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Union of two gametes to form a zygote is a defining event in the life of sexual eukaryotes, yet the mechanisms that underlie cell-cell fusion during fertilization remain poorly characterized. Here, in studies of fertilization in the green alga, Chlamydomonas, we report identification of a membrane protein on minus gametes, Minus Adhesion Receptor 1 (MAR1), that is essential for the membrane attachment with plus gametes that immediately precedes lipid bilayer merger. We show that MAR1 forms a receptor pair with previously identified receptor FUS1 on plus gametes, whose ectodomain architecture we find is identical to a sperm adhesion protein conserved throughout plant lineages. Strikingly, before fusion, MAR1 is biochemically and functionally associated with the ancient, evolutionarily conserved eukaryotic Class II fusion protein HAP2 on minus gametes. Thus, the integral membrane protein MAR1 provides a molecular link between membrane adhesion and bilayer merger during fertilization in Chlamydomonas.
We elucidate the interaction between actin and specific membrane components, using real time live cell imaging, by delivering probes that enable access to components, that cannot be accessed genetically. We initially investigated the close interplay between Phosphatidylinositol 4,5-bisphosphate (PIP2) and the F-actin network. We show that, during the early stage of cell adhesion, PIP2 forms domains within the filopodia membrane. We studied these domains alongside cell spreading and observed that these very closely follow the actin tread-milling. We show that this mechanism is associated with an active transport of PIP2 rich organelles from the cell perinuclear area to the edge, along actin fibers. Finally, mapping other phospholipids and membrane components we observed that the PIP2 domains formation is correlated with sphingosine and cholesterol rafts.
Activation of SIRT1, an NAD+-dependent protein deacetylase, ameliorates muscular pathophysiology of δ-sarcoglycan-deficient TO-2 hamsters and dystrophin-deficient mdx mice. We found that SIRT1 was highly expressed beneath the cellular membranes of muscle cells. To elucidate functional roles of SIRT1 on muscles, skeletal muscle-specific SIRT1 knockout mice (SIRT1-MKO) were generated. SIRT1-MKO mice showed muscular pathology similar to mild muscular dystrophies with increased numbers of centrally nucleated small myofibers and decreased numbers of middle-sized (2000-3001 μm2) myofibers compared to those of wild-type (WT) mice. Accordingly, SIRT1-MKO mice showed significantly decreased exercise capacity in treadmill and inverted hanging tests with higher levels of serum creatine kinase activities compared with those in WT mice. Evans blue dye uptake after exercise was greater in the muscles of SIRT1-MKO than those of WT mice, suggesting membrane fragility in SIRT1-MKO mice. Because SIRT1 was dominantly localized beneath the membranes of muscular cells, SIRT1 may have a new role in the membranes. We found that levels of fluorescent FM1-43 dye intake after laser-induced membrane disruption in C2C12 cells were significantly increased by SIRT1 inhibitors or Sirt1-siRNA compared with those of control cells. Inhibition of SIRT1 or SIRT1-knockdown severely disturbed the dynamic aggregation of membrane vesicles under the injured site but did not affect expression levels of membrane repair proteins. These data suggested that SIRT1 had a critical role in the resealing of membrane-ruptured muscle cells, which could affect phenotypes of SIRT1-MKO mice. To our knowledge, this report is the first to demonstrate that SIRT1 affected plasma-membrane repair mechanisms.
Resealing of a disrupted plasma membrane at the micron-diameter range requires Ca(2+)-regulated exocytosis. Repeated membrane disruptions reseal more quickly than the initial wound, and this potentiation of membrane resealing persists for at least 24 hours after the initial wound. Long-term potentiation of membrane resealing requires CREB-dependent gene expression, which is activated by the PKC- and p38 MAPK-dependent pathway in a wounded cell. The present study demonstrates that membrane resealing is potentiated in both wounded and neighboring cells in MDCK cells. Wounding of cells expressing CREB133, a mutant variant of CREB, does not show the potentiated response of cell membrane resealing in either wounded or neighboring cells. Furthermore, wounding of cells induces CREB phosphorylation, not only in wounded cells, but also in neighboring cells. Inhibition of the nitric oxide/PKG signaling pathway suppresses CREB phosphorylation in neighboring cells, but not in wounded cells. The potentiation of membrane resealing in neighboring cells is suppressed if the nitric oxide/PKG pathway is inhibited during the initial wound. Together, these results suggest that the nitric oxide/PKG pathway stimulates CREB phosphorylation in neighboring cells so that subsequent cell membrane disruptions of the neighboring cells reseal more quickly.
