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Cytoskeleton - Locomotion
http://gsi.semmelweis.hu
Kohidai, Laszlo MD, PhDMed. habil., Assoc. Professor
Dept. Genetics, Cell & Immunobiology, Semmelweis University
Lecture ED 2015
Main functions of cytoskeletonMain functions of cytoskeleton
• Determines the shape of the cellDetermines the shape of the cell
• Anchores organellesAnchores organelles
• Movement of organellesMovement of organelles
• Tensile strengthTensile strength
• Movement of chromosomesMovement of chromosomes
• PolarityPolarity
• MotilityMotility
CytoskeletonCytoskeleton
Microfilaments (actin)Microfilaments (actin) Microtubuli (tubulin)Microtubuli (tubulin) Intermedier filamentsIntermedier filaments
Motor proteins Motor proteins Actin and mActin and microtubule associated proteinsicrotubule associated proteins
MicrofilamentsMicrofilaments
MicrotubuliMicrotubuli
Intermedier Intermedier filamentsfilaments
SLIDINGSLIDING
Globular proteinsGlobular proteins
Fibrillar proteinsFibrillar proteins
CaCa2+2+ ATPATP Motor proteinsMotor proteins
MicrofilamentsMicrofilaments
Polymerization of actinPolymerization of actin
ATPATPADPADP
ADPADP ATPATP
PPii
Polymerization - slowPolymerization - slow
DepolymerizationDepolymerization
++
--cytochalasin – inh.cytochalasin – inh.phalloidin - stabilizerphalloidin - stabilizer
Actin - still in Prokaryots ! Actin - still in Prokaryots !
((Ent et al. Nature 2001,413, 39)
((Roeben A et al. J Mol. Biol 2006, 358, 145)
Other actin homologuesOther actin homologues
Comparison of homologuesComparison of homologues
Polymerization in both formsPolymerization in both forms
Opposite chirality !!!Opposite chirality !!!
((Wickstead and Gull J Cell. Biol 2011, 194, 513)
CyclosisCyclosis
MovingMovingcytoplasmcytoplasm
Stationary Stationary (cortical)(cortical)cytoplasmcytoplasm
Actin filamentsActin filaments
ChloroplastsChloroplastsCell-wallCell-wall
Plasma membranePlasma membrane
(Lodish, H. et al. Mol. Cell Biol. 2000, 767)(Lodish, H. et al. Mol. Cell Biol. 2000, 767)
Transitional connections between actin and myosinTransitional connections between actin and myosin
CaCa2+2+, temperature- and pH-dependent, temperature- and pH-dependent
„„Fountain” mechanismFountain” mechanismCaCa2+2+-dep.-dep.requires ATPrequires ATP
integrinsintegrins
stress-fibrillumsstress-fibrillums
Formation of pseudopodiumFormation of pseudopodium
Mono-Mono-Poly-Poly-Lobo- podialLobo- podialFilo-Filo-Reticulo-Reticulo-
Cross-linking proteins of actinCross-linking proteins of actin
contractile bundlecontractile bundle actinin – in stress fibr.actinin – in stress fibr.
„„tight” parallel bundletight” parallel bundlefimbrin – in filopodiumfimbrin – in filopodium
gel-like networkgel-like networkfilamin - cortexfilamin - cortex
Migrating keratinocyteMigrating keratinocyte
15 15 m/secm/sec
actin-networkactin-network microtubulimicrotubuli
Formation of lobopodiumFormation of lobopodium
Regulator proteins of actin Regulator proteins of actin polymerisationpolymerisation
-- ++
CofilinCofilinSeverinSeverinGelsolinGelsolin
CAP39CAP39
SeverinSeverin
GelsolinGelsolin
VillinVillin
CapZCapZ
TropomodulinTropomodulin
Actin polymerization – Actin polymerization – acrosomal-reactionacrosomal-reaction
(Lodish, H. et al. Mol. Cell Biol. 2000, 767)(Lodish, H. et al. Mol. Cell Biol. 2000, 767)
Listeria monocytogenesListeria monocytogenes • local actin polymerizationlocal actin polymerization• speed: 10 speed: 10 m/minm/min• high ability to transmit high ability to transmit in tissuesin tissues
actinactin
(Fred Soo & Julie Theriot Laboratory
Model of actin nucleation
WASP = Wiscott-Aldrich syndr. prot.WASP = Wiscott-Aldrich syndr. prot.
