Live imaging of the cytoskeleton in early cleavage-stage zebrafish embryos.

The large and transparent cells of cleavage-stage zebrafish embryos provide unique opportunities to study cell division and cytoskeletal dynamics in very large animal cells. Here, we summarize recent progress, from our laboratories and others, on live imaging of the microtubule and actin cytoskeletons during zebrafish embryonic cleavage. First, we present simple protocols for extending the breeding competence of zebrafish mating ensembles throughout the day, which ensures a steady supply of embryos in early cleavage, and for mounting these embryos for imaging. Second, we describe a transgenic zebrafish line [Tg(bactin2:HsENSCONSIN17-282-3xEGFP)hm1] that expresses the green fluorescent protein (GFP)-labeled microtubule-binding part of ensconsin (EMTB-3GFP). We demonstrate that the microtubule-based structures of the early cell cycles can be imaged live, with single microtubule resolution and with high contrast, in this line. Microtubules are much more easily visualized using this tagged binding protein rather than directly labeled tubulin (injected Alexa-647-labeled tubulin), presumably due to lower background from probe molecules not attached to microtubules. Third, we illustrate live imaging of the actin cytoskeleton by injection of the actin-binding fragment of utrophin fused to GFP. Fourth, we compare epifluorescence-, spinning-disc-, laser-scanning-, and two-photon-microscopic modalities for live imaging of the microtubule cytoskeleton in early embryos of our EMTB-3GFP-expressing transgenic line. Finally, we discuss future applications and extensions of our methods.

Antarctic notothenioid fishes: genomic resources and strategies for analyzing an adaptive radiation.

The perciform suborder Notothenoidei provides a compelling opportunity to study the adaptive radiation of a marine species-flock in the cold Southern Ocean that surrounds Antarctica. To facilitate genome-level studies of the diversification of these fishes, we present estimates of the genome sizes of 11 Antarctic species and describe the production of high-quality bacterial artificial chromosome (BAC) libraries for two, the red-blooded notothen Notothenia coriiceps and the white-blooded icefish Chaenocephalus aceratus. Our results indicate that evolution of phylogenetically derived notothenioid families (e.g., the crown group Channichthyidae [icefishes]), was accompanied by genome expansion. Six species from the basal family Nototheniidae had C-values between 0.98 and 1.20 pg, a range that is consistent with the genome sizes of proposed outgroups (e.g., percids) of the notothenioid suborder. In contrast, four icefishes had C-values in the range 1.66-1.83 pg. The BAC libraries VMRC-19 (N. coriiceps) and VMRC-21 (C. aceratus) comprise 12× and 10× coverage of the respective genomes and have average insert sizes of 138 and 168 kb. Paired BAC-end reads representing ∼0.1% of each genome showed that the repetitive element landscapes of the two genomes (13.4% of the N. coriiceps genome and 14.5% for C. aceratus) were similar. The availability of these high-quality and well-characterized BAC libraries sets the stage for targeted genomic analyses of the unusual anatomical and physiological adaptations of the notothenioids, some of which mimic human diseases. Here we consider the evolution of secondary pelagicism by various taxa of the group and illustrate the utility of Antarctic icefishes as an evolutionary-mutant model of human osteopenia (low-mineral density of bones).

Different roles of two gamma-tubulin isotypes in the cytoskeleton of the Antarctic ciliate Euplotes focardii: remodelling of interaction surfaces may enhance microtubule nucleation at low temperature.

