Raymond E. Stephens

Expression of homologues for p53 and p73 in the softshell clam ( Mya arenaria ), a naturally-occurring model for human cancer

Homologues for human p53 (Hsp53) and p73 (Hsp73) genes were cloned and expression patterns for their corresponding proteins analysed in tissues from normal and leukemic softshell clams (Mya arenaria). These are the first structural and functional data for p53 and p73 cDNAs and gene products in a naturally occurring, non-mammalian disease model. Core sequence of the predicted clam p53 (Map53) and p73 (Map73) proteins is virtually identical and includes the following highly conserved regions: the transcriptional activation domain (TAD), MDM2 binding site, ATM phosphorylation site, proline rich domain, DNA binding domains (DBDs) II-V, nuclear import and export signals and the tetramerization domain. The core sequence is a structural mosaic of the corresponding human proteins, with the TAD and DBDs resembling Hsp53 and Hsp73, respectively. This suggests that Map53 and Map73 proteins may function similarly to human proteins. Clam proteins have either a short (Map53) or long (Map73) C-terminal extension. These features suggest that Map53 and Map73 may be alternate splice variants of a p63/p73-like ancestral gene. Map73 is significantly upregulated in hemocytes and adductor muscle from leukemic clams. In leukemic hemocytes, both proteins are absent from the nucleus and sequestered in the cytoplasm. This observation suggests that a non-mutational p53/p73-dependent mechanism may be involved in the clam disease. Further studies of these gene products in clams may reveal p53/p73-related molecular mechanisms that are held in common with Burkitts lymphoma or other human cancers.

Molecular chaperones in cilia and flagella: implications for protein turnover.

The mechanisms of protein incorporation and turnover in 9+2 ciliary axonemes are not known. Previous reports of an HSP70-related protein, first in Chlamydomonas flagella and then in sea urchin embryonic cilia, suggested a potential role in protein transport or incorporation. The present study further explores this and other chaperones in axonemes from a representative range of organisms. Two-dimensional gel electrophoresis proved identity between the sea urchin ciliary 78 kDa HSP and a constitutive cytoplasmic HSP70 cognate (pI = 5.71). When isolated flagella from mature sea urchin sperm were analyzed, the same total amount and distribution of 78 kDa protein as in cilia were found. Antigens of similar size were detected in ctenophore comb plate, molluscan gill, and rabbit tracheal cilia. Absent from sea urchin sperm flagella, TCP-1alpha was detected in sea urchin embryonic and rabbit tracheal cilia; the latter also contained HSP90, detected by two distinct antibodies. Tracheal cilia were shown to undergo axonemal protein turnover while tracheal cells mainly synthesized ciliary proteins. TCP-1alpha progressively appeared in regenerating embryonic cilia only as their growth slowed, suggesting a regulatory role in incorporation or turnover. These results demonstrate that chaperones are widely distributed ciliary and flagellar components, potentially related to axonemal protein dynamics.

Tektins as structural determinants in basal bodies.

Tektins, present as three equimolar 47-55 kDa protein components, form highly insoluble protofilaments that are integral to the junctional region of outer doublet microtubules in cilia and flagella. To identify and quantify tektins in other compound microtubules such as centrioles or basal bodies, a rabbit antiserum was raised against tektin filaments isolated from Spisula solidissima (surf clam) sperm flagellar outer doublets and affinity-purified with nitrocellulose blot strips of tektins resolved by SDS- or SDS-urea-PAGE. These antibodies recognized analogous tektins in axonemes of organisms ranging from ctenophores to higher vertebrates. Quantitative immunoblotting established that outer doublet tektins occur in a 1:17 weight ratio to tubulin. Cilia and basal apparatuses were prepared from scallop gill epithelial cells; cilia and deciliated cells were prepared from rabbit trachea. Tektins were detected by immunoblotting in basal body-enriched preparations while tektins were localized to individual basal bodies by immunofluorescence. Supported by greater fluorescence in basal bodies than in adjacent axonemes in tracheal cells, analysis of basal apparatuses demonstrated both a proportionately greater ratio of tektin to tubulin (approximately 1:13) and two distinct solubility classes of tektins, consistent with tektins comprising the B-C junction of triplets in addition to the A-B junction as in doublets.

