Centrosome Regulation in Development and Dysregulation in Disease

National Heart, Lung, And Blood Institute

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Abstract

Understanding how events at the molecular and cellular scales contribute to tissue form and function is key to uncovering mechanisms driving animal development, physiology and disease. Elucidating these mechanisms has been enhanced through the study of model organisms and the use of sophisticated genetic, biochemical and imaging tools. In the past fiscal year, we have continued our longtime work aimed at understanding the underlying mechanism of centrosome duplication and maturation. One poorly aspect is how centrosome proteins achieve cell-type-specific functional differences. To gain insight into cell-type-specific differences in centrosome protein function and regulation, we investigated a duplication of Spd-2 in Drosophila willistoni, which has Spd-2A (ancestral) and Spd-2B (derived). We find that Spd-2A functions in NB mitosis, whereas Spd-2B functions in SC meiosis. Ectopically expressed Spd-2B accumulates and functions in mitotic NBs, but ectopically expressed Spd-2A failed to accumulate in meiotic SCs, suggesting cell-type-specific differences in translation or protein stability. We mapped this failure to accumulate and function in meiosis to the C-terminal tail domain of Spd-2A, revealing a novel regulatory mechanism that can potentially achieve differences in PCM function across cell types. The most counterintuitive result from our study is that although Spd-2A is the more highly conserved paralog and contains all protein-coding regions found in Spd-2, it has lost the ability to function in meiotic cells. We found that the C-terminal 119 amino acid tail of Spd-2A, which is absent in Spd-2B, is responsible for this lack of meiotic function. Our inability to achieve significant overexpression of Spd-2A in meiotic SCs using a GAL4 > UAS approach suggests that the tail domain of Spd-2A triggers protein or mRNA degradation, or inhibition of RNA translation in meiotic cells. In contrast, although Spd-2B appears to be specialized for organizing meiotic PCM, it is still capable of functioning in mitotic NBs when ectopically expressed, consistent with the idea that the tail domain functions to regulate Spd-2 levels rather than directly mediating recruitment of PCM proteins. D. melanogaster Spd-2 functions across all tissues, presumably because all cell types that require its function express and accumulate Spd-2 to functional levels. One model that would explain our results is that the Spd-2A tail domain has gained a novel function that prevents its own accumulation in meiotic SCs, and that this level-regulating function was not present in the ancestral Spd-2 tail domain. However, we do not favor this explanation as we think the evolution of a novel tail function is relatively unlikely. An alternative model could be that the ancestral Spd-2 tail domain controls the levels of Spd-2 in response to an unknown regulatory mechanism; for example, a cell-type-specific post-translational modification (PTM) or regulatory binding partner. In this alternative model, there could be two possible states for Spd-2: (1) an accumulating and activated state, where the tail allows Spd-2 to function to recruit PCM, and (2) a destabilizing or inactive state, where the tail prevents Spd-2 accumulation and activation. In this scenario, once gene duplication gave rise to SC-expressed Spd-2B, then Spd-2A would experience relaxed selection against mutations that prevent its accumulation in SCs, and possibly positive selection against the presence of two Spd-2 isoforms. Future work could extend our chimeric transgene approach to precisely map the minimal amino acid substitutions required to confer the destabilizing activity of the Spd-2A tail domain. In another study, we continue to investigate how precise regulation centrosome protein levels is critical for centrosome function. One such centrosome protein whose levels must be regulated is Pericentrin (PCNT in human, PLP in Drosophila). Increased PCNT expression and its protein accumulation are linked to clinical conditions including cancer, mental disorders, and ciliopathies. However, the mechanisms by which PCNT levels are regulated remain underexplored. Our previous study demonstrated that PLP levels are sharply down-regulated during early spermatogenesis and this regulation is essential to spatially position PLP on the proximal end of centrioles. We hypothesized that the sharp drop in PLP protein was a result of rapid protein degradation during the male germ line premeiotic G2 phase. Here, we show that PLP is subject to ubiquitin-mediated degradation and identify multiple proteins that promote the reduction of PLP levels in spermatocytes, including the UBR box containing E3 ligase Poe (UBR4), which we show binds to PLP. Although protein sequences governing posttranslational regulation of PLP are not restricted to a single region of the protein, we identify a region that is required for Poe-mediated degradation. Experimentally stabilizing PLP, via internal PLP deletions or loss of Poe, leads to PLP accumulation in spermatocytes, its mispositioning along centrioles, and defects in centriole docking in spermatids.