8.1.1. Formation :
The dimers of and tubulin bind to GTP and assemble into the (+) ends of microtubules in the GTP bound state. tubulin is present on the (+) positive end, while a-tubulin on (–) negative end. The molecules of GTP bound to -tubulin and hydrolyses into GDP, along the pro filament of microtubule. This GTP cycle provide dynamic instability to the microtubules.
8.1.2. Structure :
Microtubules are long, hollow, cylinders and formed by polymerization of and tubulin dimers.
- In profilaments, tubulin dimers polymerize end to end, which are building blocks for microtubules. 13 protofilaments associate laterally to form a single microtubule and this structure can extended, by addition of more protofilaments.
- Microtubules have a distinct polarity which is important for their biological function.
- In a protofilament one end, which have -subunits is positive end and other end having -subunits is Negative end. Elongation of microtubules typically occur form positive end.
Treatment of cells with microtubule disrupting drugs can affect the location of membranous organelles like Endoplasmic Reticulum and Golgi complex. The golgi complex is located near the center of an animal cell. By treating the cell with Nocodazole or colchicine can promote microtubule disassembly and it can disperse the golgi apparatus to the peripheral regions of the cell.
Assembly and disassembly at the positive end of a microtubule is referred to the dynamic instability. Both the a and b-subunits of tubulin dimer are bound to a GTP molecule, during polymerization. GTP bound to -subunit, hydrolyzed to GDP after assembly, which results in the addition of new dimers and GDP bound tubulin is prove to depolymerization.
When microtubules, show rapid depolymerization and shrinkage, this switch from, growth to shrinking is called as catastrophe. By addition of GTP bound tubulin to the tip of microtubules again, provides protection to microtubules from shrinking. This is called as “Rescue”.
8.1.3. Treadmilling :
The process of addition and removal of monomers, depends on the ab-tubulin dimer concentration (Cc). If tubulins concentration higher than the Cc the microtubules show polymerizations and if this concentration is less than Cc, then the microtubules show deplymerization and their length decreases. This is called as treadmilling.
It’s another member of the tubulin family. It has important role in the nucleation and polar orientation of microtubules. It is found primarily in centrosome and spindle pole bodies.
and tubulin :
They are localize at centrioles and may play role in forming the mitotic spindle during mitosis.
8.1.4. Micro Tubule Associated Proteins (MAPs)
MAPs determine that assembly, disassembly and catastrophe rates of microtubules. Generally MAPs increase the stability of microtubules and promote their assembly.
- Microtubules are nucleated and organized in Microtubule Organizing Centers (MTOCs), e.g., centrosome or basal bodies found in cilia and flagella.
- Many other proteins that binds to microtubules are motor proteins, such as kinesin and dynein, severing proteins like katanin etc.
8.1.5. Motor Proteins
Motor proteins of a cell generate the force to more cellular organelles, by converting chemical energy (ATP), into mechanical energy.
Ex.: Vesicles, mitochondria, lysosome, chromosomes and other cytoskeletal filaments are moved by there proteins.
Motor proteins can be grouped into three broad superfamilies :
In eukaryotic cells, kinesin is a protein that moves along microtubules. A typical kinesin consist of two heavy chains and two light chains. The heavy chain comprises a globular motor head domain, flexible neck linker to the stalk and a long coiled tail domain. Each head has two separate binding sites, one for microtubule and other for ATP. The tail domain binds the receptors on the membrane of cargoes.
Kinesins show many functions like : mitosis, meiosis and transport of cellular cargo (axonal transport). Kinesins walk towards the positive end of a microtubule. Which is essential to transport the cargo from center to the periphery of cell.
Dyneins are motor proteins that move towards the microtubules minus end. These are composed of two or three identical heavy chains and a intermediate and a light chains complex. Heavy chains are present in the from of homo or heterodiamer and heterotrimer.
Cytoplasmic dyneins moves progressively along microtubule toward the depolymenization end. They help in positioning the centrosome and golgi complex moving organelles and vesicles near the centre of the cell. Axonemal dyneins are highly specialized and helps in beating of cillia and flagella.
