Microtubule Regulators

 

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Katanin and Spastin, Microtubule severing proteins

We discovered a microtubule severing activity serendipitously while studying organelle transport in mitotic Xenopus egg extracts (Vale, 1991) which led subsequently to postdoc Frank McNally purifying the responsible protein (named katanin, from the Japanese sword katana)(McNally and Vale, 1993).  Jim Hartman and Frank McNally cloned the two subunits and found that one subunit has features of the AAA family of ATPases (Hartman et al., 1998) and showed that it can self-assemble into an active oligomer on the microtubule (Hartman and Vale, 1999).  Through human genetic studies to find genetic mutations that underlie spastic parapalegias, the gene spastin was identified and found to be closely related to katanin.  Antonina Roll-Mecak showed that purified spastin indeed has microtubule severing activity (Roll-Mecak and Vale, 2008) and then obtained the crystal structure of spastin, the first for a microtubule severing protein (Roll-Mecak and Vale, 2008).  The latter study also provided evidence that spastin forms a hexameric ring that may pull the disordered tubulin C-terminus through central pore of the ring in order to destabilize subunits in a polymerized microtubule.

   
movie of microtubule severing
 
spastin structure
 
model for spastin mediated microtubule disassembly
 

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See additional movies and images here.

See web sites of Frank McNally and Antonina Roll-Mecak who continue to work on this problem.

References:

  • (pdf) - Roll-Mecak, A. and Vale, R.D. (2008) Structural basis of microtubule severing by the hereditary spastic paraplegia protein spastin. Nature 451: 363 –- 367.
  • (pdf) - Roll-Mecak, A. and Vale, R.D. (2005) The Drosophila homologue of the hereditary spastic paraplegia protein, spastin, severs and disassembles microtubules. Curr Biol 15: 650-655.
  • (pdf) - Hartman, James J. and Vale, R. D. (1999) Microtubule disassembly by ATP-dependent oligomerization of the AAA enzyme katanin. Science 286: 782-785.
  • (pdf) - Hartman, James J., Mahr, Jeff, McNally, Karen, Okawa, Katsuya, Iwamatsu, Akihiro, Thomas, Susan, Cheesman, Sarah, Jeuser, John, Vale, Ronald D. and McNally, Francis J. (1998) Katanin, a microtubule-severing protein, is a novel AAA ATPase that targets to the centrosome using a WD40-containing subunit. Cell 93: 277-287.
  • (pdf) - McNally, Francis J., Okawa, K, Iwamatsu, Akihiro and Vale, Ronald D. (1996) Katanin, the microtubule-severing ATPase, is concentrated in centrosomes. J Cell Sci 109: 561-7.
  • (pdf) - McNally, F. and Vale, R.D. (1993) Identification of katanin, an ATPase that severs and disassembles stable microtubules. Cell 75: 419-429.
  • (pdf) - Vale, R.D. (1991) Severing of stable microtubules by a mitotically-activated protein in Xenopus egg extracts. Cell 64: 827-839.

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Spindly, recruiting dynein to kinetochores

 
 
     
Our first RNAi screen (conserved genes in the Drosoophila genome) yielded an interesting phenotype of S2 cells that had “spindly” rather than round shapes.  Eric Griffis, a postdoc, became to pursue this unknown gene in more depth and found that it localized to microtubules plus end in interphase and to kinetochores in mitosis (Griffis et al., 2007).  Spindly’s role in mitosis was pursued in more depth and Eric found that Spindly was recruited to the kinetochore by the RZZ complex and that Spindly was necessary for recruiting dynein to the kinetochore.  Dynein is involving in removing spindle assembly checkpoint proteins from kinetochores after metaphase alignment by minus-end-directed microtubule-based transport along kinetochore fibers. If Spindly is depleted by RNAi, then this transport does not take place and cells arrest for a prolonged time in metaphase.  Interestingly, Spindly has a very specific role in dynein recruitment to kinetochores and is not involved in other dynein functions in the cell. 

