One of the most exciting areas of cell and developmental biology today is the study of how signal transduction pathways influence or regulate cytoskeletal dynamics and likewise, how the cytoskeleton is used in the process of signal transduction. One excellent entry point into this area is the Adenomatous polyposis coli (APC) family of tumor suppressors. Loss of function mutations in one of the two human APC genes result in improper activation of the Wnt signaling pathway, and are one of the first steps in the development of colon cancer, as well as other tumors. APC is now well known as a component of the destruction complex that regulates the stability of the Wingless (Wg)/Wnt effector protein Armadillo/β-catenin (Arm). However, our studies and those from other labs indicate that APC is a multifunctional protein that may also regulate the actin and microtubule cytoskeletons. In addition to increasing our knowledge of the inner workings of the cell, studies of APC proteins will have significant implications for understanding certain cancers, and may ultimately have clinical benefits.

We are using Drosophila as a model to study the role of APC proteins in cytoskeletal organization. Drosophila is a particularly attractive model for several reasons. First, much is known about Wg signaling and cytoskeletal regulation in Drosophila. Second, the Drosophila embryo provides superb technical advantages for in vivo cell biological analyses as demonstrated in Figure 1. Finally, the molecular genetic tools available in this system are unparalleled, while the similarities to mammals at the cellular level allow rapid application to the human system.

APC2 and Armadillo mediate cytoskeletal interactions in the syncytial embryo

Expanding beyond its roles in canonical Wg signaling, we sought to decipher the relationship between Drosophila APC2 and the cytoskeleton. We therefore investigated the roles for APC2 and its known binding partners during syncytial development where interactions between actin and microtubules play an important role in spindle positioning. During syncytial development, the first ten nuclear cycles occur in the common central cytoplasm of the embryo. Nuclei then migrate to the periphery where they divide parallel to the cortex separated by pseudocleavage furrows, membrane invaginations lined with cortical actin. In the attached movie, a live syncytial stage embryo expressing moesin-GFP (labeling F-actin) undergoes three rounds of cortical nuclear division. During the cell division cycle, F-actin distribution cycles between interphase caps of microvilli over each nucleus, and pseudocleavage furrows that separate the invisible nuclei. Inhibitor studies suggest that spindles are anchored to the cortex via cortical actin; if this link is broken, spindles detach, lose their orientation parallel to the cortex, and are removed into the embryo interior. Our phenotypic analysis revealed that in either APC2, arm orzw3 mutants, a subset of spindles detach and are removed from the cortex (for an example of this phenotype see Figure 2) without apparent defects in actin or microtubule organization, suggesting a role for APC2 and Arm in linking the spindle to cortical actin. Consistent with this, APC2, Arm, and its actin-associated binding partner α-catenin all localize to sites of prospective cortical spindle attachment, and APC2 is dependent on Arm for its proper localization to the cortex. Furthermore, APC2/Arm complexes often co-localize with interphase microtubules. Zeste-white3 kinase (Zw3, fly GSK-3β), which can phosphorylate both Arm and APC, is also critical for spindle positioning (Figure 2), and regulates the localization of APC2/Arm complexes by changing their affinity for either actin-associated proteins like α-catenin, or for microtubules. Together, these data suggest that APC2, Arm and α-catenin play a novel role in the link between spindles and cortical actin, and that this link is regulated by Zw3 kinase.

Using these studies as a starting point, we are pursuing experiments designed to address the mechanisms by which APC2 complexes interact with and perhaps regulate the cytoskeleton in Drosophila. Ultimately, we will apply our findings regarding APC2 complexes to the study of mammalian systems and disease states.

Akong K, McCartney BM, Peifer M. Drosophila APC2 and APC1 have overlapping roles in the larval brain despite their distinct intracellular localizations. Developmental Biology 2002; 250(1): 71-90.

Akong K, Grevengoed E, Price M, McCartney BM, Hayden M, DeNofrio J, Peifer M. Drosophila APC2 and APC1 play overlapping roles in Wingless signaling in the embryo and imaginal discs. Developmental Biology 2002; 250(1): 91-100

McCartney B., McEwen, Grevengoed E, Maddox P, Bejsovec A, Peifer M. Drosophila APC2 and Armadillo participate in tethering mitotic spindles to cortical actin. Nature Cell Biology 2001; 3(10): 933-938.

McCartney BM, Peifer M. Teaching tumour suppressors new tricks. Nature Cell Biology 2000; 2(4): E58-E60.

McCartney BM, Dierick HA, Kirkpatrick C, Moline M, Baas A, Peifer M, Bejsovec A. Drosophila APC2 is a cytoskeletally-associated protein that regulates Wingless signaling in the embryonic epidermis. Journal of Cell Biology 1999; 146(6): 1303-1318.