Chlamydomonas reinhardtii

Spermatozopsis similis

Centrin-GFP in Chlamydomonas reinhardtii

The basal body of algae is the microtubule organizing center (MTOC) and is the equivalent to the centrosome in mammalian cells.  Over the last 30 years a wealth of data has been accumulated on the structural features of the basal apparatus (basal body and the attached fibers).  But it is until recently that the identification of the molecular components from this organelle has been approached.  Different approaches exist to identify and isolate molecules from this organelle.  Using a biochemical approach one of its components  was identified and characterized from the green alga Tetraselmis striata.  This protein, named centrin, is a highly conserved 20 kDa acidic phosphoprotein consisting of two isoforms of differing isoelectric points (4.9 and 4.8).  Densitometric scans of electropherograms indicated that their stoichiometry varied between 2:1and 1:1.  This variability seemed to be related to the state of contraction of the striated flagellar roots at the time of lysis.  Phosphate labelling suggested that phosphorylation converts one form of centrin to the other.  Both exhibited a calcium-dependent  reduction in relative electrophoretic mobility in two-dimensional gels (Salisbury et al., 1984).  At the cDNA level centrin was first cloned from Chlamydomonas and named caltractin (Huang et al., 1988).  Caltractin was immunogenetically related to the original T. striata centrin but its PI was experimentally determined to be 5.3.  It also showed the altered electrophoretic mobility in the presence of calcium (Weber et al., 1994).  Caltractin as well as some other centrins (Wiech et al., 1996) exhibits a calcium-dependent binding to the hydrophobic ligand phenyl-Sepharose suggesting that centrins, like calmodulins, expose a hydrophobic surface in their more compact calcium-bound form.  Recombinant caltractin has a distinct UV spectrum which is dominated by  9 phenylalanine residues.  This makes the ratio of 260:280 a sensitive indicator of caltractin purity.   It was found to be pH stable in the range of 6.0-8.0 and heat stable.  Based on gel filtration caltractin has an apparent molecular weight estimated at 30 kDa.  A similar property is shown by calmodulin.  This suggests that caltractin is likely to be an elongated molecule with a domain conformation similar to calmodulin.   H-NMR spectrum of centrins shows distinct changes upon calcium addition, indicative of structural or dynamic changes.  Centrins, but not calmodulin, bind with high affinity to a small peptide corresponding to the Cdc31p-binding site of Kar1p (Spang et al., 1995).  No Cdc31p-Kar1p peptide complex was observed when Cdc31p was in the calcium-free state, suggesting that binding of the Kar1p peptide is calcium-dependent (Wiech et al., 1996).  Binding of the peptide caused a decrease in the apparent molecular mass of human centrins 1 and 2 but a slight increase was observed for Cdc31p and Scherffelia. dubia centrin.  Centrin tends to form multimers at high protein concentrations whereas low calcium and peptide binding seem to reduce the formation of these multimeric structures.  Sedimentation studies showed that some centrins can form filaments but not all.  Sequence analysis of centrin family members has revealed that centrin is a closely related subfamily of the superfamily of calcium-binding proteins defined by the EF-hand motif (Lee and Huang, 1993; Errabolu et al., 1994).  The EF-hand is the motif responsible for the binding of calcium to these proteins.  Recombinant centrin from C. reinhardtii  contains two high affinity (Kd = 1.2 x 10-6 M) and two lower affinity (Kd = 1.4 x 10-4 M) sites (Weber et al., 1994).  For Chlamydomonas centrin the two likely EF-hands to bind calcium are the first and the second (Lee and Huang, 1993).  Whereas the two most likely EF-hands to bind calcium in CDC31p are the first and fourth EF-hand (Lee and Huang, 1993).

 Centrins are found in MTOC’s from phylogenetically different organisms (Levy, 1996).  Two of the organisms in which centrin function has been studied are the yeast Saccharomyces cerevisiae (Geier et al., 1996; Baum et al., 1991) and the biflagellate green alga Chlamydomonas reinhardtii  (McFadden et al., 1987; Sanders and Salisbury, 1994; Taillon et al., 1992; Wright et al., 1983).  In S. cerevisiae, centrosomal functions are provided by the spindle pole body (SPB) which is embedded in the nuclear envelope.  Three plaques can be discriminated by electron microscopy (Byers, 1981).  The outer and inner plaque organize the cytoplasmic and nuclear microtubules, while the central plaque anchors the SPB to the nuclear membrane.  An additional substructure of the SPB, the half bridge, appears along the cytoplasmic margin of the nuclear envelope.  The centrin homologue in yeast was named Cdc31p and was immunolocalized to the half-bridge (Spang et al.,  1993).  In S. cerevisiae, temperature-sensitive mutants for CDC31 are defective in the first step of duplication (Rose and Fink, 1987).  Overexpression or expression of dominant CDC31 suppressor mutations rescue KAR1 cell cycle arrest (Weber et al., 1994).  In both cases spindle pole body duplication and function is normal.  Analysis of suppressors of the effect of Kar1p mutations revealed that the carboxyl-terminal region of Cdc31p plays a critical role.  Immunofluorescence showed that in Kar1p mutants Cdc31p fails to localize to the SPB (Geier et al., 1996).  This result suggests that Cdc31p plays an important role in the cell-cycle-duplication and accumulate at the restrictive temperature with a single enlarged SPB without a satellite (Byers, 1981).

