TheSaccharomyces cerevisiae PRM1Homolog inNeurospora crassaIs Involved in Vegetative and Sexual Cell Fusion Events but Also Has Postfertilization Functions

Abstract

Cell-cell fusion is essential for a variety of developmental steps in many eukaryotic organisms, during both fertilization and vegetative cell growth. Although the molecular mechanisms associated with intracellular membrane fusion are well characterized, the molecular mechanisms of plasma membrane merger between cells are poorly understood. In the filamentous fungus Neurospora crassa, cell fusion events occur during both vegetative and sexual stages of its life cycle, thus making it an attractive model for studying the molecular basis of cell fusion during vegetative growth vs. sexual reproduction. In the unicellular yeast Saccharomyces cerevisiae, one of the few proteins implicated in plasma membrane merger during mating is Prm1p; prm1D mutants show an $50% reduction in mating cell fusion. Here we report on the role of the PRM1 homolog in N. crassa. N. crassa strains with deletions of a Prm1-like gene (Prm1) showed an $50% reduction in both vegetative and sexual cell fusion events, suggesting that PRM1 is part of the general cell fusion machinery. However, unlike S. cerevisiae, N. crassa strains carrying a Prm1 deletion exhibited complete sterility as either a male or female mating partner, a phenotype that was not complemented in a heterokaryon with wild type (WT). Crosses with DPrm1 strains were blocked early in sexual development, well before development of ascogenous hyphae. The DPrm1 sexual defect in N. crassa was not suppressed by mutations in Sad-1, which is required for meiotic silencing of unpaired DNA (MSUD). However, mutations in Sad-1 increased the number of progeny obtained in crosses with a DPrm1 (Prm1-gfp) complemented strain. These data indicate multiple roles for PRM1 during sexual development.

Membrane Organization and Cell Fusion During Mating in Fission Yeast Requires Multipass Membrane Protein Prm1

“…Out of the 96% of mutant prezygotes, an 85% exhibited fluorescence in half of the zygote body (see cell a in Figure 2B), while 11% of the prezygotes exhibited fluorescence in more or in less than half of the zygote body (see cells b-d in Figure 2B; see also Supporting Information, Figure S1). This observation suggested that intercellular bubbles/fingers, like those described for the S. cerevisiae and N. crassa prm1D mutants (Heiman and Walter 2000;Fleissner et al 2009), had been produced.…”

“…Out of the 96% of mutant prezygotes, an 85% exhibited fluorescence in half of the zygote body (see cell a in Figure 2B), while 11% of the prezygotes exhibited fluorescence in more or in less than half of the zygote body (see cells b-d in Figure 2B; see also Supporting Information, Figure S1). This observation suggested that intercellular bubbles/fingers, like those described for the S. cerevisiae and N. crassa prm1D mutants (Heiman and Walter 2000;Fleissner et al 2009), had been produced.The distribution of cell wall and plasma membrane at the cell-cell contact area of prm1D zygotes is abnormal When the mating process was analyzed in strains bearing fluorescent membrane and nuclei markers (Figure 2A), it was found that most of the control and prm1D prezygotes exhibited a flat plasma membrane separating the mating partners (an example is denoted by an arrow in Figure 3A). However, in the mutant strain a significant number of prezygotes exhibited an accumulation of membranous material (denoted by arrowheads in Figure 3A), membrane bubbles/fingers (asterisks in Figure 3A), and cell compartments delimited by two membranes (denoted by a cross) that were sometimes accompanied by a bubble.…”

“…Heiman and Walter 2000;Fleissner et al 2009). In 30% of S. pombe prm1D zygotes (Figure 4, right), there were regions where the cross wall had been partially digested and regions where it had thinned (denoted by arrowheads in the magnifications of photographs a and b).…”

“…critical for this process than that of S. cerevisiae and N. crassa PRM1 (Heiman and Walter 2000;Jin et al 2004;Fleissner et al 2009). Analysis of zygotes bearing a soluble GFP suggested that the S. pombe prm1D mutants might produce plasma membrane bubbles/fingers; however, electron microscopy revealed that the nature of the structures observed in S. pombe might be different from that described in other organisms.…”

“…It has been proposed that Prm1p is a fusion facilitator that might help to form a pore at the contact area between mating partners (White and Rose 2001; Olmo and Grote 2010a). Neurospora crassa has a PRM1 ortholog whose deletion leads to a 50% reduction in cell fusion during vegetative and sexual cell fusion, the appearance of intercellular bubbles/fingers with apposed plasma membranes during vegetative cell fusion, and defects in postmeiotic events that lead to complete sterility (Fleissner et al 2009). Intercellular bubbles/fingers have also been observed in S. cerevisiae fig1D mutants, which are defective in a claudin-like tetraspan protein that facilitates calcium influx and is required for cell polarization and fusion during mating (Erdman et al 1998;Muller et al 2003;Aguilar et al 2007).…”