Defects in G-Actin Incorporation into Filaments in Myoblasts Derived from Dysferlinopathy Patients Are Restored by Dysferlin C2 Domains

International audienceDysferlin is a transmembrane C-2 domain-containing protein involved in vesicle trafficking and membrane remodeling in skeletal muscle cells. However, the mechanism by which dysferlin regulates these cellular processes remains unclear. Since actin dynamics is critical for vesicle trafficking and membrane remodeling, we studied the role of dysferlin in Ca2+-induced G-actin incorporation into filaments in four different immortalized myoblast cell lines (DYSF2, DYSF3, AB320, and ER) derived from patients harboring mutations in the dysferlin gene. As compared with immortalized myoblasts obtained from a control subject, dysferlin expression and G-actin incorporation were significantly decreased in myoblasts from dysferlinopathy patients. Stable knockdown of dysferlin with specific shRNA in control myoblasts also significantly reduced G-actin incorporation. The impaired G-actin incorporation was restored by the expression of full-length dysferlin as well as dysferlin N-terminal or C-terminal regions, both of which contain three C2 domains. DYSF3 myoblasts also exhibited altered distribution of annexin A2, a dysferlin partner involved in actin remodeling. However, dysferlin N-terminal and C-terminal regions appeared to not fully restore such annexin A2 mislocation. Then, our results suggest that dysferlin regulates actin remodeling by a mechanism that does to not involve annexin A2

Src Kinases Regulate <i>De Novo</i> Actin Polymerization during Exocytosis in Neuroendocrine Chromaffin Cells

<div><p>The cortical actin network is dynamically rearranged during secretory processes. Nevertheless, it is unclear how <i>de novo</i> actin polymerization and the disruption of the preexisting actin network control transmitter release. Here we show that in bovine adrenal chromaffin cells, both formation of new actin filaments and disruption of the preexisting cortical actin network are induced by Ca²⁺ concentrations that trigger exocytosis. These two processes appear to regulate different stages of exocytosis; whereas the inhibition of actin polymerization with the N-WASP inhibitor wiskostatin restricts fusion pore expansion, thus limiting the release of transmitters, the disruption of the cortical actin network with cytochalasin D increases the amount of transmitter released per event. Further, the Src kinase inhibitor PP2, and cSrc SH2 and SH3 domains also suppress Ca²⁺-dependent actin polymerization, and slow down fusion pore expansion without disturbing the cortical F-actin organization. Finally, the isolated SH3 domain of c-Src prevents both the disruption of the actin network and the increase in the quantal release induced by cytochalasin D. These findings support a model where a rise in the cytosolic Ca²⁺ triggers actin polymerization through a mechanism that involves Src kinases. The newly formed actin filaments would speed up the expansion of the initial fusion pore, whereas the preexisting actin network might control a different step of the exocytosis process.</p></div

Dynamin-2 mutations linked to Centronuclear Myopathy impair actin-dependent trafficking in muscle cells

International audienceDynamin-2 is a ubiquitously expressed GTP-ase that mediates membrane remodeling. Recent findings indicate that dynamin-2 also regulates actin dynamics. Mutations in dynamin-2 cause dominant centronuclear myopathy (CNM), a congenital myopathy characterized by progressive weakness and atrophy of skeletal muscles. However, the muscle-specific roles of dynamin-2 affected by these mutations remain elusive. Here we show that, in muscle cells, the GTP-ase activity of dynamin-2 is involved in de novo actin polymerization as well as in actin-mediated trafficking of the glucose transporter GLUT4. Expression of dynamin-2 constructs carrying CNM-linked mutations disrupted the formation of new actin filaments as well as the stimulus-induced translocation of GLUT4 to the plasma membrane. Similarly, mature muscle fibers isolated from heterozygous knock-in mice that harbor the dynamin-2 mutation p.R465W, an animal model of CNM, exhibited altered actin organization, reduced actin polymerization and impaired insulin-induced translocation of GLUT4 to the sarcolemma. Moreover, GLUT4 displayed aberrant perinuclear accumulation in biopsies from CNM patients carrying dynamin-2 mutations, further suggesting trafficking defects. These results suggest that dynamin-2 is a key regulator of actin dynamics and GLUT4 trafficking in muscle cells. Our findings also support a model in which impairment of actin-dependent trafficking contributes to the pathological mechanism in dynamin-2-associated CNM

Disruption of c-Src SH2- or SH3-dependent associations inhibits de novo cortical actin assembly.

