Shape of the membrane neck around a hole during plasma membrane repair.

Plasma membrane damage and rupture occurs frequently in cells and holes must be sealed rapidly to ensure homeostasis and cell survival. The membrane repair machinery is known to involve recruitment of curvature-inducing Annexin proteins, but the connection between membrane remodeling and hole closure is poorly described. The induction of curvature by repair proteins leads to the possible formation of a membrane neck around the hole as a key intermediate structure before sealing. We formulate a theoretical model of equilibrium neck shapes to examine the potential connection to a repair mechanism. Using variational calculus, the shape equations for the membrane near a hole are formulated and solved numerically. The system is described under a condition of fixed area and a shooting approach is applied to fulfill the boundary conditions at the free membrane edge. A state-diagram of neck shapes is produced describing the variation in neck morphology with respect to the membrane area. Two distinct types of necks are predicted, one with conformations curved beyond π existing at positive excess area while flat neck conformations (curved below π) have negative excess area. The results indicate that in cells, the supply of additional membrane area and a change in edge tension is linked to the formation of narrow and curved necks. Such necks may be susceptible to passive or actively induced membrane fission as a possible mechanism for hole sealing during membrane repair in cells.

Annexins-a family of proteins with distinctive tastes for cell signaling and membrane dynamics.

Annexins are cytosolic proteins with conserved three-dimensional structures that bind acidic phospholipids in cellular membranes at elevated Ca2+ levels. Through this they act as Ca2+-regulated membrane binding modules that organize membrane lipids, facilitating cellular membrane transport but also displaying extracellular activities. Recent discoveries highlight annexins as sensors and regulators of cellular and organismal stress, controlling inflammatory reactions in mammals, environmental stress in plants, and cellular responses to plasma membrane rupture. Here, we describe the role of annexins as Ca2+-regulated membrane binding modules that sense and respond to cellular stress and share our view on future research directions in the field.

Repurposing phenothiazines for cancer therapy: compromising membrane integrity in cancer cells.

The limitations of current cancer therapies, including the increasing prevalence of multidrug resistance, underscore the urgency for more effective treatments. One promising avenue lies in the repurposing of existing drugs. This review explores the impact of phenothiazines, primarily used as antipsychotic agents, on key mechanisms driving tumor growth and metastasis. The cationic and amphiphilic nature of phenothiazines allows interaction with the lipid bilayer of cellular membranes, resulting in alterations in lipid composition, modulation of calcium channels, fluidity, thinning, and integrity of the plasma membrane. This is especially significant in the setting of increased metabolic activity, a higher proliferative rate, and the invasiveness of cancer cells, which often rely on plasma membrane repair. Therefore, properties of phenothiazines such as compromising plasma membrane integrity and repair, disturbing calcium regulation, inducing cytosolic K-RAS accumulation, and sphingomyelin accumulation in the plasma membrane might counteract multidrug resistance by sensitizing cancer cells to membrane damage and chemotherapy. This review outlines a comprehensive overview of the mechanisms driving the anticancer activities of phenothiazines derivates such as trifluoperazine, prochlorperazine, chlorpromazine, promethazine, thioridazine, and fluphenazine. The repurposing potential of phenothiazines paves the way for novel approaches to improve future cancer treatment.

Engineering a membrane-binding protein to trimerize and induce high membrane curvature.

The annexins are a family of Ca2+-dependent peripheral membrane proteins. Several annexins are implicated in plasma membrane repair and are overexpressed in cancer cells. Annexin A4 (ANXA4) and annexin A5 (ANXA5) form trimers that induce high curvature on a membrane surface, a phenomenon deemed to accelerate membrane repair. Despite being highly homologous to ANXA4, annexin A3 (ANXA3) does not form trimers on the membrane surface. Using molecular dynamics simulations, we have reverse engineered an ANXA3-mutant to trimerize on the surface of the membrane and induce high curvature reminiscent of ANXA4. In addition, atomic force microscopy images show that, like ANXA4, the engineered protein forms crystalline arrays on a supported lipid membrane. Despite the trimer-forming and curvature-inducing properties of the engineered ANXA3, it does not accumulate near a membrane lesion in laser-punctured cells and is unable to repair the lesion. Our investigation provides insights into the factors that drive annexin-mediated membrane repair and shows that the membrane-repairing property of trimer-forming annexins also necessitates high membrane binding affinity, other than trimer formation and induction of negative membrane curvature.

Annexin A7 mediates lysosome repair independently of ESCRT-III.