Gamma-tubulin belongs to the tubulin superfamily and plays an essential role in the nucleation of cellular microtubules. In the present study, we report the characterization of gamma-tubulin from the psychrophilic Antarctic ciliate Euplotes focardii. In this organism, gamma-tubulin is encoded by two genes, gamma-T1 and gamma-T2, that produce distinct isotypes. Comparison of the gamma-T1 and gamma-T2 primary sequences to a Euplotesgamma-tubulin consensus, derived from mesophilic (i.e. temperate) congeneric species, revealed the presence of numerous unique amino acid substitutions, particularly in gamma-T2. Structural models of gamma-T1 and gamma-T2, obtained using the 3D structure of human gamma-tubulin as a template, suggest that these substitutions are responsible for conformational and/or polarity differences located: (a) in the regions involved in longitudinal 'plus end' contacts; (b) in the T3 loop that participates in binding GTP; and (c) in the M loop that forms lateral interactions. Relative to gamma-T1, the gamma-T2 gene is amplified by approximately 18-fold in the macronuclear genome and is very strongly transcribed. Using confocal immunofluorescence microscopy, we found that the gamma-tubulins of E. focardii associate throughout the cell cycle with basal bodies of the non-motile dorsal cilia and of all of the cirri of the ventral surface (i.e. adoral membranelles, paraoral membrane, and frontoventral transverse, caudal and marginal cirri). By contrast, only gamma-T2 interacts with the centrosomes of the spindle during micronuclear mitosis. We also established that the gamma-T1 isotype associates only with basal bodies. Our results suggest that gamma-T1 and gamma-T2 perform different functions in the organization of the microtubule cytoskeleton of this protist and are consistent with the hypothesis that gamma-T1 and gamma-T2 have evolved sequence-based structural alterations that facilitate template nucleation of microtubules by the gamma-tubulin ring complex at cold temperatures.

Cold adaptation of microtubule assembly and dynamics. Structural interpretation of primary sequence changes present in the alpha- and beta-tubulins of Antarctic fishes.

The microtubules of Antarctic fishes, unlike those of homeotherms, assemble at very low temperatures (-1.8 degrees C). The adaptations that enhance assembly of these microtubules are intrinsic to the tubulin dimer and reduce its critical concentration for polymerization at 0 degrees C to approximately 0.9 mg/ml (Williams, R. C., Jr., Correia, J. J., and DeVries, A. L. (1985) Biochemistry 24, 2790-2798). Here we demonstrate that microtubules formed by pure brain tubulins of Antarctic fishes exhibit slow dynamics at both low (5 degrees C) and high (25 degrees C) temperatures; the rates of polymer growth and shortening and the frequencies of interconversion between these states are small relative to those observed for mammalian microtubules (37 degrees C). To investigate the contribution of tubulin primary sequence variation to the functional properties of the microtubules of Antarctic fishes, we have sequenced brain cDNAs that encode 9 alpha-tubulins and 4 beta-tubulins from the yellowbelly rockcod Notothenia coriiceps and 4 alpha-tubulins and 2 beta-tubulins from the ocellated icefish Chionodraco rastrospinosus. The tubulins of these fishes were found to contain small sets of unique or rare residue substitutions that mapped to the lateral, interprotofilament surfaces or to the interiors of the alpha- and beta-polypeptides; longitudinal interaction surfaces are not altered in the fish tubulins. Four changes (A278T and S287T in alpha; S280G and A285S in beta) were present in the S7-H9 interprotofilament "M" loops of some monomers and would be expected to increase the flexibility of these regions. A fifth lateral substitution specific to the alpha-chain (M302L or M302F) may increase the hydrophobicity of the interprotofilament interaction. Two hydrophobic substitutions (alpha:S187A in helix H5 and beta:Y202F in sheet S6) may act to stabilize the monomers in conformations favorable to polymerization. We propose that cold adaptation of microtubule assembly in Antarctic fishes has occurred in part by evolutionary restructuring of the lateral surfaces and the cores of the tubulin monomers.

Evolution, organization, and expression of alpha-tubulin genes in the antarctic fish Notothenia coriiceps. Adaptive expansion of a gene family by recent gene duplication, inversion, and divergence.

