Emergence of multiple set-points of cellular homeostatic tension.

Stress fibers (SFs), a contractile actin bundle in nonmuscle mesenchymal cells, are known to intrinsically sustain a constant level of tension or tensional stress, a process called cellular tensional homeostasis. Malfunction in this homeostatic process has been implicated in many diseases such atherosclerosis, but its mechanisms remain incompletely understood. Interestingly, the homeostatic stress in individual SFs is altered upon recruitment of α-smooth muscle actin in particular cellular contexts to reinforce the preexisting SFs. While this transition of the set-point stress is somewhat a universal process observed across different cell types, no clear explanation has been provided as to why cells end up possessing different stable stresses. To address the underlying physics, here we describe that imposing a realistic assumption on the nature of SFs yields the presence of multiple set-points of the homeostatic stress, which transition among them depending on the magnitude of the cellular tension. We analytically derive non-dimensional parameters that characterize the extent of the transition and predict that SFs tend to acquire secondary stable stresses if they are subject to as large a change in stiffness as possible or to as immediate a transition as possible upon increasing the tension. This is a minimal and simple explanation, but given the frequent emergence of force-dependent transformation of various subcellular structures in addition to that of SFs, the theoretical concept presented here would offer an essential guide to addressing potential common mechanisms governing complicated cellular mechanobiological responses.

Integrins regulate epithelial cell shape by controlling the architecture and mechanical properties of basal actomyosin networks.

Forces generated by the actomyosin cytoskeleton are key contributors to many morphogenetic processes. The actomyosin cytoskeleton organises in different types of networks depending on intracellular signals and on cell-cell and cell-extracellular matrix (ECM) interactions. However, actomyosin networks are not static and transitions between them have been proposed to drive morphogenesis. Still, little is known about the mechanisms that regulate the dynamics of actomyosin networks during morphogenesis. This work uses the Drosophila follicular epithelium, real-time imaging, laser ablation and quantitative analysis to study the role of integrins on the regulation of basal actomyosin networks organisation and dynamics and the potential contribution of this role to cell shape. We find that elimination of integrins from follicle cells impairs F-actin recruitment to basal medial actomyosin stress fibers. The available F-actin redistributes to the so-called whip-like structures, present at tricellular junctions, and into a new type of actin-rich protrusions that emanate from the basal cortex and project towards the medial region. These F-actin protrusions are dynamic and changes in total protrusion area correlate with periodic cycles of basal myosin accumulation and constriction pulses of the cell membrane. Finally, we find that follicle cells lacking integrin function show increased membrane tension and reduced basal surface. Furthermore, the actin-rich protrusions are responsible for these phenotypes as their elimination in integrin mutant follicle cells rescues both tension and basal surface defects. We thus propose that the role of integrins as regulators of stress fibers plays a key role on controlling epithelial cell shape, as integrin disruption promotes reorganisation into other types of actomyosin networks, in a manner that interferes with proper expansion of epithelial basal surfaces.

Helical structure of actin stress fibers and its possible contribution to inducing their direction-selective disassembly upon cell shortening.

Mechanisms of the assembly of actin stress fibers (SFs) have been extensively studied, while those of the disassembly-particularly cell shortening-induced ones-remain unclear. Here, we show that SFs have helical structures composed of multi-subbundles, and they tend to be delaminated upon cell shortening. Specifically, we observed with atomic force microscopy delamination of helical SFs into their subbundles. We physically caught individual SFs using a pair of glass needles to observe rotational deformations during stretching as well as ATP-driven active contraction, suggesting that they deform in a manner reflecting their intrinsic helical structure. A minimal analytical model was then developed based on the Frenet-Serret formulas with force-strain measurement data to suggest that helical SFs can be delaminated into the constituent subbundles upon axial shortening. Given that SFs are large molecular clusters that bear cellular tension but must promptly disassemble upon loss of the tension, the resulting increase in their surface area due to the shortening-induced delamination may facilitate interaction with surrounding molecules to aid subsequent disintegration. Thus, our results suggest a new mechanism of the disassembly that occurs only in the specific SFs exposed to forced shortening.

Reduction in SNAP-23 Alters Microfilament Organization in Myofibrobastic Hepatic Stellate Cells.

