Homozygous zebrafish of the mutant relaxed (red ts25 ) are paralyzed and die within days after hatching. A significant reduction of intramembrane charge movements and the lack of depolarizationinduced but not caffeine-induced Ca 2؉ transients suggested a defect in the skeletal muscle dihydropyridine receptor (DHPR). Sequencing of DHPR cDNAs indicated that the ␣1S subunit is normal, whereas the ␤1a subunit harbors a single point mutation resulting in a premature stop. Quantitative RT-PCR revealed that the mutated gene is transcribed, but Western blot analysis and immunocytochemistry demonstrated the complete loss of the ␤1a protein in mutant muscle. Thus, the immotile zebrafish relaxed is a ␤1a-null mutant. Interestingly, immunocytochemistry showed correct triad targeting of the ␣1S subunit in the absence of ␤1a. Freeze-fracture analysis of the DHPR clusters in relaxed myotubes revealed an Ϸ2-fold reduction in cluster size with a normal density of DHPR particles within the clusters. Most importantly, DHPR particles in the junctional membranes of the immotile zebrafish mutant relaxed entirely lacked the normal arrangement in arrays of tetrads. Thus, our data indicate that the lack of the ␤1a subunit does not prevent triad targeting of the DHPR ␣1S subunit but precludes the skeletal muscle-specific arrangement of DHPR particles opposite the ryanodine receptor (RyR1). This defect properly explains the complete deficiency of skeletal muscle excitationcontraction coupling in ␤1-null model organisms.calcium channels ͉ excitation-contraction coupling ͉ tetrads ͉ zebrafish E xcitation-contraction (EC) coupling is understood as the signal transduction process connecting membrane depolarization to the contraction of muscle cells. This process is initiated by the concerted action of two Ca 2ϩ channels, the plasmalemmal voltage-gated dihydropyridine receptor (DHPR) and the sarcoplasmic reticulum (SR) ryanodine receptor (RyR). In junctions of the SR with the plasma membrane (peripheral couplings) or with the transverse tubules (triads), membrane depolarization is sensed by the DHPR, which then triggers RyR opening and Ca 2ϩ release from the SR. In skeletal muscle cells, this signaltransduction is independent of Ca 2ϩ influx through the DHPR (1) but depends on protein-protein interaction between the DHPR and the RyR1 (2, 3). This physical coupling requires the coordinated arrangement of DHPRs and RyR1s in the junctions. In skeletal muscle triads and peripheral couplings, groups of four DHPRs (tetrads) are arranged in orthogonal arrays matching the opposing RyR1 arrays (4). Formation of DHPR tetrads requires the presence of RyR1.The skeletal muscle DHPR complex is composed of the voltage-sensing and pore-forming ␣ 1S subunit and the auxiliary subunits ␤ 1a , ␣ 2 ␦-1, and ␥ (5). Targeted deletions of the ␣ 2 ␦-1 and ␥ subunits do not critically interfere with EC coupling function (6, 7). In contrast, ␣ 1S and ␤ 1a subunit null-mutant mice display a lack of EC coupling and, thus, lethal muscle paralysis (8, 9). Although failure o...

