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Of the many crucial functions of the ER, homeostasis of physiological calcium increase is critical for signaling. Plasma membrane (PM) injury causes a pathological calcium influx. Here, we show that the ER helps clear this surge in cytoplasmic calcium through an ER-resident calcium pump, SERCA, and a calcium-activated ion channel, Anoctamin 5 (ANO5). SERCA imports calcium into the ER, and ANO5 supports this by maintaining electroneutrality of the ER lumen through anion import. Preventing either of these transporter activities causes cytosolic calcium overload and disrupts PM repair (PMR). ANO5 deficit in limb girdle muscular dystrophy 2L (LGMD2L) patient cells compromises their cytosolic and ER calcium homeostasis. By generating a mouse model of LGMD2L, we find that PM injury causes cytosolic calcium overload and compromises the ability of ANO5-deficient myofibers to repair. Addressing calcium overload in ANO5-deficient myofibers enables them to repair, supporting the requirement of the ER in calcium homeostasis in injured cells and facilitating PMR., (© 2021 Chandra et al.)

Mitochondria are central to the health of eukaryotic cells. While commonly known for their bioenergetic role, mitochondria also function as signaling organelles that regulate cell stress responses capable of restoring homeostasis or leading the stressed cell to eventual death. Damage to the plasma membrane is a potentially fatal stressor incurred by all cells. Repairing plasma membrane damage requires cells to mount a rapid and localized response to injury. Accumulating evidence has identified a role for mitochondria as an important facilitator of this acute and localized repair response. However, as mitochondria are organized in a cell-wide, interconnected network, it is unclear how they collectively sense and respond to a focal injury. Here we will discuss how mitochondrial shape change is an integral part of this localized repair response. Mitochondrial fragmentation spatially restricts beneficial repair signaling, enabling a localized response to focal injury. Conservation of mitochondrial fragmentation in response to cell and tissue damage across species demonstrates that this is a universal pro-survival adaptation to injury and suggests that mitochondrial fragmentation may provide cells a mechanism to facilitate localized signaling in contexts beyond repairing plasma membrane injury., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Muscle cell plasma membrane is frequently damaged by mechanical activity, and its repair requires the membrane protein dysferlin. We previously identified that, similar to dysferlin deficit, lack of annexin A2 (AnxA2) also impairs repair of skeletal myofibers. Here, we have studied the mechanism of AnxA2-mediated muscle cell membrane repair in cultured muscle cells. We find that injury-triggered increase in cytosolic calcium causes AnxA2 to bind dysferlin and accumulate on dysferlin-containing vesicles as well as with dysferlin at the site of membrane injury. AnxA2 accumulates on the injured plasma membrane in cholesterol-rich lipid microdomains and requires Src kinase activity and the presence of cholesterol. Lack of AnxA2 and its failure to translocate to the plasma membrane, both prevent calcium-triggered dysferlin translocation to the plasma membrane and compromise repair of the injured plasma membrane. Our studies identify that Anx2 senses calcium increase and injury-triggered change in plasma membrane cholesterol to facilitate dysferlin delivery and repair of the injured plasma membrane.

Plasma membrane injury can cause lethal influx of calcium, but cells survive by mounting a polarized repair response targeted to the wound site. Mitochondrial signaling within seconds after injury enables this response. However, as mitochondria are distributed throughout the cell in an interconnected network, it is unclear how they generate a spatially restricted signal to repair the plasma membrane wound. Here we show that calcium influx and Drp1-mediated, rapid mitochondrial fission at the injury site help polarize the repair response. Fission of injury-proximal mitochondria allows for greater amplitude and duration of calcium increase in these mitochondria, allowing them to generate local redox signaling required for plasma membrane repair. Drp1 knockout cells and patient cells lacking the Drp1 adaptor protein MiD49 fail to undergo injury-triggered mitochondrial fission, preventing polarized mitochondrial calcium increase and plasma membrane repair. Although mitochondrial fission is considered to be an indicator of cell damage and death, our findings identify that mitochondrial fission generates localized signaling required for cell survival., (© 2020 Horn et al.)

