Cytoskeleton - Locomotion Kohidai, Laszlo MD, PhD Med. habil., Assoc. Professor Dept. Genetics, Cell & Immunobiology, Semmelweis University Lecture ED 2014 www.dgci.sote.hu

Main functions of cytoskeleton Determines the shape of the cell Anchores organelles Movement of organelles Tensile strength Movement of chromosomes Polarity Motility

Cytoskeleton Microfilaments (actin) Microtubuli (tubulin) Intermedier filaments Motor proteins Actin and microtubule associated proteins

Microfilaments Microtubuli Intermedier filaments

SLIDING Globular proteins Ca2+ ATP Motor proteins Fibrillar proteins

Microfilaments

Polymerization of actin + ATP ADP Depolymerization - cytochalasin – inh. phalloidin - stabilizer ADP ATP Pi Polymerization - slow

Actin - still in Prokaryots ! ((Ent et al. Nature 2001,413, 39)

Other actin homologues ((Roeben A et al. J Mol. Biol 2006, 358, 145)

Comparison of homologues Polymerization in both forms Opposite chirality !!! ((Wickstead and Gull J Cell. Biol 2011, 194, 513)

Cyclosis Transitional connections between actin and myosin Moving cytoplasm Stationary (cortical) Actin filaments Chloroplasts Cell-wall Plasma membrane Transitional connections between actin and myosin Ca2+, temperature- and pH-dependent (Lodish, H. et al. Mol. Cell Biol. 2000, 767)

„Fountain” mechanism Ca2+-dep. requires ATP Mono- Poly- Lobo- podial Filo- Reticulo- Formation of pseudopodium stress-fibrillums integrins

Cross-linking proteins of actin contractile bundle a actinin – in stress fibr. gel-like network filamin - cortex „tight” parallel bundle fimbrin – in filopodium

Migrating keratinocyte 15 mm/sec Formation of lobopodium actin-network microtubuli

Regulator proteins of actin polymerisation gCAP39 Severin Gelsolin Villin CapZ Tropomodulin - +  Cofilin Severin Gelsolin

Actin polymerization – acrosomal-reaction (Lodish, H. et al. Mol. Cell Biol. 2000, 767)

Listeria monocytogenes local actin polymerization speed: 10 mm/min high ability to transmit in tissues actin (Fred Soo & Julie Theriot Laboratory

Model of actin nucleation WASP = Wiscott-Aldrich syndr. prot.

Structure of cortical region (Svitkina, TM, Borisy GG J. Cell Biol. 1999, 145, 1009)

Actin – membrane links F-Actin Myosin I. Arp2/3 Profilin - G-actin Integrin Filamin

Profilin-mechanism Tb4 = thymosin b4 Proline-rich protein (Lodish, H. et al. Mol. Cell Biol. 2000, 767)

Filamin – Membrane link actin

+ Structure of focal contact actin filament a actinin vinculin paxillin talin integrin fibronectin

A plasma membrane – cortex links Thrombocyte Muscle Epithel Spectrin tetramer Glycophorin Ankyrin ((Lux SE, 1979 Nature 281:426)

+ + + - - E Electromagnetic field induces the transformation of cytoskeleton and formation of pseudopodia Adhesion plaque + + + - -

Myosin head Ca2+-dependent phosphorylation and its effect on the 3D strcture ATP - ADP Pi light chain heavy chain a helix myosin I. 150 kD monomer myosin I I. 260 kD Head: - ATP-ase - motor dimer

Distribution of myosines in the migrating Dyctiostelium and in dividing cell myosin I. (green) myosin II. (red) (Fukui, Y. Mol. Cell Biol 2000, 785))

- + Main types of interactions between the globular and fibrillar components of cytoskeleton membrane

Non-treated F-actin blocked MT-blocked

Microtubules

Tubulin – still in Prokaryotes ! FtsZ Tubulin (Margolin Laboratory, University of Texas)

Comparison of homologues Polymerization in both forms Monomers build helical structure vs. dimers build tubulus ((Wickstead and Gull J Cell. Biol 2011, 194, 513)

