Calcium-regulated exocytosis is required for cell membrane resealing.

Bi, Guo-Qiang; Alderton, Janet M.; Steinhardt, Richard A.

doi:10.1083/jcb.131.6.1747

Cited by 266 publications

(256 citation statements)

References 57 publications

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“…In contrast, membrane repair occurred in the presence of cytochalasin D and cycloheximide. These findings were congruent with those that have been reported for the resealing of physical membrane lesions (20,21). With the exception of microinjection, methods do not exist to deliver the isolated domain to the cytosol of living cells.…”

Section: Resultssupporting

confidence: 90%

Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O

Walev

Bhakdi²,

Hofmann³

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

299

292

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The pore-forming toxin streptolysin O (SLO) can be used to reversibly permeabilize adherent and nonadherent cells, allowing delivery of molecules with up to 100 kDa mass to the cytosol. Using FITC-labeled albumin, 10 5 -10 6 molecules were estimated to be entrapped per cell. Repair of toxin lesions depended on Ca 2؉ -calmodulin and on intact microtubules, but was not sensitive to actin disruption or to inhibition of protein synthesis. Resealed cells were viable for days and retained the capacity to endocytose and to proliferate. The active domains of large clostridial toxins were introduced into three different cell lines. The domains were derived from Clostridium difficile B-toxin and Clostridium sordelli lethal toxin, which glycosylate small G-proteins, and from Clostridium botulinum C2 toxin, which ADP-ribosylates actin. After delivery with SLO, all three toxins disrupted the actin cytoskeleton to cause rounding up of the cells. Glucosylation assays demonstrated that G-proteins Rho and Ras were retained in the permeabilized cells and were modified by the respective toxins. Inactivation of these G-proteins resulted in reduced stimulus-dependent granule secretion, whereas ADP-ribosylation of actin by the C. botulinum C2-toxin resulted in enhanced secretion in cells. The presented method for introducing proteins into living cells should find multifaceted application in cell biology.protein delivery ͉ pore-forming toxin

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Section: Resultssupporting

confidence: 90%

Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O

Walev

Bhakdi²,

Hofmann³

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

299

292

View full text Add to dashboard Cite

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“…This is best demonstrated in red blood cells (40). However, most plasma membrane lesions, particularly if they are large, repair only if intracellular lipids are shuttled to the plasma membrane by an active, energy-dependent, and Ca 2ϩ -regulated process (41)(42)(43). The insertion of lipids into the plasma membrane causes a fall in plasma membrane tension, which in turn promotes "self-sealing" by lateral lipid flow (44,45).…”

Section: Molecular Mechanisms Of Plasma Membrane Repairmentioning

confidence: 99%

Hypercapnic Acidosis Impairs Plasma Membrane Wound Resealing in Ventilator-injured Lungs

Doerr

Gajić

Berríos

et al. 2005

Am J Respir Crit Care Med

118

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The objective of this study was to assess the effects of hypercapnic acidosis on lung cell injury and repair by confocal microscopy in a model of ventilator-induced lung injury. Three groups of normocapnic, hypocapnic, and hypercapnic rat lungs were perfused ex vivo, either during or after injurious ventilation, with a solution containing the membrane-impermeant label propidium iodide. In lungs labeled during injurious ventilation, propidium iodide fluorescence identifies all cells with plasma membrane wounds, both permanent and transient, whereas in lungs labeled after injurious ventilation propidium iodide fluorescence identifies only cells with permanent plasma membrane wounds. Hypercapnia minimized the adverse effects of high-volume ventilation on vascular barrier function, whereas hypocapnia had the opposite effect. Despite CO 2 -dependent differences in lung mechanics and edema the number of injured subpleural cells per alveolus was similar in the three groups (0.48 Ϯ 0.34 versus 0.51 Ϯ 0.19 versus 0.43 Ϯ 0.20 for hypocapnia, normocapnia, and hypercapnia, respectively). However, compared with normocapnia the probability of wound repair was significantly reduced in hypercapnic lungs (63 versus 38%; p Ͻ 0.02). This finding was subsequently confirmed in alveolar epithelial cell scratch models. The potential relevance of these observations for lung inflammation and remodeling after mechanical injury is discussed.Keywords: permissive hypercapnia; plasma membrane wounding and repair; ventilator-induced lung injury So-called lung protective mechanical ventilation strategies emphasize lung recruitment and the avoidance of large tidal volumes. Such strategies are often associated with hypercapnic acidosis. Many experts view "permissive hypercapnia" as a necessary evil of a low tidal ventilation strategy (1). Concern about detrimental effects of acidemia on renal and cardiovascular function have motivated attempts to enhance CO 2 removal by tracheal gas insufflation (2) and have led to unsubstantiated recommendations about the use of bicarbonate buffers in hypercapnic patients (3). More recent data suggest that hypercapnia may actually protect the lung from certain manifestations of ischemiareperfusion, endotoxin, and mechanical ventilation-related injury (4-9). Hypocapnia, in contrast, and correction of acidemia may be harmful (10-12). The specific mechanisms through which hypercapnia influences lung injury and vascular barrier function remain uncertain (13). CO 2 generates H ϩ ions, which react with titratable groups in certain amino acids and/or interacts directly (Received in original form September 3, 2003; accepted in final form January 21, 2005) Supported by grants from the National Institutes of Health (HL-63178), GlaxoSmithKline, and the Brewer Foundation. with free amine groups in proteins to form carbamate residues (14-16). CO 2 -dependent effects on the generation of reactive oxygen species and reactive nitrogen species as well as effects on ion channel conductivity have all been considered (17, 18).V...