Integral membrane protein structure determination traditionally requires extraction from cell membranes using detergents or polymers. Here, we describe the isolation and structure determination of proteins in membrane vesicles derived directly from cells. Structures of the ion channel Slo1 from total cell membranes and from cell plasma membranes were determined at 3.8 Å and 2.7 Å resolution, respectively. The plasma membrane environment stabilizes Slo1, revealing an alteration of global helical packing, polar lipid, and cholesterol interactions that stabilize previously unresolved regions of the channel and an additional ion binding site in the Ca2+ regulatory domain. The two methods presented enable structural analysis of both internal and plasma membrane proteins without disrupting weakly interacting proteins, lipids, and cofactors that are essential to biological function.
Disruption of cellular plasma membranes is a common event in many animal tissues, and the membranes are usually rapidly resealed. Moreover, repeated membrane disruptions within a single cell reseal faster than the initial wound in a protein kinase A (PKA)- and protein kinase C (PKC)-dependent manner. In addition to wounded cells, recent studies have demonstrated that wounding of Madin-Darby canine kidney (MDCK) cells potentiates membrane resealing in neighboring cells in the short-term by purinergic signaling, and in the long-term by nitric oxide/protein kinase G signaling. In the present study, real-time imaging showed that cell membrane disruption stimulated cAMP synthesis and Ca2+ mobilization from intracellular stores by purinergic signaling in neighboring MDCK cells. Furthermore, inhibition of PKA and PKC suppressed the ATP-mediated short-term potentiation of membrane resealing in neighboring cells. These results suggest that cell membrane disruption stimulates PKA and PKC via purinergic signaling to potentiate cell membrane resealing in neighboring MDCK cells.
Programmed capillary regression and remodeling are essential developmental processes. However, the cellular and molecular mechanisms that regulate vessel regression are only the beginning to be understood. Here, using in vivo, dynamic, confocal imaging of mouse transgenic reporters as well as static confocal and electron microscopy, we studied the embryonic development and postnatal regression of the transient mouse pupillary membrane (PM) vasculature. This approach allowed us to directly observe the precise temporal sequence of cellular events preceding and during the elimination of the PM from the mouse eye. Imaging of Tcf/Lef-H2B::GFP Wnt-reporter mice uncovered that, unlike the hyaloid vasculature of the posterior eye, a PM endothelial cell (EC) Wnt/β-catenin response is unlikely to be part of the regression mechanism. Live imaging of EC and macrophage dynamics revealed highly active Csf1r-GFP+ macrophages making direct contact with the Flk1-myr::mCherry+ vessel surface and with membrane protrusions or filopodia extending from the ECs. Flk1-myr::mCherry+ EC membrane particles were observed on and around ECs as well as within macrophages. Electron microscopy studies confirmed that they were in phagosomes within macrophages, indicating that the macrophages engulfed the membrane particles. Interestingly, EC plasma membrane uptake by PM macrophages did not correlate with apoptosis and was found shortly after vessel formation at mid-gestation stages in the embryo; long before vessel regression begins during postnatal development. Additionally, genetic ablation of macrophages showed that EC membrane particles were still shed in the absence of macrophages suggesting that macrophages do not induce the formation or release of EC microparticles. These studies have uncovered a novel event during programmed capillary regression in which resident macrophages scavenge endothelial cell microparticles released from the PM vessels. This finding suggests that there may be an initial disruption in vessel homeostasis embryonically as the PM forms that may underlie its ultimate regression postnatally.
During asexual replication within the Anopheles mosquito and their vertebrate host, Plasmodium parasites depend on the generation of a massive amount of new plasma membrane to produce thousands of daughter parasites. How the parasite plasma membrane (PPM) is formed has mostly been studied by electron microscopy, which does not allow an insight into the dynamics of this process. We generated a Plasmodium berghei reporter parasite line by GFP-tagging of a non-essential PPM-localized protein, and followed plasma membrane development in living parasites through the entire Plasmodium life cycle. By generating double-fluorescent parasites in which the PPM is visualized in combination with the parasite endoplasmic reticulum, we show that membrane contact sites are formed between both membrane systems during oocyst and liver stage development that might be used to deliver lipids to the dramatically expanding PPM. In conclusion, we have established a powerful tool to follow PPM development in living parasites, which promises to greatly expand our knowledge of membrane biology in the Plasmodium parasite.
Efficient cell membrane repair mechanisms are essential for maintaining membrane integrity and thus for cell life. Here we show that the Ca2+- and phospholipid-binding proteins annexin A4 and A6 are involved in plasma membrane repair and needed for rapid closure of micron-size holes. We demonstrate that annexin A4 binds to artificial membranes and generates curvature force initiated from free edges, whereas annexin A6 induces constriction force. In cells, plasma membrane injury and Ca2+ influx recruit annexin A4 to the vicinity of membrane wound edges where its homo-trimerization leads to membrane curvature near the edges. We propose that curvature force is utilized together with annexin A6-mediated constriction force to pull the wound edges together for eventual fusion. We show that annexin A4 can counteract various plasma membrane disruptions including holes of several micrometers indicating that induction of curvature force around wound edges is an early key event in cell membrane repair.
