CYTOSCLETONS AND MOVEMENT OF THE CELL:MICROFILAMENTS, INTERMEDIATE FILAMENTS AND MICROTUBULESISRA WAHIDUNIT PENELITIAN FAKULTAS KEDOKTERAN UNIVERSITAS HASANUDDIN

Motile CellsWhile most cells in the body are fixed in place by attachments to each other and basement membranes, some, like neutrophils and macrophages remain motile. Free living single cells are generally motile and cell movement plays an important in early embryogenesis. Microfilaments and microtubules interact to control cell movement.

The Cytoskeletonunique to eukaryotic cellsa dynamic three-dimensional structure that fills the cytoplasmacts as both muscle and skeleton, for movement and stability the long fibers of the cytoskeleton are polymers of subunits

Cytoskeletal ComponentsActin MicrofilamentsMicrotubulesIntermediate FilamentsIdown et al. The Histochemical Journal (2000) 32:165Scale bar represents 5mm

MicrofilamentsMicrofilaments are fine, thread-like protein fibers, 3-6 nm in diameter. Formed by polymerization of actin molecules (the most abundant cellular protein) Break down and reform rapidly, a contractile protein. Microfilaments' association with the protein myosin is responsible for muscle contraction.Microfilaments can also carry out cellular movements including gliding, contraction, and cytokinesis.

MicrotubulesMicrotubules are cylindrical tubes, 20-25 nm in diameter. Composed of subunits of the protein tubulin (alpha & beta). as a scaffold to determine cell shape, also break down and reform readilyprovide a set of "tracks" for cell organelles and vesicles to move onform the spindle fibers for separating chromosomes during mitosis. When arranged in geometric patterns inside flagella and cilia, they are used for locomotion.

Intermediate FilamentsIntermediate filaments are about 10 nm diameter Formed by polymerization of proteins such as keratin. These were thought to be very stable but is not always the caseprovide tensile strength for the cell.

Examples of the cytoskeleton in epithelial cells

In the intestine epithelial, all three types of fibers are present. Microfilaments project into the villi, giving shape to the cell surface. Microtubules grow out of the centrosome to the cell periphery. Intermediate filaments connect adjacent cells through desmosomes.

Actin MicrofilamentsThin, flexible filaments ~7nm in diameterHighly dynamicPresent in a 3-D gel throughout the cytoplasmOrganized by over 60 accessory proteinsPrimarily concentrated in structures such as stress fibers and cytoskeletal cortex Diagrams from Alberts (2002) Mol Biol Cell

MicrofilamentsForm the cortical cytoskeleton which lies under, and is attached to the plasma membrane and is involved in control of cell shape

Assist in forming the terminal web and the microvilli of epithelial cells

Cause movement of cells

4) Form bundles which form the contractile elements skeletal, cardiac and smooth muscle cells

Organisation of actinActin microfilaments are normally found as bundles. These may be networks as in the cortical cytoskeleton and in smooth muscles cells, tight, highly parallel bundles as in filopodia, microvilli and skeletal muscle or as looser bundles as in stress fibres

Actin controls cell shapeThe cortical cytoskeleton (actin plus associated proteins) not only determines the cell shape in fixed cells but also changes on shape as is the case with the platelet shown above

Function of MicrofilamentsRegulation of membrane movementProminent in growth cones (Actin)Dynamic changes in dendritic spine morphologyMuscle ContractionIn skeletal muscle (actin interacting with myosin)Local traffickingSensitive to local neuronal environment

MicrofilamentsAbundant inPresynaptic terminalsDendritic spinesGrowth conesPresent throughout cytoplasmActin cytoskelaton is universal in eukaryotes

MicrofilamentsTwo twisted strands of actin subunits4-6 nm diameter20-50 nm length (quite variable)

MicrofilamentsMultiple actin genes a-actinFour genes for four muscle types b-actin, g-actinAbundant in nervous tissueAll proteins similar (highly conserved)

MicrofilamentsProteins associated with MicrofilamentsMolecular motors (eg. myosin)Monomer actin-binding proteinsRegulate amount of actin assembled into microfilaments by sequestering actin monomersCapping proteinsAnchor microfilaments to other structuresRegulate microfilament lengthMutation in Schwann cells causes neurofibromatosis 2

