CAP350 Is Required for Apico-basal Polarisation.

<p>(<b>A</b>) Confocal Z-stack images of polarised MDCKII cells showing the distribution of α-catenin and CAP350. Sequential sections of either control (shm4, top) or CAP350-knockdown (shCAP, bottom) cells from basal to apical pole are shown. Bar = 10 μm. (<b>B</b>) Representative maximum projections of Z-stack images from either control (left) or CAP350-knockdown (right) cells stained for α-catenin and α-tubulin. Single labelling for α-tubulin and merged images are shown. (<b>C</b>) Mosaic images of polarised control (left) or CAP350-knockdown (right) MDCKII cells double labelled for ZO-1 and CAP350. Bar = 100 μm. (<b>D</b>) Quantification of the apical surface (μm²) enclosed by the ZO-1 signal in control (green) and CAP350-knockdown confluent cells (dark red). Bars represent mean values ± SD of three independent experiments (n > 20,000 for each case; **** <i>p</i> < 0.0001, two-tailed unpaired Student's <i>t</i> test). (<b>E</b>) Frequency plot showing the distribution of data represented in (D). (<b>F</b>) Quantification of the area covered by either control or CAP350-knockdown isolated cells. Data were collected from two duplicate experiments each conducted in triplicate and are represented as means ± SD. (<b>G</b>) XZ sections of control (top) and CAP350-knockdown (bottom) cells triple labelled for CAP350, α-catenin, and α-tubulin. Bar = 5 μm. The data used to make this figure are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002087#pbio.1002087.s001" target="_blank">S1 Data</a>.</p

CAP350 targets α-catenin to MTs.

<p>(<b>A</b>) Diagram of CAP350 showing the additional truncated mutants used in this part of the study. All the truncated mutants were fused to a myc tag. (<b>B</b>) MDCKII cells expressing myc-CAP350 were double labelled for myc and α-tubulin. Merged image of a low-expressing cell is shown. At right, high-magnification image of the outlined area showing binding of myc-CAP350 along cytoplasmic MTs. (<b>C</b>) Merged image of a high-expressing myc-CAP350 cell double stained for myc and α-catenin. Magnification at right (taken from the boxed area) shows a cortical MT bundle. (<b>D</b>) WB of MDCKII cells expressing myc-CAP350 showing the distribution of myc-CAP350, CAP350, and α-catenin between NP-40-soluble (S) and insoluble (P) fractions. Alpha-catenin was used as a loading control. (<b>E</b>) MDCKII cells transfected with myc-CAP1 construct and labelled for myc and γ-tubulin. High-magnification image of a MT bundle is shown at right. (<b>F</b>) Images of myc-CAP1A (top) and myc-CAP1B (bottom) transfected cells double labelled for myc and α-tubulin. (<b>G</b>) MT-pelleting assay from control or CAP350-knockdown MDCKII cell lysates. After taxol-induced polimerisation and centrifugation through a sucrose cushion, supernatants (S) and pellets (P) were analysed by WB for CAP350, α-catenin, and α-tubulin. (<b>H</b>) Densitometric analysis of three representative WB. Bar graphs represent the ratio of α-catenin and α-tubulin in MT pellets from control or CAP350-knockdown cells. * <i>p</i> = 0.0342, two-tailed unpaired Student's <i>t</i> test. Bars = 10 μm. The data used to make this figure are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002087#pbio.1002087.s001" target="_blank">S1 Data</a>.</p

Defective cadherin-based cell-cell contact formation in the absence of junctional CAP350.

