Chromobility: the rapid movement of chromosomes in interphase nuclei.
Joanna M. Bridger
Lab of Nuclear and Genomic Health, Centre of Cell & Chromosome Biology,
Biosciences, School of Health Sciences and Social Care, Brunel University, West
London, UB8 3PH.
++44-1895-266272, Joanna.bridger@brunel.ac.uk
Abstract
There are an increasing number of studies reporting the movement of gene loci and
whole chromosomes to new compartments within interphase nuclei. Some of the
movements can be rapid with relocation of parts of the genome, within less than 15
minutes, over a number of microns. Some of these studies have also revealed that
the activity of motor proteins such as actin and myosin are responsible for these long
range movements of chromatin. Within the nuclear biology field their remains some
controversy over the presence of an active nuclear acto-myosin motor in interphase
nuclei. However, with both actin and myosin isoforms being localised to the nucleus,
the requirement for rapid and directed movements of genes and whole
chromosomes and evidence for the involvement of motor proteins in this relocation,
the presence of nuclear motors for chromatin movement is an important and timely
debate to have.
Keywords
Nuclear motor, nuclear myosin, nuclear myosin 1nuclear actin, gene repositioning,
chromosome territories, nuclear bodies, chromobility.
Introduction
It is now well accepted that the genomes of eukaryotic cells are highly
spatially organised in interphase nuclei as individual chromosome territories. These
territories exhibit non-random radial positioning [1]. The non-random nuclear location
and positioning of individual chromosome territories can be differential in cells from
different origins, before and after differentiation pathway steps, or in disease [2]. This
implies that whatever state cells are in, chromosomes have preferred and regulated
locations within nuclei. However, randomness of chromosome territory positioning
can be seen as cells are reorganising or re-building their nuclei, for example, at the
very beginning of G1 [3-5].
With respect to gene movement there are now numerous examples of where
small regions of chromosomes loop away from the main body of a chromosome
territory [2]. Further studies have revealed where these genes are headed revealing
an association with a facilitating nuclear structure, such as various nuclear bodies
and transcription factories [6-9].
The relocation of individual genes to nuclear structures that may aid their
expression is perhaps easier to comprehend than why whole chromosome territories
would relocate. After mitosis, a chromosome would have the opportunity to change
its nuclear location presumably by having its chromatin modified and/or being able to
interact with different nuclear structures. This situation would not require an active
mechanism within nuclei to move chromosomes around. However, specific
chromosomes can change their nuclear location after stimuli, moving from one
nuclear location to another without rebuilding a new nucleus after mitosis. We have
demonstrated this in proliferating human dermal fibroblasts that have been placed in
low serum [10]. This situation would require a mechanism presumably within nuclei
that elicits this movement, although there could be connections with the cytoplasm.
Studying a system such as this will allow us to ask where the chromosomes are
being directed, which nuclear compartments and/or structures are functionally
important, what are the consequences of repositioning chromosomes with respect to
gene expression and silencing and what can happen to a cell/organism when this repositioning is faulty. My group are now trying to answer these questions using assay
systems where genes and chromosomes do change position rapidly after stimuli
such as removal of growth factors [10], addition of differentiation factors [11] and
parasite exposure [12]. One of the keys to understanding what is happening to
chromosomes and genes is the rate at which this repositioning can occur. Whole
chromosomes and gene loci on loops have been observed changing nuclear location
as rapidly as 1m/minute with neighbouring chromosomal regions and chromosomes
remaining stationary. It most certainly involves chromatin modification but how does
this alone allow a change in position, sometimes of over 10m?
The nucleus is full of structural proteins that could create a dynamic
scaffolding network that chromatin could bind to and be released from through
signalling and protein modification. This could mean that if chromatin were released
from one region of the nucleus it could be random or corralled diffusion that
influences where the chromatin ends up being located and/or re-anchoring. The data
from some studies do not necessarily support this serendipitous route of relocation
but favours active and directed movement to new non-random locations or specific
structures within the nucleus. If a gene needs to be located at a specific nuclear
structure such as a nuclear body, a speckle or transcription factory, then it may be
that the gene is relocated to the nearest one. However, since co-transcribed regions
of the genome are from different chromosome territories that are found co-localised
at the same nuclear entity [7,9,13,14]. This implies that there is co-ordinated
movement of genes from different chromosomes to co-occupy nuclear bodies or
factories.
