The adenomatous polyposis coli protein unambiguously localizes to microtubule plus ends and is involved in establishing parallel arrays of microtubule bundles in highly polarized epithelial cells

Loss of full-length adenomatous polyposis coli (APC) protein correlates with the development of colon cancers in familial and sporadic cases. In addition to its role in regulating β-catenin levels in the Wnt signaling pathway, the APC protein is implicated in regulating cytoskeletal organization. APC stabilizes microtubules in vivo and in vitro, and this may play a role in cell migration (Näthke, I.S., C.L. Adams, P. Polakis, J.H. Sellin, and W.J. Nelson. 1996. J. Cell Biol. 134:165–179; Mimori-Kiyosue, Y., N. Shiina, and S. Tsukita. 2000. J. Cell Biol. 148:505–517; Zumbrunn, J., K. Inoshita, A.A. Hyman, and I.S. Näthke. 2001. Curr. Biol. 11:44–49) and in the attachment of microtubules to kinetochores during mitosis (Fodde, R., J. Kuipers, C. Rosenberg, R. Smits, M. Kielman, C. Gaspar, J.H. van Es, C. Breukel, J. Wiegant, R.H. Giles, and H. Clevers. 2001. Nat. Cell Biol. 3:433–438; Kaplan, K.B., A. Burds, J.R. Swedlow, S.S. Bekir, P.K. Sorger, and I.S. Näthke. 2001. Nat. Cell Biol. 3:429–432). The localization of endogenous APC protein is complex: actin- and microtubule-dependent pools of APC have been identified in cultured cells (Näthke et al., 1996; Mimori-Kiyosue et al., 2000; Reinacher-Schick, A., and B.M. Gumbiner. 2001. J. Cell Biol. 152:491–502; Rosin-Arbesfeld, R., G. Ihrke, and M. Bienz. 2001. EMBO J. 20:5929–5939). However, the localization of APC in tissues has not been identified at high resolution. Here, we show that in fully polarized epithelial cells from the inner ear, endogenous APC protein associates with the plus ends of microtubules located at the basal plasma membrane. Consistent with a role for APC in supporting the cytoskeletal organization of epithelial cells in vivo, the number of microtubules is significantly reduced in apico-basal arrays of microtubule bundles isolated from mice heterozygous for APC

Loss of APC induces polyploidy as a result of a combination of defects in mitosis and apoptosis

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene initiate a majority of colorectal cancers. Acquisition of chromosomal instability is an early event in these tumors. We provide evidence that the loss of APC leads to a partial loss of interkinetochore tension at metaphase and alters mitotic progression. Furthermore, we show that inhibition of APC in U2OS cells compromises the mitotic spindle checkpoint. This is accompanied by a decrease in the association of the checkpoint proteins Bub1 and BubR1 with kinetochores. Additionally, APC depletion reduced apoptosis. As expected from this combination of defects, tetraploidy and polyploidy are consequences of APC inhibition in vitro and in vivo. The removal of APC produced the same defects in HCT116 cells that have constitutively active β-catenin. These data show that the loss of APC immediately induces chromosomal instability as a result of a combination of mitotic and apoptotic defects. We suggest that these defects amplify each other to increase the incidence of tetra- and polyploidy in early stages of tumorigenesis

Ultrasound facilitated marking of gastrointestinal tissue with fluorescent material

The epithelial lining of the gastrointestinal (GI) mucosal layer is an effective barrier to the contents of the gut lumen. Selective channels and tight junctions prevent contamination of the sterile internal environment of the body. Conversely, the gut barrier also prevents desired agents from entering the GI tissue. This hinders marking of tissue for further clinical follow-up. Focused ultrasound (US) may provide a potential means of overcoming the gut barrier and allowing penetration of material beyond it which was explored in a series of tests. Experiments were carried out on 14 individual postmortem-obtained murine small bowel samples for a total of 23 sonication/control paired tests. A favourable result of 80% indicated that focused US can pass a nanoscale fluorescent agent through the gut barrier. Further work is required to elucidate where the agent resides, intercellular or intracellular, post-sonication

Manipulating the Barrier Function of a Cell Monolayer Using a High-power Miniature Ultrasonic Transducer

Ultrasound (US) and cavitation agents such as microbubbles (MBs) have been demonstrated to decrease the barrier function of endothelial and epithelial layers. However, in vitro experiments that study this effect are often hindered by the inability to deliver buoyant contrast agents in proximity to cell monolayers in order to adequately control the decrease in barrier function whilst insonating a sufficiently large tissue area. We have addressed this by adapting a cell culture system and fabricating a bespoke high-power miniature unfocused US transducer. The setup was used to control the drop in barrier function and to determine how varying the mechanical index (MI) and the duty cycle affected the barrier function. It was found that buffer solution alone and buffer + MBs did not decrease the transepithelial electrical resistance (TEER) of the cell monolayer. Buffer + US decreased the TEER by ~40%, with 10% TEER recovery 9 min after switching US off. Buffer + MBs + US decreased the TEER by 80%, with little or no recovery following treatment. In the presence of MBs, the barrier function was decreased by a duty cycle = [1% - 50%] and by an MI = [0.25 - 0.5], without any recovery following treatment. Detectable amounts of fluorescent dextran were delivered across the Caco-2 monolayer only by a combination of US + MBs. These results suggest that our adapted setup and custom-built miniature transducer permits control of the decrease in barrier function for further therapeutic investigations

