Cell membrane tension gradients, membrane flows and cellular processes.

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.

Detection of membrane fission in single Bacillus subtilis cells during endospore formation with high temporal resolution.

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 ∼1 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.

PLSCR1 is a cell-autonomous defence factor against SARS-CoV-2 infection.

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.

Membrane fission during bacterial spore development requires cellular inflation driven by DNA translocation.

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.

Polybasic Patches in Both C2 Domains of Synaptotagmin-1 Are Required for Evoked Neurotransmitter Release.

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.

Rapid propagation of membrane tension at retinal bipolar neuron presynaptic terminals.

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.

Stepwise membrane binding of extended synaptotagmins revealed by optical tweezers.

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.

Optimal Detection of Fusion Pore Dynamics Using Polarized Total Internal Reflection Fluorescence Microscopy.

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 ∼15 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.

A human apolipoprotein L with detergent-like activity kills intracellular pathogens.

Activation of cell-autonomous defense by the immune cytokine interferon-γ (IFN-γ) is critical to the control of life-threatening infections in humans. IFN-γ induces the expression of hundreds of host proteins in all nucleated cells and tissues, yet many of these proteins remain uncharacterized. We screened 19,050 human genes by CRISPR-Cas9 mutagenesis and identified IFN-γ-induced apolipoprotein L3 (APOL3) as a potent bactericidal agent protecting multiple non-immune barrier cell types against infection. Canonical apolipoproteins typically solubilize mammalian lipids for extracellular transport; APOL3 instead targeted cytosol-invasive bacteria to dissolve their anionic membranes into human-bacterial lipoprotein nanodiscs detected by native mass spectrometry and visualized by single-particle cryo-electron microscopy. Thus, humans have harnessed the detergent-like properties of extracellular apolipoproteins to fashion an intracellular lysin, thereby endowing resident nonimmune cells with a mechanism to achieve sterilizing immunity.

The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores.

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.

FisB relies on homo-oligomerization and lipid binding to catalyze membrane fission in bacteria.

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.

Sorting sub-150-nm liposomes of distinct sizes by DNA-brick-assisted centrifugation.

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.

DNA-Origami-Based Fluorescence Brightness Standards for Convenient and Fast Protein Counting in Live Cells.

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.

Retromer forms low order oligomers on supported lipid bilayers.

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 (∼18%), dimer (∼35%), trimer (∼24%), and tetramer (∼13%). 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.

Leukocyte Cytoskeleton Polarization Is Initiated by Plasma Membrane Curvature from Cell Attachment.

Cell polarization is important for various biological processes. However, its regulation, particularly initiation, is incompletely understood. Here, we investigated mechanisms by which neutrophils break their symmetry and initiate their cytoskeleton polarization from an apolar state in circulation for their extravasation during inflammation. We show here that a local increase in plasma membrane (PM) curvature resulting from cell contact to a surface triggers the initial breakage of the symmetry of an apolar neutrophil and is required for subsequent polarization events induced by chemical stimulation. This local increase in PM curvature recruits SRGAP2 via its F-BAR domain, which in turn activates PI4KA and results in PM PtdIns4P polarization. Polarized PM PtdIns4P is targeted by RPH3A, which directs PIP5K1C90 and subsequent phosphorylated myosin light chain polarization, and this polarization signaling axis regulates neutrophil firm attachment to endothelium. Thus, this study reveals a mechanism for the initiation of cell cytoskeleton polarization.

A Nanodisc-Cell Fusion Assay with Single-Pore Sensitivity and Sub-millisecond Time Resolution.

During exocytosis, vesicles fuse with the plasma membrane and release their contents. The fusion pore is the initial, nanometer-sized connection between the plasma membrane and the cargo-laden vesicle. A growing body of evidence points toward the fusion pore being a regulator of exocytosis, but the shortcomings of current experimental techniques to investigate single-fusion pores make it difficult to study factors governing pore behavior. Here we describe an assay that fuses v-SNARE-reconstituted nanodiscs with cells ectopically expressing "flipped" t-SNAREs to monitor dynamics of single fusion pores in a biochemically defined system using electrical recordings. We also describe a fluorescence microscopy-based approach to monitor nanodisc-cell fusion that is much simpler to employ, but cannot resolve single pores.