8.1.6. Micro Tubule Organizing Centers (MTOCs)
Microtubules assembly and organization depends on MTOCs and the function of a microtuble in a cell depends on its location and orientation, which is also controlled by MTOCs. The “Tubulin” protein has a role in the nucleation of microtubules formation. All this occurs in association with a variety of specialized structures called as MTOCs and the MTOC in a animal cell is centrosome.
In animal cells, the nucleation of microtubules occur by the centrosome. It contains a pair of barrel shaped centrioles, which are surrounded by amorphous, electron dense material, know as Pericentrilar material (PCM) or “Centrosome Matrix”. These are typically composed of nine evenly spaced fibrils, in (9 + 0) arrangement. The centre part is called as “Hub”, which is proteinaceous in nature. Hub is connected with the peripheral triplets by “radial spokes”.
Microtubule Nucleation and Dynamic Properties
MTOCs control the number of microtubules, polarity, number of protofilaments and the assembly and location and microtubules. Microtubules show polymerization and depolymerization. The polymerization is shown by a “nucleation elongation michanism”.
The polymerization shows three phases : Lag phase growth phase and steady phase. Microtubules are stabilized by the presence of microtubules associated proteins (MAPs). Microtubules of the mitotic spindle are extremely labile i.e. more sensitive to disassembly, while microtubules of motor neurons are less labile and cilia, flagella are comparatively highly stable.
Some chemicals like : colchicine, vinblastin, nocodazole and polyphyllotoxin increase disassembly of microtubules.
Cilia and flagella
Cilia and flagella are hair like, motile organelles that are projected from the surface of variety of eukaryotic cells. Cilla and flagella are essentially same in structure and organization.
They have a control bindle of microtubules, know as axonome, in which nine outer doublet microtubules surround a central pair of singlet microtubules in (9 + 2) array (arrangement). Each peripheral doublet has one complete microtubule, the Atubule and one incomplete microtubule, B-tubule. Both have 13 and 11 proto filaments respectively. The central tubules are enclosed by central sheath, which is connected to the A tubules, of peripheral doublets by a set of radial spokes. These doublets are connected to one another by a interdouble bridge, which is composed of an elastic protein Nexin. A pair of arms, an inner arm and on outer arm. Which are projected from A tubule. The growth of a axoneme, occur at the positive ends of the microtubules. The dynein arms generate the sliding forces in axonemes, which is responsible for axonemal moment in cilia and flagella.
Mechanism of ciliary and flagellar locomotion
In an axoneme (intact), the stem of each dynein molecule is tightly anchored to the outer surface of the A tubule, with its globular heads and stalks, which are projected towards the B-tubule of its neighbouring doublet.
The dynein arms, act as swinging cross bridges which generate, forces required for ciliary and flagellar movement. Nexin protein also helps in their movement by limiting the extent, so that adjacent doublets can strike over one another. Motile cilia generate alternating power strokes and recovery strokes. These rapid power strokes moves the cell in a direction, perpendicularly to the cilium’s axis. After that a recovery stroke occurs and the cilia move. In contrast a flagellum shows wave like motion, which drives the cell in same direction.
8.2. Intermediate Filaments
Intermediate filaments are cytoskeletal components. Which are rope like and cytoplasmic in nature. They have about 10 nm of average diameter, which is between the size of microfilaments and microtubules (25 nm).
These are found in many metazoan cells, including molluscas, nematodes, vertebrates. Most types of intermediate are filaments are cytoplasmic and chemically heterogenous and differs in molecular weight, but the lamines intermediate filament are nuclear.
8.2.1. Structure :
All intermediate filaments share a common structural organization. The intermediate filament proteins appear to have a control -helical rod domain. Which is composed of four, a-helical segments (1A, 1B, 2B, 2A) and separated by three linker regions.
The NH2 (amino) and carboxy terminal of intermediate filament, are not a helical structure. They show wide variation in their lengths and sequences across the intermediate filament families. The basic building block of intermediate filaments is regid and parallel dimer. Two dimers then line up side by side to form a antiparallel tetramer of four polypeptide chains. This tetramer further show organisation to form higher level of arrangement. This tetramer is analogous to the tubulin heterodimer or G-Action filament. Intermediate filaments (IFs) lack polarity and can not participate in cell motility and intra cellular transportation.
