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J L Salisbury

Publications and source records attributed to J L Salisbury.

At least 19 recordsLinked to original sources
PubMed2001

Phosphorylation of centrin during the cell cycle and its role in centriole separation preceding centrosome duplication.

Once during each cell cycle, mitotic spindle poles arise by separation of newly duplicated centrosomes. We report here the involvement of phosphorylation of the centrosomal protein centrin in this process. We show that centrin is phosphorylated at serine residue 170 during the G(2)/M phase of the cell cycle. Indirect immunofluorescence staining of HeLa cells using a phosphocentrin-specific antibody reveals intense labeling of mitotic spindle poles during prophase and metaphase of the cell division cycle, with diminished staining of anaphase and no staining of telophase and interphase centrosomes. Cultured cells undergo a dramatic increase in centrin phosphorylation following the experimental elevation of PKA activity, suggesting that this kinase can phosphorylate centrin in vivo. Surprisingly, elevated PKA activity also resulted intense phosphocentrin antibody labeling of interphase centrosomes and in the concurrent movement of individual centrioles apart from one another. Taken together, these results suggest that centrin phosphorylation signals the separation of centrosomes at prophase and implicates centrin phosphorylation in centriole separation that normally precedes centrosome duplication.

Amino Acid Sequence↗
PubMed2001

Characterization of the X-linked murine centrin Cetn2 gene.

A multi-gene family (Cetn1, Cetn2, and Cetn3) encodes the calcium-binding protein, centrin, in the mouse. This work characterizes the Cetn2 gene. Structurally, Cetn2 consists of five exons and four introns, and contains a classical TATA-less promoter. Cetn2 has two alternate transcription start sites, and a single length 3' untranslated region. Fluorescence in situ hybridization demonstrates that Cetn2 is an X-linked gene whose alleles replicate asynchronously during S-phase. Cetn2 encodes a 172 amino acid protein, with a predicted molecular mass of 19,795 Da (pI=4.71), that contains all of the defining characteristics of centrin. Northern blot analysis indicates that Cetn2 is ubiquitously expressed in the tissues of adult mice. RT-PCR shows that Cetn2 and Cetn3, but not Cetn1, are expressed in NIH 3T3 cells. Immunofluorescence microscopy demonstrates that mouse centrin 2 protein localizes to the region immediately surrounding the centrioles in the centrosome of NIH 3T3 cells.

3T3 Cells↗
PubMed2000

GFP-centrin as a marker for centriole dynamics in living cells.

A long-standing puzzle in cell biology is the question of how cells generate one and only one new centrosome in each cell cycle and what is the role of the centriole pair in this process. In this study, the introduction of GFP-centrin into cultured cells allows direct visualization of centriole behavior in living cells and in real time. Using this method, centriole dynamics can be observed throughout the cell cycle and following a variety of experimental treatments. Our studies demonstrate that the biogenesis of new centrioles from individual members of a preexisting centriole pair is asynchronous: the older centriole initiates assembly of a new daughter centriole before the younger centriole initiates assembly of its daughter.

Calcium-Binding Proteins↗
PubMed1999

Testis-specific murine centrin, Cetn1: genomic characterization and evidence for retroposition of a gene encoding a centrosome protein.

Centrin is a centrosome component in species from yeast to humans. Here, the mouse centrin 1 gene (Cetn1) is analyzed with respect to its genomic structure, chromosome localization, tissue-specific expression, and phylogenetic relationship to the other mouse centrin genes and their human orthologs. Cetn1 is an intronless gene located on chromosome 18A2 that encodes a 172-amino-acid protein with a predicted molecular mass of 19,696 Da (pI 4.61) and all of the structural features common to centrin. Cetn1 possesses the sequence features of an expressed retroposon: the gene lacks introns, the open reading frame is not interrupted by stop codons, and the coding region is flanked by a pair of direct repeats. Reverse transcriptase-polymerase chain reaction and Northern blot analysis demonstrate that Cetn1 expression is limited exclusively to the testis in adult male mice. Cetn1 expression is first seen in the neonatal testis at 14 days postpartum, reaching adult levels by day 17. These observations provide new insight into the regulation, function, and evolutionary history of centrin in higher eukaryotes.

Amino Acid Sequence↗
PubMed1999

Centriole and centrin degeneration during mouse spermiogenesis.

Centrosome reduction during mouse spermiogenesis has been studied by immunofluorescent microscopy using anticentrin antibody (20H5) and TEM. Centrin is detected as two spots in round spermatids, corresponding to a pair of centrioles. In elongating spermatids, centrin spots colocalize with the centrioles in the neck region, while the perinuclear ring from which manchette microtubules arise, does not label with the antibody 20H5. The proximal centriole of the elongating spermatids develops a prominent adjunct, which assembles an aster of microtubules. TEM studies after immunogold labeling revealed that centrin is associated with the distal and the proximal centrioles, but not with the adjunct. Centrin labeling in the neck region diminishes after spermiation stage, although it is not completely lost from all testicular sperm. Mature epididymal sperm do not display centrin labeling. Mouse sperm lose both distal and proximal centrioles at maturity. Loss of centrin staining appears to correlate with the degeneration of centrioles during mouse spermiogenesis.

