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. 2003 Jul;8(3):258–264. doi: 10.1379/1466-1268(2003)008<0258:mcmcap>2.0.co;2

Molt cycle–dependent molecular chaperone and polyubiquitin gene expression in lobster

Jeffrey L Spees 1,*, Sharon A Chang 1, Donald L Mykles 2, Mark J Snyder 1, Ernest S Chang 1,2
PMCID: PMC514879  PMID: 14984059

Abstract

Lobster claw muscle undergoes atrophy in correlation with increasing ecdysteroid (steroid molting hormone) titers during premolt. In vivo molecular chaperone (constitutive heat shock protein 70 [Hsc70], heat shock protein 70 [Hsp70], and Hsp90) and polyubiquitin messenger ribonucleic acid (mRNA) levels were examined in claw and abdominal muscles from individual premolt or intermolt lobsters. Polyubiquitin gene expression was assayed as a marker for muscle atrophy. Both Hsc70 and Hsp90 mRNA levels were significantly induced in premolt relative to intermolt lobster claw muscle, whereas Hsp70 mRNA levels were not. Hsp90 gene expression was significantly higher in premolt claw muscle when compared with abdominal muscle. Polyubiquitin mRNA levels were elevated in premolt when compared with intermolt claw muscle and significantly elevated relative to premolt abdominal muscle.

INTRODUCTION

Crustaceans shed their protective exoskeletons many times during their lives to grow. The American lobster, Homarus americanus, undergoes 20–25 molts from the time of hatching to sexual maturity (about 500 g wet weight) (Hughes et al 1972). Lobsters 15–20 kg in weight have been found in nature and are estimated to be 50–70 years old (Herrick 1895; Cooper and Uzmann 1980).

Although possession of a protective exoskeleton has proven to be an extremely successful evolutionary strategy for marine crustaceans, the protection it affords does come with some trade-offs. For juvenile H americanus, molting (ecdysis) may last 30 minutes or longer. After pulling out of and shedding the old exoskeleton, they are capable of only limited movement and are highly vulnerable. The simultaneous release of molting fluid containing lubricants, proteases, and other organic molecules is a potent attractant for predators. Immediately after ecdysis, the new exocuticle is soft and pliable, offering little protection. Accordingly, the tips of the chelipeds (large front claws) and the cutting and tearing edges of the mandibles and maxillipeds are the first structures to harden on the new exoskeleton because they are critical for defense and feeding (Waddy et al 1995).

Although they are magnificent adaptations for predation and protection, the lobster's large claws pose an anatomical dilemma for molting. The diameters of the proximal leg segments and the exoskeletal openings at the base of the claws (the basis-ischium joint) are too narrow to allow the large front claw muscles to fit through (the largest cross-sectional area of the claws is approximately 10 times that of its base). Juvenile and adult lobsters therefore risk losing these crucial structures every time they molt. This predicament appears to be solved by way of hormonal mediation. In correlation with increasing titers of ecdysteroids (steroid molting hormones), molting initiates a cascade of poorly understood events that culminate in the selective degradation of myofilaments in claw muscle fibers (Mykles 1997). This process does not occur in the walking leg muscles (Mykles and Skinner 1982) or in the lobster's large abdominal (tail) muscle, all of which easily fit through their designated openings in the exoskeleton. Originally identified over 30 years ago in the land crab, Gecarcinus lateralis (Skinner 1966), molt-induced claw muscle atrophy is now understood to involve Ca2+-dependent enzymatic proteolysis (Mykles 1992; Beyette and Mykles 1997) as well as the adenosine triphosphate-ubiquitin–dependent degradation system (Shean and Mykles 1995; reviewed by Mykles 1998).

Despite remarkable atrophy during the crustacean premolt stages (30–60% atrophy of the claw muscle protein content in G lateralis; Skinner 1966), protein synthesis rates are actually elevated in premolt compared with intermolt claw muscle tissues (Mykles 1992; El Haj et al 1996; El Haj and Whiteley 1997). This increase in protein synthesis is attributed to the synthesis of proteinases (Mykles and Skinner 1990), although the end result is a net loss of muscle mass due to subsequent proteolysis and myofibrillar degradation.

Molecular chaperones (heat shock proteins [Hsps] and constitutive heat shock proteins [Hscs]) that aid in folding nascent proteins at the ribosome may be involved in crustacean muscle atrophy. Chaperones provide a favorable folding environment while preventing aggregation by binding to exposed hydrophobic residues on newly synthesized proteins (Feder and Hofmann 1999; Hartl and Hayer-Hartl 2002). Frequently studied because of their induction during cellular perturbations resulting in protein denaturation, Hscs and Hsps are also involved in many cellular processes including mitochondrial and endoplasmic recticular transport (Elston 2000), the cell cycle (Milarski and Morimoto 1986; Taira et al 1997; Helmbrecht et al 2000), chaperoning of steroid hormone receptors (Pratt 1997, 1998), and protein turnover (Connell et al 2000).