So the hypothesis we present here is that whole chromosomes and subchromosomal regions can be relocated rapidly and directed to specific nuclear
locations. We believe that these regions of chromatin are moved around the nucleus
on a network of molecular motors, similar to those found in the cytoplasm. Indeed,
the components of such a transport system could include nuclear actin and nuclear
myosins working together in concert to move DNA/chromatin cargo around the
nucleus.
Nuclear presence of actin and myosin
Both actin and myosin have been located in the nucleus from early on in the
study of nuclear biology but the belief in the presence of either molecule was
blighted by accusations of cytoplasmic contamination. However, with more
sophisticated methodologies and technologies the acceptance of both these proteins
in the nucleus is gaining credence.
The first papers to discuss the presence of nucleoplasmic actin showed the
actin in networks in Xenopus oocytes. Other reports followed describing actin in the
nuclei of cells from a range of different species such as bovine lymphocytes, and
slime mould (see [15]). With respect to the different forms of actin, G-actin and Factin have been revealed in the nucleus using anti-actin antibodies [16, 17], as well
as in FRAP studies using GFP-actin [18]. -actin was shown to by bind the
chromatin modelling protein BAF in vivo [19]. However, they may be less
conventional forms of actin in the nucleus, which are not polymeric in form and are
revealed with specially designed antibodies. These antibodies reveal accumulations
of actin and a concentration of actin throughout the nucleoplasm [20]. With respect to
the movement of chromatin perhaps the most important study so far revealing
nuclear actin employment in chromatin movement is that a -actin dominant negative
mutant expressed in Hela cells blocked the movement of gene loci to Cajal bodies
[6].
Myosin isoforms were originally found in the nucleus of Acanthamoeba cells
when an antibody to myosin I revealed myosin in the nucleoplasm [21]. Further,
myosin II heavy chain-like protein was found proximal to nuclear pore complexes in
Drosophila [22]. More recently different myosin isoforms have been located in nuclei.
These are nuclear myosin 123, 24], myosin VI [25], myosin 16b [26], myosin Va
[27], and myosin Vb [28]. Myosin VI has been found to have a role in the DNA
damage response [29] and myosin 16b appears to be involved in DNA replication
[26]. So far nuclear myosin 1 (NM1) has been the myosin candidate most
investigated for transposing chromatin around the nucleus [10, 15].
NM1 has a single globular head and a single heavy chain structure [15].
NM1 also contains a unique 16-residue amino acid extension at its N-terminus
which is required for nuclear import [24]. The nuclear distribution of NM1 is
throughout the nucleoplasm with a concentration at nucleoli [10, 23, 24, 30, and 31].
In addition, NM1 has also been found localised at the nuclear envelope in
proliferating human dermal fibroblasts (HDF) [10, 15]. In non-proliferating HDF NM1
is re-organised into large aggregates deep within the nucleoplasm [10, 15, Mehta IS;
Meaburn KJ, Figgitt M, Kill IR manuscript in preparation]. Interestingly these large
aggregates are found in proliferating HDF from Hutchinson-Gilford Progeria
Syndrome patients but a normal distribution of NM1 is restored after treatment with
drugs that ameliorate some of the abnormalities in these cells [32].
Re-positioning of gene loci and whole chromosomes
Individual chromosomes and gene loci have been shown to change their
nuclear location upon stimuli, whether that be stimuli to induce differentiation [11, 33,
34 35, 36], signals that change growth status [10, 37, 38], environmental stimuli [12]
or compounds to induce exogenous gene expression [39]. The consensus
hypothesis in the field is that whole chromosomes are moved to allow some control
over genes that are housed upon that chromosome, whether for expression or for
repression. Gene loci may be relocated to a new region of the nucleus with the
movement of the whole chromosome territory but also genes can be repositioned by
chromatin looping out side the main body of the chromosome territory. Chromatin
looped out of territories has been shown to become associated with nuclear
structures such as Cajal bodies [6], splicing speckles/SC35 domains [7, 40] and
transcription factories [9]. Individual genes and genes from different chromosomes
that are co-regulated can be found at the same transcription factories [9].
Evidence for directed chromosome and gene relocation elicited through
nuclear motor activity.
