Cytoskeleton and locomotion
Láng, Orsolya MD, PhD
Dept. Genetics, Cell & Immunobiology, Semmelweis University
Lecture EPh 2014
www.dgci.sote.hu
Main components of the eukaryotic cytoskeleton
Microfilaments:
 actin
7nm
Microtubules:
 tubulins (a, b)
25 nm
Intermediate filaments:
 lamin
 cell specific prot. (e.g. vimentin)
8-12 nm
+ Associated proteins (e.g. motor proteins)
Filamentous structures in the cytoplasm of walled bacteria
weak sequence homology but
crystallography appreciate
their striking structural
properties and similarity to
eukaryotic cytoskeletal
elements
Microfilament like:
 MreB
Microtubule like:
 FtsZ, TubZ
Intermediate filament like:
 Crescentin, Parm
Nature Cell Biology12, 731–733 (2010)
Function of the cytoskeleton
Tissue level:
 muscle movement
Cellular level:
 determines shape of the cell
 motility of the cells
 cell adhesion
 mitosis, meiosis
Dynamic
Subcellular level:
 anchors organelles
Adaptable
 organization of organelles
Stable
 provides tensile strength
Strong
 movement of chromosomes
 organizing cell polarity
 Intracellular movement of vesicles
- Endocytosis – clathrin-mediated endocytosis and phagocytosis
Cytoskeletal filaments are dynamic and adaptable
Stability of cytoskeletal filaments
Strong cytoskeletal filaments
Intermediate filaments- resistant to stretching forces
Microfilaments
Molecular structure of actin
G-actin
F-actin
( Garland Science Molecular Biology of the Cell 2008)
Polymerazition of actin
ATP
ADP
+
Depolymerization
ATP
ADP
Pi
Polymerization - slow
Drugs: cytochalasin – inhibition (cap at + end)
phalloidin - stabilizer
Treadmilling of actin filament
Video: http://csls-text.c.u-tokyo.ac.jp/flash/0611_1.html
Actin and accessory proteins
Molecular Biology of the Cell (© Garland Science 2008)
Actin binding proteins (ABPs)
3 groups:
 banding and cross linking proteins
 regulatory proteins:
polymerization/depolymerization,
severing proteins,capping proteins

Organisation of
actin filaments
Motor proteins
- sliding on MF (myosin)
Sliding
Organisation of actin filaments
Molecular Biology of the Cell (© Garland Science 2008)
Cross-linking proteins I.
Contractile bundle
Parallel bundle
Molecular Biology of the Cell (© Garland Science 2008)
Cross-linking proteins II.
plasma membrane
Gel-like network
Molecular Biology of the Cell (© Garland Science 2008)
Regulatory proteins
gCAP39
Tropomodulin
Severin
Gelsolin
-
+
Villin

CapZ
Cofilin
Severin
Gelsolin
Profilin: G actin pool
Thymosin: actin sequestring
Formin: actin polymerizing
protein
Actin polymerisation – moition I.
Acrosomal reaction
(Lodish, H. et al. Mol. Cell Biol. 2000, 767)
Actin polymerisation – motion II.
Listreia Monocytogenes
infection
Actin – membrane links
membrane
Myozin I.
Arp2/3
F-Actin
Profilin
- G-aktin
Filamin
Integrin
Model of actin nucleation
WASP = Wiscott-Aldrich syndr. prot.
Profilin-mechanism
Tb4 = thymozin b4
Proline-rich
protein
(Lodish, H. et al. Mol. Cell Biol. 2000, 767)
Filamin – Membrane link
filamin
actin
Structure of focal contact
actin filament
a actinin
vinculin
+
paxillin
talin
integrin
fibronectin
A plasma membrane – cortex links
Thrombocyte: filamin
Glycophorin
Ankyrin
Spectrin
tetramer
Muscle: dystrophin
Epithel: ezrin
(Lux SE, 1979 Nature 281:426)
Microvilli
At bottom: (spectrin, myosin, intermediate filaments)
It is called: terminal web
Motor proteins: myosins
General structure:
 Globular head and
fibrillar tail
 Heavy chains and light
chains
 Head: motor domain with
ATP-ase activity
 ADP- straight
Direction: + end motors
Myosin II molecule
Molecular Biology of the Cell (© Garland Science 2008)
Distribution of myosines in the migrating
Dyctiostelium and in dividing cell
myozin I
(green)
myozin II
(red)
(Fukui, Y. Mol. Cell Biol 2000, 785))
Microtubules
Molecular structure of tubulin dimers and microtubules
GTP
Molecular Biology of the Cell (© Garland Science 2008)
Polymerisation of microtubules
Drugs:
 Colchicine and colcemid– inhibition (binds subunit)
 Taxol - stabilizer
Molecular Biology of the Cell (© Garland Science 2008)
Polymerisation of microtubules
Dynamic instability
and treadmilling
Video: http://csls-text.c.u-tokyo.ac.jp/flash/0612_1.html
Molecular Biology of the Cell (© Garland Science 2008)
Role of g-tubulin in nucleation of microtubules at - end
Molecular Biology of the Cell (© Garland Science 2008)
Microtubule associated proteins (MAPs)

