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Microtubules, Actin (and intermediate filaments). Macrophage microtubulin green Intermediate filaments red DNA blue. Mitochondria, actin. Cytoplasmic microtubules Cytoplasmic m tubules brkdwn Mitotic spindle Differentiated cell types have stable m tubule structures

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Microtubules, Actin

(and intermediate filaments)

Macrophage microtubulin green

Intermediate filaments red

DNA blue



Cytoplasmic microtubules

Cytoplasmic mtubules

brkdwn

Mitotic spindle

Differentiated cell types have

stable mtubule structures

Cilia, flagella form from

basal bodies


So what are Microtubules??

… hollow, rigid tubes composed of a/b heterodimers

POLAR.. w/o polarity, it can not function.


Each subunit binds GTP

… but the beta

subunit has intrinsic

GTPase activity.


Microtubule networks assemble and disassemble

Assembly is initiated at MTOCs

MicroTubule Organizing Centers

(aka centrosomes)


MTOC

100’s of g subunits serve to nucleate

mtubules,

Centriole pair

(sometimes)

Similar to

Basal bodies


Anti a tubulin, anti g tubulin


Growth vs. shrinking

is related to the amt.

of free tubulin (Cc)

Growth when balanced

tipped toward >[Cc]


Microtubule assembly and disassemble is

CATASTROPHIC!!

Occurs preferentially at the plus ends

Critical Concentration, Cc, above grows/below shrinks

Does not require GTP hydrolysis for assembly

The rapid nature of disassembly is due to its constrained

internal structure (GDP tubulin)


Autumn Crocus

Toolbox:

Colchicine (from crocus) binds free tubulin

Taxol (from yew) stabilizes microtubules

Both cancer therapy drugs, both block mitosis.

How? If opposite modes of action?

Yew


Microtubules in the mitotic spindle of a mouse fibroblast in culture.  

Image by Steve Rogers, UNC Dept. Biology.  (Confocal Microscope)


Organelles, vesicles with culture.  

cargo move around the cell.

Why’s that?


How do we know? culture.  

Pulse-chase with radioactive amino acids injected into rat ganglion

Assay, slice up axon over time, SDS PAGE autoradiography


Microtubules are needed for trafficking vesicles (10 cm/day)

but this is dramatically faster than diffusion in the axon.

…in a differentiated cell- dynamic instability is suppressed… by capping proteins.


In vitro,

Extruded

Exoplasm

ATP-dependent



Video microscopy

increases resolution and

visualizes movement.

This movement requires ATP.

But AMP-PNP (analog)

Competitively inhibits


Ron Vale, UCSF

… and AMP-PNP

Michael Sheetz, Columbia

17.5 kinesin.mov

Movie from Jeff Gelles lab






Filaments (F actin) are another important

means by which the cytoskeleton is used for trafficking.

It is important for cell shape, location and contraction


Abundant:

5% of total protein,

half as filaments

Actin monomers

in 4 colors


F actin grows at BOTH ends but the plus ends grows faster.

ATP hydrolysis is not required for assembly.


Phalloidin binds F actin, from Death Angel

Cytochalasins and lantrunculin

bind G actin


Actin filaments are rarely single …

Nets and bundles.

Profilin

thymosin

filamin

gelsolin

myosin



Actin-related proteins

promote branching

More distally


Racs (Cdc42 is a member) are small GTPases related to the Ras members

discussed before.

These can be activated by recptors and in turn activate actin assembly


Myosin is a motor that runs along (or tugs at) actin. Ras members

Actin-based movement of vesicles

Myosin-driven cell shape changes

Muscle contraction

Myosin I and myosin I



Dimer Ras members

300 heads here, bind actin


Best understood example … contraction in muscle cells. Ras members

Multinucleate (formed by cell fusions

50 mm diameter and centimeters long)


… just one sarcomere Ras members .


Plus ends Ras members


Each filament has ~300 heads. Ras members

Each head binds 5X/s

32 mm in 0.1 s

Attached, No ATP, “rigor”

Released, binds ATP

Cocked, hydrolysis

Loose Pi

Power stroke, loose ADP

Attached again,



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