The plasma membrane of eukaryotic cells forms the essential barrier to the extracellular environment, and thus plasma membrane disruptions pose a fatal threat to cells. Here, using invasive breast cancer cells we show that the Ca 2+ - and phospholipid-binding protein annexin A7 is part of the plasma membrane repair response by enabling assembly of the endosomal sorting complex required for transport (ESCRT) III. Following injury to the plasma membrane and Ca 2+ flux into the cytoplasm, annexin A7 forms a complex with apoptosis linked gene-2 (ALG-2) to facilitate proper recruitment and binding of ALG-2 and ALG-2-interacting protein X (ALIX) to the damaged membrane. ALG-2 and ALIX assemble the ESCRT III complex, which helps excise and shed the damaged portion of the plasma membrane during wound healing. Our results reveal a novel function of annexin A7 - enabling plasma membrane repair by regulating ESCRT III-mediated shedding of injured plasma membrane.

The plasma membrane forms the physical barrier between the cytoplasm and extracellular space, allowing for biochemical reactions necessary for life to occur. Plasma membrane damage needs to be rapidly repaired to avoid cell death. This relies upon the coordinated action of the machinery that polarizes the repair response to the site of injury, resulting in resealing of the damaged membrane and subsequent remodeling to return the injured plasma membrane to its pre-injury state. As lipids comprise the bulk of the plasma membrane, the acts of injury, resealing, and remodeling all directly impinge upon the plasma membrane lipids. In addition to their structural role in shaping the physical properties of the plasma membrane, lipids also play an important signaling role in maintaining plasma membrane integrity. While much attention has been paid to the involvement of proteins in the membrane repair pathway, the role of lipids in facilitating plasma membrane repair remains poorly studied. Here we will discuss the current knowledge of how lipids facilitate plasma membrane repair by regulating membrane structure and signaling to coordinate the repair response, and will briefly note how lipid involvement extends beyond plasma membrane repair to the tissue repair response., (© 2019 Elsevier Inc. All rights reserved.)

Plasma membrane forms the barrier between the cytoplasm and the environment. Cells constantly and selectively transport molecules across their plasma membrane without disrupting it. Any disruption in the plasma membrane compromises its selective permeability and is lethal, if not rapidly repaired. There is a growing understanding of the organelles, proteins, lipids, and small molecules that help cells signal and efficiently coordinate plasma membrane repair. This review aims to summarize how these subcellular responses are coordinated and how cellular signals generated due to plasma membrane injury interact with each other to spatially and temporally coordinate repair. With the involvement of calcium and redox signaling in single cell and tissue repair, we will discuss how these and other related signals extend from single cell repair to tissue level repair. These signals link repair processes that are activated immediately after plasma membrane injury with longer term processes regulating repair and regeneration of the damaged tissue. We propose that investigating cell and tissue repair as part of a continuum of wound repair mechanisms would be of value in treating degenerative diseases.

Dystrophin deficiency is the genetic basis for Duchenne muscular dystrophy (DMD), but the cellular basis of progressive myofiber death in DMD is not fully understood. Using two dystrophin-deficient mdx mouse models, we find that the mitochondrial dysfunction is among the earliest cellular deficits of mdx muscles. Mitochondria in dystrophic myofibers also respond poorly to sarcolemmal injury. These mitochondrial deficits reduce the ability of dystrophic muscle cell membranes to repair and are associated with a compensatory increase in dysferlin-mediated membrane repair proteins. Dysferlin deficit in mdx mice further compromises myofiber cell membrane repair and enhances the muscle pathology at an asymptomatic age for dysferlin-deficient mice. Restoring partial dystrophin expression by exon skipping improves mitochondrial function and offers potential to improve myofiber repair. These findings identify that mitochondrial deficit in muscular dystrophy compromises the repair of injured myofibers and show that this repair mechanism is distinct from and complimentary to the dysferlin-mediated repair of injured myofibers., Competing Interests: The authors declare no conflict of interest.

Mechanical activity of cells and the stress imposed on them by extracellular environment is a constant source of injury to the plasma membrane (PM). In invasive tumor cells, increased motility together with the harsh environment of the tumor stroma further increases the risk of PM injury. The impact of these stresses on tumor cell plasma membrane and mechanism by which tumor cells repair the PM damage are poorly understood. Ca(2+) entry through the injured PM initiates repair of the PM. Depending on the cell type, different organelles and proteins respond to this Ca(2+) entry and facilitate repair of the damaged plasma membrane. We recently identified that proteins expressed in various metastatic cancers including Ca(2+)-binding EF hand protein S100A11 and its binding partner annexin A2 are used by tumor cells for plasma membrane repair (PMR). Here we will discuss the involvement of S100, annexin proteins and their regulation of actin cytoskeleton, leading to PMR. Additionally, we will show that another S100 member--S100A4 accumulates at the injured PM. These findings reveal a new role for the S100 and annexin protein up regulation in metastatic cancers and identify these proteins and PMR as targets for treating metastatic cancers.

