RESUMO
Understanding protective immunity to COVID-19 facilitates preparedness for future pandemics and combats new SARS-CoV-2 variants emerging in the human population. Neutralizing antibodies have been widely studied; however, on the basis of large-scale exome sequencing of protected versus severely ill patients with COVID-19, local cell-autonomous defence is also crucial1-4. Here we identify phospholipid scramblase 1 (PLSCR1) as a potent cell-autonomous restriction factor against live SARS-CoV-2 infection in parallel genome-wide CRISPR-Cas9 screens of human lung epithelia and hepatocytes before and after stimulation with interferon-γ (IFNγ). IFNγ-induced PLSCR1 not only restricted SARS-CoV-2 USA-WA1/2020, but was also effective against the Delta B.1.617.2 and Omicron BA.1 lineages. Its robust activity extended to other highly pathogenic coronaviruses, was functionally conserved in bats and mice, and interfered with the uptake of SARS-CoV-2 in both the endocytic and the TMPRSS2-dependent fusion routes. Whole-cell 4Pi single-molecule switching nanoscopy together with bipartite nano-reporter assays found that PLSCR1 directly targeted SARS-CoV-2-containing vesicles to prevent spike-mediated fusion and viral escape. A PLSCR1 C-terminal ß-barrel domain-but not lipid scramblase activity-was essential for this fusogenic blockade. Our mechanistic studies, together with reports that COVID-associated PLSCR1 mutations are found in some susceptible people3,4, identify an anti-coronavirus protein that interferes at a late entry step before viral RNA is released into the host-cell cytosol.
Assuntos
COVID-19 , Proteínas de Transferência de Fosfolipídeos , SARS-CoV-2 , Animais , Humanos , Camundongos , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Quirópteros , COVID-19/imunologia , COVID-19/metabolismo , COVID-19/prevenção & controle , COVID-19/virologia , Sequenciamento do Exoma , Hepatócitos/imunologia , Hepatócitos/metabolismo , Interferon gama/imunologia , Pulmão/imunologia , Pulmão/metabolismo , Fusão de Membrana , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/imunologia , Proteínas de Transferência de Fosfolipídeos/metabolismo , SARS-CoV-2/classificação , SARS-CoV-2/imunologia , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Internalização do VírusRESUMO
Cell membrane tension affects and is affected by many fundamental cellular processes, yet it is poorly understood. Recent experiments show that membrane tension can propagate at vastly different speeds in different cell types, reflecting physiological adaptations. Here we briefly review the current knowledge about membrane tension gradients, membrane flows, and their physiological context.
Assuntos
Membrana Celular , Membrana Celular/fisiologia , Membrana Celular/metabolismo , Humanos , AnimaisRESUMO
Extended synaptotagmins (E-Syts) mediate lipid exchange between the endoplasmic reticulum (ER) and the plasma membrane (PM). Anchored on the ER, E-Syts bind the PM via an array of C2 domains in a Ca2+- and lipid-dependent manner, drawing the two membranes close to facilitate lipid exchange. How these C2 domains bind the PM and regulate the ER-PM distance is not well understood. Here, we applied optical tweezers to dissect PM binding by E-Syt1 and E-Syt2. We detected Ca2+- and lipid-dependent membrane-binding kinetics of both E-Syts and determined the binding energies and rates of individual C2 domains or pairs. We incorporated these parameters in a theoretical model to recapitulate salient features of E-Syt-mediated membrane contacts observed in vivo, including their equilibrium distances and probabilities. Our methods can be applied to study other proteins containing multiple membrane-binding domains linked by disordered polypeptides.
