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Monoclonal and polyclonal antibodies raised against algal centrin, a protein of algal striated flagellar roots, were used to characterize the occurrence and distribution of this protein in interphase and mitotic Chlamydomonas cells. Chlamydomonas centrin, as identified by Western immunoblot procedures, is a low molecular (20,000-Mr) acidic protein. Immunofluorescence and immunogold labeling demonstrates that centrin is a component of the distal fiber. In addition, centrin-based flagellar roots link the flagellar apparatus to the nucleus. Two major descending fibers extend from the basal bodies toward the nucleus; each descending fiber branches several times giving rise to 8-16 fimbria which surround and embrace the nucleus. Immunogold labeling indicates that these fimbria are juxtaposed to the outer nuclear envelope. Earlier studies have demonstrated that the centrin-based linkage between the flagellar apparatus and the nucleus is contractile, both in vitro and in living Chlamydomonas cells (Wright, R. L., J. Salisbury, and J. Jarvik. 1985. J. Cell Biol. 101:1903-1912; Salisbury, J. L., M. A. Sanders, and L. Harpst. 1987. J. Cell Biol. 105:1799-1805). Immunofluorescence studies show dramatic changes in distribution of the centrin-based system during mitosis that include a transient contraction at preprophase; division, separation, and re-extension during prophase; and a second transient contraction at the metaphase/anaphase boundary. These observations suggest a fundamental role for centrin in motile events during mitosis.

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.

A human cDNA expression library was screened using anti-centrin antibodies to obtain a cDNA clone encoding human centrin. The cDNA clone contains an open reading frame of 516 base pairs and predicts a product of 172 amino acids with a calculated molecular mass of 19,528 and a pI of 4.61. Sequence analysis demonstrates that human centrin and centrins from higher plants, protozoa, algae, Xenopus and the yeast CDC31 gene product are closely related members of a subfamily of the EF-hand superfamily of calcium-binding proteins. The human centrin sequence has four putative calcium-binding domains as defined by the EF-hand consensus. Immunoprecipitation and western blot studies from HeLa cells confirm that human centrin is a protein of approximately 20,000 M(r) as predicted from the cDNA clone. Indirect immunofluorescence analysis of HeLa cells demonstrates that centrin is localized at the centrosome of interphase cells and that it redistributes to the region of the spindle poles during mitosis. When taken together with earlier genetic studies, these results demonstrate that centrin is a ubiquitous component of centrosomes and mitotic spindle poles of diverse organisms and suggest that centrin plays a role in centrosome separation at the time of mitosis.

In this study, we demonstrate that manipulations of temperature and free calcium alter the morphology of the centrin-containing pericentriolar lattice of PtK2 cells. Immunofluorescence microscopy reveals that low-temperature incubation (4 degrees C) causes anti-centrin-labeled pericentrosomal spots to coalesce in the peripheral cytoplasm, and fuses small spots into larger spots near the cell center. At electron microscopic resolution, well-formed pericentriolar satellites appear around the centrioles in response to incubation at 4 degrees C. Elevated free calcium enhances these low-temperature-dependent effects. The data suggest that pericentrosomal spots correspond to one or more pericentriolar satellites, and that pericentriolar satellites and centrosomal matrix are interconvertable forms of the same material. Transient elevation of intracellular free calcium at 37 degrees C from a basal level of 3.7 x 10(-8) M to a peak level of 2.0 x 10(-7) M within 30 seconds with ionomycin results in a 35% increase in pericentrosomal spot number throughout the cytoplasm. The number of pericentrosomal spots is 50% larger 2 minutes after ionomycin addition; these spots are also nearer to the cell center as compared to 30 seconds after ionomycin addition. As intracellular free calcium returns to a basal level over 5 minutes, the number of spots and their cellular distribution resume a pretreatment value and pattern. We interpret these observations to indicate movement of pericentrosomal spots toward the cell center in response to the flux in intracellular free calcium. Alternatively, it is possible that no movement has occurred, but that the rise in free calcium has unmasked an epitope responsive to our anti-centrin antiserum. Regardless of the interpretation, we conclude that the pericentriolar lattice exhibits calcium-modulated behavior.

