4 research outputs found
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The role of EHD proteins in caveolae, and the role of caveolae in adipocytes
Caveolae are 50-60 nm flask-shaped invaginations of the plasma membrane that protect the plasma membrane from damage under stretch forces. They are highly abundant in cells that experience high levels of stress forces such as adipocytes, endothelial cells and muscle cells. Caveolae are generated by the oligomerisation and association of caveolin and cavin proteins, which form the caveolar coat complex at the caveolar bulb and are progressively well characterised. However, less is known about the proteins that localise to the caveolar neck. Using the CRIPSR/Cas9 system to generate gene knock-in and knockout cell lines, the role of EHD proteins at caveolae was investigated. It was found that, in addition to EHD2 being at the neck, both EHD1 and EHD4 were also present. The recruitment of other EHD proteins was markedly increased in the absence of EHD2. This functional redundancy was confirmed by the generation of EHD1, 2 and 4 triple knockout cell lines, which displayed two striking sets of phenotypes. Firstly, the characteristic higher-order clusters of caveolae are lost in the absence of EHD proteins. And secondly, caveolae are destabilised and the plasma membrane is more likely to rupture when the EHD1,2,4 knockout cells are subjected to cycles of stretch forces. The data identify the first molecular components that cluster caveolae into a membrane ultrastructure that potentially extends stretch buffering capacity. A second series of experiments tested different ideas about the function of caveolae in adipocytes. The insulin receptor and CD36 were found to at most partially colocalise with caveolae, and the role of caveolae in regulating signalling processes remains unclear. In contrast, the plasma membrane of adipocytes without caveolae is clearly more prone to rupture, confirming a mechanoprotective function
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Vesicular trafficking plays a role in centriole disengagement and duplication
Centrosomes are the major microtubule-nucleating and microtubule-organizing centers of cells and play crucial roles in microtubule anchoring, organelle positioning, and ciliogenesis. At the centrosome core lies a tightly associated or "engaged" mother-daughter centriole pair. During mitotic exit, removal of centrosomal proteins pericentrin and Cep215 promotes "disengagement" by the dissolution of intercentriolar linkers, ensuring a single centriole duplication event per cell cycle. Herein, we explore a new mechanism involving vesicular trafficking for the removal of centrosomal Cep215. Using small interfering RNA and CRISPR/ Cas9 gene-edited cells, we show that the endocytic protein EHD1 regulates Cep215 transport from centrosomes to the spindle midbody, thus facilitating disengagement and duplication. We demonstrate that EHD1 and Cep215 interact and show that Cep215 displays increased localization to vesicles containing EHD1 during mitosis. Moreover, Cep215-containing vesicles are positive for internalized transferrin, demonstrating their endocytic origin. Thus, we describe a novel relationship between endocytic trafficking and the centrosome cycle, whereby vesicles of endocytic origin are used to remove key regulatory proteins from centrosomes to control centriole duplication.National Institute of General Medical Sciences (NIGMS) [R01GM074876, P30GM106397]; National Cancer Institute [P30 CA23074]; NIH/NIGMS [R01GFM110166, R01GM126035]This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]