Biology subjects

Ravi, R. T.

Publications and source records attributed to Ravi, R. T..

3 recordsLinked to original sources

bioRxiv2021

Membrane remodeling and matrix dispersal intermediates during mammalian acrosomal exocytosis

To become fertilization-competent, mammalian sperm must undergo a complex series of biochemical and morphological changes in the female reproductive tract. These changes, collectively called capacitation, culminate in the exocytosis of the acrosome, a large vesicle overlying the nucleus. Acrosomal exocytosis is not an all-or-nothing event, but rather a regulated process in which vesicle cargo disperses gradually. However, the structural mechanisms underlying this controlled release remain undefined. In addition, unlike other exocytotic events, fusing membranes are shed as vesicles; the cell thus loses the entire anterior two-thirds of its plasma membrane and yet remains intact while the remaining non-vesiculated plasma membrane becomes fusogenic. Precisely how cell integrity is maintained through-out this drastic vesiculation process is unclear, as is how it ultimately leads to the acquisition of fusion competence. Here, we use cryo-electron tomography to visualize these processes in unfixed, unstained, fully-hydrated sperm. We show that crystalline structures within the acrosome disassemble during capacitation and acrosomal exocytosis, representing a plausible mechanism for gradual dispersal of the acrosomal matrix. We find that the architecture of the sperm head supports an atypical membrane fission-fusion pathway that maintains cell integrity. Finally, we detail how the acrosome reaction transforms both the micron-scale topography and the nano-scale protein landscape of the sperm surface, thus priming the sperm for fertilization. SignificanceMammalian sperm must undergo a complex series of biochemical and morphological changes in the female reproductive tract in order to become fertilization-competent. These changes culminate in acrosomal exocytosis, during which multiple membrane fusions destabilize the acrosomal vesicle and liberate its contents, which include proteins implicated in penetrating and binding to the egg vestments. Here, we use cryo-electron tomography to visualize acrosomal exocytosis intermediates in unfixed, unstained sperm. Our results suggest structural bases for how gradual dispersal of acrosome contents is regulated, as well as for how the cell remains intact after losing much of its plasma membrane, We also show that acrosomal exocytosis transforms both the micron-scale topography and the nano-scale molecular landscape of the sperm surface, thus priming it for interaction and fusion with the egg. These findings yield important insights into sperm physiology and contribute to our understanding of the fundamental yet enigmatic process of mammalian fertilization.

cell biology↗

bioRxiv2021

In-cell structures of a conserved supramolecular array at the mitochondria-cytoskeleton interface in mammalian sperm

Mitochondria-cytoskeleton interactions modulate cellular physiology by regulating mitochondrial transport, positioning, and immobilization. However, there is very little structural information defining mitochondria-cytoskeleton interfaces in any cell type. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image mammalian sperm, where mitochondria wrap around the ciliary cytoskeleton. We find that mitochondria are tethered to their neighbors through inter-mitochondrial linkers and are anchored to the cytoskeleton through ordered arrays on the outer mitochondrial membrane. We use subtomogram averaging to resolve in-cell structures of these arrays from three mammalian species, revealing they are conserved across species despite variations in mitochondrial dimensions and cristae organization. We find that the arrays consist of boat-shaped particles anchored on a network of membrane pores whose arrangement and dimensions are consistent with voltage dependent anion channels. Proteomics and in-cell cross-linking mass spectrometry suggest that the conserved arrays are composed of glycerol kinase-like proteins. Ordered supramolecular assemblies may serve to stabilize similar contact sites in other cell types where mitochondria need to be immobilized in specific subcellular environments, such as in muscles and neurons.

cell biology↗

bioRxiv2020

The multi-scale architecture of mammalian sperm flagella and implications for ciliary motility

Motile cilia are molecular machines used by a myriad of eukaryotic cells to swim through fluid environments. However, available molecular structures represent only a handful of cell types, limiting our understanding of how cilia are modified to support motility in diverse media. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image sperm flagella from three mammalian species. We resolve in-cell structures of centrioles, axonemal doublets, central pair apparatus, and endpiece singlets, revealing novel protofilament-bridging microtubule inner proteins throughout the flagellum. We present native structures of the flagellar base, which is crucial for shaping the flagellar beat. We show that outer dense fibers are directly coupled to microtubule doublets in the principal piece but not in the midpiece. Thus, mammalian sperm flagella are ornamented across scales, from protofilament-bracing structures rein-forcing microtubules at the nano-scale to accessory structures that impose micron-scale asymmetries on the entire assembly. Our structures provide vital foundations for linking molecular structure to ciliary motility and evolution.

cell biology↗