Assuntos
Cálcio , Pinças Ópticas , Cálcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Lipídeos/análiseRESUMO
Little is known about mechanisms of membrane fission in bacteria despite their requirement for cytokinesis. The only known dedicated membrane fission machinery in bacteria, fission protein B (FisB), is expressed during sporulation in Bacillus subtilis and is required to release the developing spore into the mother cell cytoplasm. Here, we characterized the requirements for FisB-mediated membrane fission. FisB forms mobile clusters of approximately 12 molecules that give way to an immobile cluster at the engulfment pole containing approximately 40 proteins at the time of membrane fission. Analysis of FisB mutants revealed that binding to acidic lipids and homo-oligomerization are both critical for targeting FisB to the engulfment pole and membrane fission. Experiments using artificial membranes and filamentous cells suggest that FisB does not have an intrinsic ability to sense or induce membrane curvature but can bridge membranes. Finally, modeling suggests that homo-oligomerization and trans-interactions with membranes are sufficient to explain FisB accumulation at the membrane neck that connects the engulfment membrane to the rest of the mother cell membrane during late stages of engulfment. Together, our results show that FisB is a robust and unusual membrane fission protein that relies on homo-oligomerization, lipid binding, and the unique membrane topology generated during engulfment for localization and membrane scission, but surprisingly, not on lipid microdomains, negative-curvature lipids, or curvature sensing.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Lipídeos de Membrana/metabolismo , Multimerização Proteica , Proteínas de Bactérias/química , Catálise , Clostridium perfringens/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana/metabolismo , Modelos Moleculares , Proteínas Mutantes/metabolismo , Ligação Proteica , Domínios ProteicosRESUMO
Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release, composed of a single-pass transmembrane domain linked to two C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is poorly understood. Calcium binding to C2B is critical for synchronous release, and C2B additionally binds the SNARE complex. The C2A domain is also required for Syt1 function, but it is not clear why. Here, we asked what critical feature of C2A may be responsible for its functional role and compared this to the analogous feature in C2B. We focused on highly conserved poly-lysine patches located on the sides of C2A (K189-192) and C2B (K324-327). We tested effects of charge-neutralization mutations in either region (Syt1K189-192A and Syt1K326-327A) side by side to determine their relative contributions to Syt1 function in cultured cortical neurons from mice of either sex and in single-molecule experiments. Combining electrophysiological recordings and optical tweezers measurements to probe dynamic single C2 domain-membrane interactions, we show that both C2A and C2B polybasic patches contribute to membrane binding, and both are required for evoked release. The size of the readily releasable vesicle pool and the rate of spontaneous release were unaffected, so both patches are likely required specifically for synchronization of release. We suggest these patches contribute to cooperative membrane binding, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release.SIGNIFICANCE STATEMENT Synaptotagmin-1 is a vesicular calcium sensor required for synchronous neurotransmitter release. Its tandem cytosolic C2 domains (C2A and C2B) bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. How calcium binding to Synaptotagmin-1 leads to release and the relative contributions of the C2 domains are unclear. Combining electrophysiological recordings from cultured neurons and optical tweezers measurements of single C2 domain-membrane interactions, we show that conserved polybasic regions in both domains contribute to membrane binding cooperatively, and both are required for evoked release, likely by increasing the overall affinity of Synaptotagmin-1 for acidic membranes.
Assuntos
Domínios C2 , Cálcio , Neurotransmissores , Sinaptotagmina I , Animais , Cálcio/metabolismo , Lipídeos , Camundongos , Neurotransmissores/metabolismo , Proteínas SNARE/metabolismo , Sinaptotagmina I/genética , Sinaptotagmina I/metabolismoRESUMO
Retromer orchestrates the selection and export of integral membrane proteins from the endosome via retrograde and plasma membrane recycling pathways. Long-standing hypotheses regarding the retromer sorting mechanism posit that oligomeric interactions between retromer and associated accessory factors on the endosome membrane drives clustering of retromer-bound integral membrane cargo prior to its packaging into a nascent transport carrier. To test this idea, we examined interactions between components of the sorting nexin 3 (SNX3)-retromer sorting pathway using quantitative single particle fluorescence microscopy in a reconstituted system. This system includes a supported lipid bilayer, fluorescently labeled retromer, SNX3, and two model cargo proteins, RAB7, and retromer-binding segments of the WASHC2C subunit of the WASH complex. We found that the distribution of membrane-associated retromer is predominantly comprised of monomer (â¼18%), dimer (â¼35%), trimer (â¼24%), and tetramer (â¼13%). Unexpectedly, neither the presence of membrane-associated cargo nor accessory factors substantially affected this distribution. The results indicate that retromer has an intrinsic propensity to form low order oligomers on a supported lipid bilayer and that neither membrane association nor accessory factors potentiate oligomerization. The results support a model whereby SNX3-retromer is a minimally concentrative coat protein complex adapted to bulk membrane trafficking from the endosomal system.
