Confocal interferometric scattering microscopy reveals 3D nanoscopic structure and dynamics in live cells.

Bright-field light microscopy and related phase-sensitive techniques play an important role in life sciences because they provide facile and label-free insights into biological specimens. However, lack of three-dimensional imaging and low sensitivity to nanoscopic features hamper their application in many high-end quantitative studies. Here, we demonstrate that interferometric scattering (iSCAT) microscopy operated in the confocal mode provides unique label-free solutions for live-cell studies. We reveal the nanometric topography of the nuclear envelope, quantify the dynamics of the endoplasmic reticulum, detect single microtubules, and map nanoscopic diffusion of clathrin-coated pits undergoing endocytosis. Furthermore, we introduce the combination of confocal and wide-field iSCAT modalities for simultaneous imaging of cellular structures and high-speed tracking of nanoscopic entities such as single SARS-CoV-2 virions. We benchmark our findings against simultaneously acquired fluorescence images. Confocal iSCAT can be readily implemented as an additional contrast mechanism in existing laser scanning microscopes. The method is ideally suited for live studies on primary cells that face labeling challenges and for very long measurements beyond photobleaching times.

Corneal confocal microscopy identifies corneal nerve fibre loss and increased dendritic cells in patients with long COVID.

BACKGROUND/AIMS: Long COVID is characterised by a range of potentially debilitating symptoms which develop in at least 10% of people who have recovered from acute SARS-CoV-2 infection. This study has quantified corneal sub-basal nerve plexus morphology and dendritic cell (DC) density in patients with and without long COVID. METHODS: Forty subjects who had recovered from COVID-19 and 30 control participants were included in this cross-sectional comparative study undertaken at a university hospital. All patients underwent assessment with the National Institute for Health and Care Excellence (NICE) long COVID, Douleur Neuropathique 4 (DN4) and Fibromyalgia questionnaires, and corneal confocal microscopy (CCM) to quantify corneal nerve fibre density (CNFD), corneal nerve branch density (CNBD), corneal nerve fibre length (CNFL), and total, mature and immature DC density. RESULTS: The mean time after the diagnosis of COVID-19 was 3.7±1.5 months. Patients with neurological symptoms 4 weeks after acute COVID-19 had a lower CNFD (p=0.032), CNBD (p=0.020), and CNFL (p=0.012), and increased DC density (p=0.046) compared with controls, while patients without neurological symptoms had comparable corneal nerve parameters, but increased DC density (p=0.003). There were significant correlations between the total score on the NICE long COVID questionnaire at 4 and 12 weeks with CNFD (ρ=-0.436; p=0.005, ρ=-0.387; p=0.038, respectively) and CNFL (ρ=-0.404; p=0.010, ρ=-0.412; p=0.026, respectively). CONCLUSION: Corneal confocal microscopy identifies corneal small nerve fibre loss and increased DCs in patients with long COVID, especially those with neurological symptoms. CCM could be used to objectively identify patients with long COVID.

Corneal Cellular and Neuroinflammatory Changes After SARS-CoV-2 Infection.

PURPOSE: The purpose of this study was to evaluate corneal cellular and ultrastructural changes and to quantify the neuroinflammatory process in patients after mild severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: Thirty patients after SARS-CoV-2 infection and 41 age-matched controls were examined. All subjects underwent in vivo confocal microscopy of the corneal cell layers and subbasal nerve fibers with the Heidelberg Retina Tomograph II. Semiautomated analysis of basal epithelial, anterior and posterior stromal keratocyte, and endothelial cell density was performed. Dendritic cell (DC) density and area were also calculated, and subbasal nerve plexus morphology was analyzed. RESULTS: The posterior stromal keratocyte density was significantly lower in patients after SARS-CoV-2 infection ( P = 0.0006). DC density in the central cornea was significantly higher in patients after SARS-CoV-2 infection ( P = 0.0004). There was a significant difference in the DC area between the 2 groups ( P < 0.0001). Significantly altered subbasal nerve fiber morphology was detected in patients after SARS-CoV-2 infection compared with healthy volunteers ( P < 0.05). CONCLUSIONS: Corneal cellular and ultrastructural changes demonstrated in this study suggest neuroinflammatory consequences of COVID-19 in the cornea in the absence of ophthalmoscopic alterations.

