Brooke M. McCartney

Drosophila APC2 and Armadillo participate in tethering mitotic spindles to cortical actin

Proper positioning of mitotic spindles ensures equal allocation of chromosomes to daughter cells. This often involves interactions between spindle and astral microtubules and cortical actin. In yeast and Caenorhabditis elegans, some of the protein machinery that connects spindles and cortex has been identified but, in most animal cells, this process remains mysterious. Here, we report that the tumour suppresser homologue APC2 and its binding partner Armadillo both play roles in spindle anchoring during the syncytial mitoses of early Drosophila embryos. Armadillo, α-catenin and APC2 all localize to sites of cortical spindle attachment. APC2–Armadillo complexes often localize with interphase microtubules. Zeste-white 3 kinase, which can phosphorylate Armadillo and APC, is also crucial for spindle positioning and regulates the localization of APC2–Armadillo complexes. Together, these data suggest that APC2, Armadillo and α-catenin provide an important link between spindles and cortical actin, and that this link is regulated by Zeste-white 3 kinase.

Cell regulation by the Apc protein Apc as master regulator of epithelia.

The adenomatous polyposis coli (Apc) protein participates in many of the fundamental cellular processes that govern epithelial tissues: Apc is directly involved in regulating the availability of beta-catenin for transcriptional de-repression of Tcf/LEF transcription factors, it contributes to the stability of microtubules in interphase and mitosis, and has an impact on the dynamics of F-actin. Thus Apc contributes directly and/or indirectly to proliferation, differentiation, migration, and apoptosis. This particular multifunctionality can explain why disruption of Apc is especially detrimental for the epithelium of the gut, where Apc mutations are common in most cancers. We summarise recent data that shed light on the molecular mechanisms involved in the different functions of Apc.

Testing hypotheses for the functions of APC family proteins using null and truncation alleles in Drosophila

Adenomatous polyposis coli (APC) is mutated in colon cancers. During normal development, APC proteins are essential negative regulators of Wnt signaling and have cytoskeletal functions. Many functions have been proposed for APC proteins, but these have often rested on dominant-negative or partial loss-of-function approaches. Thus, despite intense interest in APC, significant questions remain about its full range of cellular functions and about how mutations in the gene affect these. We isolated six new alleles of Drosophila APC2. Two resemble the truncation alleles found in human tumors and one is a protein null. We generated ovaries and embryos null for both APC2 and APC1, and assessed the consequences of total loss of APC function, allowing us to test several previous hypotheses. Surprisingly, although complete loss of APC1 and APC2 resulted in strong activation of Wingless signaling, it did not substantially alter cell viability, cadherin-based adhesion, spindle morphology, orientation or selection of division plane, as predicted from previous studies. We also tested the hypothesis that truncated APC proteins found in tumors are dominant negative. Two mutant proteins have dominant effects on cytoskeletal regulation, affecting Wnt-independent nuclear retention in syncytial embryos. However, they do not have dominant-negative effects on Wnt signaling.

Cytoskeletal dynamics and cell signaling during planar polarity establishment in the Drosophila embryonic denticle

Many epithelial cells are polarized along the plane of the epithelium, a property termed planar cell polarity. The Drosophila wing and eye imaginal discs are the premier models of this process. Many proteins required for polarity establishment and its translation into cytoskeletal polarity were identified from studies of those tissues. More recently, several vertebrate tissues have been shown to exhibit planar cell polarity. Striking similarities and differences have been observed when different tissues exhibiting planar cell polarity are compared. Here we describe a new tissue exhibiting planar cell polarity – the denticles, hair-like projections of the Drosophila embryonic epidermis. We describe in real time the changes in the actin cytoskeleton that underlie denticle development, and compare this with the localization of microtubules, revealing new aspects of cytoskeletal dynamics that may have more general applicability. We present an initial characterization of the localization of several actin regulators during denticle development. We find that several core planar cell polarity proteins are asymmetrically localized during the process. Finally, we define roles for the canonical Wingless and Hedgehog pathways and for core planar cell polarity proteins in denticle polarity.

A novel role for an APC2-Diaphanous complex in regulating actin organization in Drosophila

The rearrangement of cytoskeletal elements is essential for many cellular processes. The tumor suppressor Adenomatous polyposis coli (APC) affects the function of microtubules and actin, but the mechanisms by which it does so are not well understood. Here we report that Drosophila syncytial embryos null for Apc2 display defects in the formation and extension of pseudocleavage furrows, which are cortical actin structures important for mitotic fidelity in early embryos. Furthermore, we show that the formin Diaphanous (DIA) functions with APC2 in this process. Colocalization of APC2 and DIA peaks during furrow extension, and localization of APC2 to furrows is DIA-dependent. Furthermore, APC2 binds DIA directly through a region of APC2 not previously shown to interact with DIA-related formins. Consistent with these results, reduction of dia enhances actin defects in Apc2 mutant embryos. Thus, an APC2-DIA complex appears crucial for actin furrow extension in the syncytial embryo. Interestingly, EB1, a microtubule +TIP and reported partner of vertebrate APC and DIA1, may not function with APC2 and DIA in furrow extension. Finally, whereas DIA-related formins are activated by Rho family GTPases, our data suggest that the APC2-DIA complex might be independent of RHOGEF2 and RHO1. Furthermore, although microtubules play a role in furrow extension, our analysis suggests that APC2 and DIA function in a novel complex that affects actin directly, rather than through an effect on microtubules.