Identification, Characterization, and Localization of a Novel Kidney Polycystin-1-Polycystin-2 Complex

The functions of the two proteins defective in autosomal dominant polycystic kidney disease, polycystin-1 and polycystin-2, have not been fully clarified, but it has been hypothesized that they may heterodimerize to form a "polycystin complex" involved in cell adhesion. In this paper, we demonstrate for the first time the existence of a native polycystin complex in mouse kidney tubular cells transgenic for PKD1, non-transgenic kidney cells, and normal adult human kidney. Polycystin-1 is heavily N-glycosylated, and several glycosylated forms of polycystin-1 differing in their sensitivity to endoglycosidase H (Endo H) were found; in contrast, native polycystin-2 was fully Endo H-sensitive. Using highly specific antibodies to both proteins, we show that polycystin-2 associates selectively with two species of full-length polycystin-1, one Endo H-sensitive and the other Endo H-resistant; importantly, the latter could be further enriched in plasma membrane fractions and co-immunoprecipitated with polycystin-2. Finally, a subpopulation of this complex co-localized to the lateral cell borders of PKD1 transgenic kidney cells. These results demonstrate that polycystin-1 and polycystin-2 interact in vivo to form a stable heterodimeric complex and suggest that disruption of this complex is likely to be of primary relevance to the pathogenesis of cyst formation in autosomal dominant polycystic kidney disease

Deletion of a Single beta-Catenin Allele in Osteocytes Abolishes the Bone Anabolic Response to Loading

The Wnt/β-catenin signaling pathway is essential for bone cell viability and function and for skeletal integrity. To determine if β-catenin in osteocytes plays a role in the bone anabolic response to mechanical loading, 18- to 24-week-old osteocyte β-catenin haploinsufficient mice (Dmp1-Cre × β-catenin fl/ + ; HET cKO) were compared with their β-catenin fl/fl (control) littermates. Trabecular bone volume (BV/TV) was significantly less (58.3%) in HET cKO females versus controls, whereas male HET cKO and control mice were not significantly different. Trabecular number was significantly less in HET cKO mice compared with controls for both genders, and trabecular separation was greater in female HET cKO mice. Osteoclast surface was significantly greater in female HET cKO mice. Cortical bone parameters in males and females showed subtle or no differences between HET cKO and controls. The right ulnas were loaded in vivo at 100 cycles, 2 Hz, 2500 µϵ, 3 days per week for 3 weeks, and the left ulnas served as nonloaded controls. Calcein and alizarin complexone dihydrate were injected 10 days and 3 days before euthanization, respectively. Micro-computed tomography (µCT) analysis detected an 8.7% and 7.1% increase in cortical thickness in the loaded right ulnas of male and female control mice, respectively, compared with their nonloaded left ulnas. No significant increase in new cortical bone formation was observed in the HET cKO mice. Histomorphometric analysis of control mice showed a significant increase in endocortical and periosteal mineral apposition rate (MAR), bone-formation rate/bone surface (BFR/BS), BFR/BV, and BFR/TV in response to loading, but no significant increases were detected in the loaded HET cKO mice. These data show that deleting a single copy of β-catenin in osteocytes abolishes the anabolic response to loading, that trabecular bone in females is more severely affected and suggest that a critical threshold of β-catenin is required for bone formation in response to mechanical loading

Nanotechnology in multimodal theranostic capsule endoscopy

Video capsule endoscopy (VCE) has become a clinically accepted diagnostic modality in the last 20 years and has established a technological roadmap for other capsule endoscopy (CE) devices, incorporating microscale technology, a local power supply and wireless communication. However, VCE does not provide a therapeutic function and research in therapeutic capsule endoscopy (TCE) has been limited. This paper proposes a new route towards viable TCE based on multiple CE devices including essential nanoscale components. A first device is used for multimodal diagnosis, with quantitative microultrasound as a complement to video imaging. Ultrasound-enhanced fluorescent marking of sites of pathology allows follow-up with a second device for therapy. This is based on fluorescence imaging and ultrasound-mediated targeted drug delivery. Subsequent treatment verification and monitoring with a third device exploits the minimally invasive nature of CE. Clinical implementation of a complete patient pathway remains the subject of research but several key components have been prepared in early prototype form. These are described, along with gaps that remain to be filled