Hélio Roque

DSas-6 and Ana2 Coassemble into Tubules to Promote Centriole Duplication and Engagement

Summary Centrioles form cilia and centrosomes, organelles whose dysfunction is increasingly linked to human disease. Centriole duplication relies on a few conserved proteins (ZYG-1/Sak/Plk4, SAS-6, SAS-5/Ana2, and SAS-4), and is often initiated by the formation of an inner “cartwheel” structure. Here, we show that overexpressed Drosophila Sas-6 and Ana2 coassemble into extended tubules (SAStubules) that bear a striking structural resemblance to the inner cartwheel of the centriole. SAStubules specifically interact with centriole proximal ends, but extra DSas-6/Ana2 is only recruited onto centrioles when Sak/Plk4 kinase is also overexpressed. This extra centriolar DSas-6/Ana2 induces centriole overduplication and, surprisingly, increased centriole cohesion. Intriguingly, we observe tubules that are structurally similar to SAStubules linking the engaged centrioles in normal wild-type cells. We conclude that DSas-6 and Ana2 normally cooperate to drive the formation of the centriole inner cartwheel and that they promote both centriole duplication and centriole cohesion in a Sak/Plk4-dependent manner.

Drosophila Cep135/Bld10 maintains proper centriole structure but is dispensable for cartwheel formation.

Summary Cep135/Bld10 is a conserved centriolar protein required for the formation of the central cartwheel, an early intermediate in centriole assembly. Surprisingly, Cep135/Bld10 is not essential for centriole duplication in Drosophila, suggesting either that Cep135/Bld10 is not essential for cartwheel formation, or that the cartwheel is not essential for centriole assembly in flies. Using electron tomography and super-resolution microscopy we show that centrioles can form a cartwheel in the absence of Cep135/Bld10, but centriole width is increased and the cartwheel appears to disassemble over time. Using 3D structured illumination microscopy we show that Cep135/Bld10 is localized to a region between inner (SAS-6, Ana2) and outer (Asl, DSpd-2 and D-PLP) centriolar components, and the localization of all these component is subtly perturbed in the absence of Cep135/Bld10, although the ninefold symmetry of the centriole is maintained. Thus, in flies, Cep135/Bld10 is not essential for cartwheel assembly or for establishing the ninefold symmetry of centrioles; rather, it appears to stabilize the connection between inner and outer centriole components.

CP110 exhibits novel regulatory activities during centriole assembly in Drosophila.

Although loss of CP110 is tolerated in Drosophila, CP110 is important for limiting centriole length, limiting centriolar microtubule length, and suppressing centriole overduplication when duplication proteins are overexpressed.

SAS-6 oligomerization: the key to the centriole?

Centrioles are among the most beautiful of biological structures. How their highly conserved nine-fold symmetry is generated is a question that has intrigued cell biologists for decades. Two recent structural studies provide the tantalizing suggestion that the self-organizing properties of the SAS-6 protein hold the answer.

Mechanical design principles of a mitotic spindle

An organised spindle is crucial to the fidelity of chromosome segregation, but the relationship between spindle structure and function is not well understood in any cell type. The anaphase B spindle in fission yeast has a slender morphology and must elongate against compressive forces. This ‘pushing’ mode of chromosome transport renders the spindle susceptible to breakage, as observed in cells with a variety of defects. Here we perform electron tomographic analyses of the spindle, which suggest that it organises a limited supply of structural components to increase its compressive strength. Structural integrity is maintained throughout the spindles fourfold elongation by organising microtubules into a rigid transverse array, preserving correct microtubule number and dynamically rescaling microtubule length. DOI: http://dx.doi.org/10.7554/eLife.03398.001

