THE BASAL APPARATUS OF THE QUADRIFLAGELLATE SPERMATOZOPSIS EXSULTANS(CHLOROPHYCEAE):NUMBERING OF BASAL BODY TRIPLETS REVEALS TRIPLET INDIVIDUALITY AND DEVELOPMENTAL MODIFICATIONS1

Though there are no detectable structural differences between each of the axonemal doublets of Spermatozopsis exsultans Korshikov, basal body triplets do show structural peculiarities: one triplet consistently has an electron-dense patch appressed to its proximal part. This triplet is labeled No. 1, and all triplets are numbered in accordance to the numbering system used for other flagellate green algae. We present a detailed analysis of the basal apparatus (basal bodies and attached cytoskeletal elements) of S. exsultans and describe how basal apparatus elements are attached, very specifically, to particular basal body triplets. The analysis includes immunogold detection of centrin-containing structures and characterization of their sites of attachment to basal bodies. The sequence of basal body development in S. exsultans is deduced from what we know of other green algae. With this, we describe how the cytoskeletal structures associated with the separate basal bodies, particularly those attached to the right side of a basal body, undergo apparent morphological modifications from cell generation to generation. The data indicate that basal body triplets are truly different from one another and that this subsequent basal body asymmetry, combined with the developmental differences between basal bodies themselves, presumably accounts for the heterogeneity in the basal apparatus and any asymmetry in the cell as a whole.     

Localization of Tubulin and a Centrin-Homologue in Vegetative Cells and Developing Gametangia of Ectocarpus siliculosus (Dillw.) Lyngb. (Phaeophyceae,Ectocarpales)

The distribution of tubulin and centrin in vegetative cells and during gametogenesis of Ectocarpus siliculosus was studied by immunofluorescence. In interphase cells bundles of microtubules are focused on the centriolar region near the nuclear surface. Some of the bundles ensheath the nucleus while others traverse the cytoplasm in various directions, sometimes reaching the cell cortex. Evaluation of serial optical sections by confocal laser scanning microscopy (CLSM) revealed that the perinuclear and “cytoplasmic” microtubule bundles presumably constitute a single complex. In interphase cells centrin is localized as a single bright spot in the centriolar region. In dividing cells duplication and separation of the microtubular complex and the centrin spot takes place. In post-mitotic cells with two nuclei, the centrioles are located at opposite cell poles, short microtubule bundles emanate from them and partially encompass the nucleus. During gametogenesis a gradual transformation of the vegetative cytoskeleton to the gametic flagellar apparatus occurs.     

The respective contributions of the mother and daughter centrioles to centrosome activity and behavior in vertebrate cells

We have generated several stable cell lines expressing GFP-labeled centrin. This fusion protein becomes concentrated in the lumen of both centrioles, making them clearly visible in the living cell. Time-lapse fluorescence microscopy reveals that the centriole pair inherited after mitosis splits during or just after telophase. At this time the mother centriole remains near the cell center while the daughter migrates extensively throughout the cytoplasm. This differential behavior is not related to the presence of a nucleus because it is also observed in enucleated cells. The characteristic motions of the daughter centriole persist in the absence of microtubules (Mts). or actin, but are arrested when both Mts and actin filaments are disrupted. As the centrioles replicate at the G1/S transition the movements exhibited by the original daughter become progressively attenuated, and by the onset of mitosis its behavior is indistinguishable from that of the mother centriole. While both centrioles possess associated gamma-tubulin, and nucleate similar number of Mts in Mt repolymerization experiments. during G1 and S only the mother centriole is located at the focus of the Mt array. A model, based on differences in Mt anchoring and release by the mother and daughter centrioles, is proposed to explain these results.     

An EF-hands protein, centrin-1, is an EGTA-sensitive SUMO-interacting protein in mouse testis

A multifunctional calcium‐binding protein, centrin‐1, is specifically expressed in male germ cells, certain neurons and ciliated cells. We identified centrin‐1 as a protein interacting with SUMO‐2/3 using yeast two‐hybrid screening of a mouse testicular cDNA library. In bead halo assays, the interaction between centrin‐1 and SUMO‐2/3 was reduced in the presence of EGTA and facilitated by the addition of CaCl_2. immunostaining of seminiferous tubules in 35‐day‐old mouse testes revealed that cells in the layer containing spermatogonia showed colocalization of SUMO‐2/3 with centrin‐1 in cytoplasmic spots. Identification of centrin‐1 as the EGTA‐sensitive SUMO‐2/3‐interacting protein indicates the possible role of calcium in modulating the centrin‐1–SUMO‐2/3 interaction and suggests the importance of this interaction in mouse testis. Copyright © 2010 John Wiley & Sons, Ltd.     