Despite the well-established role of actin polymerization as a driving mechanism for cell protrusion, upregulated actin polymerization alone does not initiate protrusions. Using a combination of theoretical modeling and quantitative live-cell imaging experiments, we show that local depletion of actin-membrane links is needed for protrusion initiation. Specifically, we show that the actin-membrane linker ezrin is depleted prior to protrusion onset and that perturbation of ezrin's affinity for actin modulates protrusion frequency and efficiency. We also show how actin-membrane release works in concert with actin polymerization, leading to a comprehensive model for actin-driven shape changes. Actin-membrane release plays a similar role in protrusions driven by intracellular pressure. Thus, our findings suggest that protrusion initiation might be governed by a universal regulatory mechanism, whereas the mechanism of force generation determines the shape and expansion properties of the protrusion.
The cell plasma membrane, the natural barrier of a cell, plays critical roles in a mass of cell physiological and pathological processes. Therefore, revealing and monitoring the local status of the cell plasma membrane are of great significance. Herein, using a near-infrared (NIR) fluorescence probe BTCy, microenvironmental polarity in the cell plasma membrane was in situ monitored. BTCy showed sensitive and selective fluorescence decrease response at 706 nm with the increase of polarity as its polarity-responsive D-π-A structure. Most importantly, BTCy showed unexpected cell plasma membrane-targeting ability, probably due to its amphiphilic structure. With BTCy, the distinguishing imaging of cancer and normal cells was done, in which cancer cells exhibited significantly stronger signals due to their lower cell plasma membrane polarity. In addition, with the imaging of BTCy, the ferroptosis process was revealed with no significant cell plasma membrane polarity variation for the first time. Furthermore, BTCy was employed for in vivo imaging of tumor tissue in the 4T1-tumor-bearing mice. The polarity-responsive and cell plasma membrane-targeting properties of BTCy make it a useful tool for monitoring cell plasma membrane polarity variation, providing an efficient and simple method for tumor diagnosis.
Membrane fusion is an energy-consuming process that requires tight juxtaposition of two lipid bilayers. Little is known about how cells overcome energy barriers to bring their membranes together for fusion. Previously, we have shown that cell-cell fusion is an asymmetric process in which an "attacking" cell drills finger-like protrusions into the "receiving" cell to promote cell fusion. Here, we show that the receiving cell mounts a Myosin II (MyoII)-mediated mechanosensory response to its invasive fusion partner. MyoII acts as a mechanosensor, which directs its force-induced recruitment to the fusion site, and the mechanosensory response of MyoII is amplified by chemical signaling initiated by cell adhesion molecules. The accumulated MyoII, in turn, increases cortical tension and promotes fusion pore formation. We propose that the protrusive and resisting forces from fusion partners put the fusogenic synapse under high mechanical tension, which helps to overcome energy barriers for membrane apposition and drives cell membrane fusion.
Lysosome-associated membrane glycoprotein 2 (LAMP-2) is a target protein for glycosylation by N-acetylglucosaminyltransferase IV (GnT-IV), which catalyzes the formation of β1,4GlcNAc branches on the mannose core of N-glycans in choriocarcinoma cells. However, the role of LAMP-2, especially when it is expressed in the cell surface membrane of choriocarcinoma cells, has not been well investigated in the progression of choriocarcinoma. This study aimed to elucidate the function of the cell surface membrane LAMP-2 in the malignancy of choriocarcinoma.
Membrane proteins are a widespread class of bio-macromolecules responsible for numerous vital biological processes and serve as therapeutic targets for a vast array of contemporary medications. For membrane protein isolation and purification, detergents have historically been used. Despite this, detergents frequently result in protein instability. Consequently, their application was limited. Recent detergent-free approaches have been invented. Among these, styrene-maleic acid lipid particle (SMALP), diisobutylene-maleic acid lipid particle (DIBMALP), and native cell membrane nanoparticle (NCMN) systems are the most prevalent. The NCMN system intends to create a library of membrane-active polymers suitable for high-resolution structure determination of membrane protein. Design, synthesis, characterization, and comparative application evaluations of three novel classes of NCMN polymers, NCMNP13-x, NCMNP21-x, and NCMNP21b-x, are presented in this article. Although each NCMN polymer can solubilize distinct model membrane proteins and retain native lipids in NCMN particles, only the NCMNP21b-x family produces lipid-protein particles with ideal buffer compatibility and high homogeneity suitable for single-particle cryo-EM analysis. NCMNP21b-x polymers that generate high-quality NCMN particles are particularly desirable for membrane protein structural biology.