Toward a unified picture of the exocytotic fusion pore.

Neurotransmitter and hormone release involve calcium-triggered fusion of a cargo-loaded vesicle with the plasma membrane. The initial connection between the fusing membranes, called the fusion pore, can evolve in various ways, including rapid dilation to allow full cargo release, slow expansion, repeated opening-closing and resealing. Pore dynamics determine the kinetics of cargo release and the mode of vesicle recycling, but how these processes are controlled is poorly understood. Previous reconstitutions could not monitor single pores, limiting mechanistic insight they could provide. Recently developed nanodisc-based fusion assays allow reconstitution and monitoring of single pores with unprecedented detail and hold great promise for future discoveries. They recapitulate various aspects of exocytotic fusion pores, but comparison is difficult because different approaches suggested very different exocytotic fusion pore properties, even for the same cell type. In this Review, I discuss how most of the data can be reconciled, by recognizing how different methods probe different aspects of the same fusion process. The resulting picture is that fusion pores have broadly distributed properties arising from stochastic processes which can be modulated by physical constraints imposed by proteins, lipids and membranes.

Single-molecule force spectroscopy of protein-membrane interactions.

Many biological processes rely on protein-membrane interactions in the presence of mechanical forces, yet high resolution methods to quantify such interactions are lacking. Here, we describe a single-molecule force spectroscopy approach to quantify membrane binding of C2 domains in Synaptotagmin-1 (Syt1) and Extended Synaptotagmin-2 (E-Syt2). Syts and E-Syts bind the plasma membrane via multiple C2 domains, bridging the plasma membrane with synaptic vesicles or endoplasmic reticulum to regulate membrane fusion or lipid exchange, respectively. In our approach, single proteins attached to membranes supported on silica beads are pulled by optical tweezers, allowing membrane binding and unbinding transitions to be measured with unprecedented spatiotemporal resolution. C2 domains from either protein resisted unbinding forces of 2-7 pN and had binding energies of 4-14 kBT per C2 domain. Regulation by bilayer composition or Ca2+ recapitulated known properties of both proteins. The method can be widely applied to study protein-membrane interactions.

Regulation of Exocytotic Fusion Pores by SNARE Protein Transmembrane Domains.

Calcium-triggered exocytotic release of neurotransmitters and hormones from neurons and neuroendocrine cells underlies neuronal communication, motor activity and endocrine functions. The core of the neuronal exocytotic machinery is composed of soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs). Formation of complexes between vesicle-attached v- and plasma-membrane anchored t-SNAREs in a highly regulated fashion brings the membranes into close apposition. Small, soluble proteins called Complexins (Cpx) and calcium-sensing Synaptotagmins cooperate to block fusion at low resting calcium concentrations, but trigger release upon calcium increase. A growing body of evidence suggests that the transmembrane domains (TMDs) of SNARE proteins play important roles in regulating the processes of fusion and release, but the mechanisms involved are only starting to be uncovered. Here we review recent evidence that SNARE TMDs exert influence by regulating the dynamics of the fusion pore, the initial aqueous connection between the vesicular lumen and the extracellular space. Even after the fusion pore is established, hormone release by neuroendocrine cells is tightly controlled, and the same may be true of neurotransmitter release by neurons. The dynamics of the fusion pore can regulate the kinetics of cargo release and the net amount released, and can determine the mode of vesicle recycling. Manipulations of SNARE TMDs were found to affect fusion pore properties profoundly, both during exocytosis and in biochemical reconstitutions. To explain these effects, TMD flexibility, and interactions among TMDs or between TMDs and lipids have been invoked. Exocytosis has provided the best setting in which to unravel the underlying mechanisms, being unique among membrane fusion reactions in that single fusion pores can be probed using high-resolution methods. An important role will likely be played by methods that can probe single fusion pores in a biochemically defined setting which have recently become available. Finally, computer simulations are valuable mechanistic tools because they have the power to access small length scales and very short times that are experimentally inaccessible.