MICROTUBULES, MICROFILAMENTS AND INTERMEDIATE FILAMENTS

Guided by-

Dr. MURALIDHAR T.SPresented byDWIJENDRA TRIVEDI

CONTENTS DEFINATION INTRODUCTION MICROTUBULES STRUCTURE

CHEMICAL COMPOSITION MAPs ASSEMBLY & DISASSEMBLY GTP HYDROLYSIS TREDMILLING FUNCTIONS

MICROFILAMENTS STRUCTURE POLYMERISATION OF ACTIN TREDMILLING GTP HYDROLYSIS

FUNCTIONS

INTERMEDIATE FILAMENTS STRUCTURE CHARACTERISTICS ASSEMBLY FUNCTIONS

COMPARISONS

ABSTRACTS & CONCLUSION REFERENCES

Introduction Cytoskeleton, an intracellular network of protein

filaments, involved in determining cell shape, cell movement, and intracellular movements. Postulated in 1928 by koltzoff The cytoskeleton helps to support the cell and

maintain its shape It interacts with motor proteins to produce motility

Introduction Contd.. It is composed of three types of molecular structures: Microtubules Microfilaments

Intermediate filaments

>The ability of eukaryotic cells to adopt a variety of shapes and to carry out coordinated and directed movements depends upon CYTOSKELETON.

MICROTUBULE

MICROTUBULES Microtubules are polymers of tubulin First observed in axoplasm of myelinated nerve fibres by

ROBERTIS and FRANCHI. OCCURANCE-

With rare exception such as the human microtubules are found in all eukaryotic

erythrocytes, and flagella. Microtubules

cells,either free in cytoplasm or forming part of centrioles,cilia

are

dynamically

unstable

they

can

be

assembled and disassembled rapidly Tubules are organized by the centrosome complex

Structure

Column of tubulin

dimers

25 nm

Tubulin dimer

The beta-tubulin of one dimer in contact with alfa-

tubulin of the next. Since all the 13 protofilaments are aligned parallely with

same polarity,the microtubule are the polar structurehaving PLUS or FAST GROWING END & MINUS or SLOWGROWING END. The minus ends of cytoplasmic microtubules in cells are

bound

tightly

to

microtubule

organizing

centres

{MTOCs} from which their assembly or polymerization

starts. NOTE-MTOCs acts as a template for the initiation of

polymerisation

--Microtubules are stiff,

Chemical composition

cylindrical polymers of tubulin. --Made up of alpha & beta tubulin heteromers containingGDP

450 amino acid.--TUBULIN is a acidic protein with molecular

weight of 55 Kd and asedimentation constant of 6s.

Microtubule associated proteins These are proteins that associate with the surface of microtubles. Two major class of MAPs have been isolated:-

HMW proteins Tau proteins Both classes of proteins have two domains,one of which binds to microtubule

and tends to speed up the nucleation step of tubulin polymerisation. The other domain is believed to be involved in linking the microtubule to

other cell components.

NOTE-HMW-HIGH MOLECULAR WEIGHT PROTEINS

Assembly and disassembly of microtubules Cytoplasmic

microtubules

are

highly

dynamic

structures,constantly forming & disappearing dependent on cells activities They,like the microfilaments,grow by the reversible

addition of subunits,accompanied by nucleotide{GTP} hydrolysis & conformational change. The assembly of microtubules from the tubulin dimers

is a specifically oriented & programmed process.

In the cell,the sites of orientation are MTOCs from which the

polymerisation is directed. Microtubules

polymerisation

occurs

by

a

nucleation-

elongation mechanism, in which the formation of shortmicrotubule nucleus is followed by growth of the microtubule at both its ends by the reversible non covalent addition of tubulin subunits. Within the cell, microtubules are in equilibrium with free

tubulin. Phosphorylation of the tubulin monomers by a cyclic AMP_ dependent KINASE favouring the polymerisation In a microtubule,the assembly of tubulin dimers takes place at

one end ,while disassembly is common at other end.

Whole course of polymerisation is divided into

three phases

The lag phase corresponds to time taken for nucleation.