Assuntos
Bicamadas Lipídicas/química , Complexos Multiproteicos/química , Proteínas de Ligação a Fosfato/química , Nexinas de Classificação/química , Proteínas rab de Ligação ao GTP/química , Humanos , Complexos Multiproteicos/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Nexinas de Classificação/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Fluorescence microscopy has been one of the most discovery-rich methods in biology. In the digital age, the discipline is becoming increasingly quantitative. Virtually all biological laboratories have access to fluorescence microscopes, but abilities to quantify biomolecule copy numbers are limited by the complexity and sophistication associated with current quantification methods. Here, we present DNA-origami-based fluorescence brightness standards for counting 5-300 copies of proteins in bacterial and mammalian cells, tagged with fluorescent proteins or membrane-permeable organic dyes. Compared to conventional quantification techniques, our brightness standards are robust, straightforward to use, and compatible with nearly all fluorescence imaging applications, thereby providing a practical and versatile tool to quantify biomolecules via fluorescence microscopy.
Assuntos
DNA , Corantes Fluorescentes , Animais , Microscopia de Fluorescência , ProteínasRESUMO
How bacteria catalyze membrane fission during growth and differentiation is an outstanding question in prokaryotic cell biology. Here, we describe a protein (FisB, for fission protein B) that mediates membrane fission during the morphological process of spore formation in Bacillus subtilis. Sporulating cells divide asymmetrically, generating a large mother cell and smaller forespore. After division, the mother cell membranes migrate around the forespore in a phagocytic-like process called engulfment. Membrane fission releases the forespore into the mother cell cytoplasm. Cells lacking FisB are severely and specifically impaired in the fission reaction. Moreover, GFP-FisB forms dynamic foci that become immobilized at the site of fission. Purified FisB catalyzes lipid mixing in vitro and is only required in one of the fusing membranes, suggesting that FisB-lipid interactions drive membrane remodeling. Consistent with this idea, the extracytoplasmic domain of FisB binds with remarkable specificity to cardiolipin, a lipid enriched in the engulfing membranes and regions of negative curvature. We propose that membrane topology at the final stage of engulfment and FisB-cardiolipin interactions ensure that the mother cell membranes are severed at the right time and place. The unique properties of FisB set it apart from the known fission machineries in eukaryotes, suggesting that it represents a new class of fission proteins.
Assuntos
Bacillus subtilis/citologia , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Esporos Bacterianos/citologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Ligação ProteicaRESUMO
SNARE proteins are the core of the cell's fusion machinery and mediate virtually all known intracellular membrane fusion reactions on which exocytosis and trafficking depend. Fusion is catalyzed when vesicle-associated v-SNAREs form trans-SNARE complexes ("SNAREpins") with target membrane-associated t-SNAREs, a zippering-like process releasing â¼65 kT per SNAREpin. Fusion requires several SNAREpins, but how they cooperate is unknown and reports of the number required vary widely. To capture the collective behavior on the long timescales of fusion, we developed a highly coarse-grained model that retains key biophysical SNARE properties such as the zippering energy landscape and the surface charge distribution. In simulations the â¼65-kT zippering energy was almost entirely dissipated, with fully assembled SNARE motifs but uncomplexed linker domains. The SNAREpins self-organized into a circular cluster at the fusion site, driven by entropic forces that originate in steric-electrostatic interactions among SNAREpins and membranes. Cooperative entropic forces expanded the cluster and pulled the membranes together at the center point with high force. We find that there is no critical number of SNAREs required for fusion, but instead the fusion rate increases rapidly with the number of SNAREpins due to increasing entropic forces. We hypothesize that this principle finds physiological use to boost fusion rates to meet the demanding timescales of neurotransmission, exploiting the large number of v-SNAREs available in synaptic vesicles. Once in an unfettered cluster, we estimate ≥15 SNAREpins are required for fusion within the â¼1-ms timescale of neurotransmitter release.
Assuntos
Exocitose , Fusão de Membrana , Modelos Biológicos , Proteínas SNARE/metabolismo , Entropia , Método de Monte CarloRESUMO
Flickering of fusion pores during exocytotic release of hormones and neurotransmitters is well documented, but without assays that use biochemically defined components and measure single-pore dynamics, the mechanisms remain poorly understood. We used total internal reflection fluorescence microscopy to quantify fusion-pore dynamics in vitro and to separate the roles of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and lipid bilayer properties. When small unilamellar vesicles bearing neuronal v-SNAREs fused with planar bilayers reconstituted with cognate t-SNARES, lipid and soluble cargo transfer rates were severely reduced, suggesting that pores flickered. From the lipid release times we computed pore openness, the fraction of time the pore is open, which increased dramatically with cholesterol. For most lipid compositions tested, SNARE-mediated and nonspecifically nucleated pores had similar openness, suggesting that pore flickering was controlled by lipid bilayer properties. However, with physiological cholesterol levels, SNAREs substantially increased the fraction of fully open pores and fusion was so accelerated that there was insufficient time to recruit t-SNAREs to the fusion site, consistent with t-SNAREs being preclustered by cholesterol into functional docking and fusion platforms. Our results suggest that cholesterol opens pores directly by reducing the fusion-pore bending energy, and indirectly by concentrating several SNAREs into individual fusion events.