Brain tumor specifies intermediate progenitor cell identity by attenuating β-catenin/Armadillo activity

During asymmetric stem cell division, both the daughter stem cell and the presumptive intermediate progenitor cell inherit cytoplasm from their parental stem cell. Thus, proper specification of intermediate progenitor cell identity requires an efficient mechanism to rapidly extinguish the activity of self-renewal factors, but the mechanisms remain unknown in most stem cell lineages. During asymmetric division of a type II neural stem cell (neuroblast) in the Drosophila larval brain, the Brain tumor (Brat) protein segregates unequally into the immature intermediate neural progenitor (INP), where it specifies INP identity by attenuating the function of the self-renewal factor Klumpfuss (Klu), but the mechanisms are not understood. Here, we report that Brat specifies INP identity through its N-terminal B-boxes via a novel mechanism that is independent of asymmetric protein segregation. Brat-mediated specification of INP identity is critically dependent on the function of the Wnt destruction complex, which attenuates the activity of β-catenin/Armadillo (Arm) in immature INPs. Aberrantly increasing Arm activity in immature INPs further exacerbates the defects in the specification of INP identity and enhances the supernumerary neuroblast mutant phenotype in brat mutant brains. By contrast, reducing Arm activity in immature INPs suppresses supernumerary neuroblast formation in brat mutant brains. Finally, reducing Arm activity also strongly suppresses supernumerary neuroblasts induced by overexpression of klu. Thus, the Brat-dependent mechanism extinguishes the function of the self-renewal factor Klu in the presumptive intermediate progenitor cell by attenuating Arm activity, balancing stem cell maintenance and progenitor cell specification.

Apical constriction and invagination downstream of the canonical Wnt signaling pathway require Rho1 and Myosin II

The tumor suppressor Adenomatous polyposis coli (APC) is a negative regulator of Wnt signaling and functions in cytoskeletal organization. Disruption of human APC in colonic epithelia initiates benign polyps that progress to carcinoma following additional mutations. The early events of polyposis are poorly understood, as is the role of canonical Wnt signaling in normal epithelial architecture and morphogenesis. To determine the consequences of complete loss of APC in a model epithelium, we generated APC2 APC1 double null clones in the Drosophila wing imaginal disc. APC loss leads to segregation, apical constriction, and invagination that result from transcriptional activation of canonical Wnt signaling. Further, we show that Wnt-dependent changes in cell fate can be decoupled from Wnt-dependent changes in cell shape. Wnt activation is reported to upregulate DE-cadherin in wing discs, and elevated DE-cadherin is thought to promote apical constriction. We find that apical constriction and invagination of APC null tissue are independent of DE-cadherin elevation, but are dependent on Myosin II activity. Further, we show that disruption of Rho1 suppresses apical constriction and invagination in APC null cells. Our data suggest a novel link between canonical Wnt signaling and epithelial structure that requires activation of the Rho1 pathway and Myosin II.

Self-association of the APC tumor suppressor is required for the assembly, stability, and activity of the Wnt signaling destruction complex

The colon cancer tumor suppressor adenomatous polyposis coli (APC) negatively regulates Wnt signaling in the β-catenin destruction complex by binding to β-catenin and facilitating its phosphorylation and degradation. APC self-association plays an integral role in the assembly, stability, and activity of the destruction complex.

Teaching tumour suppressors new tricks

Examination of the tumour suppressor adenomatous polyposis coli (APC) has shown that it may be multifunctional. Recent work has demonstrated dynamic interactions of APC with the microtubule cytoskeleton, supporting the idea that APC has an important function in cell migration.

Fluorescent DNA Nanotags Featuring Covalently Attached Intercalating Dyes: Synthesis, Antibody Conjugation and Intracellular Imaging

We have synthesized fluorescent DNA duplexes featuring multiple thiazole orange (TO) intercalating dyes covalently attached to the DNA via a triazole linkage. The intercalating dyes stabilize the duplex against thermal denaturation and show bright fluorescence in the green region of the spectrum. The emission color can be changed to orange or red by addition of energy-accepting Cy3 or Cy5 dyes attached covalently to the DNA duplex. The dye-modified DNA duplexes were then attached to a secondary antibody for intracellular fluorescence imaging of centrosomes in Drosophila embryos. Bright fluorescent foci were observed at the centrosomes in both the donor (TO) and acceptor (Cy5) channels, because the energy transfer efficiency is moderate. Monitoring the Cy5 emission channel significantly minimized the background signal because of the large shift in emission wavelength allowed by energy transfer.