Figure 2 Subcellular localization and possible function of MUPP1 in spermatozoa. (A) Confocal laser scanning micrographs showing the subcellular localization of MUPP1 in spermatozoa of different mammalian species. Isolated spermatozoa from different mammalian species (mouse, rat, bull and man) were fixed and incubated with a specific antibody recognizing MUPP1. Antibody binding was visualized using a fluorescein-conjugated anti-rabbit IgG (green). Sperm nuclei were counter-stained with propidium iodide (PI, red). Note that MUPP1 expression in all analyzed species does not overlap with the propidium iodide labeling of the nucleus but is restricted to the acrosomal region of the sperm head and the equatorial segment (bull, man). The inserts indicate a region shown at higher magnification in the lower parts. Photomicrographs show overlays of the green and red channel and the transmitted light channel. Scale bars for the upper parts: mouse, rat: 20 µm; bull, man: 10 µm. Scale bars for the lower parts: mouse, rat and bull: 5 µm; man: 2 µm. (B) working model illustrating a possible functional role of a MUPP1/CaMKIIα complex in preventing spontaneous acrosome reaction during the sequential fusion steps of SNARE-promoted acrosomal exocytosis. The model summarizes the molecular rearrangements and modifications of proteins and lipids involved in the acrosomal fusion process, first depicting a pre-fusion state (upper part) and second at the time after fusion of the outer acrosomal membrane and the plasma membrane (lower part). After capacitation, the acrosomal vesicle has been suggested to be in a pre-fusion stage which is reflected by pre-assembled trans-SNARE complexes which force the outer acrosomal membrane (oam, grey) and the adjacent plasma membrane (pm, dark grey) to close proximity (see also insert on the left). At this stage, a catalytically active CaMKIIα, recruited by the PDZ domains 10 and/or 11 of MUPP1, is “freezing” the acrosomal exocytotic process in this intermediate fusion state, thereby preventing “accidental” spontaneous acrosomal secretion. whether this CaMKIIα/MUPP1 mediated fusion clamp is the result of a CaMKII-catalyzed phosphorylation of proteins participating in the sequential Ca2+-controlled secretion pathway, like SNARE-components and/or ion channels, is currently not known. Furthermore, it is not clear whether the observed co-localization of CaMKIIα and MUPP1 in detergent resistant membrane platforms (green) is necessary to position the kinase and possibly also its “interlocking” target/s to a defined space between the outer acrosomal membrane and the plasma membrane. Upon sperm/egg interaction, Zona pellucida ligand/s bind to complementary receptors on the capacitated sperm plasma membrane (ZP-R), thus inducing an initial transient influx of Ca2+ mediated by voltage-gated channels (Cav) and a subsequent sustained increase in Ca2+ concentration mediated by the interplay of inositol-1,4,5-trisphosphate receptors (IP3-R) on the outer acrosomal membrane and transient receptor potential channels (TRP) in the plasma membrane (bottom part). A local increase in Ca2+ (blue spots) and the formation of Ca2+ occupied calmodulin (CaM) might then be able to release the kinase from the PDZ scaffolding protein, thereby unfreezing the CaMKIIα/MUPP1 clamp. At this point, Ca2+ also triggers the final steps of membrane fusion, coinciding with the formation of hybrid vesicles at multiple sites of the membrane (see insert on the left), controlled by the calcium sensor protein synaptotagmin (synt). For the sake of simplicity, not all known molecules, which are functionally operative in the acrosome reaction (e.g., cAMP, Enkurin, EPAC, NSF, PLCδ, PI3K, Rab3A) are depicted. Localization of Q- and R-SNAREs is plotted according to reference Citation125. Cav, voltage-activated Ca2+ channel; CaMKIIα, Ca2+/calmodulin-dependent kinase IIα; IP3-R, inositol 1,4,5-trisphosphate receptor; MUPP1, Multi PDZ domain Protein 1; TRP, transient receptor potential; VAMP, vesicle-associated membrane protein/synaptobrevin; ZP-R, ZP-receptor.