Search

Summary Centrosomes are the main microtubule (MT)-organizing centers in animal cells, but they also influence the actin/myosin cytoskeleton [1]. The Drosophila CP190 protein is nuclear in interphase, interacts with centrosomes during mitosis, and binds to MTs directly in vitro [2, 3]. CP190 has an essential function in the nucleus as a chromatin insulator [4], but centrosomes and MTs appear unperturbed in Cp190 mutants [4, 5]. Thus, the centrosomal function of CP190, if any, is unclear. Here, we examine the function of CP190 in Cp190 mutant germline clone embryos. Mitosis is not perturbed in these embryos, but they fail in axial expansion, an actin/myosin-dependent process that distributes the nuclei along the anterior-to-posterior axis of the embryo. Myosin organization is disrupted in these embryos, but actin appears unaffected. Moreover, a constitutively activated form of the myosin regulatory light chain can rescue the axial expansion defect in mutant embryos, suggesting that CP190 acts upstream of myosin activation. A CP190 mutant that cannot bind to MTs or centrosomes can rescue the lethality associated with Cp190 mutations, presumably because it retains its nuclear functions, but it cannot rescue the defects in myosin organization in embryos. Thus, CP190 has distinct nuclear and centrosomal functions, and it provides a crucial link between the centrosome/MT and actin/myosin cytoskeletal systems in early embryos.

Summary Compromising the activity of the spindle checkpoint permits mitotic exit in the presence of unattached kinetochores and, consequently, greatly increases the rate of aneuploidy in the daughter cells [1–3]. The metazoan checkpoint mechanism is more complex than in yeast in that it requires additional proteins and activities besides the classical Mads and Bubs. Among these are Rod, Zw10, and Zwilch, components of a 700 Kdal complex (Rod/Zw10) [4–6] that is required for recruitment of dynein/dynactin to kinetochores [7, 8] but whose role in the checkpoint is poorly understood. The dynamics of Rod and Mad2, examined in different organisms, show intriguing similarities as well as apparent differences [7, 9] . Here we simultaneously follow GFP-Mad2 and RFP-Rod and find they are in fact closely associated throughout early mitosis. They accumulate simultaneously on kinetochores and are shed together along microtubule fibers after attachment. Their behavior and position within attached kinetochores is distinct from that of BubR1; Mad2 and Rod colocalize to the outermost kinetochore region (the corona), whereas BubR1 is slightly more interior. Moreover, Mad2, but not BubR1, Bub1, Bub3, or Mps1, requires Rod/Zw10 for its accumulation on unattached kinetochores. Rod/Zw10 thus contributes to checkpoint activation by promoting Mad2 recruitment and to checkpoint inactivation by recruiting dynein/dynactin that subsequently removes Mad2 from attached kinetochores.

gp63 is a highly abundant glycosylphosphatidylinositol (GPI)-anchored membrane protein expressed in both the promastigote and the amastigote forms of Leishmania species. In Leishmania mexicana , gp63 exists as a heterogeneous family of proteins that are differentially processed and localized during the 2 developmental stages. In this study we determined the molecular organization of the L. mexicana gp63 gene family, demonstrating that the gp63 genes fall into 3 linked families of tandemly repeated, but structurally distinct, entities designated as C1, C2 and C3. The C1 and C2 gene clusters contain 4–5 copies each, while the C3 gene may be single copy. Whilst promastigotes contain transcripts from all 3 gene classes, the intracellular amastigote only expressed detectable transcript from the C1 gene class. Moreover, the sequence of the C1 genes predicts a unique carboxy terminus substantially different from the GPI anchor addition signal sequence found in other Leishmania spp. and which has characteristics incompatible with substitution with a GPI anchor. These findings have significance for both the diversity of gp63 and for the regulation of tightly clustered, tandem gene arrays.

In this issue of Structure, Civril et al. (2010) describe an unexpected structural similarity between the kinetochore component Rod-Zw10-Zwilch (RZZ) and the vesicle transport component Nag-Rint1-Zw10 (NRZ).

The white-ivory ( w i ) mutation, an unstable allele of the white locus in Drosophila, reverts to wild-type at frequencies of 5 × 10 −5 in homozygous females, and 5 × 10 −6 in males and deletion heterozygous females. We show by molecular cloning and Southern blot analysis of DNA from w i flies that a 2.9 kilobase tandem duplication within the white locus is responsible for the mutation. Phenotypic reversion appears, in most cases, to be due to an exact excision of the extra copy of the sequence. Two derivative alleles of w i , one phenotypically wild-type, the other a partial revertant, carry insertions of moderately repetitive DNA from outside the locus, in addition to suffering deletions of some white locus DNA. Earlier genetic data preclude unequal crossing-over between homologs as an explanation for the precise reversions. Rather, an intrachromosomal meiotic event seems to be responsible. Our results suggest that intrachromosomal recombination may be responsible in other systems for a larger number of rearrangements than has been suspected, and that interallelic recombination frequencies in Drosophila do not always correlate in a simple way with DNA length or extent of homology.