To assess the organization and expression of tubulin genes in ectothermic vertebrates, we have chosen the Antarctic yellowbelly rockcod, Notothenia coriiceps, as a model system. The genome of N. coriiceps contains approximately 15 distinct DNA fragments complementary to alpha-tubulin cDNA probes, which suggests that the alpha-tubulins of this cold-adapted fish are encoded by a substantial multigene family. From an N. coriiceps testicular DNA library, we isolated a 13.8-kilobase pair genomic clone that contains a tightly linked cluster of three alpha-tubulin genes, designated NcGTbalphaa, NcGTbalphab, and NcGTbalphac. Two of these genes, NcGTbalphaa and NcGTbalphab, are linked in head-to-head (5' to 5') orientation with approximately 500 bp separating their start codons, whereas NcGTbalphaa and NcGTbalphac are linked tail-to-tail (3' to 3') with approximately 2.5 kilobase pairs between their stop codons. The exons, introns, and untranslated regions of the three alpha-tubulin genes are strikingly similar in sequence, and the intergenic region between the alphaa and alphab genes is significantly palindromic. Thus, this cluster probably evolved by duplication, inversion, and divergence of a common ancestral alpha-tubulin gene. Expression of the NcGTbalphac gene is cosmopolitan, with its mRNA most abundant in hematopoietic, neural, and testicular tissues, whereas NcGTbalphaa and NcGTbalphab transcripts accumulate primarily in brain. The differential expression of the three genes is consistent with distinct suites of putative promoter and enhancer elements. We propose that cold adaptation of the microtubule system of Antarctic fishes is based in part on expansion of the alpha- and beta-tubulin gene families to ensure efficient synthesis of tubulin polypeptides.

The major adult alpha-globin gene of antarctic teleosts and its remnants in the hemoglobinless icefishes. Calibration of the mutational clock for nuclear genes.

The icefishes of the Southern Ocean (family Channichthyidae, suborder Notothenioidei) are unique among vertebrates in their inability to synthesize hemoglobin. We have shown previously (Cocca, E., Ratnayake-Lecamwasam, M., Parker, S. K., Camardella, L., Ciaramella, M., di Prisco, G., and Detrich, H. W., III (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1817-1821) that icefishes retain inactive genomic remnants of adult notothenioid alpha-globin genes but have lost the gene that encodes adult beta-globin. Here we demonstrate that loss of expression of the major adult alpha-globin, alpha1, in two species of icefish (Chaenocephalus aceratus and Chionodraco rastrospinosus) results from truncation of the 5' end of the notothenioid alpha1-globin gene. The wild-type, functional alpha1-globin gene of the Antarctic yellowbelly rockcod, Notothenia coriiceps, contains three exons and two A + T-rich introns, and its expression may be controlled by two or three distinct promoters. Retained in both icefish genomes are a portion of intron 2, exon 3, and the 3'-untranslated region of the notothenioid alpha1-globin gene. The residual, nonfunctional alpha-globin gene, no longer under positive selection pressure for expression, has apparently undergone random mutational drift at an estimated rate of 0.12-0.33%/million years. We propose that abrogation of hemoglobin synthesis in icefishes most likely resulted from a single mutational event in the ancestral channichthyid that deleted the entire beta-globin gene and the 5' end of the linked alpha1-globin gene.

The cloche and spadetail genes differentially affect hematopoiesis and vasculogenesis.

In vertebrates, hematopoietic and vascular progenitors develop from ventral mesoderm. The first primitive wave of hematopoiesis yields embryonic red blood cells, whereas progenitor cells of subsequent definitive waves form all hematopoietic cell lineages. In this report we examine the development of hematopoietic and vasculogenic cells in normal zebrafish and characterize defects in cloche and spadetail mutant embryos. The zebrafish homologs of lmo2, c-myb, fli1, flk1, and flt4 have been cloned and characterized in this study. Expression of these genes identifies embryonic regions that contain hematopoietic and vascular progenitor cells. The expression of c-myb also identifies definitive hematopoietic cells in the ventral wall of the dorsal aorta. Analysis of b316 mutant embryos that carry a deletion of the c-myb gene demonstrates that c-myb is not required for primitive erythropoiesis in zebrafish even though it is expressed in these cells. Both cloche and spadetail mutant embryos have defects in primitive hematopoiesis and definitive hematopoiesis. The cloche mutants also have significant decreases in vascular gene expression, whereas spadetail mutants expressed normal levels of these genes. These studies demonstrate that the molecular mechanisms that regulate hematopoiesis and vasculogenesis have been conserved throughout vertebrate evolution and the clo and spt genes are key regulators of these programs.

