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Cytoskeleton and locomotion
www.dgci.sote.huLecture EPh 2014
Láng, Orsolya MD, PhDDept. Genetics, Cell & Immunobiology, Semmelweis University
Main components of the eukaryotic cytoskeleton
Microfilaments:
actin
7nm
Microtubules:
tubulins (
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
Microfilament like:
MreB
Microtubule like:
FtsZ, TubZ
Intermediate filament like:
Crescentin, Parm
weak sequence homology butcrystallography appreciate their striking structural properties and similarity to eukaryotic cytoskeletal elements
Nature Cell Biology12, 731–733 (2010)Nature Cell Biology12, 731–733 (2010)
Tissue level:
muscle movement
Cellular level:
determines shape of the cell
motility of the cells
cell adhesion
mitosis, meiosis
Subcellular level:
anchors organelles
organization of organelles
provides tensile strength
movement of chromosomes
organizing cell polarity
Intracellular movement of vesicles
- Endocytosis – clathrin-mediated endocytosis and phagocytosis
Function of the cytoskeleton
Dynamic
Adaptable
Stable
Strong
Cytoskeletal filaments are dynamic and adaptable
Stability of cytoskeletal filaments
Strong cytoskeletal filaments
Intermediate filaments- resistant to stretching forces
Microfilaments
( Garland Science Molecular Biology of the Cell 2008)
Molecular structure of actin
F-actinG-actin
ATPATPADPADP
ADPADP ATPATP
PiPolymerization - slow
Depolymerization
+
-
Drugs: cytochalasin – inhibition (cap at + end) phalloidin - stabilizer
Polymerazition of actin
Treadmilling of actin filament
Video: http://csls-text.c.u-tokyo.ac.jp/flash/0611_1.html
Molecular Biology of the Cell (© Garland Science 2008)
Actin and accessory proteins
3 groups: 3 groups:
banding and cross linking proteins banding and cross linking proteins
regulatory proteins: regulatory proteins: polymerization/depolymerization, polymerization/depolymerization, severing proteins,capping proteins severing proteins,capping proteins
Motor proteinsMotor proteins -- sliding on MF (myosin)sliding on MF (myosin)
Organisation of Organisation of actin filamentsactin filaments
SlidingSliding
Actin binding proteins (ABPs)
Molecular Biology of the Cell (© Garland Science 2008)
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.
Gel-like network
plasma membrane
Regulatory proteins
-- ++
CofilinCofilinSeverinSeverinGelsolinGelsolin
CAP39CAP39
SeverinSeverin
GelsolinGelsolin
VillinVillin
CapZCapZ
TropomodulinTropomodulin
Profilin: G actin pool
Thymosin: actin sequestring
Formin: actin polymerizing protein
Actin polymerisation – moition I.Actin polymerisation – moition I.
(Lodish, H. et al. Mol. Cell Biol. 2000, 767)(Lodish, H. et al. Mol. Cell Biol. 2000, 767)
Acrosomal reaction
Listreia Monocytogenes infection
Actin polymerisation – motion II.Actin polymerisation – motion II.
Actin – membrane links
F-ActinF-Actin
IntegrinIntegrinFilaminFilamin
ProfilinProfilin- G-aktin- G-aktin
Myozin I.Myozin I.Arp2/3Arp2/3
membranemembrane
Model of actin nucleation
WASP = Wiscott-Aldrich syndr. prot.WASP = Wiscott-Aldrich syndr. prot.