Assuntos
Colesterol/metabolismo , Fusão de Membrana , Proteínas SNARE/química , Proteínas SNARE/metabolismo , Cinética , Microscopia de Fluorescência , Modelos Moleculares , Conformação Proteica , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismoRESUMO
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes are the core molecular machinery of membrane fusion, a fundamental process that drives inter- and intracellular communication and trafficking. One of the questions that remains controversial has been whether and how SNAREs cooperate. Here we show the use of self-assembled DNA-nanostructure rings to template uniform-sized small unilamellar vesicles containing predetermined maximal number of externally facing SNAREs to study the membrane-fusion process. We also incorporated lipid-conjugated complementary ssDNA as tethers into vesicle and target membranes, which enabled bypass of the rate-limiting docking step of fusion reactions and allowed direct observation of individual membrane-fusion events at SNARE densities as low as one pair per vesicle. With this platform, we confirmed at the single event level that, after docking of the templated-SUVs to supported lipid bilayers (SBL), one to two pairs of SNAREs are sufficient to drive fast lipid mixing. Modularity and programmability of this platform makes it readily amenable to studying more complicated systems where auxiliary proteins are involved.
Assuntos
DNA/metabolismo , Proteínas SNARE/metabolismo , Proteínas de Transporte Vesicular/metabolismo , DNA de Cadeia Simples/química , Bicamadas Lipídicas/química , Lipossomos/química , Fusão de Membrana , Ligação ProteicaRESUMO
Membrane fission is an essential process in all domains of life. The underlying mechanisms remain poorly understood in bacteria, partly because suitable assays are lacking. Here, we describe an assay to detect membrane fission during endospore formation in single Bacillus subtilis cells with a temporal resolution of â¼1 min. Other cellular processes can be quantified and temporally aligned to the membrane fission event in individual cells, revealing correlations and causal relationships. For complete details on the use and execution of this protocol, please refer to Landajuela et al.1.
Assuntos
Bacillus subtilis , Membrana Celular , Esporos Bacterianos , Bacillus subtilis/metabolismo , Bacillus subtilis/citologia , Esporos Bacterianos/metabolismo , Membrana Celular/metabolismo , Análise de Célula Única/métodosRESUMO
Over the past 15 years, hundreds of previously undiscovered bacterial small open reading frame (sORF)-encoded polypeptides (SEPs) of fewer than fifty amino acids have been identified, and biological functions have been ascribed to an increasing number of SEPs from intergenic regions and small RNAs. However, despite numbering in the dozens in Escherichia coli, and hundreds to thousands in humans, same-strand nested sORFs that overlap protein coding genes in alternative reading frames remain understudied. In order to provide insight into this enigmatic class of unannotated genes, we characterized GndA, a 36-amino acid, heat shock-regulated SEP encoded within the +2 reading frame of the gnd gene in E. coli K-12 MG1655. We show that GndA pulls down components of respiratory complex I (RCI) and is required for proper localization of a RCI subunit during heat shock. At high temperature GndA deletion (ΔGndA) cells exhibit perturbations in cell growth, NADH+/NAD ratio, and expression of a number of genes including several associated with oxidative stress. These findings suggest that GndA may function in maintenance of homeostasis during heat shock. Characterization of GndA therefore supports the nascent but growing consensus that functional, overlapping genes occur in genomes from viruses to humans.
RESUMO
Almost all known intracellular fusion reactions are driven by formation of trans-SNARE complexes through pairing of vesicle-associated v-SNAREs with complementary t-SNAREs on target membranes. However, the number of SNARE complexes required for fusion is unknown, and there is controversy about whether additional proteins are required to explain the fast fusion which can occur in cells. Here we show that single vesicles containing the synaptic/exocytic v-SNAREs VAMP/synaptobrevin fuse rapidly with planar, supported bilayers containing the synaptic/exocytic t-SNAREs syntaxin-SNAP25. Fusion rates decreased dramatically when the number of externally oriented v-SNAREs per vesicle was reduced below 5-10, directly establishing this as the minimum number required for rapid fusion. Docking-to-fusion delay time distributions were consistent with a requirement that 5-11 t-SNAREs be recruited to achieve fusion, closely matching the v-SNARE requirement.
