Microtubules, Actin(and intermediate filaments)
Macrophage microtubulin greenIntermediate filaments redDNA blue
Mitochondria, actin
Cytoplasmic microtubules
Cytoplasmic tubulesbrkdwn
Mitotic spindle
Differentiated cell types have stable tubule structures
Cilia, flagella form from basal bodies
So what are Microtubules??
… hollow, rigid tubes composed of heterodimers
POLAR.. w/o polarity, it can not function.
Each subunit binds GTP
… but the betasubunit has intrinsicGTPase activity.
Microtubule networks assemble and disassemble
Assembly is initiated at MTOCs
MicroTubule Organizing Centers(aka centrosomes)
MTOC100’s of subunits serve to nucleatetubules,
Centriole pair(sometimes)
Similar to Basal bodies
Anti Anti tubulin tubulin, , anti anti tubulin tubulin
Growth vs. shrinkingis 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 endsCritical Concentration, Cc, above grows/below shrinks
Does not require GTP hydrolysis for assemblyThe rapid nature of disassembly is due to its constrained
internal structure (GDP tubulin)
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?
YewYew
Autumn CrocusAutumn Crocus
Microtubules in the mitotic spindle of a mouse fibroblast in culture. Microtubules in the mitotic spindle of a mouse fibroblast in culture. Image by Steve Rogers, UNC Dept. Biology. (Confocal Microscope)Image by Steve Rogers, UNC Dept. Biology. (Confocal Microscope)
Organelles, vesicles with cargo move around the cell.
Why’s that?
Pulse-chase with radioactive amino acids injected into rat ganglionPulse-chase with radioactive amino acids injected into rat ganglionAssay, slice up axon over time, SDS PAGE autoradiographyAssay, slice up axon over time, SDS PAGE autoradiography
How do we know?How do we know?
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,ExtrudedExoplasm
ATP-dependent
Centrosomes …… away from
Video microscopy Video microscopy increases resolution and increases resolution and visualizes movement. visualizes movement.
This movement requires ATP.This movement requires ATP.
But AMP-PNP (analog)But AMP-PNP (analog)Competitively inhibitsCompetitively inhibits
Ron Vale, UCSF
Michael Sheetz, Columbia
… and AMP-PNP
Movie from Jeff Gelles lab
17.5 kinesin.mov
Sliding microtubule assay- used to purify kinesin. Sliding microtubule assay- used to purify kinesin.
Video microscopy can be used to track the movement of a Video microscopy can be used to track the movement of a single kinesin moleculesingle kinesin molecule
One example of microtubules in action…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
Actin monomersin 4 colors
Abundant:5% of total protein, half as filaments
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 lantrunculinbind G actin
Actin filaments are rarely single …
Nets and bundles.
Profilinthymosin
filamin
myosin
gelsolin
Filopodium (spikes) are similarly formed
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.
Actin-based movement of vesicles
Myosin-driven cell shape changes
Muscle contraction
Myosin I and myosin I
One head (ATPase) and one tail, tail varies
Dimer
300 heads here, bind actin
Best understood example … contraction in muscle cells.
Multinucleate (formed by cell fusions50 m diameter and centimeters long)
… just one sarcomere.
Plus ends
Each filament has ~300 heads.Each head binds 5X/s32 m in 0.1 s Attached, No ATP, “rigor”
Released, binds ATP
Cocked, hydrolysisLoose Pi
Power stroke, loose ADP
Attached again,
T-tubules bring PM up close to the SR …T-tubules bring PM up close to the SR …