Abstract
Microtubule-based centrioles in the centrosome mediate accurate bipolar cell division, spindle orientation, and primary cilia formation. Cellular checkpoints ensure that the centrioles duplicate only once in every cell cycle and achieve precise dimensions, dysregulation of which results in genetic instability and neuro- and ciliopathies. The normal cellular level of centrosomal protein 4.1-associated protein (CPAP), achieved by its degradation at mitosis, is considered as one of the major mechanisms that limits centriole growth at a predetermined length. Here we show that CPAP levels and centriole elongation are regulated by centrobin. Exogenous expression of centrobin causes abnormal elongation of centrioles due to massive accumulation of CPAP in the cell. Conversely, CPAP was undetectable in centrobin-depleted cells, suggesting that it undergoes degradation in the absence of centrobin. Only the reintroduction of full-length centrobin, but not its mutant form that lacks the CPAP binding site, could restore cellular CPAP levels in centrobin-depleted cells, indicating that persistence of CPAP requires its interaction with centrobin. Interestingly, inhibition of the proteasome in centrobin-depleted cells restored the cellular and centriolar CPAP expression, suggesting its ubiquitination and proteasome-mediated degradation when centrobin is absent. Intriguingly, however, centrobin-overexpressing cells also showed proteasome-independent accumulation of ubiquitinated CPAP and abnormal, ubiquitin-positive, elongated centrioles. Overall, our results show that centrobin interacts with ubiquitinated CPAP and prevents its degradation for normal centriole elongation function. Therefore, it appears that loss of centrobin expression destabilizes CPAP and triggers its degradation to restrict the centriole length during biogenesis.
Highlights
The mechanism by which centriole dimensions are regulated is not understood
To understand the underlying molecular mechanism by which centrobin-mediates centriole elongation, we examined the cellular expression levels of centrosomal protein 4.1-associated-protein (CPAP) and CP110 in centrobinoverexpressing cells. 293T cells were transfected with control or myc-centrobin expression vectors, and cell lysates were prepared 72 h post-transfection and subjected to immunoblotting to detect the endogenous levels of CPAP, CP110, and tubulin
In association with our previous report that centrobin and CPAP interact directly [48], this study demonstrates that loss of centrobin-CPAP interaction results in targeting of ubiquitinated CPAP for proteasome-mediated degradation, a potential mechanism for restricting the centriole length to ϳ500 nm
Summary
The mechanism by which centriole dimensions are regulated is not understood. Results: The absence of centrobin leads to degradation of centrosomal protein 4.1-associated-protein (CPAP). The normal cellular level of centrosomal protein 4.1-associated protein (CPAP), achieved by its degradation at mitosis, is considered as one of the major mechanisms that limits centriole growth at a predetermined length. Identifying the upstream targets and underlying signals that govern cellular CPAP levels will shed light on how the centriole length is restricted In this regard, we recently reported that centrobin interacts with tubulin and CPAP, and these interactions are critical during the elongation stage of centriole duplication [47, 48]. Centrobin has a crucial role in centriole assembly, bipolar mitotic spindle assembly, microtubule polymerization, and asymmetric cell division (5, 36, 49 –52) It is considered as a daughter centriole protein, centrobin was detected on mother centrioles of G2/M-arrested cells [47], suggesting that its levels are regulated on the centrioles at different stages of cell cycle. Once the right length of centrioles is achieved at mitosis [39], an unknown event, perhaps loss of centrobin, triggers CPAP degradation to limit centriole growth during biogenesis
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