Structural MAP-s
- MT-assembly
-links to MF and to IF
(eg. tau, MAPs1 and MAPs2)

Motor proteins
- sliding on MT
(e.g. kinesin and dynein family)

Enzymes, signal molecules
- glycolytic enzymes
- kinases
Shape and polarity
of the cell
Vesicular transports
Assembly of molecules
Structure of motor-proteins
assoc.
motor domain
polypeptides
motor
domain
„stalk”
assoc.
polypeptides
Kinesin
„stalk”
assoc.
polypeptides
Myozin
Dynein
Motor proteins
-
+
heavy
chain
light chain
dynein
+
kinesin
?
kinesin
-
+
dynein
cAMP
cAMP
pigment cells
Kinesin
ADP
ATP
ADP
ADP
ATP
ATP
ADP
ADP-Pi
Microtubular systems in the cells
-Centrosome
-Cilia / flagellum
Interphase cell
centrosome
Cilla
Basal body
Dividing cell
spindle
-Mitotic system
Neuron
centrosome
- Vesicular transport
axon
In cells: MTOC = Microtubule organizing center
g-tubulin
9x3 microtubules A,B,C
2 centrioles at a right angle
Organisator:
Gamma-tubulin in pericentriolar matrix
- end of forming microtubule
(Brinkley, B.R. Encyclop. Neurosci. 1987, 665)
Centrosome
Molecular Biology of the Cell (© Garland Science 2008)
Cilia
cilia
flagellum
Paramecium
Structure of cilia
tubulin
(13 ill. 11 protofilaments)
B
A
dynein-arms
nexin
Molecules composing the cilia
more than
250 types of molecules
 70% α and β tubulin
 dynein arms
- outer - 9 polypeptides - ATP-ase
- inner – composition varies
 radial spokes - 17 polypeptides
Role of the dynein arms in beating cilia
Telescopic effect
Beating
Microtubules of mitotic spindle and kinetochore
How motor proteins can organise the position of cell
organelles (ER, Golgi) ?
(Hirokawa, N. Science 1998, 279:519
Dynein – membrane relations
(Hirokawa, N. Science 1998, 279:519)
Intermediate filaments
Structure of an intermediate filaments
Monomer
Parallel dimer
Antiparallel
tetramer
Protofilaments
Intermedaite
filaments
Molecular Biology of the Cell (© Garland Science 2008)
Mechanical characterization of cytoskeleton components
intermedier filament
i.e. vimentin
deformation
microtubule
= rupture
actin filament
force
Role of intermedier filaments
Buffer against external mechanical stress
Tissue specificity !!!
Epithel – keratin
Connective tissue
Muscles
Neuroglia
}
Vimentin
vimentin-like
Neurones(axon) - neurofilaments
Exception:
Nucleus – lamines (A,B,C) →(lamina fibrosa)
Desmin
Glial protein
Domain structures of intermedier filamentums
Head
a helical domain
H2N-
keratins
vimentin
neurofilam. prot.
nuclear prot.
Tail
COOH
Axon of a neuron
Glial cell
Neurofilament (NF-H)
Glial filaments
Cross bridges are formed
by non-helical C terminus
Axon cross-section
few cross bridges
Molecular Biology of the Cell (© Garland Science 2008)
Cell locomotion/ movement
Cellular level:
 Ciliar movement
 Amoeboid mocevent
Mesenchymal migration
Collective migration
Tissue level:
 muscle movement – skeletal muscle
Cilia - flagellum
Amoeboid movement
chemoattractant
Composition of thick filament in a sarcomer
Composition of sarcomer
 Working unit bordered by two Z-lines/disc
 Z line – a-actinin + desmin
 Thin filament – a-helical actin
molecules + tropomyosins + troponin
 Thick filament – myosin II molecules
+ MBP (myosin binding proteins)
Other proteins: titin, distrophin
Troponin:
Tn-T binds tropomyosin
Tn-C binds Ca2+(4 Ca2+/mol = calmodulin)
Tn-I inhibitor
Skeletal muscle - Sarcomere
Skeletal muscle - Sarcoplasmic reticulum
X-linked recessive inheritance
Duschenne muscular distrophy
Useful videos and linkes:
http://csls-text.c.u-tokyo.ac.jp/active/06_01.html
http://www.microscopyu.com/moviegallery/livecellimaging/index.html
http://www.cellmigration.org/science/