Microtubule assembly in cold-adapted organisms: functional properties and structural adaptations of tubulins from antarctic fishes.

Fishes native to the coastal waters of the Antarctic have adapted to habitat and body temperatures in the range -1.8 to +2 degrees C. Their cytoplasmic microtubules, unlike those of mammals and temperate poikilotherms, have evolved to assemble efficiently at these low temperatures. To learn about the underlying molecular adaptations, my laboratory is studying microtubule proteins [tubulin alpha beta dimers and microtubule-associated proteins (MAPs)] and tubulin genes from several Antarctic fishes, including the rockcods Notothenia coriiceps and Gobionotothen gibberifrons. We find that the assembly-enhancing adaptations of the fish microtubule proteins are intrinsic to the tubulin subunits themselves. Furthermore, microtubule formation by Antarctic fish tubulins is strongly entropy driven, due in part to an increased reliance, relative to tubulins from other species, on hydrophobic interactions. Based on analyses of tubulin polypeptides and cDNAs, we suggest that the structural adaptations of Antarctic fish tubulins most likely involve alterations in the primary sequences of tubulin isotypes. With respect to neural beta tubulins from other vertebrates, for example, the class II beta-tubulin isotype of N. coriiceps brain contains seven unique amino acid substitutions and one novel insertion in its 446-residue primary sequence. Most of these changes are located in a structural domain that forms contacts between tubulin dimers during microtubule assembly and would be expected to enhance polypeptide flexibility, thereby facilitating addition of tubulin to microtubule ends. The acidic carboxy-terminal tails of the alpha and beta tubulins, by contrast, appear not to be sites of cold adaptation of polymerization. We have also found that brain and egg tubulins from Antarctic fishes differ strikingly in their polymerization efficiencies, which demonstrates, in agreement with the multitubulin hypothesis, that tissue-specific tubulin isoforms can possess distinct functional properties. Thus, study of microtubule proteins from organisms, such as the Antarctic fishes, that have adapted to extreme thermal regimes should contribute significantly to an understanding of the quaternary interactions that control microtubule assembly in all eukaryotes.

Solar UVB-induced DNA damage and photoenzymatic DNA repair in antarctic zooplankton.

The detrimental effects of elevated intensities of mid-UV radiation (UVB), a result of stratospheric ozone depletion during the austral spring, on the primary producers of the Antarctic marine ecosystem have been well documented. Here we report that natural populations of Antarctic zooplankton also sustain significant DNA damage [measured as cyclobutane pyrimidine dimers (CPDs)] during periods of increased UVB flux. This is the first direct evidence that increased solar UVB may result in damage to marine organisms other than primary producers in Antarctica. The extent of DNA damage in pelagic icefish eggs correlated with daily incident UVB irradiance, reflecting the difference between acquisition and repair of CPDs. Patterns of DNA damage in fish larvae did not correlate with daily UVB flux, possibly due to different depth distributions and/or different capacities for DNA repair. Clearance of CPDs by Antarctic fish and krill was mediated primarily by the photoenzymatic repair system. Although repair rates were large for all species evaluated, they were apparently inadequate to prevent the transient accumulation of substantial CPD burdens. The capacity for DNA repair in Antarctic organisms was highest in those species whose early life history stages occupy the water column during periods of ozone depletion (austral spring) and lowest in fish species whose eggs and larvae are abundant during winter. Although the potential reduction in fitness of Antarctic zooplankton resulting from DNA damage is unknown, we suggest that increased solar UV may reduce recruitment and adversely affect trophic transfer of productivity by affecting heterotrophic species as well as primary producers.

Inner and outer arm axonemal dyneins from the Antarctic rockcod Notothenia coriiceps.

