Annexin A2 is involved in Ca2 +-dependent plasma membrane repair in primary human endothelial cells

BBAMCR-18008; No. of pages: 8; 4C: 2, 3, 5, 6 Biochimica et Biophysica Acta xxx (2016) xxx–xxx

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Annexin A2 is involved in Ca2 +-dependent plasma membrane repair in primary human endothelial cells Sophia Nina Koerdt, Volker Gerke ⁎ Institute of Medical Biochemistry, Center for Molecular Biology of Inflammation, University of Muenster, Von-Esmarch-Str. 56, 48149 Münster, Germany

a r t i c l e

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Article history: Received 9 October 2016 Received in revised form 18 November 2016 Accepted 8 December 2016 Available online xxxx Keywords: Annexin Calcium Endothelium Membrane

a b s t r a c t Many cells in an organism are exposed to constant and acute mechanical stress that can induce plasma membrane injuries. These plasma membrane wounds have to be resealed rapidly to guarantee cell survival. Plasma membrane resealing in response to mechanical strain has been studied in some detail in muscle, where it is required for efficient recovery after insult. However, less is known about the capacity of other cell types and tissues to perform membrane repair and the underlying molecular mechanisms. Here we show that vascular endothelial cells, which are subject to profound mechanical burden, can reseal plasma membrane holes inflicted by laser ablation. Resealing in endothelial cells is a Ca2+-dependent process, as it is inhibited when cells are wounded in Ca2+-free medium. We also show that annexin A1 (AnxA1), AnxA2 and AnxA6, Ca2+-regulated membrane binding proteins previously implicated in membrane resealing in other cell types, are rapidly recruited to the site of plasma membrane injury. S100A11, a known protein ligand of AnxA1, is also recruited to endothelial plasma membrane wounds, albeit with a different kinetic. Mutant expression experiments reveal that Ca2+ binding to AnxA2, the most abundant endothelial annexin, is required for translocation of the protein to the wound site. Furthermore, we show by knock-down and rescue experiments that AnxA2 is a positive regulator of plasma membrane resealing. Thus, vascular endothelial cells are capable of active, Ca2+-dependent plasma membrane resealing and this process requires the activity of AnxA2. © 2016 Elsevier B.V. All rights reserved.

1. Introduction To survive and to maintain tissue integrity, several types of cells have been shown to be able to repair (or reseal) plasma membrane holes that can be as large as several μm2 [1–3]. Such holes can form in different ways. In infectious scenarios, membrane wounds can be inflicted by pore-forming toxins that are inserted into the plasma membrane [4]. In the course of repair, such wounds can be physically eliminated by endocytosis or the shedding of microparticles containing the pore [5]. Membrane ruptures that occur in non-infected cells independent of toxin action are usually the consequence of mechanical stress. This is particularly pronounced in skeletal muscle, where membrane damage is observed in up to 20% of muscle fibers during normal exercise, and is increased in diseases such as Duchenne muscular dystrophy [6]. Although differences are likely to exist between the repair mechanisms of pore induced and physical membrane wounds, both require Ca2+ entering through the plasma membrane hole as a crucial initiator of resealing. In the case of mechanical wounds, published evidence suggests that the increase in intracellular Ca2+ induces the formation of a ⁎ Corresponding author. E-mail addresses: [email protected] (S.N. Koerdt), [email protected] (V. Gerke).

membrane patch underneath the site of injury, most likely through the recruitment and homotypic fusion of internal membranes. This patch then fuses with the plasma membrane, thereby replacing the hole with a new continuous membrane [3]. However, alternative mechanisms of membrane repair have been proposed and shown to occur in certain scenarios. These include endocytic retrieval of damaged membrane and ESCRT-dependent shedding of membrane vesicles containing the ruptured site [7–9]. Most likely, the mechanism chosen depends on the type and size of membrane wound and could differ between cell types, but in any case is likely to also involve a Rho-GTPase-driven remodeling of the cortical actin cytoskeleton [10]. Despite the fundamental importance of membrane repair for cell survival and tissue integrity, little is known about underlying molecular mechanisms und components of the resealing machinery. By analyzing sarcolemma repair and focusing on proteins altered in patients suffering from muscular dystrophies, dysferlin, a member of the ferlin protein family containing Ca2+-sensing C2 domains, was identified. Dysferlin is mutated in limb-girdle muscular dystrophy type 2B and Miyoshi myopathy, and experiments involving cultured cells as well as developing zebrafish embryos implicate the protein as a Ca2 +-regulated fusogen in the course of exocytotic events occurring during membrane resealing [11–13]. Several dysferlin interacting proteins have been identified, among them two members of the annexin (Anx) family of Ca2 + and