Animals↗
PubMed1999

Regulation and regulatory activities of centrosomes.

The centrosome functions in the organization of the cytoskeleton, in specification of cell polarity, and in the assembly of the bipolar spindle during mitosis. These activities are largely the result of microtubule nucleation activity and the centrosome's structural influence on the form of the microtubule array that it anchors. Centrosome duplication and microtubule nucleation activity are precisely regulated during development and the cell cycle. Loss of normal centrosome regulation and function may lead to alterations in cell polarity and to chromosomal instability through mitotic defects resulting in aneuploidy. This is particularly true for many malignant tumors. Here, we review the regulation and regulatory activities of centrosomes and consider some of the questions of current interest in this area. J. Cell. Biochem. Suppls. 32/33:192-199, 1999.

Animals↗
PubMed1999

Altered centrosome structure is associated with abnormal mitoses in human breast tumors.

Centrosomes are the major microtubule organizing center in mammalian cells and establish the spindle poles during mitosis. Centrosome defects have been implicated in disease and tumor progression and have been associated with nullizygosity of the p53 tumor suppressor gene. In the present ultrastructural analysis of 31 human breast tumors, we found that centrosomes of most tumors had significant alterations compared to centrosomes of normal breast tissue. These alterations in included 1) supernumerary centrioles, 2) excess pericentriolar material, 3) disrupted centriole barrel structure, 4) unincorporated microtubule complexes, 5) centrioles of unusual length, 6) centrioles functioning as ciliary basal bodies, and 7) mispositioned centrosomes. These alterations are associated with changes in cell polarity, changes in cell and tissue differentiation, and chromosome missegregation through multipolar mitoses. Significantly, the presence of excess pericentriolar material was associated with the highest frequency of abnormal mitoses. Centrosome abnormalities may confer a mutator phenotype to tumors, occasionally yielding cells with a selective advantage that emerge and thrive, thus leading the tumor to a more aggressive state.

Adenocarcinoma↗
PubMed1999

Biparental inheritance of gamma-tubulin during human fertilization: molecular reconstitution of functional zygotic centrosomes in inseminated human oocytes and in cell-free extracts nucleated by human sperm.

Human sperm centrosome reconstitution and the parental contributions to the zygotic centrosome are examined in mammalian zygotes and after exposure of spermatozoa to Xenopus laevis cell-free extracts. The presence and inheritance of the conserved centrosomal constituents gamma-tubulin, centrin, and MPM-2 (which detects phosphorylated epitopes) are traced, as is the sperm microtubule-nucleating capability on reconstituted centrosomes. gamma-Tubulin is biparentally inherited in humans (maternal >> than paternal): Western blots detect the presence of paternal gamma-tubulin. Recruitment of maternal gamma-tubulin to the sperm centrosome occurs after sperm incorporation in vivo or exposure to cell-free extract, especially after sperm "priming" induced by disulfide bond reduction. Centrin is found in the proximal sperm centrosomal region, demonstrates expected calcium sensitivity, but appears absent from the zygotic centrosome after sperm incorporation or exposure to extracts. Sperm centrosome phosphorylation is detected after exposure of primed sperm to egg extracts as well as during the early stages of sperm incorporation after fertilization. Finally, centrosome reconstitution in cell-free extracts permits sperm aster microtubule assembly in vitro. Collectively, these results support a model of a blended zygotic centrosome composed of maternal constituents attracted to an introduced paternal template after insemination.

Animals↗
PubMed1999

Microtubule nucleating capacity of centrosomes in tissue sections.

We used a novel adaptation of methods for microtubule polymerization in vitro to assess the MTOC activity of centrosomes in frozen-sectioned tissues. Remarkably, centrosomes of tissue sections retain the ability to nucleate microtubules even after several years of storage as frozen tissue blocks. Adaptations of these methods allow accurate counts of microtubules from individual cells and the quantitative estimation the MTOC activity of the intact tissue. These methods can be utilized to characterize MTOC activity in normal and diseased tissues and in particular tissues at different stages of development. (J Histochem Cytochem 47:1265-1273, 1999)

Adenocarcinoma↗
PubMed1999

Centrosomes and cancer.