Recent studies indicate that the cell membrane, interacting with its attached cytoskeleton, is an important regulator of cell function, exerting and responding to forces. We investigate this relationship by looking for connections between cell membrane elastic properties, especially surface tension and bending modulus, and cell function. Those properties are measured by pulling tethers from the cell membrane with optical tweezers. Their values are determined for all major cell types of the central nervous system, as well as for macrophage. Astrocytes and glioblastoma cells, which are considerably more dynamic than neurons, have substantially larger surface tensions. Resting microglia, which continually scan their environment through motility and protrusions, have the highest elastic constants, with values similar to those for resting macrophage. For both microglia and macrophage, we find a sharp softening of bending modulus between their resting and activated forms, which is very advantageous for their acquisition of phagocytic functions upon activation. We also determine the elastic constants of pure cell membrane, with no attached cytoskeleton. For all cell types, the presence of F-actin within tethers, contrary to conventional wisdom, is confirmed. Our findings suggest the existence of a close connection between membrane elastic constants and cell function.
The targeted delivery of therapeutics using antibodies or nanomaterials has improved the precision and safety of cancer therapy. However, the paucity and heterogeneity of identified molecular targets within tumours have resulted in poor and uneven distribution of targeted agents, thus compromising treatment outcomes. Here, we construct a cooperative targeting system in which synthetic and biological nanocomponents participate together in the tumour cell membrane-selective localization of synthetic receptor-lipid conjugates (SR-lipids) to amplify the subsequent targeting of therapeutics. The SR-lipids are first delivered selectively to tumour cell membranes in the perivascular region using fusogenic liposomes. By hitchhiking with extracellular vesicles secreted by the cells, the SR-lipids are transferred to neighbouring cells and further spread throughout the tumour tissues where the molecular targets are limited. We show that this tumour cell membrane-targeted delivery of SR-lipids leads to uniform distribution and enhanced phototherapeutic efficacy of the targeted photosensitizer.
First recognized more than 30 years ago, glycine protects cells against rupture from diverse types of injury. This robust and widely observed effect has been speculated to target a late downstream process common to multiple modes of tissue injury. The molecular target of glycine that mediates cytoprotection, however, remains elusive. Here, we show that glycine works at the level of NINJ1, a newly identified executioner of plasma membrane rupture in pyroptosis, necrosis, and post-apoptosis lysis. NINJ1 is thought to cluster within the plasma membrane to cause cell rupture. We demonstrate that the execution of pyroptotic cell rupture is similar for human and mouse NINJ1 and that NINJ1 knockout functionally and morphologically phenocopies glycine cytoprotection in macrophages undergoing lytic cell death. Next, we show that glycine prevents NINJ1 clustering by either direct or indirect mechanisms. In pyroptosis, glycine preserves cellular integrity but does not affect upstream inflammasome activities or accompanying energetic cell death. By positioning NINJ1 clustering as a glycine target, our data resolve a long-standing mechanism for glycine-mediated cytoprotection. This new understanding will inform the development of cell preservation strategies to counter pathologic lytic cell death.
Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension.
The role of cell membrane dynamics in cell migration is unclear. To examine whether total cell surface area changes are required for cell migration, Dictyostelium cells were flattened by agar-overlay. Scanning electron microscopy demonstrated that flattened migrating cells have no membrane reservoirs such as projections and membrane folds. Similarly, optical sectioning fluorescence microscopy showed that the cell surface area does not change during migration. Interestingly, staining of the cell membrane with a fluorescent lipid analogue demonstrated that the turnover rate of cell membrane is closely related to the cell migration velocity. Next, to clarify the mechanism of cell membrane circulation, local photobleaching was separately performed on the dorsal and ventral cell membranes of rapidly moving cells. The bleached zones on both sides moved rearward relative to the cell. Thus, the cell membrane moves in a fountain-like fashion, accompanied by a high membrane turnover rate and actively contributing to cell migration.
T cells play an integral role in the generation of an effective immune response and are responsible for clearing foreign microbes that have bypassed innate immune system defenses and possess cognate antigens. The immune response can be directed toward a desired target through the selective priming and activation of T cells. Due to their ability to activate a T cell response, dendritic cells and endogenous vesicles from dendritic cells are being developed for cancer immunotherapy treatment. However, current platforms, such as exosomes and synthetic nanoparticles, are limited by their production methods and application constraints. Here, we engineer nanovesicles derived from dendritic cell membranes with similar properties as dendritic cell exosomes via nitrogen cavitation. These cell-derived nanovesicles are capable of activating antigen-specific T cells through direct and indirect mechanisms. Additionally, these nanovesicles can be produced in large yields, overcoming production constraints that limit clinical application of alternative immunomodulatory vesicle or nanoparticle-based methods. Thus, dendritic cell-derived nanovesicles generated by nitrogen cavitation show potential as an immunotherapy platform to stimulate and direct T cell response.
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