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Please cite this article as: S.N. Koerdt, V. Gerke, Annexin A2 is involved in Ca2 +-dependent plasma membrane repair in primary human endothelial cells, Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbamcr.2016.12.007

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Fig. 1. HUVEC reseal plasma membrane injuries in the presence of Ca2+. A) HUVEC were injured at t = 0 by irradiating a 2 μm2 circular region of interest (ROI) with 820 nm laser light in the presence of 5 μg/ml FM4-64 in Na-Tyrode's buffer supplemented with either 2.5 mM Ca2+ or 100 μM EGTA. Scale bars = 10 μm. B) FM4-64 dye influx into the wounded cell was quantified over time for wounding ROIs of either 2 μm2 or 4.5 μm2. The increase in fluorescence intensity is depicted, normalized to both fluorescence intensity before wounding and fluorescence intensity of an uninjured neighbouring cell. n = 10–15 cells per condition from 2 to 3 independent experiments. Error bars are SEM.

phospholipid binding proteins, namely AnxA1 and A2 [14]. This is of special interest because both annexins are also dysregulated in dysferlinopathic patients [15] and because several annexins are recruited to sites of plasma membrane injury in a Ca2+-regulated manner [13, 16–19]. Moreover, while a functional contribution of AnxA2 to the

membrane repair process has not been elucidated, AnxA1 has been shown to be required for efficient membrane resealing [16]. AnxA5 and AnxA6 are two other annexins that also participate in membrane resealing in certain cell types [13,20]. Mechanistically, AnxA5 has been shown to erect a semi-crystalline membrane scaffold at the site of

Fig. 2. AnxA1-GFP and AnxA6-GFP are recruited to sites of endothelial plasma membrane injury. A) A ROI of 2 μm2 of a HUVEC transfected with AnxA1-GFP was irradiated with 820 nm laser light 24 h post transfection. Success of plasma membrane rupture was assessed by FM4-64 imaging. B) Wounding of a HUVEC overexpressing AnxA6-GFP was performed as above. Scale bars = 20 μm.

Please cite this article as: S.N. Koerdt, V. Gerke, Annexin A2 is involved in Ca2 +-dependent plasma membrane repair in primary human endothelial cells, Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbamcr.2016.12.007

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injury, thereby preventing an expansion of the wound [20–22]. Other factors implicated in membrane resealing are the Trim72 protein MG53 [23], the giant protein AHNAK [24], the Ca2+-regulated protease calpain [25] and the C2 domain protein synaptotagmin VII, which most likely functions by supporting secretory lysosome exocytosis in the course of endocytosis-mediated membrane repair [26]. Plasma membrane ruptures have been identified in several mammalian tissues that experience mechanical stress. They include skeletal muscle, the gastrointestinal and respiratory tracts and the vascular system [1,27]. However, an active, Ca2+-dependent resealing of such membrane ruptures has only been shown in a limited number of cell types of these tissues, e.g. skeletal muscle cells and placental trophoblasts, as well as some cultured cell lines such as HeLa and MCF7 breast cancer cells [1,16,18,21]. As such active membrane resealing has not been assessed in vascular endothelial cells that are known to experience substantial mechanical stress and show frequent ruptures [28], we employed microscopy assays to analyze membrane resealing in primary human endothelial cells. Our data show that endothelial cells are capable of repairing laser-induced plasma membrane wounds in a rapid, Ca2+-dependent process, that resealing is accompanied by the recruitment of AnxA1, AnxA2 and AnxA6 to the wound site and that AnxA2 is functionally required for resealing.

