Donald L. Mykles

Regulation of crustacean molting: a review and our perspectives.

Molting is a highly complex process that requires precise coordination to be successful. We describe the early classical endocrinological experiments that elucidated the hormones and glands responsible for this process. We then describe the more recent experiments that have provided information on the cellular and molecular aspects of molting. In addition to providing a review of the scientific literature, we have also included our perspectives.

Intracellular proteinases of invertebrates: calcium-dependent and proteasome/ubiquitin-dependent systems.

Cytosolic proteinases carry out a variety of regulatory functions by controlling protein levels and/or activities within cells. Calcium-dependent and ubiquitin/proteasome-dependent pathways are common to all eukaryotes. The former pathway consists of a diverse group of Ca(2+)-dependent cysteine proteinases (CDPs; calpains in vertebrate tissues). The latter pathway is highly conserved and consists of ubiquitin, ubiquitin-conjugating enzymes, deubiquitinases, and the proteasome. This review summarizes the biochemical properties and genetics of invertebrate CDPs and proteasomes and their roles in programmed cell death, stress responses (heat shock and anoxia), skeletal muscle atrophy, gametogenesis and fertilization, development and pattern formation, cell-cell recognition, signal transduction and learning, and photoreceptor light adaptation. These pathways carry out bulk protein degradation in the programmed death of the intersegmental and flight muscles of insects and of individuals in a colonial ascidian; molt-induced atrophy of crustacean claw muscle; and responses of brine shrimp, mussels, and insects to environmental stress. Selective proteolysis occurs in response to specific signals, such as in modulating protein kinase A activity in sea hare and fruit fly associated with learning; gametogenesis, differentiation, and development in sponge, echinoderms, nematode, ascidian, and insects; and in light adaptation of photoreceptors in the eyes of squid, insects, and crustaceans. Proteolytic activities and specificities are regulated through proteinase gene expression (CDP isozymes and proteasomal subunits), allosteric regulators, and posttranslational modifications, as well as through specific targeting of protein substrates by a diverse assemblage of ubiquitin-conjugases and deubiquitinases. Thus, the regulation of intracellular proteolysis approaches the complexity and versatility of transcriptional and translational mechanisms.

CRUSTACEAN MUSCLE PLASTICITY : MOLECULAR MECHANISMS DETERMINING MASS AND CONTRACTILE PROPERTIES

Two crustacean models for understanding molecular mechanisms of muscle plasticity are reviewed. Metabolic changes underlying muscle protein synthesis and degradation have been examined in the Bermuda land crab, Gecarcinus lateralis. During proecdysis, the claw closer muscle undergoes a programmed atrophy, which results from a highly controlled breakdown of myofibrillar proteins by Ca(2+)-dependent and, possibly, ATP/ubiquitin-dependent proteolytic enzymes. The advantage of this model is that there is neither fiber degeneration nor contractile-type switching, which often occurs in mammalian skeletal muscles. The second model uses American lobster, Homarus americanus, to understand the genetic regulation of fiber-type switching. Fibers in the claw closer muscles undergo a developmentally-regulated transformation as the isomorphic claws of larvae and juveniles differentiate into the heteromorphic cutter and crusher claws of adults. This switching occurs at the boundary between fast- and slow-fiber regions, and thus the transformation of a specific fiber is determined by its position within the muscle. The ability to predict fiber switching can be exploited to isolate and identify putative master regulatory factors that initiate and coordinate the expression of contractile proteins.

