Elaine Y. Lai

Centrin is a conserved protein that forms diverse associations with centrioles and MTOCs in Naegleria and other organisms

Centrin, a approximately or equal to 20 kDa calcium-binding protein also known as caltractin, is a component of centrosome-associated algal flagellar roots capable of calcium-mediated contraction, and is also found in the centrosomes of vertebrate cells. Our analysis of a centrin gene from a protist, the amoeboflagellate Naegleria gruberi, reveals conserved features that distinguish centrins from calmodulin. Antibodies to bacterially expressed Naegleria centrin, which also recognize yeast Cdc31p, were employed to localize centrin immunoreactivity in selected organisms possessing specialized microtubule-organizing centers (MTOCs) or accessory structures. There is a striking morphological diversity of such structures. In the simplest associations, as found in Naegleria flagellates and vertebrates tracheal epithelium, centrin is intimately associated with the cylinder of the basal bodies. In cells with unfocused mitotic spindles, Naegleria amoebae and onion root tips, no localization of centrin was detected. In Dictyostelium discoideum and Saccharomyces cerevisiae, which lack centrioles, centrin immunoreactivity was observed as punctate cytoplasmic bodies but not associated with spindle pole MTOCs. In Paramecium multimicronucleatum, centrin immunoreactivity is localized to the infraciliary lattice, previously shown to exhibit calcium-mediated contraction. In Vorticella microstoma, known for the calcium-induced rapid contraction of its stalk, centrin immunoreactivity is localized to the contractile spasmoneme and myonemes. Similar antigens from Paramecium and Vorticella are detected by anti-centrin and anti-spasmin. The pattern of localization of centrin immunoreactivity supports the conjecture that a contractile system involving centrin, initially associated with centriolar structures, was recruited during evolution to build specialized organelles in different organisms and cell types.

Programmed appearance of translatable flagellar tubulin mRNA during cell differentiation in Naegleria.

The programmed de novo synthesis of flagellar tubulin during the hour-long differentiation of Naegleria gruberi from amoebae to flagellates is our paradigm for the study of gene expression during cell differentiation. This paper reports the efficient translation of flagellar tubulin mRNA in the wheat germ cell-free system directed by total or polyadenylated RNA extracted from differentiating cells. The tubulin in the in vitro product has a subunit molecular weight of 55,000, separates into alpha and beta subunits under suitable conditions of polyacrylamide gel electrophoreis and co-polymerizes with calf brain tubulin. At least half of the tubulin synthesized in vitro is precipitated by antibodies specific to flagellar tubulin, and the immunoprecipitated tubulin subunits yield peptide maps similar to those of outer doublet tublin. Flagellar tubulin is the predominant protein synthesized in the cell-free system, and amounts to about 5% of the polypeptides whose synthesis is directed by total RNA from differentiating cells. In contrast, little or no flagellar tubulin is synthesized when the cell-free system is directed by RNA extracted from amoebae prior to differentiation. Translation assays show that at least 92% of the flagellar tubulin mRNA appears during differentiation. The time course of appearance of this mRNA was measured by quantitative immunoprecipitation of the cell-free products. Under conditions where cells from flagella 60 min after initiation of differentiation, translatable flagellar tubulin mRNA was first detected at 20 min, reached a maximum at about 60 min and then declined. An excellent correlation was observed between the amount of translatable flagellar tubulin mRNA and the previously measured rates of flagellar tubulin synthesis in vivo. These results indicate that synthesis of flagellar tubulin is a direct reflection of the abundance of its mRNA, and provide the molecular techniques for dissection of the factors that regulate the rapid appearance of this structural protein during differentiation.

CENTRIN IS SYNTHESIZED AND ASSEMBLED INTO BASAL BODIES DURING NAEGLERIA DIFFERENTIATION

During differentiation of Naegleria from vegetative amoebae to temporary flagellates, the microtubular cytoskeleton, including two basal bodies and flagella, is assembled de novo. Centrin is an integral component of these basal bodies [Levy et al., 1996, Cell Motil. Cytoskeleton 33: 298-323]. In many organisms, centrin appears to be a constitutive protein, but in Naegleria centrin gene expression occurs only during differentiation. Centrin mRNA, which has not been detected in amoebae, appears and disappears earlier in differentiation than a coordinately regulated set of differentiation-specific mRNAs encoding flagellar tubulin and calmodulin. Centrin antigen accumulates during differentiation, and then decreases in abundance as the flagellates mature and revert to amoebae. No localization of centrin has been detected in amoebae. During differentiation, centrin becomes localized to the basal bodies as soon as these structures are detected with anti-tubulin antibodies, first as a single dot and finally as two basal bodies. During reversion of flagellates to amoebae, centrin remains localized to the basal bodies for as long as they are present. When assembly of tubulin-containing structures during differentiation is prevented using oryzalin, centrin localization is prevented as well, yet inhibition of assembly does not affect accumulation of centrin antigen. Apparently in Naegleria, the role of centrin is primarily for a differentiation- or flagellate-specific function. The temporary presence of centrin is concurrent with the presence of centriolar basal bodies, which supports the conjecture that in Naegleria centrin may be needed only when these organelles are present.