2. Materials and methods 2.1. Cell culture and transfection HUVEC were purchased from Promocell or prepared from umbilical cords as described before [29] and either used directly or cryo-conserved for later use. Cells were cultured at 37 °C and 5% CO2 for up to 5 passages in mixed endothelial growth medium, a 1:1 mixture of ECGM-2 (C22111, Promocell) and M199 (F0615, Biochrom) + 100 i.u. heparin + 10%

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FCS (Sigma), supplemented with 0.015 μg/ml amphotericin B and 30 μg/ml gentamycin. HUVEC were transfected with plasmid DNA (1–10 μg per 20 cm2 nearly confluent cells) using electroporation as described before [29] and used for live-cell microscopy or lysate preparation 20–50 h post transfection. The plasmids used in this study were described before: AnxA1-FP and AnxA2-FP in [30], AnxA6-GFP in [31], S100A11 in [32], and AnxA2-sires in [29]; the AnxA2CM mutation is described in [33]. siRNA targeting AnxA2 (CUU UGA UGC UGA GCG GGA UdTdT) was obtained from Microsynth. For control experiments, non-targeting AllStars Neg. Control siRNA (1027281, Qiagen) was used. For siRNA transfections, 20 cm2 nearly confluent HUVEC were transfected with 400 pmol of siRNA, incubated for 48 h and transfected in the same manner again. The second transfection included the siRNA-resistant AnxA2sires-GFP (6 μg) or empty EGPF-N1 vector (0.3 μg). Cells were used for lysate preparation or the laser wounding assay 48 h after the second transfection. 2.2. Laser wounding assay HUVEC (transfected or non-transfected) were seeded onto 8-well glass bottom μ-slides (80827, ibidi) pretreated with collagen. Cell density was adjusted to reach approx. 80% confluency at the time of imaging. Microscopy was performed on a LSM780 confocal microscope (Zeiss) equipped with a Chameleon Vision tunable NLO laser (Coherent) and a 63×, NA 1.4 oil immersion objective (Plan-Apochromat, Zeiss). Cells were maintained in mixed endothelial growth medium + 20 mM HEPES at 37 °C in the incubation chamber of the microscope during the assay. Individual wells of the μ-slide were washed with Tyrode's buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES, pH 7.4) [34] supplemented with either 2.5 mM Ca2 + or 100 μM EGTA once and then incubated with the same buffer + 5 μg/ml FM4-64FX (F34653, Life Technologies, Molecular Probes) and subjected

Fig. 3. Recruitment of AnxA2 to endothelial plasma membrane wounds requires Ca2+ binding. HUVEC transfected with either AnxA2-GFP or AnxA2CM-GFP were subjected to laser wounding as described in Fig. 1 with an ROI of 4.5 μm2 in the presence of 2.5 mM Ca2+ or 100 μM EGTA. Scale bars = 20 μm.

Please cite this article as: S.N. Koerdt, V. Gerke, Annexin A2 is involved in Ca2 +-dependent plasma membrane repair in primary human endothelial cells, Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbamcr.2016.12.007

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Please cite this article as: S.N. Koerdt, V. Gerke, Annexin A2 is involved in Ca2 +-dependent plasma membrane repair in primary human endothelial cells, Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbamcr.2016.12.007

