MICROTUBULES

-- - - - -MOHAMMED ADNAN M.HANEEF

QURESHI

HISTORY
It was first observed by K.R.Portar and

M.C.Ledbetter in plants in the year 1963.

The term MICROTUBULES was introduced by

Slauterback (1963).

Its nature was established by Sabatini , Bensch

and Barnett by conducting cytological studies.

[The studies were conducted using Glutaraldehyde

fixation in Electron Microscopy]

OCCURENCE
In Animal Cell grow out from CENTROSOME. Almost all eukaryotic cells show presence of

microtubules.

Present in free Cytoplasm , Centrioles , Basal

Bodies , Axonemes of Cilia , Flagella , Sperm tails, Mitotic Spindle apparatus and Kinetochores.

Microtubules present in free cytoplasm are more

dynamic in nature than that of Flagella and Cilia.

FUNCTION
Microtubules are stiff enough to resist forces

that might compress or bend the fiber. This property enables them to provide mechanical support to the cell. The distribution of cytoplasmic microtubules in a cell helps them determine the cell shape. The role of microtubules as skeletal elements is evident in certain highly elongated cell processes, such as cilia, flagella and the axons of the nerve
Microtubules are also thought to play a role in

cells.

maintaining the internal organization of the cell organelles.

STRUCTURE
Hollow Tubular Unbranched Outer Diameter

24 - 25 nm

Wall thickness 5 6 nm

 Composed of GLOBULAR PROTEINS arranged in

longitudinal rows termed as PROTOFILAMENTS.

Microtubules are composed of 13 protofilaments

arranged in circular patter.

Each protofilament is assembled from dimeric

building blocks consisting of one -tubulin and one -tubulin globular sub unit.

-tubulin and -tubulin are acidic proteins , each

having around 450 units of amino acids , a molecular weight of approx. 55ooo daltons.

They are highly conserved proteins.

 Each protofilament has a structural polarity

with -tubulin exposed at one end , called the minus[-] end and -tubulin at the other , called the [+] end.

All microtubules are oriented in such a way

that + end is always directed towards the margins of the cell and ends towards the interior sides of the cell.

POLYMERIZATION AND DEPOLYMERIZATION

Microtubule is a dynamic protein, subjected to

assembly and disassembly according to the needs of the cell.

At any given time point, there is dynamic

equilibrium between free tubulin monomers and polymerised tubulin in the cell.

ASSEMBLY
The assembly of Microtubules occurs in 2

phases:1. Nucleation 2. Elongation

NUCLEATION
Microtubules are formed by outgrowth from

specialized organizing centres MTOCs (MicroTubule Organizing Centers) which control their location and orientation in the cell.

Peri-Centriolar Matrix (PCM) surrounding the

MTOCs consist of ring shaped -tubulin oriented in a specific manner. - tubulin serves as nucleation sites for growth of one microtubule. Only -tublulin can associate with -tubulin and thus polarity is maintained.
GTP and Mg2+ are required.

ELONGATION

Formation of the tubular structure from the

initial curled sheet, GTP gets hydrolyzed to GDP once the monomer is incorporated into the microtubule assembly.

 tubulin
Discovered in the mid 80s.
Fluorescent antibodies to -tubulin stain all

types of MTOCs including the peri-centriolar matrix suggesting that -tubulin is a critical component in the microtubule nucleation.

Serve as attachment sites for the -tubulin in

the PCM.

DISASSEMBLY
At a given temperature and concentration of

tubulin , Ca2+ , Mg2+ and MAPs (Microtubule Associated Proteins), a point is reached at which no further growth of microtubule is observed. at the + end and lost from the end .

The new subunits are incorporated preferentially

Consequently there is a movement or flux of

individual subunits through the linear polymer from one end to the other a phenomenon known as THREADMILLING.

 Treadmilling is driven by hydrolysis of GTP.

Thus a subunit disassembly from the end has a bound GDP. immediately get phosphorylated to GTP dimers for reuse. level favor depolymerisation.

After disassembly free GDP dimers

Calcium ions when present above a critical

Addition and deletion takes place together

but at different rates according to the need of the cell.

ROLE OF GTP IN ASSEMBLY AND DISASSEMBLY

GTP gets attached to -tubulin and GTP-bound

-tubulin dimers are added at the + end of the microtubules.

GTP hydrolysis is not required for the actual

incorporation of the dimer at the + end. It is hydrolyzed shortly after addition of GTPdimer into the microtubule.
in the microtubule.

After the hydrolysis GDP dimer remains bound

 During disassembly GDP dimers are lost to the

cytoplasm. These are then recharged by conversion of GDP to GTP.

Now GTP can again participate in the

polymerization.

The cell has chosen such an expensive

pathway because it allows the cell to control the rate of two opposite reactions, which are constantly occurring in any given cell at any time.

MOTOR PROTEINS
The motor proteins of cell convert chemical

energy into mechanical energy , which is used to carry the cell cargo attached to the motor.

Cell Cargos carried are Vesicles, Mitochondria,

Lysosomes, Chromosomes and other cytoskeletal filaments.

Each motor protein is presumably specialized

for the movement of particular type of cargo in a particular region of the cell.

 Motor Proteins can broadly be classified into

3 groups :-

Kinesins Dyneins

Myosins

Kinesins and Dyneins move along microtubules whereas myosins move along microfilaments

 Motor proteins move unidirectionally along

their cytoskeletal track in stepwise manner from one binding site to next.

As protein moves along, it undergoes

conformational change that constitute a mechanical cycle.

The steps of mechanical cycle are coupled

with the chemical cycle, which provides the energy necessary to fuel the motors activity.

KINESINS
Kinesin is a class of motor proteins, constructed from

two identical heavy chains and two identical light chains.

A kinesins consists of several parts 1.

2.
3. 4.

A globular head that binds microtubules and acts as ATP-hydrolizing force generating engines A neck that connects the head and Stalk A long coiled Stalk A tail (fan shaped) that binds the cargo to be carried. Kinesins move along microtubules toward their plus end and therefore said to be plus end-directed

microtubular motor.

DYNEIN
These are protein responsible for the

movement of cilia and flagella. However cytoplasmic dynein exist but was purified and characterized after almost 20 years.. It is present throughout the animal kingdom but there is a controversy whether it is present or not in plants. It is a huge protein (approx 1.5 million daltons) composed of two identical heavy chains and a variety of intermediate and light chains.

 Dynein-drivencargo include endosomes,

lysosomes, ER derived vesicles heading toward the golgi complex and the HIV virus which is transported to the nucleus of an infected cell.

Cytoplasmic dynein does not interact directly

with the membrane bound cargo but requires an intervening multisubunit adaptor called

Dynactin

MTOCs
The function of a microtubule within a living

cell depends on its location and orientation which makes it important to understand why a microtubule assembles at place as opposed to another.

Example: Centrosomes

Basal bodies of Cilia and Flagella

MITOSIS

TOXIN AND DRUGS

Some toxins and drugs (all of which inhibit mitosis) affect polymerization or depolymerization of tubulin:
Taxol Binds tightly to microtubules and prevents

them from losing subunits.Since new subunits can still be added the microtubule can grow but cannot shrink.

Colchicine It binds tightly to free tubulin and

prevents its polymerisation into microtubules.The mitotic spindle rapidly disappears and the cell stalls in the middle of mitosis, unable to partition its chromosomes into two groups.

 Vinblastine The vinca alkaloid-vinblastine tends

to produce crystal like structure of tubulin in the cytoplasm and in homogenates it produces precipitation of this protein. microtubules.

Nocodazole causes depolymerization of

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