Drosophila Ana1 is required for centrosome assembly and centriole elongation

ABSTRACT Centrioles organise centrosomes and cilia, and these organelles have an important role in many cell processes. In flies, the centriole protein Ana1 is required for the assembly of functional centrosomes and cilia. It has recently been shown that Cep135 (also known as Bld10) initially recruits Ana1 to newly formed centrioles, and that Ana1 then recruits Asl (known as Cep152 in mammals) to promote the conversion of these centrioles into centrosomes. Here, we show that ana1 mutants lack detectable centrosomes in vivo, that Ana1 is irreversibly incorporated into centrioles during their assembly and appears to play a more important role in maintaining Asl at centrioles than in initially recruiting Asl to centrioles. Unexpectedly, we also find that Ana1 promotes centriole elongation in a dose-dependent manner: centrioles are shorter when Ana1 dosage is reduced and are longer when Ana1 is overexpressed. This latter function of Ana1 appears to be distinct from its role in centrosome and cilium function, as a GFP–Ana1 fusion lacking the N-terminal 639 amino acids of the protein can support centrosome assembly and cilium function but cannot promote centriole over-elongation when overexpressed. Highlighted Article: Ana1 is a conserved centriole protein that we show is required for centrosome and cilium assembly and that also helps to promote centriole elongation in a dose-dependent manner.

Drosophila sensory cilia lacking MKS proteins exhibit striking defects in development but only subtle defects in adults.

ABSTRACT Cilia are conserved organelles that have important motility, sensory and signalling roles. The transition zone (TZ) at the base of the cilium is crucial for cilia function, and defects in several TZ proteins are associated with human congenital ciliopathies such as nephronophthisis (NPHP) and Meckel–Gruber syndrome (MKS). In several species, MKS and NPHP proteins form separate complexes that cooperate with Cep290 to assemble the TZ, but flies seem to lack core components of the NPHP module. We show that MKS proteins in flies are spatially separated from Cep290 at the TZ, and that flies mutant for individual MKS genes fail to recruit other MKS proteins to the TZ, whereas Cep290 seems to be recruited normally. Although there are abnormalities in microtubule and membrane organisation in developing MKS mutant cilia, these defects are less apparent in adults, where sensory cilia and sperm flagella seem to function quite normally. Thus, localising MKS proteins to the cilium or flagellum is not essential for viability or fertility in flies. Highlighted Article: An analysis of Meckel–Gruber syndrome (MKS) fly mutants reveals that cilium structure is perturbed during development, but that cilia are largely structurally and functionally normal by adulthood.

Drosophila PLP assembles pericentriolar clouds that promote centriole stability, cohesion and MT nucleation

Pericentrin is a conserved centrosomal protein whose dysfunction has been linked to several human diseases. It has been implicated in many aspects of centrosome and cilia function, but its precise role is unclear. Here, we examine Drosophila Pericentrin-like-protein (PLP) function in vivo in tissues that form both centrosomes and cilia. Plp mutant centrioles exhibit four major defects: (1) They are short and have subtle structural abnormalities; (2) They disengage prematurely, and so overduplicate; (3) They organise fewer cytoplasmic MTs during interphase; (4) When forming cilia, they fail to establish and/or maintain a proper connection to the plasma membrane—although, surprisingly, they can still form an axoneme-like structure that can recruit transition zone (TZ) proteins. We show that PLP helps assemble “pericentriolar clouds” of electron-dense material that emanate from the central cartwheel spokes and spread outward to surround the mother centriole. We propose that the partial loss of these structures may largely explain the complex centriole, centrosome and cilium defects we observe in Plp mutant cells.

Drosophila PLP forms centriolar-clouds that promote centriole stability, cohesion and MT nucleation

Pericentrin is a conserved centrosomal protein whose dysfunction has been linked to several human diseases. The precise function of Pericentrin, however, is controversial. Here, we examine Drosophila Pericentrin-like- protein (PLP) function in vivo, in tissues that form both centrosomes and cilia. PLP mutant centrioles exhibit four major defects: (1) They are too short and have subtle structural defects; (2) They separate prematurely, and so overduplicate; (3) They organise fewer MTs during interphase; (4) They fail to establish and/or maintain a proper connection to the plasma membrane— although, surprisingly, mutant centrioles can still form an axoneme and recruit transition zone (TZ) proteins. We show that PLP helps to form “ pericentriolar clouds” of electron-dense material that emanate from the central cartwheel spokes and spread outward to surround the mother centriole. The partial loss of these structures may explain the complex centriole, centrosome and cilium defects we observe in PLP mutant cells.

Meeting report - imaging in cell biology: where next?

The Company of Biologists Workshop entitled ‘Imaging in Cell Biology: Where Next?’ was held in October 2012 at Cumberland Lodge in Windsor, UK. The meeting was a forum for leaders in different areas of single-molecule analysis, high- and super-resolution imaging and data processing both at