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

!!