McNally Lab Research Interests

We are interested in the biochemical mechanisms used to regulate microtubule dynamics and microtubule organization in cells. We are particularly interested in the microtubule-severing ATPase, katanin, and in the microtubule-dependent mechanisms used to segregate chromosomes during female meiosis.

Katanin

Microtubule dynamics and organization can be regulated by several different classes of microtubule-binding proteins. Dyneins and kinesin-related proteins use ATP hydrolysis to generate forces on microtubules and may be used to form cross-bridges between anti-parallel microtubule bundles within mitotic and meiotic spindles. Several discrete families of proteins regulate the polymerization/depolymerization dynamics at microtubule ends. These include the KinI (kinesin internal motor domain) family, the Dis1/TOG family, the OP18/stathmin family and many others. Katanin is unique in that it uses ATP hydrolysis to generate internal breaks along the length of a microtubule. The mechanism by which katanin severs microtubules and the purpose of this severing in cells are two fundamental questions addressed by our research.

Katanin subunits

Katanin was originally identified through purification of a heterodimeric microtubule-severing ATPase from sea urchin eggs (McNally and Vale, 1993). Analysis of recombinant katanin subunits revealed that the p60 subunit of katanin is responsible for the microtubule-severing and ATPase activities of katanin. The p80 subunit activates and targets the p60 subunit (Hartman et al., 1998). The p60 subunit of sea urchin katanin is a member of the AAA family of ATPases. This subunit undergoes a cycle of oligomerization from monomer to hexamer that is coupled to microtubule binding and the ATPase cycle (Hartman and Vale, 1999). The AAA region of p60 katanin is very similar to the AAA domains of several unrelated protein families. The VPS4 family is conserved between humans and budding yeast and includes proteins essential in vacuolar/lysosomal membrane traffic (Babst et al., 1998). Mutations in the conserved fidgetin gene cause the "fidget" phenotype in mouse (Cox et al., 2000). Mutations in the conserved spastin or SPG4 gene cause dominant hereditary spastic paraplegia in humans (Hazan et al., 1999). "True" p60 katanin subunits show weak sequence homology in the N-terminal non-AAA region. This region is responsible for microtubule binding (Hartman and Vale, 1998) and binding to the p80 subunit (McNally et al., 2000). The "true" p60 katanin subunits are found in the genomes of humans, plants and algae but have not been identified in fungi. The C. elegans p60 katanin subunit is the most divergent p60 so far (Srayko et al., 2000). As shown in the dendrogram below, Drosophila has a p60 subunit (CG10229) closely related to sea urchin (sp) and other p60s as well as a highly divergent p60-related protein (CG1193). The biochemical and cellular activities of p60-related proteins like dm.CG1193, fidgetin and spastin are currently (3/26/03) unknown, although spastin may have microtubule disassembly activity (Errico et al., 2002).

 The p80 subunit of most katanin's has an N-terminal WD-repeat domain and a C-terminal "conserved" domain which is unique to p80 katanin's (Hartman et al., 1998) and which is sufficient to bind and activate p60 katanin (McNally et al., 2000). In C. elegans, the p80 ortholog is MEI-2, a protein that only shares sequence homology with other p80 subunits in this "conserved" domain (Srayko et al., 2000).

In vivo functions of katanin

Functional inactivation of katanin has resulted in the following phenotypes as of 3/26/03:

Cultured rat neurons - failure in axon outgrowth (Ahmad et al., 1999).

Chlamydomonas - failure to assemble central pair microtubules in the axoneme (Smith and Lefbvre, 1998).

Cultured monkey fibroblasts - altered localization of g-tubulin on mitotic spindle poles and altered kinetics of mitotic spindle disassembly in nocodazole (Buster et al., 2002).

Arabidopsis - abnormal cortical microtubule organization, abnormal deposition of cellulose and failure in anisotropic cell growth (Burk et al., 2001 and many others).

C. elegans - The p60 and p80 katanin orthologs, MEI-1 and MEI-2, are specifically required for assembly of the female meiotic spindle but are not required for male meiosis or mitosis (Yang et al., 2003).

The relationship between microtubule-severing activity and these cellular functions is not understood. We are pursuing this question in C. elegans.