<p>ACCs were permeabilized in the presence of Alexa Fluor 488 G-actin, 10 µM free Ca²⁺ and 5 µM GST alone or growing concentrations of cSrc SH2-GST (SH2) or cSrc SH3-GST (SH3). (<b>A–B</b>) Representative images showing the new formation of cortical actin in cells permeabilized without peptides (non-treated) and in the presence of GST (A), or with different amounts of SH2 (B, left panel) or SH3 (B, right panel). Scale bar  = 10 µm. (<b>C</b>) Quantification demonstrates that 0.1 µM of c-Src SH2 or SH3 domain is sufficient to significantly disrupt Ca²⁺-dependent actin polymerization in ACCs. Data are means ± SEM of fluorescence intensity from at least 10 cells per each condition. *p<0.05 compared to GST.</p

Ca²⁺-dependent cortical actin polymerization colocalizes with secretory granules in permeabilized chromaffin cells.

<p>(<b>A–B</b>) Cultured ACCs were permeabilized in KGEP buffer for 6 minutes with 20 µM digitonin in the presence of increasing concentrations of free Ca²⁺ and 0.3 µM of Alexa Fluor 488 G-actin, fixed and visualized by confocal microscopy (top panel). Under these conditions, the actin ring corresponds to recently polymerized actin filaments. To evaluate the effect of growing free Ca²⁺ concentrations on the preexisting cortical actin ring, digitonin-permeabilized ACCs were fixed and stained with 1 µM phalloidin-rhodamine B (bottom panel). (<b>B</b>) Quantification of the cortical actin fluorescence intensity shows the coexistence of assembly (green line) and disassembly (red line) of the cortical actin network at 10 µM of free Ca² compared to basal Ca²⁺ levels (0.1 µM). Data are means ± SEM from 20–22 cells. (<b>C</b>) ACCs permeabilized in the presence of Alexa Fluor 488 G-actin and 10 µM of free Ca²⁺ were fixed and immunolabeled with a specific antibody against the chromaffin granule marker chromogranin A. F-actin and chromogranin A images were thresholded and colocalization sites were automatically detected (white spots). A colocalization site is highlighted in yellow. Note that the newly polymerized actin (green) colocalizes with chromogranin A (red). The mean score for the Pearson Correlation Coefficient between chromogranin A and F-actin at these sites was 0.89±0.03 (n = 12). Scale bar  = 10 µm.</p

Cytochalasin D does not affect the quantal release in cells injected with the cSrc SH3 domain.

<p>ACCs were injected with 5 µM GST or c-Src SH3-GST (SH3), and 20 min later incubated with 4 µM cytochalasin D (CytoD) or its vehicle DMSO for 10 min at 37°C. Then, exocytosis was induced with 20 µM ionomycin and monitored by amperometry. Data show average values ± S.E.M. of Imax, Q, t_1/2, tP, foot duration, foot amplitude, foot frequency and number of events during the recording from cells injected with GST and treated with DMSO (n = 15) or CytoD (n = 16) or injected with SH3 treated with DMSO (n = 24) or CytoD (n = 14). All amperometric parameter values correspond to the median values of the events from individual cells, which were subsequently averaged per treatment group. *p<0.05 compared with cells injected with GST and treated with DMSO. Note that SH3 and CytoD have common effects in tP, foot duration and foot frequency, but show dissimilar effects on Imax and Q. In the latter parameters, the effects of c-Src-SH3 prevail over those of CytoD.</p

Src kinase inhibition slows down the fusion pore expansion.

<p>Exocytosis was induced with 20 µM ionomycin and monitored by amperometry. Cells were incubated with 10 µM PP2 or its inactive isomer PP3 for 20 min before the exocytosis induction. These agents were present during the recording. GST, c-Src SH2-GST (SH2) or c-Src SH3-GST (SH3) was injected 30 min before cell stimulation. (<b>A</b>) Scheme of an amperometric spike with the analyzed parameters: peak amplitude (Imax), quantal size (Q), half-width (t_1/2), rise time (tP) and food duration. (<b>B</b>) Representative amperometric spikes from cells treated with PP3 or PP2, or injected with GST, SH2 or SH3. (<b>C</b>) Data show average values ± S.E.M. of Imax, Q, t_1/2, tP, foot frequency and foot duration of amperometric events in control cells (n = 35) or cells treated with PP3 (n = 15), PP2 (n = 20) or injected with GST (n = 13), SH2 (n = 12), SH3 (n = 15). All amperometric parameter values correspond to the median values of the events from individual cells, which were subsequently averaged per treatment group. ^&p<0.05 compared with control; *p<0.05 compared with PP3; ^†p<0.05 compared with GST.</p

c-Src-SH3 diminishes the F-actin disruption induced by cytochalasin D.