Lysosomes are crucial organelles essential for various cellular processes, and any damage to them can severely compromise cell viability. This study uncovers a previously unrecognized function of the calcium- and phospholipid-binding protein Annexin A7 in lysosome repair, which operates independently of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Our research reveals that Annexin A7 plays a role in repairing damaged lysosomes, different from its role in repairing the plasma membrane, where it facilitates repair through the recruitment of ESCRT-III components. Notably, our findings strongly suggest that Annexin A7, like the ESCRT machinery, is dispensable for membrane contact site formation within the newly discovered phosphoinositide-initiated membrane tethering and lipid transport (PITT) pathway. Instead, we speculate that Annexin A7 is recruited to damaged lysosomes and promotes repair through its membrane curvature and cross-linking capabilities. Our findings provide new insights into the diverse mechanisms underlying lysosomal membrane repair and highlight the multifunctional role of Annexin A7 in membrane repair.

Interplay of membrane crosslinking and curvature induction by annexins.

Efficient plasma membrane repair (PMR) is required to repair damage sustained in the cellular life cycle. The annexin family of proteins, involved in PMR, are activated by Ca2+ influx from extracellular media at the site of injury. Mechanistic studies of the annexins have been overwhelmingly performed using a single annexin, despite the recruitment of multiple annexins to membrane damage sites in living cells. Hence, we investigate the effect of the presence of the crosslinking annexins, annexin A1, A2 and A6 (ANXA1, ANXA2 and ANXA6) on the membrane curvature induction of annexin A4 (ANXA4) in model membrane systems. Our data support a mechanistic model of PMR where ANXA4 induced membrane curvature and ANXA6 crosslinking promotes wound closure. The model now can be expanded to include ANXA1 and ANXA2 as specialist free edge membrane crosslinkers that act in concert with ANXA4 induced curvature and ANXA6 crosslinking.

Plasma Membrane Wounding and Repair Assays for Eukaryotic Cells.

Damage to the plasma membrane and loss of membrane integrity are detrimental to eukaryotic cells. It is, therefore, essential that cells possess an efficient membrane repair system to survive. However, the different cellular and molecular mechanisms behind plasma membrane repair have not been fully elucidated. Here, we present three complementary methods for plasma membrane wounding, and measurement of membrane repair and integrity. The first protocol is based on real time imaging of cell membrane repair kinetics in response to laser-induced injury. The second and third protocols are end point assays that provide a population-based measure of membrane integrity, after either mechanical injury by vortex mixing with glass beads, or by detergent-induced injury by digitonin in sublytic concentrations. The protocols can be applied to most adherent eukaryotic cells in culture, as well as cells in suspension.

Thermoplasmonic nano-rupture of cells reveals annexin V function in plasma membrane repair.

Maintaining the integrity of the cell plasma membrane (PM) is critical for the survival of cells. While an efficient PM repair machinery can aid survival of healthy cells by preventing influx of extracellular calcium, it can also constitute an obstacle in drug delivery and photothermal therapy. We show how nanoscopic holes can be created in a controlled fashion to the cell's plasma membrane, thus allowing identification of molecular components which have a pivotal role in PM repair. Cells are punctured by laser induced local heating of gold nanostructures at the cell surface which causes nano-ruptures in cellular PMs. Recruitment of annexin V near the hole is found to locally reshape the ruptured plasma membrane. Experiments using model membranes, containing recombinant annexin V, provide further biophysical insight into the ability of annexin V to reshape edges surrounding a membrane hole. The thermoplasmonic method provides a general strategy to monitor the response to nanoscopic injuries to the cell surface which offer new insight into how cells respond to photothermal treatment.

Filopodia rotate and coil by actively generating twist in their actin shaft.

Filopodia are actin-rich structures, present on the surface of eukaryotic cells. These structures play a pivotal role by allowing cells to explore their environment, generate mechanical forces or perform chemical signaling. Their complex dynamics includes buckling, pulling, length and shape changes. We show that filopodia additionally explore their 3D extracellular space by combining growth and shrinking with axial twisting and buckling. Importantly, the actin core inside filopodia performs a twisting or spinning motion which is observed for a range of cell types spanning from earliest development to highly differentiated tissue cells. Non-equilibrium physical modeling of actin and myosin confirm that twist is an emergent phenomenon of active filaments confined in a narrow channel which is supported by measured traction forces and helical buckles that can be ascribed to accumulation of sufficient twist. These results lead us to conclude that activity induced twisting of the actin shaft is a general mechanism underlying fundamental functions of filopodia.

Erratum: CHIP-dependent regulation of the actin cytoskeleton is linked to neuronal cell membrane integrity.

[This corrects the article DOI: 10.1016/j.isci.2021.102878.].

