Molecular Diffusion in Sperm Plasma Membranes: Rafts, Fences, and Filters.

Mammalian spermatozoa are transcriptionally inactive cells and hence have a limited repertoire of responses to external signals. At the same time they have to regulate their fertilizing capacity so that important molecules for egg recognition and fusion are available only at the appropriate time and place. Two basic strategies can be identified. Either egg recognition molecules are enclosed within a membrane bound vesicle (e.g. the acrosome) and only released when the sperm is in the vicinity of the egg, or they are sequestered to regions on the sperm surface where they are inactive but, on receiving the appropriate signal, migrate to new locations thereby acquiring novel properties through association with other membrane components. This causes formation of multimolecular assemblies that subsequently mediate fertilization. To understand the mechanistic details of the processes involved we have employed a range of high resolution microscopy and video imaging techniques that enable us to measure diffusion of single molecules and molecular assemblies within the plasma membrane. We have addressed questions regarding the type of diffusion of lipids and proteins within a membrane compartment (e.g. random versus constrained diffusion), how signalling complexes (e.g. lipid rafts) are formed and dispersed, how some molecules are corralled within surface domains whereas others are free to exchange between domains, and the nature of the barriers or fences between domains. Techniques to be described include Atomic Force Microscopy (AFM), Scanning Ion Conductance Microscopy (SICM), Fluorescence Recovery after Photobleaching (FRAP), Fluorescence Loss in Photobleaching (FLIP), Single Molecule Tracking (SMT) and Single Particle Tracking (SPT). A new subdomain is described, the equatorial subsegment, that is enriched in Hsp70 and tyrosine phosphorylated proteins. The equatorial subsegment may function as a co-ordinating center for intracellular signalling. We propose a model in which external signals, such as those inducing capacitation or exocytosis of the acrosomal vesicle, cause rearrangement of surface membrane components that confer on spermatozoa the ability to recognise the zona pellucida and acquire fusion competence for the oolemma.