Multiple protein differences distinguish clam leukemia cells from normal hemocytes: evidence for the involvement of p53 homologues

In coastal locations, marine invertebrates, primarily molluscs, develop fatal leukemias in their blood or hemolymph. In the clam Mya arenaria, non-adhesive, mitotic, spherical leukemia cells replace adhesive, motile, normal hemocytes as leukemia progresses. End-stage leukemia cells express a unique antigen, IE10, while normal cells express the 2A4 marker. The goals of this work were to further differentiate the normal and leukemia specific antigens relative to protein structure, determine if other protein distinctions exist, and examine p53 gene family expression in both cell types. Recognized by the monoclonal antibody 2A4, normal cells express a 185-kDa glycoprotein that may have multiple forms. Detected by the monoclonal antibody 1E10, leukemic cells express a very hydrophobic 252-kDa glycoprotein that is likely to be a transmembrane protein with spectrin/dystrophin-like characteristics. After normalization to the major cytoskeletal protein actin, sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals major distinguishing protein and glycoprotein differences between the two cell types. Most obvious is the near-absence of tubulin in the non-mitotic normal hemocytes. We have also characterized the expression of p53 gene family members in normal and end-stage leukemia cells, finding shifts in expression of the p53 gene homologues p73 and p97 coincident with leukemia-specific protein synthesis.

Preferential incorporation of tubulin into the junctional region of ciliary outer doublet microtubules: a model for treadmilling by lattice dislocation.

Even in the presence of colchicine or Taxol(R), sea urchin embryonic cilia undergo substantial steady-state turnover, with a rate of tubulin incorporation approaching half that seen in full regeneration [Stephens: Mol Biol Cell 8:2187-2198, 1997]. Preliminary experiments suggest that tubulin incorporates differentially into the most stable portion of the outer doublet, the junctional protofilaments [Stephens: Cell Struct Funct 24:413-418, 1999]. To explore this possibility further, embryos of the sea urchin Tripneustes gratilla, a ciliary length inducible system [Stephens: J Exp Zool 269:106-115, 1994a], were pulse labeled with (3)H leucine during steady-state turnover or induced elongation, followed by regeneration in the presence of unlabeled leucine. Cilia were isolated by hypertonic shock and fractionated into detergent-soluble membrane plus matrix, thermally-solubilized microtubule walls, and insoluble 9-fold symmetric remnants of A-B junctional protofilaments plus associated architectural elements. The fractions were resolved by SDS-PAGE and the specific activity of alpha-tubulin was determined. In cilia undergoing turnover or elongation during an isotope pulse, the specific activity of tubulin in the junctional region approximated that of precursor membrane plus matrix tubulin but surpassed that of the tubule wall by a factor of approximately 1.5. In cilia regenerated during an isotope chase, the specific activity of junctional tubulin exceeded that of both the membrane plus matrix and the tubule wall by a similar factor. These data indicate that tubulin is preferentially incorporated into junctional protofilaments during steady-state turnover, induced elongation and regeneration. A model for directional incorporation based on surface lattice discontinuities in the outer doublet is proposed.

Ciliogenesis, Ciliary Function, and Selective Isolation

In addition to their classic role in cell motility, certain cilia have sensory or signaling functions. In sea urchin embryos, short motile cilia randomly propel the early embryo, while a group of long, immotile cilia appear later, coincident with directional swimming and localized within a region that gives rise to the larval nervous system. Motile cilia can be selectively removed by treatment with a novel derivative of dillapiol, leaving the putative sensory cilia for comparative investigation and a gently deciliated embryo ready for studies of regeneration signaling.

POLYCHLORINATED BIPHENYLS ARE SELECTIVELY NEUROTOXIC IN THE DEVELOPING SPISULA SOLIDISSIMA EMBRYO

Polychlorinated biphenyls (PCBs) are ubiquitous environmental pollutants that accumulate to toxic levels in the food chain. Using Spisula solidissima (surf clam) embryos as a developmental model, it was shown that Aroclor 1254 specifically targets two neuronal structures during embryonic development. Embryos were exposed to 1, 10, or 100 ppm Aroclor 1254 or an acetone vehicle control posthatching for 24, 48, and 72 h. Embryos labeled with a serotonin antibody or a neural antigen antibody and a rhodamine-conjugated secondary antibody were viewed by confocal microscopy. The cerebropleural ganglion showed a decrease both in serotonin production and in the size of the serotonin-synthesizing region upon exposure to 10 and 100 ppm Aroclor 1254. These decreases were detectable as early as 48 h postfertilization. When exposed to 100 ppm Aroclor 1254, the primitive neural plexus, which coordinates the movements of the mouth and velum, showed a delay in onset and cessation of expression of a molluscan-specific neural antigen. Exposure to Aroclor 1254 did not affect the overall growth and morphology of the embryos. In addition, analyses of total protein profiles and heat-shock protein 70 levels showed that exposure to Aroclor 1254 did not trigger protein degradation or cause a stress or shock response. These results show that exposure of Spisula embryos to Aroclor 1254 specifically targets neurogenesis while having no effect on the overall growth of the embryo.

