Dynamin-2 Regulates Fusion Pore Expansion and Quantal Release through a Mechanism that Involves Actin Dynamics in Neuroendocrine Chromaffin Cells

<div><p>Over the past years, dynamin has been implicated in tuning the amount and nature of transmitter released during exocytosis. However, the mechanism involved remains poorly understood. Here, using bovine adrenal chromaffin cells, we investigated whether this mechanism rely on dynamin’s ability to remodel actin cytoskeleton. According to this idea, inhibition of dynamin GTPase activity suppressed the calcium-dependent <i>de novo</i> cortical actin and altered the cortical actin network. Similarly, expression of a small interfering RNA directed against dynamin-2, an isoform highly expressed in chromaffin cells, changed the cortical actin network pattern. Disruption of dynamin-2 function, as well as the pharmacological inhibition of actin polymerization with cytochalasine-D, slowed down fusion pore expansion and increased the quantal size of individual exocytotic events. The effects of cytochalasine-D and dynamin-2 disruption were not additive indicating that dynamin-2 and F-actin regulate the late steps of exocytosis by a common mechanism. Together our data support a model in which dynamin-2 directs actin polymerization at the exocytosis site where both, in concert, adjust the hormone quantal release to efficiently respond to physiological demands.</p></div

Dynamin-2 and actin polymerization regulate the fusion pore expansion and quantal size in BCC.

<p>Chromaffin cells were incubated with 4 µM CytoD during 10 minutes at 37°C. After that the exocytosis was evoked with 10 µM DMPP. <b>A–C:</b> Data show average values ± SEM of Q (A), t_1/2 (B) and foot duration (C) of amperometric spikes induced by 10 µM DMPP in cells transfected with pEGFP (n = 27), Dyn2K44A (n = 13) or iRNADyn2 (n = 16). All amperometric parameter values correspond to the median values of the events from individual cells, which were subsequently averaged per treatment group. Thus, n correspond to the number of cells in each treatment group. Note that the CytoD treatment (grey bars) significantly increased Q, t_1/2 and foot duration of the exocytotic events in cells transfected with pEGFP, without additional effects in cells transfected with Dyn2K44A or iRNADyn2. * p<0.05 compared with the untreated cells (Kruskal-Wallis test).</p

Amperometric parameters of exocytotic events induced by 10 µM DMPP in cells transfected with pEGFP, UnR-iRNA, iRNADyn2, Dyn2WT or Dyn2K44A.

<p>Data are means ± SEM of averages, where n is the number of cells. Imax corresponds to the spike amplitude.</p>*<p>p<0.05 compared with pEGFP-transfected cells;</p>†<p>p<0.05 compared with Dyn2 WT-transfected cells (Kruskal-Wallis test).</p

Impaired function or expression of dynamin-2 change F-actin organization pattern.

<p>Cells were transfected with Life-act-ruby (n = 11) or co-transfected with Life-act-ruby and pEGFP (n = 34), iRNA-UnR (n = 9) Dyn2WT (n = 21), Dyn2K44A (n = 31), iRNADyn2 (n = 38) or Eps15ED95/295 (n = 17) plasmids and visualized by TIRF microscopy 48 h later. To evaluate the effects of a pharmacological inhibition of dynamin, cells transfected with Life-act-ruby were treated with 100 µM dynasore (n = 28), or the vehicle DMSO (n = 25) during 1 hr at 37°C. The 81.8% of control cells exhibited a “normal” pattern with clear cortical actin fibers. This value was not significantly different in cells expressing pEGFP (73.6%), iRNA-UnR (88.9%) or Dyn2WT (85.7%) constructs. However, the expression of Dyn2K44A or iRNADyn2, as well as the treatment with dynasore, modified the cortical actin organization and 80.6%, 92.1% and 71.4% of the cells, respectively, exhibited a “punctuate” pattern. The treatment with 4 µM CytoD during 10 minutes at 37°C produced exactly the same effect: 84.6% of the cells displayed a “punctuate” pattern. Eps15ED95/295 expression did not alter actin organization (82.4% of cells exhibited a normal pattern), indicating that dynamin, but not of endocytosis disruption, modified the actin cytoskeleton pattern. Scale bar = 5 µm.</p

Calcium-dependent cortical actin polymerization in permeabilized chromaffin cells.

<p>Cultured chromaffin cells were permeabilized in buffer KGEP (mM: 139 K⁺-glutamate, 20 Pipes, 5 EGTA, 2 ATP-Mg and 0.01 free calcium, pH 6.6) during 6 minutes with 20 µM digitonin in the presence of 0.3 µM Alexa-Fluor488-G-actin conjugate (AF488-G-actin), fixed and visualized by confocal microscopy. <b>A:</b> Total F-actin was stained using 1 µM phalloidin-rodhamine B (red) and nuclei were stained with 5 µg/ml DAPI (blue). Note that newly synthesized actin was incorporated into pre-existing cortical filaments. <b>B–C:</b> The new formation of cortical actin filaments was assessed by quantifying AF488-G-actin staining mean intensity at the cell periphery in the presence of increasing free Ca²⁺ concentrations. Note that maximal cortical actin polymerization was observed at a range of 1–10 µM of free Ca^2+. Scale = 10 µm. Data are means of cortical actin fluorescence intensity from at least 12 cells per each Ca²⁺ concentration (12 cells for 0.01 µM Ca²⁺, 13 cells for 0.1 µM Ca²⁺, 15 cells for 1 µM Ca²⁺,and 18 cells for 10 µM Ca²⁺).</p

Inhibition of dynamin GTP-ase activity suppresses Ca²⁺-dependent <i>de novo</i> cortical actin polymerization.