The hypothesis that a nuclear motor mechanism is required to relocate a
gene/genomic regions or whole chromosome has been tested in a few studies. The
involvement of actin and/or myosin in the directed movement of a chromosome or a
region of a chromosome, can be assayed by employing drugs that interfere with the
polymerisation and the activity of actin and myosins, by introducing mutant nuclear
motor proteins into cells, or using interference RNA technology to knock-down
specific nuclear motor proteins. Indeed, Belmont used both drugs to treat cells and
transfected with mutant actin, to test whether an activated exogenous lac operator
integrated array, tagged by bound GFP-lac repressor protein, was relocated and
whether this was due to motor activity [39]. These experiments were performed in
live cells using time-lapse microscopy and showed that the activated gene moved
from the nuclear periphery towards the nuclear interior, whether or not transcription
was activated. The loci on average moved 3m in 2 hours but there were short
bursts of rapid directed movement of 1m/min. Furthermore transfection of an actin
mutant, lacking the ability to polymerise, also blocked the repositioning. However,
when the authors used an actin mutant that permitted F-actin to polymerise the array
was still able to move, implying that it is the polymeric F-actin that is required to
move chromatin.
A further study by the Matera group has shown that tagged U2 snRNA
repeats containing the lac operator arrays collide with GFP labelled Cajal bodies.
Using sophisticated imaging and analysis it appeared that there was a final directed
collision of array and body which resulted in association. The U2 snRNA array was
often found on a loop emanating away from the main chromosome territory directed
towards the Cajal body. This movement of the chromatin loop was inhibited when a
polymerisation deficient actin mutant was expressed in the cells. In this situation
neither chromatin looping of the U2 array was seen nor was array:Cajal body
association observed [6].
We were one of the first groups to show that whole chromosome movement in
interphase nuclei relies on the activity of actin and nuclear myosin. We have shown
that although chromosomes are positioned non-randomly in nuclei some of them
change nuclear location when cells become arrested either irreversibly in replicative
senescence or reversibly in quiescence [10, 41, 42]. Indeed when serum is removed
from proliferating cultures chromosomes move towards the nuclear periphery,
whereas some move away to the nuclear interior and some chromosomes do not
move at all. These findings suggested that there must be specific individual targeted
chromosome movement occurring. One of the most interesting chromosomes was
chromosome 10. It was found at the nuclear periphery in quiescent HDF, at an
intermediate position in proliferating HDF and in the nuclear interior in senescent
HDF [10,42, Mehta IS, Meaburn KJ, Figgitt M, Kill IR, Bridger JM manuscript in
prep]. In order to determine the rate of chromosome relocation proliferating cells
were placed in low serum and chromosome positioning analysed to reveal when the
relocation of chromosomes took place i.e. from 0 minutes to 7 days. Chromosomes
did not take days to relocate but moved within 15 minutes of the high serum being
removed. This indicated that the movement was active and directed so given recent
evidence of actin and myosin in the nucleus it was pertinent to determine if these
nuclear motor proteins were involved. This was achieved by using drugs to inhibit
actin and/or myosin polymerisation and activity. Three drugs did prevent the
chromosomal movement; 2-3 butane-dionemonoxime, latrunculin A and
jasplakinolide. However, phalloidin oleate did not block the movement but it was
employed in such a way as to only affect cytoplasmic actin emphasising further that
it is probable that nuclear motors proteins that are affected and chromosome
relocation is not a consequence of long range actions from the cytoplasmic motors
[10,15].
The involvement of NM1 in the whole chromosome relocation was
specifically assessed by using RNA interference to knock-down MYO1C expression.
Again this intervention blocked the rapid chromosomal movement upon serum
removal, putting NM1in the frame as the nuclear myosin involved. From this study
and studies from the Belmont’s laboratory indicate F-actin and NM1are active
members of the molecular motor responsible for moving chromatin around the
nucleus.
Nuclear Structure meets Nuclear Motor
In the cytoplasm in order to create force myosin 1 moves over actin filaments
and contains actin binding domains in its globular head and with sites in its tail
domain [15]. But how does a nuclear motor comprising actin and myosin work?