Blood identified and quantified in formalin fixed paraffin embedded lung sections using eosin fluorescence.

Eosin Y is a common stain in histology. Although usually used for colourimetric imaging where the dye is used to stain pink/red a range of structures in the tissue, Eosin Y is also a fluorochrome, and has been used in this manner for decades. In this study our aim was to investigate the fluorescence properties of the dye to enable quantification of structures within formalin-fixed paraffin-embedded (FFPE) tissue sections. To do this, FFPE sections of hamster tissue were prepared with haematoxylin and eosin Y dyes. Spectral detection on a confocal laser scanning microscope was used to obtain the fluorescence emission spectra of the eosin Y under blue light. This showed clear spectral differences between the red blood cells and congealed blood, compared to the rest of the section. The spectra were so distinct that it was possible to discern these in fluorescence and multi-photon microscopy. An image analysis algorithm was used to quantify the red blood cells. These analyses could have broad applications in histopathology where differentiation is required, such as the analysis of clotting disorders to haemorrhage or damage from infectious disease.

Novel aspects of Raman spectroscopy in skin research.

The analytical technology of Raman spectroscopy has an almost 100-year history. During this period, many modifications and developments happened in the method like discovery of laser, improvements in optical elements and sensitivity of spectrometer and also more advanced light detection systems. Many types of the innovative techniques appeared (e.g. Transmittance Raman spectroscopy, Coherent Raman Scattering microscopy, Surface-Enhanced Raman scattering and Confocal Raman spectroscopy/microscopy). This review article gives a short description about these different Raman techniques and their possible applications. Then, a short statistical part is coming about the appearance of Raman spectroscopy in the scientific literature from the beginnings to these days. The third part of the paper shows the main application options of the technique (especially confocal Raman spectroscopy) in skin research, including skin composition analysis, drug penetration monitoring and analysis, diagnostic utilizations in dermatology and cosmeto-scientific applications. At the end, the possible role of artificial intelligence in Raman data analysis and the regulatory aspect of these techniques in dermatology are briefly summarized. For the future of Raman Spectroscopy, increasing clinical relevance and in vivo applications can be predicted with spreading of non-destructive methods and appearance with the most advanced instruments with rapid analysis time.

A novel liquid biopsy-based approach for highly specific cancer diagnostics: mitigating false responses in assaying patient plasma-derived circulating microRNAs through combined SERS and plasmon-enhanced fluorescence analyses.

Studies have shown that microRNAs, which are small noncoding RNAs, hold tremendous promise as next-generation circulating biomarkers for early cancer detection via liquid biopsies. A novel, solid-state nanoplasmonic sensor capable of assaying circulating microRNAs through a combined surface-enhanced Raman scattering (SERS) and plasmon-enhanced fluorescence (PEF) approach has been developed. Here, the unique localized surface plasmon resonance properties of chemically-synthesized gold triangular nanoprisms (Au TNPs) are utilized to create large SERS and PEF enhancements. With careful modification to the surface of Au TNPs, this sensing approach is capable of quantifying circulating microRNAs at femtogram/microliter concentrations. Uniquely, the multimodal analytical methods mitigate both false positive and false negative responses and demonstrate the high stability of our sensors within bodily fluids. As a proof of concept, microRNA-10b and microRNA-96 were directly assayed from the plasma of six bladder cancer patients. Results show potential for a highly specific liquid biopsy method that could be used in point-of-care clinical diagnostics to increase early cancer detection or any other diseases including SARS-CoV-2 in which RNAs can be used as biomarkers.

Highly Efficient SARS-CoV-2 Infection of Human Cardiomyocytes: Spike Protein-Mediated Cell Fusion and Its Inhibition.