A beta-tubulin gene of Naegleria encodes a carboxy-terminal tyrosine: aromatic amino acids are conserved at carboxy termini

A gene that directs the programmed synthesis of flagellar beta-tubulin during the rapid differentiation of Naegleria gruberi from amoebae to flagellates has been cloned and sequenced. The intronless gene is one of 8 to 10 similar but non-identical genes that are dispersed in the genome. beta-Tubulin mRNA homologous to this gene family is expressed transiently during differentiation, and has not been detected in amoebae. The encoded beta-tubulin is strongly conserved, with features that closely resemble the beta-tubulins of diverse organisms, especially organisms that, like Naegleria, use tubulin to assemble flagellar axonemes. In most sequenced alpha-tubulins, the encoded carboxy-terminal amino acid is tyrosine, which undergoes post-translational removal and readdition, conserved processes of unknown function. In N. gruberi, unusually, the terminus of alpha-tubulin is encoded as glutamine while that of beta-tubulin is tyrosine. The presence of these divergent termini on subunits of a conserved tubulin provoked us to re-examine aromatic amino acids at the termini of alpha- and beta-tubulins. Although evolution has tinkered extensively with the carboxy-terminal domains of tubulin subunits, we find an unexpected conservation. In every organism or cell type for which both tubulin subunits have been sequenced, except the ciliate Stylonychia lemnae, at least one tubulin subunit of some or all tubulin heterodimers terminates in an aromatic amino acid, either tyrosine or phenylalanine. This remarkable conservation of carboxy-terminal aromatic amino acids suggests that these residues serve some crucial function.

A calcineurin-B-encoding gene expressed during differentiation of the amoeboflagellate Naegleria gruberi contains two introns

One of two similar genes in the unicellular eukaryote Naegleria gruberi is shown to encode calcineurin B (CnB), the regulatory subunit of calcium-calmodulin-regulated protein phosphatase 2B. Over a span of 156 amino acids, excluding divergent N-termini, the encoded sequence shows 62% identity with vertebrate CnB, and also shows sequence elements specific, among calcium-binding proteins, to CnB. In contrast, the sequence shows only 23% identity with N. gruberi flagellar calmodulin. CNB mRNA is readily detected in amoebae; its abundance increases fourfold during differentiation to flagellates, reaches a peak at 50-70 min, when flagella are forming, and then declines. A genomic clone matches an expressed cDNA, except that it is interrupted by two phase I introns. The position of one intron, which separates the divergent N-terminal domain from the four calcium-binding domains (EF hands), is shared with a yeast CNB gene; the other is located in the central helix between the two pairs of calcium-binding loops; features that support an ancient origin. These introns, the first found in protein-coding genes of Naegleria, are flanked by characteristic splice junction sequences. N. gruberi CnB also shares similarities with recoverins. The finding in a protist of a CNB gene that contains two introns separating functional domains, shares similarities to recoverins and shows increased expression during differentiation is provocative. If the phylogeny of major groups derived from ribosomal RNA is accepted, Naegleria is among the earliest branching eukaryotes known to contain canonical pre-mRNA introns.

Rapid centriole assembly in Naegleria reveals conserved roles for both de novo and mentored assembly

Centrioles are eukaryotic organelles whose number and position are critical for cilia formation and mitosis. Many cell types assemble new centrioles next to existing ones (“templated” or mentored assembly). Under certain conditions, centrioles also form without pre‐existing centrioles (de novo). The synchronous differentiation of Naegleria amoebae to flagellates represents a unique opportunity to study centriole assembly, as nearly 100% of the population transitions from having no centrioles to having two within minutes. Here, we find that Naegleria forms its first centriole de novo, immediately followed by mentored assembly of the second. We also find both de novo and mentored assembly distributed among all major eukaryote lineages. We therefore propose that both modes are ancestral and have been conserved because they serve complementary roles, with de novo assembly as the default when no pre‐existing centriole is available, and mentored assembly allowing precise regulation of number, timing, and location of centriole assembly.

Structure of a eukaryotic thiaminase I.

Significance Thiaminases, enzymes that cleave vitamin B1 into its pyrimidine and thiazole ring moieties, are sporadically distributed among prokaryotes and eukaryotes. Thiaminase I enzymes accomplish this reaction through substitution of the thiazole ring with a nitrogenous base or sulfhydryl compound. A thiaminase I of the single-celled amoeboflagellate Naegleria gruberi is the first eukaryotic thiaminase I to have been examined structurally. The crystal structures in both apo form and bound to 3-deazathiamin, a noncleavable thiamin analog and inhibitor of the enzyme, define the mode of thiamin binding to this class of thiaminases and indicate the residues important for catalysis. Comparison with thiaminase II argues for convergent evolution between these two enzymes. Thiaminases, enzymes that cleave vitamin B1, are sporadically distributed among prokaryotes and eukaryotes. Thiaminase I enzymes catalyze the elimination of the thiazole ring moiety from thiamin through substitution of the methylene group with a nitrogenous base or sulfhydryl compound. In eukaryotic organisms, these enzymes are reported to have much higher molecular weights than their bacterial counterparts. A thiaminase I of the single-celled amoeboflagellate Naegleria gruberi is the only eukaryotic thiaminase I to have been cloned, sequenced, and expressed. Here, we present the crystal structure of N. gruberi thiaminase I to a resolution of 2.8 Å, solved by isomorphous replacement and pseudo–two-wavelength multiwavelength anomalous diffraction and refined to an R factor of 0.231 (Rfree, 0.265). This structure was used to solve the structure of the enzyme in complex with 3-deazathiamin, a noncleavable thiamin analog and enzyme inhibitor (2.7 Å; R, 0.233; Rfree, 0.267). These structures define the mode of thiamin binding to this class of thiaminases and indicate the involvement of Asp272 as the catalytic base. This enzyme is able to use thiamin as a substrate and is active with amines such as aniline and veratrylamine as well as sulfhydryl compounds such as l-cysteine and β-mercaptoethanol as cosubstrates. Despite significant differences in polypeptide sequence and length, we have shown that the N. gruberi thiaminase I is homologous in structure and activity to a previously characterized bacterial thiaminase I.