There is evidence that levels of Hsps change during the course of the crustacean molt cycle. Chang et al (1999) observed that the expression of Hsp90 messenger ribonucleic acid (mRNA) was increased in the hepatopancreas (a digestive tissue) after injection of the molting hormone 20-hydroxyecdysone. This hormone mediates the progression of the molt cycle and peaks at late premolt. Elevated levels of Hsp70 protein were observed in whole lobster larvae during premolt and early postmolt relative to intermolt levels (Snyder and Mulder 2001).

Regarding the elevated protein synthesis rates observed in premolt compared with intermolt claw muscle, the regulation of steroid hormone receptors by Hsps, and the role of Hsps-Hscs in protein degradation, we hypothesized that molecular chaperones (Hsc70, Hsp70, and Hsp90) would be expressed over the molt cycle and in particular during premolt atrophy. To address this question, we examined mRNA levels of molecular chaperones and polyubiquitin, a marker for protein degradation and premolt muscle atrophy (Shean and Mykles 1995; Koenders et al 2002). For comparison, we analyzed the mRNA levels for each of these genes in abdominal muscle from the same animals. We hypothesized that abdominal muscle would not induce molecular chaperone or polyubiquitin expression during premolt because it does not undergo premolt atrophy.

MATERIALS AND METHODS

Juvenile (nonreproductive) lobsters (full-sibling males with a mean weight of 240.1 ± 35.2 g) were reared at the Bodega Marine Laboratory and molt-staged by recording the timing of several molts and by examining the degree of cuticular separation and setal development in the pleopods (Aiken 1973). The molt interval was approximately 120 days. Lobsters were sacrificed, and crusher claw and abdominal muscle samples were dissected, frozen with liquid N2, and stored at −70°C. Before their selection, lobsters were maintained in a flow-through aquaculture system supplied with ambient seawater, held in individual compartments, and fed shrimp thrice weekly. Detailed descriptions of the aquaculture system and our lobster culture techniques are discussed elsewhere (Chang and Conklin 1993; Conklin and Chang 1993).

Northern analyses

Total RNA was isolated from the claw and abdominal muscle samples (RNAgents kit, Promega, Madison, WI, USA), quantified with a spectrophotometer, and equally loaded (15 μg total RNA) onto denaturing 1% agarose gels. These gels were washed (15 minutes, diethyl pyrocarbonate water) and blotted overnight onto nylon membranes (Magnagraph, Osmonics, Minnetonka, MN, USA). After ultraviolet cross-linking (UV Stratalinker 1800, Stratagene, La Jolla, CA, USA), blots were prehybridized (2 hours) in 5× sodium chloride, sodium phosphate, ethylenediaminetetracetic acid (SSPE buffer) (0.75 M NaCl, 50 mM NaH2PO4, 5 mM ethylenediamine-tetraacetic acid, pH 7.4), 50% (w/v) formamide, 5× Denhardt, 1% sodium dodecyl sulfate, and 100 μg/mL denatured sheared salmon sperm deoxyribonucleic acid (DNA). A partial lobster hsp70 clone (499 bp) was 32P-labeled (Prime-It RmT, Stratagene), added directly to the prehybridization solution, and allowed to hybridize overnight at 42°C. After hybridization, the blots were washed twice with 2× SSPE buffer and placed on film overnight at −70°C.

After exposure of the film, the blots were stripped with several washes with 0.1× standard saline citrate buffer (15 mM NaCl, 1.5 mM sodium citrate, pH 7.0) and 0.1% sodium dodecyl sulfate at 65°C until background was minimal and prehybridized, hybridized, and washed as above, except that a partial lobster hsc70 (600 bp, primarily 3′ untranslated region), hsp90 (350 bp), or polyubiquitin (690 bp; Shean and Mykles 1995) complementary DNA (cDNA) probe was added. The blots were then probed with a partial lobster β-actin cDNA to check for equal loading of RNA (700 bp; Harrison and El Haj 1994). Reverse transcriptase–polymerase chain reaction and cloning of the hsc70, hsp70, and hsp90 probes has been described previously (Chang et al 1999; Spees et al 2002). The hsc70 probe, cloned primarily from the 3′ untranslated region of the Hsc70 mRNA, does not hybridize with the hsp70 transcript (Spees et al 2002). Films were scanned on a high-resolution scanner, and densitometry was performed with NIH Image software; units for the optical density data are relative.