Severe cardiovascular complications can occur in coronavirus disease of 2019 (COVID-19) patients. Cardiac damage is attributed mostly to the aberrant host response to acute respiratory infection. However, direct infection of cardiac tissue by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also occurs. We examined here the cardiac tropism of SARS-CoV-2 in spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These cardiomyocytes express the angiotensin-converting enzyme 2 (ACE2) receptor but not the transmembrane protease serine 2 (TMPRSS2) that mediates spike protein cleavage in the lungs. Nevertheless, SARS-CoV-2 infection of hiPSC-CMs was prolific; viral transcripts accounted for about 88% of total mRNA. In the cytoplasm of infected hiPSC-CMs, smooth-walled exocytic vesicles contained numerous 65- to 90-nm particles with canonical ribonucleocapsid structures, and virus-like particles with knob-like spikes covered the cell surface. To better understand how SARS-CoV-2 spreads in hiPSC-CMs, we engineered an expression vector coding for the spike protein with a monomeric emerald-green fluorescent protein fused to its cytoplasmic tail (S-mEm). Proteolytic processing of S-mEm and the parental spike were equivalent. Live cell imaging tracked spread of S-mEm cell-to-cell and documented formation of syncytia. A cell-permeable, peptide-based molecule that blocks the catalytic site of furin and furin-like proteases abolished cell fusion. A spike mutant with the single amino acid change R682S that disrupts the multibasic furin cleavage motif was fusion inactive. Thus, SARS-CoV-2 replicates efficiently in hiPSC-CMs and furin, and/or furin-like-protease activation of its spike protein is required for fusion-based cytopathology. This hiPSC-CM platform enables target-based drug discovery in cardiac COVID-19. IMPORTANCE Cardiac complications frequently observed in COVID-19 patients are tentatively attributed to systemic inflammation and thrombosis, but viral replication has occasionally been confirmed in cardiac tissue autopsy materials. We developed an in vitro model of SARS-CoV-2 spread in myocardium using induced pluripotent stem cell-derived cardiomyocytes. In these highly differentiated cells, viral transcription levels exceeded those previously documented in permissive transformed cell lines. To better understand the mechanisms of SARS-CoV-2 spread, we expressed a fluorescent version of its spike protein that allowed us to characterize a fusion-based cytopathic effect. A mutant of the spike protein with a single amino acid mutation in the furin/furin-like protease cleavage site lost cytopathic function. Of note, the fusion activities of the spike protein of other coronaviruses correlated with the level of cardiovascular complications observed in infections with the respective viruses. These data indicate that SARS-CoV-2 may cause cardiac damage by fusing cardiomyocytes.

Synthetic virions reveal fatty acid-coupled adaptive immunogenicity of SARS-CoV-2 spike glycoprotein.

SARS-CoV-2 infection is a major global public health concern with incompletely understood pathogenesis. The SARS-CoV-2 spike (S) glycoprotein comprises a highly conserved free fatty acid binding pocket (FABP) with unknown function and evolutionary selection advantage1,2. Deciphering FABP impact on COVID-19 progression is challenged by the heterogenous nature and large molecular variability of live virus. Here we create synthetic minimal virions (MiniVs) of wild-type and mutant SARS-CoV-2 with precise molecular composition and programmable complexity by bottom-up assembly. MiniV-based systematic assessment of S free fatty acid (FFA) binding reveals that FABP functions as an allosteric regulatory site enabling adaptation of SARS-CoV-2 immunogenicity to inflammation states via binding of pro-inflammatory FFAs. This is achieved by regulation of the S open-to-close equilibrium and the exposure of both, the receptor binding domain (RBD) and the SARS-CoV-2 RGD motif that is responsible for integrin co-receptor engagement. We find that the FDA-approved drugs vitamin K and dexamethasone modulate S-based cell binding in an FABP-like manner. In inflammatory FFA environments, neutralizing immunoglobulins from human convalescent COVID-19 donors lose neutralization activity. Empowered by our MiniV technology, we suggest a conserved mechanism by which SARS-CoV-2 dynamically couples its immunogenicity to the host immune response.

Emerging Phospholipid Nanobiomaterials for Biomedical Applications to Lab-on-a-Chip, Drug Delivery, and Cellular Engineering.