The centrosome of animal cells differs from that of yeast in that it contains centrioles.  Centrin has been immunolocalized to the centrosome or to the centrioles depending on the antibody used (Lee and Huang, 1993; Errabolu et al., 1994; and Middendorp et al., 1997).  Although centrin plays an important function in C. reinhardtii  and S. cerevisiae, its function in mammalian cells is unknown.

 In C. reinhardtii, each flagellum is organized by a basal body.  The stellate fiber serves as a transition region between the basal body and the flagellum.  A distal striated fiber interconnects each basal body and two other connectors link the basal bodies to the nucleus.  Analysis of C. reinhardtii centrin mutants (vfl-2 which is a substitution of the glutamic acid at position 101 to alanine) revealed a variable flagellar number, abnormal positioning of basal bodies and no calcium-induced excision of flagella (Wright et al., 1983; Taillon et al., 1992; Jarvik and Suhan, 1991).  In this mutant, centrin was absent from the centrin-containing structures.  A number of revertants have been isolated and studied.  These revertants show changes at the central helix and suggest that aa 101 is critical for centrin function.  It was shown that even though the flagellar number phenotype was suppressed some defects on the distal striated fibers and transition region could be detected.  On the other handthe NBBC are functional and the basal apparatus has been restored suggesting that the NBBC is necessary for proper basal body number.   Flagellar excision occurs when calcium in the medium is increased, and also when there is a pH shock.  When calcium is elevated, the stellate fibers contract and subject the axonemal doublets to mechanical shear forces and torsional loads, which cause the local severing of MTs just distal to the flagellar transition zone.  It was shown (Salisbury, 1987) that the effect of Ca2+ on centrin contraction requires ATP.  It was suggested that the high energy of the phosphate bond is preserved for later use by protein phosphorylation and that centrin could be the target for the phosphorylation (Martindale, 1990).  On the other hand recent data on the severing activity of katanin, which is a member of the AAA family of ATPases (ATPases Associated with various cellular Activities), during deflagellation showed that there is a significant overlap of katanin and centrin both localizing to the distal and proximal end of the transition zone and in association with the distal straiated fibers connecting the basal bodies (Lohret, 1999).  Taken all together these data suggest that centrin may be important for accurate basal body number, localization, and organization and in flagellar excision.

We are interested in isolating Centrin Binding Proteins (CBP’s).  We have performed centrin overlay assays on highly purified basal apparatuses to identify putative CBP’s.  These assays showed a reproducible pattern of bands.  Using the same technique on a cDNA library of Spermatozopsis similis we have isolated and characterized several putative CBP’s.  Currently we are developing antibodies to further characterize these proteins.

On a second approach we are using molecular genetics to characterize these proteins at the molecular level.  With the recent development of a GFP that is expressed in Chlamydomonas, fusions between wt and mutant centrin or CBP’s with GFP will allow us to localize these proteins and to study their function(s) (Ruiz Binder et al. 2002).
 

References:

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Huang, B., Mergensen, A., and Lee, V. D. (1988). Molecular cloning of cDNA for caltractin, a basal body-associated Ca2+-binding protein:  Homology in its protein sequence with calmodulin and the yeast CDC31p gene product. J. Cell Biol. 107, 133-140.

Weber, C., Lee, V., Chazin, W. J., and Huang, B. (1994). High level expression in E. coli  and characterization of the EF-hand Ca2+-binding protein caltractin. J. Biol. Chem. 269, 15729-15802.

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Spang, A., Courtney, I., Fackler, U., Matzner, M., and Schiebel, E. (1993). The calcium-binding protein cell division cycle 31 of Saccharomyces cervisiae is a component of the half bridge of the spindle pole body. J. Cell Biol. 123, 405-416.

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Middendorp, S., Paoletti, A., Schiebel, E., and Bornens, M. (1997). Identification of a new mammalian centrin gene, more closely related to Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA  94, 9141-9146.

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Lohret, T. A., Zhao, L., and Quarmby, M.  (1999).  Cloning of Chlamydomonas p60 Katanin and Localization to the site of Outer doublet Severing During Deflagellation.  Cell Motil. Cytoskeleton 43, 221-2331.

Ruiz-Binder, N.E., Geimer, S., and Melkonian, M. (2002): In vivo localization of centrin in the green alga Chlamydomonas reinharttii. Cell Motil. Cytoskeleton (in press).