RESULTS

Northern analysis of individual lobsters over the molt cycle indicated that polyubiquitin mRNA levels were reduced during late premolt and on the day of molt in abdominal muscle (Fig 1). This expression pattern was in contrast to that of claw muscle samples isolated from the same animals. Claw muscle polyubiquitin mRNA levels appeared to increase during premolt and on the day of molt compared with late postmolt and intermolt (Fig 1). β-Actin mRNA levels were relatively constant over the molt cycle for both abdominal muscle and claw muscle, despite the atrophy that occurs in claw muscle before ecdysis (Fig 1). Therefore, we chose to measure levels of the β-actin transcript to normalize the hybridization signals from the other cDNA probes.

Fig 1.

Fig 1.

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Northern analysis of polyubiquitin messenger ribonucleic acid (mRNA) levels expressed over the molt cycle in (A) abdominal muscle and (B) claw muscle. Samples from 3 intermolt (stage C) animals are shown (I). Single samples are shown for premolt stages (D0, D1, D2, D3), day of molt (M), and 1, 2, and 4 weeks postmolt (labeled 1, 2, and 4, respectively). For each molt stage (each lane), abdominal muscle and claw muscle were dissected from the same animal. Note that β-actin mRNA levels are relatively constant over the molt cycle for both claw and abdominal muscles, despite the atrophy that occurs in premolt claw muscle

Hsp90 gene expression was significantly induced in premolt compared with intermolt claw muscle (Mann-Whitney rank sum test; P = 0.002; Fig 2 A,B). There was no significant difference between intermolt and premolt Hsp70 mRNA levels in claw muscle (Student's t-test; P = 0.969; Fig 2A,B). Unlike Hsp70, Hsc70 mRNA levels were significantly higher in premolt compared with intermolt claw tissues (Mann-Whitney rank sum test; P = 0.004; Fig 2 A,B). Polyubiquitin mRNA levels, although higher in premolt relative to intermolt samples, were not significantly different (Student's t-test; P = 0.067; Fig 2 A,B).

Fig 2.

Fig 2.

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(A) Northern analysis of molecular chaperone and polyubiquitin messenger ribonucleic acid (mRNA) levels in intermolt (stage C; lanes 1–6) and premolt (stage D2; lanes 7–12) claw muscle from juvenile lobster. Each lane represents a single animal (n = 6). Actin mRNA levels are shown as an indicator of equal loading. Data are derived from a single blot that was serially hybridized with 32P-labeled complementary deoxyribonucleic acid probes. (B) Relative mRNA levels for molecular chaperones and polyubiquitin in intermolt compared with premolt claw muscle. Data from (A) were normalized against the actin signal; n = 6 for each bar. Absolute expression levels for one transcript should not be compared with any other because of potential differences in probe strength and film exposure. Significant difference between the intermolt and premolt stages is indicated; ** P ≤ 0.01

There were no significant differences in molecular chaperone expression between premolt compared with intermolt abdominal muscle (Student's t-test; Hsp90, P = 0.058; Hsp70, P = 0.256; Hsc70, P = 0.0534; Fig 3 A,B). There was, however, a significant difference in polyubiquitin mRNA levels. In contrast to claw muscle expression, abdominal muscle showed significantly higher polyubiquitin mRNA levels in intermolt rather than premolt tissues (Mann-Whitney rank sum test; P = 0.002; Fig 3 A,B).

Fig 3.

Fig 3.

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(A) Northern analysis of molecular chaperone and polyubiquitin messenger ribonucleic acid (mRNA) levels in intermolt (stage C; lanes 1–6) and premolt (stage D2; lanes 7–12) abdominal muscles from juvenile lobster. Each lane represents a single animal (n = 6). Actin mRNA levels are shown as an indicator of equal loading. Data are derived from a single blot that was serially hybridized with 32P-labeled complementary deoxyribonucleic acid probes. (B) Relative mRNA levels for molecular chaperones and polyubiquitin in intermolt compared with premolt abdominal muscle. Data from (A) were normalized against the actin signal; n = 6 for each bar. Absolute expression levels for one transcript should not be compared with any other because of potential differences in probe strength and film exposure. Significant difference between the intermolt and premolt stages is indicated; ** P ≤ 0.01

Comparison of premolt claw and abdominal muscle gene expression profiles revealed significant differences between tissues. Hsp90 mRNA levels were significantly higher in premolt claw compared with premolt abdominal muscle (Student's t-test; P = 0.01; Fig 4 A,B), although neither Hsp70 (P = 0.764) nor Hsc70 (P = 0.729) mRNA levels were significantly different between tissues. Differences in polyubiquitin expression were highly significant between muscle types: premolt claw muscle had higher polyubiquitin mRNA levels than premolt abdominal muscle (Student's t-test; P = 0.002; Fig 4 A,B).

