Myosin and paramyosin of Caenorhabditis elegans embryos assemble into nascent structures distinct from thick filaments and multi-filament assemblages

Journal of Cell Science

2012

SummaryAssembly of contractile apparatuses in striated muscle requires precisely regulated reorganization of the actin cytoskeletal proteins into sarcomeric organization. Regulation of actin filament dynamics is one of the essential processes of myofibril assembly, but the mechanism of actin regulation in striated muscle is not clearly understood. Actin depolymerizing factor (ADF)/cofilin is a key enhancer of actin filament dynamics in striated muscle in both vertebrates and nematodes. Here, we report that CAS-1, a cyclase-associated protein in Caenorhabditis elegans, promotes ADF/cofilin-dependent actin filament turnover in vitro and is required for sarcomeric actin organization in striated muscle. CAS-1 is predominantly expressed in striated muscle from embryos to adults. In vitro, CAS-1 binds to actin monomers and enhances exchange of actin-bound ATP/ADP even in the presence of UNC-60B, a muscle-specific ADF/cofilin that inhibits the nucleotide exchange. As a result, CAS-1 and UNC-60B cooperatively enhance actin filament turnover. The two proteins also cooperate to shorten actin filaments. A cas-1 mutation is homozygous lethal with defects in sarcomeric actin organization. cas-1-mutant embryos and worms have aggregates of actin in muscle cells, and UNC-60B is mislocalized to the aggregates. These results provide genetic and biochemical evidence that cyclase-associated protein is a critical regulator of sarcomeric actin organization in striated muscle.

Section: Fluorescence Microscopymentioning

confidence: 99%

CAS-1, a C. elegans cyclase-associated protein, is required for sarcomeric actin assembly in striated muscle

Nomura

Journal of Cell Science

2012

“…For immunofluorescent staining of adult body wall muscle in Figure 3, adult worms were cut in halves near the vulva by needles on poly-lysine-coated slides and permeabilized by a freeze-crack method (Epstein et al, 1993). The gonads were dissected by cutting adult hermaphrodites at the level of pharynx on poly-lysine-coated slides as described previously (Rose et al, 1997).…”

Section: Fluorescence Microscopymentioning

confidence: 99%

Caenorhabditis elegansKettin, a Large Immunoglobulin-like Repeat Protein, Binds to Filamentous Actin and Provides Mechanical Stability to the Contractile Apparatuses in Body Wall Muscle

Mohri

et al. 2006

MBoC

Kettin is a large actin-binding protein with immunoglobulin-like (Ig) repeats, which is associated with the thin filaments in arthropod muscles. Here, we report identification and functional characterization of kettin in the nematode Caenorhabditis elegans. We found that one of the monoclonal antibodies that were raised against C. elegans muscle proteins specifically reacts with kettin (Ce-kettin). We determined the entire cDNA sequence of Ce-kettin that encodes a protein of 472 kDa with 31 Ig repeats. Arthropod kettins are splice variants of much larger connectin/titin-related proteins. However, the gene for Ce-kettin is independent of other connectin/titin-related genes. Ce-kettin localizes to the thin filaments near the dense bodies in both striated and nonstriated muscles. The C-terminal four Ig repeats and the adjacent non-Ig region synergistically bind to actin filaments in vitro. RNA interference of Ce-kettin caused weak disorganization of the actin filaments in body wall muscle. This phenotype was suppressed by inhibiting muscle contraction by a myosin mutation, but it was enhanced by tetramisole-induced hypercontraction. Furthermore, Ce-kettin was involved in organizing the cytoplasmic portion of the dense bodies in cooperation with ␣-actinin. These results suggest that kettin is an important regulator of myofibrillar organization and provides mechanical stability to the myofibrils during contraction. INTRODUCTIONIn muscle cells, the actin cytoskeleton is highly differentiated to form the myofibrils that are specialized for producing contractile forces. In addition to actin and myosin as the essential contractile components, regulatory components for the contractile activity are integrated into the structure (Squire, 1997). Despite the fact that many myofibrillar components have been identified, little is known about how these components functionally interact to assemble and maintain the myofibrils in living cells (Littlefield and Fowler, 1998;Gregorio and Antin, 2000;Clark et al., 2002). In particular, the assembly process of actin filaments is complex. During development, actin-monomer binding proteins, including profilin, and actin depolymerizing factor/cofilin regulate actin filament dynamics (Obinata, 1993;Obinata et al., 1997; Ono, 2003a,b). However, in mature myofibrils, these proteins are no longer major components of the thin filaments, and filament binding proteins, such as tropomyosin and ␣-actinin, and end-capping proteins, such as CapZ and tropomodulin, become the major actin-associated proteins. However, the list of actin binding proteins in muscle is still growing and cellular functions of many of these proteins are not clearly understood.Kettin is a large protein of 500-700 kDa found in the Z-discs and the I-bands of arthropod muscles (Bullard et al., 2000;Bullard et al., 2002Bullard et al., , 2006. Kettin directly binds to actin filaments with high-affinity (Lakey et al., 1993;Maki et al., 1995;van Straaten et al., 1999). Proteolytic removal of kettin by calpain from the Z-discs causes ...

“…Immunofluorescence microscopy Worm embryos were obtained by cutting gravid adults on poly-lysinecoated slides, freeze-cracked as described previously (Epstein et al, 1993) and fixed with methanol at -20°C for 5 minutes. They were washed with phosphate-buffered saline (PBS) for 10 minutes and stained with antibodies diluted in 1% bovine serum albumin in PBS.…”

Section: Northern Blotmentioning

confidence: 99%

Specific requirement for two ADF/cofilin isoforms in distinct actin-dependent processes inCaenorhabditis elegans

Journal of Cell Science

Parast

Alberico

et al. 2003

109

Actin depolymerizing factor (ADF)/cofilin is an essential enhancer of actin turnover. Multicellular organisms express multiple ADF/cofilin isoforms in different patterns of tissue distribution. However, the functional significance of different ADF/cofilin isoforms is not understood. The Caenorhabditis elegans unc-60 gene generates two ADF/cofilins,UNC-60A and UNC-60B, by alternative splicing. These two ADF/cofilin proteins have different effects on actin dynamics in vitro, but their functional difference in vivo remains unclear. Here, we demonstrate that the two isoforms are expressed in different tissues and are required for distinct morphogenetic processes. UNC-60A was ubiquitously expressed in most embryonic cells and enriched in adult gonads, intestine and oocytes. In contrast, UNC-60B was specifically expressed in the body wall muscle, vulva and spermatheca. RNA interference of UNC-60A caused embryonic lethality with variable defects in cytokinesis and developmental patterning. In severely affected embryos, a cleavage furrow was formed and progressed but reversed before completion of the cleavage. Also, in some affected embryos, positioning of the blastomeres became abnormal, which resulted in embryonic arrest. In contrast, an unc-60B-null mutant was homozygous viable, underwent normal early embryogenesis and caused disorganization of actin filaments specifically in body wall muscle. These results suggest that the ADF/cofilin isoforms play distinct roles in specific aspects of actin reorganization in vivo.