Hepatic stellate cells (HSC) are critical effector cells of liver fibrosis. In the injured liver, HSC differentiate into a myofibrobastic phenotype. A critical feature distinguishing myofibroblastic from quiescent HSC is cytoskeletal reorganization. Soluble NSF attachment receptor (SNARE) proteins are important in trafficking of newly synthesized proteins to the plasma membrane for release into the extracellular environment. The goals of this project were to determine the expression of specific SNARE proteins in myofibroblastic HSC and to test whether their alteration changed the HSC phenotype in vitro and progression of liver fibrosis in vivo. We found that HSC lack the t-SNARE protein, SNAP-25, but express a homologous protein, SNAP-23. Downregulation of SNAP-23 in HSC induced reduction in polymerization and disorganization of the actin cytoskeleton associated with loss of cell movement. In contrast, reduction in SNAP-23 in mice by monogenic deletion delayed but did not prevent progression of liver fibrosis to cirrhosis. Taken together, these findings suggest that SNAP-23 is an important regular of actin dynamics in myofibroblastic HSC, but that the role of SNAP-23 in the progression of liver fibrosis in vivo is unclear.

An Abl-FBP17 mechanosensing system couples local plasma membrane curvature and stress fiber remodeling during mechanoadaptation.

Cells remodel their structure in response to mechanical strain. However, how mechanical forces are translated into biochemical signals that coordinate the structural changes observed at the plasma membrane (PM) and the underlying cytoskeleton during mechanoadaptation is unclear. Here, we show that PM mechanoadaptation is controlled by a tension-sensing pathway composed of c-Abl tyrosine kinase and membrane curvature regulator FBP17. FBP17 is recruited to caveolae to induce the formation of caveolar rosettes. FBP17 deficient cells have reduced rosette density, lack PM tension buffering capacity under osmotic shock, and cannot adapt to mechanical strain. Mechanistically, tension is transduced to the FBP17 F-BAR domain by direct phosphorylation mediated by c-Abl, a mechanosensitive molecule. This modification inhibits FBP17 membrane bending activity and releases FBP17-controlled inhibition of mDia1-dependent stress fibers, favoring membrane adaptation to increased tension. This mechanoprotective mechanism adapts the cell to changes in mechanical tension by coupling PM and actin cytoskeleton remodeling.

Analyzing valve interstitial cell mechanics and geometry with spatial statistics.

Understanding cell geometric and mechanical properties is crucial to understanding how cells sense and respond to their local environment. Moreover, changes to cell mechanical properties under varied micro-environmental conditions can both influence and indicate fundamental changes to cell behavior. Atomic Force Microscopy (AFM) is a well established, powerful tool to capture geometric and mechanical properties of cells. We have previously demonstrated substantial functional and behavioral differences between aortic and pulmonary valve interstitial cells (VIC) using AFM and subsequent models of VIC mechanical response. In the present work, we extend these studies by demonstrating that to best interpret the spatially distributed AFM data, the use of spatial statistics is required. Spatial statistics includes formal techniques to analyze spatially distributed data, and has been used successfully in the analysis of geographic data. Thus, spatially mapped AFM studies of cell geometry and mechanics are analogous to more traditional forms of geospatial data. We are able to compare the spatial autocorrelation of stiffness in aortic and pulmonary valve interstitial cells, and more accurately capture cell geometry from height recordings. Specifically, we showed that pulmonary valve interstitial cells display higher levels of spatial autocorrelation of stiffness than aortic valve interstitial cells. This suggests that aortic VICs form different stress fiber structures than their pulmonary counterparts, in addition to being more highly expressed and stiffer on average. Thus, the addition of spatial statistics can contribute to our fundamental understanding of the differences between cell types. Moving forward, we anticipate that this work will be meaningful to enhance direct analysis of experimental data and for constructing high fidelity computational of VICs and other cell models.

Filamin A mediates isotropic distribution of applied force across the actin network.

Cell sensing of externally applied mechanical strain through integrin-mediated adhesions is critical in development and physiology of muscle, lung, tendon, and arteries, among others. We examined the effects of strain on force transmission through the essential cytoskeletal linker talin. Using a fluorescence-based talin tension sensor (TS), we found that uniaxial stretch of cells on elastic substrates increased tension on talin, which was unexpectedly independent of the orientation of the focal adhesions relative to the direction of strain. High-resolution electron microscopy of the actin cytoskeleton revealed that stress fibers (SFs) are integrated into an isotropic network of cortical actin filaments in which filamin A (FlnA) localizes preferentially to points of intersection between SFs and cortical actin. Knockdown (KD) of FlnA resulted in more isolated, less integrated SFs. After FlnA KD, tension on talin was polarized in the direction of stretch, while FlnA reexpression restored tensional symmetry. These data demonstrate that a FlnA-dependent cortical actin network distributes applied forces over the entire cytoskeleton-matrix interface.