Fig 4.

Fig 4.

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(A) Northern analysis of molecular chaperone and polyubiquitin messenger ribonucleic acid (mRNA) levels in premolt abdominal muscle (stage D2; lanes 1–6) and premolt claw muscle (stage D2; lanes 7–12) from juvenile lobster (n = 6). The 2 tissues were dissected from the same animals (eg, lanes 1 and 7 represent RNA isolated from abdominal and claw muscles of the same lobster). Actin mRNA levels are shown as an indicator of equal loading. Data are derived from a single blot that was serially hybridized with 32P-labeled complementary deoxyribonucleic acid probes. (B) Relative mRNA levels for molecular chaperones in premolt abdominal and claw muscles. Data from (A) were normalized against the actin signal; n = 6 for each bar. Absolute expression levels for one transcript should not be compared with any other because of potential differences in probe strength and film exposure. Significant difference between the intermolt and premolt stages is indicated; ** P ≤ 0.01

DISCUSSION

We found significant in vivo differences in Hsc70, Hsp90, and polyubiquitin mRNA levels for lobster claw and abdominal muscle types at different molt stages. These changes in expression were evident both among and between tissues. Fundamental physiological changes required for molting such as premolt-driven claw muscle atrophy are likely to account for the differences we observed. Molt cycle–dependent muscle atrophy is a novel example of molecular chaperone (Hsp90 and Hsc70) mRNA induction in a differentiated somatic tissue that is not undergoing environmental stress. In the absence of protein-denaturing stress such as heat shock or transition metal exposure, human Hsp70 may be induced by oncogenes and the synthetic phase of the cell cycle (Milarski and Morimoto 1986; Taira et al 1997; Helmbrecht et al 2000). During Drosophila development, pulses of ecdysteroids are known to induce the expression of heat shock genes such as hsp70 and hsp22 (White et al 1999). In tobacco hornworm (Manduca sexta) development, Hsc70 mRNA levels are elevated during the major premetamorphic peak of ecdysteroid synthesis during the last larval instar and are responsive to 20-hydroxyecdysone (Rybczynski and Gilbert 2000).

Arbeitman and Hogness (2000) have recently shown that both Hsc70 and Hsp90 form a complex with the Drosophila ecdysteroid receptor (EcR) in vivo that is required for the active receptor's assembly. We found significant induction of both Hsc70 and Hsp90 mRNAs in premolt (stage D2) compared with intermolt (stage C) lobster claw tissues. EcR immunoreactivity has been previously demonstrated in both walking leg extensor muscle and eyestalk neural tissues of premolt H americanus using antisera against the Drosophila EcR (El Haj et al 1994). Ecdysteroid titers quantified by radioimmunoassay of hemolymph from premolt H americanus rise during stages D1 and D2 and fall during D3 and D4 (immediately before ecdysis) (Chang and Bruce 1980; Snyder and Chang 1991). Because premolt claw muscle atrophy correlates with increasing titers of ecdysteroids, the significant induction in Hsc70 and Hsp90 mRNA we observed may be involved in the regulation of ecdysteroid receptors in claw muscle cells responsive to molting hormones. We found no such induction of Hsc70 or Hsp90 gene expression in premolt abdominal muscle samples from animals undergoing claw muscle atrophy. The cells of abdominal muscle tissues may thus express lower concentrations or different isoforms of ecdysteroid receptors than claw muscles. Functional differences have been found between isoforms of the Drosophila EcR, and tissues expressing different EcR isoforms have been shown to exhibit distinct metamorphic responses to ecdysone (Talbot et al 1993; Bender et al 1997).

Chung et al (1998a) reported the cloning of crustacean ecdysteroid receptor gene homologues from the fiddler crab, Uca pugilator. These transcripts were significantly elevated in premolt (stage D1–D4) samples of muscle from the large cheliped relative to intermolt samples. The increases in receptor mRNA correlated with premolt peaks in hemolymph ecdysteroid levels (Chung et al 1998b). The significant increases in lobster claw muscle mRNAs for Hsc70 and Hsp90 that we observed during the same period in the molt cycle may correspond with the enhanced transcription of a lobster ecdysteroid receptor isoform.

