1. 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

2. 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

3. 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

4. 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 + -

5. Other Microtubule Geometries Cytoplasmic: (10-16 pf) 13 pf is typical Axomeme (Cilia and Flagella) Centriole or Basal Body

6. Microtubule Organization in Interphase and Mitosis 25  m 5  m Centrosome

7. Polarized Microtubule Organization in vivo Interphase Mitosis Centrosome + + + + + ++ + +

8. Microtubules are Abundant in Neurons:Brain is the best source of tubulin -+-+

9. 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  

10. 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 37  C [tub] in polymer Cc (can “seed” MTs using preformed cross-linked MT fragments for example:

11. Microtubule Ends Exhibit Dynamic Instability, Not Simple Equilibrium Assembly + End

12. At an end: dL/dt ~ [f g (ka g (S-Cc g )) –f s kd s ]  

13. Microtubule Ends Change Conformation Between Growth and Shortening Phases

14. Recording Microtubule Dynamics in Living Cells Or, express Tubulin-GFP

15. Digital-Imaging Fluorescence Microsocpy CCD

16. Hamamatsu Orca ER CCD Camera Low readout noise (~8 electrons) High Quantum Efficiency Broad spectral response Fast readout: ~14MHz

17. Yokogawa Scanning HeadNikon TE300 inverted microscope Orca ER CCD Focus Controller 80 mW Argon-Krypton Laser Input (fiber optic) Filter Wheel

18. Schematic of the CSU-10

19. Microtubule Polarity and Dynamics Dynamic Instability In vitro mitotic cell extract + centrosome Living interphase animal cell; cell edge Fluorescent Speckle Microscopy MTs green; actin red (Salmon and Waterman)

20.  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. 2000 Nat. Cell Biol. 2:358

21. 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

22. Centrosome is a Microtubule Organizing Center (MTOC) Centrosomes contain: Peri-centrosomal:  TuRC nucleation complexes bound to pericentrin and centrin fibrous material plus many kinases

23. Centrioles within a pair of centrosomes before mitosis

24. Basal Bodies are Nucleation Centers for Cilia and Flagella

25. Microtubule Associated Proteins (MAPs) Control Microtubule Assembly and Many Stabilizing MAPs Bind to Outer Surface of Protofilaments

26. Neuronal MAPs Initially Most Studied

27. 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 cycle by phosphorylation or during cell motility *Note: In budding yeast, microtubule motors can effect plus end dynamics: destabilize-Dyn1, Kar3, Kip3 stabilize-Kip2

28. (Cyclin B/Cdk1 kinase) (Inactive) (Active) MT Assembly Dynamics is Regulated in the Cell Cycle Cat. Rescue

29. A Catastrophe Factor:

30. 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.

31. Alexa-488-EB1 Bound to the Growing Ends (10  m/min) of Microtubules in Early Prometaphase Spindle in Xenopus Egg Extracts Jennifer Tirnauer

32. Microtubule Drugs Bind tubulin dimer and blocks assembly: Colchicine, nocodazole, vinblastine, podophilotoxin, vincristine Binds dimer in microtubule lattice and stabilizes microtubules: Taxol