Adaptive compensation of enzymatic activities is common among cold-living poikilotherms. Their enzymes often demonstrate higher activities at low temperatures than do homologs from temperate or thermophilic species. To understand the molecular features necessary for cold adaptation of microtubule motor proteins, we have initiated studies of the flagellar dynein ATPases of Antarctic fishes (body temperature range = -1.8 to +2 degrees C). Dyneins were isolated by high-salt extraction of demembranated sperm axonemes from the Antarctic yellowbelly rockcod, Notothenia coriiceps. Although solubilization of inner arms was incomplete, an inner arm dynein was recognized as a discrete complex containing one major dynein heavy chain (DHC) and sedimenting through sucrose gradients at approximately 12 S. Like inner arm dyneins from Chlamydomonas, the fish complex contained an actin-immunoreactive protein of 43 kDa and a 30-kDa protein. One isoform of the inner arm DHC gene family of N. coriiceps was detected by the polymerase chain reaction, and Southern analysis established that this DHC gene is present at one copy per haploid genome. Outer arm dynein was extracted quantitatively by high-salt treatment, contained two DHCs (one major, one minor), and sedimented through sucrose gradients as a polydisperse, aggregating system. Associated with the outer arm DHCs were five presumptive intermediate chains (ICs) of 66-91 kDa, immunologically defined by their cross-reactivity to four monoclonal antibodies specific for ICs from other organisms. The basal (non-microtubule-stimulated) specific ATPase activities of the N. coriiceps inner and outer arm dyneins were approximately 0.07 and approximately 0.04 micromol of P(i) min(-1) mg(-1), respectively, at 0 degrees C, attained their maxima (approximately 0.1 micromol of P(i) min(-1) mg(-1)) at 9 and 19 degrees C, respectively, and at higher temperatures declined substantially. Furthermore, the activities of the fish dyneins at temperatures < or = 15 degrees C were significantly larger than that of outer arm dynein from the mesophile Tetrahymena. These results suggest that the greater catalytic efficiencies of N. coriiceps inner and outer arm dyneins at low temperatures are due to enhanced polypeptide flexibility in the active sites of their protein subunits. We conclude that temperature adaptation of flagellar dyneins from Antarctic fishes is compatible with substantial conservation of primary and quaternary structure.

MAP 0, a 400-kDa microtubule-associated protein unique to teleost fish.

Microtubules from neural tissues of the Atlantic cod, Gadus morhua, and of several species of Antarctic teleosts are composed of tubulin and several microtubule-associated proteins (MAPs), one of which has an apparent molecular weight of approximately 400-430 kDa. Because its apparent molecular weight exceeds those of the MAP 1 proteins, we designate this high molecular weight teleost protein MAP 0. Cod MAP 0 failed to cross-react with antibodies specific for MAPs 1A, 1B and 2 of mammalian brain, for MAP H1 of squid optic lobe, and for chicken erythrocyte syncolin, which suggests that it has a novel structure. Similarly, MAP 0 from the Antarctic fish was not recognized by an antibody specific for bovine MAP 2. Together, these observations suggest that MAP 0 is a novel MAP that may be unique to fish. To determine the tissue specificity and phylogenetic distribution of this protein, we generated a rabbit polyclonal antibody against cod MAP 0. Using this antibody, we found that MAP 0 was present in microtubule proteins isolated from cod brain tissues and spinal cord but was absent in microtubules from heart, liver, and spleen. At the subcellular level, MAP 0 was distributed in cod brain cells in a punctate pattern coincident with microtubules but was absent in skin cells. MAP 0 was also detected in cells of the peripheral nervous system. A survey of microtubule proteins from chordates and invertebrates showed that anti-MAP 0-reactive homologs were present in five teleost species but not in more primitive fish and invertebrates or in higher vertebrates. MAP 0 bound to cod microtubules by ionic interaction at a site recognized competitively by bovine MAP 2. Although its function is unknown, MAP 0 does not share the microtubule-binding properties of the motor proteins kinesin and dynein. We propose that MAP 0 is a unique, teleost-specific MAP.

