Microtubules are dynamic polymers composed of tubulin that are central in determining cell shape, cell polarity and cell division. There are many proteins that regulate the microtubule cytoskeleton, primarily by controlling microtubule dynamics. We have been interested in discovering new microtubule regulatory proteins and then understanding their cell biological functions and dissecting their mechanisms through in vitro assays and structural approaches. More recently we have also started working on the post-translational modifications of tubulin and their roles in regulation of microtubule motor activity.
Over the past 20 years we have identified and characterized a number of microtubule regulatory proteins including Katanin/Spastin, Spindly, Augmin and Patronin. Katanin was identified from Xenopus extract as a protein exhibiting microtubule severing activity. Subsequent studies from the lab showed that Katanin, and its human homolog Spastin, self-assemble into an active oligomer on the microtubule that destabilize subunits in a polymerized microtubule.
Spindly, Augmin and Patronin were all identified as proteins required for normal spindle assembly in our RNAi screens in Drosophila S2 cells. Spindly is necessary to recruit dynein to the kinetochore to facilitate the removal of spindle assembly checkpoint proteins from kinetochores after metaphase alignment. Augmin recruits the gamma-tubulin ring complex to nucleate microtubules throughout the spindle and form a robust mitotic spindle. Patronin acts to protect microtubule minus ends from the microtubule-depolymerizing kinesin-13 family members.
See bottom of the page for the stories of the discovery and follow up studies of these proteins.
Current questions we are interested in the lab include:
- How does post-translational modification of microtubules affect motor activity and microtubule organization in cellular processes?
- How is microtubule nucleation regulated at the microtubule-organization center (MTOC)?
- How do motor proteins regulate microtubule organization during mitosis?
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, 2005) 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.
(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) – Vale, R.D. (1991) Severing of stable microtubules by a mitotically-activated protein in Xenopus egg extracts. Cell 64: 827-839.
Spindly, recruiting dynein to kinetochores
Our first RNAi screen (conserved genes in the Drosophila 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.
(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.
Augmin, recruiting gamma-tubulin ring complex to nucleate microtubules throughout the spindle
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. She demonstrated microtubule nucleation from the sides of existing microtubules in meiotic Xenopus egg extracts. Branching microtubule nucleation depends upon augmin and gamma tubulin, and is stimulated by Ran GTPase and its effector TPX2. This amplification of microtubule density may contribute to efficient and faithful spindle assembly.
(pdf) – Petry, S., Groen, A.C., Ishihara, K., Mitchison, T.J., Vale, R.D. (2013) Branching microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2. Cell 1523: 768-777. PMCID: PMC3680348.
(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 assembly in Drosophila S2 cells. Science 316: 417-421.
Patronin, protecting microtubule minus ends
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. Melissa Hendershott, a previous graduate student, further showed that CAMSAP family members, the mammalian homologues of Patronin, have evolved distinct effects on microtubule dynamics. CAMSAP1 does not interfere with polymerization and tracks along growing minus-ends. CAMSAP2 and CAMSAP3 decrease the rate of tubulin incorporation and remain bound, thereby creating stretches of decorated MT minus-ends.