<p>(<b>A</b>) Time-lapse microscopy of MDCKII cells infected with either shm4 (left) or shCAP lentiviruses (right) and transfected with GFP-α-catenin. Cells were treated with 4 mM EGTA for 2 h to disrupt cell-cell contacts. EGTA was washed out and cells allowed recovery time in complete culture media. Time after EGTA removal is shown. Coloured arrows indicate representative examples of regions of unstable contact formation over time. (<b>B</b>) Control (top) or CAP350-knockdown (bottom) cells were treated with nocodazole for 2 h and incubated at 4°C for 30 min in the presence of the drug. At the indicated times after washout, cells were double labelled for α-catenin and α-tubulin. (<b>C</b>) Live-cell imaging of control and CAP350-knockdown subconfluent MDCKII cells, inducibly expressing Ruby-End binding protein 3 (EB3). Single images of GFP expression were taken at time 0 to confirm the effectiveness of shCAP viral transduction (upper panels). Maximal projections from the first four frames were overlaid to better visualise Ruby-EB3 tracks (lower panels). (<b>D</b>) Same as in (C), but cells were allowed to polarise for four days. Bars = 5 μm. (<b>E</b>) Quantification of the number of EB3 comets per cell under the conditions shown in (C) and (D). Bars represent mean values ± SD of three independent experiments (n > 10 for each case; **** <i>p</i> < 0.0001, one-way ANOVA followed by multiple comparisons test). The data used to make this figure are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002087#pbio.1002087.s001" target="_blank">S1 Data</a>.</p

CAP350 peripheral labelling is dependent on E-cadherin-mediated cell adhesion.

<p>(<b>A</b>) Control cells were treated with 4 mM EGTA for 2 h to disrupt cell-cell contacts, fixed at the indicated times after washout, and stained with anti-E-cadherin and CAP350r (top) or anti-α-catenin and CAP350r (bottom) antibodies. Single labellings are shown. (<b>B</b>) Low-magnification images of MDCKII cells treated with either a control immunoglobulin G (IgG) antibody (Ab) or the E-cadherin blocking Ab (DECMA-1) for 72 h. Treated cells were then fixed and stained for CAP350. The presence of DECMA-1 antibody bound to E-cadherin at the cell surface was visualised with a secondary antibody. (<b>C</b>) Representative images of cells treated as in (B) and double labelled for α-catenin and CAP350. E-cadherin in DECMA-1-treated cells was revealed by incubation with a secondary antibody. Merged images are shown at right. High-magnification images of boxed areas are shown in the panels underneath. (<b>D</b>, <b>E</b>) MDCKII cells processed as in (B) but stained with zonula occludens protein 1 (ZO-1) (<b>D</b>) or α-tubulin antibodies (<b>E</b>). Bars = 10 μm.</p

CAP350 is required for epithelial morphogenesis in medaka embryos.

<p>(<b>A</b>) Schematic representation of <i>cap350</i> exons 5–7 indicating the E5-I5 junction targeted by the morpholino Mcap350 (red arrowhead). Exon and intron sizes as well as intron retention and cryptic donor and acceptor sites are indicated by red dotted lines. (<b>B</b>) RT-PCRs show <i>cap350</i> aberrant splicing products in morpholino-injected embryos as compared with normal splicing (350 bp band) in wild-type and mock-injected embryos. PCR products derived from intron 5 retention (I5_Reten) and cryptic donors at exon 5 (E5_C.donor) and intron 5 (I5_C.donor) are indicated (red arrows). (<b>C–D</b>) Lateral views showing the progression of the epiboly front (arrow heads and dotted line) in Mock (<b>C</b>, <b>C'</b>) and Mcap350 (<b>D–D'</b>) injected embryos at stage 15. Fluorescent signal associated to the injected membrane-tagged tracer Lyn_tdTomato is shown in <b>C'</b> and <b>D'</b>. (<b>E</b>–<b>J</b>) Confocal microscopy analysis of α-tubulin (<b>E–H</b>) and α-catenin (<b>I</b>, <b>J</b>) whole mount immunostainings (green) in Mock and Mcap350 injected embryos at stage 15. Lyn_tdTomato (here in blue) injected embryos were also labelled with DAPI (here in red for simplicity). The nuclei of enveloping layer (EVLn) and deep cells (Dn) are indicated. Note the disorganized MT network (<b>F</b>, <b>H</b>) and the deficient recruitment of α-catenin to the cortex (<b>J</b>) in Mcap350-injected embryos. Bar = 100 μm (<b>C</b> and <b>D</b>) and 50 μm (<b>E–J</b>).</p

CAP350 is a cell-cell junction protein.