Sodium dodecyl sulfate and heat induce two distinct forms of lobster muscle multicatalytic proteinase : the heat-activated form degrades myofibrillar proteins

A multicatalytic proteinase (MCP) purified from lobster claw and abdominal muscles degrades a variety of peptide and protein substrates. The enzyme is activated by low concentrations (0.03%) of sodium dodecyl sulfate (SDS) and brief (1 min) heating at 60 degrees C. The lobster MCP can assume three stable and functionally distinct states in vitro; these are classified as the basal, heat-activated, and SDS-activated forms. The basal MCP possessed high trypsin-like peptidase activity and low chymotrypsin-like peptidase, peptidylglutamyl-peptide hydrolase, and caseinolytic activities; incubation of the basal form with SDS stimulated the peptidylglutamyl-hydrolase activity about 30-fold and inhibited the other three activities 80% to 100%. Heating the basal form stimulated caseinolytic activity about 6-fold with little effect on the peptidase activities. The heat-activated enzyme also degraded myosin, tropomyosin, troponin, and actin depolymerizing factor; alpha-actinin was resistant to proteolysis. Incubation of the heat-activated MCP with SDS inhibited the trypsin-like, chymotrypsin-like, and proteinase activities 95 to 100% and stimulated the peptidylglutamyl-hydrolase activity about 16-fold. Incubation of myosin with either the basal or the heat-activated forms in the presence of SDS generated identical proteolytic fragments of the myosin heavy chain, suggesting that SDS induced a third form that can be produced from either the basal or the heat-activated forms. The heat-activated form produced proteolytic fragments of myosin heavy chain different from those generated by either basal or heat-activated enzymes in the presence of SDS. Furthermore, 100 mM KCl stimulated the caseinolytic activity of the heat-activated form 24% and inhibited the trypsin-like and peptidylglutamyl-hydrolase activities 56 and 20%, respectively. These results, though indirect, suggest that heating induced a proteinase activity that was distinct from the three peptidase activities. Activation of the basal form with SDS was reversible, since precipitation of dodecyl sulfate with 100 mM KCl restored trypsin-like activity and inhibited peptidylglutamyl-hydrolase activity. In contrast, removal of dodecyl sulfate from the SDS-activated form that was derived from the heat-activated MCP induced its conversion to the basal form. Thus, although heat-activation was irreversible, the heat-activated form was converted back to the basal form via the SDS-activated form.

MULTIPLE VARIANTS OF MYOFIBRILLAR PROTEINS IN SINGLE FIBERS OF LOBSTER CLAW MUSCLES: EVIDENCE FOR TWO TYPES OF SLOW FIBERS IN THE CUTTER CLOSER MUSCLE

SDS-polyacrylamide gel electrophoresis of myofibrillar proteins in single fibers of lobster claw closer muscles distinguished three types of fibers: one fast and two slow. The fibers differed both qualitatively and quantitatively in the variants of paramyosin and troponin present. There were four proteins unique to fast fibers (paramyosin1, a 75-kD protein, troponin-I3, and -I5) and two proteins unique to slow fibers (troponin-I4 and -C1). Fast fibers were found only in the cutter claw. The major type of slow fibers (S1) appeared to account for the entire muscle mass in the crusher claw as well as ~85% of the slow fibers in the cutter claw. Another type (S2) comprised 10-15% of the slow fibers in the cutter claw. The S1 and S2 fibers differed in the variants of troponin-I and -T. The S2 fibers contained troponin-T, and I2 as the major variant of troponin-I; S1 fibers lacked T1 and contained I4 as the major isoform. These data indicate that the heterogeneity of myofibrillar proteins observed in actomyosins extracted from whole muscle (Mykles, 1985) is due to three populations of fibers, each containing its own assemblage of regulatory and contractile isoforms. More than one variant of a myofibrillar protein can be expressed in a single fiber, forming unique assemblages by which subgroups can be discriminated within the broader categories of fast and slow fibers.

Cloning of tropomyosins from lobster (Homarus americanus) striated muscles: fast and slow isoforms may be generated from the same transcript.

