REFERENCES
 Cowden R.R., Curtis S.K.,: In vitro (supravital) fluorescence cytochemistry. Bas.
Appl. Histochem. 30: 7, 1986
 Wang Y.L., Taylor D.L.: Methods in cell biology. Vol. 29: Fluorescence microscopy
of living cells in culture. Academic Press, New York, 1989
 Horobin R.W., Rashid F.: Interaction of molecular probes with living cells and
tissues. Histochemistry 94: 205, 1990
 Barni S., Sciola L., Spano A., Pippia P.: Static cytofluorometry and fluorescence
morphology of mitochondria and DNA in proliferating fibroblasts: supravital double
staining. Biotech. Histochem. 71: 66, 1996
 Johnson I.: Fluorescent probes for living cells. Histochem. J. 30: 123, 1998
57
Speakers
Sergio Barni
Laboratorio di Anatomia Comparata e Citologia
Dipartimento di Biologia Animale, Università di Pavia
Piazza Botta, 10 - 27100 Pavia
Tel. +39-0382-506.316 FAX +39-0382-506.406
E-mail: anatcomp@unipv.it
Giuseppe Biamonti
Istituto di Genetica Biochimica ed Evoluzionistica del CNR
Via Abbiategrasso 207 - 27100 Pavia
Tel +39-0382-546322 FAX +39-0382-422286
E-mail: biamonti@igbe.pv.cnr.it
Marco Biggiogera
Laboratorio di Biologia Cellulare
Dip. di Biologia Animale and Centro di Studio per l'Istochimica del CNR
Università di Pavia
Piazza Botta 10 - 27100 Pavia
Tel.: +39-0382.506322 FAX: +39-0382.506325
E-mail: marcobig@unipv.it
Marie-Christine Dabauvalle
Department of Cell and Developmental Biology
Biocenter of the University of Würzburg
Am Hubland, D-97074, Germany.
Tel.: +49-931.8884273 FAX: +49-931.8884252
E-mail: dabauvalle@biozentrum.uni-wuerzburg.de
Luciana Dini
Dipartimento di Biologia, Università di Lecce
Via per Monteroni - 73100 Lecce
Tel.: +39-0832.320614 FAX: +39-0832.320626
E-mail: ldini@ilenic.unile.it
58
Heinz Gundlach
Carl Zeiss GmbH
Carl Zeiss Str. 1
D-7347 Oberkochen, Germany
phone: + 49 - (0)7364 - 202071
FAX: + 49 - (0)7364 - 204013
e-mail: gundlach@zeiss.de
Peter J.S. Hutzler
GSF - Research Center
Postfach 1129, D-85758 Neuherberg
Tel.: +49-89.3187-2410 FAX: +49-89.3187-3349
E-mail: hutzler@gsf.de
Gabriele A. Losa
Laboratorio di Patologia Cellulare, Istituto di Patologia
Via In Selva 24, CH-6601 Locarno Switzerland
Tel : +41-091.7562680 FAX : +41-091. 7562691;
E-mail: glosa@guest.cscs.ch
Pier Carlo Marchisio
DIBIT, Department of Biological and Technological Research
San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milano
Tel.: +39-02.26434834 FAX: +39-02.26434855
E-mail: marchisio.piercarlo@hsr.it
Carlo Pellicciari
Laboratorio di Biologia Cellulare
Dipartimento di Biologia Animale, University of Pavia
Piazza Botta 10
Tel. +39-0382.506420 FAX: +39-0382.506325
E-mail: pelli@unipv.it
Paola Ramoino
Dipartimento per lo Studio del Territorio e delle sue Risorse
(DIP.TE.RIS.)
Via Balbi 5, 16126 Genova
Tel.: +39-010.2099459 FAX: +39-010.2099323
E-mail: zoologia@unige.it
59
Rosario Rizzuto
Dept. of Experimental and Diagnostic Medicine
Section of General Pathology
Via Borsari 46, 44100 Ferrara
Tel. +39-0532-291361 E-mail: rzr@unife.it
Patrizia Rovere
Laboratory of Tumor Immunology, H.S. Raffaele
Via Olgettina 60 - 20132 Milano
Tel. +39-02.2643.2612 FAX: +39-02.2643.2611
E-mail: Rovere.Patrizia@hsr.it
Luigi Sciola
Dipartimento di Scienze Fisiologiche, Biochimiche e Cellulari
Università di Sassari
Tel. +39-079-228651 FAX +39-079-228615;
E-mail: sciola@ssmain.uniss.it
Jürgen Wehland
Department of Cell Biology
Gesellschaft für Biotechnologische Forschung
D-38124 Braunschweig, Germany
Tel: +49-531.6181.415 FAX: +49-531.6181.444
E-mail: jwe@gbf.de
Michael RH White
School of Biological Sciences, University of Liverpool
Crown Street, Liverpool, L69 3BX, UK
Tel. +44-0151.7944371 FAX. +44-0151.7944349
E-mail: mwhite@liv.ac.uk
60
ACKNOWLEDGEMENTS
We gratefully acknowledge the collaboration and support of several persons who
have contributed to organize and manage this Symposium and the VI Basic Course
on Light Microscopy and Micrography Techniques. In particular:
Prof. Maria Gabriella Manfredi Romanini, President of the Italian Society of
Histochemistry under whose auspices this Symposium was held;
Prof. Giuseppe Gerzeli, Director of the Department of Animal Biology and
Coordinator of the Research Doctorate in Cell and Animal Biology, who encouraged
and supported our initiative from its very beginning;
The "Divisione Strumenti" of Carl Zeiss S.p.A., Milan, and in particular Dr. Thomas
Beller for his constant help and technical assistance;
Our colleagues of the Laboratory of Zoology, who allowed us to use their teaching
facilities, and the colleagues of the Laboratories of Comparative Anatomy and
Cytology, Cell Biology and Zoology for allowing us to use their research microscopes
during the Basic Course and the Symposium Workshops;
Prof. Alberto Calligaro and Dr. Patrizia Vaghi who organized the practical session on
confocal microscopy at the Institute of Histology and General Embryology, Faculty of
Medicine, Pavia University;
Mrs. Paola Veneroni and Mr. Maurino Bosio for their help in the preparation of cell
samples used in the Workshops, the development of films, and the technical
organization;
Drs. Maria Grazia Bottone, Simona Fracchiolla, Maria B. Pisu, Vittorio Bertone and
Luigi Sciola who served as co-tutors during the workshop sessions
Mrs. Teresa Bosini, , Mrs. Lucia Riva, Mr. Teresio Sanga and Mr. Massimo Barbetta
from the Administrative staff of the Department;
Prof. Andrea Belvedere, Dean of the Collegio Ghislieri, Dr. Paola Bernardi, Dean of
the Collegio Nuovo, and Dr. Graziano Leonardelli, President of I.S.U. who gave
hospitality to some of the speakers of the Symposium.
61