<p>(<b>A</b>) Diagram of CAP350 showing the different antigen recognition sites for all CAP350 antibodies used in this study. Continuous and dotted lines represent antibodies used for immunofluorescence (IF) and western blot (WB), respectively. (<b>B</b>) IF analysis of CAP350 localisation in either non-extracted (left) or Triton extracted (right) MDCKII cells using the anti-CAP350 goat antibody (CAP350g). Enlarged image of the outlined area is shown at right. Hereafter, white arrows indicate the CTR. (<b>C</b>) Merged image of control MDCKII cells double labelled for CAP350 and α-catenin. Note the co-localisation of both signals at cell-cell contacts and the absence of signals at the free edge (yellow arrows). Single labelling for CAP350 is also shown at right. (<b>D</b>) Control MDCKII cells processed by IF with either anti-CAP350 rabbit antibody (CAP350r, top panels) or anti-CAP350 mouse antibody (CAP350m, bottom) and α-catenin antibodies. At right, high-magnification images of the boxed areas showing single labellings and merged images. (<b>E</b>) Images of control MDCKII cells showing CAP350 co-localisation with E-cadherin and β-catenin, respectively. (<b>F</b>) Representative WB of MDCKII cells with CAP350m and CAP350r antibodies showing efficient depletion of CAP350 in cells infected with shCAP350 (shCAP) lentiviruses compared to those infected with control shm4 lentivirus. GMAP210 was used as a loading control. Alpha-catenin levels were also examined. (<b>G</b>) Same as in (D) but in shCAP lentivirus-infected cells. (<b>H</b>) Quantification of centrosomal CAP350 fluorescence intensity. Data represent mean ± standard deviation (SD) of three independent experiments. AU = arbitrary units. (<b>I</b>) Control (top) or CAP350-knockdown (bottom) MDCKII cells triple labelled for CAP350, FOP, and γ-tubulin. Single images are shown. (<b>J</b>) Same as in (I), but cells were stained for CAP350 and pericentrin (PCNT). (<b>K</b>) IF analysis showing the absence of peripheral CAP350 staining in human dermal fibroblasts (HDF) in which CAP350 is restricted to the centrosome (HDF, left). WB analysis confirming the absence of both E-cadherin and CAP350 signals in total extracts of HDF (right). Bars = 10 μm. The data used to make this figure are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002087#pbio.1002087.s001" target="_blank">S1 Data</a>.</p

CAP350 directly interacts with α-catenin.

<p>(<b>A</b>) Summary of α-catenin clones isolated from a human PAZ-6 cell line complementary DNA (cDNA) library in a yeast two-hybrid screening, using as baits CAP2 and CAP4 fragments of CAP350. Eighteen positive clones (seven independent) of α-catenin were obtained when the CAP2 fragment was used as bait (dark red), whereas three positive clones were isolated with the bait fragment CAP4 (yellow). The mapped positions of clones are shown in amino acids. VH1: vinculin homology domain 1. (<b>B</b>) Schematic diagram of CAP350 showing the truncated mutants used in these assays and their relative positions. Hereafter, numbers represent amino acid positions in the full-length protein (top). A293T cells were double transfected with GFP-α-catenin and either myc-CAP1A, myc-CAP2, or myc-CAP4 truncated mutants. After IP with an anti-myc antibody, blots were revealed for GFP and myc (bottom). (<b>C</b>) Schematic representation of human α-catenin and the truncated mutants used in this work (top). Cells co-expressing myc-CAP2/myc-CAP4 and either GFP-VH1cat or GFP-VH2cat fragments were immunoprecipitated with an anti-myc antibody. Immunoprecipitates were then tested for the presence of myc-CAP2/myc-CAP4 or GFP (bottom). (<b>D</b>) Co-IP from GFP-α-catenin expressing cells using an anti-CAP350 antibody. Blots were revealed for CAP350 and GFP (top). Extracts from nontransfected MDCKII cells were incubated with anti-CAP350 antibody, and immunoprecipitates were analysed by WB for CAP350 and α-catenin (bottom).</p

CAP350 knockdown leads to defective cystogenesis.

