Microtubules. Forms a sheet of protofilaments that folds to a tube Both tubulins bind GTP

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1 Microtubules Polymerize from a-tubulin/ß-tubulin dimers Hollow tube of 13 protofilaments In vitro- filament number varies In vivo- always 13 protofilaments Forms a sheet of protofilaments that folds to a tube Both tubulins bind GTP E-site on ß-tubulin can exchange GTP and hydrolyzes GTP to GDP N-site on a-tubulin is always GTP bound (non-exchangable)

2 Microtubule structure (23-1)

3 Microtubule assembly Only GTP tubulin can assemble Below the critical concentration of tubulin there is no assembly Above the critical, filaments assemble and elongate There is a lag phase while oligomers assemble If a seed is provided, there is no lag phase Elongation continues until the critical concentration is reached At equilibrium There is continuous addition to and release from the ends

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5 Assembly Kinetics

6 Filament formation at equilibrium Filament Mass Monomer C c Concentration of tubulin

7 The ends of the filaments are different The off rates and on rates are different at the two ends k on (+)end k on (-) end Cc=k off /k on Because the overall G for the reactions is the same: The fast assembling end is the fast disassembling end The slow assembling end is the slow disassembling end As assembly begins, elongation occurs rapidly to the (+) end At equilibrium, subunits are exchanged rapidly at the (+) end and slowly at the (-) end

8 How do you distinguish the ends? Use a seed- axoneme- a fragment of a flagellum with a static stable microtubule network Add tubulin Polymerize Fix and do EM or use fluorescent tubulin and microscope Can also orient with hook assay add tubulin in high salt and tubulin polymerizes from sides curve of sheet determines which end you are looking from

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10 Nucleotide Hydrolysis leads to treadmilling The dimer adds as ß-tubulin-GTP complex After addition, GTP is hydrolyzed to GDP The GDP bound form is much less stably bound than GTP form Now the kinetics of the two ends changes so that at steady state: Addition is primarily at the (+) end Loss is primarily at the (-) end This is called Treadmilling

11 The two ends have different critical concentrations Filament (+) end Monomer C c Monomer Filament (-) end C c Concentration

12 Does treadmilling happen in vivo? Waterman-Storer et al. J. Cell Biol. Volume 139, Number 2, October 20,

13 Treadmilling

14 Waterman-Storer J. Cell Biol. 139, Inject cells with rho-tubulin at low concentration so not uniform. Image a broken microtubule fragment. Can see treadmillingboth + and - ends move while mark in middle is fixed!

15 Treadmilling of microtubules occurs in cells In this case, the filament moved as a result Could a particle attached be moved on a stationary tubule? Does treadmilling happen in a normal cell? Yes, during mitosis

16 Which end is which? Is the (+) end ß-tubulin or α-tubulin? Add GTP fluorescent beads to tubules and they bind the (+) end α-tubulin antibody binds only the (-) end

17 Dynamic Instability GTP-tubulin is more stably bound to the filament GDP-tubulin ends fray and then depolymerize If GTP-tubulin is abundant, polymerization is rapid at (+) end-gtp cap If assembly slows or GTP cleavage catches up with the end rapid dissassembly occurs at the (+) end Dynamic Instability- Microtubules go through repeated cycles of rapid polymerization and depolymerization in cells and in vitro

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21 GTP cap (23-14)

22 Dynamic instability in vitro

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24 Microtubules in Cells They do not nucleate de novo In most cells they radiate from a single region near the nucleus- The Centrosome/Microtubule Organizing Region (MTOC) There can be multiple nucleating points in some cells epithelial cells have mt s running from apical to basolateral plant cells have mt s running around the circumference under the plasma membrane Cells with cilia and flagella have basal bodies in addition to centrosomes

25 Treat cells with an agent the depolymerizes microtubules nocodazole, colchicine, colcemid Stain for microtubules with antibody Wash out drug Stain for microtubules Can see regrowth of microtubules from MTOC

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27 Microtubule s in cells

28 Centrosomes In most animal cells, the centrosome has two centrioles plus pericentriolar material Centrioles have 9 triplet microtubules Plants have no centioles Epithelial MTOC s have no centrioles γ-tubulin is part of pericentriolar material forms part of a ring complex that nucleates mt s has been found in epithelial MTOC s as well as centrosomes

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34 Microtubules in Cells

35 Epithelial cells The organization of microtubules is different Tubules are arranged with the MTOC (-end) near the apical end stretching toward the basal surface (+end) These cells can specifically sort vesicles and proteins to apical or basal domains

