A Splice Variant of Centrosomin Converts Mitochondria to Microtubule-Organizing Centers.

Non-centrosomal microtubule organizing centers (MTOCs) direct microtubule (MT) organization to exert diverse cell-type-specific functions. In Drosophila spermatids, the giant mitochondria provide structural platforms for MT reorganization to support elongation of the extremely long sperm. However, the molecular basis for this mitochondrial MTOC and other non-centrosomal MTOCs has not been discerned. Here we report that Drosophila centrosomin (cnn) expresses two major protein variants: the centrosomal form (CnnC) and a non-centrosomal form in testes (CnnT). CnnC is established as essential for functional centrosomes, the major MTOCs in animal cells. We show that CnnT is expressed exclusively in testes by alternative splicing and localizes to giant mitochondria in spermatids. In cell culture, CnnT targets to the mitochondrial surface, recruits the MT nucleator γ-tubulin ring complex (γ-TuRC), and is sufficient to convert mitochondria to MTOCs independent of core pericentriolar proteins that regulate MT assembly at centrosomes. We mapped two separate domains in CnnT: one that is necessary and sufficient to target it to mitochondria and another that is necessary and sufficient to recruit γ-TuRCs and nucleate MTs. In elongating spermatids, CnnT forms speckles on the giant mitochondria that are required to recruit γ-TuRCs to organize MTs and support spermiogenesis. This molecular characterization of the mitochondrial MTOC defines a minimal molecular requirement for MTOC generation and implicates the potent role of Cnn (or its related) proteins in the direct regulation of MT assembly and organization of non-centrosomal MTOCs.

Rootletin organizes the ciliary rootlet to achieve neuron sensory function in Drosophila.

Cilia are essential for cell signaling and sensory perception. In many cell types, a cytoskeletal structure called the ciliary rootlet links the cilium to the cell body. Previous studies indicated that rootlets support the long-term stability of some cilia. Here we report that Drosophila melanogaster Rootletin (Root), the sole orthologue of the mammalian paralogs Rootletin and C-Nap1, assembles into rootlets of diverse lengths among sensory neuron subtypes. Root mutant neurons lack rootlets and have dramatically impaired sensory function, resulting in behavior defects associated with mechanosensation and chemosensation. Root is required for cohesion of basal bodies, but the cilium structure appears normal in Root mutant neurons. We show, however, that normal rootlet assembly requires centrioles. The N terminus of Root contains a conserved domain and is essential for Root function in vivo. Ectopically expressed Root resides at the base of mother centrioles in spermatocytes and localizes asymmetrically to mother centrosomes in neuroblasts, both requiring Bld10, a basal body protein with varied functions.

Cytokinesis: RhoGEFs control a developmental cleavage switch.

New findings provide evidence that developmentally staged RhoGEFs control assembly of two alternative forms of cleavage furrows: the 'Rappaport' furrows, which govern division of Drosophila syncytial cortical division cycles, and conventional spindle-directed furrows.

CDK5RAP2 regulates centriole engagement and cohesion in mice.

Centriole duplication occurs once per cell cycle, ensuring that each cell contains two centrosomes, each containing a mother-daughter pair of tightly engaged centrioles at mitotic entry. Loss of the tight engagement between mother and daughter centrioles appears to license the next round of centriole duplication. However, the molecular mechanisms regulating this process remain largely unknown. Mutations in CDK5RAP2, which encodes a centrosomal protein, cause autosomal recessive primary microcephaly in humans. Here we show that CDK5RAP2 loss of function in mice causes centriole amplification with a preponderance of single, unpaired centrioles and increased numbers of daughter-daughter centriole pairs. These results indicate that CDK5RAP2 is required to maintain centriole engagement and cohesion, thereby restricting centriole replication. Early in mitosis, amplified centrosomes assemble multipolar spindles in CDK5RAP2 mutant cells. Moreover, both mother and daughter centrioles are amplified and the excess mother centrioles template multiple primary cilia in CDK5RAP2 mutant cells.

Centrocortin cooperates with centrosomin to organize Drosophila embryonic cleavage furrows.

In the Drosophila early embryo, the centrosome coordinates assembly of cleavage furrows. Currently, the molecular pathway that links the centrosome and the cortical microfilaments is unknown. In centrosomin (cnn) mutants, in which the centriole forms but the centrosome pericentriolar material (PCM) fails to assemble, actin microfilaments are not organized into furrows at the syncytial cortex [6]. Although CNN is required for centrosome assembly and function, little is known of its molecular activities. Here, we show the novel protein Centrocortin (CEN), which associates with centrosomes and also with cleavage furrows in early embryos, is required for cleavage furrow assembly. CEN binds to CNN within CNN Motif 2 (CM2), a conserved 60 amino acid domain at CNN's C terminus. The cnn(B4) allele, which contains a missense mutation at a highly conserved residue within CM2, blocks the binding of CEN and disrupts cleavage furrow assembly. Together, these findings show that the C terminus of CNN coordinates cleavage furrow formation through binding to CEN, thereby providing a molecular link between the centrosome and cleavage furrow assembly.

Biochemical characterization and cloning of transglutaminases responsible for hemolymph clotting in Penaeus monodon and Marsupenaeus japonicus.

To investigate the shrimp blood clotting enzyme, a transglutaminase in the hemocytes of Penaeus monodon (abbreviated as TGH) was purified. TGH is an abundant homodimeric cytosolic protein with 84.2 kDa subunits. It clotted shrimp plasma and incorporated fluorescent dansylcadaverine into succinyl casein upon activation by CaCl(2) in vitro. IC(50) for the activation was 3 mM, which is below the shrimp plasma Ca(2+) level. Showing similar properties as other type II transglutaminase, TGH was particularly unstable after activation. MALDI-TOF/TOF mass-analyses of tryptic peptides of P. monodon TGH confirmed its identity to STG I (AY074924) previously cloned. A possible allele of the other isozyme STG II (AY771615) has also been cloned from the P. monodon cDNA and designated as PmTG. The predicted PmTG protein sequence is 58% similar to that of STG I and 99.2% to that of STG II. Likewise, a novel enzyme Mj-TGH was purified and cloned from Marsupenaeus japonicus hemocytes. Results of sequence alignment and phylogenetic analyses of these transglutaminases suggest that STG I and Mj-TGH are 83% identical and orthologous to each other, while PmTG/STG II and a previously cloned M. japonicus transglutaminase (AB162767) are their paralogs. Protein of the latter two could not be isolated, their regulated expression was discussed.