Unexpected beta2-microglobulin sequence diversity in individual rainbow trout.

For mammals beta2-microglobulin (beta2m), the light chain of major histocompatibility complex (MHC) class I molecules, is invariant (or highly conserved) and is encoded by a single gene unlinked to the MHC. We find that beta2m of a salmonid fish, the rainbow trout (Oncorhynchus mykiss), does not conform to the mammalian paradigm. Ten of 12 randomly selected beta2m cDNA clones from an individual fish have different nucleotide sequences. A complex restriction fragment length polymorphism pattern is observed with rainbow trout, suggesting multiple beta2m genes in the genome, in excess of the two genes expected from the ancestral salmonid tetraploidy. Additional duplication and diversification of the beta2m genes might have occurred subsequently. Variation in the beta2m cDNA sequences is mainly at sites that do not perturb the structure of the mature beta2m protein, showing that the observed diversity of the trout beta2m genes is not primarily a result of pathogen selection.

Intraembryonic hematopoietic cell migration during vertebrate development.

Vertebrate hematopoietic stem cells are derived from vental mesoderm, which is postulated to migrate to both extra- and intraembryonic positions during gastrula and neurula stages. Extraembryonic migration has previously been documented, but the origin and migration of intraembryonic hematopoietic cells have not been visualized. The zebrafish and most other teleosts do not form yolk sac blood islands during early embryogenesis, but instead hematopoiesis occurs solely in a dorsal location known as the intermediate cell mass (IM) or Oellacher. In this report, we have isolated cDNAs encoding zebrafish homologs of the hematopoietic transcription factors GATA-1 and GATA-2 and have used these markers to determine that the IM is formed from mesodermal cells in a posterior-lateral position on the yolk syncytial layer of the gastrula yolk sac. Surprisingly, cells of the IM then migrate anteriorly through most of the body length prior to the onset of active circulation and exit onto the yolk sac. These findings support a hypothesis in which the hematopoietic program of vertebrates is established by variations in homologous migration pathways of extra- and intraembryonic progenitors.

Cloche, an early acting zebrafish gene, is required by both the endothelial and hematopoietic lineages.

Endothelial and hematopoietic cells appear synchronously on the extra-embryonic membranes of amniotes in structures known as blood islands. This observation has led to the suggestion that these two ventral lineages share a common progenitor. Recently, we have shown in the zebrafish, Danio rerio, that a single cell in the ventral marginal zone of the early blastula can give rise to both endothelial and blood cells as well as to other mesodermal cells (Stainier, D. Y. R., Lee, R. K. and Fishman, M. C. (1993). Development 119, 31-40; Lee, R. K. K., Stainier, D. Y. R., Weinstein, B. M. and Fishman, M. C. (1994). Development 120, 3361-3366). Here we describe a zebrafish mutation, cloche, that affects both the endothelial and hematopoietic lineages at a very early stage. The endocardium, the endothelial lining of the heart, is missing in mutant embryos. This deletion is selective as evidenced by the presence of other endothelial cells, for example those lining the main vessels of the trunk. Early cardiac morphogenesis proceeds normally even in the absence of the endocardium. The myocardial cells form a tube that is demarcated into chambers, beats rhythmically, but exhibits a reduced contractility. This functional deficit is likely due to the absence of the endocardial cells, although it may be a direct effect of the mutation on the myocardial cells. Cell transplantation studies reveal that the endothelial defect, i.e. the endocardial deletion, is a cell-autonomous lesion, consistent with the possibility that cloche is part of a signal transduction pathway. In addition, the number of blood cells is greatly reduced in cloche mutants and the hematopoietic tissues show no expression of GATA-1 or GATA-2, two key hematopoietic transcription factors that are first expressed during early embryogenesis. These results show that cloche is involved in the genesis and early diversification of the endothelial and blood lineages, possibly by affecting a common progenitor cell population.

Genomic remnants of alpha-globin genes in the hemoglobinless antarctic icefishes.

