Proteolytic processes underlying molt-induced claw muscle atrophy in decapod crustaceans

Proteolytic Processes Underlying Molt-Induced Claw Muscle Atrophy in Decapod Crustaceans1

SYNOPSIS. The claw muscles of large-clawed decapod crustaceans undergo a programmed atrophy in preparation for molting, or ecdysis. This is mediated by five cytosolic proteinases organized into two proteolytic pathways: calcium-dependent and ubiquitin/proteasome-dependent. The calcium-dependent system consists of four calcium-dependent cysteine proteinases (CDPs I, IIa, IIb, and III; native masses 310, 125, 195, and 59 kDa, respectively) that completely degrade myofibrillar proteins and are activated in atrophic muscles. Immunological analysis shows that the active-site sequence in CDP IIa (60-kDa subunit mass) is similar to that in mammalian CDPs (calpains), and that CDP IIb is homologous to a calpain-like gene isolated from Drosophila cDNA libraries. Increased intracellullar Ca^sup 2+^ stimulates proteolysis in situ, indicating CDPs play an important role in muscle protein catabolism. The ubiquitin/proteasome-dependent system involves the ATP-dependent conjugation of multi-ubiquitin chains to protein by ubiquitin-conjugating enzymes. This acts as a signal for substrate degradation by the 26S proteasome, a multi-subunit complex consisting of a 20S proteasome catalytic "core" and two PA700 (19S) regulatory complexes. Polyubiquitin mRNA, ubiquitin-protein conjugates, and 20S proteasome are elevated about 5-, 8-, and 2-fold, respectively, during atrophy. A heat-induced form of the 20S proteasome hydrolyzes myosin, troponin, and tropomyosin to large fragments in vitro. Biochemical studies identified the branched-chain amino acid-preferring (BrAAP) activity, one of six distinct catalytic components in the complex, as the activity that carries out these initial cleavages. These results indicate that the ubiquitin/proteasome pathway is involved, but its precise role remains to be resolved.

INTRODUCTION

The mechanical problem of pulling out the large distally-enlarged chelipeds of decapod crustaceans at molting was widely recognized by l9th century biologists (see Mykles and Skinner, 1990; Skinner and Cook, 1991; Mykles, 1992 for reviews). Reamur (1718a, b) described the process of molting, or ecdysis, in the freshwater crayfish, but it was Couch (1837, 1843) who first described claw muscle atrophy and proposed that it occurs to enable withdrawal of the chelipeds from the old exoskeleton.

These observations were forgotten until the rediscovery of premolt atrophy by Skinner in 1966. In studies on the land crab, Gecarcinus lateralis, she found that the mass of the claw muscle decreased about 40% in the 3-4 weeks, before ecdysis; mass is restored by 3 weeks after successful exuviation (Skinner, 1966). This reduction was not due to cell death, since the DNA content was unchanged. Later work showed that atrophy is associated with a decrease in myofibrillar cross-sectional area (Mykles and Skinner, 1981). Thus, there is a loss of myofilaments so that the numbers of myofibrils and muscle fibers remain relatively constant throughout the molting process. A similar atrophy occurs in the claw muscles of the yabby (Cherax destructor) during premolt (West et al., 1995; West, 1997). In both land and fiddler crabs the extent of atrophy is enhanced by the regeneration load; the reduction in myofibrillar cross-sectional area is greater (78% vs. 43-52%) in animals regenerating 7-8 walking legs than in animals regenerating 1 walking leg (Mykles and Skinner, 1982a; Ismail and Mykles, 1992). During atrophy there is a decrease in the thin-to-thick myofilament ratio from approximately 9:1 to 6:1, resulting in a dramatic increase in thick myofilament packing density (Mykles and Skinner, 1981, 1982b; Ismail and Mykles, 1992). This has two effects: (1) the muscles, though weaker, retain contractile function and (2) maintenance of the sarcomeric structure would accelerate restoration of atrophic fibers to the intermolt condition. Retention of the thick myofilaments may facilitate assembly of newly-synthesized myofilaments into the contractile apparatus.

Atrophy is specific to the claw muscle. Thoracic muscles atrophy after a walking leg is autotomized, but this is in response to unweighting and not to molt induction (Moffett, 1987; Schmiege et al., 1992). Muscles in the walking legs, which lack an enlarged distal segment, do not atrophy (Mykles and Skinner, 1982a). Further specificity is observed in animals with dimorphic claws that differ greatly in size. In male fiddler crabs, for example, the closer muscle of the major claw undergoes a greater atrophy than that of the minor claw (Ismail and Mykles, 1992). This provides strong evidence supporting the hypothesis that atrophy facilitates withdrawal of the claw at ecdysis. Furthermore, this differential atrophy is associated with the fiber type composition. Slow type 1 (S^sub 1^) fibers, which are restricted to the major claw, atrophy to greater degree than slow type 2 (S^sub 2^) fibers in the minor claw (Ismail and Mykles, 1992; see also Mykles, 1997a for review). This suggests that fiber types respond differently to hormones, presumably ecdysteroids, regulating premolt processes. This difference in sensitivity may result from differential expression of ecdysteroid receptors.