Ultrastructural plasma membrane asymmetries in tension and curvature promote yeast cell fusion

Cell–cell fusion is central for sexual reproduction, and generally involves gametes of different shapes and sizes. In walled fission yeast Schizosaccharomyces pombe, the fusion of h+ and h− isogametes requires the fusion focus, an actin structure that concentrates glucanase-containing vesicles for cell wall digestion. Here, we present a quantitative correlative light and electron microscopy (CLEM) tomographic dataset of the fusion site, which reveals the fusion focus ultrastructure. Unexpectedly, gametes show marked asymmetries: a taut, convex plasma membrane of h− cells progressively protrudes into a more slack, wavy plasma membrane of h+ cells. Asymmetries are relaxed upon fusion, with observations of ramified fusion pores. h+ cells have a higher exo-/endocytosis ratio than h− cells, and local reduction in exocytosis strongly diminishes membrane waviness. Reciprocally, turgor pressure reduction specifically in h− cells impedes their protrusions into h+ cells and delays cell fusion. We hypothesize that asymmetric membrane conformations, due to differential turgor pressure and exocytosis/endocytosis ratios between mating types, favor cell–cell fusion.

Why are SNAREpins rod-shaped?

SNARE proteins are the core components of the cellular machineries that fuse membranes for neurotransmitter or hormone release and other fundamental processes. Fusion is accomplished when SNARE proteins hosted by apposing membranes form SNARE complexes called SNAREpins, but the mechanism of fusion remains unclear. Computational simulations of SNARE-mediated membrane fusion are challenging due to the millisecond timescales of physiological membrane fusion. Here we used ultra-coarse-grained (UCG) simulations to investigate the minimal requirements for a molecular intracellular fusogen, and to elucidate the mechanisms of SNARE-mediated fusion. We find fusion by simple body forces that push vesicles together is highly inefficient. Inter-vesicle fusogens with different aspect ratios can fuse vesicles only if they are rodlike, of sufficient length to clear the fusogens from the fusion site by entropic forces. Simulations with rod-shaped SNAREpin-like fusogens fused 50-nm vesicles on ms timescales, driven by entropic forces along a reproducible fusion pathway. SNARE-SNARE and SNARE-membrane entropic forces cleared the fusion site and pressed the vesicles into an extended contact zone (ECZ), drove stalk nucleation at the high curvature ECZ boundary, and expanded the stalk into a long-lived hemifusion diaphragm in which a simple pore completed fusion. Our results provide strong support for the entropic hypothesis of SNARE-mediated membrane fusion, and implicate the rodlike structure of the SNAREpin complex as a necessity for entropic force production and fusion.

SNAREs and Synaptotagmin cooperatively determine the Ca2+ sensitivity of neurotransmitter release in fixed stoichiometry modules

Neurotransmitter release is accomplished by a multi-component machinery including the membrane-fusing SNARE proteins and Ca2+-sensing Synaptotagmin molecules. However, the Ca2+ sensitivity of release was found to increase or decrease with more or fewer SNARE complexes at the release site, respectively, while the cooperativity is unaffected (Acuna et al., 2014; Arancillo et al., 2013), suggesting that there is no simple division of labor between these two components. To examine the mechanisms underlying these findings, we developed molecular dynamics simulations of the neurotransmitter release machinery, with variable numbers of Synaptotagmin molecules and assembled SNARE complexes at the release site. Ca2+ uncaging simulations showed that increasing the number of SNARE complexes at fixed stoichiometric ratio of Synaptotagmin to SNAREs increased the Ca2+ sensitivity without affecting the cooperativity. The physiological cooperativity of ~4-5 was reproduced with 2-3 Synaptotagmin molecules per SNARE complex, suggesting that Synaptotagmin and SNAREs cooperate in fixed stoichiometry modules. In simulations of action potential-evoked release, increased numbers of Synaptotagmin-SNARE modules increased release probability, consistent with experiment. Our simulations suggest that the final membrane fusion step is driven by SNARE complex-mediated entropic forces, and by vesicle-tethering forces mediated by the long Synaptotagmin linker domains. In consequence, release rates are increased when more SNARE complexes and Synaptotagmin monomers are present at the fusion site.