MicrotubulesThick, rigid tube ~25 nm in diameterHighly dynamicPresent throughout the cytoplasmHigher order structures are not observed Diagrams from Alberts (2002) Mol Biol Cell

Microtubules1) Organise the endoplasmic reticulum and the Golgi apparatus2) Act as a railroad connecting the trans golgi network to the cell surface and the early endosome compartments to the late ones3) Form the spindle apparatus in mitotic cells4) Act as motile elements in cilia and flagella

Function of MicrotubulesCell movementFunctional core of cilia and flagellaMitotic spindleOrganelle involved in cell divisionInhabitants of axons and dendritesIntracellular transportEssential for fast-axonal transportCell Morphology

MicrotubulesSmallest subunit is tubulin10% of total brain protein a and b tubulin50 kDa proteinsMultiple genes for both typesDifferent gene products are enriched or specific to neuronsDifferent gene products are expressed at specific times in development

MicrotubulesSecond smallest subunit is "gobule"Heterodimer of a and b tubulinProtofilamentsLinear arrangement of globular subunits12-14 protofilaments form microtubule25 nm diameter, hollow tubeUp to hundreds mm lengthPolarized: +(fast) and (slow-growing) ends

Microtubules

Intermediate FilamentsSemi-flexible filaments ~10nm in diameterVery stable proteinsForms a 3-D gel throughout the cytoplasmProtects the cell from overloading Diagrams from Alberts (2002) Mol Biol Cell

Intermediate FilamentsAlso called NeurofilamentsFive classesType I, II: Keratin (hair and nails)Type V: nuclear laminsType III, IV: neuronal function

Intermediate Filaments1) Form cables which stretch across the cell from desmosomes on one side to desmosomes on the other so giving strength to the cells2) Hold the nucleus in place3) May play a role in organising permanent cell extensions such as nerve axons4) The nuclear lamina is formed by proteins called lamins whichare closelyrelated to intermediate filament proteins

Intermediate filamentsconcentrated in the region round the nucleus and holding the nucleus in place

Others raddiate to the cell surface and attach to desmosomes / hemi-desmosomes giving strength to the cell.

Reinforced tissueDistribution of the cytokeratin filaments (green) of cultured epithelial cells as compared with the plasma membrane (blue).

Desmosome bind both stains and appear pale blue.

Molecular MotorsMolecules that hydrolyze ATP (ATPase)Drive cell movement such as axonal transportThree typesMyosinMuscle contraction via interaction with microfilamentsDyneinKinesin

Cellular Motors

KinesinResponsible for fast axonal transport toward distal (terminal) endHead attaches to microtubuleTail attaches to organelleHydrolysis of ATP moves kinesin head distally, toward plus end of microtubuleStrongly inhibited by adenylyl-imidodiphosphate (AMP-PNP; nonhydrolyzable ATP analog)

KinesinSome kinesins areMonomers (KIF1A)Trimers (KIF3A/B)Head contains ATP binding and microtubule binding domainsHeadTail

DyneinMicrotubule transportAnterograde directionSubstrate is actin filaments or long microtubulesRetrograde directionSubstrate is long microtubulesMAP1c is one type40 nm in lengthWeakly inhibited by AMP-PNP

Dynein Structure

MyosinsFirst identified in skeletal musclesMyosin I, II and V found in nervous systemMyosin VI and VIIA also in nervous systemImplicated incongenital deafnessLikely role in growth cone motility

Myosin Structure

Myosin IStructureSingle heavy chainFunctionInteracts directly with membrane surfacesMay generate movement of plasma membrane componentsMechanotransduction (myosin IB expressed in stereocilia of hair cells)

Myosin IIStructureDimer composed of two heavy chains Two dimers may form bipolar filamentsFunctionContractile ring in mitosisUnknown role in neurons

Myosin VStructureDimer composed of two heavy chainsMultiple calmodulin binding sitesFunctionFound in growth cones"Dilute" mutation results in seizures in adult mice

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