The design of advanced nanobiomaterials to improve analytical accuracy and therapeutic efficacy has become an important prerequisite for the development of innovative nanomedicines. Recently, phospholipid nanobiomaterials including 2-methacryloyloxyethyl phosphorylcholine (MPC) have attracted great attention with remarkable characteristics such as resistance to nonspecific protein adsorption and cell adhesion for various biomedical applications. Despite many recent reports, there is a lack of comprehensive review on the phospholipid nanobiomaterials from synthesis to diagnostic and therapeutic applications. Here, we review the synthesis and characterization of phospholipid nanobiomaterials focusing on MPC polymers and highlight their attractive potentials for applications in micro/nanofabricated fluidic devices, biosensors, lab-on-a-chip, drug delivery systems (DDSs), COVID-19 potential usages for early diagnosis and even treatment, and artificial extracellular matrix scaffolds for cellular engineering.

Small fiber neuropathy in the cornea of Covid-19 patients associated with the generation of ocular surface disease.

PURPOSE: To describe the association between Sars-CoV-2 infection and small fiber neuropathy in the cornea identified by in vivo corneal confocal microscopy. METHODS: Twenty-three patients who had overcome COVID-19 were recruited to this observational retrospective study. Forty-six uninfected volunteers were also recruited and studied as a control group. All subjects were examined under in vivo confocal microscopy to obtain images of corneal subbasal nerve fibers in order to study the presence of neuroma-like structures, axonal beadings and dendritic cells. The Ocular Surface Disease Index (OSDI) questionnaire and Schirmer tear test were used as indicators of Dry Eye Disease (DED) and ocular surface pathology. RESULTS: Twenty-one patients (91.31%) presented alterations of the corneal subbasal plexus and corneal tissue consistent with small fiber neuropathy. Images from healthy subjects did not indicate significant nerve fiber or corneal tissue damage. Eight patients reported increased sensations of ocular dryness after COVID-19 infection and had positive DED indicators. Beaded axons were found in 82.60% of cases, mainly in patients reporting ocular irritation symptoms. Neuroma-like images were found in 65.22% patients, more frequently in those with OSDI scores >13. Dendritic cells were found in 69.56% of patients and were more frequent in younger asymptomatic patients. The presence of morphological alterations in patients up to 10 months after recovering from Sars-CoV-2 infection points to the chronic nature of the neuropathy. CONCLUSIONS: Sars-CoV-2 infection may be inducing small fiber neuropathy in the ocular surface, sharing symptomatology and morphological landmarks with DED and diabetic neuropathy.

Ultrasensitive Detection of COVID-19 Causative Virus (SARS-CoV-2) Spike Protein Using Laser Induced Graphene Field-Effect Transistor.

COVID-19 is a highly contagious human infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the war with the virus is still underway. Since no specific drugs have been made available yet and there is an imbalance between supply and demand for vaccines, early diagnosis and isolation are essential to control the outbreak. Current nucleic acid testing methods require high sample quality and laboratory conditions, which cannot meet flexible applications. Here, we report a laser-induced graphene field-effect transistor (LIG-FET) for detecting SARS-CoV-2. The FET was manufactured by different reduction degree LIG, with an oyster reef-like porous graphene channel to enrich the binding point between the virus protein and sensing area. After immobilizing specific antibodies in the channel, the FET can detect the SARS-CoV-2 spike protein in 15 min at a concentration of 1 pg/mL in phosphate-buffered saline (PBS) and 1 ng/mL in human serum. In addition, the sensor shows great specificity to the spike protein of SARS-CoV-2. Our sensors can realize fast production for COVID-19 rapid testing, as each LIG-FET can be fabricated by a laser platform in seconds. It is the first time that LIG has realized a virus sensing FET without any sample pretreatment or labeling, which paves the way for low-cost and rapid detection of COVID-19.

Mammalian hybrid pre-autophagosomal structure HyPAS generates autophagosomes.

The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2 nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.

Characterisation of factors contributing to the performance of nonwoven fibrous matrices as substrates for adenovirus vectored vaccine stabilisation.

