Centrioles and centrosomes are conserved organelles in animals that are essential for cell signaling, cell division, and for specialized roles in differentiated cells, impacting organ function in whole animals. Mutations in proteins that function in centriole/basal body assembly or function, or which impact the cilia that grow from basal bodies, comprise the etiological basis for an expanding category of developmental and disease genes. Mutations in genes for other centrosomal proteins like CDK5RAP2, Pericentrin, sas4 and Aspm, are the root cause of related syndromes that affect brain and/or body size, yet an understanding of the connection between function at the centrosome and the causes of disease are unclear. To understand the disease or developmental process, we must first understand the functions of the proteins affected. Here we focus on the Centrosomin family of proteins. A key breakthrough came with mutation studies of Drosophila Centrosomin (CNN), showing that it is required in an early process of mitotic centrosome assembly. Centrosomes are the major microtubule-organizing centers (MTOCs) in animal cells and are required for efficient assembly of microtubules into the bipolar spindle apparatus at mitosis. The molecular functions of centrosome and centriole proteins are largely uncharacterized;indeed, the centrosome "parts list" is still being compiled. We employ Drosophila as a model system to investigate the molecular function of Centrosomin (Cnn) and the proteins and processes it impacts. With this model we combine classical and molecular genetic approaches with cell biology and biochemistry, capitalizing on the efficiency and rich tool chest that this model affords. The estimated 70% of human disease genes with homologs in Drosophila validate its use as a model for vertebrate biology. Yet, by employing a mouse mutant for CDK5RAP2, a mouse CNN family member, we bridge these systems and achieve a closer understanding of the pathology that underlies mutations in CDK5RAP2, which cause microcephaly in humans. CNN contains two conserved modules. One domain, near the N-terminus, regulates microtubule assembly at centrosomes, while the second domain at the C-terminus regulates actin organization into cleavage furrows. The first domain functions to recruit a microtubule assembly factor to centrosomes, while the second domain binds directly to Centrocortin (Cen), a novel factor required for cleavage furrow assembly. In mice, CDK5RAP2 mutant cells lose centriole engagement and cohesion, inducing centriole amplification. At mitosis, these excess centrosomes induce multipolar spindle assembly, implicating a role for CDK5RAP2 in centrosome clustering at mitosis. The multiple centrioles also template multiple primary cilia in CDK5RAP2 mutant cells. These findings pave a path toward a deeper understanding of the key processes that centrosomes regulate and that govern the regulation of centrosome replication.
Our specific aims are to: 1. Determine the mechanisms of MTOC regulation by CNN, 2. Define the role of Centrocortin and its cooperation with CNN in cleavage furrow assembly, and 3. Define the functions of CDK5RAP2 in the mouse.
Mutations in genes that function at centrioles and centrosomes cause a host of human disorders due to their importance in so many developmental and physiological processes. These ailments include polycystic kidney disease, deafness, hydrocephaly, obesity, microcephaly, and more. The goal of this proposal is to gain molecular and mechanistic understanding of centrioles and centrosomes, structures found in nearly every cell, in flies and mice and discern the pathology of autosomal recessive primary microcephaly (MCPH).
|Galletta, Brian J; Guillen, Rodrigo X; Fagerstrom, Carey J et al. (2014) Drosophila pericentrin requires interaction with calmodulin for its function at centrosomes and neuronal basal bodies but not at sperm basal bodies. Mol Biol Cell 25:2682-94|
|Chen, Jieyan V; Megraw, Timothy L (2014) Spermitin: a novel mitochondrial protein in Drosophila spermatids. PLoS One 9:e108802|
|Megraw, Timothy L; Sharkey, James T; Nowakowski, Richard S (2011) Cdk5rap2 exposes the centrosomal root of microcephaly syndromes. Trends Cell Biol 21:470-80|
|Barrera, Jose A; Kao, Ling-Rong; Hammer, Robert E et al. (2010) CDK5RAP2 regulates centriole engagement and cohesion in mice. Dev Cell 18:913-26|
|Kao, Ling-Rong; Megraw, Timothy L (2009) Centrocortin cooperates with centrosomin to organize Drosophila embryonic cleavage furrows. Curr Biol 19:937-42|
|Mottier-Pavie, Violaine; Megraw, Timothy L (2009) Drosophila bld10 is a centriolar protein that regulates centriole, basal body, and motile cilium assembly. Mol Biol Cell 20:2605-14|
|Kotadia, S; Kao, L R; Comerford, S A et al. (2008) PP2A-dependent disruption of centrosome replication and cytoskeleton organization in Drosophila by SV40 small tumor antigen. Oncogene 27:6334-46|
|Zhang, Jiuli; Megraw, Timothy L (2007) Proper recruitment of gamma-tubulin and D-TACC/Msps to embryonic Drosophila centrosomes requires Centrosomin Motif 1. Mol Biol Cell 18:4037-49|
|Kao, Ling-Rong; Megraw, Timothy L (2004) RNAi in cultured Drosophila cells. Methods Mol Biol 247:443-57|