Scale bar, 5?m. Source Data File. Other data that support the findings of this study are available from your corresponding author on request. A zip file of the Python library, the Jupyter notebook, the natural data and a full environment specification of the installed packages EPZ005687 utilized for analysis of TrackMate output data can be found at 10.5281/zenodo.2605576. Abstract Following fertilization, cortical granules exocytose ovastacin, a metalloendopeptidase that cleaves ZP2 in the zona pellucida surrounding mouse eggs to prevent additional sperm binding. Using high- and super-resolution imaging with ovastacinmCherry as a fluorescent marker, we characterize cortical granule dynamics at single granule resolution in transgenic mouse eggs. Newly-developed imaging protocols provide an unprecedented view of vesicular dynamics near the plasma membrane in mouse eggs. We discover that cortical granule anchoring in the cortex is dependent on maternal MATER and document that myosin IIA is required for biphasic trafficking to the plasma membrane. We observe local clearance of cortical actin during exocytosis and determine that pharmacologic or genetic disruption of trafficking to the plasma membrane impairs secretion of cortical granules and results in polyspermy. Thus, the regulation of cortical granule dynamics at the cortex-plasma membrane interface is critical for exocytosis and the post-fertilization block to sperm binding that ensures monospermic fertilization. agglutinin) lectin. In additional, mouse CGs are ~200?nm which is near the diffraction limit of standard light microcopy8. However, total internal fluorescence microscopy (TIRFM) combined with super-resolution structured illumination microscopy (SIM) can overcome these limitations and provide sub-diffractive, live-imaging of Rabbit Polyclonal to GPR174 fluorescently labeled subcellular organelles9C11. We have previously reported a subcortical maternal complex (SCMC) that forms during oogenesis and, when disrupted, delays or abrogates cleavage-stage embryogenesis12C16. Its biological function(s) during oogenesis have been less well investigated and its peripheral location suggests potential involvement in membrane-associated processes including vesicle trafficking and exocytosis. We also have reported on ovastacin, a zinc metalloendopeptidase that was identified as a pioneer marker of mammalian CGs. It is released during exocytosis and cleaves ZP2 in the extracellular zona pellucida to prevent sperm binding which provides a potent post-fertilization block to polyspermy17,18. Here, we seek to gain insight into CG biology using multiple live-cell imaging modalities with enhanced spatiotemporal resolution and transgenic mice expressing fluorescently tagged ovastacinmCherry as a marker of CGs. We genetically document the involvement of the SCMC component MATER in anchoring CGs at the egg cortex and demonstrate a role for myosin IIA in CG trafficking and clearance of cortical actin prior to exocytosis. Perturbation of CG trafficking at the cortex-plasma membrane interface prospects to polyspermy and adversely affects in vivo female fertility. Results Mouse CGs accumulate in the egg cortex To perform live-diffraction limited and super-resolution (instant TIRF-SIM) imaging of the cortical cytoskeleton and plasma membrane, the 8?m solid zona pellucida was removed (Supplementary Fig.?1aCd). Live confocal microscopy of stimulated wild-type eggs in the presence of plasma membrane (CellMask) and actin-binding fluorogenic (SiR-actin) dyes documented an uneven plasma membrane topography and enrichment of filamentous actin (F-actin, referred to as actin hereafter) as a cytoskeletal scaffold. Single XZ optical sections spanning the membrane clusters showed individual microvilli-like structures and actin fibers that extended EPZ005687 in the axial direction. 3D recordings further documented dynamic disappearance and reappearance of actin in a single lateral plane (Supplementary Fig.?2aCf). CGs in the beginning were detected by confocal microscopy in fixed eggs from transgenic mice expressing ovastacinmCherry (agglutinin (LCA) or ovastacinmCherry (Fig.?1a). To determine whether CGs were enriched in the cortex prior to exocytosis, we imaged zona-free eggs EPZ005687 at single granule resolution. Live confocal microscopy showed accumulation of CGs in the egg cortex (Fig.?1b). Surprisingly, 3D confocal recordings that sampled the cortex of zona-free eggs every 5?s (Fig.?1c, inset; Supplementary Movies?1, 2) revealed that CGs not only EPZ005687 co-localized and moved along actin (Fig.?1c, C2), but actin also polymerized and extended into the imaging plane towards a single CG (Fig.?1c, C1; arrowhead). Following attachment, actin retracted and EPZ005687 pulled the CG towards an area, which accumulated a new membrane microdomain (Fig.?1c, C1; dashed area). This contrasted with a stationary CG (Fig.?1c, C1; arrow) which appeared near the membrane at the onset of imaging. The proportion of CGs that associated with actin was higher than those without contact suggesting that CGs interact with the cortical actin scaffold for movement (Fig.?1d). The co-localization and associated movement with cortical actin also were confirmed in fixed samples using super-resolution instant structured illumination microscopy iSIM (Fig.?1e, f). Open in a separate windows Fig. 1 Cortical granules in the mouse egg. a Confocal cross-sections of z-projections of individual and merged channels of ovastacinmCherry eggs (reddish) fixed and stained with LCA-FITC (green). Arrows, cortical granule free zone. Scale bar, 20?m. b XZ projection of XY optical sections (0.2?m) of zona-free ovastacinmCherry eggs stimulated to exocytose cortical granules (red). Microvillar plasma membrane (green), actin (gray). Scale.