Structure of cortical regionStructure of cortical region
(Svitkina, TM, Borisy GG J. Cell Biol. 1999, 145, 1009)(Svitkina, TM, Borisy GG J. Cell Biol. 1999, 145, 1009)
Actin – membrane linksActin – membrane links
F-ActinF-Actin
IntegrinIntegrinFilaminFilamin
ProfilinProfilin- G-actin- G-actin
Myosin I.Myosin I.Arp2/3Arp2/3
membranemembrane
Proline-richprotein
Profilin-mechanismProfilin-mechanism
(Lodish, H. et al. Mol. Cell Biol. 2000, 767)(Lodish, H. et al. Mol. Cell Biol. 2000, 767)
TT44 = = thymosin thymosin 44
Filamin – Membrane linkFilamin – Membrane link
filaminfilamin
actinactin
actin filamentactin filament actininactinin
vinculinvinculin
paxillinpaxillin
talintalin
integrinintegrinfibronectinfibronectin
Structure of focal contactStructure of focal contact
++
ThrombocyteThrombocyte
MuscleMuscle
EpithelEpithel
A plasma membrane – cortex linksA plasma membrane – cortex links
SpectrinSpectrintetramertetramer
GlycophorinGlycophorin
AnkyrinAnkyrin
((Lux SE, 1979 Nature 281:426)
EE
-- --
++ ++++
Adhesion plaqueAdhesion plaque
Electromagnetic fieldElectromagnetic field induces the transformationinduces the transformation of cytoskeleton and of cytoskeleton and formation of pseudopodiaformation of pseudopodia
MyosinMyosin
myosin I.myosin I.150 kD150 kD
myosin I I.myosin I I.260 kD260 kD
headhead
heavy chainheavy chain helixhelix
light chainlight chain
ATPATP - - ADPADP
PPii
Head: - ATP-aseHead: - ATP-ase - motor- motor
CaCa2+2+-dependent phosphorylation-dependent phosphorylationand its effect on the 3D strctureand its effect on the 3D strcture
dimerdimer
monomermonomer
Distribution of myosines in the Distribution of myosines in the migrating migrating DyctiosteliumDyctiostelium and in and in
dividing celldividing cell
myosin I.myosin I.(green)(green)
myosin II.myosin II.
(red)(red)(Fukui, Y. Mol. Cell Biol 2000, 785))
++--
Main types of interactions Main types of interactions between the between the globular globular and fibrillar and fibrillar componentscomponents of cytoskeletonof cytoskeleton
membranemembrane
Non-treatedNon-treated F-actin blocked F-actin blocked MT-blockedMT-blocked
MicrotubulesMicrotubules
FtsZFtsZ TubulinTubulin
Tubulin – Tubulin – still in Prokaryotes !still in Prokaryotes !
(Margolin Laboratory, University of Texas)
Comparison of homologuesComparison of homologues
Polymerization in both formsPolymerization in both forms
Monomers build helical structure vs. dimers build tubulusMonomers build helical structure vs. dimers build tubulus
((Wickstead and Gull J Cell. Biol 2011, 194, 513)
Polymerization of tubulinPolymerization of tubulin
Protofilament (strait)Protofilament (strait)
Protofilament (curved)Protofilament (curved)
GTPGTPGTPGTP GTPGTP
GTPGTP
GDPGDPGDPGDPGDPGDP
GDPGDP
Polymerization - fastPolymerization - fast
DepolymerizationDepolymerization
Dynamics of microtubule-assemblyDynamics of microtubule-assembly
incorporationincorporation
balancedbalanced
releaserelease
++ --
NucleationNucleation Elongation Elongation
Role of Role of -tubulin in nucleation-tubulin in nucleation
(Wiease et al. Curr.Opin.Struct.Biol. 1999, 9, 250)
Interphase cellInterphase cell
Dividing cellDividing cell
NeuronNeuron
centrosomecentrosome
Basal bodyBasal body
CillaCilla
spindlespindle
centrosomecentrosome
axonaxon
MicrotubularMicrotubular systemssystems in the cellsin the cells
-- CentrosomeCentrosome
- Cilia / flagellum- Cilia / flagellum
- Mitotic system- Mitotic system
- Vesicular transport- Vesicular transport
MTOC = MTOC = MMicroicrottubulus ubulus oorganizing rganizing ccenterenter
specificregion of the cortex
((Brinkley, B.R. Encyclop. Neurosci. 1987, 665)
-tubulin-tubulin
Network of microtubuli Network of microtubuli
ProtofilamentsProtofilaments
a-b dimerdimer
alpha tubulintubulin
beta tubulintubulin
24 nm
FibroblastFibroblast
Cilia
ciliacilia
flagellumflagellum
ParameciumParamecium
AABB dynein-armsdynein-arms
nexinnexin
tubulin tubulin (13 ill. 11 protofilaments)(13 ill. 11 protofilaments)
ATP-independent bindingATP-independent binding
ATP-dependent hydrolATP-dependent hydrolyysissis
The arm moves toward the The arm moves toward the - - pole pole
Composition of dynein-armsComposition of dynein-arms
The role of dynein arms The role of dynein arms in beating of ciliain beating of cilia
„„Telescoping”Telescoping”
BendingBending
ProteolysisProteolysis
Molecules composing the ciliaMolecules composing the cilia
70% 70% and and tubulin tubulin dynein armsdynein arms
outer - 9 polypeptides - ATP-aseouter - 9 polypeptides - ATP-aseinner – composition variesinner – composition varies
radial spokes - 17 polypeptidesradial spokes - 17 polypeptides
more than more than 250250 types of molecules types of molecules
Microtubules of mitotic spindle Microtubules of mitotic spindle and kinetochoreand kinetochore
Arrangement of actin during cell-divisionArrangement of actin during cell-division
Intermedier filamentsIntermedier filaments
CrescentinCrescentin
Mechanical characterization Mechanical characterization of cytoskeleton componentsof cytoskeleton componentsde
form
atio
nde
form
atio
n
forceforce
actin filamentactin filament
intermedier filamentintermedier filamenti.e. vimentini.e. vimentin
microtubulemicrotubule
= rupture= rupture
Role of intermedier filamentsRole of intermedier filaments
Buffer against external mechanical stressBuffer against external mechanical stress
Tissue specificityTissue specificity
Epithel –Epithel – keratin keratin
Connective tissueConnective tissueMusclesMusclesNeurogliaNeuroglia
Neurones - Neurones - neurofilaments neurofilaments
}} vimentinvimentin
Nucleus – Nucleus – lamineslamines(lamina fibrosa)(lamina fibrosa)
Structure of intermedier filamentumsStructure of intermedier filamentums
(Lodish, H. et al. Mol. Cell Biol. 2000, 767)(Lodish, H. et al. Mol. Cell Biol. 2000, 767)
HH22N-N- --COOHCOOH
keratinskeratins
vimentinvimentin
neurofilam. prot.neurofilam. prot.