The growth phase occurs as monomers add to the

exposed ends of the growing filament causingfilament elongation. The steady phase which is reached when the growth

of the filament due to monomer addition is exactly balanced by depolymerisation from the ends.

Assembly of microtubules.--Tubulin

hetrodimers

forms Oligomers of tubulin and further forms a single 13 tubulin PROTOFILAMENT. --Several such proto filaments attaches & forms sheets of potofilaments. --Later these protofilament sheets folds by conformational changes into a functional

microtubule.

GTP hydrolysis Soluble tubulin binds GTP reversibly at a site on the beta- subunit &

the GTP becomes hydrolysed to GDP & Pi as, or shortly after,the

tubulin polymerizes onto growing microtubule ends. Hydrolysis of the bound nucleotide reduces the binding affinity of

subunit for neighbouring subunits & makes it more likely to dissociate from each other in the ends of the filament. Thus the normal role of GTP hydrolysis is apparently to allow

microtubules to depolymerize by weakening the bonds between tubulin subunits in the microtubule. NOTE- High concentration of GTP bound tubulin makes microtubule

stable by providing GTP cap,but low concentration of GTP causes the microtubule to dissociate.

GTP hydrolysis of microtubules

Function; Mechanical function:-The shape of some cell processes

has been correlated to the orientation & distribution of microtubules. Eg.Axons & Dendrites of neurons Morphogenesis:-Microtubule shape the cell during cell differentiation. Eg. The morphogenetic changes that occur during spermiogenesis. Cellular polarity & motility:- The determination of the intrinsic polarity of certain cells is also related to the microtubules. Eg. Membrane ruffling,endocytosis. Contraction:- microtubule play a role in the contraction of spindle and movement of chromosomes & centrioles as well as in cilliary & flagellar movements.

MICROFILAMENTS

STRUCTURE Microfilaments are solid rods about 7 nm in

diameter, built as a twisted double chain of actin subunits Each actin molecule is usually bound to either ATP or ADP. The G-Actin subunits assemble head-to-tail to generate F-actins with a distinct structural polarity. Two parallel F-Actins twist around each other in a right-handed helix to form an actin filament. Consists of plus & minus ends.

"-" end = "pointed" end "+" end = "barbed" end

NOTE-"Pointed" and "barbed" refer to appearance

of filament when decorated with globular heads of myosin Each G-actin monomer has intimate contact with four others (one on top, one on bottom and two to one side). Filament is tightly-wound helix of two "strands. NOTE- (strands are not stable in isolation).

Actin subunit7 nm

POLYMERISATION OF ACTIN

The polymerization of actin filaments proceeds in three phases: Nucleation Elongation and Steady-state.

TREDMILLINGTreadmilling in actin filaments (uncapped) results when rate of assembly at ATP (plus) end equals rate of depolymerization at ADP (minus) end.

--Binding of subunits to ends of

filament is stabilized when ATP isbound.--However, it is thought that when an

ATP HYDROLYSIS

actin molecule is incorporated into afilament, interactions between it and adjoining subunits causes a conformational change, which "closes" the cleft and triggers ATP hydrolysis. --ADP is trapped in the cleft by interactions with other subunits, and

the ADP molecule cannot be releasedand replaced by another ATP molecule until depolymerization occurs.

Functions--The structural role of microfilaments is to bear tension, resisting pulling forces within the cell

--They form a 3-D network called the cortex justinside the plasma membrane to help support the

cells shape--Bundles of microfilaments make up the core of microvilli of intestinal cells --Involved in cell migration during embryonic development & muscle contraction.

INTERMEDIATE FILAMENTS

Keratin proteins Fibrous subunit (keratins coiled together)812 nm

Intermediate Filaments Intermediate filaments range in diameter from 812

nanometers, larger than microfilaments but smaller than microtubules which is intermediate between

thick & thin filaments They are tough and durable protein fibres in the

cytoplasm of most EUKARYOTIC CELLS. They support cell shape and fix organelles in place Intermediate filaments are of four types- TYPE-I,TYPE-

II,TYPE-III & TYPE-IV.