Using anti-myosin 1 antibodies NM1 is found throughout the nucleoplasm but with
a definite concentration at nucleoli and the nuclear envelope. Also actin has been
described as being throughout the nucleus [43]. If actin is part of a nucleoskeleton,
that although dynamic, can take tension and force then by NM1binding to
DNA/chromatin genomic cargo could be envisaged moving over short actin
filaments, even if they are localised to compartments within the nucleus. There are
some interesting binding capabilities between NM1, actin and other members of a
nuclear structure such as nuclear lamins. Nuclear lamins are at the nuclear envelope
in the nuclear lamina which underlies the nuclear membrane and throughout the
nucleoplasm [44, 45]. Actin will bind to A-type lamins [46]. Emerin, an integral
membrane protein embedded into the nuclear membrane, is an interesting protein
with respect to its binding since it binds A-type lamins [47] but also NM1and F-actin
[48]. These proteins at the nuclear envelope could be the link to the LINC complex
that would connect a nuclear motor to the cytoskeleton [49].
In conclusion, there a number of examples where whole chromosomes and
gene loci move rapidly and apparently directionally through interphase nuclei, in
response to stimuli. The evidence is starting to accumulate of these movements
being elicited by a nuclear motor complex that employs actin as a filamentous base
for myosins to work against. Both actin and myosin maybe anchored at various
points around the nucleus by being bound to nucleoskeleton proteins such as
nuclear lamins and emerin. However more evidence of nuclear motors in moving
chromatin in a directed manner is required.
Acknowledgements
I would like to thank Dr Christopher Eskiw and Dr Ian Kill for helpful discussions and
proof reading of this manuscript. Our work on chromosome positioning in HGPS cells
was supported in part by the Brunel Progeria Research Fund.
Abbreviations
ARP Actin Related Proteins
BDM 2-3 butane-dionemonoxime
DNA Deoxy nucleic acid
DSB Double strand breaks
FRAP Fluorescence recovery after photobleaching
GFP Green fluorescent protein
HDF Human dermal fibroblasts
NM Nuclear myosin
RNA ribose nucleic acid
SnRNA small nuclear RNA
References
1. Meaburn KJ, Misteli T. (2007). Cell biology: chromosome territories. Nature.
445:379-381.
2. Foster HA, Bridger JM. (2005). The genome and the nucleus: a marriage
made by evolution. Genome organisation and nuclear architecture.
Chromosoma.114:212-229.
3. Croft JA, Bridger JM, Boyle S, Perry P, Teague P, Bickmore WA. (1999).
Differences in the localization and morphology of chromosomes in the human
nucleus. J Cell Biol. 145:1119-1131.
4. Chubb JR, Boyle S, Perry P, Bickmore WA. (2002). Chromatin motion is
constrained by association with nuclear compartments in human cells. Curr
Biol. 12:439-445.
5. Walter J, Schermelleh L, Cremer M, Tashiro S, Cremer T. (2003).
Chromosome order in HeLa cells changes during mitosis and early G1, but is
stably maintained during subsequent interphase stages. J Cell Biol. 160:685697.
6. Dundr M, Ospina JK, Sung MH, John S, Upender M, Ried T, et al. (2007).
Actin-dependent intranuclear repositioning of an active gene locus in vivo. J
Cell Biol. 179:1095-1103.
7. Szczerbal I, Bridger JM. (2010). Association of adipogenic genes with SC-35
domains during porcine adipogenesis. Chromosome Res. 18:887-895.
8. Gong H, Wang Z, Zhao GW, Lv X, Wei GH, Wang L, Liu DP, Liang CC.
(2009). SATB1 regulates beta-like globin genes through matrix related nuclear
relocation of the cluster. Biochem Biophys Res Commun. 383:11-15.
9. Eskiw CH, Cope NF, Clay I, Schoenfelder S, Nagano T, Fraser P. (2010).
Transcription factories and nuclear organization of the genome. Cold Spring
Harb Symp Quant Biol. 75:501-506.
10. Mehta IS, Amira M, Harvey AJ, Bridger JM. (2010). Rapid chromosome
territory relocation by nuclear motor activity in response to serum removal in
primary human fibroblasts. Genome Biol. 11(1):R5
11. Szczerbal I, Foster HA, Bridger JM. (2009). The spatial repositioning of
adipogenesis genes is correlated with their expression status in a porcine
mesenchymal stem cell adipogenesis model system. Chromosoma. 118:647663.
