microtubules (mts) e. d. salmon biology tsalmon@email.unc.edu reading: lodish et al., molecular cell...

Microtubules (MTs)

E. D. Salmon

Biology

tsalmon@email.unc.edu

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