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to microscopy. Once FM4-64 had been added, cells were used for no more than 40 min to limit dye uptake by endocytic processes and to ensure sufficient concentration of free FM4-64 in the buffer. The wounding protocol was set up with the Zen2 software (Zeiss). NLO Laser power at 820 nm was set to 12% and bleaching was performed for 2 iterations with a pixel dwell of 77 μs on circular ROIs in the “zoom bleach” mode of Zen2. ROIs were 20 or 30 pixels in diameter, corresponding to 2 μm2 or 4.5 μm2 surface area, respectively. A time series of 100 images was recorded, with the wounding taking place after the second frame had been acquired. Image analysis was performed in Fiji [35,36]. A maximum intensity projection of the acquired XYT image stack was used to draw the surface area of the wounded cell of interest (COI), while part of an uninjured cell was selected for background (BG) correction purposes to compensate for any endocytic uptake of FM4-64 during acquisition. The first image of the FM4-64 channel was subtracted from all other images in this channel within the XYT stack. Using the “plot Z profile” function of Fiji, fluorescence intensity changes were assessed for both COI and BG. The resulting table of intensity values was processed by subtracting BG from COI intensity at each timepoint and averaging the corrected fluorescence intensities obtained thus within timepoints and experimental conditions. 2.3. Western blotting and antibodies HUVEC lysates were prepared by harvesting 20 cm2 cells with trypsin/EDTA, washing the cell pellet in PBS once, and lysing the cells by sonication for 1 min in 40 μl lysis buffer (20 mM HEPES, 150 mM NaCl, 0.5% Triton X-100,1.5 mM PMSF and complete EDTA-free protease inhibitor cocktail (11873580001, Roche) added according to the manufacturer's instructions). After incubation on ice for 15 min, cellular debris was pelleted (10 min, 1250 × g, 4 °C) and the cleared lysate was processed for and subjected to SDS-PAGE and western blotting according to standard protocols. The primary antibodies used and their dilutions were: rabbit α-GFP 1:1000 (Invitrogen, A6455), mouse αcalnexin (1:1000) (BD Transduction Laboratories, 610524) and mouse α-AnxA2 HH7 1:2000, which was described previously [37]. Secondary antibodies were goat α-mouse-IRdye800CW (926-32210, LICOR) and goat α-rabbit-IRdye680RD (925-68071, LICOR). Quantification of AnxA2 knock-down efficiency was performed with the Odyssey Infrared Imaging System Application Software version 3.0 (LICOR) with settings for median background method, border width 3 and right/left segmentation. 3. Results 3.1. Endothelial cells repair laser-induced plasma membrane wounds We first determined whether primary human endothelial cells isolated from umbilical veins (HUVEC) are capable of resealing plasma membrane injuries. Laser ablation was employed to generate plasma membrane holes of defined sizes and resealing of these holes was recorded microscopically by following uptake of the fluorescent dye FM4-64. The FM dye is excluded from entering the cytosol as long as the plasma membrane is intact, but rapidly accumulates in intracellular membranes when plasma membrane injuries occur. Resealing of a plasma membrane hole is thus reflected in the block of dye uptake after an initial phase of rapid dye internalization. Fig. 1 shows this typical twophase behavior for HUVEC after laser-induced plasma membrane wounding. Membrane resealing in other tissues and cells has been shown to be a strictly Ca2+-dependent process requiring extracellular

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Ca2+ that enters through the wound to initiate the repair [1]. Therefore, we next analyzed whether Ca2+ is also necessary for the laser-induced plasma membrane repair in HUVEC by carrying out laser wounding in the absence of extracellular Ca2 +. As depicted in Fig. 1, FM dye continues to enter the wounded cell in the absence of extracellular Ca2+. Thus, HUVEC are capable of sealing laser-induced plasma membrane wounds and this process requires extracellular Ca2+ most likely entering through the wound. We also altered the size of the laser-induced wound and could show that efficient resealing is observed regardless of whether the actual wound size was 2 or 4.5 μm2 (Fig. 1B). 3.2. Annexins are recruited to endothelial plasma membrane wounds with different kinetics Previous work has revealed that different members of the annexin family translocate from the cytosol to the site of plasma membrane wounds in a Ca2+-dependent manner. This has been studied in great detail in the case of plasma membrane holes introduced by bacterial pore-forming toxins [5,7,17]. To extend these studies to endothelial cells and laser-induced plasma membrane wounds that are larger than toxin pores, we expressed fluorescent protein-tagged versions of different annexins in HUVEC and recorded their dynamic distribution in response to laser injury. In these analyses, we first concentrated on annexins A1 and A6 because these members of the family are expressed in endothelial cells [38,39] and have been shown previously to be recruited to toxin-induced pores and mechanical membrane wounds in other cells [1,5]. Fig. 2 shows that both AnxA1 and AnxA6 translocate to the site of plasma membrane injury within 10 s. Interestingly, the translocation occurs in a wave-like manner where the respective annexin concentrates in a front deeper in the cytosol and then moves towards the wound. The annexin front that migrates towards the wound often has a dot-like appearance suggesting that the annexin in this front is associated with membrane vesicles, possibly those involved in forming a membrane patch that is then applied to seal the wound [3]. No recruitment of AnxA1 or AnxA6 to the laser wound is observed when the wounding experiment is performed in the absence of extracellular Ca2+ (not shown) indicating that the annexin translocation is Ca2+-dependent, as expected from previous experiments in other cells [40]. An annexin that has not been studied with respect to a function in the resealing of mechanically induced plasma membrane wounds is AnxA2. As it translocates to the plasma membrane in response to raised intracellular Ca2+ in different types of cells and is also recruited to holes inflicted by pore-forming toxins [17,34,41], we next recorded the dynamic intracellular distribution of fluorescent protein-tagged AnxA2 following laser wounding of HUVEC. Fig. 3 shows that similar to AnxA1 and A6, AnxA2 is recruited to the laser-induced wound. This recruitment also occurs in a wave-like fashion and requires the presence of extracellular Ca2+. To verify that the Ca2+-dependency of this translocation is due to direct binding of Ca2+ to AnxA2 and not the result of an intermediate Ca2 +-sensitive factor that interacts with AnxA2, we carried out the same live cell experiments with an AnxA2 mutant incapable of binding Ca2+. This mutant, referred to as AnxA2CM (or Ca2+ minus), has suffered single amino acid replacements of the so-called acidic cap residues of the type II Ca2+ binding sites that render these sites inactive [33]. Live cell recordings after laser wounding show that AnxA2CM is not recruited to the site of plasma membrane injury (Fig. 3). Thus, direct Ca2+-binding to AnxA2 triggers its recruitment to plasma membrane wounds. Moreover, as plasma membrane repair is not affected by the expression of AnxA2CM, this mutant does not act in a dominant-negative manner over endogenous AnxA2, at least not at the expression levels achieved in our experiments.

