QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 1 August 2006
doi: 10.1242/jcs.03088

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Eisman, R. C. Articles by Kaufman, T. C. Search for Related Content PubMed PubMed Citation Articles by Eisman, R. C. Articles by Kaufman, T. C.

centrosomin's beautiful sister (cbs) encodes a GRIP-domain protein that marks Golgi inheritance and functions in the centrosome cycle inDrosophila

Department of Biology, Indiana University, Bloomington, IN 47405, USA

View larger version (37K): [in a new window]   Fig. 1. Structure of the cbs gene and its protein product. The cbs gene is located in the 50A region on the right arm of the second chromosome in Drosophila. (A) Transcript map of cbs, showing the location of cnn (green), the two promoters (P1 and P2) and the exon structure (red) of five transcripts that potentially produce three different protein isoforms. UTRs are in pink, except the unique UTR in the short transcript, which is in yellow. Both promoters produce transcripts that contain a GRIP domain spanning exons 5 and 6. (B) Alignment of the GRIP domains from Cbs and Golgin-97 showing identical residues in dark grey and similar residues in boxes. The ten amino acids that are eliminated from the non-GRIP, long isoform produced by two transcripts are underlined. (C) The predicted secondary structure of Cbs is 60% coiled-coil (red), with short disordered regions (blue) interrupting the coils. No coiled-coil predictions were provided for the -helical parts at either end (black). The GRIP domain (yellow) is near the C-terminus of Cbs, followed by 18 amino acids. Putative post-translational modifications of Cbs include nine threonine phosphorylation sites (P) and three myristylation sites (purple line). (D) Western blot showing the bands identified by guinea pig anti-Cbs whole serum and rabbit anti--tubulin 84B as a control. The upper band in the Cbs lane probably represents both of the larger isoforms, which would be expected to migrate together. The lower, fainter band runs in a position predicted for the truncated shorter isoform.

View larger version (111K): [in a new window]   Fig. 2. Cbs cycles from the cytoplasm to DNA during mitosis. Mitotic series (PDI::GFP stock) immunostained for GFP to mark the ER lumen (green), Cbs (red), and stained for either Cnn, microtubles or DNA TOTO-3 (blue) during mitosis in syncytial embryos. From early prophase (A-D) to late prophase (E-H), Cbs rapidly changes from a diffuse cytoplasmic haze, to small particles that accumulate near centrosomes and begin to migrate into the nucleus. During prometaphase Cbs is present in elongating spindles and at chromatin (I-L), but is present only at chromatin by late metaphase (M-P). The ER forms an envelope around the spindle during these stages, but is absent from the spindle proper. Throughout anaphase (Q-T) Cbs remains associated with DNA, although, as the ER begins to disperse, small cytoplasmic Cbs particles are present. At late telophase (U-X) Cbs moves towards centrosomes and astral microtubules, and rapidly fragments into small particles that disperse into the cytoplasm. The ER is present throughout the cytoplasm and at the spindle mid-bodies during telophase, but does not colocalize with Cbs. Bar, 20 µM.

View larger version (35K): [in a new window]   Fig. 3. The fluorescence intensity of Cbs cycles during syncytial mitotic cycles. In wild-type embryos (green bars) Cbs FI is the highest during prophase and drops by approximately 50% during metaphase and early anaphase, before increasing during late anaphase and telophase. In Cbs RNAi depletion experiments (red bars) embryos with severe defects all have FI levels that are significantly lower at all stages of mitosis. The variation among nuclei in both wild-type and Cbs RNAi embryos fluctuates in a similar pattern. In Arf72A1-mutant embryos (blue bars) FI values are relatively constant throughout mitosis, as is the variation among nuclei.

View larger version (125K): [in a new window]   Fig. 4. Cbs concentrates in the pericentriolar region between centrosomes at cellularization. (A-D) At the start of cellularization immediately after centrosomes have replicated, Cbs concentrates on the apical surface of nuclei, filling the region between separated centrosomes and persists throughout cellularization. We detect low levels of colocalization between Cbs and Cnn (arrowheads B-D) and between Cbs and the ER at this stage. (E-H) The concentration of Cbs at the pericentriolar region persists in most cells during early gastrulation, lasting the longest in cells along segmental boundaries and embryonic furrows. There is weak colocalization between Cbs and the ER near nuclei (white in merged image). (I-L) By late gastrulation the levels of Cbs are reduced to multiple foci, randomly distributed throughout cells in close association with the most compact microtubule staining. There is no detectable colocalization between Cbs and the ER in these embryos. Embryos are immunostained as in Fig. 2. Bar, 10 µM.