Hsc70 and Hsp90 mRNA levels for lobster claw muscle were induced during atrophy, whereas the Hsp70 mRNA level was not. This may also indicate a specialized need for particular molecular chaperones in receptor regulation or substrate degradation compared with a more generalized function such as chaperoning during protein synthesis. In vivo rates of protein synthesis are significantly elevated in premolt leg, claw, and abdominal muscle relative to intermolt or postmolt tissues (El Haj et al 1996). Increased protein synthesis rates in claw muscle undergoing atrophy are believed to result from enhanced synthesis of myofibrillar-degradation enzymes. Despite possible changes in protein synthesis rates over the molt cycle (and levels of chaperoning for protein synthesis), we did not find significant differences in Hsp70 mRNA expression between intermolt and premolt claw or abdominal muscles. Additionally, no significant difference was found between Hsp70 or Hsc70 mRNA levels for premolt claw compared with abdominal muscle from the same individuals.

Polyubiquitin mRNA levels were significantly higher in premolt claw muscle compared with abdominal muscle, and premolt claw also exhibited greater levels of polyubiquitin gene expression relative to intermolt claw tissues. Our data are in agreement with those of Shean and Mykles (1995) and Koenders et al (2002) who found similarly elevated polyubiquitin mRNA levels in premolt claw tissues of G lateralis and H americanus, respectively. We expected increased polyubiquitin expression in premolt claw muscle because of protein degradation during muscle atrophy. Unexpectedly, we found significant differences in polyubiquitin mRNA levels in intermolt compared with premolt abdominal muscle. In contrast to lobster claw muscle, intermolt abdominal muscle had significantly higher levels of polyubiquitin mRNA than premolt muscle. Although we do not have data on ubiquitin conjugation, reduced polyubiquitin gene expression in premolt compared with intermolt abdominal muscle hypothetically indicates reduced protein turnover in premolt abdominal muscle. Increased polyubiquitin expression and ubiquitin production occurs in mammalian skeletal muscle in a variety of catabolic states (for review see Attaix and Taillandier 1998). With elevated protein synthesis rates in premolt claw and abdominal muscles and a potential reduction in protein turnover in abdominal muscle, this should correspond to an overall deposition of muscle mass in premolt abdominal muscle compared with an overall loss of muscle mass in premolt claw muscle. El Haj et al (1996) discussed muscle-specific mass increases in preparation for the molt, as indicated by a lengthening in both premolt leg and abdominal muscles.

Notably, the elevated molecular chaperone mRNAs we observed may be accounted for by protein degradation rather than by protein synthesis. Under some conditions, binding of Hsc70 and Hsp90 to substrates (either alone or in concert) may target them for degradation instead of refolding, if the chaperones do not dissociate from the complex. In vitro, Hsc70 is required for the ubiquitin-dependent degradation of actin, α-crystallin, glyceraldehydes-3-phosphate dehydrogenase, α-lacalbumin, and histone 2A (Bercovich et al 1997). The cochaperone CHIP (carboxyl terminus of Hsc70-interacting protein) has been shown to interact with Hsp90 in addition to Hsc70 and to mediate the actions of Hsps to chaperone substrates or to elicit their degradation via ubiquitylation (Connell et al 2000). By northern analysis, the mRNA for CHIP was found to be highly expressed in skeletal muscle and to a lesser extent in cardiac muscle as compared with other tissues (Ballinger et al 1999). Thus, although we do not have data on molecular chaperone or CHIP protein levels, the increased levels of Hsc70 and Hsp90 mRNAs we observed may precede the production of chaperone proteins that bind substrates destined for degradation. The patterns of molt-induced molecular chaperone and polyubiquitin gene expression we observed provide the basis for further study regarding the regulation of crustacean muscle atrophy during premolt.

Acknowledgments

We thank Dr James S. Clegg for helpful discussions and W.A. Hertz for assistance with animal care. This work was funded in part by a grant from the National Sea Grant College System, National Oceanic and Atmospheric Administration (NOAA), US Department of Commerce, under grant R/A-108, project NA66RG0477, through the California Sea Grant College system and in part by the California State Resources Agency (to M.J.S. and E.S.C.), project LR/LR-1 through the Connecticut Sea Grant College Program (to E.S.C.), and Bodega Marine Laboratory student travel grants (to J.L.S.). The views expressed in this study are those of the authors and do not necessarily reflect the views of NOAA or any of its subagencies. The US Government is authorized to reproduce and distribute for governmental purposes. Contribution Number 2177, Bodega Marine Laboratory, University of California at Davis.

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