A novel NINJ1-mediated regulatory step is essential for active membrane rupture and common to different cell death pathways.

Plasma membrane rupture (PMR), the final event in lytic cell death that is in part responsible for the release of pro-inflammatory signals, was believed to be a passive event that followed osmotic swelling. Kayagaki et al. 1 have discovered that PMR is, in fact, mediated by ninjurin-1 (NINJ1), adding a novel regulatory step that is conserved across different types of lytic cell death, such as pyroptosis, necroptosis, and apoptosis. PMR is dependent on NINJ1 oligomerization, which is mediated by its highly conserved putative N-terminal α-helix. In vivo data suggest that the NINJ1-dependent secretome that is released upon PMR is likely to modulate antimicrobial host defense, suggesting this additional regulatory step also has physiological relevance.

Actin Cytoskeletal Dynamics in Single-Cell Wound Repair.

The plasma membrane protects the eukaryotic cell from its surroundings and is essential for cell viability; thus, it is crucial that membrane disruptions are repaired quickly to prevent immediate dyshomeostasis and cell death. Accordingly, cells have developed efficient repair mechanisms to rapidly reseal ruptures and reestablish membrane integrity. The cortical actin cytoskeleton plays an instrumental role in both plasma membrane resealing and restructuring in response to damage. Actin directly aids membrane repair or indirectly assists auxiliary repair mechanisms. Studies investigating single-cell wound repair have often focused on the recruitment and activation of specialized repair machinery, despite the undeniable need for rapid and dynamic cortical actin modulation; thus, the role of the cortical actin cytoskeleton during wound repair has received limited attention. This review aims to provide a comprehensive overview of membrane repair mechanisms directly or indirectly involving cortical actin cytoskeletal remodeling.

Short-term transcriptomic response to plasma membrane injury.

Plasma membrane repair mechanisms are activated within seconds post-injury to promote rapid membrane resealing in eukaryotic cells and prevent cell death. However, less is known about the regeneration phase that follows and how cells respond to injury in the short-term. Here, we provide a genome-wide study into the mRNA expression profile of MCF-7 breast cancer cells exposed to injury by digitonin, a mild non-ionic detergent that permeabilizes the plasma membrane. We focused on the early transcriptional signature and found a time-dependent increase in the number of differentially expressed (> twofold, P < 0.05) genes (34, 114 and 236 genes at 20-, 40- and 60-min post-injury, respectively). Pathway analysis highlighted a robust and gradual three-part transcriptional response: (1) prompt activation of immediate-early response genes, (2) activation of specific MAPK cascades and (3) induction of inflammatory and immune pathways. Therefore, plasma membrane injury triggers a rapid and strong stress and immunogenic response. Our meta-analysis suggests that this is a conserved transcriptome response to plasma membrane injury across different cell and injury types. Taken together, our study shows that injury has profound effects on the transcriptome of wounded cells in the regeneration phase (subsequent to membrane resealing), which is likely to influence cellular status and has been previously overlooked.

CHIP-dependent regulation of the actin cytoskeleton is linked to neuronal cell membrane integrity.

CHIP is an E3-ubiquitin ligase that contributes to healthy aging and has been characterized as neuroprotective. To elucidate dominant CHIP-dependent changes in protein steady-state levels in a patient-derived human neuronal model, CHIP function was ablated using gene-editing and an unbiased proteomic analysis conducted to compare knock-out and wild-type isogenic induced pluripotent stem cell (iPSC)-derived cortical neurons. Rather than a broad effect on protein homeostasis, loss of CHIP function impacted on a focused cohort of proteins from actin cytoskeleton signaling and membrane integrity networks. In support of the proteomics, CHIP knockout cells had enhanced sensitivity to induced membrane damage. We conclude that the major readout of CHIP function in cortical neurons derived from iPSC of a patient with elevate α-synuclein, Parkinson's disease and dementia, is the modulation of substrates involved in maintaining cellular "health". Thus, regulation of the actin cytoskeletal and membrane integrity likely contributes to the neuroprotective function(s) of CHIP.

Restructuring of the plasma membrane upon damage by LC3-associated macropinocytosis.

The plasma membrane shapes and protects the eukaryotic cell from its surroundings and is crucial for cell life. Although initial repair mechanisms to reseal injured membranes are well established, less is known about how cells restructure damaged membranes in the aftermath to restore homeostasis. Here, we show that cells respond to plasma membrane injury by activating proteins associated with macropinocytosis specifically at the damaged membrane. Subsequent to membrane resealing, cells form large macropinosomes originating from the repair site, which eventually become positive for autophagy-related LC3B protein. This process occurs independent of ULK1, ATG13, and WIPI2 but dependent on ATG7, p62, and Rubicon. Internalized macropinosomes shrink in the cytoplasm, likely by osmotic draining, and eventually fuse with lysosomes. We propose that a form of macropinocytosis coupled to noncanonical autophagy, which we term LC3-associated macropinocytosis (LAM) functions to remove damaged material from the plasma membrane and restore membrane integrity upon injury.