Fig. 4. Relative recruitment dynamics of AnxA1, AnxA2 and S100A11. A) HUVEC transfected with AnxA1-GFP and AnxA2-mCherry were incubated in mixed endothelial growth medium and laser irradiated at 820 nm with an ROI of 2 μm2. No FM4-64 dye was added as spectral dye characteristics do not allow for combining FM4-64 and mCherry. B,C) HUVEC expressing S100A11YFP and AnxA1-CFP (B) or AnxA2-CFP (C) were laser wounded in the presence of 5 μg/ml FM4-64 and 2.5 mM Ca2+ with an ROI of 4.5 μm2 (B) or 2 μm2 (C), respectively. Scale bars = 10 μm.

Please cite this article as: S.N. Koerdt, V. Gerke, Annexin A2 is involved in Ca2 +-dependent plasma membrane repair in primary human endothelial cells, Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbamcr.2016.12.007

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In a next set of experiments, we compared the kinetics of membrane wound recruitment for AnxA1 and AnxA2. We focused on these annexins because they are closely related within the annexin family and thus could potentially serve redundant functions in membrane repair. HUVEC expressing AnxA1-EGFP and AnxA2-mCherry were subjected to membrane wounding and protein translocation to the wound was recorded as above. Fig. 4A shows stills of the respective live cell videos revealing that recruitment of the two annexins occurs in a successive manner. Whereas AnxA1 appears at the site of injury few seconds after wounding, AnxA2 usually arrives approximately 4– 8 s later than AnxA1. Interestingly, this order of appearance at the wound site differs from the kinetics of their Ca2+-dependent recruitment to the plasma membrane after ionomycin treatment of cultivated neuroblastoma cells [41] and that observed in zebrafish muscle fibers [13,20]. AnxA1 has been shown to interact with the EF hand type Ca2+ binding protein S100A11 [42,43] and this S100 protein has been shown to participate in plasma membrane repair in migrating MCF7 cells [18]. Therefore, we also analyzed whether S100A11 is recruited to plasma membrane wounds in endothelial cells by employing the same experimental set-up. Fig. 4 shows that S100A11 also translocates to the site of laser injury. Similar to what is observed for the annexins, this translocation occurs in a wave-like fashion and is dependent on the presence of extracellular Ca2+ (not shown). Interestingly, the kinetics of S100A11 recruitment do not replicate those of its known interaction partner AnxA1 (Fig. 4B). Instead, the S100A11 wave appears at the same cellular location as the initial AnxA2 accumulation, and both proteins arrive at the repair site at the same time. This is reminiscent of the situation in MCF7 tumor cells where S100A11 seems to be recruited to membrane wounds together with AnxA2 [18]. 3.3. AnxA2 is functionally required for plasma membrane resealing in HUVEC As AnxA2 is the most highly expressed annexin in endothelium [38] and is recruited to laser-induced membrane wounds in endothelial cells, we next addressed its functional involvement in the resealing process. Therefore, HUVEC were depleted of AnxA2 by a specific siRNA and the effect of this downregulation on membrane resealing was assessed by the FM dye uptake assay. The siRNA-mediated downregulation of AnxA2 worked very efficiently in HUVEC (Fig. 5) and quantification of five independent experiments revealed that the cellular AnxA2 levels were reduced by at least 90%. FM dye uptake recordings show that HUVEC depleted of AnxA2 have a severe defect in repairing laser-induced plasma membrane wounds. To verify the specificity of the knock-down effect, we expressed in the AnxA2-depleted cells AnxA2sires-GFP, an AnxA2 variant that was rendered insensitive to