The centrosome functions as the major microtubule organizing center (MTOC) of the cell and as such it determines the number, polarity, and organization of interphase and mitotic microtubules. Cytoplasmic organization, cell polarity and the equal partition of chromosomes into daughter cells at the time of cell division are all dependent on the normal function of the centrosome and on its orderly duplication, once and only once, in each cell cycle. Malignant tumor cells show characteristic defects in cell and tissue architecture and in chromosome number that can be attributed to inappropriate centrosome behavior during tumor progression. In this review, we will summarize recent observations linking centrosome defects to disruption of normal cell and tissue organization and to chromosomal instability found in malignant tumors.

Aneuploidy↗
PubMed1998

A novel immediate early response gene, IEX-1, is induced by ultraviolet radiation in human keratinocytes.

Skin cancer is the most common human malignancy and is strongly associated with exposure to ultraviolet radiation (UVR). Several mechanisms including an increase in immediate early gene activation have been postulated to be involved in UVR-mediated carcinogenesis. We show that in a dose-dependent manner, UVR induces the expression of messenger RNA of a novel immediate early response gene, IEX-1, in human keratinocytes. Human keratinocytes and mouse fibroblasts transfected with an expression plasmid for IEX-1 grow at a faster rate than keratinocytes transfected with a similar plasmid that does not contain the IEX-1 sequence. IEX-1 protein is localized predominantly in the nucleus of keratinocytes by fluorescent antibody methods and by examination of the location of a green fluorescence IEX-1 fusion protein. Epidermal growth factor (EGF), a major mitogen of keratinocytes, and a tumor-promoting phorbol ester increase IEX-1 mRNA expression. IEX-1 may play a role in keratinocyte proliferation especially following UVR.

3T3 Cells↗
PubMed1998

Expression of centrin isoforms in the mammalian retina.

Centrin is a calcium-binding phosphoprotein of centrosomes, mitotic spindle poles, and flagellar basal apparatus. Indirect immunofluorescence studies in human and rat retinas reveal centrin localization in two distinct cellular structures: at centrosomes of nonciliated neuronal cells as well as in basal bodies, and in larger amounts in the highly modified cilium--the connecting cilium--of photoreceptor cells. Western blot analyses of mammalian retinal proteins show two closely migrating centrin bands at about 20 kDa, the previously described molecular weight of centrins. Using isoform specific primers in PCR, the expression of two related but distinct forms of centrin (centrin 1 and centrin 2), can be identified in the retina of human and rat as well as in the mammalian testis, tissues where cilia are present. However, only one isoform (centrin 2) is expressed in nondifferentiated, nonciliated retinal cells (retinoblastoma cells), as well as in rat liver, skeletal muscle, and cardiac muscle. These observations suggest centrin 2 message may be universally expressed while centrin 1 message may be restricted to retina and testis which contain cells that have differentiated cilia or flagella, or their modifications.

Animals↗
PubMed1998

Centrosome hypertrophy in human breast tumors: implications for genomic stability and cell polarity.

The centrosome plays an important role in maintenance of cell polarity and in progression through the cell cycle by determining the number, polarity, and organization of interphase and mitotic microtubules. By examining a set of 35 high grade human breast tumors, we show that centrosomes of adenocarcinoma cells generally display abnormal structure, aberrant protein phosphorylation, and increased microtubule nucleating capacity in comparison to centrosomes of normal breast epithelial and stromal tissues. These structural and functional centrosome defects have important implications for understanding the mechanisms by which genomic instability and loss of cell polarity develop in solid tumors.

Adenocarcinoma↗
PubMed1997

A transformation-associated complex involving tyrosine kinase signal adapter proteins and caldesmon links v-erbB signaling to actin stress fiber disassembly.

The avian erythroblastosis viral oncogene (v-erbB) encodes a receptor tyrosine kinase that possesses sarcomagenic and leukemogenic potential. We have expressed transforming and nontransforming mutants of v-erbB in fibroblasts to detect transformation-associated signal transduction events. Coimmunoprecipitation and affinity chromatography have been used to identify a transformation-associated, tyrosine phosphorylated, multiprotein complex. This complex consists of Src homologous collagen protein (Shc), growth factor receptor binding protein 2 (Grb2), son of sevenless (Sos), and a novel tyrosine phosphorylated form of the cytoskeletal regulatory protein caldesmon. Immunofluorescence localization studies further reveal that, in contrast to the distribution of caldesmon along actin stress fibers in normal fibroblasts, caldesmon colocalizes with Shc in plasma membrane blebs in transformed fibroblasts. This colocalization of caldesmon and Shc correlates with actin stress fiber disassembly and v-erbB-mediated transformation. The tyrosine phosphorylation of caldesmon, and its association with the Shc-Grb2-Sos signaling complex directly links tyrosine kinase oncogenic signaling events with cytoskeletal regulatory processes, and may define one mechanism regulating actin stress fiber disassembly in transformed cells.

Actins↗
PubMed1997

Centrin and the cytoskeleton of the protist Holomastigotoides.

