RESUMO
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 nonvesiculated plasma membrane becomes fusogenic. Precisely how cell integrity is maintained throughout this drastic vesiculation process is unclear, as is how it ultimately leads to the acquisition of fusion competence. Here, we use cryoelectron tomography to visualize these processes in unfixed, unstained, fully hydrated sperm. We show that paracrystalline 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 nanoscale protein landscape of the sperm surface, thus priming the sperm for fertilization.
RESUMO
Assembly of the bacterial flagellar rod, hook, and filament requires penetration through the peptidoglycan (PG) sacculus and outer membrane. In most ß- and γ-proteobacteria, the protein FlgJ has two functional domains that enable PG hydrolyzing activity to create pores, facilitating proper assembly of the flagellar rod. However, two distinct proteins performing the same functions as the dual-domain FlgJ are proposed in δ- and ε-proteobacteria as well as spirochetes. The Lyme disease spirochete Borrelia burgdorferi genome possesses a FlgJ and a PG lytic SLT enzyme protein homolog (BB0259). FlgJ in B. burgdorferi is crucial for flagellar hook and filament assembly but not for the proper rod assembly reported in other bacteria. However, BB0259 has never been characterized. Here, we use cryo-electron tomography to visualize periplasmic flagella in different bb0259 mutant strains and provide evidence that the E580 residue of BB0259 is essential for PG-hydrolyzing activity. Without the enzyme activity, the flagellar hook fails to penetrate through the pores in the cell wall to complete assembly of an intact periplasmic flagellum. Given that FlgJ and BB0259 interact with each other, they likely coordinate the penetration through the PG sacculus and assembly of a functional flagellum in B. burgdorferi and other spirochetes. Because of its role, we renamed BB0259 as flagellar-specific lytic transglycosylase or LTaseBb.
RESUMO
Vinculin plays a fundamental role in integrin-mediated cell adhesion. Activated by talin, it interacts with diverse adhesome components, enabling mechanical coupling between the actin cytoskeleton and the extracellular matrix. Here we studied the interactions of activated full-length vinculin with actin and the way it regulates the organization and dynamics of the Arp2/3 complex-mediated branched actin network. Through a combination of surface patterning and light microscopy experiments we show that vinculin can bundle dendritic actin networks through rapid binding and filament crosslinking. We show that vinculin promotes stable but flexible actin bundles having a mixed-polarity organization, as confirmed by cryo-electron tomography. Adhesion-like synthetic design of vinculin activation by surface-bound talin revealed that clustered vinculin can initiate and immobilize bundles from mobile Arp2/3-branched networks. Our results provide a molecular basis for coordinate actin bundle formation at nascent adhesions.