Adenovirus vectors offer a platform technology for vaccine development. The value of the platform has been proven during the COVID-19 pandemic. Although good stability at 2-8 °C is an advantage of the platform, non-cold-chain distribution would have substantial advantages, in particular in low-income countries. We have previously reported a novel, potentially less expensive thermostabilisation approach using a combination of simple sugars and glass micro-fibrous matrix, achieving excellent recovery of adenovirus-vectored vaccines after storage at temperatures as high as 45 °C. This matrix is, however, prone to fragmentation and so not suitable for clinical translation. Here, we report an investigation of alternative fibrous matrices which might be suitable for clinical use. A number of commercially-available matrices permitted good protein recovery, quality of sugar glass and moisture content of the dried product but did not achieve the thermostabilisation performance of the original glass fibre matrix. We therefore further investigated physical and chemical characteristics of the glass fibre matrix and its components, finding that the polyvinyl alcohol present in the glass fibre matrix assists vaccine stability. This finding enabled us to identify a potentially biocompatible matrix with encouraging performance. We discuss remaining challenges for transfer of the technology into clinical use, including reliability of process performance.

DNA-Directed Patterning for Versatile Validation and Characterization of a Lipid-Based Nanoparticle Model of SARS-CoV-2.

Lipid-based nanoparticles have been applied extensively in drug delivery and vaccine strategies and are finding diverse applications in the coronavirus disease 2019 (COVID-19) pandemic-from vaccine-component encapsulation to modeling the virus, itself. High-throughput, highly flexible methods for characterization are of great benefit to the development of liposomes featuring surface proteins. DNA-directed patterning is one such method that offers versatility in immobilizing and segregating lipid-based nanoparticles for subsequent analysis. Here, oligonucleotides are selectively conjugated onto a glass substrate and then hybridized to complementary oligonucleotides tagged to liposomes, patterning them with great control and precision. The power of DNA-directed patterning is demonstrated by characterizing a novel recapitulative lipid-based nanoparticle model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-S-liposomes-that presents the SARS-CoV-2 spike (S) protein on its surface. Patterning a mixture of S-liposomes and liposomes that display the tetraspanin CD63 to discrete regions of a substrate shows that angiotensin-converting enzyme 2 (ACE2) specifically binds to S-liposomes. Subsequent introduction of S-liposomes to ACE2-expressing cells tests the biological function of S-liposomes and shows agreement with DNA-directed patterning-based assays. Finally, multiplexed patterning of S-liposomes verifies the performance of commercially available neutralizing antibodies against the two S variants. Overall, DNA-directed patterning enables a wide variety of custom assays for the characterization of any lipid-based nanoparticle.

Neutrophil Extracellular Traps Contribute to COVID-19 Hyperinflammation and Humoral Autoimmunity.

The coronavirus disease 2019 (COVID-19) is related to enhanced production of NETs, and autoimmune/autoinflammatory phenomena. We evaluated the proportion of low-density granulocytes (LDG) by flow cytometry, and their capacity to produce NETs was compared with that of conventional neutrophils. NETs and their protein cargo were quantified by confocal microscopy and ELISA. Antinuclear antibodies (ANA), anti-neutrophil cytoplasmic antibodies (ANCA) and the degradation capacity of NETs were addressed in serum. MILLIPLEX assay was used to assess the cytokine levels in macrophages' supernatant and serum. We found a higher proportion of LDG in severe and critical COVID-19 which correlated with severity and inflammatory markers. Severe/critical COVID-19 patients had higher plasmatic NE, LL-37 and HMGB1-DNA complexes, whilst ISG-15-DNA complexes were lower in severe patients. Sera from severe/critical COVID-19 patients had lower degradation capacity of NETs, which was reverted after adding hrDNase. Anti-NET antibodies were found in COVID-19, which correlated with ANA and ANCA positivity. NET stimuli enhanced the secretion of cytokines in macrophages. This study unveils the role of COVID-19 NETs as inducers of pro-inflammatory and autoimmune responses. The deficient degradation capacity of NETs may contribute to the accumulation of these structures and anti-NET antibodies are related to the presence of autoantibodies.

[First results of clinical diagnostic analysis of post-COVID peripheral neuropathy]. / Pervye rezul'taty kliniko-diagnosticheskogo analiza postkovidnoi perifericheskoi nevropatii.