Functions of intermediate filaments
Keratin filaments constitute the primary structure proteins of epithelial cells. e.g., linear hepatocytes, epidermal cells, pancreatic aciner cells etc. Intermediate filaments network serve as scaffold for organizing and maintaining cellular architecture and absorbs mechanical stresses, which are applied by the extracellular environment. Intermediate filaments provide mechanical strength to cells, situated in epithelial layers. Desmin plays role in maintaining the alignment of the myofibril of muscle cells. Intermediate filaments have tissue specific functions also which are more important in some cells than in others.
8.3. Micro filament
Micro filaments are the thinnest filaments of the cytoskeleton found in the cytoplasm of eukaryotic cells. These are linear polymers of actin subunits.
Micro filaments are highly versatile, show amoeboid movement functioning in cytokinesis and it changes the cell shape. They function as part of actomyosin driven contractile molecular motors and including cell motility.
Actin filaments show assembly in two general types of structures : bundles and networks. Bundles are composed of polar filament arrays or non polar arrays. Cross linking proteins, determine filament orientation and spacing in the networks and bundles. These structures are regulated by many other classes of proteins.
8.3.1. Actin filament
Actin is the major cytoskeletal protein of most cells. Which is polymerized to form the actin filaments. Thus filaments are thin and flexible which are approximately 7 nm in diameter and may be up to several mm in length. Action filaments are particularly abundant beneath the plasma membrane, where they form a network, which provides mechanical support and allow movement of the cell surface.
Assembly and Disassembly of Actin Filaments
Actin filament was first isolated from muscle cells. Each actin filament is made up of G-actions (globular actin), which is of 43 kDa (375 amino acids) and has ATPase power.
These actin monomers polymerize and form F actin filament (filamentous). Two parallel F-actins, twist around each others (in a right handed helix) and form an actin filament. An actin filament is a polar structure, which has a slow growing negative end and a faster growing (barbed end) positive end. The rate of their polymerization into filaments equals to the rate of dissociation. So the monomers and filaments show apparent equilibrium.
The actin monomers also bind with ATP, which is hydrolyzed to ADP for assembles of filament. Actin monomers, which are ATP bound, polymerize more rigidity, than those which are ADP bond. Actin polymerization is reversible. The filaments show depolymerization by dissociation of G-actin subunits. All this process occur in 3 steps : Nucleation, elongation and steady state phase. There is no changes in the total length of filaments.
There are some drugs, which affect polymerization of actin. They bind with positive ends of acting filaments and block their elongation. eg. cytochalasin D. Another drug is Phalloidin, which binds tightly with actin filament and stabilize them against depolymerization.
Myosin was first isolated from mammalian skeletal muscle tissue. Myosin is motor protein and actin filaments are tracks, along which myosin show movement. Myosis have different major classes. Myosins have two classes. Each class has its own specialization functions. Interaction of myosin with actin show a special form of movement called as “contraction”.
Myosin I and Myosin II, are most abundant proteins, present in nearly all eukaryotic cells. All myosins are composed of one or two identical heavy chains and a variety of low molecular weight (light) Chains. The each heavy chain has three structurally and functionally differs domains. A globular head domain is present at N-termines, contains actin and ATP binding sites. The head hydrolyzes ATP. By the energy of hydrolyzed ATP myosin filaments walk towards the end of an actin filament. The head is followed by a -helical reck region. Which associates with light chains and show regulation of the activity of the head domain. The tail domain has the binding sites which allow the molecules to polymerize into bipolar filaments and determine the specific activities of a particular myosin. The tail-tail interactions forms large bipolar filaments, that contains several myosin heads.
Many other types of myosin I, III, IV, XIV are also found in non-muscle cells. Myosins may be involved in a variety of other kinds of cell movements and vesicles transport, Phagocytosis, extension of pseudopodia in amoeba cell locomotion etc.