Cell-cell fusion: a mystery with few molecular players

Sexual reproduction begins with the act of membrane fusion, forming the first cellular unit of the organism by merging two gamete cells with different genetic and cytoplasmic contents.  Cell fusion can involve somatic or sexual cells and requires bringing two lipid bilayers into close proximity, formation of a fusion pore and cytoplasmic mixing. Fusogens are mediators of fusion that rearrange the lipid bilayers and lead to the formation of pores. Somatic cell fusion is still poorly understood, with only a handful being thoroughly established as authentic fusogens. One such example is EFF-1, a fusogen in the worm C. elegans that is necessary for fusion in organ development, but we still do not know the equivalent fusogens in other important model organisms including fusion in sperm-egg fertilization and the mating of fungi such as baking yeast (see Fig. 1).

Figure 1: Cell fusion in various organisms. (A) A model of Epithelial fusion failure 1 (EFF-1) in action during membrane merging and pore formation. EFF-1 fuses epithelial cells of the worm C. elegans during tissue development. In the fusion of gamete cells, by contrast, a few genes are known to be to either partially or fully necessary for gamete fusion. For example, (B) in the yeast S. cerevisiae where the haploid a and 𝛂 cells fuse to form a diploid, Prm1 is localized to the site of cell fusion and enhances it, but is ultimately not essential for fusion. In C. elegans, (C) sperm-egg fusion requires the expression of EGG-1; EGG-2 in the egg, and SPE-9, SPE-38 and SPE-42 in the sperm. However, these are likely adhesion molecules which do not participate directly in fusion.  Schematic figures kindly provided by B. Podbilewicz.


Cell-cell fusion in yeast mating

Our research efforts focus on the underlying biochemistry of the mysterious cell fusion machinery of the yeast S. cerevisiae (Fig. 2). Historically, loss-of-function genetic analysis has been employed to identify genes that are necessary for cell-cell fusion in yeast, specifically at the level of the plasma membranes (between steps C and D in Fig. 2). However, none appear to play a direct and essential role in the final fusion step.

Figure 2: The different steps required for the successful mating and fusion of yeast cells of opposite mating types a and 𝛂.  (A) Pheromone signaling; (B) cell polarization and shmoo formation; (C) cell wall remodeling; and (D) plasma membrane fusion.  We focus on understanding what happens at the level of the proteins at the plasma membrane in the transition between steps C and D.

Our approaches

Taking into account the many difficulties encountered so far, we are following two alternative strategies to identify the machinery responsible for cell-cell membrane fusion in yeast.  In one strategy, we have conducted a proteomics analysis of highly-enriched fractions of the yeast plasma membrane.  In particular, we are quantitively comparing changes in the protein composition of the plasma membrane as the cell transits from a vegetative into a fusion-ready state after pheromone response.  With this analysis, we have uncovered a handful of membrane proteins which have previously not been analyzed in connection to cell-cell membrane fusion.  Our efforts are now centered on conducting genetic, cellular and biochemical reconstitution experiments to evaluate any role in fusion of these proteins.

We are also exploring a second strategy by using yeast spheroplasts, which are cells which have had their cells walls enzymatically digested.  The basic idea here is to develop a biochemical system in which we can “bypass” the cell wall, seen in this context as a physical barrier which masks the biochemical analysis of the underlying plasma membrane.  For this, we are testing if spheroplasts can fuse in a pheromone-dependent manner, thus demonstrating that a fusion machinery is present at the plasma membrane.