Assuntos
Recuperação de Fluorescência Após Fotodegradação/métodos , Fusão de Membrana , Proteínas SNARE/metabolismo , Animais , Humanos , Proteínas SNARE/química , Proteína 25 Associada a Sinaptossoma/química , Proteína 25 Associada a Sinaptossoma/metabolismo , Lipossomas Unilamelares/químicaRESUMO
Many cellular activities, such as cell migration, cell division, phagocytosis, and exo-endocytosis, generate and are regulated by membrane tension gradients. Membrane tension gradients drive membrane flows, but there is controversy over how rapidly plasma membrane flow can relax tension gradients. Here, we show that membrane tension can propagate rapidly or slowly, spanning orders of magnitude in speed, depending on the cell type. In a neuronal terminal specialized for rapid synaptic vesicle turnover, membrane tension equilibrates within seconds. By contrast, membrane tension does not propagate in neuroendocrine adrenal chromaffin cells secreting catecholamines. Stimulation of exocytosis causes a rapid, global decrease in the synaptic terminal membrane tension, which recovers slowly due to endocytosis. Thus, membrane flow and tension equilibration may be adapted to distinct membrane recycling requirements.
RESUMO
Bacteria require membrane fission for both cell division and endospore formation. In Bacillus subtilis, sporulation initiates with an asymmetric division that generates a large mother cell and a smaller forespore that contains only a quarter of its genome. As the mother cell membranes engulf the forespore, a DNA translocase pumps the rest of the chromosome into the small forespore compartment, inflating it due to increased turgor. When the engulfing membrane undergoes fission, the forespore is released into the mother cell cytoplasm. The B. subtilis protein FisB catalyzes membrane fission during sporulation, but the molecular basis is unclear. Here, we show that forespore inflation and FisB accumulation are both required for an efficient membrane fission. Forespore inflation leads to higher membrane tension in the engulfment membrane than in the mother cell membrane, causing the membrane to flow through the neck connecting the two membrane compartments. Thus, the mother cell supplies some of the membrane required for the growth of the membranes surrounding the forespore. The oligomerization of FisB at the membrane neck slows the equilibration of membrane tension by impeding the membrane flow. This leads to a further increase in the tension of the engulfment membrane, promoting its fission through lysis. Collectively, our data indicate that DNA translocation has a previously unappreciated second function in energizing the FisB-mediated membrane fission under energy-limited conditions.
Assuntos
Proteínas de Bactérias , Esporos Bacterianos , Bacillus subtilis , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Divisão Celular , DNA/metabolismo , Esporos Bacterianos/genéticaRESUMO
Analysis of particle trajectories in images obtained by fluorescence microscopy reveals biophysical properties such as diffusion coefficient or rates of association and dissociation. Particle tracking and lifetime measurement is often limited by noise, large mobilities, image inhomogeneities, and path crossings. We present Speckle TrackerJ, a tool that addresses some of these challenges using computer-assisted techniques for finding positions and tracking particles in different situations. A dynamic user interface assists in the creation, editing, and refining of particle tracks. The following are results from application of this program: 1), Tracking single molecule diffusion in simulated images. The shape of the diffusing marker on the image changes from speckle to cloud, depending on the relationship of the diffusion coefficient to the camera exposure time. We use these images to illustrate the range of diffusion coefficients that can be measured. 2), We used the program to measure the diffusion coefficient of capping proteins in the lamellipodium. We found values â¼0.5 µm(2)/s, suggesting capping protein association with protein complexes or the membrane. 3), We demonstrate efficient measuring of appearance and disappearance of EGFP-actin speckles within the lamellipodium of motile cells that indicate actin monomer incorporation into the actin filament network. 4), We marked appearance and disappearance events of fluorescently labeled vesicles to supported lipid bilayers and tracked single lipids from the fused vesicle on the bilayer. This is the first time, to our knowledge, that vesicle fusion has been detected with single molecule sensitivity and the program allowed us to perform a quantitative analysis. 5), By discriminating between undocking and fusion events, dwell times for vesicle fusion after vesicle docking to membranes can be measured.