the siRNA by silent nucleotide mutations [29]. This reexpression rescued the siRNA induced phenotype, i.e. restored the membrane resealing capacity to control levels (Fig. 5). Together, our results add AnxA2 to the list of annexins functionally involved in membrane resealing, and also show that at least one annexin participates in membrane repair in primary endothelial cells. 4. Discussion Endothelial cells are subject to different kinds of mechanical stress. Stressors include shear forces of the circulation, stretch forces induced by underlying smooth muscle cells, and pulling forces that are exerted when circulating leukocytes start to roll on and finally adhere to activated endothelium. Although not shown directly so far, such forces are likely to trigger wounds in the endothelial plasma membrane that need to be repaired rapidly to ensure cellular integrity and survival. Indeed, inspection of rat aortas revealed that N 5% of endothelial cells lining the vessel appeared wounded [28]. Thus, it seems likely that endothelial cells, like cells in other tissues, must have developed an efficient membrane resealing machinery to cope with insults. Here, we show that this is indeed the case. Using laser ablation to generate membrane wounds of defined dimensions, we show that primary human endothelial cells actively reseal their injured membrane in a Ca2 +dependent process. We also show that different annexins are recruited to the wound site and that this recruitment depends on Ca2+ binding sites of the annexin, at least in the case of AnxA2. Several annexins have been shown to be recruited to mechanical or laser ablation-induced plasma membrane wounds, and some have also been identified as functional components of the resealing machinery [1]. Most prominently, AnxA5 is required for the resealing of membrane wounds in perivascular cells, trophoblasts and skeletal muscle cells [20– 22]. In this case, triggered by Ca2 + influx through the wound, the annexin most likely binds to negatively charged phospholipids of the plasma membrane in the area of the wound and then forms a 2D array on the membrane that is thought to stabilize the torn membrane edges [44]. Other annexins shown to be functionally involved are AnxA1, which is required for Ca2+-dependent resealing in HeLa cells [16], and AnxA1 and AnxA6, which appear to form a scaffold during the repair of skeletal muscle in zebrafish [13]. Here we extend this list to AnxA2 and show by specific knock-down and rescue experiments that it is functionally required for plasma membrane resealing in endothelial cells. Except for AnxA5, the mechanistic basis of the annexin action in membrane wound repair is not known. It appears likely, however, that a Ca2+-dependent binding to cellular membranes is involved. As shown here for AnxA1, A2 and A6, the translocation of these proteins from the cytosol to the site of injury occurs in a wave-like manner

Fig. 5. AnxA2 is a positive regulator of plasma membrane resealing. A) HUVEC were transfected with siAnxA2 or siCtrl for 2 × 48 h. The second transfection also included either siRNAresistant AnxA2sires-GFP or empty GFP vector. Cells were then subjected to laser wounding and results were quantified. n = ≥ 12 cells per condition from 3 independent experiments. Error bars = 95% CI. B) Cells transfected as in A) were used to prepare lysates that were subjected to Western blot analysis using anti-AnxA2, anti-GFP or anti-calnexin (used as loading control) antibodies. Quantifying the intensities of the respective protein bands revealed that endogenous AnxA2 was reduced to 1.7 ± 1.0% (n = 5 independent experiments) in the siAnxA2 + GFP samples compared to the siCtrl + GFP condition.