Selective incorporation of architectural proteins into terminally differentiated molluscan gill cilia.

Incubation of excised gills from the bay scallop Aequipecten irradians with 3H-leucine demonstrates that many ciliary structural proteins can attain a degree of labeling approaching that previously reported for sea urchin or surf clam embryos undergoing ciliary turnover or regeneration. This labeling is not a consequence of any predominant incorporation into new cilia at the meristematic growth tips of the gill since tissue regions of varying maturity incorporate label into the same proteins at similar levels, with the most mature region having the highest incorporation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorographic analysis of isolated cilia, separated into detergent-soluble membrane/matrix and detergent-insoluble 9+2 axoneme fractions, reveals that 1) tubulin in the membrane/matrix fraction is labeled whereas tubulin in the axoneme is not; 2) no labeled dynein heavy chains are seen in either fraction; 3) the most heavily labeled axonemal components do not appear to any significant extent in the membrane/matrix fraction; and 4) after thermal depolymerization of the microtubules, nearly all labeled proteins reside in the in-soluble ninefold ciliary remnant, the most prominent being tektin A, an integral component of outer doublet microtubules. Further fractionation of the remnant with sarkosyl-urea to produce tektin filaments demonstrates two solubility classes of tekin A, only the more soluble of which is labeled. Very similar selective architectural protein labeling patterns have been reported for steady-state cilia of sea urchin embryos, and this may indicate a widespread turnover or exchange mechanism characteristic of cilia heretofore considered static.

Isolation of molluscan gill cilia, sperm flagella, and axonemes.

Publisher Summary The detailed methods presented in this chapter on the isolation of molluscan sperm flagella are based on those outlined by Stephens and Prior for the comparison of activation mechanisms of mussel or clam gill cilia with those of sperm flagella. Blue mussels and several clam species are generally quite suitable for gill cilia preparations, but it musst be made sure that the animals are alive and healthy. The isolation of molluscan sperm flagella is also derived from methods first used with sea urchins, the basis of which is simple mechanical shear to sever the head from the tail, followed by differential centrifugation to selectively sediment the heads. In most of the isolation procedures for marine cilia and flagella, 9 + 2 axonemes are produced by solubilization of the membrane with detergents such as Triton X-100 and Nonidet P-40. In the case of sperm preparations, animals shipped during their breeding season retain a good deal of sperm within their gonads during cold shipment; only when later placed in seawater will they spawn, especially if that water is warm. The chapter discusses preparation of gill cilia and sperm flagella—the solutions needed and the procedure. There is description of the preparation of 9+2 axonemes—the solutions and the procedure involved.

Preparation of ciliary and flagellar remnants.

Publisher Summary Gentle heating selectively depolymerizes or “melts” the microtubules of axonemes in vivo and in vitro. In both cases, considerable mass remains after thermal treatment. When the proper conditions are chosen, the tubulin derived from thermal fractionation is polymerization competent, suggesting that selective tubulin removal is not simply a denaturation artifact. The ninefold symmetry of the organelle is retained, as is its full length, from basal body to tapering tip, after solubilization of most of the tubulin of the 9 + 2 structure. This “ciliary remnant” retains most of the structural or architectural (nontubulin, nondynein) proteins of the axoneme, perhaps most significant among them being the tektins, the integral microtubule proteins that form the A-B junctional protofilaments of the outer doublets. In the case of flagella, the ninefold cylindrical structure is generally not retained, although under low-shear conditions, sheets of nine doublets will fractionate to sheets of nine singlets and further fractionation will yield insoluble long, fibrous material that will retain the same kinds of proteins as in cilia but having no ordered cylindrical structure. There is a “centriolar rim” structure that retained the basic ninefold configuration after removal of the triplet microtubules from isolated centrioles by either high ionic strength or extremes of pH. Considering that both the ninefold symmetry and the final length may be a function of the proteins that form these remnants or skeletons, study of remnants may provide some insight into the mechanism of centriole or basal body formation, ciliogenesis, and length regulation. Identification of remnant-specific proteins is useful for exploring pathways for their differential incorporation into growing cilia. This chapter presents the experiment on remnant fractionation. In cilia, the A-tubule quickly follows but the A-tubule depolymerization is generally slower in flagella. The flagellar B-subfiber is far more stable than the ciliary B-subfiber to low-ionic-strength dialysis.