<p><b>A:</b> Representative images of F-actin formation in cells permeabilized in the presence of 10 µM free Ca²⁺. Note that no new polymerized cortical actin was observed when the permeabilization was performed in the absence of ATP-Mg (n = 16) or in the presence of 4 µM CytoD (n = 27) or 100 µM dynasore (n = 28) Scale bar = 10 µm <b>B:</b> Quantification of G-actin staining mean intensity at the cell periphery. Data are means of cortical actin fluorescence intensity *p<0.05 compared with cells treated with DMSO (ANOVA).</p

Intracellular distribution of endogenous NECC2 in PC12 cells.

<p><i>A</i>. Representative confocal images of a PC12 cell immunolabeled with the anti-NECC2 antiserum. NECC2 distributes throughout the cytoplasm and in close apposition to the cell membrane. Specificity of the signal was tested by preadsorption of the anti-NECC2 antibody with excess of antigen. <i>B</i>. PC12 cells were double-stained with antibodies against NECC2 (red) and caveolin-1 or actin (green) (top and bottom panels respectively). Significant overlap between markers at the cell periphery is shown in the binary mask at the rightmost panels. <i>C</i>. Prior to double immunostaining with anti-NECC2 and anti-caveolin-1, PC12 cells were treated with 5 µmol/L of LatB for 30 min at 37°C. Scale bars, 10 µm.</p

NECC2 expression and distribution are regulated by NGF.

<p><i>A</i>. Representative confocal images of HA-<i>TrkA</i> transfected PC12 cells under basal conditions or treated with NGF at the indicated time points. After treatment, cells were subjected to double-immunofluorescence using anti-NECC2 and anti-HA antibodies. The colocalization channel was isolated using Imaris 6.4 (Bitplane) and shown alone in the images on the far bottom. Mander’s coefficient A (between NECC2 and TkA) was calculated to quantify the degree of colocalization and represented as the mean ±SEM of at least 5 cells per experimental group. a, <i>P</i> < 0.05 <i>vs</i>. control; b, <i>P</i> < 0.05 <i>vs</i>. 5 min; c, <i>P</i> < 0.05 <i>vs</i>. 30 min (unpaired, 2-tailed t test). <i>B</i>. Representative micrographs of PC12 cells immunolabeled with anti-NECC2 during long-term (1-4 days) stimulation with NGF. NECC2 staining localizes beneath the plasma membrane as well as in puncta/vesicular-like structures in growing neurites and tips. <i>C</i>. Double-immunostaining of NGF-differentiated PC12 cells with anti-NECC2 and anti-caveolin-1 sera. Scale bars, 10 µm. <i>D</i>. Protein extracts from NGF-stimulated PC12 cells during differentiation (1-4 days) were analyzed by immunoblotting using the anti-NECC2 antibody. Quantitative data were represented as ratio of NECC2 vs. Ponceau. The data represent the mean (± SEM) of three independent experiments. a, <i>P</i> < 0.05 <i>vs</i>. corresponding control; b, <i>P</i> < 0.05 <i>vs</i>. 1- or 2-days treated cells (one-way ANOVA followed by <i>Newman–Keuls</i> test).</p

In silico analysis of rat NECC2 sequence.

<p><i>A</i>. Schematic representation of the structural and functional motifs predicted in rat NECC2 amino acid sequence. <i>B</i>. Schematic representation of the genomic structure of rat <i>Necc2</i> coding for the <i>Necc2</i> isoform containing the HR domain and a newly identified transcript lacking this domain (<i>Necc2</i>α). Arrows indicate the location of the paired primers used to amplify both <i>Necc2</i> transcripts. <i>C</i>. Standard PCR amplification in PC12 cells shows two PCR products with the expected sizes for the two rat <i>Necc2</i> transcripts (left panel; primers a and b). Nested PCR amplification of <i>Necc2</i> transcript (central panel; primer c) or the <i>Necc2</i>α transcript (rightmost panel; primer d) using specific internal reverse primers. Non-DNA samples (C-) are shown as controls for exogenous contamination.</p

Overexpression of NECC2 inhibits NGF-mediated TrkA signaling pathway.

<p><i>A</i>. Representative confocal images of PC12 cells transfected with cMyc-<i>Necc2</i>ΔHR and double-stained with anti-cMyc and anti-TrkA antibodies. Immunofluorescent signals significantly overlap at the cell periphery and intracellularly as shown in the binary mask (right panels). Scale bars, 10 µm. <i>B</i>, <i>C</i>, <i>D</i> and <i>E</i>. PC12 cells transiently transfected with full-length cMyc-<i>Necc2</i>, cMyc-<i>Necc2</i>ΔHR, or the empty vector (mock) were grown to 90% confluence and exposed for 4 h to serum-low differentiation media before NGF stimulation for the indicated time points. Whole cell protein extracts were then subjected to immunoblot with Akt and phospho-Akt (pAkt) antibodies (<i>B</i> and <i>C</i>) or with ERK and phosphor-ERK (pERK) antibodies (<i>D</i> and <i>E</i>). Quantitative data were represented as ratio of pAkt <i>vs</i>. Akt or pERK vs. ERK, respectively. The data represent the means (± SEM) of three independent experiments. <i>P</i> < 0.05 <i>vs</i>. corresponding control (unpaired, 2-tailed t test).</p