Complementary DNAs encoding fibre-type-specific isoforms of tropomyosin (Tm) have been isolated from lobster (Homarus americanus) striated muscle expression libraries made from poly(A)+ RNA purified from deep abdominal (fast-type) and crusher-claw closer (slow-type) muscles. A cDNA of slow-muscle Tm (sTm1), containing a complete open reading frame (ORF) and portions of the 5 prime; and 3 prime untranslated regions (UTRs), encodes a protein of 284 amino acid residues with a predicted mass of 32950, assuming acetylation of the amino terminus. The nucleotide sequence of a fast-muscle tropomyosin (fTm cDNA), which includes the entire ORF and part of the 3 prime UTR, is identical to that of sTm1 cDNA, except in the region encoding amino acid residues 39-80 (equivalent to exon 2 of mammalian and Drosophila muscle tropomyosin genes). The deduced amino acid sequences, which display the heptameric repeats of nonpolar and charged amino acids characteristic of alpha-helical coiled-coils, are highly homologous to tropomyosins from rabbit, Drosophila, and shrimp (57% to 99% identities, depending on species). Northern blot analysis showed that two transcripts (1.1 and 2.1kb) are present in both fibre types. Mass spectrometry indicated that fast muscle contains one major isoform (fTm: 32903), while slow muscle contains two major isoforms (sTm1 and sTm2: 32950 and 32884 respectively). Both Tm preparations contained minor species with a mass of about 32830. Sequences of peptides derived from purified slow and fast Tms were identical to the deduced amino acid sequences of the sTm1 and fTm cDNAs, respectively, except in the C-terminal region of fTm. The difference in mass between that predicted by the deduced sequence (32880) and that measured by mass spectrometry (32903) suggests that fTm is post-translationally modified, in addition to acetylation of the N-terminal methionine. These data are consistent with the hypothesis that the fTm and sTm1 are generated by alternative splicing of two mutually-exclusive exons near the 5 prime end of the same gene.

Purification and characterization of a multicatalytic proteinase from crustacean muscle: Comparison of latent and heat-activated forms

A high-molecular-weight (Mr 740,000) multicatalytic proteinase (MCP) was purified over 3100-fold from soluble extracts of lobster claw and abdominal muscles. The enzyme was extracted from muscle in a latent state; brief (3 min) heating of an ammonium sulfate fraction (45-65% saturation) at 60 degrees C irreversibly activated the proteinase while denaturing about 55% of the protein. MCP was further purified by chromatography on two sequential arginine-Sepharose columns and a Mono Q column with a yield of 60%. About 1.12 mg MCP was obtained for every 100 g tissue. In addition to [14C]methylcasein, the MCP hydrolyzed synthetic peptide substrates of trypsin and chymotrypsin at pH 7.75. Serine protease inhibitors (diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, aprotinin, benzamidine, soybean trypsin inhibitor, chloromethyl ketones), leupeptin, antipain, hemin, sulfhydryl-blocking reagents (N-ethylmaleimide, mersalyl acid, p-chloromercurisulfonic acid, iodoacetamide) suppressed activity while Ep-475, a specific inhibitor of cysteine proteinases, had no effect, suggesting the MCP is a serine proteinase with one or more cysteine residues indirectly involved in catalysis. The latent MCP was purified using the same procedure as that for the active form, except that thermal activation was omitted. The elution characteristics of latent MCP from the arginine-Sepharose and Mono Q columns were identical to those of active MCP. Since the purified latent form could still be activated by heating, activation did not involve denaturation of an endogenous inhibitor or substrate. Subunit compositions of both forms were identical in two-dimensional polyacrylamide gels; each was composed of eight polypeptides with molecular weights between 25,000 and 32,500 and a ninth polypeptide with a molecular weight of 41,000. Electron microscopy of negatively stained material showed that each form was a cylinder-shaped particle (approximately 10 x 15 nm) consisting of a stack of four rings with a hollow center; no differences in shape, dimensions, or submolecular structure were observed. These results suggest that activation probably involved small conformational changes rather than covalent modifications or rearrangement of subunits within the complex.