12. Knight M, Ittiprasert W, Odoemelam EC, Adema CM, Miller A, Raghavan N,
Bridger JM. (2011). Non-random organization of the Biomphalaria glabrata
genome in interphase Bge cells and the spatial repositioning of activated
genes in cells co-cultured with Schistosoma mansoni. Int J Parasitol. 41:6170.
13. Spilianakis CG, Lalioti MD, Town T, Lee GR, Flavell RA. (2005).
Interchromosomal associations between alternatively expressed loci. Nature.
435:637-645.
14. Brown JM, Green J, das Neves RP, Wallace HA, Smith AJ, Hughes J, et al.
(2008). Association between active genes occurs at nuclear speckles and is
modulated by chromatin environment. J Cell Biol. 182:1083-1097.
15. Bridger JM and Mehta IS. (2011). Nuclear Molecular Motors for Active,
Directed Chromatin Movement in Interphase Nuclei. In: Adams, Niall M.;
Freemont, Paul S. Eds. Advances in Nuclear Architecture. Springer 2011
16. Gonsior SM, Platz S, Buchmeier S, Scheer U, Jockusch BM, Hinssen H
(1999). Conformational difference between nuclear and cytoplasmic actin as
detected by a monoclonal antibody. J Cell Sci. 112:797-809.
17. Schoenenberger CA, Buchmeier S, Boerries M, Sütterlin R, Aebi U, Jockusch
BM. (2005) Conformation-specific antibodies reveal distinct actin structures in
the nucleus and the cytoplasm. J Struct Biol. 152:157-68.
18. McDonald D, Carrero G, Andrin C, de Vries G, Hendzel MJ. (2006).
Nucleoplasmic beta-actin exists in a dynamic equilibrium between low-mobility
polymeric species and rapidly diffusing populations. J Cell Biol. 172:541-552.
19. Zhao K, Wang W, Rando OJ, Xue Y, Swiderek K, Kuo A, Crabtree GR.
(1998). Rapid and phosphoinositol-dependent binding of the SWI/SNF-like
BAF complex to chromatin after T lymphocyte receptor signaling. Cell.
95:625-636.
20. Jockusch BM, Schoenenberger CA, Stetefeld J, Aebi U. (2006). Tracking
down the different forms of nuclear actin.Trends Cell Biol. 16:391-396.
21. Hagen SJ, Kiehart DP, Kaiser DA, Pollard TD. (1986). Characterization of
monoclonal antibodies to Acanthamoeba myosin-I that cross-react with both
myosin-II and low molecular mass nuclear proteins. J Cell Biol 103:2121–
2128
22. Berrios M, Fisher PA, Matz EC. (1991). Localization of a myosin heavy chainlike polypeptide to Drosophila nuclear pore complexes. Proc Natl Acad Sci U
S A 88:219–223
23. Nowak G, Pestic-Dragovich L, Hozak P, Philimonenko A, Simerly C et al
(1997). Evidence for the presence of myosin I in the nucleus. J Biol Chem
272:17176–17181
24. Pestic-Dragovich L, Stojiljkovic L, Philimonenko AA, Nowak G, Ke Y et al.
(2000). A myosin I isoform in the nucleus. Science 290:337–341
25. Vreugde S, Ferrai C, Miluzio A, Hauben E, Marchisio PC et al. (2006).
Nuclear myosin VI enhances RNA polymerase II-dependent transcription. Mol
Cell 23:749–755
26. Cameron RS, Liu C, Mixon AS, Pihkala JP, Rahn RJ et al. (2007).
Myosin16b: The COOH-tail region directs localization to the nucleus and
overexpression delays S-phase progression. Cell Motil Cytoskeleton 64:19–
48
27. Pranchevicius MC, Baqui MM, Ishikawa-Ankerhold HC, Lourenco EV, Leao
RM et al. (2008). Myosin Va phosphorylated on Ser1650 is found in nuclear
speckles and redistributes to nucleoli upon inhibition of transcription. Cell Motil
Cytoskeleton 65:441–456
28. Lindsay AJ, McCaffrey MW. (2009). Myosin Vb localises to nucleoli and
associates with the RNA polymerase I transcription complex. Cell Motil
Cytoskeleton 66:1057–1072
29. Jung EJ, Liu G, Zhou W, Chen X. (2006). Myosin VI is a mediator of the p53dependent cell survival pathway. Mol Cell Biol 26:2175–2186
30. Philimonenko VV, Zhao J, Iben S, Dingová H, Kyselá K, Kahle M, et al.
(2004). Nuclear actin and myosin I are required for RNA polymerase I
transcription. Nat Cell Biol. 6:1165-1172.