nuclear protnuclear prot
helical domainhelical domain
Domain structures of intermedier filamentumsDomain structures of intermedier filamentums
Intermedier filaments
Keratin filamentsKeratin filaments Vimentin-like filamentsVimentin-like filaments
! They DO NOT co-polymeri! They DO NOT co-polymerizze !e !
MicrovilliMicrovilli
actinactinmyosin myosin I.I.
villinvillin
„„terminal web”terminal web”
• a rigida rigid bundle composed bybundle composed by 20-30 actin mol.s20-30 actin mol.s• actin + on the apical partactin + on the apical part• villin is the linker molecule of actinsvillin is the linker molecule of actins• „ „terminal web” = intermedterminal web” = intermedier ier fil. + spectrinfil. + spectrin• anchoreanchoreb by b by myosin I. and calmodulin to the surface membranemyosin I. and calmodulin to the surface membrane
SEM structure of microvilliSEM structure of microvilli
„„terminal web”terminal web”
actin bundleactin bundle
linker moleculeslinker molecules
Intermedier filamentsIntermedier filaments
The number of protein cross-links between theThe number of protein cross-links between the intermedier filaments intermedier filaments varies varies in different tissues in different tissues
Neuro-filaments Neuro-filaments – – many many cross-linkerscross-linkers
Glial filamentsGlial filaments
– – fewfew cross-linkers cross-linkers
Microtubuli associated proteinsMicrotubuli associated proteins(MAP-s)(MAP-s)
Groups of MAP-sGroups of MAP-s
• Structural MAP-sStructural MAP-s -- MT-assemblyMT-assembly
-- links to MF and to IF links to MF and to IF
• Motor proteinsMotor proteins -- sliding on MTsliding on MT
• Enzymes, signal moleculesEnzymes, signal molecules- glycolytic enzymek- glycolytic enzymek
- kinases- kinases
Shape and polarityShape and polarityof the cellof the cell
Membrane transportsMembrane transports
Assembly of moleculesAssembly of molecules
Motor-proteinsMotor-proteins
Structure of motor-proteinsStructure of motor-proteins
motor domain
motor domain
assoc.polypeptides
assoc.
polypeptides
assoc.
polypeptides
„stalk”
„stalk”
Kinesin Myosin Dynein
Motor proteinsMotor proteins
kinesinkinesin dyneindynein
microtubule
light chainlight chain
heavyheavychainchain
kinesinkinesin dyneindynein++ --
++--
kinesinkinesin
dyneindynein
cAMPcAMPcAMPcAMP
pigment cellspigment cells
ATPATPADPADP
ATPATP
ADPADPADPADP
ATPATP
ADPADP
ADP-PiADP-Pi
KinesinKinesin
MT-motor proteins and the transported elementsMT-motor proteins and the transported elements
(Hirokawa, N. Science 1998, 279:519
Dynein – membrane relationsDynein – membrane relations
(Hirokawa, N. Science 1998, 279:519)
There are other mechanismsThere are other mechanismsover sliding …over sliding …
Locomotion – with spasmoneme ofLocomotion – with spasmoneme ofVorticellaVorticella
Spasmoneme springSpasmoneme spring
Contracts Contracts 40%40% in few msecs in few msecsVelocity: Velocity: 8 cm˛/sec8 cm˛/sec
NegativeNegativechargescharges
Neutralization Neutralization with Cawith Ca2+2+
Actin spring in sperm of horse-Actin spring in sperm of horse-shoe crab shoe crab Limulus polyphemusLimulus polyphemus
• The extension does not involve a myosin The extension does not involve a myosin motor or actin polymerizationmotor or actin polymerization• The bundle is crystalline in its coiled and The bundle is crystalline in its coiled and uncoiled statesuncoiled states
acrosomeacrosome
actin bundleactin bundle
!!