Polymerization of tubulin GTP GTP GTP GTP Polymerization - fast Protofilament (strait) GDP GDP GDP GDP Protofilament (curved) Depolymerization

Dynamics of microtubule-assembly Nucleation Elongation - + incorporation balanced release

Role of g-tubulin in nucleation (Wiease et al. Curr.Opin.Struct.Biol. 1999, 9, 250)

Microtubular systems in the cells - Centrosome - Cilia / flagellum Interphase cell Dividing cell Neuron centrosome Basal body Cilla spindle axon Microtubular systems in the cells - Centrosome - Cilia / flagellum - Mitotic system - Vesicular transport

MTOC = Microtubulus organizing center g-tubulin specific region of the cortex ((Brinkley, B.R. Encyclop. Neurosci. 1987, 665)

Network of microtubuli Protofilaments a-b dimer alpha tubulin beta tubulin 24 nm Fibroblast

Cilia cilia flagellum Paramecium

A B dynein-arms nexin tubulin (13 ill. 11 protofilaments)

Composition of dynein-arms ATP-independent binding ATP-dependent hydrolysis The arm moves toward the - pole

The role of dynein arms in beating of cilia Bending „Telescoping” Proteolysis

Molecules composing the cilia more than 250 types of molecules 70% a and b tubulin dynein arms outer - 9 polypeptides - ATP-ase inner – composition varies radial spokes - 17 polypeptides

Microtubules of mitotic spindle and kinetochore

Arrangement of actin during cell-division

Intermedier filaments

Crescentin

Mechanical characterization of cytoskeleton components intermedier filament i.e. vimentin microtubule = rupture deformation actin filament force

Role of intermedier filaments Buffer against external mechanical stress Tissue specificity Epithel – keratin Connective tissue Muscles Neuroglia Neurones - neurofilaments Nucleus – lamines (lamina fibrosa) } vimentin

Structure of intermedier filamentums (Lodish, H. et al. Mol. Cell Biol. 2000, 767)

Domain structures of intermedier filamentums H2N- a helical domain -COOH keratins vimentin neurofilam. prot. nuclear prot

Intermedier filaments Keratin filaments Vimentin-like filaments ! They DO NOT co-polymerize !

Microvilli myosin I. actin villin „terminal web” a rigid bundle composed by 20-30 actin mol.s actin + on the apical part villin is the linker molecule of actins „terminal web” = intermedier fil. + spectrin anchoreb by myosin I. and calmodulin to the surface membrane

SEM structure of microvilli actin bundle linker molecules „terminal web”

Intermedier filaments Neuro-filaments – many cross-linkers Glial filaments – few cross-linkers The number of protein cross-links between the intermedier filaments varies in different tissues

Microtubuli associated proteins (MAP-s)

Groups of MAP-s Structural MAP-s - MT-assembly - links to MF and to IF Motor proteins - sliding on MT Enzymes, signal molecules - glycolytic enzymek - kinases Shape and polarity of the cell Membrane transports Assembly of molecules

Motor-proteins

Structure of motor-proteins motor domain motor domain assoc. polypeptides „stalk” Kinesin Myosin Dynein

Motor proteins - + kinesin dynein microtubule kinesin dynein heavy chain light chain kinesin dynein

kinesin - + dynein cAMP cAMP pigment cells

Kinesin ADP ATP ADP ATP ADP ATP ADP ADP-Pi

MT-motor proteins and the transported elements (Hirokawa, N. Science 1998, 279:519

Dynein – membrane relations (Hirokawa, N. Science 1998, 279:519)

There are other mechanisms over sliding …

Locomotion – with spasmoneme of Vorticella

Spasmoneme spring Contracts 40% in few msecs Velocity: 8 cm˛/sec Negative charges Neutralization with Ca2+

Actin spring in sperm of horse-shoe crab Limulus polyphemus ! acrosome actin bundle The extension does not involve a myosin motor or actin polymerization The bundle is crystalline in its coiled and uncoiled states