Phenothiazines alter plasma membrane properties and sensitize cancer cells to injury by inhibiting annexin-mediated repair.

Repair of damaged plasma membrane in eukaryotic cells is largely dependent on the binding of annexin repair proteins to phospholipids. Changing the biophysical properties of the plasma membrane may provide means to compromise annexin-mediated repair and sensitize cells to injury. Since, cancer cells experience heightened membrane stress and are more dependent on efficient plasma membrane repair, inhibiting repair may provide approaches to sensitize cancer cells to plasma membrane damage and cell death. Here, we show that derivatives of phenothiazines, which have widespread use in the fields of psychiatry and allergy treatment, strongly sensitize cancer cells to mechanical-, chemical-, and heat-induced injury by inhibiting annexin-mediated plasma membrane repair. Using a combination of cell biology, biophysics, and computer simulations, we show that trifluoperazine acts by thinning the membrane bilayer, making it more fragile and prone to ruptures. Secondly, it decreases annexin binding by compromising the lateral diffusion of phosphatidylserine, inhibiting the ability of annexins to curve and shape membranes, which is essential for their function in plasma membrane repair. Our results reveal a novel avenue to target cancer cells by compromising plasma membrane repair in combination with noninvasive approaches that induce membrane injuries.

Simultaneous membrane binding of Annexin A4 and A5 suppresses 2D lattice formation while maintaining curvature induction.

HYPOTHESIS: Annexin A4 and A5 (ANXA4, ANXA5), both shown to be required for efficient plasma membrane repair (PMR) in living cells, bind as trimers to anionic membranes in the presence of calcium. Both annexins induce membrane curvature and self-assemble into crystal arrays on membranes, observations that have been associated with PMR. However, in-vitro studies of annexins have traditionally been performed using single annexins, despite the recruitment of multiple annexins to the damage site in cells. Hence, we study the potential cooperativity of ANXA4 and ANXA5 during membrane binding. EXPERIMENTS: Laser injury experiments were performed on MCF7 cells transfected to transiently express labelled ANXA4 and ANXA5 to study the localization of the proteins at the damage site. Using free-edged DOPC/DOPS (9:1) membranes we investigated the annexin-induced membrane rolling by fluorescence microscopy and the lateral arrangement of annexin trimers on the membrane surface by atomic force microscopy (AFM). FINDING: ANXA4 and ANXA5 colocalise at the damage site of MCF7 cells during repair. A (1:1) mixture of ANXA4 and ANXA5 induces membrane rolling with a time constant intermediate between the value for the pure annexins. While binding of the pure annexins creates crystal lattices, the (1:1) mixture generates a random arrangement of trimers. Thus, curvature induction remains as a functional property of annexin mixtures in PMR rather than crystal formation.

Annexins A1 and A2 Accumulate and Are Immobilized at Cross-Linked Membrane-Membrane Interfaces.

Rapid membrane repair is required to ensure cell survival after rupture of the plasma membrane. The annexin family of proteins is involved in plasma membrane repair (PMR) and is activated by the influx of Ca2+ from the extracellular medium at the site of injury. Annexins A1 and A2 (ANXA1 and ANXA2, respectively) are structurally similar and bind to negatively charged phosphatidylserine (PS) to induce membrane cross-linking and to promote fusion, which are both essential processes that occur during membrane repair. The degree of annexin accumulation and the annexin mobility at cross-linked membranes are important aspects of ANXA1 and ANXA2 function in repair. Here, we quantify ANXA1- and ANXA2-induced membrane cross-linking between giant unilamellar vesicles (GUVs). Time-lapse measurements show that ANXA1 and ANXA2 can induce membrane cross-linking on a time scale compatible with PMR. Cross-linked membrane-membrane interfaces between the GUVs persist in time without fusion, and quantification of confocal microscopy images demonstrates that ANXA1, ANXA2, and, to a lesser extent, PS lipids accumulate at the double membrane interface. Fluorescence recovery after photobleaching shows that the annexins are fully immobilized at the double membrane interface, whereas PS lipids display a 75% decrease in mobility. In addition, the complete immobilization of annexins between two membranes indicates a high degree of network formation between annexins, suggesting that membrane cross-linking is mainly driven by protein-protein interactions.