microtubules (mts) e. d. salmon biology [email protected] reading: lodish et al., molecular cell...
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Microtubules (MTs)
E. D. Salmon
Biology
Reading: Lodish et al., Molecular Cell Biology; Alberts et al., Molecular Biology of
the Cell
Some Microtubule Functions
• Provide structure, acting as an internal skeleton• Act as a polarized tracks for microtubule motor
(cytoplasmic kinesins and dyneins) driven movements within cells
• With axonemal dynein produce motility of cilia and flagella
• With microtubule motors segregate chromosomes in mitosis
• Give cell polarity and produce polarized organization of organelles
• Regulate activity of actin cytoskeleton in cell motility and cell division
•Each tubulin is about 50 kD and structurally very similar to each other and to FtsZ in bacteria•Microtubules are composed of and tubulins
•Each tubulin binds GTP: the GTP is non-exchangeable and the dimer is very stable, Kd = 10-10; the GTP is exchangable in the dimer•The intracellular tubulin concentration is about 20 M; about half in microtubules and half as dimers in the cytosol
The Tubulin Dimer
Microtubules are Polarized Polymers of Tubulin
• heterodimers bind head-to-tail along protofilaments
•13 protofilaments form a hollow tube-the microtubule: 25 nm OD, 14 nm ID•Microtubules are polar-they have a plus and a minus end
*1625 dimers/m length
*A mammalian tissue cell (6 pL) has 16,500 m total microtubule length; a frog egg (1 L) has 2,500 m
+
-
Other Microtubule Geometries
Cytoplasmic:(10-16 pf)13 pf is typical
Axomeme (Cilia and Flagella)
Centriole orBasal Body
Microtubule Organization in Interphase and Mitosis
25 m 5 m
Centrosome
Polarized Microtubule Organization in vivo
Interphase Mitosis
Centrosome
++
++
+
+ +
+
+
Microtubules are Abundant in Neurons:Brain is the best source
of tubulin
-
+
Microtubule Assembly Occurs By End-Dependent Association-Dissociation Reactions
•In vitro, microtubules that self-assemble from pure tubulin can grow and shorten at both ends
•The plus end grows faster and is more dynamic than the minus end
Microtubule Self-Assembly From Pure
Tubulin In Vitro: • 2 mg/ml tubulin in 1 M PIPES,
1mMEGTA, 1mM MgCl2, 1mM GTP pH = 6.8 and 37C
[tu
b]
in p
oly
me
rCc
(can “seed” MTs using preformed cross-linked MT fragments for example:
Microtubule Ends Exhibit Dynamic Instability, Not Simple
Equilibrium Assembly
+ End
GROWTH PHASE
RESCUECATASTROPHE
GTP-TUBULIN GDP-TUBULIN
SHORTENING PHASE
Microtubule Dynamic Instability
GDPGTP
At an end: dL/dt ~ [fg(kag(S-Ccg)) –fskds]
Microtubule Ends Change Conformation Between Growth
and Shortening Phases
Recording Microtubule Dynamics in Living Cells
Or, expressTubulin-GFP
Digital-Imaging Fluorescence Microsocpy
CCD
Hamamatsu Orca ER CCD Camera
• Low readout noise (~8 electrons)
• High Quantum Efficiency
• Broad spectral response
• Fast readout: ~14MHz
Yokogawa Scanning Head Nikon TE300 inverted microscope
Orca ER CCD
Focus Controller
80 mW Argon-Krypton Laser Input (fiber optic)
Filter Wheel
Schematic of the CSU-10
Microtubule Polarity and DynamicsDynamic Instability
In vitro mitotic cell extract + centrosome
Living interphase animal cell; cell edgeFluorescent Speckle MicroscopyMTs green; actin red (Salmon and Waterman)
tubulin Ring Complexes Nucleate Plus-End Growth in Cells; Self-Assembly and Minus-End Growth Is Rare
TuRC~12-14 tubulins,Xgrips72,109,110,133,195
Tubulin Ring Complex
+
-
Zheng et al. 2000Nat. Cell Biol. 2:358
Centrosome is A Microtubule Organizing Center (MTOC)
MTOC’s control where microtubules are formed
Centrosomes contain peri-centrosomal nucleation complexes surrounding pair of centrioles
Centrioles within centrosomes become basal bodies, which are nucleation centers for cilia and flagella
Centrosome is a Microtubule Organizing Center (MTOC)
Centrosomes contain:
Peri-centrosomal: TuRC nucleation complexes bound to pericentrin and centrin fibrous material plus many kinases
Centrioles within a pair of centrosomes before mitosis
Basal Bodies are Nucleation Centers for Cilia and Flagella
Microtubule Associated Proteins (MAPs) Control Microtubule Assembly and Many Stabilizing MAPs Bind to Outer Surface of
Protofilaments
Neuronal MAPs Initially Most Studied
Catastrophe Factors and Rescue Factors: Many Concentrate at
MT Plus Ends•Catastrophe: Op18, Stathmin; End-binding protein
Kin I (e.g.XKCM1, hMCAK); Kip3•Rescue: Stabilizing Microtubule Associated Proteins (MAPs)
-XMAP215 (hTog, S.c. Stu2 ): antagonizes KinI-Brain Maps (MAP2, Tau)-Growing End Binding proteins: EB1,CLIP170
*Note: Activity of all these factors regulated in the cell cycleby phosphorylation or during cell motility
*Note: In budding yeast, microtubule motors can effect plus end dynamics: destabilize-Dyn1, Kar3, Kip3
stabilize-Kip2
(Cyclin B/Cdk1 kinase)
(Inactive)
(Active)
MT Assembly Dynamics is Regulated in the Cell Cycle
Cat. Rescue
A Catastrophe Factor:
Proteins Which Transiently Bind Growing Ends
• EB1: Links to APC which can bind Beta catenin at cortex; EB1 binds Ncd kinesin motor
• Bim1 in yeast binds Kar 9 at cortex• Clip 170: Links to dynein/dynactin, CLASPS• Dynein/dynactin-links to cortex (binds beta-catenin• Ncd: minus kinesin• MCAK: KinI depolymerase• Review: Vaughan KT, 2004, Surfing, regulating and
capturing: are all microtubule-tip-tracking proteins created equal? Trends Cell Biol. 2004 14:491-6.
Alexa-488-EB1 Bound to the Growing Ends(10 m/min)of Microtubulesin Early PrometaphaseSpindle in Xenopus EggExtracts
Jennifer Tirnauer
Microtubule Drugs
Bind tubulin dimer and blocks assembly: Colchicine, nocodazole, vinblastine, podophilotoxin, vincristine
Binds dimer in microtubule lattice and stabilizes microtubules: Taxol