Female Meiosis in C. elegans

Microtubules and conserved microtubule-dependent mechanisms are used to segregate both meiotic and mitotic chromosomes in all eukaryotes. However, there are some mechanisms that are unique to female meiotic chromosome segregation in animals that are distinct from mitotic and male meiotic mechanisms. In females of both animals and plants, 3/4 of the meiotic products are eliminated and only 1 of the 4 meiotic products is inherited by a female germ cell. The apparent purpose of this conserved tendency is to maximize the cytoplasmic resources donated to one of the 4 meiotic products. In contrast, each of the 4 products of male meiosis is inherited by a germ cell. In many animals, the unequal inheritance in females is mediated by a meiotic spindle that attaches to the oocyte cortex in a perpendicular orientation at anaphase. In mouse, Xenopus and C. elegans, this attachment and perpendicular spindle orientation minimize the volume of oocyte cytoplasm that is lost with the chromosomes that are eliminated during asymmetric cytokinesis. This perpendicular spindle orientation, however, is also conserved in Drosophila which does not utilize asymmetric cytokinesis as a method for chromosome disposal. Another unique feature of female meiosis in many animals is the absence of centriole-based centrosomes. This feature of female meiosis is important because positioning of spindles relative to the cell cortex in many mitotic and male meiotic cells is mediated by interactions between the cell cortex and the plus ends of centrosome-nucleated microtubule asters. Thus acentriolar female meiotic spindles must have unique mechanisms for attachment and orientation at the oocyte cortex.

In C. elegans, the unique movements of the female meiotic spindle can be monitored in living worms by time-lapse imaging of GFP-histone or GFP-tubulin. We are interested in elucidating the mechanisms by which the female meiotic spindle assembles in the absence of centrosomes, translocates to the cortex and rotates to the perpendicular orientation at anaphase. MEI-1/MEI-2, katanin, is required for spindle assembly, translocation to the cortex, spindle disassembly during anaphase and chromosome segregation (Yang et al., 2003). See our movies: mcnallymovies.htm. We hope to elucidate the microtubule-dependent mechanisms that distinguish acentriolar female meiosis from both mitotic and male meiotic chromosome segregation.

References

Ahmad, F.J., Yu, W., McNally, F.J. and P.W. Baas. (1999). An essential role for katanin in severing microtubules in the neuron. J. Cell Biol. 145: 305-315.

Babst, M., Wendland, B., Estepa, E.J. and Emr, S.D. (1998). The VPS4 AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function. EMBO J. 17: 2982-2993.

Burk DH, Liu B, Zhong R, Morrison WH, Ye ZH. (2001). A katanin-like protein regulates normal cell wall biosynthesis and cell elongation. Plant Cell. 13(4):807-27.

Buster, D.W., McNally, K., and F.J. McNally. 2002. Katanin inhibition prevents the redistribution of g-tubulin at mitosis. J. Cell Science 115: 1083-1092.

Cox GA, Mahaffey CL, Nystuen A, Letts VA, Frankel WN. (200). The mouse fidgetin gene defines a new role for AAA family proteins in mammalian development. Nat Genet. 26(2):198-202.

Errico A, Ballabio A, Rugarli EI. (2002). Spastin, the protein mutated in autosomal dominant hereditary spastic paraplegia, is involved in microtubule dynamics. Hum Mol Genet. 11(2):153-63.

Hartman, J.J., Mahr, J., McNally, K., Okawa, K., Iwamatsu, A., Thomas, S., Cheesman, S., Heuser, J., Vale, R.D. and McNally, F.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.

Hartman, J.J. and Vale, R.D. (1999). Microtubule disassembly by ATP-dependent oligomerization of the AAA enzyme katanin. Science 286: 782-785.

Hazan J, Fonknechten N, Mavel D, Paternotte C, Samson D, Artiguenave F, Davoine CS, Cruaud C, Durr A, Wincker P, Brottier P, Cattolico L, Barbe V, Burgunder JM, Prud'homme JF, Brice A, Fontaine B, Heilig B, Weissenbach J. (1999). Spastin, a new AAA protein, is altered in the most frequent form of autosomal dominant spastic paraplegia. Nat Genet. 23(3):296-303.

McNally, K.P., Bazirgan, O.A. and McNally, F.J. (2000). Two domains of p80 katanin regulate microtubule severing and spindle pole targeting by p60 katanin. J. Cell Sci. 113, 1623-1633.

McNally, F. and Vale, R. (1993). Identification of katanin, an ATPase that severs and disassembles stable microtubules. Cell 75, 419-429.

Smith, E. and Lefebvre, P. (1998). PF15 is required for central microtubule assembly and is homologous to katanin P80. Mol. Biol. Cell 9 SUPPL: 278A.

Srayko, M., Buster, D.W., Bazirgan, O.A., McNally, F.J. and Mains, P.E. (2000). MEI-1/MEI-2 katanin-like microtubule severing activity is required for Caenorhabditis elegans meiosis. Genes and Devel. 14, 1072-1084.

Yang, H., McNally, K., and F.J. McNally. 2003. MEI-1/katanin is required for translocation of the meiosis I spindle to the oocyte cortex in C. elegans. Dev. Biol. in press.