title: Characterization of Adenomatous Polyposis Coli Protein Dynamics and Localization at the Centrosome.
abstract: The adenomatous polyposis coli (APC) tumor suppressor is a multifunctional regulator of Wnt signaling and acts as a mobile scaffold at different cellular sites. APC was recently found to stimulate microtubule (MT) growth at the interphase centrosome; however, little is known about its dynamics and localization at this site. To address this, we analysed APC dynamics in fixed and live cells by fluorescence microscopy. In detergent-extracted cells, we discovered that APC was only weakly retained at the centrosome during interphase suggesting a rapid rate of exchange. This was confirmed in living cells by fluorescence recovery after photobleaching (FRAP), which identified two pools of green fluorescent protein (GFP)-APC: a major rapidly exchanging pool (~86%) and minor retained pool (~14%). The dynamic exchange rate of APC was unaffected by C-terminal truncations implicating a targeting role for the N-terminus. Indeed, we mapped centrosome localization to N-terminal armadillo repeat (ARM) domain amino acids 334-625. Interestingly, the rate of APC movement to the centrosome was stimulated by intact MTs, and APC dynamics slowed when MTs were disrupted by nocodazole treatment or knockdown of γ-tubulin. Thus, the rate of APC recycling at the centrosome is enhanced by MT growth, suggesting a positive feedback to stimulate its role in MT growth.
authors.affiliation: Centre for Cancer Research, The Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW 2145, Australia. christina.lui@sydney.edu.au.
authors.forename: Christina
authors.initials: C
authors.lastname: Lui
authors.affiliation: Centre for Cancer Research, The Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW 2145, Australia. mythmok@mythmok.com.|School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong SAR, China. mythmok@mythmok.com.|Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China. mythmok@mythmok.com.
authors.forename: Myth T S
authors.initials: MT
authors.lastname: Mok
authors.affiliation: Centre for Cancer Research, The Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW 2145, Australia. beric.henderson@sydney.edu.au.
authors.forename: Beric R
authors.initials: BR
authors.lastname: Henderson
keywords: APC; FRAP; Wnt signaling; centrosome; colorectal cancer; protein dynamics