36 Nocodazole treatment of Epithelial Cells

37 Summary of Mt (+) and (-) ends behave differently Dynamic instability occurs at (+) end Treadmilling is possible MT s originate from MTOC s in cells Not all MTOC s have centrioles

38 Gamma tubulin acts as the nucleator for cellular microtubules Identified in genetic screen for ß-tubulin interacting proteins Found in MTOC s by IMF Now found to assemble into short spiral structure Binds to the (-) ends of mt s Prevents growth at the (-) end Ideal structure for nucleating mt assembly

39 γ α β α β α β MTOC α β γ γ γ γ γ α β α β α β α β α β α β α β α β α β α β α β α β α β α β α β α α β (b) β α β

40 Microtubules in Cells The free concentration of tubulin in cells is 5-10 µm which is about Cc No evidence for proteins that affect ends If you inject tubulin, more polymer is made Not regulated by monomer sequestration

41 Accessory Proteins MAP s bind to Mt s most identified in neurons Cross-link microtubules in axons Can stabilize microtubules to prevent depolymerization Katenin severs microtubules

42 Microtubule Modifications Acetylation of a-tubulin found on stable microtubules in chlamydomonas- flagellar mt s are acetylated and cytoplasmic are not cytoplasm has enzyme to remove acetyl group Detyrosylation removes C-terminal tyrosine of a-tubulin another enzyme can add it back stable mt s tend to have a-tubulin detyrosinated Whether either is cause or effect and reason for the modifications unclear

43 Transport on Microtubules In neurons there is visible transport of vesicles from cell body to growth cone Transcription and translation and membrane biosynthesis in cell body Need to get material to growth cone to elongate Axonal transport Fast anterograde (3µm/sec)-vesicles Intermediate anterograde (0.6µm/sec)-mitochondria Slow anterograde- ( µm/sec) -proteins Retrograde- 2µm/sec

44 Fast axonal transport movie

45 Vesicles on microtubule s in vitro

46 Kinesin animation

47 Giant Squid axoplasm can be extruded and watched under microscope Can watch vesicles move on mt s Now use brain mt s and squid axoplasm vesicles move with ATP added (2µm/sec) vesicles bind but don t move with AMPPNP now isolate proteins that bind to mt s in the presence of AMPPNP but elute with ATP! Kinesin is discovered

48 Kinesins Moves toward the (+) end 2 x 124kd + 64kd complex Double headed ATP motor with a tail that binds cargo 4 families involved in vesicle movement 3 families involved in spindle function Some are actually (-) end directed

49 Heavy chains Light chain Flexible hinge (a) Head Stalk Tail α β α β α β α β (b)

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51 Dynein Huge protein complex kd proteins several intermediate and light chains dynactin complex 4 proteins including an actin-related protein (ARP) regulates dynein? (-) end directed ATPase motor Three classes of cytoplasmic plus flagellar One looks vesicular One is near Golgi One is in punctate structures of unknown origin

52 Heavy chain Light chains (a) Cell body Dynein Kinesin + Axon terminus (b)

53 Microtubule motors in vitro

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55 Terasaki et al. DiOC6 stains mitochondria +ER Shows a reticular network in cells Co-localizes with mt s Depolymerize mt s and it collapses, but slower than mt s During regrowth, the ER follows the mt s

56 Dabora and Sheetz Make an membrane prep from CEF cells Add to mt s on coverslip the vesicles are pulled out into a reticular network requires ATP inhibited by AMPPNP and vanadate (requires kinesin and dynein motors) looks like Terasaki s ER Recent- Kinesin binding protein found on cytoplasmic face of ER- Kinectin

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59 Turner and Tartakoff Depolymerize mt s with nocodazole Golgi vesiculates required energy Now remove nocodazole mt s reform Golgi coalescence requires energy

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62 Mitochondria also coalign with Mt s in cell They are elongated into tubules along the length of mt s Recently, a kinesin homolog (Kif1B) has been found to be specific for mitochondria

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65 Lysosomes and Micrtotubules Label endosomal system see extensive network of vesicles and tubules clustered around MTOC Nocodazole causes dispersal Movements of individual vesicles ceases when mt s depolymerized repolymerize mt s and they recluster at MTOC

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67 Melanophores Vesicles more bidirectionally on Mt s to change the color of cells in fish scales

68 Future? Challenge is to figure out the specific function of each motor Where is it? What is it s cargo? What turns the motor off and on? Organization of Golgi, ER, lysosomes by motors and how function is interrelated