In muscle and other mechanically active tissue, cell membranes are constantly injured, and their repair depends on the injury-induced increase in cytosolic calcium. Here, we show that injury-triggered Ca(2+) increase results in assembly of ESCRT III and accessory proteins at the site of repair. This process is initiated by the calcium-binding protein-apoptosis-linked gene (ALG)-2. ALG-2 facilitates accumulation of ALG-2-interacting protein X (ALIX), ESCRT III and Vps4 complex at the injured cell membrane, which in turn results in cleavage and shedding of the damaged part of the cell membrane. Lack of ALG-2, ALIX or Vps4B each prevents shedding, and repair of the injured cell membrane. These results demonstrate Ca(2+)-dependent accumulation of ESCRT III-Vps4 complex following large focal injury to the cell membrane and identify the role of ALG-2 as the initiator of sequential ESCRT III-Vps4 complex assembly that facilitates scission and repair of the injured cell membrane.

Cell migration and invasion require increased plasma membrane dynamics and ability to navigate through dense stroma, thereby exposing plasma membrane to tremendous physical stress. Yet, it is largely unknown how metastatic cancer cells acquire an ability to cope with such stress. Here we show that S100A11, a calcium-binding protein upregulated in a variety of metastatic cancers, is essential for efficient plasma membrane repair and survival of highly motile cancer cells. Plasma membrane injury-induced entry of calcium into the cell triggers recruitment of S100A11 and Annexin A2 to the site of injury. We show that S100A11 in a complex with Annexin A2 helps reseal the plasma membrane by facilitating polymerization of cortical F-actin and excision of the damaged part of the plasma membrane. These data reveal plasma membrane repair in general and S100A11 and Annexin A2 in particular as new targets for the therapy of metastatic cancers.

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

Total internal reflection fluorescence (TIRF) microscopy is a high-contrast imaging technique suitable for observing biological events that occur on or near the cell membrane. The improved contrast is accomplished by restricting the thickness of the excitation field to over an order of a magnitude narrower than the z-resolution of an epi-fluorescence microscope. This technique also increases signal-to-noise, making it a valuable tool for imaging cellular events such as vesicles undergoing exocytosis or endocytosis, viral particle formation, cell signaling, and dynamics of membrane proteins. This protocol describes the basic procedures for setting up a through-the-objective TIRF illuminator and a prism-based TIRF illuminator. In addition, an alternate protocol for incorporating an automated deflection system into through-the-objective TIRF is given. This system can be used to decrease aberrations in the illumination field, to quickly switch between epi- and TIRF illumination, and to adjust the penetration depth during multicolor TIRF applications. In the commentary, a description of the total internal reflection phenomenon is given, critical parameters of a TIRF microscope are discussed, and technical challenges and considerations are reviewed.

The ability to sense and respond to the environment is a hallmark of living systems. These processes occur at the levels of the organism, cells and individual molecules. Sensing of extracellular changes could result in a structural or chemical alteration in a molecule, which could in turn trigger a cascade of intracellular signals or regulated trafficking of molecules at the cell surface. These and other such processes allow cells to sense and respond to environmental changes. Often, these changes and the responses to them are spatially and/or temporally localized, and visualization of such events necessitates the use of high-resolution imaging approaches. Here we discuss optical imaging approaches and tools for imaging individual events at the cell surface with improved speed and resolution.

The development of new imaging probes to noninvasively detect, diagnose and guide therapy for a large variety of diseases is needed. Quantum dots are highly fluorescent and photostable nanoparticles that are finding increasing applications as imaging probes. We have conjugated quantum dots to annexin A5, in order to develop a sensor for detecting apoptotic cells. The sensor specifically recognizes phosphatidylserine on the extracellular leaflet of the plasma membrane through the annexin A5. This quantum dot-based sensor allows increased sensitivity of detection and continuous monitoring of apoptotic cells over time periods that are longer than previously possible.

Similar to its role in secretory cells, calcium triggers exocytosis in nonsecretory cells. This calcium-dependent exocytosis is essential for repair of membrane ruptures. Using total internal reflection fluorescence microscopy, we observed that many organelles implicated in this process, including ER, post-Golgi vesicles, late endosomes, early endosomes, and lysosomes, were within 100 nm of the plasma membrane (in the evanescent field). However, an increase in cytosolic calcium led to exocytosis of only the lysosomes. The lysosomes that fused were predominantly predocked at the plasma membrane, indicating that calcium is primarily responsible for fusion and not recruitment of lysosomes to the cell surface.