Alone among piscine taxa, the antarctic icefishes (family Channichthyidae, suborder Notothenioidei) have evolved compensatory adaptations that maintain normal metabolic functions in the absence of erythrocytes and the respiratory oxygen transporter hemoglobin. Although the uniquely "colorless" or "white" condition of the blood of icefishes has been recognized since the early 20th century, the status of globin genes in the icefish genomes has, surprisingly, remained unexplored. Using alpha- and beta-globin cDNAs from the antarctic rockcod Notothenia coriiceps (family Nototheniidae, suborder Notothenioidei), we have probed the genomes of three white-blooded icefishes and four red-blooded notothenioid relatives (three antarctic, one temperate) for globin-related DNA sequences. We detect specific, high-stringency hybridization of the alpha-globin probe to genomic DNAs of both white- and red-blooded species, whereas the beta-globin cDNA hybridizes only to the genomes of the red-blooded fishes. Our results suggest that icefishes retain inactive genomic remnants of alpha-globin genes but have lost, either through deletion or through rapid mutation, the gene that encodes beta-globin. We propose that the hemoglobinless phenotype of extant icefishes is the result of deletion of the single adult beta-globin locus prior to the diversification of the clade.

Polymerization of Antarctic fish tubulins at low temperatures: role of carboxy-terminal domains.

We have proposed previously that the efficient polymerization of tubulins from Antarctic fishes at low, physiological temperatures (-1.8 to +2 degrees C) may result in part from adaptations (e.g., reductions in acidic residues) located in their carboxy-terminal (C-terminal) tails [Detrich & Overton (1986) J. Biol. Chem. 261, 10922-10930]. To test this hypothesis, we have examined the polymerization of Antarctic fish neural tubulins modified at their C termini by proteolysis or by neutralization of carboxyl groups. Addition of subtilisin to low concentrations of Notothenia coriiceps tubulin induced a biphasic assembly reaction: stage I corresponded to the C-terminal cleavage of beta chains to produce alpha beta s dimers, and stage II coincided with the slower, C-terminal cleavage of alpha chains to yield alpha s beta s. Both stage I and stage II polymers consisted of protofilament sheets and microtubules with attached sheets. The critical concentration for assembly of the stage II polymer was at least 10-fold lower than that of untreated tubulin. Neutralization of Glu and Asp carboxyls in Gobionotothen gibberifrons microtubules by the carbodiimide-catalyzed incorporation of glycine ethyl ester (GEE) moieties produced a tubulin, modified largely in its C termini, that assembled more readily than did control tubulin. When 12 GEE groups were incorporated per dimer, the critical concentrations for assembly of modified tubulin at 5-10 degrees C were 2-3-fold smaller than those for the unmodified protein. Comparably modified bovine tubulin (10 GEE/dimer) assembled at 37 degrees C with a critical concentration 2.6-fold lower than that for the unmodified tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamic instability of microtubules from cold-living fishes.

The dynamic instability of microtubules free of microtubule-associated proteins from two genera of cold-living fishes was measured, by means of video-enhanced differential-interference-contrast microscopy, at temperatures near those of their habitats. Brain microtubules were isolated from the boreal Atlantic cod (Gadus morhua; habitat temperature approximately 2-15 degrees C) and from two austral Antarctic rockcods (Notothenia gibberifrons and N. coriiceps neglecta; habitat temperature approximately -1.8 to + 2 degrees C). Critical concentrations for polymerization of the fish tubulins were in the neighborhood of 1 mg/ml, consistent with high interdimer affinities. Rates of elongation and frequencies of growth-to-shortening transitions ("catastrophes") for fish microtubules were significantly smaller than those for mammalian microtubules. Slow dynamics is therefore an intrinsic property of these fish tubulins, presumably reflecting their adaptation to low temperatures. Two-dimensional electrophoresis showed striking differences between the isoform compositions of the cod and the rockcod tubulins, which suggests that the cold-adapted microtubule phenotypes of northern and southern fishes may have arisen independently.