During endocytic vesicle formation, distinct subdomains along the membrane invagination are specified by different proteins, which bend the membrane and drive scission. Bin-Amphiphysin-Rvs (BAR) and Fer-CIP4 homology-BAR (F-BAR) proteins can induce membrane curvature and have been suggested to facilitate membrane invagination and scission. Two F-BAR proteins, Syp1 and Bzz1, are found at budding yeast endocytic sites. Syp1 arrives early but departs from the endocytic site before formation of deep membrane invaginations and scission. Using genetic, spatiotemporal, and ultrastructural analyses, we demonstrate that Bzz1, the heterodimeric BAR domain protein Rvs161/167, actin polymerization, and the lipid phosphatase Sjl2 cooperate, each through a distinct mechanism, to induce membrane scission in yeast. Additionally, actin assembly and Rvs161/167 cooperate to drive formation of deep invaginations. Finally, we find that Bzz1, acting at the invagination base, stabilizes endocytic sites and functions with Rvs161/167, localized along the tubule, to achieve proper endocytic membrane geometry necessary for efficient scission. Together, our results reveal that dynamic interplay between a lipid phosphatase, actin assembly, and membrane-sculpting proteins leads to proper membrane shaping, tubule stabilization, and scission.

The mechanisms that stabilize synaptic strength are enigmatic. Goel et al. demonstrate that the abundance and nanostructure of scaffolds at presynaptic active zones are bidirectionally scaled to homeostatically calibrate global neurotransmitter release at the Drosophila melanogaster neuromuscular junction.

SummaryHigh-pressure freezing followed by freeze substitution and plastic embedding is becoming a more widely used method for TEM sample preparation. Here, we have investigated the influence of solvents, fixative concentrations and water content in the substitution medium on the sample quality of high-pressure frozen, freeze-substituted and plastic embedded mammalian cell culture monolayers. We found that the visibility of structural details was optimal with acetone and that extraction increased with both increasing and decreasing solvent polarity. Interestingly, the addition of water to polar solvents increased the sample quality, while being destructive when added to apolar solvents. The positive effect of water addition is saturable in acetone and ethanol at 5%(v/v), but even addition of up to 20% water has no negative effect on the sample structure. Therefore, a medium based on acetone containing fixatives and 5% water is most optimal for the substitution of mammalian cell cultures. In addition, our results suggest that the presence of water is critical for the retention of structure at temperatures around -60• C.

We have investigated the morphogenesis of human and murine cytomegalovirus by transmission electron microscopy after high-pressure freezing, freeze substitution, and plastic embedding. We observed large tubular infoldings of the inner nuclear membrane that were free of lamina and active in primary envelopment and subsequent transport of capsids to the nuclear periphery. Semiquantitative determinations of the enlarged inner nuclear membrane area and the location of the primary envelopment of nucleocapsids demonstrated that this structure represents a virus-induced specialized membrane domain at which the particles are preferentially enveloped. This is a previously undescribed structural element relevant in cytomegalovirus morphogenesis.Murine cytomegalovirus (MCMV) and human CMV (HCMV) are members of the Betaherpesvirinae. Both viruses encode more than 200 open reading frames (15). The morphogenesis of herpesviruses is a complex process involving multiple interactions between viral and cellular components, especially membranes, and thus is of high interest for both virology and cell biology. The stepwise assembly of the virion has been studied extensively in alphaherpesviruses (10,11,18,20). However, much less is known about the morphogenesis of CMVs (3,12). When the findings from alphaherpesviruses are compared with those from cytomegaloviruses, it has to be kept in mind that the sequence homology is only partial and that the latter viruses have a larger coding capacity. Since the size of all herpesvirus capsids prevents their transport into the cytoplasm through the nuclear pore complex, nuclear egress requires the penetration of the nuclear membranes and the nuclear lamina, probably through an envelopment/de-envelopment process, which is still under debate (11,24). This may also be different for CMVs, since the involved alphaherpesviral kinase US3 is not conserved in betaherpesviruses (7, 16). For MCMV, it has been shown that the viral protein M50 inserts into the inner nuclear membrane and is aggregated by a second viral protein, M53, to form the putative capsid docking site (13). M50 then recruits cellular protein kinases for phosphorylation and dissolution of the nuclear lamina. Additionally, it has been shown for HCMV that pUL97 in concert with the cellular p32 acts by the redistribution of lamina components (9). While the list of viral proteins involved in this process is growing, little ultrastructural information on nuclear egress is available. After release to the cytoplasm, CMV capsids are tegumented and enveloped at Golgi apparatus-derived cisternae (6) by a wrapping process and released by fusion with the plasma membrane.We have investigated cytomegalovirus nuclear egress by electron microscopy using high-pressure freezing, freeze substitution, and plastic embedding. Fibroblast cell monolayers (3T3 murine fibroblasts for MCMV or human foreskin fibroblasts for HCMV) were grown on carbon-coated sapphire discs (3-mm diameter; Engineering Office M. Wohlwend GmbH, Switzerland) and infected at a multiplic...