Nucleus Basal Body Connector of Dunaliella: Threshold Concentration of Calcium Necessary for in vitro Contraction*

Detergent-isolated flagellar apparatuses of the flagellate green alga Dunaliella bioculata retain remnants of the nucleus (the karyoskeleton) which are linked to the basal bodies by the centrin-containing nucleus basal body connectors (NBBC). Such complexes were subjected to different calcium concentrations (1 × 10^?9 M ? 5 × 10^?4 M Ca²⁺) and the distance between the basal bodies and the karyoskeleton was measured by light microscopy. The threshold concentration of Ca²⁺ for NBBC contraction was determined to be around 5 × 10^?8 M Ca²⁺. At > 10^?7 M Ca²⁺ NBBC were maximally contracted and the distance between the basal bodies and the karyoskeleton was only about 50% of the initial distance. Using a polyclonal antibody generated against centrin (Salisbury et al., 1984), the NBBC were visualized by indirect immunofluorescence in both the extended and contracted state. Our results demonstrate that in vitro contraction of centrin-containing filaments in green algae is initiated at about the same free Ca²⁺ concentration in three different centrin-containing basal apparatus components (i.e. the distal connecting fibre, the flagellar transitional region and the NBBC).     

IMMUNOLOCALIZATION OF CENTER IN THE FLAGELLAR APPARATUS OF MALE GAMETES OF ECTOCARPUS SILICULOSUS (PHAEOPHYCEAE) AND OTHER BROWN ALGAL MOTILE CELLS1

Centrin or a centrin homologue was localized using immunofluorescence in the flagellar basal body region in zoids of five brown algal species: Ectocarpus siliculosus (Dillw.) Lyngb., Scytosiphon lomentaria (Lyngb.) Link, Laminaria digitata (Huds.) Lamour., Sphacelaria rigidula (Kütz.) Prud'homme van Reine, and Fucus serratus L. The antigen is restricted to short rods extending along the basal body(ies) and towards the nucleus, which always remains firmly linked to the flagellar apparatus in isolated cytoskeletons. To identify these antigenic sites, pre- and postembedding immunogold electron microscopy was applied to male gametes of E. siliculosus. At least three different structures associated with the basal bodies were antigenic: a fibrous structure connecting the proximal end of the posterior basal body to the nucleus (nucleus-basal body connector), a striated band that links the two basal bodies to each other and is located in the angel formed by them, and amorphous material interconnecting the basal bodies in their most proximal regions. In addiction, specific labeling occurs along the external surface and within the lumen of both basal bodies and in the flagellar transitional region. The possible function of these centrin-containing structures is discussed.     

Insights into the Morphology of Haplozoan Parasites (Dinoflagellata) using Confocal Laser Scanning Microscopy

We describe new insights into the morphology and life history of the bizarre parasite Haplozoon axiothellae (Dinoflagellata) using light microscopy (LM), scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Trophonts were isolated from the intestines of host maldanid polychaetes, Axiothella rubrocincta, collected from San Juan Island, Washington, USA. LM and SEM confirmed features previously observed, such as amphiesmal projections, mature and immature junctions between the nucleated compartments of the vermiform syncytium and visible polygonal alveoli. CLSM of adult trophonts fluorescently stained for DNA, tubulin, centrin, and plasma membrane demonstrated several new ultrastructural traits: (1) an extensive basket of parallel microtubules within the trophomere used for host attachment, (2) two physically separated MTOCs (i.e. putative pairs of basal bodies) beneath pores on the ventral side of each compartment, (3) robust mitotic and/or meiotic spindles associated with one to four nuclei in each compartment, (4) spindles with polar bodies that are disconnected from the MTOCs, (5) a centrin-stained fibril within the trophomeres that potentially functions to retract the motile stylet, and (6) cytokinesis in the posterior-most compartments. This study renames haplozoan compartments using the suffix “-mere” rather than “-cyte” (i.e. trophomere, gonomere, sporomere) to reflect their status within a single syncytium.     

Roots1

ABSTRACT Many unicellular eukaryotic organisms possess complex fiber systems that organize and anchor the flagellar basal apparatus in the cell [20, 24]. In 1978 we first published the observation that one of these fiber systems, the striated flagellar root of the quadriflagellate green alga Tetraselmis subcordiformis (=Platymonas subcordiformis), is a contractile organelle [31]. We subsequently found that striated flagellar roots are composed, in part, of the Ca²⁺-binding protein centrin [30]. Since that time, centrin has been found to be a ubiquitous component of the flagellar basal apparatus, basal bodies and centrioles, and centrosomes and mitotic spindle poles of eukaryotic cells (for general reviews see [28, 34]). While we have learned a great deal about centrin from other organisms, our earliest success in understanding the biology of centrin was in large part due to the extraordinary extent to which Tetraselmis cells have elaborated their centrin-based organelles. In this paper, I will return attention to several unanswered questions concerning Tetraselmis striated flagellar root behavior and I will suggest several new directions that students may wish to pursue in order to tease fresh insights from this fascinating organism.