Phosphorylation Regulates CAP1 (Cyclase-Associated Protein 1) Functions in the Motility and Invasion of Pancreatic Cancer Cells.

Pancreatic cancer has the worst prognosis among major malignancies, largely due to its highly invasive property and difficulty in early detection. Mechanistic insights into cancerous transformation and especially metastatic progression are imperative for developing novel treatment strategies. The actin-regulating protein CAP1 is implicated in human cancers, while the role still remains elusive. In this study, we investigated roles for CAP1 and its phosphor-regulation in pancreatic cancer cells. No evidence supports remarkable up-regulation of CAP1 in the panel of cancer cell lines examined. However, knockdown of CAP1 in cancer cells led to enhanced stress fibers, reduced cell motility and invasion into Matrigel. Phosphorylation of CAP1 at the S308/S310 tandem regulatory site was elevated in cancer cells, consistent with hyper-activated GSK3 reported in pancreatic cancer. Inhibition of GSK3, a kinase for S310, reduced cell motility and invasion. Moreover, phosphor mutants had defects in alleviating actin stress fibers and rescuing the reduced invasiveness in the CAP1-knockdown PANC-1 cells. These results suggest a required role for transient phosphorylation for CAP1 function in controlling cancer cell invasiveness. Depletion of CAP1 also reduced FAK activity and cell adhesion, but did not cause significant alterations in ERK or cell proliferation. CAP1 likely regulates cancer cell invasiveness through effects on both actin filament turnover and cell adhesion. Finally, the growth factor PDGF induced CAP1 dephosphorylation, suggesting CAP1 may mediate extracellular signals to control cancer cell invasiveness. These findings may ultimately help develop strategies targeting CAP1 or its regulatory signals for controlling the invasive cycle of the disease.

Group I metabotropic glutamate receptor activation induces TRPC6-dependent calcium influx and RhoA activation in cultured human kidney podocytes.

Researches have shown that mice lacking the metabotropic glutamate receptor 1 (mGluR) showed albuminuria, remodeling of F-actin, with loss of stress fibers. Selective group I mGluRs agonist (S)-3,5-dihydroxyphenylglycine (DHPG) attenuated albuminuria in several rodent models of nephrotic syndrome. However, the molecular mechanism is obscure. Using a human podocyte cell line, we here investigated the molecular mechanisms of group I mGluRs-induced calcium influx and the formation of stress fibers. Our data showed that group I mGluRs activation by DHPG induced a significant calcium influx, and promoted cytoskeletal stress fiber formation and focal adhesions in podocytes. Pre-incubating podocytes with non-selective inhibitor of transient receptor potential channels (TRPC), or the knockdown of TRPC6 attenuated the calcium influx and the stress fiber formation induced by DHPG. Further, DHPG resulted in an increase of active RhoA expression. However, the knockdown of RhoA by siRNA abolished the DHPG-induced increase in stress fibers. Additionally, nonselective inhibitors of TRPC or TRPC6 knockdown clearly inhibited RhoA activation induced by DHPG, as assessed by Glutathione-S-transferase pull-down assay followed by Western blotting. Taken together, our findings suggest TRPC6 regulates actin stress fiber formation and focal adhesions via the RhoA pathway in response to group I mGluRs activation. Our data can potentially explain the mechanism of protective action of group I mGluRs in glomerular podocyte injury.

Calponin-3 is critical for coordinated contractility of actin stress fibers.

