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8/12/2019 Nov1 Lecture for Biology
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THE CYTOSKELETON III:
Microtubules
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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
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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
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http://www.youtube.com/watch?feature=player_embedded&v=QGAm6hMysTA
Parameciumcilia for swimming
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Microtubules: Common Structure
15 nm
0.2-25 m
ProtofilamentTubulin subunits
25 nm
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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
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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
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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
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Kinetics of Microtubule Assembly
0
100
%t
ubulindimersinpolymer
Time, at 37oC
Nucleation
Nucleation(formation of small MT)
Elongation
Elongation
Steady-State
Equilibrium
Steady-State
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The two ends of a
MT are NOT equal.
- Plus-end, favored
for elongation.
- Minus-end, biased
against elongation.
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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)+
-
+ ++
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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
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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
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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
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Dynamic Instability
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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).
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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
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Cycle of Dynamic
Instability:
- punctuated by changes in
behavior
1) elongation to stall
2) stall to catastrophe
3) catastrophe to rescue
4) repeat
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FRAP: Fluorescence Recovery After Photobleaching
Dynamic measure of protein movement:
- How stable are existing structures (MTs)?
- How often do the structures turn-over?
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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
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Gamma Tubulin Ring Complex: -TRC
-TRC is similar to split lock washer - 1 helical turn
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Centrosome:
Evolutionarily- conserved structure.
Unchanged from protozoa to man.Contains a pair of centrioles.
Recruits pericentriolar material.
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The Centrosome (MTOC)
Centrioles
PericentriolarMaterial
-Tubulinpericentrin
+
+
+
Microtubules
++
+
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MTOCs Organize MTs by Organizing -Tubulin Ring Complexes