KAP, the accessory subunit of kinesin-2, binds the predicted coiled-coil stalk of the motor subunits.

Kinesin-2 is an anterograde motor involved in intraflagellar transport and certain other intracellular transport processes. It consists of two different motor subunits and an accessory protein KAP (kinesin accessory protein). The motor subunits were shown to bind each other through the coiled-coil stalk domains, while KAP was proposed to bind the tail domains of the motor subunits. Although several genetic studies established that KAP plays an important role in kinesin-2 functions, its exact role remains unclear. Here, we report the results of a systematic analysis of the KAP binding sites by using recombinant Drosophila kinesin-2 subunits as well as the endogenous proteins. These show that at least one of the coiled-coil stalks is sufficient to bind the N-terminal region of DmKAP. The soluble complex involving the recombinant kinesin-2 fragments is reconstituted in vitro at high salt concentrations, suggesting that the interaction is primarily nonionic. Furthermore, independent distant homology modeling indicated that DmKAP may bind along the coiled-coil stalks through a combination of predominantly hydrophobic interactions and hydrogen bonds. These observations led us to propose that KAP would stabilize the motor subunit heterodimer and help assemble a greater kinesin-2 complex in vivo.

Over-elongation of centrioles in cancer promotes centriole amplification and chromosome missegregation.

Centrosomes are the major microtubule organising centres of animal cells. Deregulation in their number occurs in cancer and was shown to trigger tumorigenesis in mice. However, the incidence, consequence and origins of this abnormality are poorly understood. Here, we screened the NCI-60 panel of human cancer cell lines to systematically analyse centriole number and structure. Our screen shows that centriole amplification is widespread in cancer cell lines and highly prevalent in aggressive breast carcinomas. Moreover, we identify another recurrent feature of cancer cells: centriole size deregulation. Further experiments demonstrate that severe centriole over-elongation can promote amplification through both centriole fragmentation and ectopic procentriole formation. Furthermore, we show that overly long centrioles form over-active centrosomes that nucleate more microtubules, a known cause of invasiveness, and perturb chromosome segregation. Our screen establishes centriole amplification and size deregulation as recurrent features of cancer cells and identifies novel causes and consequences of those abnormalities.

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.

Biochemical and molecular dynamic simulation analysis of a weak coiled coil association between kinesin-II stalks.

DEFINITION: Kinesin-2 refers to the family of motor proteins represented by conserved, heterotrimeric kinesin-II and homodimeric Osm3/Kif17 class of motors. BACKGROUND: Kinesin-II, a microtubule-based anterograde motor, is composed of three different conserved subunits, named KLP64D, KLP68D and DmKAP in Drosophila. Although previous reports indicated that coiled coil interaction between the middle segments of two dissimilar motor subunits established the heterodimer, the molecular basis of the association is still unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present a detailed heterodimeric association model of the KLP64D/68D stalk supported by extensive experimental analysis and molecular dynamic simulations. We find that KLP64D stalk is unstable, but forms a weak coiled coil heteroduplex with the KLP68D stalk when coexpressed in bacteria. Local instabilities, relative affinities between the C-terminal stalk segments, and dynamic long-range interactions along the stalks specify the heterodimerization. Thermal unfolding studies and independent simulations further suggest that interactions between the C-terminal stalk fragments are comparatively stable, whereas the N-terminal stalk reversibly unfolds at ambient temperature. CONCLUSIONS/SIGNIFICANCE: Results obtained in this study suggest that coiled coil interaction between the C-terminal stalks of kinesin-II motor subunits is held together through a few hydrophobic and charged interactions. The N-terminal stalk segments are flexible and could uncoil reversibly during a motor walk. This supports the requirement for a flexible coiled coil association between the motor subunits, and its role in motor function needs to be elucidated.

BLD10/CEP135 is a microtubule-associated protein that controls the formation of the flagellum central microtubule pair.

Cilia and flagella are involved in a variety of processes and human diseases, including ciliopathies and sterility. Their motility is often controlled by a central microtubule (MT) pair localized within the ciliary MT-based skeleton, the axoneme. We characterized the formation of the motility apparatus in detail in Drosophila spermatogenesis. We show that assembly of the central MT pair starts prior to the meiotic divisions, with nucleation of a singlet MT within the basal body of a small cilium, and that the second MT of the pair only assembles much later, upon flagella formation. BLD10/CEP135, a conserved player in centriole and flagella biogenesis, can bind and stabilize MTs and is required for the early steps of central MT pair formation. This work describes a genetically tractable system to study motile cilia formation and provides an explanation for BLD10/CEP135's role in assembling highly stable MT-based structures, such as motile axonemes and centrioles.

Heterotrimeric kinesin-II is necessary and sufficient to promote different stepwise assembly of morphologically distinct bipartite cilia in Drosophila antenna.

Structurally diverse sensory cilia have evolved from primary cilia, a microtubule-based cellular extension engaged in chemical and mechanical sensing and signal integration. The diversity is often associated with functional specialization. The olfactory receptor neurons in Drosophila, for example, express three distinct bipartite cilia displaying different sets of olfactory receptors on them. Molecular description underlying their assembly and diversification is still incomplete. Here, we show that the branched and the slender olfactory cilia develop in two distinct step-wise patterns through the pupal stages before the expression of olfactory receptor genes in olfactory neurons. The process initiates with a thin procilium growth from the dendrite apex, followed by volume increment in successive stages. Mutations in the kinesin-II subunit genes either eliminate or restrict the cilia growth as well as tubulin entry into the developing cilia. Together with previous results, our results here suggest that heterotrimeric kinesin-II is the primary motor engaged in all type-I sensory cilia assembly in Drosophila and that the cilia structure diversity is achieved through additional transports supported by the motor during development.