The cytoskeleton of the parabasalid protozoan Holomastigotoides was investigated by epifluorescence, scanning confocal, and transmission electron microscopy using antibodies to centrin, tubulin, and MPM-2 epitopes. Previous microscopic analysis of Holomastigotoides spp. has shown that up to 10,000 flagella are arranged in 2-8 spiral bands encircling the cell. Spindle poles are associated with two flagellar bands. Sheets of cytoplasmic microtubules (MTs) called axostyles originate in the cell apex and extend to the cell base. Antibodies to centrin, a member of the EF-hand family of calcium-binding proteins, labeled a number of structures in Holomastigotoides, namely axostyles, the mitotic spindle, and portions of flagellar bands. The identity of these structures was confirmed by transmission electron microscopy and by immunofluorescence microscopy using antibodies to tubulin and MPM-2 epitopes. Antibodies to tubulin labeled MTs in basal bodies, flagella, axostyles, and the mitotic spindle. MPM-2 antibodies labeled spindle poles and short segments of flagellar bands to which the spindle poles are attached. Centrin is known to show calcium-sensitive contractile behavior. The pattern of flagellar band staining by antibodies to centrin was affected by [Ca2+]. In detergent-extracted cell fragments, the centrin staining pattern could be changed by changing [Ca2+]. This Ca2+ effect was modulated by a monoclonal antibody to centrin (20H5), indicating that centrin plays a role in altering flagellar band structure. These results show that centrin is located in key positions for maintaining cell polarity and directing cell movement through interactions with other cytoskeletal elements. Calcium may regulate the morphology of centrin-containing structures.

Animals↗
PubMed1996

Most of centrin in animal cells is not centrosome-associated and centrosomal centrin is confined to the distal lumen of centrioles.

Centrin is a member of the calcium-binding EF-hand protein superfamily present in centrosomes of widely divergent species. Investigating the cellular distribution of human centrin by both immunofluorescence and cell fractionation, we report that centrin is biochemically complex in human cells, displaying as much as ten isoforms in 2-D electrophoresis. This suggests that centrin may be subject to multiple regulations. Strikingly, more than 90% of centrin is not associated with the centrosome fraction. The centrosome-associated centrin, however, displays a specific pattern in 2-D electrophoresis and is concentrated within the distal lumen of the centrioles, where a complex structure has been previously described. This precise localization allows the resolution of centrioles at the optical level throughout the cell cycle and provides a valuable tool for monitoring centriole duplication. To get insights on centrin function, we performed injection experiments of recombinant heterologous centrin in two-cell stage frog embryos in an attempt to produce dominant negative effects. We report that green algae and human centrin delay cleavage and promote the formation of abnormal blastomeres in which the distribution of microtubule asters and of nuclei is dramatically impaired. This suggests that centrin could be involved in the centrosome reproduction cycle, in the coordination of cytoplasmic and nuclear division or in cytokinesis.

Animals↗
PubMed1995

Identification of a complex between centrin and heat shock proteins in CSF-arrested Xenopus oocytes and dissociation of the complex following oocyte activation.

Coimmunoprecipitation experiments using a monoclonal anti-centrin antibody (20H5) and cytostatic factor (CSF)-arrested Xenopus oocyte extracts specifically precipitates oocyte centrin (20-kDa) and two associated proteins of 70- and 90-kDa. Microsequence analysis of a tryptic peptide fragment of the 70-kDa protein reveals 100% identity with a 13-amino-acid peptide sequence from Xenopus heat shock protein hsp-70. Western blot analysis of immunoprecipitates using anti-hsp monoclonal antibodies (N27 and AC-88) confirms the identity of the 70-kDa protein as hsp-70 and identifies the 90-kDa protein as hsp-90. The centrin/hsp complex is also immunoprecipitated when anti-hsp-70 or anti-hsp-90 monoclonal antibodies (BB70 and 4F3, respectively) are used as primary antibodies during immunoprecipitation. The centrin/hsp complex is sensitive to pH and Ca2+ concentration. The complex shows differential dissociation of hsp-70 and hsp-90 under a variety of conditions, suggesting that each hsp can bind to centrin independently of the other. When oocytes are first activated by electric shock or ionophore treatment, followed by immunoprecipitation using anti-centrin monoclonal antibody 20H5, centrin precipitates with significantly reduced levels of hsp-70 in the complex, and these complexes contain no apparent hsp-90. We conclude that, in CSF-arrested oocytes, the centrosomal protein, centrin, is associated as a complex with the heat shock proteins, hsp-70 and hsp-90, and that this complex dissociates upon activation of the oocyte. The functional consequences of the formation of complexes between centrin and these hsps are unknown. However, based on the roles that have been defined for heat shock proteins in other systems, several possibilities are suggested.

Amino Acid Sequence↗