Please cite this article as: S.N. Koerdt, V. Gerke, Annexin A2 is involved in Ca2 +-dependent plasma membrane repair in primary human endothelial cells, Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbamcr.2016.12.007

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with punctate structures inside the wavefront. This suggests that the annexins move together with and possibly bound to internal membranes that eventually form the membrane patch underneath the actual wound. However, due to our level of optical resolution and the rather flat architecture of endothelial cells it is also possible that the annexins are first recruited to the plasma membrane and then move laterally to the wound site, possibly undergoing dissociation and Ca2+-dependent reassociation to the membrane during the process, as observed before in cells suffering from bacterial toxin pores [45]. This movement could appear dot-like or cloudy, as the plasma membrane is likely to be uneven. The speed of the movement could at least in part be determined by the Ca2+ sensitivity of the respective annexin [45]. Thus, annexins might be involved in recruiting internal membranes to the wound site and/or scaffolding and stabilizing the membrane patch close to the wound. A scaffolding function of these annexins could be similar to what has been proposed for the annexin scaffold that is required for severing membrane blebs and microvesicles, including those containing toxin pores from cells [5]. Our data identify AnxA2 as a positive regulator of plasma membrane resealing in HUVEC. Although AnxA2 is the most abundant annexin in endothelium, other annexins including AnxA1 and AnxA5 are expressed as well [38]. While AnxA5 appears to exert a rather specific role in forming the 2D arrays at torn membranes, other annexins have been discussed to be functionally redundant. However, the highly specific effects obtained here by AnxA2 knock-down and re-expression indicate that no other annexin can compensate for AnxA2 in endothelial plasma membrane repair. Whether AnxA2 functions as a monomer or in complex with an EF hand-type S100 protein in this process has not been elucidated. A known S100 ligand of AnxA2 is S100A10, and AnxA2S100A10 complexes are present in endothelial cells [29]. However, recent evidence obtained in MCF7 tumor cells suggests that AnxA2 might also function in conjunction with a related S100 protein, S100A11, in the course of membrane repair [18]. In this respect, it is interesting to note that our kinetic analyses reveal a simultaneous recruitment of AnxA2 and S100A11 to the wound site. Thus, AnxA2 could potentially engage in Ca2 + dependent complex formation with S100A11 in the course of membrane resealing and such complexes could participate in recruiting and/or stabilizing a patch of internal membranes at the wound site, possibly by providing links between the membrane surfaces. Based on the known high resolution structures of S100A10 and S100A11 in complex with peptides of their annexin ligands reported so far, AnxA2 and AnxA1, such an AnxA2-S100A11 complex could exist [46–48]. Future experiments have to reveal whether AnxA2-S100A11 complexes indeed form following membrane wounding and possibly function in the resealing process. 5. Concluding remarks We show here that vascular endothelial cells are capable of resealing laser-induced plasma membrane wounds in an autonomous, active and Ca2 +-dependent process. Membrane repair in endothelial cells is accompanied by a recruitment of several annexins to the wound site nd requires the activity of AnxA2. Together, these data suggest endothelial cells can safeguard the integrity of their plasma membrane, which is subject to numerous shear, stretch and pulling forces. Transparency document The Transparency document associated with this article can be found in online version. Acknowledgements We thank Anthony Bouter (University of Bordeaux) for introducing SNK to the laser wounding assay, Ursula Rescher (University of Münster) for the AnxA2CM-GFP and AnxA2-sires-GFP plasmids, and

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Sarah Hardebeck for technical help. This work has been supported by grants from the German Research Council (DFG, SFB1009, project A06) and the Interdisciplinary Center for Clinical Research (Münster Medical School) to VG. SNK is a member of the joint graduate school Cells-in-Motion (EXC 1003 – CiM)/IMPRS, Münster, Germany. References [1] S.T. Cooper, P.L. McNeil, Membrane repair: mechanisms and pathophysiology, Physiol. Rev. 95 (2015) 1205–1240. [2] V. Idone, C. Tam, J.W. Goss, D. Toomre, M. Pypaert, N.W. Andrews, Repair of injured plasma membrane by rapid Ca2+-dependent endocytosis, J. Cell Biol. 180 (2008) 905–914. [3] P.L. McNeil, R.A. Steinhardt, Plasma membrane disruption: repair, prevention, adaptation, Annu. Rev. Cell Dev. Biol. 19 (2003) 697–731. [4] M. Bischofberger, I. Iacovache, F.G. van der Goot, Pathogenic pore-forming proteins: function and host response, Cell Host Microbe 12 (2012) 266–275. [5] E.B. Babiychuk, A. 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Please cite this article as: S.N. Koerdt, V. Gerke, Annexin A2 is involved in Ca2 +-dependent plasma membrane repair in primary human endothelial cells, Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbamcr.2016.12.007