Research

A molecular solution to centrosome structure and function 

Genetic and molecular approaches have identified numerous components of the centrosome. Thus far, our knowledge of where, when and how these proteins are assembled represents just the tip of the iceberg. One major goal of our research is to assemble a dynamic 3D landscape of the centrosome, pinpointing the spatiotemporal organization of its individual structural components in dividing and differentiating cells. We use various super-resolution microscopy techniques, as the size of the centrosome is at the diffraction limit of conventional light microscopy. Determining when centrosome proteins are recruited and how they are spatially arranged provides important insight into their functions and the molecular interactions that govern centrosome assembly.

Figure 1. 3D-SIM reveals zone-based architecture of centrosome. (A) Left and middle: Drosophila centrosome stained for Dplp and resolved by conventional (conv) or 3D-SIM mode. Right: centrosome resolved by electron microscopy (EM). Bars, 200 nm. (B,C) Representative 3D-SIM staining patterns of centrosome proteins in (B), summarized as a zone-based architectural model in (C).

Full text: http://rsob.royalsocietypublishing.org/content/2/8/120104.long

Figure 2. A nine-fold molecular model of the centriole core. (A) STED imaging defines radial span of core centriole proteins in native centrioles. Bar, 200 nm. (B) U-ExM/3D-SIM resolves nine-fold symmetry and shows that Ana1 and Asl align along common radial axes. These axes extend between acetylated-tubulin-marked microtubule blades, whereas Ana3 occupies offset positions between Asl axes. Bar, 500 nm. (C) Integrated model showing how radial scaffold proteins and inter-spoke components together build centriole core symmetry.

Full text: https://rupress.org/jcb/article/220/4/e202005103/211748/Superresolution-characterization-of-core-centriole

Centriole maturation to motherhood – competent to duplicate and recruit PCM

The centriole duplicates once every cell cycle in concert with the DNA duplication. The newly assembled daughter centriole gradually recruits its components and eventually gains the ability to become the mother. We are interested in how this process takes place and how it is regulated. Our recent finding showed that in both Drosophila and human cells, three molecules Cep135, Ana1/Cep295 and Asl/Cep152 are sequentially loaded onto daughter centrioles from late interphase to mitosis, and constitute an architectural network that is key for centriole to mature to motherhood. Notably, the three molecules show an elongated distribution and interact through adjacent regions to generate a molecular network spanning from the inner- to the outer-most parts of the centriole. Ana1 provides a molecular link between the inner and outer parts of the centriole, and positions Asl at an appropriate radial position. Asl, in turn, is the recruiting partner of the master regulator of centriole duplication, Plk4, and also needed to recruit PCM. This finding accounts for the final stages in the assembly of the daughter centriole that convert it into a mature mother able to duplicate and recruit PCM.

Figure 3. Sequential loading of centrosome proteins to daughter centriole to facilitate its conversion to mature mother in Drosophila cultured cells (A). Also note the spatial distribution of these factors (B). Bar, 500 nm.

Full text: https://www.nature.com/articles/ncb3274

Centrosome function and disease implications

Centrosomes and their derivative basal bodies template cilia and flagella, and defects in these structures are associated with developmental disorders, ciliopathies, and infertility. We are extending our structural and regulatory studies to understand how specific centriolar architectures support organismal function, and how their disruption leads to disease-relevant phenotypes. In Drosophila testes, we found that Ana1/CEP295 acts as a radial scaffold spanning the centriolar microtubule wall and regulates microtubule remodeling from doublets to triplets. Its N-terminal region localizes near the A- and B-tubules and promotes their elongation, whereas its C-terminal region extends outward, recruits Centrobin, and supports C-tubule assembly. Loss of Ana1 disrupts triplet microtubule formation, shortens basal bodies, destabilizes sperm axonemes, and causes male infertility. These findings show how a defined centriolar architecture is translated into the structural integrity of sperm and reproductive capacity.

Figure 4. Ana1-dependent centriole microtubule remodeling during spermatogenesis. (A) EM analysis of wild-type and ana1 mutant Drosophila spermatogonia, spermatocytes, and sperm. Bars, 100 nm. (B) Model for Ana1-dependent doublet-to-triplet conversion and centriole elongation. Ana1 spans from the lumen of the centriolar microtubule wall to the outside. Its N-terminal region promotes modest A/B-tubule elongation, whereas its C-terminal region recruits Centrobin and supports C-tubule assembly. These activities may occur in repeated rounds as the microtubule wall extends. Loss of Ana1 disrupts triplet microtubule integrity, basal body elongation, and axoneme stability, ultimately impairing male fertility.

Full text: https://rupress.org/jcb/article-abstract/225/7/e202508192/282583/Ana1-CEP295-regulates-centriolar-doublet-to?redirectedFrom=fulltext