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See additional movies and images here.

See web site of Eric Griffis who continues to work on this problem.

References:

  • (pdf) - Griffis, E.R., Stuurman, N. and Vale, R.D. (2007) Spindly, a novel protein essential for silencing the spindle assembly checkpoint, recruits dynein to the kinetochore. J Cell Biol 177: 1005-1015.

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Augmin, recruiting gamma-tubulin ring complex to nucleate microtubules throughout the spindle

 
 
 
A model for augmin-gamma-tubulin mediated microtubule nucleation
 
 

Augmin emerged from our 2007 full genome Drosophila RNAi screen on mitotic spindle assembly.  In that work, we identified 5 genes (called dim gamma tubulin genes), which after RNAi-mediated protein depletion, gave rise to a very specific phenotype of the loss of gamma-tubulin staining from the body of the mitotic spindle but not the centrosomes (note- other gene RNAi gave the opposite phenotype and these were involved in building centrosomes or recruiting gamma-tubulin to the centrosome).  The RNAi depletion of the genes also produced interesting phenotypes of loss of microtubule staining in the body of the spindle (consistent with a  role in microtubule nucleation) and chromosome alignment defects, consistent with impaired kinetochore fiber formation. 

Subsequent work by Gohta Goshima et al. showed that the dgt genes indeed form a large protein complex and that there are 8 subunits (three were missed in the original screen).  There is also a similar protein complex in human cells that is also involved in recruiting the gamma-tubulin ring complex to the mitotic spindle and in building microtubule density, also the sequence conservation of the subunits between human and flies is low or even unidentifiable for some subunits.  Because of its role in augmenting microtubule density in the spindle, we named this complex “augmin” from the Latin augmentare (to add).

Sabine Petry has been studying the role of the augmin complex in Xenopus egg extracts, a cell-free system that allows one to study function and perform microscopy in ways that can be difficult in living cells.  She (together with the Francois Nedelec and his laboratory) found that augmin is indeed important for the generating microtubules in Xenopus meiotic spindles and also plays a role is facilitating bipolarity of the spindle (Petry et al., 2011).  This work led to a model for how microtubule nucleating activities might contribute to spindle formation.  More recently, Sabine (in collaboration with Aaron Groen and Tim Mitchison) have been investigating the detailed microtubule nucleating events that are promoted by augmin together with gamma-tubulin.

Our future work is focusing on completely in vitro systems and trying to establish a reconstituted assay for microtubule nucleation using purified augmin.

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See additional movies and images here.

See web site of Gohta Goshima at Nagoya University who continues to work on this problem (independently and in some cases collaboratively with our lab).

References:

  • (pdf) - Petry, S., Pugieux, C., Nedelec, F.J., Vale, R.D. (2011) Augmin promotes meiotic spindle formation and bipolarity in Xenopus egg extracts. Proc Natl Acad Sci 108: 14473-14478.
  • (pdf) - Uehara, R., Nozawa, R., Tomioka, A., Petry, S., Vale, R.D., Obuse, C. and Goshima, G. (2009) The augmin complex plays a critical role in spindle microtubule generation for mitotic progression and cytokinesis in human cells. Proc Natl Acad Sci USA 106: 6998-7003.
  • (pdf) - Goshima, G., Mayer, M., Zhang, N., Stuurman, N. and Vale, R.D. (2008). Augmin: a protein complex required for centrosome-independent microtubule generation within the spindle. J Cell Biol 181: 421-429.
  • (pdf) - Goshima, G., Wollman, R., Goodwin, S.S., Zhang, N., Scholely, J.M., Vale, R.D. and Stuurman, N. (2007) Genes required for mitotic spindle assembl in Drosophila S2 cells. Science 316: 417-421.

