Minimal mutation of the cytoplasmic tail inhibits the ability of E-cadherin to activate Rac but not phosphatidylinositol 3-kinase - Direct evidence of a role for cadherin-activated Rac signaling in adhesion and contact formation

Classic cadherins are adhesion-activated cell signaling receptors. In particular, homophilic cadherin ligation can directly activate Rho family GTPases and phosphatidylinositol 3-kinase (PI3-kinase), signaling molecules with the capacity to support the morphogenetic effects of these adhesion molecules during development and disease. However, the molecular basis for cadherin signaling has not been elucidated, nor is its precise contribution to cadherin function yet understood. One attractive hypothesis is that cadherin-activated signaling participates in stabilizing adhesive contacts ( Yap, A. S., and Kovacs, E. M. ( 2003) J. Cell Biol. 160, 11-16). We now report that minimal mutation of the cadherin cytoplasmic tail to uncouple binding of p120-ctn ablated the ability of E-cadherin to activate Rac. This was accompanied by profound defects in the capacity of cells to establish stable adhesive contacts, defects that were rescued by sustained Rac signaling. These data provide direct evidence for a role of cadherin-activated Rac signaling in contact formation and adhesive stabilization. In contrast, cadherin-activated PI3-kinase signaling was not affected by loss of p120-ctn binding. The molecular requirements for E-cadherin to activate Rac signaling thus appear distinct from those that stimulate PI3-kinase, and we postulate that p120-ctn may play a central role in the E-cadherin-Rac signaling pathway.

Direct continuities between cisternae at different levels of the Golgi complex in glucose-stimulated mouse islet beta cells

Direct continuity between the membranes of cisternae in the Golgi complex in mammalian cells rarely has been observed; when seen, its documentation has been equivocal. Here we have used dual-axis electron microscope tomography to examine the architecture of the Golgi in three dimensions at approximate to6-nm resolution in rapidly frozen, freeze-substituted murine cells that make and secrete insulin in response to glucose challenge. Our data show three types of direct connections between Golgi cisternae that are normally distinct from one another. These connections all bypass interceding cisternae. We propose that when pancreatic beta cells are stimulated to synthesize and secrete insulin rapidly in vivo, such connections provide a continuous lumen that facilitates the rapid transit of large amounts of newly made protein for secretion. The heterotypic fusion of cisternae, even transiently, raises important questions about the molecular mechanisms that (i) facilitate the fusion/fission of cisternal membranes and control the directionality and specificity of such events, and (it) retain Golgi processing enzymes at specific places within individual cisternae when two cisternae at different levels in the Golgi have fused, maintaining the sequential processing hierarchy that is a hallmark of Golgi organization.

Cadherin/catenin complex appears to be intact in hepatocellular carcinomas from Australia and South Africa

Background and aim: E-cadherin binds to beta-catenin to form the cadherin/catenin complex required for strong cell adhesion. Inactivation of this complex in tumors facilitates invasion into surrounding tissues. Alterations of both proteins have been reported in hepatocellular carcinomas (HCC). However, the interactions between E-cadherin and beta-catenin in HCC from different geographical groups have not been explored. The aim of the present study was to assess the role of E-cadherin and beta-catenin in Australian and South African patients with HCC. Methods: DNA was extracted from malignant and non-malignant liver tissue from 37 Australian and 24 South African patients, and from histologically normal liver from 20 transplant donors. Chromosomal instability at 16q22, promoter methylation at E-cadherin, beta-catenin mutations and E-cadherin and beta-catenin protein expression was assessed using loss of heterozygosity, methylation-specific polymerase chain reaction, denaturing high-performance liquid chromatography and immunohistochemistry, respectively. Results: Loss of heterozygosity at 16q22 was prevalent in South African HCC patients (50%vs 11%; P < 0.05, chi(2)). In contrast, E-cadherin promoter hypermethylation was common in Australian cases in both malignant (30%vs 13%; P = not significant, chi(2)) and non-malignant liver (57%vs 8%, respectively, P < 0.001, chi(2)). Methylation of non-malignant liver was more likely to be detected in patients over the age of 50 years (P < 0.001, chi(2)), the overall mean age for our cohort of patients. Only one beta-catenin mutation was identified. E-cadherin protein expression was reduced in one HCC, while abnormalities in protein expression were absent in beta-catenin. Conclusion: Contrary to previous observations in HCC from other countries, neither E-cadherin nor beta-catenin appears to play a role in hepatocarcinogenesis in Australian and South African patients with HCC. (C) 2004 Blackwell Publishing Asia Pty Ltd.