31. Fomproix N, Percipalle P (2004). An actin-myosin complex on actively
transcribing genes. Exp Cell Res 294:140–148
32. Mehta IS, Eskiw CH, Arican HD, Kill IR, Bridger JM. (2011).
Farnesyltransferase inhibitor treatment restores chromosome territory
positions and active chromosome dynamics in Hutchinson-Gilford Progeria
Syndrome cell. Genome Biol In press.
33. Morey C, Da Silva NR, Perry P, Bickmore WA. (2007). Nuclear reorganisation
and chromatin decondensation are conserved, but distinct, mechanisms
linked to Hox gene activation. Development. 134:909-919.
34. Martou G, Park PC, De Boni U (2002). Intranuclear relocation of the Plc beta3
sequence in cerebellar purkinje neurons: temporal association with de novo
expression during development. Chromosoma. 110:542-549.
35. Takizawa T, Gudla PR, Guo L, Lockett S, Misteli T. (2008). Allele-specific
nuclear positioning of the monoallelically expressed astrocyte marker GFAP.
Genes Dev. 22:489-498.
36. Strasák L, Bártová E, Harnicarová A, Galiová G, Krejcí J, Kozubek S. (2009).
H3K9 acetylation and radial chromatin positioning. J Cell Physiol. 220:91-101.
37. Bridger JM, Boyle S, Kill IR, Bickmore WA. (2000). Re-modelling of nuclear
architecture in quiescent and senescent human fibroblasts. Curr Biol. 10:149152.
38. Cook PR. (2010) A model for all genomes: the role of transcription factories.
J Mol Biol. 395:1-10.
39. Chuang CH, Carpenter AE, Fuchsova B, Johnson T, de Lanerolle P, Belmont
AS. (2006). Long-range directional movement of an interphase chromosome
site. Curr Biol. 16:825-831
40. Brown JM, Green J, das Neves RP, Wallace HA, Smith AJ, Hughes J, et al.
(2008). Association between active genes occurs at nuclear speckles and is
modulated by chromatin environment. J Cell Biol. 182:1083-1097.
41. Meaburn KJ, Cabuy E, Bonne G, Levy N, Morris GE, Novelli G, Kill IR, Bridger
JM. (2007). Primary laminopathy fibroblasts display altered genome
organization and apoptosis. Aging Cell. 6:139-153.
42. Mehta IS, Figgitt M, Clements CS, Kill IR, Bridger JM. (2007). Alterations to
nuclear architecture and genome behavior in senescent cells. Ann N Y Acad
Sci. 1100:250-263.
43. Dingová H, Fukalová J, Maninová M, Philimonenko VV, Hozák P. (2009).
Ultrastructural localization of actin and actin-binding proteins in the nucleus.
Histochem Cell Biol. 131:425-434.
44. Bridger JM, Kill IR, O'Farrell M, Hutchison CJ. (1993). Internal lamin
structures within G1 nuclei of human dermal fibroblasts. J Cell Sci. 104:297306.
45. Hozák P, Sasseville AM, Raymond Y, Cook PR. (1995). Lamin proteins form
an internal nucleoskeleton as well as a peripheral lamina in human cells. J
Cell Sci. 108:635-644.
46. Sasseville AM, Langelier Y. (1998). In vitro interaction of the carboxy-terminal
domain of lamin A with actin. FEBS Lett. 425:485-489.
47. Holt I, Ostlund C, Stewart CL, Man N, Worman HJ, Morris GE. (2003). Effect
of pathogenic mis-sense mutations in lamin A on its interaction with emerin in
vivo. J Cell Sci. 116:3027-3035.
48. Holaska JM, Wilson KL. (2007). An emerin “proteome”: purification of distinct
emerin-containing complexes from HeLa cells suggests molecular basis for
diverse roles including gene regulation, mRNA splicing, signaling,
mechanosensing, and nuclear architecture. Biochemistry 46:8897–8908
49. Mellad JA, Warren DT, Shanahan CM. (2011). Nesprins LINC the nucleus
and cytoskeleton. Curr Opin Cell Biol. 23:47-54.