Assuntos
Actinas/química , Fusão de Membrana , Microscopia de Fluorescência/métodos , Multimerização Proteica , Interface Usuário-Computador , Proteínas de Capeamento de Actina/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular , Movimento Celular , Difusão , Corantes Fluorescentes/metabolismo , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Estrutura Quaternária de Proteína , Pseudópodes/metabolismo , Proteínas SNARE/metabolismoRESUMO
The fusion pore is the initial narrow connection that forms between fusing membranes. During vesicular release of hormones or neurotransmitters, the nanometer-sized fusion pore may open-close repeatedly (flicker) before resealing or dilating irreversibly, leading to kiss-and-run or full-fusion events, respectively. Pore dynamics govern vesicle cargo release and the mode of vesicle recycling, but the mechanisms are poorly understood. This is partly due to a lack of reconstituted assays that combine single-pore sensitivity and high time resolution. Total internal reflection fluorescence (TIRF) microscopy offers unique advantages for characterizing single membrane fusion events, but signals depend on effects that are difficult to disentangle, including the polarization of the excitation electric field, vesicle size, photobleaching, orientation of the excitation dipoles of the fluorophores with respect to the membrane, and the evanescent field depth. Commercial TIRF microscopes do not allow control of excitation polarization, further complicating analysis. To overcome these challenges, we built a polarization-controlled total internal reflection fluorescence (pTIRF) microscope and monitored fusion of proteoliposomes with planar lipid bilayers with single molecule sensitivity and â¼15 ms temporal resolution. Using pTIRF microscopy, we detected docking and fusion of fluorescently labeled small unilamellar vesicles, reconstituted with exocytotic/neuronal v-SNARE proteins (vSUVs), with a supported bilayer containing the cognate t-SNAREs (tSBL). By varying the excitation polarization angle, we were able to identify a dye-dependent optimal polarization at which the fluorescence increase upon fusion was maximal, facilitating event detection and analysis of lipid transfer kinetics. An improved algorithm allowed us to estimate the size of the fusing vSUV and the fusion pore openness (the fraction of time the pore is open) for every event. For most events, lipid transfer was much slower than expected for diffusion through an open pore, suggesting that fusion pore flickering limits lipid release. We find a weak correlation between fusion pore openness and vesicle area. The approach can be used to study mechanisms governing fusion pore dynamics in a wide range of membrane fusion processes.
RESUMO
All membrane fusion reactions proceed through an initial fusion pore, including calcium-triggered release of neurotransmitters and hormones. Expansion of this small pore to release cargo is energetically costly and regulated by cells, but the mechanisms are poorly understood. Here, we show that the neuronal/exocytic calcium sensor Synaptotagmin-1 (Syt1) promotes expansion of fusion pores induced by SNARE proteins. Pore dilation relied on calcium-induced insertion of the tandem C2 domain hydrophobic loops of Syt1 into the membrane, previously shown to reorient the C2 domain. Mathematical modelling suggests that C2B reorientation rotates a bound SNARE complex so that it exerts force on the membranes in a mechanical lever action that increases the height of the fusion pore, provoking pore dilation to offset the bending energy penalty. We conclude that Syt1 exerts novel non-local calcium-dependent mechanical forces on fusion pores that dilate pores and assist neurotransmitter and hormone release.
Assuntos
Proteínas SNARE/metabolismo , Sinaptotagmina I/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Cálcio/metabolismo , Fusão Celular , Membrana Celular , Regulação da Expressão Gênica/fisiologia , Células HeLa , Humanos , Lipoproteínas , Modelos Biológicos , Modelos Moleculares , Nanoestruturas , Conformação Proteica , Proteínas SNARE/genética , Sinaptotagmina I/genética , Proteína 2 Associada à Membrana da Vesícula/genéticaRESUMO
In cells, myriad membrane-interacting proteins generate and maintain curved membrane domains with radii of curvature around or below 50 nm. To understand how such highly curved membranes modulate specific protein functions, and vice versa, it is imperative to use small liposomes with precisely defined attributes as model membranes. Here, we report a versatile and scalable sorting technique that uses cholesterol-modified DNA 'nanobricks' to differentiate hetero-sized liposomes by their buoyant densities. This method separates milligrams of liposomes, regardless of their origins and chemical compositions, into six to eight homogeneous populations with mean diameters of 30-130 nm. We show that these uniform, leak-resistant liposomes serve as ideal substrates to study, with an unprecedented resolution, how membrane curvature influences peripheral (ATG3) and integral (SNARE) membrane protein activities. Compared with conventional methods, our sorting technique represents a streamlined process to achieve superior liposome size uniformity, which benefits research in membrane biology and the development of liposomal drug-delivery systems.