View larger version (83K): [in a new window]   Fig. 5. Cbs colocalizes with Lva during chromosome congression and cellularization. The localization of Cbs and Lva in wild-type (OreR) syncytial embryos was determined with antibodies directed against Lva (green), Cbs (red) and DNA was stained with TOTO-3. (A-D) Lva colocalizes with a subset of Cbs (yellow in B,C) on DNA during chromosome congression and is sensitive to microtubule stability. Both, our initial fixation method (A,B) and the formaldehyde preparations that are pretreated with taxol (C,D) preserve this colocalization, although the FI is strongest with our initial method. (E,F) In a top view of an embryo during early cellularization Cbs becomes concentrated in the pericentriolar region (E) where Cbs colocalizes with Lva (yellow in F). (G,H) In a lateral view of the slow phase of cellularization, nuclei become columnar in shape and Cbs extends down over the lateral surface of nuclei (H), as Lva moves in a basal to apical direction (Sisson et al., 2000). Cbs and Lva colocalize weakly along the lateral sides of nuclei, and strongly at the apical surface of nuclei (yellow in G). We do not find large Lva vesicles in the lateral domain of Cbs, and Cbs is not present above nuclei at the region where Lva is incorporated into membranes of growing furrows. Bar, 20 µM.

View larger version (97K): [in a new window]   Fig. 6. Loss of Cbs causes centrosome replication and maturation defects. To determine the mutant phenotype the Gal4/UAS-inducible expression system was used to trigger RNAi during early embryogenesis. Embryos were immunostained for Cnn (green, A-D) or microtubles (green, E,H,I,K,L), Cbs (red, A-L) and Cnn (blue, E-J), or DNA staining with TOTO-3 (blue A,D,L). During prophase a moderate decrease in the levels of Cbs (A,B) leads to minor centrosome defects and occasional acentrosomal nuclei, whereas the loss of Cbs (C,D) results in small centrosomes, an increased number of acentrosomal nuclei and aberrant nuclear morphology. During metaphase, a moderate decrease in the levels of Cbs (E,F) causes aberrant spindle formation and a reduction in centrosome size (arrowheads, left), and collapsed spindles (arrowheads, lower right). When Cbs is significantly depleted during metaphase (G,H) centrosome replication is blocked, resulting in monopolar spindles, although in some examples (arrows) the loss of centrosomes may be due to poor attachment to spindles. At the start of cellularization (I,J), centrosome maturation defects become more prevalent (arrowheads), and Cbs fails to form a dense pericentriolar structure, remaining as an amorphous haze above nuclei. As cellularization continues (K,L) Cbs fails to move down over nuclei and many nuclei fall into the syncytium, creating large gaps in the embryonic cortex. Bars, 20 µM (A-D,I,J); 40 µM (E-H,K,L).

View larger version (121K): [in a new window]   Fig. 7. Cbs fails to localize to DNA during mitosis in Arf72A mutants. The mutant allele Arf72A1, an embryonic recessive lethal mutation, was used to investigate the loss of GRIP function during embryogenesis. Embryos that were immunostained with antibodies against microtubules (green), Cbs (red), and Cnn (blue) show that during prophase (A), metaphase (B), and telophase (C), Cbs forms cytoplasmic particles, but never associate with DNA during mitosis. Centrosome replication and maturation is usually normal in early embryos, although some defects (arrowheads in B) are present. Most Arf72A1 mutants fail during cycle 13 and 14, as shown in embryos stained for Cnn (D,F, green) or Lva (E, green), Cbs (A-F, red), and DNA (A-F, blue). (D) In embryos with low levels of Cbs, adjacent centrosomes fuse during anaphase (arrowheads), and the start of telophase is delayed. (E) At cellularization Cbs is present as a weak haze above nuclei and Lva is undetectable. In addition, nuclear fallout is extremely high, although it is not as severe as the RNAi phenotype. (F) Cellular embryos have a significant reduction in cell number, posses many aneuploid nuclei and mitotic domains have many monopolar spindles (arrowheads). Arf72A1-mutant embryos all fail prior to the completion of cuticle secretion. Bars, 40 µM (A-C,E); 20 µM (D,F).

View larger version (128K): [in a new window]   Fig. 8. Ectopic expression of the GRIP domain causes centrosome hypertrophy. To investigate the function of the Cbs GRIP domain we expressed a GFP::GRIP fusion protein during embryogenesis under control of nanos::Gal4 and immunostained embryos for Cnn (green, A,E,I) or microtubules (green, M), GFP (red, B,F,J,N), and Cbs (blue, O) or DNA stained with TOTO-3 (blue, C,G,K). (A-D) During early prophase the GRIP domain initially localizes to one centrosome, resulting in an asymmetric accumulation of Cnn at replicated centrosomes (arrowheads). A cytoplasmic pool of the GRIP domain is also present around nuclei during prophase. (E-H) When the GRIP domain remains concentrated at centrosomes during prophase, individual centrosomes (arrowhead) and pairs of centrosomes (arrow), undergo additional rounds of replication and frequently become dissociated from nuclei. (I-P) During metaphase the GRIP domain accumulates as cytoplasmic particles and localizes strongly at centrosomes and the microtubule spindle, but does not cause further accumulation of Cnn at centrosomes or centrosome hypertrophy. Low levels of the GRIP domain are present at DNA (J,N), but the transport efficiency and DNA-binding affinity of endogenous Cbs appears higher (O). However, the GRIP domain does cause excessive accumulation of cytoplasmic Cbs particles that are not seen in wild-type embryos during mitosis. Bars, 20 µM (A-D, I-P); 5 µM (E-H).