Probing Membrane Domains and Diffusion Barriers in Live Sperm Cells Using Fluorescent Particle Tracking

Abstract

Polarized cells, e.g. neurones and spermatozoa, characteristically contain compositionally distinct domains in their plasma membranes that are commensurate with specialized function. Elucidating the mechanisms that generate and maintain these heterogeneities is fundamental to understanding many of the processes involved in cell differentiation. Spermatozoa are excellent models for studying membrane compartmentalization as several distinct domains are present on the surface of the head [1,2]. In this work we probe the nature of the barriers that separate these regions by analyzing the trajectories of individual fluorophore-labelled lipids and proteins as they diffuse within and between domains.The probes used were wheat germ (WGA) lectin, DOPE, DiIC16 and cholera toxin β-subunit (CTXB) and were delivered either as single molecules from a nanopipette [2] or from suspension. Results showed that single protein and lipid molecules exchanged freely between all domains on the head plasma membrane. Conversely, particles of DiIC16 and clusters of CTXB cross-linked GM1 gangliosides, ranging in size from 0.5 to 2.0 microns, showed confinement and were unable to traverse domain boundaries. We hypothesise that a mass filter is present within the membrane that is permissive to single molecules but not multimolecular complexes. Relocation and assembly of these molecules and complexes on the sperm head in response to external stimuli is likely to be important in the developmental processes that lead to successful fertilization.[1] James et al. (2004) Journal of Cell Science 117 6485-6495.[2] Bruckbauer et al. (2007) Biophys. J. 93 3120-3131.