Dynamin controls neuropeptide secretion by organizing dense-core vesicle fusion sites

Synaptic vesicles (SVs) release neurotransmitters at specialized active zones, but release sites and organizing principles for the other major secretory pathway, neuropeptide/neuromodulator release from dense-core vesicles (DCVs), remain elusive. We identify dynamins, yeast Vps1 orthologs, as DCV fusion site organizers in mammalian neurons. Genetic or pharmacological inactivation of all three dynamins strongly impaired DCV exocytosis, while SV exocytosis remained unaffected. Wild-type dynamin restored normal exocytosis but not guanosine triphosphatase–deficient or membrane-binding mutants that cause neurodevelopmental syndromes. During prolonged stimulation, repeated use of the same DCV fusion location was impaired in dynamin 1-3 triple knockout neurons. The syntaxin-1 staining efficiency, but not its expression level, was reduced. αSNAP (α–soluble N-ethylmaleimide–sensitive factor attachment protein) expression restored this. We conclude that mammalian dynamins organize DCV fusion sites, downstream of αSNAP, by regulating the equilibrium between fusogenic and non-fusogenic syntaxin-1 promoting its availability for SNARE (SNAP receptor) complex formation and DCV exocytosis.

VAMP2 AND SYNAPTOTAGMINS ARE RELATIVELY IMMOBILE ON CHROMAFFIN GRANULE MEMBRANES: IMPLICATIONS FOR MEMBRANE FUSION AND FUSION PORE EXPANSION

Although many of the proteins of secretory granules have been identified, little is known about their molecular organization and diffusion characteristics. Granule-plasma membrane fusion can only occur when proteins that enable fusion are present at the granule-plasma membrane contact. Thus, the mobility of granule membrane proteins may be an important determinant of fusion pore formation and expansion. To address this issue, we measured the mobility of (fluorophore-tagged) vesicle associated membrane protein 2 (VAMP2), synaptotagmin 1 (Syt1), and synaptotagmin 7 (Syt7) in chromaffin granule membranes in living chromaffin cells. We used a method that is not limited by standard optical resolution. A bright flash of strongly decaying evanescent field (∼80 nm exponential decay constant) produced by total internal reflection (TIR) was used to photobleach GFP-labeled proteins in the granule membrane. Fluorescence recovery occurs as unbleached protein in the granule membrane distal from the glass interface diffuses into the more bleached proximal regions, thereby enabling the measurement of diffusion coefficients. The studies revealed that VAMP2, Syt1, and Syt7 are relatively immobile in chromaffin granules membranes with diffusion constants of ≤ 3 × 10−10 cm2/s. Utilizing these diffusion parameters and the known density of VAMP2 and Syt 1 on synaptic vesicles, we estimated the time required for these proteins to arrive at a nascent fusion site to be tens of milliseconds. We propose that the mobilities of secretory granule SNARE and Syt proteins, heretofore unappreciated factors, influence the kinetics of exocytosis and protein discharge.Significance StatementIn eukaryotic cells, secretory vesicles fuse with the plasma membrane to secrete chemical transmitters, hormones and proteins that enable diverse physiological functions including neurotransmission. Fusion proteins need to be assembled at the fusion site in sufficient number in order to enable membrane fusion. However, the diffusion characteristics of fusogenic proteins on secretory vesicles remained unknown. Here we used a novel method not limited by standard optical resolution to measure the diffusion of VAMP2 and synaptotagmins on chromaffin granule membranes. We found they have limited mobility. The time required for these proteins to reach the granule-plasma membrane contact site suggests that their limited mobility likely influences the kinetics of membrane fusion and subsequent fusion pore expansion.

