Building Mitotic Spindles

 

 
 
 
Anaphase in Drosophila S2 cell
 
 

Cell division is the complex and tightly orchestrated process of separating duplicated chromosomes to each new cell, a process known as mitosis. Microtubules are essential for the mechanical action of pulling chromosomes apart; this process is mediated by a structure known as the mitotic spindle.  We began to study the process of building the mitotic spindle in Drosophila S2 cells because it provides a very tractable system for studying the loss of gene function through highly efficient RNAi as well as good sample for fluorescence time-lapse microscopy.

Gohta Goshima first began to study the function of molecular motors (kinesins and dynein) through this approach of examining RNAi phenotypes by microscopy and coupling an understanding of these phenotypes with computational analysis (with Roy Wollman and Francois Nedelec) (Goshima et al. 2005, 2007). He, Nico Stuurman and Roy Wollman then took on the challenge of moving away from studying known genes to finding new genes that might be involved in mitosis, specifically in constructing the metaphase mitotic spindle.  This work involved in screening the entire Drosophila genome for RNAi phenotypes using an automated microscope to acquire four-color images of what amounted to be millions of spindles.  The analysis was non-trivial and involved both unsupervised computational analysis of spindles as well as human (Gohta) assessment of galleries of spindle images (found and organized by a computer program).  The result was the identification of ~200 genes that contribute to mitotic spindle assembly, many of which were already known, some that were known genes but not associated previously with mitotic spindle function, and many unknown genes.  The screen was a goldmine of information and led to the identification of the augmin complex, patronin, and sentin (studied in the Goshima lab).  There is undoubtedly more cell biology to mine from the results of this screen.

We also have been particularly interested in microtubule nucleation in the spindle.  Most of the early literature focused upon microtubule nucleation from centrosomes or in the vicinity of chromosomes.  However, in S2 cells where centrosome function was knocked out, we found out that new microtubule growth occurred throughout the mitotic spindle and did not appear to be selectively concentrated around chromosomes (Mahoney et al., 2006).  We favored a model where gamma-tubulin ring complex was nucleating microtubules from the sides of existing microtubules throughout the spindle.  Later, through the whole genome RNAi screen, the augmin complex came into the picture as a factor that can recruit gamma-tubulin ring complex to spindle microtubules and have investigated the role of this complex in generating spindle microtubules through a microtubule-dependent microtubule nucleation process.  We are continuing to study augmin-mediated microtubule nucleation and its relationship to chromatin-dependent nucleation.

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See additional movies and images here.

See web site of of Gohta Goshima who continues to work on these problems in his lab at Nagoya University.

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.
  • (pdf) - Mahoney, N.M., Goshima, G., Douglass, A.D., Vale, R.D. (2006) Making microtubules and mitotic spindles in cells without functional centrosomes. Curr Biol 16: 564-569.
  • (pdf) - Goshima, G., Wollman, R., Stuurman, N., Scholey, J.M. and Vale, R.. (2005) Length control of the metaphase spindle. Curr. Biol. 15: 1979-1988.

 

updated 9/07/2012