Proline-richprotein
Profilin-mechanism
(Lodish, H. et al. Mol. Cell Biol. 2000, 767)(Lodish, H. et al. Mol. Cell Biol. 2000, 767)
TT44 = = thymozin thymozin 44
Filamin – Membrane link
filaminfilamin
actinactin
actin filamentactin filament actininactinin
vinculinvinculin
paxillinpaxillin
talintalin
integrinintegrinfibronectinfibronectin
Structure of focal contact
++
Thrombocyte: Thrombocyte: filaminfilamin
Muscle: Muscle: dystrophindystrophin
Epithel: Epithel: ezrinezrin
A plasma membrane – cortex links
SpectrinSpectrintetramertetramer
GlycophorinGlycophorin
AnkyrinAnkyrin
(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
Molecular Biology of the Cell (© Garland Science 2008)
Myosin II molecule
Distribution of myosines in the migrating Distribution of myosines in the migrating DyctiosteliumDyctiostelium and in dividing celland in dividing cell
myozin Imyozin I(green)(green)
myozin IImyozin II
(red)(red)
(Fukui, Y. Mol. Cell Biol 2000, 785))
Microtubules
Molecular Biology of the Cell (© Garland Science 2008)
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 tubulin in nucleation of microtubules at - end
Structural MAP-sStructural MAP-s-- MT-assemblyMT-assembly-links to MF and to IF links to MF and to IF
(eg. tau, MAPs1 and MAPs2)(eg. tau, MAPs1 and MAPs2)
Motor proteinsMotor proteins -- sliding on MTsliding on MT(e.g. kinesin and dynein family)(e.g. kinesin and dynein family)
Enzymes, signal moleculesEnzymes, signal molecules- glycolytic enzymes- glycolytic enzymes
- kinases- kinases
Shape and polarityShape and polarityof the cellof the cell
Vesicular transportsVesicular transports
Assembly of moleculesAssembly of molecules
Microtubule associated proteins (MAPs)
Structure ofStructure of motor-proteinsmotor-proteins
motor domain
motor domain
assoc.polypeptides
assoc.
polypeptides
assoc.
polypeptides
„stalk”
„stalk”
Kinesin Myozin Dynein
Motor proteins
++
--
dyneindynein
++
kinesinkinesin
light chainlight chain
heavyheavychainchain
?
++--
kinesinkinesin
dyneindyneincAMPcAMP cAMPcAMP
pigment cellspigment cells
ATPATPADPADP
ATPATP
ADPADPADPADP
ATPATP
ADPADP
ADP-PiADP-Pi
KinesinKinesin
Interphase cellInterphase cell
Dividing cellDividing cell
NeuronNeuron
centrosomecentrosome
Basal bodyBasal body
CillaCilla
spindlespindle
centrosomecentrosome
axonaxon
Microtubular systems in the cells
-Centrosome
-Cilia / flagellum
-Mitotic system
- Vesicular transport
In cells: MTOC = Microtubule organizing center
(Brinkley, B.R. Encyclop. Neurosci. 1987, 665)
-tubulin-tubulin
9x3 microtubules A,B,C
2 centrioles at a right angle
Organisator:
Gamma-tubulin in pericentriolar matrix
- end of forming microtubule
Molecular Biology of the Cell (© Garland Science 2008)
Centrosome
Cilia
ciliacilia
flagellumflagellum
Paramecium
AABB dynein-armsdynein-arms
nexinnexin
tubulin tubulin (13 ill. 11 protofilaments)(13 ill. 11 protofilaments)
Structure of cilia
Molecules composing the cilia
70% α and β tubulin dynein arms - outer - 9 polypeptides - ATP-ase
- inner – composition varies radial spokes - 17 polypeptides
more than more than 250250 types of molecules types of molecules
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 relationsDynein – membrane relations
(Hirokawa, N. Science 1998, 279:519)
Intermediate filaments
Molecular Biology of the Cell (© Garland Science 2008)
Structure of an intermediate filaments
Monomer
Parallel dimer
Antiparallel tetramer
Protofilaments
Intermedaite
filaments
Mechanical characterization of cytoskeleton components
defo
rmat
ion
force
actin filament
intermedier filamenti.e. vimentin
microtubule
= rupture
Role of intermedier filaments
Buffer against external mechanical stress
Tissue specificity !!!
Epithel – keratin
Connective tissueMusclesNeuroglia
Neurones(axon) - neurofilaments
} vimentin-like
Exception:Nucleus – lamines (A,B,C) →(lamina fibrosa)
Vimentin
Desmin
Glial protein
HH22N-N- --COOHCOOH
keratinskeratins
vimentinvimentin
neurofilam. prot.neurofilam. prot.
nuclear protnuclear prot..
helical domainhelical domain
Domain structures of intermedier filamentumsDomain structures of intermedier filamentums
HeadHead TailTail
Molecular Biology of the Cell (© Garland Science 2008)
Cross bridges are formed by non-
helical C terminus
few cross bridges
Axon of a neuron
Neurofilament (NF-H)
Glial cell
Glial filaments
Axon cross-section
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 – -actinin + desmin Thin filament – -helical actin
molecules + tropomyosins + troponin Thick filament – myosin II molecules
+ MBP (myosin binding proteins)Other proteins: titin, distrophin
Troponin: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.htmlhttp://www.microscopyu.com/moviegallery/livecellimaging/index.htmlhttp://www.cellmigration.org/science/