<p>ACCs were digitonin-permeabilized for 6 minutes in the presence of 10 µM free Ca²⁺, 4 µM cytochalasin D (CytoD) and GST or c-Src-SH3-GST (SH3) at 0.1 or 5 µM. Then cells were fixed and stained with 1 µM of phalloidin-rhodamine B. (<b>A</b>) Representative images of each experimental condition. Scale bar  = 10 µm. (<b>B</b>) Quantification of the cortical actin intensity fluorescence. Data are means ± SEM for at least 18 cells from 3 different cultures per each condition. Note a significantly higher cortical F-actin intensity in cells treated with CytoD in the presence of c-Src-SH3, at either 0.1 or 5 µM, as compared with cells treated with CytoD in the presence of GST (*p<0.05).</p

Influence of the different treatments on actin dynamics.

<p>(<b>A</b>) A stimulus that induces an increase in the cytosolic Ca²⁺ hastens SNARE-mediated fusion of secretory vesicles with the plasma membrane, but it also promotes both disruption of the preexisting actin network (green rosary beads) and the formation of new actin filaments (yellow rosary beads). This actin dynamics appears to favor the expansion of the fusion pore, but also prevents the collapse of the vesicle in the plasma membrane. This mechanism allows the fast release of soluble catecholamines (see the purple spike at the right). (<b>B</b>) The N-WASP inhibitor wiskostatin (Wks) strongly inhibits the new actin polymerization, while it slightly disturbs the preexisting cortical F-actin network (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099001#pone-0099001-g009" target="_blank">Figure 9</a>). Such effect could be due to a reduction of the slow-rate of actin polymerization/depolymerization in resting conditions. Wsk also gives rise to incomplete release events (see the small green spike), suggesting that the lack of actin polymerization hinders the expansion of the fusion pore. Wsk could also perturb membrane transport, decreasing cellular ATP levels and Ca²⁺ entry <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099001#pone.0099001-Guerriero1" target="_blank">[52]</a>. These effects could also contribute to the failure of the fusion pore expansion. (<b>C</b>) Cytochalasin D (CytoD) greatly disrupts the preexisting F-actin network, as well as the new F-actin formation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099001#pone-0099001-g007" target="_blank">Figure 7</a>). CytoD also slows down the fusion pore expansion and increases the quantal size of the release events (see the big brown spike with a long foot). Probably the loss of actin meshwork favors the collapse of the vesicle in the plasma membrane, as previously proposed by Doreian <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099001#pone.0099001-Doreian1" target="_blank">[8]</a>. (<b>D</b>) The inhibition of Src kinases with PP2 suppresses the <i>de novo</i> F-actin formation, but it does not disturb the pre-existing cortical actin network (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099001#pone-0099001-g002" target="_blank">Fig. 2</a>). PP2 also slows down the fusion pore expansion, and reduces the quantal size of the release events (note the small blue spike with a long foot). Similar effects were observed with the microinjections of cSrc-SH2 or -SH3 domains. These findings support the idea that the new F-actin formation favors the expansion of the fusion pore.</p

Inhibition of N-WASP activation suppresses de novo actin polymerization and reduces the quantal release of transmitters.

<p>(<b>A–B</b>) ACCs were permeabilized in the presence of 10 µM free Ca²⁺ and 5 µM of the N-WASP inhibitor wiskostatin (Wsk), or the vehicle DMSO. The new F-actin formation was evaluated in the presence of 0.3 µM of AF488-G-actin. Note that Wsk treatment significantly disrupted the new F-actin polymerization. (<b>C–F</b>) Cultured ACC were incubated with the N-WASP inhibitor Wsk for 5 minutes at 37°C, then maintained in resting conditions (C–D) or stimulated with 20 µM ionomycin (E–F), fixed and stained with phalloidin-rhodamine B for confocal visualization. Note that Wsk slightly disturbs the cortical actin ring in resting cells, but it did not induce an additional F-actin disruption in ionomycin treated cells. A, C and E show representative confocal images for each condition. Scale bar  = 10 µm. B, D and F correspond to quantification of the cortical actin fluorescence intensity, where data are means ± SEM from at least 11 cells per each condition. *p<0.05 compared to DMSO. (<b>G–J</b>) Cultured ACCs were incubated with 5 µM Wsk for 5 min prior to the exocytosis induction, then this agent was kept during the amperometric recording. Representative amperometric spikes are shown in panel G. Data show average values ± S.E.M. of the number of cumulative exocytotic events (H), Imax (I) or Q (J) from cells treated with DMSO (n = 29) or Wsk (n = 15). The amperometric parameter values correspond to the median values of the events from individual cells, which were subsequently averaged per treatment group. Note that the inhibition of N-WASP activation with Wsk reduced the number of exocytotic events (D), and decreased the amplitude (E) and quantal size (F).*p<0.05 compared to DMSO.</p