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, whereas 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.

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.

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.

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.

Annexin A4 trimers are recruited by high membrane curvatures in giant plasma membrane vesicles.

The plasma membrane (PM) of eukaryotic cells consists of a crowded environment comprised of a high diversity of proteins in a complex lipid matrix. The lateral organization of membrane proteins in the PM is closely correlated with biological functions such as endocytosis, membrane budding and other processes which involve protein mediated shaping of the membrane into highly curved structures. Annexin A4 (ANXA4) is a prominent player in a number of biological functions including PM repair. Its binding to membranes is activated by Ca2+ influx and it is therefore rapidly recruited to the cell surface near rupture sites where Ca2+ influx takes place. However, the free edges near rupture sites can easily bend into complex curvatures and hence may accelerate recruitment of curvature sensing proteins to facilitate rapid membrane repair. To analyze the curvature sensing behavior of curvature inducing proteins in crowded membranes, we quantifify the affinity of ANXA4 monomers and trimers for high membrane curvatures by extracting membrane nanotubes from giant PM vesicles (GPMVs). ANXA4 is found to be a sensor of negative membrane curvatures. Multiscale simulations, in which we extract molecular information from atomistic scale simulations as input to our macroscopic scale simulations, furthermore predicted that ANXA4 trimers generate membrane curvature upon binding and have an affinity for highly curved membrane regions only within a well defined membrane curvature window. Our results indicate that curvature sensing and mobility of ANXA4 depend on the trimer structure of ANXA4 which could provide new biophysical insight into the role of ANXA4 in membrane repair and other biological processes.

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.

ErbB2-driven breast cancer cell invasion depends on a complex signaling network activating myeloid zinc finger-1-dependent cathepsin B expression.

Aberrant ErbB2 receptor tyrosine kinase activation in breast cancer is strongly linked to an invasive disease. The molecular basis of ErbB2-driven invasion is largely unknown. We show that cysteine cathepsins B and L are elevated in ErbB2 positive primary human breast cancer and function as effectors of ErbB2-induced invasion in vitro. We identify Cdc42-binding protein kinase beta, extracellular regulated kinase 2, p21-activated protein kinase 4, and protein kinase C alpha as essential mediators of ErbB2-induced cysteine cathepsin expression and breast cancer cell invasiveness. The identified signaling network activates the transcription of cathepsin B gene (CTSB) via myeloid zinc finger-1 transcription factor that binds to an ErbB2-responsive enhancer element in the first intron of CTSB. This work provides a model system for ErbB2-induced breast cancer cell invasiveness, reveals a signaling network that is crucial for invasion in vitro, and defines a specific role and targets for the identified serine-threonine kinases.

Using Liprotides to Deliver Cholesterol to the Plasma Membrane.

Cholesterol (chol) is important in all mammalian cells as a modulator of membrane fluidity. However, its low solubility is a challenge for controlled delivery to membranes. Here we introduce a new tool to deliver chol to membranes, namely, liprotides, i.e., protein-lipid complexes composed of a fatty acid core decorated with partially denatured protein. We focus on liprotides prepared by incubating Ca2+-depleted α-lactalbumin with oleic acid (OA) for 1 h at 20 °C (lip20) or 80 °C (lip80). The binding and membrane delivery properties of liprotides is compared to the widely chol transporter methyl-ß-cyclodextrin (mBCD). Both lip20 and lip80 increase the solubility of chol ~ 50% more than mBCD and deliver chol to membranes with comparable efficiency. Although OA is cytotoxic at high concentrations, its effects are counterbalanced by chol. Further, cytotoxicity is strongly reduced when OA is replaced by cis-palmitoleic acid or cis-vaccenic acid. This makes liprotides good tools to deliver chol to membranes and cells.

Annexins are instrumental for efficient plasma membrane repair in cancer cells.

Plasma membrane stress can cause damage to the plasma membrane, both when imposed by the extracellular environment and by enhanced oxidative stress. Cells cope with these injuries by rapidly activating their plasma membrane repair system, which is triggered by Ca(2+) influx at the wound site. The repair system is highly dynamic, depends on both lipid and protein components, and include cytoskeletal reorganization, membrane replacements, and membrane fusion events. Cancer cells experience enhanced membrane stress when navigating through dense extracellular matrix, which increases the frequency of membrane injuries. In addition, increased motility and oxidative stress further increase the risk of plasma membrane lesions. Cancer cells compensate by overexpressing Annexin proteins including Annexin A2 (ANXA2). Annexin family members can facilitate membrane fusion events and wound healing by binding to negatively charged phospholipids in the plasma membrane. Plasma membrane repair in cancer cells depends on ANXA2 protein, which is recruited to the wound site and forms a complex with the Ca(2+)-binding EF-hand protein S100A11. Here they regulate actin accumulation around the wound perimeter, which is required for wound closure. In this review, we will discuss the requirement for Annexins, S100 proteins and actin cytoskeleton in the plasma membrane repair response of cancer cells, which reveals a novel avenue for targeting metastatic cancers.