Contractile actomyosin bundles, stress fibers, contribute to morphogenesis, migration, and mechanosensing of non-muscle cells. In addition to actin and non-muscle myosin II (NMII), stress fibers contain a large array of proteins that control their assembly, turnover, and contractility. Calponin-3 (Cnn3) is an actin-binding protein that associates with stress fibers. However, whether Cnn3 promotes stress fiber assembly, or serves as either a positive or negative regulator of their contractility has remained obscure. Here, we applied U2OS osteosarcoma cells as a model system to study the function of Cnn3. We show that Cnn3 localizes to both NMII-containing contractile ventral stress fibers and transverse arcs, as well as to non-contractile dorsal stress fibers that do not contain NMII. Fluorescence-recovery-after-photobleaching experiments revealed that Cnn3 is a dynamic component of stress fibers. Importantly, CRISPR/Cas9 knockout and RNAi knockdown studies demonstrated that Cnn3 is not essential for stress fiber assembly. However, Cnn3 depletion resulted in increased and uncoordinated contractility of stress fibers that often led to breakage of individual actomyosin bundles within the stress fiber network. Collectively these results provide evidence that Cnn3 is dispensable for the assembly of actomyosin bundles, but that it is required for controlling proper contractility of the stress fiber network.

Rotation of stress fibers as a single wheel in migrating fish keratocytes.

Crawling migration plays an essential role in a variety of biological phenomena, including development, wound healing, and immune system function. Keratocytes are wound-healing cells in fish skin. Expansion of the leading edge of keratocytes and retraction of the rear are respectively induced by actin polymerization and contraction of stress fibers in the same way as for other cell types. Interestingly, stress fibers in keratocytes align almost perpendicular to the migration-direction. It seems that in order to efficiently retract the rear, it is better that the stress fibers align parallel to it. From the unique alignment of stress fibers in keratocytes, we speculated that the stress fibers may play a role for migration other than the retraction. Here, we reveal that the stress fibers are stereoscopically arranged so as to surround the cytoplasm in the cell body; we directly show, in sequential three-dimensional recordings, their rolling motion during migration. Removal of the stress fibers decreased migration velocity and induced the collapse of the left-right balance of crawling migration. The rotation of these stress fibers plays the role of a "wheel" in crawling migration of keratocytes.

Amelogenesis imperfecta: A novel FAM83H mutation and characteristics of periodontal ligament cells.

OBJECTIVE: To delineate orodental features, dental mineral density, genetic aetiology and cellular characteristics associated with amelogenesis imperfecta (AI). MATERIALS AND METHODS: Three affected patients in a family were recruited. Whole-exome sequencing was used to identify mutations confirmed by Sanger sequencing. The proband's teeth were subjected for mineral density analysis by microcomputerised tomography and characterisation of periodontal ligament cells (PDLCs). RESULTS: The patients presented yellow-brown, pitted and irregular enamel. A novel nonsense mutation, c.1261G>T, p.E421*, in exon 5 of the FAM83H was identified. The mineral density of the enamel was significantly decreased in the proband. The patient's PDLCs (FAM83H cells) exhibited reduced ability of cell proliferation and colony-forming unit compared with controls. The formation of stress fibres was remarkably present. Upon cultured in osteogenic induction medium, FAM83H cells, at day 7 compared to day 3, had a significant reduction of BSP, COL1 and OCN mRNA expression and no significant change in RUNX2. The upregulation of ALP mRNA levels and mineral deposition were comparable between FAM83H and control cells. CONCLUSIONS: We identified the novel mutation in FAM83H associated with autosomal dominant hypocalcified AI. The FAM83H cells showed reduced cell proliferation and expression of osteogenic markers, suggesting altered PDLCs in FAM83H-associated AI.

The Bacterial Protein CNF1 as a Potential Therapeutic Strategy against Mitochondrial Diseases: A Pilot Study.

The Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1), which acts on the Rho GTPases that are key regulators of the actin cytoskeleton, is emerging as a potential therapeutic tool against certain neurological diseases characterized by cellular energy homeostasis impairment. In this brief communication, we show explorative results on the toxin's effect on fibroblasts derived from a patient affected by myoclonic epilepsy with ragged-red fibers (MERRF) that carries a mutation in the m.8344A>G gene of mitochondrial DNA. We found that, in the patient's cells, besides rescuing the wild-type-like mitochondrial morphology, CNF1 administration is able to trigger a significant increase in cellular content of ATP and of the mitochondrial outer membrane marker Tom20. These results were accompanied by a profound F-actin reorganization in MERRF fibroblasts, which is a typical CNF1-induced effect on cell cytoskeleton. These results point at a possible role of the actin organization in preventing or limiting the cell damage due to mitochondrial impairment and at CNF1 treatment as a possible novel strategy against mitochondrial diseases still without cure.

Transforming growth factor-ß modulates pancreatic cancer associated fibroblasts cell shape, stiffness and invasion.

