The sarcomere is the contractile unit within cardiomyocytes driving heart muscle contraction. We sought to test the mechanisms regulating actin and myosin filament assembly during sarcomere formation. Therefore, we developed an assay using human cardiomyocytes to monitor sarcomere assembly. We report a population of muscle stress fibers, similar to actin arcs in non-muscle cells, which are essential sarcomere precursors. We show sarcomeric actin filaments arise directly from muscle stress fibers. This requires formins (e.g., FHOD3), non-muscle myosin IIA and non-muscle myosin IIB. Furthermore, we show short cardiac myosin II filaments grow to form ~1.5 μm long filaments that then ‘stitch’ together to form the stack of filaments at the core of the sarcomere (i.e., the A-band). A-band assembly is dependent on the proper organization of actin filaments and, as such, is also dependent on FHOD3 and myosin IIB. We use this experimental paradigm to present evidence for a unifying model of sarcomere assembly.
G protein–coupled receptors (GPCRs) that couple to Gi/o proteins modulate neurotransmission presynaptically by inhibiting exocytosis. Release of Gβγ subunits from activated G proteins decreases the activity of voltage-gated Ca2+ channels (VGCCs), decreasing excitability. A less understood Gβγ-mediated mechanism downstream of Ca2+ entry is the binding of Gβγ to SNARE complexes, which facilitate the fusion of vesicles with the cell plasma membrane in exocytosis. Here, we generated mice expressing a form of the SNARE protein SNAP25 with premature truncation of the C terminus and that were therefore partially deficient in this interaction. SNAP25Δ3 homozygote mice exhibited normal presynaptic inhibition by GABAB receptors, which inhibit VGCCs, but defective presynaptic inhibition by receptors that work directly on the SNARE complex, such as 5-hydroxytryptamine (serotonin) 5-HT1b receptors and adrenergic α2a receptors. Simultaneously stimulating receptors that act through both mechanisms showed synergistic inhibitory effects. SNAP25Δ3 homozygote mice had various behavioral phenotypes, including increased stress-induced hyperthermia, defective spatial learning, impaired gait, and supraspinal nociception. These data suggest that the inhibition of exocytosis by Gi/o-coupled GPCRs through the Gβγ-SNARE interaction is a crucial component of numerous physiological and behavioral processes.
Objective. To determine novel genes regulated by tumor necrosis factor ␣ (TNF␣) signaling in primary rheumatoid arthritis synovial fibroblasts (RASFs).Methods. Oligonucleotide microarrays were used to measure gene expression levels in 6 independent replicate samples of RASFs. RASFs were transfected for 18 hours with AdIB-dominant negative (AdIB-DN) (n ؍ 3) or with control AdTet expressing the reverse tetracycline trans-activator (n ؍ 3). The cells were stimulated for 3 hours with TNF␣, and total RNA was prepared. Several novel parametric and nonparametric methods were used to rank genes in terms of the magnitude and significance of intergroup differences. Microarray expression differences were confirmed by real-time quantitative reverse transcription-polymerase chain reaction. Small interfering RNA (siRNA) was used to specifically down-modulate microarrayidentified genes to demonstrate their role in the promotion of apoptosis, proliferation, or matrix metalloproteinase (MMP) expression. Rheumatoid arthritis (RA) is associated with inflammation of the synovium and development of RA synovial fibroblasts (RASFs) that undergo hyperplasia and invade cartilage and bone (1). RASFs exhibit an increased ability to enter into the cell cycle and, therefore, to undergo hyperplasia (2,3), and they show a decreased ability to undergo apoptosis (4,5). RASFs produce proinflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor ␣ (TNF␣), which provide further stimulation for the inflammation in RA (6), as well as enzymes, including stromelysin and collagenase, which are capable of invading cartilage and bone (7,8). Thus, RASFs are unique, and their altered properties of growth and production of proinflammatory and matrix-degrading proteins are central to the erosion of cartilage and bone that is seen in RA. Results. Blocking of NF-B byThe cytokine TNF␣ is central to the development and continued growth and invasion of RASFs (9).
Spatial and temporal regulation of neurotransmitter release is a complex process accomplished by the exocytotic machinery working in tandem with numerous regulatory proteins. G-protein ␥ dimers regulate the core process of exocytosis by interacting with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins soluble N-ethylmaleimide-sensitive factor attachment protein-25 (SNAP-25), syntaxin 1A, and synaptobrevin. G␥ binding to ternary SNAREs overlaps with calcium-dependent binding of synaptotagmin, inhibiting synaptotagmin-1 binding and fusion of the synaptic vesicle. To further explore the binding sites of G␥ on SNAP-25, peptides based on the sequence of SNAP-25 were screened for G␥ binding. Peptides that bound G␥ were subjected to alanine scanning mutagenesis to determine their relevance to the G␥-SNAP-25 interaction. Peptides from this screen were tested in protein-protein interaction assays for their ability to modulate the interaction of G␥ with SNAP-25. A peptide from the C terminus, residues 193 to 206, significantly inhibited the interaction. In addition, Ala mutants of SNAP-25 residues from the C terminus of SNAP-25, as well as from the amino-terminal region decreased binding to G 1 ␥ 1 . When SNAP-25 with eight residues mutated to alanine was assembled with syntaxin 1A, there was significantly reduced affinity of this mutated t-SNARE for G␥, but it still interacted with synaptotagmin-1 in a Ca 2ϩ -dependent manner and reconstituted evoked exocytosis in botulinum neurotoxin E-treated neurons. However, the mutant SNAP-25 could no longer support 5-hydroxytryptamine-mediated inhibition of exocytosis.
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