<p>(<b>A</b>) Sequential confocal z sections of a control MDCKII cyst in a 3-D culture from the top (left) to the middle (right) of the cyst. MDCKII cells were plated in matrigel for four days to allow cystogenesis, and cysts were fixed and double labelled for CAP350 and FOP. Single staining with CAP350g is shown. Magnifications at the right of the outlined area show inverted images of CAP350g and FOP labelling. (<b>B</b>) Images showing sequential cross sections of a representative MDCKII control cyst stained for GP135 (blue) as an apical marker, α-catenin (red) as a basolateral marker, and CAP350 (green). Enlarged views of the boxed areas are shown on the lower panels. Note increasing accumulation of CAP350 towards the basal pole of cells (yellow arrows). (<b>C</b>) Box-and-whisker plot showing quantification of diameter in control or CAP350-knockdown MDCKII cysts. Top and bottom end boxes represent 75th and 25th percentiles, and whiskers represent 90th and 10th percentiles. The black line within the box marks the median. **** <i>p</i> < 0.0001 (two-tailed unpaired Student's <i>t</i> test). Data were collected from three independent experiments. (<b>D</b>) Single confocal sections through the middle region of either control (shm4) or CAP350-knockdown (shCAP) cysts fixed at day 4 and labelled for FOP, CAP350, and vinculin. High magnifications of boxed areas are shown at the far-right panels as indicated. (<b>E</b>) Quantification of lumen formation in both control and CAP350-knockdown cysts. Data come from four independent experiments. (<b>F</b>) Single confocal sections through the middle region of either the control (top) or CAP350-knockdown (bottom) cysts labelled for β-catenin, α-catenin, GP135, and ZO-1 as indicated. Rhodamine-phalloidin was used to label filamentous actin (F-actin). DNA was counterstained with 4 ',6-diamino-2-fenilindol (DAPI). Bars = 10 μm. The data used to make this figure are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002087#pbio.1002087.s001" target="_blank">S1 Data</a>.</p

CAP350 depletion disrupts MT organisation during cystogenesis.

<p>(<b>A, B</b>) Sequential confocal sections of representative cysts formed from MDCKII cells infected with lentiviruses encoding either control shm4 (<b>A</b>) or shCAP (<b>B</b>) RNAs and stained for α-catenin and α-tubulin. Enlarged views of the boxed areas are shown at the bottom. (<b>C</b>) Single confocal section through the middle region of either control (left) or CAP350-knockdown (right) cysts fixed at day 4 and double labelled for acetylated-tubulin and ZO-1. Bars = 10 μm.</p

A New Mint1 Isoform, but Not the Conventional Mint1, Interacts with the Small GTPase Rab6

<div><p>Small GTPases of the Rab family are important regulators of a large variety of different cellular functions such as membrane organization and vesicle trafficking. They have been shown to play a role in several human diseases. One prominent member, Rab6, is thought to be involved in the development of Alzheimer’s Disease, the most prevalent mental disorder worldwide. Previous studies have shown that Rab6 impairs the processing of the amyloid precursor protein (APP), which is cleaved to β-amyloid in brains of patients suffering from Alzheimer’s Disease. Additionally, all three members of the Mint adaptor family are implied to participate in the amyloidogenic pathway. Here, we report the identification of a new Mint1 isoform in a yeast two-hybrid screening, Mint1 826, which lacks an eleven amino acid (aa) sequence in the conserved C-terminal region. Mint1 826, but not the conventional Mint1, interacts with Rab6 via the PTB domain. This interaction is nucleotide-dependent, Rab6-specific and influences the subcellular localization of Mint1 826. We were able to detect and sequence a corresponding proteolytic peptide derived from cellular Mint1 826 by mass spectrometry proving the absence of aa 495–505 and could show that the deletion does not influence the ability of this adaptor protein to interact with APP. Taking into account that APP interacts and co-localizes with Mint1 826 and is transported in Rab6 positive vesicles, our data suggest that Mint1 826 bridges APP to the small GTPase at distinct cellular sorting points, establishing Mint1 826 as an important player in regulation of APP trafficking and processing.</p></div