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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(and intermediate filaments)
Macrophage microtubulin green
Intermediate filaments red
Differentiated cell types have
stable mtubule structures
Cilia, flagella form from
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
Microtubule networks assemble and disassemble
Assembly is initiated at MTOCs
MicroTubule Organizing Centers
100’s of g subunits serve to nucleate
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
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)
Colchicine (from crocus) binds free tubulin
Taxol (from yew) stabilizes microtubules
Both cancer therapy drugs, both block mitosis.
How? If opposite modes of action?
Microtubules in the mitotic spindle of a mouse fibroblast in culture.
Image by Steve Rogers, UNC Dept. Biology. (Confocal Microscope)
Organelles, vesicles with
cargo move around the cell.
How do we know?
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.
Centrosomes …… away from
increases resolution and
This movement requires ATP.
But AMP-PNP (analog)
Ron Vale, UCSF
… and AMP-PNP
Michael Sheetz, Columbia
Movie from Jeff Gelles lab
Sliding microtubule assay- used to purify kinesin.
Video microscopy can be used to track the movement of a
single kinesin molecule
One example of microtubules in action…
Attached ciliary dynein
Filaments (F actin) are another important
means by which the cytoskeleton is used for trafficking.
It is important for cell shape, location and contraction
5% of total protein,
half as filaments
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.
Filopodium (spikes) are similarly formed
Racs (Cdc42 is a member) are small GTPases related to the Ras members
These can be activated by recptors and in turn activate actin assembly
Myosin is a motor that runs along (or tugs at) actin.
Actin-based movement of vesicles
Myosin-driven cell shape changes
Myosin I and myosin I
One head (ATPase) and one tail, tail varies
300 heads here, bind actin
Best understood example … contraction in muscle cells.
Multinucleate (formed by cell fusions
50 mm diameter and centimeters long)
… just one sarcomere.
Each filament has ~300 heads.
Each head binds 5X/s
32 mm in 0.1 s
Attached, No ATP, “rigor”
Released, binds ATP
Power stroke, loose ADP
T-tubules bring PM up close to the SR …