BACKGROUND: Tumor microenvironment consists of the extracellular matrix (ECM), stromal cells, such as fibroblasts (FBs) and cancer associated fibroblasts (CAFs), and a myriad of soluble factors. In many tumor types, including pancreatic tumors, the interplay between stromal cells and the other tumor microenvironment components leads to desmoplasia, a cancer-specific type of fibrosis that hinders treatment. Transforming growth factor beta (TGF-ß) and CAFs are thought to play a crucial role in this tumor desmoplastic reaction, although the involved mechanisms are unknown. METHODS: Optical/fluorescence microscopy, atomic force microscopy, image processing techniques, invasion assay in 3D collagen I gels and real-time PCR were employed to investigate the effect of TGF-ß on normal pancreatic FBs and CAFs with regard to crucial cellular morphodynamic characteristics and relevant gene expression involved in tumor progression and metastasis. RESULTS: CAFs present specific myofibroblast-like characteristics, such as α-smooth muscle actin expression and cell elongation, they also form more lamellipodia and are softer than FBs. TGF-ß treatment increases cell stiffness (Young's modulus) of both FBs and CAFs and increases CAF's (but not FB's) elongation, cell spreading, lamellipodia formation and spheroid invasion. Gene expression analysis shows that these morphodynamic characteristics are mediated by Rac, RhoA and ROCK expression in CAFs treated with TGF-ß. CONCLUSIONS: TGF-ß modulates CAFs', but not FBs', cell shape, stiffness and invasion. GENERAL SIGNIFICANCE: Our findings elucidate on the effects of TGF-ß on CAFs' behavior and stiffness providing new insights into the mechanisms involved.

Nanopillar force measurements reveal actin-cap-mediated YAP mechanotransduction.

A robust nanopillar platform with increased spatial resolution reveals that perinuclear forces, originating from stress fibres spanning the nucleus of fibroblasts, are significantly higher on these nanostructured substrates than the forces acting on peripheral adhesions. Many perinuclear adhesions embrace several nanopillars at once, pulling them into ß1-integrin- and zyxin-rich clusters, which are able to translocate in the direction of cell motion without losing their tensile strength. The high perinuclear forces are greatly reduced upon inhibition of cell contractility or actin polymerization and disruption of the actin cap by KASH dominant-negative mutant expression. LMNA null fibroblasts have higher peripheral versus perinuclear forces, impaired perinuclear ß1-integrin recruitment, as well as YAP nuclear translocation, functional alterations that can be rescued by lamin A expression. These highly tensed actin-cap fibres are required for YAP nuclear signalling and thus play far more important roles in sensing nanotopographies and mechanochemical signal conversion than previously thought.

Selective hemangioma cell dysfunction and apoptosis triggered by in vitro treatment with imiquimod.

Infantile hemangiomas are the most common benign tumors of infancy, characterized by unregulated angiogenesis and endothelial cells with high mitotic rate. Although spontaneous regression occurs, sometimes treatment is required and alternatives to corticosteroids should be considered to reduce side effects. Imiquimod is an imidazoquinoline, approved for some skin pathologies other than hemangioma. It is proposed that the effectiveness of imiquimod comes from the activation of immune cells at tumor microenvironment. However, the possibility to selectively kill different cell types and to directly impede angiogenesis has been scarcely explored in vitro for endothelial cells. In this work we showed a dramatic cytotoxicity on hemangioma cell, with a significant lower IC50 value in hemangioma compared to normal endothelial cells and melanoma (employed as a non-endothelial tumor cell line). Nuclear morphometric and flow-cytometry assays revealed imiquimod-induced apoptosis on hemangioma and melanoma cells but a small percentage of senescence on normal endothelial cells. At sub-lethal conditions, cell migration, a key step in angiogenesis turned out to be inhibited in a tumor-selective manner along with actin cytoskeleton disorganization on hemangioma cells. Altogether, these findings pointed out the selective cytotoxic effects of imiquimod on transformed endothelial cells, evidencing the potential for imiquimod to be a therapeutic alternative to reduce extensive superficial hemangioma lesions.

UNC-45a promotes myosin folding and stress fiber assembly.