Inhibition of protein interactions with the beta(2) sliding clamp of Escherichia coli DNA polymerase III by peptides from beta(2)-binding proteins

The sliding clamp of the Escherichia coli replisome is now understood to interact with many proteins involved in DNA synthesis and repair. A universal interaction motif is proposed to be one mechanism by which those proteins bind the E. coli sliding clamp, a homodimer of the beta subunit, at a single site on the dimer. The numerous beta(2)-binding proteins have various versions of the consensus interaction motif, including a related hexameric sequence. To determine if the variants of the motif could contribute to the competition of the beta-binding proteins for the beta(2) site, synthetic peptides derived from the putative beta(2)-binding motifs were assessed for their abilities to inhibit protein-beta(2) interactions, to bind directly to beta(2), and to inhibit DNA synthesis in vitro. A hierarchy emerged, which was consistent with sequence similarity to the pentameric consensus motif, QL(S/D)LF, and peptides containing proposed hexameric motifs were shown to have activities comparable to those containing the consensus sequence. The hierarchy of peptide binding may be indicative of a competitive hierarchy for the binding of proteins to beta(2) in various stages or circumstances of DNA replication and repair.

Targeting of a tropomyosin isoform to short microfilaments associated with the Golgi complex

A growing body of evidence suggests that the Golgi complex contains an actin-based filament system. We have previously reported that one or more isoforms from the tropomyosin gene Tm5NM (also known as gamma-Tm), but not from either the alpha- or beta-Tm genes, are associated with Golgi-derived vesicles (Heimann et al., (1999). J. Biol. Chem. 274, 10743-10750). We now show that Tm5NM-2 is sorted specifically to the Golgi complex, whereas Tm5NM-1, which differs by a single alternatively spliced internal exon, is incorporated into stress fibers. Tm5NM-2 is localized to the Golgi complex consistently throughout the G1 phase of the cell cycle and it associates with Golgi membranes in a brefeldin A-sensitive and cytochalasin D-resistant manner. An actin antibody, which preferentially reacts with the ends of microfilaments, newly reveals a population of short actin filaments associated with the Golgi complex and particularly with Golgi-derived vesicles. Tm5NM-2 is also found on these short microfilaments. We conclude that an alternative splice choice can restrict the sorting of a tropomyosin isoform to short actin filaments associated with Golgi-derived vesicles. Our evidence points to a role for these Golgi-associated microfilaments in vesicle budding at the level of the Golgi complex.

Predicting function from structure: 3D structure studies of the mammalian golgi complex

3D electron tomography studies of the structure of the mammalian Golgi complex have led to four functional predictions (1). The sorting and exit site from the Golgi comprises two or three distinct trans-cisternae (2). The docking of vesicular-tubular clusters at the cis-face and the fragmentation of trans-cisternae are coordinated (3). The mechanisms of transport through, and exit from, the Golgi vary with physiological state, and in different cells and tissues (4). Specialized trans-ER functions in the delivery of ceramide to sphingomyelin synthase in the trans-Golgi membrane, for the regulated sorting via sphingolipid-cholesterol-rich domains. These structure-based predictions can now be tested using a variety of powerful cell and molecular tools.

The effect of complexation on characteristics and drug release from PLGA microspheres loaded by cyclosporine-cyclodextrin complex

The purpose of this study was to evaluate the effect of cyclosporine (CyA)-cyclodextrin (CD) complex incorporated within PLGA inicrospheres on microsphere characteristics, with particular emphasis on drug release kinetics. For this purpose, microspheres encapsulated with CyA and those loaded by CyA-CD complex were prepared by solvent evaporation and multiple emulsification solvent evaporation methods, respectively. Morphology, size, encapsulation efficiency and drug release pattern from microspheres were evaluated. Also, physicochemical properties of drug inside microspheres were characterized by differential scanning calorimetry (DSC) and infrared spectroscopy (IR) studies. Scanning electron microscopy (SEM) studies showed that microspheres encapsulated with CyA had islands on the microsphere surface but the islands were not seen on the surface of microspheres loaded by complex. Size range varied from 1 to 25 mu m for CyA encapsulated microspheres and 1 to 50 mu m for complex loaded microspheres. The release of CyA was biphasic with an initial more rapid release phase followed by a slower phase but drug release was twice as fast for complex loaded microspheres. IR studies did not indicate any chemical interaction between the components of microspheres and DSC thermograms revealed that CyA was present either in its amorphous state in microspheres or the presence of CyA as an inclusion complex within microspheres loaded by complex. In conclusion, using CyA as an inclusion complex with CD within microspheres can affect microsphere characteristics and drug release and it is possible to modify microsphere properties like drug release by incorporating CDs as complexing agents.

Inhibition of transforming growth factor-beta 1 signaling attenuates ataxia telanglectasia mutated activity in response to genotoxic stress

Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor beta (TGF beta)-1, which is activated by radiation, is a potent and pleiotropic mediator of physiologic and pathologic processes. Here we show that TGF beta inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgf beta 1 nail murine epithelial cells or human epithelial cells treated with a small-molecule inhibitor of TGF beta type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17, and p53; reduced gamma H2AX radiation-induced foci; and increased radiosensitivity compared with TGF beta competent cells. We determined that loss of TGF beta signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGF beta restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM, which directs epithelial cell stress responses, cell fate, and tissue integrity. Thus, Tgf beta 1, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGF beta may be used to advantage in cancer therapy.