The minus end directed motor Kar3p drives nuclear congression through spindle pole body cytoplasmic microtubule interactions

Submitted by jzlynch on Fri, 25/11/2011 - 16:39

Date:
31 Jan 2012, 16:00 - 17:00

After cell fusion during mating in Saccharomyces cerevisiae the two nuclei are pulled towards each other through a microtubule-dependent process called nuclear congression. This process has been intensively studied using genetics and live cell fluorescence microscopy analysis of various mutants displaying specific defects in the mating process. However, the organisation and the interaction between cytoplasmic microtubules nucleated from opposite spindle pole bodies (SPBs) as well as the mode of force generation for nuclear migration remained unclear (Rose, 1996; Molk et al., 2006; Molk and Bloom, 2006). To approach this question we resolved the three-dimensional microtubule organisation during nuclear congression using electron tomography carried out on mating cells at different stages in the process. The models show no particular connections between microtubules emanating from opposite SPBs, for instance no microtubule overlaps or microtubule-end interactions. Strikingly, in these models we always observed long microtubules passing the opposite SPB with an average distance between the SPB and these microtubules compatible with the size of a molecular motor. The minus end-directed kinesin Kar3p is known to be essential for nuclear congression (Meluh and Rose, 1990; Endow et al., 1994). Therefore, we used a fully functional GFP labelled Kar3p for live cell imaging and confirmed its localization at the cytoplasmic side of the SPB. We also validated the significance of long cytoplasmic microtubules during nuclear congression. Subsequent work using genetics and live cell microscopy demonstrated that in fact no inter-microtubule interaction between mating partners is necessary for efficient nuclear congression. We identified the molecular anchor of Kar3p at the cytoplasmic side of the SPB and found an additional dependency on Cik1p, one of the two light chains of Kar3p. Altogether, our results support a model in which Kar3p drives nuclear congression by directly connecting SPBs through microtubules nucleated at the opposite SPB.

Endow S. A., Kang S. J., Satterwhite L. L., Rose M. D., Skeen V. P., Salmon E. D. (1994). Yeast Kar3 is a minus-end microtubule motor protein that destabilizes microtubules preferentially at the minus ends. EMBO J. 13(11):2708-2713.

Meluh, P. B. and Rose, M. D. (1990). KAR3, a kinesin-related gene required for yeast nuclear fusion. Cell 60, 1029-1041.

Molk, J. N., Salmon, E. D. and Bloom, K. (2006). Nuclear congression is driven by cytoplasmic microtubule plus end interactions in S. cerevisiae. J. Cell Biol. 172, 27- 39.

Molk, J. N. and Bloom, K. (2006). Microtubule dynamics in the budding yeast mating pathway. J. Cell Sc. 119, 3485-3490.

Rose, M. D. (1996). Nuclear fusion in the yeast Saccharomyces cerevisiae. Ann. Rev. Cell Dev. Biol. 12, 663-695.

Type of Event:
Seminar

Speaker:
Mr Romain Gibeaux

Location:
WTB Seminar Rooms