This analysis of ocular manifestations of the COVID-19 that can occur both in the acute and the post-COVID period focuses on pathological changes associated with hypercoagulation: thrombosis of the central retinal vein, occlusion of the central retinal artery, and decreased peripapillary perfusion. Potential changes in corneal nerve fibers (CNF) associated with coronavirus infection remain poorly understood. Meanwhile, previous studies have shown the possibility of using CNF as biomarkers of polyneuropathy (PNP). PURPOSE: To study clinical and diagnostic features of post-COVID PNP. MATERIAL AND METHODS: We examined 9 female patients aged 32 to 51 years who had suffered an infection caused by SARS-CoV-2 of varying severity. The patients were divided into two groups: the first consisted of 6 patients with clinical manifestations of PNP in the form of neuropathic pain, the second - 3 patients without clinical manifestations. Patients of group 1 underwent comprehensive examination including neurological examination, confocal microscopy of the cornea (in vivo CM), electroneuromyography (ENMG) of the nerves of the upper and lower extremities, quantitative sensory testing in the zone of painful sensations, while patients of group 2 only underwent in vivo CM. RESULTS: All patients of the first group, within 5-40 days after the onset of infection caused by SARS-CoV-2, exhibited symptoms of damage to various parts of the nervous system (peripheral nerves, thoracic spinal roots, trigeminal nerve). The main - and in most cases the only - symptom of the disease was neuropathic pain syndrome. In Guillain-Barré syndrome, for the first time using in vivo CM, the following changes were revealed: enlargement of in Langerhans cells, shortening of CNF and their processes, presence of neuromas. CONCLUSION: The standard neurological examination of patients with post-COVID neuropathy should be supplemented with special diagnostic methods for assessment of structural and functional state of the peripheral nerves. The in vivo CM method is capable of non-invasive assessment of changes in thin CNF and, with accumulation of empirical data, can be included in the algorithm for diagnosing post-COVID PNP.

A digital single-molecule nanopillar SERS platform for predicting and monitoring immune toxicities in immunotherapy.

The introduction of immune checkpoint inhibitors has demonstrated significant improvements in survival for subsets of cancer patients. However, they carry significant and sometimes life-threatening toxicities. Prompt prediction and monitoring of immune toxicities have the potential to maximise the benefits of immune checkpoint therapy. Herein, we develop a digital nanopillar SERS platform that achieves real-time single cytokine counting and enables dynamic tracking of immune toxicities in cancer patients receiving immune checkpoint inhibitor treatment - broader applications are anticipated in other disease indications. By analysing four prospective cytokine biomarkers that initiate inflammatory responses, the digital nanopillar SERS assay achieves both highly specific and highly sensitive cytokine detection down to attomolar level. Significantly, we report the capability of the assay to longitudinally monitor 10 melanoma patients during immune inhibitor blockade treatment. Here, we show that elevated cytokine concentrations predict for higher risk of developing severe immune toxicities in our pilot cohort of patients.

Probe-based confocal laser endomicroscopy in COVID-19.

Probe-based confocal laser endomicroscopy (pCLE) is a method that produces microscopic imaging of a lung tissue during bronchoscopy. We report a case ot a patient with negative nasopharyngeal swabs and suspected lung cancer who underwent pCLE. The diagnosis of COVID-19 was confirmed by PCR analyses of lavage fluid and transbrohial biopsy. The pCLE image shows density of alveolar thickened fibres, disorganization of elastin network, and multiple large drops of intraalveolar secretions. As far as we know, this is the first pCLE image discribed in patient with COVID-19 at that moment.

ATG9A protects the plasma membrane from programmed and incidental permeabilization.

The integral membrane protein ATG9A plays a key role in autophagy. It displays a broad intracellular distribution and is present in numerous compartments, including the plasma membrane (PM). The reasons for the distribution of ATG9A to the PM and its role at the PM are not understood. Here, we show that ATG9A organizes, in concert with IQGAP1, components of the ESCRT system and uncover cooperation between ATG9A, IQGAP1 and ESCRTs in protection from PM damage. ESCRTs and ATG9A phenocopied each other in protection against PM injury. ATG9A knockouts sensitized the PM to permeabilization by a broad spectrum of microbial and endogenous agents, including gasdermin, MLKL and the MLKL-like action of coronavirus ORF3a. Thus, ATG9A engages IQGAP1 and the ESCRT system to maintain PM integrity.