The intracellular localization of calcium during contraction of striated flagellar roots of the green alga Tetrasehnis subcordiformis was investigated using calcium pyroantimonate and calcium oxalate cytochemistry and energy-dispersive X-ray microanalysis. Vesicles (0·25-0·50 μm diameter) located in the anterior-most region of the cell are calcium-sequestering organelles. These vesicles exhibit a consistent and reproducible localization of electron-opaque pyroantimonate and oxalate salts of calcium. Striated contractile flagellar roots (CFR) show a periodic pattern of calcium antimonate (Ca-Sb) deposits following ‘calcium shock’ stimulation of contraction of this organelle. When CFRs are in a fully extended state they do not show Ca-Sb deposits. Deposits of Ca-Sb precipitate are present throughout the cytosol when CFRs are stimulated to contract. The implications of these findings with respect to the regulation of intracellular levels of ‘free ‘Ca2+ and to the mechanism of CFR contraction are discussed.

Antisera raised against centrin (Salisbury, J.L., A.T. Baron, B. Surek, and M. Melkonian. 1984. J. Cell Biol. 99:962-970) have been used, here, to identify a centrosome-associated protein with an Mr of 165,000. Immunocytochemistry indicates that this protein is a component of pericentriolar satellites, basal feet, and pericentriolar matrix of interphase cells. These components of pericentriolar material are, in part, composed of 3-8-nm-diam filaments, which interconnect to form a three-dimensional pericentriolar lattice. We conclude that the 165,000-Mr protein is immunologically related to centrin, and that it is a component of a novel centrosome-associated cytoskeletal filament system. Microtubule organizing centers such as the flagellar apparatus of algal cells, spindle pole body of yeast cells, and centrosome of mammalian cells are homologous structures essential for cytoplasmic organization and cellular proliferation. Molecular cloning studies have recently shown that the cell cycle gene product CDC31, required for spindle pole body duplication, shares 50% sequence homology with centrin (Huang, B., A. Mengersen, and V.D. Lee. 1988. J. Cell Biol. 107:133-140). The evolutionary conservation of centrin-related sequences and immunologic epitopes to microtubule organizing centers of divergent phylogeny suggests that a functional attribute(s) may have been conserved as well. Elucidation of a common thread between these related molecules may be fundamental to our understanding of cell structure and function.

A rapid, Ca2+-dependent change in the angle between basal bodies (up to 180 degrees) is associated with light-induced reversal of swimming direction (the "photophobic" response) in a number of flagellated green algae. In isolated, detergent-extracted, reactivated flagellar apparatus complexes of Spermatozopsis similis, axonemal beat form conversion to the symmetrical/undulating flagellar pattern and basal body reorientation (from the antiparallel to the parallel configuration) are simultaneously induced at greater than or equal to 10(-7) M Ca2+. Basal body reorientation, however, is independent of flagellar beating since it is induced at greater than or equal to 10(-7) M Ca2+ when flagellar beating is inhibited (i.e., in the presence of 1 microM orthovanadate in reactivation solutions; in the absence of ATP or dithiothreitol in isolation and reactivation solutions), or when axonemes are mechanically removed from flagellar apparatuses. Although frequent axonemal beat form reversals were induced by varying the Ca2+ concentration, antiparallel basal body configuration could not be restored in isolated flagellar apparatuses. Observations of the photophobic response in vivo indicate that even though the flagella resume the asymmetric, breaststroke beat form 1-2 s after photostimulation, antiparallel basal body configuration is not restored until a few minutes later. Using an antibody generated against the 20-kD Ca2+-modulated contractile protein of striated flagellar roots of Tetraselmis striata (Salisbury, J. L., A. Baron, B. Surek, and M. Melkonian, 1984, J. Cell Biol., 99:962-970), we have found the distal connecting fiber of Spermatozopsis similis to be immunoreactive by indirect immunofluorescence and immunogold electron microscopy. Electrophoretic and immunoblot analysis indicates that the antigen of S. similis flagellar apparatuses consists, like the Tetraselmis protein, of two acidic isoforms of 20 kD. We conclude that the distal basal body connecting fiber is a contractile organelle and reorients basal bodies during the photophobic response in certain flagellated green algae.