Characteristics of types of IF proteinsTypes of intermediate filaments1.TYPE I

Component polypeptide (mass in daltons)Acidic keratins (40,000-70,000) Neutral or basic keratins (40,000-70,000)

Cellular location

Epithelial cells & epidermal derivatives such as hair & nail.

2. TYPE II

Vimentin (53,000) Desmin (52,000) Glial fibrillar acidic protein (45,000) Synemin(230,000)Neurofilament proteins (about 130,000,100,000 & 60,000) Nuclear lamins A, B & C (65,000-75,000)

Many cells of mesenchymal origin Muscle cells Glial cells(astrocytes & some schwann cells) Muscles cellsNeurons

3. TYPE III

4. TYPE 1V

Nuclear lamina of all cells

Structure of IFs Despite the large differences in their size, all cytoplasmic IF

proteins are encoded by members of the same multigene family. Their amino acid sequences indicate that each IF polypeptide

chain contains a homologous central region of about 310 amino acids residues that forms an extended alpha helix with

three short alpha helical interruptions

Assembly of IFs

Functions The main function of most intermediate

filaments is to provide mechanical support to the cell & its nucleus. The neurofilaments in the nerve cell axons probably resists stresses caused by the motion of the animal which would other wise break these long,thin cylinders of cytoplasm. Desmin filaments provide mechanical support for the sarcomeres in muscle cells & vimentin filaments surrounds & probably support the large fat droplets in the fat cells.

Comparisons of some properties of elements of CYTOSKELETON Intermediate Microfilaments Property Microtubulesfilaments 1. STRUCTURE Hollow with walls made up of 13 PROTOFILAMENTS 24-25 nm Alpha & beta tubulin. Present in Dyniein arms -Motility -Chromosome movements -Movements of intracellular materials. -maintainance of cell shape Hollow with walls made up of 4-5 Solid made up of polymerised acin(FActin) 7-9 nm G- Actin

Protofilaments.10 nm Five types of protein defining five major classes None -Integrate contractile units in muscles. -Cytoskeletal structural function in cytoplasm.

2. DIAMETER 3. MONOMER UNIT 4.ATPase ACTIVITY 5. Functions

None -Muscle contraction -Cell shape changes -Protoplasmic streaming -cytokinesis.

CONCLUSION Within the cell cytoplasm or cytoplasmic matrix,contains

protein filaments, involved in determining cell shape &

structure. The cytoskeleton helps to support the cell and maintain its

shape. The ability of eukaryotic cells to adopt a variety of shapes

and to carry out coordinated and directed movements depends upon CYTOSKELETON. It is composed of three types of molecular structures viz.

Microtubules, Microfilaments, Intermediate filaments.

CONCLUSION Cont. The main protein present in cytoskeleton are :-

TUBULIN,ACTIN,MYOSIN,TROPOMYOSIN and others as MICROFILAMENTS. This network of filaments helps to support the large

volume of cytoplasm in a eukaryotic cell, a function that is very important in animal cells which have no walls. The interior of the cell is in constant motion and the

cytoskeleton provides the machinery for intracellular movements such as the transport of organelles from one place to another.

ABSTRACT-1Regulation of T-cell activation by the cytoskeleton

AUTHOR- Daniel D. Billadeau , Jeffrey C. Nolz & Timothy S. Gomez JOURNAL- Nature Reviews Immunology 7, 131143 (1 February 2007) | doi:10.1038/nri2021

Abstract---To become activated, T cells must efficiently recognize antigen-presenting cells or target cells through several complex cytoskeleton-dependent processes, including integrinmediated adhesion, immunological-synapse formation, cellular polarization, receptor sequestration and signalling.

The actin and microtubule systems provide the dynamic cellular framework that is required to orchestrate these processes and ultimately contol T-cell activation.

Here, we discuss recent advances that have furthered our understanding of the crucial importance of the T-cell cytoskeleton in controlling these aspects of T-cell immune recognition.