Conserved role of cyclic nucleotides in the regulation of ecdysteroidogenesis by the crustacean molting gland

Molting processes in crustaceans are regulated by ecdysteroids produced in the molting gland (Y-organ), and molting is indirectly controlled by circulating factors that inhibit the production of these polyhydroxylated steroids. Two of these regulatory factors are the neuropeptides molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH). CHH appears to inhibit ecdysteroidogenesis in the Y-organ through the activation of a receptor guanylyl cyclase. The signaling pathway activated by MIH, however, remains a subject of controversy. It is clear that neuropeptides inhibit ecdysteroidogenesis by simultaneously suppressing ecdysteroid biosynthetic processes, protein synthesis, and uptake of high density lipoproteins. Data demonstrate that cAMP is the primary regulator of critical catabolic, anabolic, and transport processes, which ultimately support the capacity for ecdysteroid production by the Y-organ. While cAMP also regulates acute ecdysteroidogenesis to some extent, data indicate that cGMP is the primary signaling molecule responsible for acute inhibition by neuropeptides. It is clear that the regulatory roles filled by cAMP and cGMP are conserved among decapod crustaceans. It is unknown if these complementary second messengers are linked in a single signaling pathway or are components of independent pathways activated by different factors present in extracts of eyestalk ganglia.

Atrophy of crustacean somatic muscle and the proteinases that do the job. A review

As much as 60% of the muscle in the propodus of both chelae of some brachyurans atrophies during proecdysis and is restored during metecdysis. We describe here the structural changes that occur during atrophy of such chelae muscles as well as atrophy of somatic muscle from a number of species of Crustacea in response to one or more of several physiological stimuli. Atrophy is not random and whole fibers are not lost; the remaining cellular structures provide a framework into which newly synthesized myofibrillar proteins are packed during metecdysis. As seen in electron micrographs as well as in gel electropherograms, more thin filament proteins (actin, troponin, and tropomyosin) are degraded when compared to those from thick filaments

Grand Challenges in Comparative Physiology: Integration Across Disciplines and Across Levels of Biological Organization

Schwenketal.(2009)providedanover-view of five major challenges in organ-ismal biology: (1) understanding theorganism’s role in organism–environ-ment linkages; (2) utilizing the func-tional diversity of organisms; (3)integrating living and physical systemsanalysis; (4) understanding howgenomes produce organisms; and (5)understanding how organisms walkthe tightrope between stability andchange. Subsequent ‘‘GrandChallenges’’ papers have expanded onthese topics from different viewpoints,including ecomechanics (Denny andHelmuth 2009), endocrinology(Denver et al. 2009), development ofadditional model organisms (Satterlieet al. 2009), and development oftheoretical and financial resources(Halanych and Goertzen 2009). This isthe sixth paper in the ‘‘GrandChallenges’’ series, which offers theview from comparative physiology.In this article, we expand upon threemajor challenges facing comparativephysiology in the 21st century: verticalintegration of physiological processesacross organizational levels within or-ganisms, horizontal integration ofphysiological processes across organ-isms within ecosystems, and temporalintegration of physiological pro-cesses during evolutionary change.‘‘Integration’’ is a key. It defines thescope of the challenges and must beconsidered in any solution. Reductiveand inductive approaches both havebeen used with great success in biology.The reductive approach employs a sim-plified system to study a complexprocess. There is no question thatsuch an approach has yielded a greaterunderstanding of the molecular mech-anismsofcellularprocesses.Theinduc-tive approach depends on observationto develop universal principles. CharlesDarwin, after all, used this approach todevelop the theory of natural selection.All too often these approaches areviewed as mutually exclusive, when, infact, they are complementary and areused, to varying extents, by most biol-ogists working today. Yet, we havefallen short of full integration acrossdisciplinesandlevelsofbiologicalorga-nization. A major impediment for fur-ther advancement has been thelimitations in tools and resources.However, recent technological ad-vances (e.g., systems biology) give usan opportunity to combine reductiveand inductive approaches to studyemergent properties (Boogerd et al.2007) and now allow us to entertain