Annexins in plasma membrane repair.

Disruption of the plasma membrane poses deadly threat to eukaryotic cells and survival requires a rapid membrane repair system. Recent evidence reveal various plasma membrane repair mechanisms, which are required for cells to cope with membrane lesions including membrane fusion and replacement strategies, remodeling of cortical actin cytoskeleton and vesicle wound patching. Members of the annexin protein family, which are Ca2+-triggered phospholipid-binding proteins emerge as important components of the plasma membrane repair system. Here, we discuss the mechanisms of plasma membrane repair involving annexins spanning from yeast to human cancer cells.

Hsp70 stabilizes lysosomes and reverts Niemann-Pick disease-associated lysosomal pathology.

Heat shock protein 70 (Hsp70) is an evolutionarily highly conserved molecular chaperone that promotes the survival of stressed cells by inhibiting lysosomal membrane permeabilization, a hallmark of stress-induced cell death. Clues to its molecular mechanism of action may lay in the recently reported stress- and cancer-associated translocation of a small portion of Hsp70 to the lysosomal compartment. Here we show that Hsp70 stabilizes lysosomes by binding to an endolysosomal anionic phospholipid bis(monoacylglycero)phosphate (BMP), an essential co-factor for lysosomal sphingomyelin metabolism. In acidic environments Hsp70 binds with high affinity and specificity to BMP, thereby facilitating the BMP binding and activity of acid sphingomyelinase (ASM). The inhibition of the Hsp70-BMP interaction by BMP antibodies or a point mutation in Hsp70 (Trp90Phe), as well as the pharmacological and genetic inhibition of ASM, effectively revert the Hsp70-mediated stabilization of lysosomes. Notably, the reduced ASM activity in cells from patients with Niemann-Pick disease (NPD) A and B-severe lysosomal storage disorders caused by mutations in the sphingomyelin phosphodiesterase 1 gene (SMPD1) encoding for ASM-is also associated with a marked decrease in lysosomal stability, and this phenotype can be effectively corrected by treatment with recombinant Hsp70. Taken together, these data open exciting possibilities for the development of new treatments for lysosomal storage disorders and cancer with compounds that enter the lysosomal lumen by the endocytic delivery pathway.

Modulation of Annexin-Induced Membrane Curvature by Cholesterol and the Anionic Lipid Headgroup during Plasma Membrane Repair.

Annexins (ANXAs), calcium-sensitive phospholipid-binding proteins, are pivotal for cellular membrane repair, which is crucial for eukaryotic cell survival under membrane stress. With their unique trimeric arrangements and crystalline arrays on the membrane surface, ANXA4 and ANXA5 induce membrane curvature and rapidly orchestrate plasma membrane resealing. However, the influence of cholesterol and anionic lipid headgroups on annexin-induced membrane curvature remains poorly understood at the molecular level. Using all-atom molecular dynamics simulations, we measured the local curvature-induced underneath ANXA4 and ANXA5 monomers and trimers when they bind to lipid bilayers of distinct lipid compositions: PSPC (20% POPS, 80% POPC), PAPC (20% POPA, 80% POPC), and PSPCCHL (14% POPS, 56% POPC, 30% cholesterol). Laser injury experiments were conducted on MCF7 cells transfected to transiently express fluorescently labeled ANXA4 or ANXA5 to facilitate the examination of protein and lipid accumulation at the damage site. Annexin trimers induce higher curvature than monomers, particularly with cholesterol present. Annexin trimers induce similar curvatures on both PAPC and PSPC membranes. Notably, among monomers, ANXA5 induces the highest curvature on PAPC, suggesting more efficient recruitment of ANXA5 compared with ANXA4 in the early stages of membrane repair near a lesion. Laser injury experiments confirm rapid coaccumulation of phosphatidic acid lipids with ANXA4 and ANXA5 at repair sites, potentially enhancing the accumulation of annexins in the early stages of membrane repair.

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.

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.

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.

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.

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.].

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.