Nov1 Lecture for Biology

8/12/2019 Nov1 Lecture for Biology

1/30

1

THE CYTOSKELETON III:

Microtubules


2/30

2

Cytoskeletal Filament Systems

I. Intermediate Filaments - (size) - numerous subunits, Form long fibrous

filaments (8-12nm)

II. Microtubules - Tubulin &Associated Proteins - Tubulins subunits, Long Stiff

Cylindrical Polymers(25nm, diameter)

III. Microfilaments - Actin &

Associated Proteins - Actins subunits, Form long thin-

filaments & meshworks (6-8nm)

- Membrane-Cytoskeleton (RBCs &All Eukaryotic Cells)

- Spectrin-Actin Meshwork


3/30

3

Microtubules:

General & SpecializedFunctions

Cytoplasmic Microtubules: - All Eukaryotic Cells:

Brain rich source (25% of protein)

- Cell Architecture & Vesicle

Trafficking: *Originate frommicrotubule orgainizing center (MTOC)

Centrosome / Spindle Poles

> Define & Maintain cell shape &

polarity, Form the Mitotic Spindle.

> Determine: Distribution of other the

cytoskeletal filaments (IFs).

> Position of organelles (ER, Golgi,

mitochondria).

> Provide tracks for vesicles &chromosomes.

Cilia & Axonemal

Microtubules:

- Cilia: (protozoans, epithelial

linings, sensory organs). - Flagella: (gametes, flagellates,

etc...).

* Originate from specialized

centrioles , termedbasal bodies.

> Structural Role &

> Motile Role - Bending /

Sliding


4/30

4

http://www.youtube.com/watch?feature=player_embedded&v=QGAm6hMysTA

Parameciumcilia for swimming


5/30

5

Microtubules: Common Structure

15 nm

0.2-25 m

ProtofilamentTubulin subunits

25 nm


6/30

6


7/30

7

Microtubules: Tubulin

Microtubules are composed of tubulin subunits:

tubulins (Present in all MT structures)

-subunit: 450 amino acids (~55 kD)

-subunit: 455 amino acids (~50 kD) Encoded by separate genes

tubulin (Specific:MTOC / Centrosome / Spindle Poles) Multiple isotypes of each tubulin exist:

Each isotype encoded by separate gene

6& 6in mammals


8/30

8

Tubulin: Synthesis & Assembly

tubulin dimer: basic building

block of MTs

GTP(exchangeable)GTP

Monomers synthesized

Rapidly Self-associate

to form dimers with:

Bound GTP

Protofilament

formation

hydrolysis toGDP


9/30

9


10/30

10

In vitroMicrotubule Assembly

MTs form by reversible polymerization of tubulin dimers:

Self-assembly is stimulated by addition of Mg+2/ GTP @ 37oC

I. Nucleation(The Lag phase): MTOC serves as nucleation site in cell

- Tubulin dimersself associate to form rings. - Rings uncoil forming smallprotofilaments that laterally associate to form

a closed tube with 13 protofilaments making up the wall.

II. Elongation (The Growth phase)

Nucleation

Elongation


11/30

11

Kinetics of Microtubule Assembly

0

100

%t

ubulindimersinpolymer

Time, at 37oC

Nucleation

Nucleation(formation of small MT)

Elongation

Elongation

Steady-State

Equilibrium

Steady-State


12/30

12

The two ends of a

MT are NOT equal.

- Plus-end, favored

for elongation.

- Minus-end, biased

against elongation.


13/30

13

In Vivo, Tubulin Assembly Differs at the

Two Ends of a Microtubule

-Minus(Slow Growing End)---

SEED - Nucleus for Assembly(Axoneme, centrioles, centrosomes)

Plus(Fast Growing End)+

-

+ ++


14/30

14

0

100

%t

ubulind

imersinpolymer

Time, at 37oC

Nucleation

Elongation

Steady-State

Equilibrium

Drugs that Influence MT Polymerization

1) Colchicine, Nocodazole: Monomer Sequestering2) Taxol: Polymer Stabilizing


15/30

15

0

100

%t

ubulind

imersinpolymer

Nucleation

Elongation

Steady-State

Equilibrium

Proteins that Influence MT Polymerization

1) OP18, Stathmin, XKCM1: MT Plus-end Disrupter2) MAPs (tau, MAP2): Polymer Stabilizing


16/30

16

Microtubules Display

Dynamic Instability

In cells, microtubule arrays turn-

over rapidly:

1/2 life = ~10 min (fibroblast)

Direct Observations demonstratemicrotubules growat a constant rate

then rapidly begin toshrinkin vitro

& in vivo.

Thetransitionbetween the growingand shrinking phase is due to

Dynamic Instability, a process

linked to : GTP hydrolysis


17/30

17


18/30

18

Dynamic Instability


19/30

19

Tubulin is a GTPase:

GTP statefavors polymerization

GDP statefavors depolymerization

1) GTP tubulin polymerizes into MTs.

2) Incorporation in MTs stimulates GTP

hydrolysis (now GDP state).

3) GDP state triggers depolymerization

(delayed).


20/30

20

Dynamic Instability:A Consequence of GTP Hydrolysis

Dynamic Instability: A consequence of delayed hydrolysis of

GTP during MT assembly:

Growth Phase: Tubulin assembly more rapid than GTP hydrolysis leads

to formation of: GTP-Tubulin Cap- GTP-Tubulin Cap, is stable because subunits have higher affinity for one

another, hence growth is favored.

- If rate of polymerization slows, GTP hydrolysis will catch up to the rate of

assembly exposing GDP-Tubulinends.

Shrinking Phase: Since GDP-Tubulinsubunits have a lower affinity for

one another their interactions become unstable and the microtubule

begins to rapidly depolymerize. Catastrophe often followed by

Rescue


21/30

21

Cycle of Dynamic

Instability:

- punctuated by changes in

behavior

1) elongation to stall

2) stall to catastrophe

3) catastrophe to rescue

4) repeat


22/30

22


23/30

23


24/30

24

FRAP: Fluorescence Recovery After Photobleaching

Dynamic measure of protein movement:

- How stable are existing structures (MTs)?

- How often do the structures turn-over?


25/30

25

In Cells: The Minus-Endof Microtubules areLinked to MT-Organizing Centers (MTOCs)

+

+N

Ciliates / Flagellates

Neurons

basal bodies

N++

+

++

+

+

+ ++

++

+

++

+

+

++

+

+

++

+

+

+

+

+

+

+

Interphase

Mitosis

Spindle Poles

Centrosomes

Centrosomes


26/30

26


27/30

27

Gamma Tubulin Ring Complex: -TRC

-TRC is similar to split lock washer - 1 helical turn


28/30

28

Centrosome:

Evolutionarily- conserved structure.

Unchanged from protozoa to man.Contains a pair of centrioles.

Recruits pericentriolar material.


29/30

29

The Centrosome (MTOC)

Centrioles

PericentriolarMaterial

-Tubulinpericentrin

+

+

+

Microtubules

++

+


30/30

30

MTOCs Organize MTs by Organizing -Tubulin Ring Complexes

Documents

Nov1 Lecture for Biology