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Patronin, protecting microtubule minus ends

 
 
 
Figure by Graham Johnson (graham@grahamj.com)
 
 

Patronin was first uncovered in the whole genome RNAi screen as unknown gene whose depletion resulted in a short spindle phenotype (initially named ssp4) and also short microtubule fragments in the cytoplasm of interphase cells (Goshima et al., 2007).  In followup studies of this proteins, Sarah Goodwin found that the short microtubules were produced as a result of instability of the microtubule minus end (which are normally very stable in wildtype cells)(Goodwin and Vale, 2010).  In the absence of ssp4 in cells, the depolymerizing kinesin-13 can attack and depolymerize the microtubule minus, suggesting that ssp4 counteracts kinesin-13 and protects microtubule minus ends.  This phenomenon was recapitulated in vitro with purified ssp4, kinesin-13, and microtubules, where it could be shown that ssp4 selectively binds to and protects the minus end but does not appear to interact with the plus end.  Because of ssp4’s protective role, we named the protein “Patronin” from the protective patronus in the Harry Potter novels (Harry Potter’s patronus was a stag, and hence the stag protecting the microtubule minus end in Graham Johnson’s illustration shown here).  Melissa Hendershott in the lab is continuing to study the Patronin protein.

 
Patronin bound to microtubule minus ends in vitro

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See additional movies and images here.

References:

  • (pdf) - Goodwin, S.S. and Vale, R.D. (2010) Patronin regulates the microtubule network by protecting microtubule minus ends. Cell 143: 263-274.

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Microtubule Plus End Binding Proteins

Certain proteins, most notably EB1, XMAP and Clip170, bind to the growing plus end of microtubule and can recruit other proteins to microtubule tips.  These plus end binding proteins and their cargo are involved in many aspects of microtubule cell biology, such as signaling and interactions with actin and the cell cortex.  Although we are currently not working on plus end binding proteins, we studied their structure and cell biology in the past decade.  We identified several new cargos of EB1 including RhoGEF2 (Rogers et al., 2004), spectraplakins (microtubule-actin crosslinking factors)(Slep et al., 2005), and the kinesin 14 motor Ncd (Goshima et al., 2005).  We also solved the atomic resolution structure of the C-terminal cargo binding domain of EB1 (Slep et al., 2005) and the microtubule binding domains of EB1, Clip170, and Stu2 and minispindles, two members of the XMAP-215 family (Slep et al., 2007). Our initial studies on S2 cells also began by studying the cell biological functions of EB1 by RNAi (Rogers et al., 2002).  Steve Rogers made the finding that EB1 promotes microtubule dynamicity in interphase cells (frequent transitions between growth and shrinkage) and enhances the stability of astral microtubules in mitosis and is necessary for normal mitotic spindle formation.

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See additional movies and images here.

See web site of Steve Rogers and Kevin Slep who continue to work on related problems.

References:

  • (pdf) - Slep, K.C. and Vale, R.D. (2007) Structural basis of microtubule plus end tracking by XMAP215, CLIP-170, and EB1. Mol. Cell 27: 976-991
  • (pdf) - Goshima, G., Nédélec, F., and Vale, R.D. (2005) Mechanisms for focusing mitotic spindle poles by minus-end-directed motor proteins. J Cell Biol 171: 229-240.
  • (pdf) - Slep, K.C., Rogers, S.L., Elliott, S.L., Ohkura, H., Kolodziej, P.A. and Vale, R.D. (2005) Structural determinants for EB1-mediated recruitment of APC and spectraplakins to the microtubule plus end. J Cell Biol 168: 587-598.
  • (pdf) - Rogers, S.L., Weidemann, U., Haecker, U. and Vale, R.D. (2004) Drosophila DRhoGEF2 associates with microtubule plus ends in an EB1-dependent manner. Curr Biol 14: 1827-1833.
  • (pdf) - Rogers, S.L., Rogers, G.C., Sharp, D.J. and Vale, R.D. (2002) Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle. J Cell Biol 158: 873-884
updated 9/07/2012