Poster Presentation 25th Lorne Cancer Conference 2013

The characterisation of Adenomatous polyposis coli (APC) at the centrosome (#258)

Christina Lui 1 , Cahora Palazon 1 , Manisha Sharma 1 , Mariana Brocardo 1 , Beric Henderson 1
  1. Westmead Millenium Institute, Westmead , NSW, Australia

Background: Adenomatous polyposis coli (APC) gene mutations are linked to Familial Adenomatous Polyposis (FAP), a syndrome that manifests as multiple polyps in the bowel. Mutations in both alleles the APC gene predispose to high risk of colorectal cancer, with generation of shortened APC isoforms that have abnormal functions and cell localization patterns.  APC is known for its tumour suppressing role in the Wnt signalling pathway, but in addition contributes to cytoskeletal regulation and control of mitosis and cell cycle. The centrosome nucleates assembly of the microtubule network in interphase cells and also the alignment, orientation and separation of chromosomes during mitosis. Improper chromosome separation can lead to chromosome instability typical of cancers. Currently there is little known of the targeting, dynamics or function of APC at the centrosomes.

Aim and Method: This study employs a range of techniques including confocal microscopic imaging of fluorescent-tagged APC in fixed and living cells, and mass spectrometry analysis, to elucidate the function and targeting of APC at the centrosome.

Results: We mapped the minimal APC targeting sequence required for centrosome localisation to amino acids 334-625, a part of the ARM domain implicated in protein interactions and APC localization to mitochondria and plasma membrane. Furthermore, fluorescence recovery after photobleaching (FRAP) assays identified two pools of the APC cancer mutant 1309 at centrosomes: a dominant dynamic pool and a smaller immobile pool. Preliminary mass spectrometry screening for proteins that bound the GST-ARM domain of APC identified a range of potential partners, and at present we are evaluating the role of the integral centrosomal protein, pericentrin. Discussion: APC displays an unexpectedly dynamic exchange at the centrosome in living cells. This could allow for rapid fluctuations in its activity in response to signalling cues. Experiments are underway to define specific functions of APC at the centrosome.