Molekulare Mechanismen der Membranfusion
Fusogens Mediate Fusion
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. Most of our knowledge on the mechanism of cell fusion comes from studies on intracellular vesicle fusion and viral-host cell fusion. 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 (Figure 1). No sexual fusogens are presently known, and so identifying the true fusogens of sexual gametes remains crucial for unravelling the molecular basis of fertilization.
Our research efforts focus on the underlying biochemistry of the yet-to-be-identified cell fusion machinery of the yeast S. cerevisiae (Figure 2). Historically, loss-of-function genetic analysis has been employed to identify genes that are necessary for cell-cell fusion in yeast, more specifically at the level of the plasma membranes (between steps 3 and 4 in Fig.2). However, none appear to play a direct and essential role in the final fusion step. Because of the difficulty in identifying key fusion components that act on the plasma membranes, it has been proposed that cell fusion may be predominantly driven by physical forces originating from the cytoskeleton rather than from specialized proteins at the PM. In addition, or alternatively, there may be several proteins contributing redundantly to membrane fusion, providing a possible explanation as to why so few single factors have been found to be essential for plasma membrane fusion.
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 state into a fusion-ready state following 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 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 re-investigating an old idea and testing if spheroplasts can fuse in a pheromone-dependent manner, thus demonstrating that a fusion machinery is present at the plasma membrane (Figure 3). One problem with this approach is that the spheroplasts lose their ability to adhere to each other in a specific manner (this is one of the roles of the cell wall), and so we are devising different biochemical tricks to bring the spheroplasts into tight apposition. Finally, continuing with the use of spheroplasts as a biochemical model, we are devising “synthetic hybrid” fusion assays with proteoliposomes to reconstitute candidate fusogen proteins identified in the first strategy to test for fusion sufficiency, thus proving that they are genuine fusogens.