APC Truncation Alters Cytoskeletal Structure and Microtubule Stability in Early Intestinal Tumorigenesis.
Abstracts
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Partial loss of function of APC protein by truncation of its COOH-terminus is an early factor in the development of many spontaneous colorectal cancers. In the C57BL-6J-Min/+ (Min/+) mouse, an animal with a germline mutation of Apc, we found that APC truncation is associated with reduced enterocyte migration and loss of association and membrane expression of adherens junction proteins. We hypothesized that these changes resulted from cytoskeletal dysfunction caused by loss of microtubule stabilization, an activity mediated by binding sites in the COOH-terminus of APC. In this study, we tested this hypothesis by determining whether APC truncation produced in vivo changes in: 1) actin cytoskeleton structure and 2) microtubule stability. The actin cytoskeleton of histologically normal enterocytes from mice with truncated APC (ApcMin/+) were compared to those of mice with wild-type APC (Apc+/+) by confocal microscopy of paraffin-embedded tissue stained with Alexa 488-conjugated phalloidin. We found a significant loss of actin localization at the plasma membrane in ApcMin/+ enterocytes (Fig 1). Microtubule instability is associated with high levels of tyrosinated tubulin isoforms (Tyr-tubulin) in polarized cells. Relative amounts of a-tubulin indicate overall tubulin turnover rate. We therefore determined the relative amounts of Tyr-tubulin and a-tubulin by immunoblotting of enterocytes isolated from ApcMin/+ and Apc+/+ mice. This experiment showed greatly increased levels of Tyr-tubulin and a-tubulin in enterocytes containing truncated APC (Fig 2). These studies suggest that loss of microtubule stability is a dominant negative effect of truncated APC, which may lead to the defects in enteroctye migration and junctional complex formation observed in early colorectal tumorigenesis. The first two authors contributed equally to this work. |