Ca2+ is considered a key element in multiple steps during regulated exocytosis. During the postfusion phase, an elevated cytoplasmic Ca 2+ concentration ([Ca 2+ ]) c leads to fusion pore dilation. In neurons and neuroendocrine cells, this results from activation of voltage-gated Ca 2+ channels in the plasma membrane. However, these channels are activated in the prefusion stage, and little is known about Ca 2+ entry mechanisms during the postfusion stage. This may be particularly important for slow and nonexcitable secretory cells. We recently described a "fusion-activated" Ca 2+ entry (FACE) mechanism in alveolar type II (ATII) epithelial cells. FACE follows initial fusion pore opening with a delay of 200-500 ms. The site, molecular mechanisms, and functions of this mechanism remain unknown, however. Here we show that vesicle-associated Ca 2+ channels mediate FACE. Using RT-PCR, Western blot analysis, and immunofluorescence, we demonstrate that P2X 4 receptors are expressed on exocytotic vesicles known as lamellar bodies (LBs). Electrophysiological, pharmacological, and genetic data confirm that FACE is mediated via these vesicular P2X 4 receptors. Furthermore, analysis of fluorophore diffusion into and out of individual vesicles after exocytotic fusion provides evidence that FACE regulates postfusion events of LB exocytosis via P2X 4 . Fusion pore dilation was clearly correlated with the amplitude of FACE, and content release from fused LBs was accelerated in fusions followed by FACE. Based on these findings, we propose a model for regulation of the exocytotic postfusion phase in nonexcitable cells in which Ca 2+ influx via vesicular Ca 2+ channels regulates fusion pore expansion and vesicle content release.egulated secretion is a fundamental cellular process in many different types of eukaryotic cells, with Ca 2+ -triggered exocytosis being the key element (1-4). Multiple Ca 2+ -dependent steps have been elucidated that ultimately lead to fusion of exocytic vesicles with the plasma membrane, resulting in formation of an aqueous channel, the fusion pore, through which vesicle contents are released (5-8). Although the molecular composition of the fusion pore remains elusive, there is a general acceptance that fusion pores are not merely passive structures, but that their opening and closure are highly regulated and control, or even fine-tune, vesicle content secretion (9-14). Voltage-gated Ca 2+ channels are not present (25). After LB fusion with the plasma membrane, surfactant, a water-insoluble bulky complex, largely remains entrapped within the fused vesicles (26) in which the fusion pores behave as regulated valves or mechanical barriers for release (16,27). As a result, in vitro full content release can be delayed for minutes up to hours (28).We recently reported a "fusion-activated" Ca 2+ entry (FACE) mechanism as a phenomenon in the postfusion phase of surfactant secretion (29). Given that this Ca 2+ signal occasionally spreads throughout the cell, we speculated that it might be important for triggering...

SummaryThe C3 transferases from Clostridium botulinum (C3bot) and Clostridium limosum (C3lim) mono-ADP-ribosylate and thereby inactivate RhoA, -B and -C of eukaryotic cells. Due to their extremely poor cellular uptake, C3 transferases were supposed to be exoenzymes rather than exotoxins, challenging their role in pathogenesis. Here, we report for the first time that low concentrations of both C3lim and C3bot are selectively internalized into macrophages/monocytes in less than 3 h, inducing the reorganization of the actin cytoskeleton by ADP-ribosylation of Rho. We demonstrate that C3 transferases are internalized into the cytosol of macrophages/monocytes via acidified early endosomes. Bafilomycin A1, an inhibitor of endosomal acidification, protected J774A.1 macrophages and human promyelotic leukaemia cells (HL-60) from intoxication by C3. Moreover, confocal laser scanning microscopy revealed colocalization of C3 with early endosomes. An extracellular acidic pulse enabled direct translocation of cell surface-bound C3 across the cytoplasmic membrane to the cytosol. In line with this finding, both C3 proteins exhibited membrane activity in lipid bilayer membranes only under acidic conditions (pH < 5.5). In conclusion, we identified macrophages/monocytes as target cells for clostridial C3 transferases and shed light on their selective uptake mechanism, which might contribute to understand the role of C3 transferases in pathogenesis.

Type II pneumocytes secrete surfactant, a lipoprotein-like substance reducing the surface tension in the lung, by regulated exocytosis of secretory vesicles termed lamellar bodies (LBs). This secretory process is characterized by a protracted postfusion phase in which fusion pores open slowly and may act as mechanical barriers for release. Combining dark-field with fluorescence microscopy, we show in ss-actin green fluorescent protein-transfected pneumocytes that LB fusion with the plasma membrane is followed by actin coating of the fused LB. This is inhibited by cytoplasmic Ca(2+) chelation or the phospholipase D inhibitor C2 ceramide. Actin coating occurs by polymerization of actin monomers, as evidenced by staining with Alexa 568 phalloidin. After actin coating of the fused LB, it either shrinks while releasing surfactant ("kiss-coat-and-release"), remains in this fused state without further action ("kiss-coat-and-wait"), or is retrieved and pushed forward in the cell on top of an actin tail ("kiss-coat-and-run"). In the absence of actin coating, no release or run was observed. These data suggest that actin coating creates a force needed for either extrusion of vesicle contents or retrieval and intracellular propulsion.