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“…Small disruptions on the order of micron-diameter evoke the Ca 2ϩ -dependent exocytosis of vesicles near the wound site, which is essential for successful membrane resealing (2)(3)(4)(5)(6)(7)(8). The recruitment of vesicles to docking sites near the disruption is dependent on the motor proteins, kinesin and myosin, and the activity of Ca 2ϩ /calmodulin-dependent kinase (CaMK) 1 (2,4,9).…”

Section: From the Misaki Marine Biological Station The University Ofmentioning

confidence: 99%

Long-term Potentiation of Wound-induced Exocytosis and Plasma Membrane Repair Is Dependant on cAMP-response Element-mediated Transcription via a Protein Kinase C- and p38 MAPK-dependent Pathway

Togo¹

2004

Journal of Biological Chemistry

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Ca 2؉ -regulated exocytosis is required for rapid resealing of disrupted plasma membranes. It has been previously demonstrated that repeated membrane disruptions reseal more quickly than the initial wound and that this facilitated response requires the transcription factor cAMP-response element-binding protein (CREB). This study examines the signaling pathway between membrane disruption and CREB-dependent gene expression in 3T3 fibroblasts. A reporter gene assay using pCRE-d2EGFP revealed that membrane disruption induced CRE-mediated transcription. Immunofluorescence observations suggested that membrane disruption activated CREB, p38 mitogen-activated protein kinase (p38 MAPK), and MAPK kinase3/6, the kinase responsible for activation of p38 MAPK. CREB phosphorylation upon membrane disruption was inhibited by a specific p38 MAPK inhibitor, SB203580. Both CRE-mediated transcription and long-term potentiation of membrane resealing and wound-induced exocytosis were suppressed when cells were wounded in the presence of either SB203580 or Gö -6976, a specific protein kinase C (PKC) inhibitor. Furthermore, activation of MAPK kinase3/6 was impaired by PKC inhibition during membrane disruption. These results suggest that PKC mediates the stimulation of CREB-dependent gene expression through a p38 MAPK pathway upon membrane disruption.Mechanical stress induces disruptions of plasma membranes in many animal tissues under physiological conditions, and a cell survives these disruptions by rapidly resealing its cell membrane (1). Small disruptions on the order of micron-diameter evoke the Ca 2ϩ -dependent exocytosis of vesicles near the wound site, which is essential for successful membrane resealing (2-8). The recruitment of vesicles to docking sites near the disruption is dependent on the motor proteins, kinesin and myosin, and the activity of Ca 2ϩ /calmodulin-dependent kinase (CaMK) 1 (2, 4, 9). It has been proposed that wound-induced exocytosis promotes resealing by decreasing membrane tension (10).Expression of genes having a cAMP-response element (CRE) can be activated through a variety of signaling cascades that include protein kinase A (PKA), CaMK, and mitogen-activated protein kinase (MAPK) pathways. These pathways are reported to be involved in complex and diverse processes such as learning, memory, synaptic plasticity, development, and stress responses (11)(12)(13)(14). Induction of these signaling pathways activates the transcription factor cAMP-response element binding protein (CREB) by specifically phosphorylating amino acid Ser-133.Wound-induced exocytosis is potentiated following an initial wound so that subsequent cell membrane disruptions reseal more quickly (6, 15). It has also been indicated that the potentiation of membrane resealing and wound-induced exocytosis last at least 24 h after the initial wound and require CREB because a dominant-negative CREB mutant inhibited these processes (15). Furthermore, it has been demonstrated that inhibition of PKA during the initial wound does not affect long-term p...

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Calcium-regulated exocytosis is required for cell membrane resealing.

Cited by 266 publications

References 57 publications

Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O

Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O

Hypercapnic Acidosis Impairs Plasma Membrane Wound Resealing in Ventilator-injured Lungs

Long-term Potentiation of Wound-induced Exocytosis and Plasma Membrane Repair Is Dependant on cAMP-response Element-mediated Transcription via a Protein Kinase C- and p38 MAPK-dependent Pathway

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