ABSTRACT-2Transmembrane crosstalk between the extracellular matrix and the cytoskeleton

AUTHOR- Benjamin Geiger , Alexander Bershadsky , Roumen Pankov & Kenneth M. Yamada

JOURNAL- Nature Reviews Molecular Cell Biology 2, 793805 (1 November 2001) |

doi:10.1038/35099066

Abstract--Integrin-mediated cell adhesions provide dynamic, bidirectional links between the extracellular matrix and the cytoskeleton.

Besides having central roles in cell migration and morphogenesis, focal adhesions and related structures convey information across the cell membrane, to regulate extracellularmatrix assembly, cell proliferation, differentiation, and death.

This review describes integrin functions, mechanosensors, molecular switches and signaltransduction pathways activated and integrated by adhesion, with a unifying theme being the importance of local physical forces.

ABSTRACT-3actin cytoskeleton Direct dynaminactin interactions regulate the

AUTHOR- Changkyu Gu, Suma Yaddanapudi1, Astrid Weins, Teresia Osborn, Jochen Reiser, Martin Pollak,

John Hartwig and Sanja Sever

JOURNAL - EMBO Journal(2010) 29,3593-3606 ABSTRACT-----

The large GTPase dynamin assembles into higher order structures that are thought to promote endocytosis.

Dynamin also regulates the actin cytoskeleton through an unknown, GTPase-dependent mechanism. Here, we identify a highly conserved site in dynamin that binds directly to actin filaments and aligns them into bundles.

Point mutations in the actin-binding domain cause aberrant membrane ruffling and defective actin stress fibre formation in cells. Short actin filaments promote dynamin assembly into higher order structures, which in turn efficiently release the actin-capping protein (CP) gelsolin from barbed actin ends in vitro, allowing for elongation of actin filaments.

Together, our results support a model in which assembled dynamin, generated through interactions with short actin filaments, promotes actin polymerization via displacement of actin-CPs.

Conclusions for abstracts- CONCLUSION (ABSTRACT-1):- For the activation of T cells, recognition ofantigen-presenting cells or target cells by several complex cytoskeletondependent processes is required. The cytoskeleton ultimately contol T-cell activation. provides the dynamic cellular framework that is required to orchestrate these processes and

CONCLUSION (ABSTRACT-2):-The data presented in this paper suggest anovel, dynamin-dependent mechanism that promotes actin polymerization. In this model, proteins such as Gsn first fragment and cap F-actin filaments; the resulting short filaments bind and promote dynamin assembly into rings; dynamin rings in turn displace CPs; finally, the uncapped barbed actin ends undergo polymerization.

CONCLUSION (ABSTRACT-3):- Cell proliferation is subject to many levelsof control, but it is becoming clear that mechanical signaling through the cytoskeleton linkage between FAs and regulators of cellular contractility contribute to the regulation of cell proliferation.

REFERENCES CELL BIOLOGY- P.C VERMA & V.K AGARWAL-14th Edition,2009,(Pg. 294-298):-MICROTUBULES. MOLECULAR BIOLOGY OF THE CELLALBERTS,JOHNSON,ROBERTS,LEWIS & RAFF-5th Edition,2008,(Pg. 976-

980):-TREDMILLING & DYNAMIC INSTABILITY. CELL & MOLECULAR BIOLOGY- DE ROBERTS-8th

Edition,1999,(Pg.174-176):- MICROFILAMENTS. BIOLOGICAL SCIENCES- PRANAV KUMAR(Pg.279-282):- GTP HYDROLYSIS PUBMED.COM: Regulation of T-cell activation by the cytoskeleton(ABSTRACT -1) Nature Reviews Immunology 7, 131143 (1 February 2007) | doi:10.1038/nri2021 Transmembrane crosstalk between the extracellular matrix and the cytoskeleton (ABSTRACT-2) Nature Reviews Molecular Cell Biology 2, 793805 (1 November 2001) | doi:10.1038/35099066 Direct dynaminactin interactions regulate the actin cytoskeleton(ABSTRACT-3) EMBO Journal(2010) 29,3593-3606 www.wikipidia.com:- Definations