Orbit/CLASP determines centriole length by antagonising Klp10A in Drosophila spermatocytes.

After centrosome duplication, centrioles elongate before M phase. To identify genes required for this process and to understand the regulatory mechanism, we investigated the centrioles in Drosophila premeiotic spermatocytes expressing fluorescently tagged centriolar proteins. We demonstrated that an essential microtubule polymerisation factor, Orbit (the Drosophila CLASP orthologue, encoded by chb), accumulated at the distal end of centrioles and was required for the elongation. Conversely, a microtubule-severing factor, Klp10A, shortened the centrioles. Genetic analyses revealed that these two proteins functioned antagonistically to determine centriole length. Furthermore, Cp110 in the distal tip complex was closely associated with the factors involved in centriolar dynamics at the distal end. We observed loss of centriole integrity, including fragmentation of centrioles and earlier separation of the centriole pairs, in Cp110-null mutant cells either overexpressing Orbit or depleted of Klp10A Excess centriole elongation in the absence of the distal tip complex resulted in the loss of centriole integrity, leading to the formation of multipolar spindle microtubules emanating from centriole fragments, even when they were unpaired. Our findings contribute to understanding the mechanism of centriole integrity, disruption of which leads to chromosome instability in cancer cells.

Drosophila Ana1 is required for centrosome assembly and centriole elongation.

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.

With Age Comes Maturity: Biochemical and Structural Transformation of a Human Centriole in the Making.

Centrioles are microtubule-based cellular structures present in most human cells that build centrosomes and cilia. Proliferating cells have only two centrosomes and this number is stringently maintained through the temporally and spatially controlled processes of centriole assembly and segregation. The assembly of new centrioles begins in early S phase and ends in the third G1 phase from their initiation. This lengthy process of centriole assembly from their initiation to their maturation is characterized by numerous structural and still poorly understood biochemical changes, which occur in synchrony with the progression of cells through three consecutive cell cycles. As a result, proliferating cells contain three structurally, biochemically, and functionally distinct types of centrioles: procentrioles, daughter centrioles, and mother centrioles. This age difference is critical for proper centrosome and cilia function. Here we discuss the centriole assembly process as it occurs in somatic cycling human cells with a focus on the structural, biochemical, and functional characteristics of centrioles of different ages.

Studying Centriole Duplication and Elongation in Human Cells.

Centrioles assemble centrosomes and cilia/flagella, which are microtubule-based structures with key roles in cell division, polarity, motility, and signaling. Centriole biogenesis is a tightly regulated process, and deregulation of centriole numbers and structure can have dramatic consequences for cellular function and integrity. However, their small size poses a challenge to study them. Here, we describe protocols that allow the identification and assessment of true centrioles and that provide straightforward strategies to study the role of new candidate proteins in centriole duplication and elongation.

Functional Analysis of Hydrolethalus Syndrome Protein HYLS1 in Ciliogenesis and Spermatogenesis in Drosophila.

Cilia and flagella are conserved subcellular organelles, which arise from centrioles and play critical roles in development and reproduction of eukaryotes. Dysfunction of cilia leads to life-threatening ciliopathies. HYLS1 is an evolutionarily conserved centriole protein, which is critical for ciliogenesis, and its mutation causes ciliopathy-hydrolethalus syndrome. However, the molecular function of HYLS1 remains elusive. Here, we investigated the function of HYLS1 in cilia formation using the Drosophila model. We demonstrated that Drosophila HYLS1 is a conserved centriole and basal body protein. Deletion of HYLS1 led to sensory cilia dysfunction and spermatogenesis abnormality. Importantly, we found that Drosophila HYLS1 is essential for giant centriole/basal body elongation in spermatocytes and is required for spermatocyte centriole to efficiently recruit pericentriolar material and for spermatids to assemble the proximal centriole-like structure (the precursor of the second centriole for zygote division). Hence, by taking advantage of the giant centriole/basal body of Drosophila spermatocyte, we uncover previously uncharacterized roles of HYLS1 in centriole elongation and assembly.

PPP1R35 is a novel centrosomal protein that regulates centriole length in concert with the microcephaly protein RTTN.

Centrosome structure, function, and number are finely regulated at the cellular level to ensure normal mammalian development. Here, we characterize PPP1R35 as a novel bona fide centrosomal protein and demonstrate that it is critical for centriole elongation. Using quantitative super-resolution microscopy mapping and live-cell imaging we show that PPP1R35 is a resident centrosomal protein located in the proximal lumen above the cartwheel, a region of the centriole that has eluded detailed characterization. Loss of PPP1R35 function results in decreased centrosome number and shortened centrioles that lack centriolar distal and microtubule wall associated proteins required for centriole elongation. We further demonstrate that PPP1R35 acts downstream of, and forms a complex with, RTTN, a microcephaly protein required for distal centriole elongation. Altogether, our study identifies a novel step in the centriole elongation pathway centered on PPP1R35 and elucidates downstream partners of the microcephaly protein RTTN.

Aurora A inhibition by MNL8054 promotes centriole elongation during Drosophila male meiosis.

Aurora A kinase plays an important role in several aspects of cell division, including centrosome maturation and separation, a crucial step for the correct organization of the bipolar spindle. Although it has long been showed that this kinase accumulates at the centrosome throughout mitosis its precise contribution to centriole biogenesis and structure has until now not been reported. It is not surprising that so little is known, due to the small size of somatic centrioles, where only dramatic structural changes may be identified by careful electron microscopy analysis. Conversely, centrioles of Drosophila primary spermatocytes increase tenfold in length during the first prophase, thus making any change easily detectable. Therefore, we examined the consequence of the pharmacological inhibition of Aurora A by MLN8054 on centriole biogenesis during early Drosophila gametogenesis. Here, we show that depletion of this kinase results in longer centrioles, mainly during transition from prophase to prometaphase of the first meiosis. We also found abnormal ciliogenesis characterized by irregularly growing axonemal doublets. Our results represent the first documentation of a potential requirement of Aurora A in centriole integrity and elongation.