Abstract #13

Following the steps of nuclear envelope fusion in budding yeast. Patricia Melloy¹, Shu Shen¹, Erin White², Mark Winey², J. Richard McIntosh², Mark Rose¹. 1) Molecular Biology, Princeton University, Princeton, NJ; 2) MCD Biology, University of Colorado at Boulder, CO.
   Nuclear envelope fusion is the last step in diploid formation during yeast mating. Little is known about the steps of nuclear fusion, including whether distinct membrane fusion intermediates are detectable, and the specific timing of spindle pole body (SPB) fusion in relation to membrane fusion. Two models for nuclear fusion have been proposed, differing in whether one or three fusion events occur. We are using time-lapse microscopy of cells with different fluorescent protein-tagged compartments to follow the steps of nuclear fusion during mating. We have followed an SPB marker, Spc42-RFP, and a nuclear envelope/ER luminal marker, GFP-HDEL, during nuclear fusion. We see close juxtaposition of the two parental SPBs just prior to nuclear envelope fusion. To distinguish between outer and inner nuclear envelope fusion, we have followed transfer of the GFP-HDEL marker and an RFP-tagged nucleoplasm marker, Pap1-mRFP, over time. Using one min timepoints, we detect nuclear envelope luminal transfer prior to nucleoplasm transfer in 25% of zygotes. Using 30 sec timepoints, we observe prior nuclear envelope transfer in 50% of zygotes. We conclude that outer and inner membrane fusion occur in distinct steps, supporting the three step model. Examination of transfer kinetics in karyogamy mutants indicate that they block at distinct fusion stages. We have also conducted electron microscopic tomography of wildtype and mutant mating yeast cells. We have generated detailed three-dimensional models that show the details of the outer and inner membranes, SPBs, and microtubules during nuclear fusion. In wildtype zygotes, we have observed nuclear fusion intermediates indicating that outer nuclear envelope fusion precedes inner nuclear envelope fusion, both of which precede SPB fusion. Different karyogamy mutants become blocked at different fusion steps, often with membrane bridges whose properties are consistent with the different kinetics of luminal protein transfer.

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