Contractile actomyosin bundles, stress fibers, are crucial for adhesion, morphogenesis, and mechanosensing in nonmuscle cells. However, the mechanisms by which nonmuscle myosin II (NM-II) is recruited to those structures and assembled into functional bipolar filaments have remained elusive. We report that UNC-45a is a dynamic component of actin stress fibers and functions as a myosin chaperone in vivo. UNC-45a knockout cells display severe defects in stress fiber assembly and consequent abnormalities in cell morphogenesis, polarity, and migration. Experiments combining structured-illumination microscopy, gradient centrifugation, and proteasome inhibition approaches revealed that a large fraction of NM-II and myosin-1c molecules fail to fold in the absence of UNC-45a. The remaining properly folded NM-II molecules display defects in forming functional bipolar filaments. The C-terminal UNC-45/Cro1/She4p domain of UNC-45a is critical for NM-II folding, whereas the N-terminal tetratricopeptide repeat domain contributes to the assembly of functional stress fibers. Thus, UNC-45a promotes generation of contractile actomyosin bundles through synchronized NM-II folding and filament-assembly activities.

Automated segmentation and quantification of actin stress fibres undergoing experimentally induced changes.

The actin cytoskeleton is a main component of cells and it is crucially involved in many physiological processes, e.g. cell motility. Changes in the actin organization can be effected by diseases or vice versa. Due to the nonuniform pattern, it is difficult to quantify reasonable features of the actin cytoskeleton for a significantly high cell number. Here, we present an approach capable to fully segment and analyse the actin cytoskeleton of 2D fluorescence microscopic images with a special focus on stress fibres. The extracted feature data include length, width, orientation and intensity distributions of all traced stress fibres. Our approach combines morphological image processing techniques and a trace algorithm in an iterative manner, classifying the segmentation result with respect to the width of the stress fibres and in nonfibre-like actin. This approach enables us to capture experimentally induced processes like the condensation or the collapse of the actin cytoskeleton. We successfully applied the algorithm to F-actin images of cells that were treated with the actin polymerization inhibitor latrunculin A. Furthermore, we verified the robustness of our algorithm by a sensitivity analysis of the parameters, and we benchmarked our algorithm against established methods. In summary, we present a new approach to segment actin stress fibres over time to monitor condensation or collapse processes.

Diallyl trisulfide inhibits cell migration and invasion of human melanoma a375 cells via inhibiting integrin/facal adhesion kinase pathway.

Melanoma is the leading cause of death from skin disease due to its propensity for metastasis. Studies have shown that integrin-mediated focal adhesion kinase (FAK) signal pathway is implicated in cell proliferation, survival and metastasis of tumor cells. Our previous results indicated that diallyl trisulfide (DATS) provided its antimelanoma activity via inducing cell cycle arrest and apoptosis. The aim of this study was to explore DATS mediated antimetastatic effect and the corresponding mechanism in human melanoma A375 cells. We found that DATS exhibited an inhibitory effect on the abilities of migration and invasion in A375 cells under noncytotoxic concentrations analyzed by wound healing assays and Matrigel invasion chamber system. DATS attenuated invasion of A375 cells with characteristic of decreased activities and protein expressions of matrix metalloproteinase-2 (MMP-2) and MMP-9. Moreover, DATS exerted an inhibitory effect on cell adhesion of A375 cells, which is in correlation with the change in integrin signaling pathway. Results of Western blotting showed that DATS decreased the levels of several integrin subunits, including α4, α5, αv, ß1, ß3 and ß4. Subsequently, DATS induced a strong decrease in total FAK, phosphorylated FAK Tyr-397,-576, -577, and disorganized F-actin stress fibers, resulting in a nonmigratory phenotype. These results suggest that the antimetastatic potential of DATS for human melanoma cells might be due to the disruption of integrin/FAK signaling pathway.

Membrane wound healing at single cellular level.

We report a nano-technological method of creating a micrometer sized hole on the live cell membrane using atomic force microscope (AFM) and its resealing process at the single cellular level as a model of molecular level wound healing. First, the cell membrane was fluorescently labeled with Kusabira Orange (KO) which was tagged to a lipophilic membrane-sorting peptide. Then a glass bead glued on an AFM cantilever and modified with phospholipase A2 was made to contact the cell membrane. A small dark hole (4-14 µm2 in area) was created on the otherwise fluorescent cell surface often being accompanied by bleb formation. Refilling of holes with KO fluorescence proceeded at an average rate of ~0.014µm2s-1. The fluorescent lumps which initially surrounded the hole were gradually lost. We compared the present result with our previous ones on the repair processes of artificially damaged stress fibers (Graphical Abstract: Figure S2).