Failure in Lumbar Spinal Fusion and Current Management Modalities

Lumbar spinal fusion is a commonly performed procedure to stabilize the spine, and the frequency with which this operation is performed is increasing. Multiple factors are involved in achieving successful arthrodesis. Systemic factors include patient medical comorbidities—such as rheumatoid arthritis and osteoporosis—and smoking status. Surgical site factors include choice of bone graft material, number of fusion levels, location of fusion bed, adequate preparation of fusion site, and biomechanical properties of the fusion construct. Rates of successful fusion can vary from 65 to 100%, depending on the aforementioned factors. Diagnosis of pseudoarthrosis is confirmed by imaging studies, often a combination of static and dynamic radiographs and computed tomography. Once pseudoarthrosis is identified, patient factors should be optimized whenever possible and a surgical plan implemented to provide the best chance of successful revision arthrodesis with the least amount of surgical risk.

The vesicular release probability sets the strength of individual Schaffer collateral synapses

Information processing in the brain is controlled by quantal release of neurotransmitters, a tightly regulated process. Even in a single axon, presynaptic boutons differ in the number of docked vesicles, but it is not known if the vesicular release probability (pves) is homogenous or variable between individual boutons. We optically measured evoked transmitter release at individual Schaffer collateral synapses using the genetically encoded glutamate sensor iGluSnFR, localizing the fusion site on the bouton with high spatiotemporal precision. Fitting a binomial model to measured response amplitude distributions allowed us to extract the quantal parameters N, pves, and q. Schaffer collateral boutons typically released only a single vesicle under low pves conditions and switched to multivesicular release in high calcium saline. We found that pves was highly variable between individual boutons and had a dominant impact on presynaptic output.

The vesicular release probability sets the strength of individual Schaffer collateral synapses

Information processing in the brain is controlled by quantal release of neurotransmitters, a tightly regulated process. Even in a single axon, presynaptic boutons differ in the number of docked vesicles, but it is not known if the vesicular release probability (pves) is homogenous or variable between individual boutons. We optically measured evoked transmitter release at individual Schaffer collateral synapses using the genetically encoded glutamate sensor iGluSnFR, localizing the fusion site on the bouton with high spatiotemporal precision. Fitting a binomial model to measured response amplitude distributions allowed us to extract the quantal parameters N, pves, and q. Schaffer collateral boutons typically released only a single vesicle under low pves conditions and switched to multivesicular release in high calcium saline. We found that pves was highly variable between individual boutons and had a dominant impact on presynaptic output.

Unveiling a novel function of CD9 in surface compartmentalization of oocytes

ABSTRACTGamete fusion is an indispensable process for bearing offspring. In mammals, sperm IZUMO1–oocyte JUNO recognition essentially carries out the primary step of this process. In oocytes, CD9 is also known to play a crucial role in gamete fusion. In particular, microvilli biogenesis through CD9 involvement appears to be a key event for successful gamete fusion, because CD9-disrupted oocytes produce short and sparse microvillous structures, resulting in almost no fusion ability with spermatozoa. In order to determine how CD9 and JUNO cooperate in gamete fusion, we analyzed the molecular profiles of each molecule in CD9- and JUNO-disrupted oocytes. Consequently, we found that CD9 is crucial for the exclusion of GPI-anchored proteins, such as JUNO and CD55, from the cortical actin cap region, suggesting strict molecular organization of the unique surface of this region. Through distinct surface compartmentalization due to CD9 governing, GPI-anchored proteins are confined to the appropriate fusion site of the oocyte.

Arthroscopic Bone Grafting and Arthrodesis of Partial Lunotriquetral Coalition

Background Lunotriquetal coalition is the most common carpal coalition that can be symptomatic if trauma disrupts the syndesmosis or synchondrosis or if degenerative changes develop between the abnormal articulating surfaces. Case Description A 15-year-old boy presented with a symptomatic lunotriquetral coalition after a fall 2 years prior. Following appropriate investigation, he was managed via arthroscopic debridement, bone grafting, and lunotriquetral arthrodesis. Literature Review The majority of symptomatic lunotriquetral coalitions have been managed with open arthrodesis. There is only one prior report of arthroscopic arthrodesis of this articulation that did not utilize bone graft. Relevance This report details the procedure to allow arthroscopic lunotriquetral arthrodesis with bone grafting, conveying osteogenic properties, and encouraging incorporation at the fusion site while maintaining the dorsal ligaments.