A role for the TGFbeta-Par6 polarity pathway in breast cancer progression.

The role of polarity signaling in cancer metastasis is ill defined. Using two three-dimensional culture models of mammary epithelial cells and an orthotopic mouse model of breast cancer, we reveal that Par6 signaling, which is regulated directly by TGFbeta, plays a role in breast cancer metastasis. Interference with Par6 signaling blocked TGFbeta-dependent loss of polarity in acini-like structures formed by non-transformed mammary cells grown in three-dimensional structures and suppressed the protrusive morphology of mesenchymal-like invasive mammary tumor cells without rescuing E-cadherin expression. Moreover, blockade of Par6 signaling in an in vivo orthotopic model of metastatic breast cancer induced the formation of ZO-1-positive epithelium-like structures in the primary tumor and suppressed metastasis to the lungs. Analysis of the pathway in tissue microarrays of human breast tumors further revealed that Par6 activation correlated with markers of the basal carcinoma subtype in BRCA1-associated tumors. These studies thus reveal a key role for polarity signaling and the control of morphologic transformation in breast cancer metastasis.

Arf1-GTP-induced tubule formation suggests a function of Arf family proteins in curvature acquisition at sites of vesicle budding.

ADP-ribosylation factor (Arf) and related small GTPases play crucial roles in membrane traffic within the exo- and endocytic pathways. Arf proteins in their GTP-bound state are associated with curved membrane buds and tubules, frequently together with effector coat proteins to which they bind. Here we report that Arf1 is found on membrane tubules originating from the Golgi complex where it colocalizes with COPI and GGA1 vesicle coat proteins. Arf1 also induces tubulation of liposomes in vitro. Mutations within the amino-terminal amphipathic helix (NTH) of Arf1 affect the number of Arf1-positive tubules in vivo and its property to tubulate liposomes. Moreover, hydrophilic substitutions within the hydrophobic part of its NTH impair Arf1-catalyzed budding of COPI vesicles in vitro. Our data indicate that GTP-controlled local induction of high curvature membranes is an important property of Arf1 that might be shared by a subgroup of Arf/Arl family GTPases.

Quantitative proteomics analysis of detergent-resistant membranes from chemical synapses: evidence for cholesterol as spatial organizer of synaptic vesicle cycling.

Synaptic vesicles (SVs) in the central nervous system upon stimulation undergo rapid calcium-triggered exoendocytic cycling within the nerve terminal that at least in part depends on components of the clathrin- and dynamin-dependent endocytosis machinery. How exocytic SV fusion and endocytic retrieval are temporally and spatially coordinated is still an open question. One possibility is that specialized membrane microdomains characterized by their high content in membrane cholesterol may assist in the spatial coordination of synaptic membrane protein recycling. Quantitative proteomics analysis of detergent-resistant membranes (DRMs) isolated from rat brain synapses or cholesterol-depleted control samples by liquid chromatography-tandem mass spectrometry identified a total of 159 proteins. Among these 122 proteins were classified as cholesterol-dependent DRM or DRM-associated proteins, many of which with proven or hypothesized functions in exoendocytic vesicle cycling including clathrin, the clathrin adaptor complex AP-2, and a variety of SV proteins. In agreement with this, SV membrane and endocytic proteins displayed a partial resistance to extraction with cold Triton X-100 in cultured rat hippocampal neurons where they co-localized with labeled cholera toxin B, a marker for cholesterol-enriched DRMs. Moreover SV proteins formed cholesterol-dependent complexes in CHAPS-extracted synaptic membrane lysates. Our combined data suggest that lipid microdomains may act as spatial coordinators for exoendocytic vesicle cycling at synapses.

Visualization of a functionally enhanced GFP-tagged galanin R2 receptor in PC12 cells: constitutive and ligand-induced internalization.

Trafficking of the galanin R2 receptor (GALR2) fused with enhanced GFP (EGFP) was studied by using confocal fluorescence microscopy. The fusion protein was predominantly localized on the plasma membrane with some intracellular fluorescent structures (vesicles), mainly in the perinuclear region. Incubation with galanin resulted in a concentration-dependent increase in intracellular Ca2+ concentration levels, suggesting that the GALR2-EGFP conjugate is functional. After blocking endocytosis with methyl-beta-cyclodextrin GALR2-EGFP expression was increased on the surface and decreased in the cytoplasm. Blocking endocytic recycling with monensin caused an increase of intracellular GALR2-EGFP accumulation and a decrease of fluorescence on the plasma membrane. GALR2-EGFP on the plasma membrane was internalized within 5-10 min after treatment with galanin or AR-M1896, a selective GALR2 agonist, with a dramatic reduction in plasma membrane localization and appearance in intracellular vesicles. Neither M35 nor M40, two galanin analogues with putative antagonistic action, prevented GALR2 agonist-induced internalization of GALR2-EGFP, suggesting that they are not antagonists at this receptor under the present circumstances. Galanin stimulation at low temperature caused GALR2-EGFP aggregation and clustering on the surface but no translocation to cytoplasm. After coincubation with galanin the GALR2-EGFP was colocalized with internalized Texas red-transferrin, a marker of the clathrin endocytic pathway. Hyperosmotic sucrose inhibited internalization of GALR2-EGFP. Taken together these findings indicate that GALR2 undergoes constitutive endocytosis and recycling and that both ligand-independent and ligand-dependent internalization use the clathrin-dependent endocytic recycling pathway.