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Rab25 and CLIC3 Collaborate to Promote Integrin Recycling from Late Endosomes/Lysosomes and Drive Cancer Progression

Developmental Cell (2012) 22(1) 131-145
DOI: 10.1016/j.devcel.2011.11.008
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Abstract

Here we show that Rab25 permits the sorting of ligand-occupied, active-conformation α5β1 integrin to late endosomes/lysosomes. Photoactivation and biochemical approaches show that lysosomally targeted integrins are not degraded but are retrogradely transported and recycled to the plasma membrane at the back of invading cells. This requires CLIC3, a protein upregulated in Rab25-expressing cells and tumors, which colocalizes with active α5β1 in late endosomes/lysosomes. CLIC3 is necessary for release of the cell rear during migration on 3D matrices and is required for invasion and maintenance of active Src signaling in organotypic microenvironments. CLIC3 expression predicts lymph node metastasis and poor prognosis in operable cases of pancreatic ductal adenocarcinoma (PDAC). The identification of CLIC3 as a regulator of a recycling pathway and as an independent prognostic indicator in PDAC highlights the importance of active integrin trafficking as a potential drive to cancer progression invivo. © 2012 Elsevier Inc.

Figures

  • Figure 1. CLIC3 Is Upregulated in Rab25-Expressing A2780 Cells
  • Figure 2. Rab25 Is Required for Sorting of Active a5b1 to CLIC3-Positive Late Endosomes/Lysosomes (A–D) A2780-Rab25 cells were transfected with the indicated fluorescently tagged proteins and incubated with Alexa647 fibronectin (2.5 mg/ml) or Alexa488 transferrin (10 mg/ml) as indicated. The distribution of the fluorescent proteins was determined by live confocal microscopy. In (A)–(C) single images were acquired, in (D) confocal sections were captured at 2 s intervals over a period of 2 min, and six time points from this sequence (0, 4, 8, 12, 16, and 20 s) are shown. Scale bars, 10mm. The number and duration of contact events between CLIC3-positive structures and Rab25 (black filled circles) or Rab11a (red filled circles) vesicles were quantified and are presented as a scatterplot and a histogram. The stills in (D) are extracted from Movie S1. See also Figure S2. (E) A2780-Rab25 or A2780-DNA3 cells were transfected with Cherry-CLIC3 (red) and either GFP-a5 or GFP-CA-a5 and incubated with or without fibronectin (FN, 2.5 mg/ml). ImageJ was used to quantify colocalization between CLIC3 and a5, as indicated in the lower panel. Colocalization is expressed as a percentage of yellow pixels versus red pixels. Values are mean ± SEM from at least four experiments (n > 30 cells). Scale bars, 10 mm.
  • Figure 3. CLIC3 Is Required for Recycling of Activated a5b1 Integrin (A) A2780-Rab25 cells were either left untransfected or were transfected with nontargeting siRNA (siRNA-NT) or with siRNA targeting CLIC3 (siRNA CLIC3#4). They were then surface labeled with 0.2 mg/ml NHS-S-S-Biotin at 4 C, and internalization was then allowed to proceed for 30 min at 37 C in the presence or absence of 3 mg/ml FN, bafilomycin (100 nM), or vehicle control (DMSO). Biotin remaining at the cell surface was removed by exposure to MesNa at 4 C, and the cells were incubated for the indicated times at 37 C. The quantity of biotinylated receptors remaining within the cells was determined by capture ELISA using microtiter wells coated with monoclonal antibodies recognizing a5 or active b1 (9EG7). Values are mean ± SEM (n = 3 independent experiments). (B) A2780-Rab25 cells were transfected with nontargeting (siRNA-NT) or siRNA oligonucleotide targeting CLIC3 (siRNA-CLIC3#4), in conjunction with GFP-a5. Recycling assays were performed in the presence of fibronectin (3.0 mg/ml) as for (A). Biotinylated integrins were detected by capture ELISA using antibodies recognizing GFP or active b1 (9EG7). Values are mean ± SEM (n = 3 independent experiments).
  • Figure 4. Conformationally Active Integrins Are Trafficked Retrogradely in Cells Migrating on CDMs A2780-Rab25 cells were transfected with Cherry-Rab25 in conjunction with photoactivatable a5 integrin (A; paGFP-wt-a5) or a constitutively active version of the probe (C and D; paGFP-CA-a5). Cells were plated onto CDMs 6 hr prior to imaging and either left untreated (A and C) or treated with bafilomycin (D). Integrin recycling from Rab25 vesicles toward the tips of extending pseudopods was visualized using photoactivation, which was performed with a 405 nm laser aimed at Rab25-positive vesicles, as denoted by the white arrows. Images were captured with a confocal microscope over a period of 140 s. Movies were generated from these time-lapse images, and stills presented correspond to frames prior to photoactivation, immediately after photoactivation, and subsequently at intervals as indicated. Scale bars, 10 mm. Yellow arrow indicates the direction of migration. The integrated fluorescence intensity of the photoactivated region and plasma membrane regions at the cell front and cell rear (yellow dotted lines) was calculated for each frame of the movie. Relative fluorescence intensity (vesicle) or percent increase in fluorescence intensity (region) was plotted against time (B and E). Values are mean ± SEM from more than three individual experiments (n = 6 for paGFP-wt-a5, n = 19 for paGFP-CA-a5 +DMSO, n = 11 for paGFP-CA-a5 +BAF). See also Movies S4, S5, and S6.
  • Figure 5. Knockdown of CLIC3 Causes Migrating Cells to Pause and Suppresses Invasion into Fibronectin-Supplemented Matrigel (A–D) A2780-Rab25 cells were transfected with nontargeting siRNA (si-NT) or with an siRNA targeting CLIC3 (oligo#4) (si-CLIC3), in combination with a5 integrin (a5b1), constitutively active a5 integrin (CAa5b1), and an siRNA-resistant CLIC3 rescue vector (rescue) as indicated. Transfected cells were plated onto CDM in the presence or absence of soluble fibronectin (2.5 mg/ml), with or without bafilomycin (10 nM), and allowed to adhere for 24 hr prior to time-lapse microscopy. Images were captured every 5min over an 8 hr period, and movies were generated from these. Excerpts from thesemovies are displayed in (B) (Movie S7). Scale bar in (B) is 100 mm. The position of the cell nucleus was followed using cell-tracking software, and the averagemigration speed (A) and the distance between the center of the nucleus and the cell front (with respect to the direction of migration) (B; pseudopod length) were measured. We used in-house software to identify periods during which cells moved less than 3 mm in 90 min and defined this as a ‘‘pause’’ in cell migration. The number and duration of pausing events are plotted in (C). The speed that cells move when they are not pausing is termed the ‘‘momentary velocity,’’ and these values are also plotted in (C). Values are mean ± SEM (n > 90 track plots); ****p < 0.001, **p < 0.005, Mann-Whitney U test. Representative migration tracks are displayed and aligned (with cell movement running from
  • Figure 6. CLIC3 Is Required to Maintain Src Signaling and for Growth and Survival of Cells in Organotypic Matrices
  • Figure 7. CLIC3 Expression Predicts Poor Patient Survival

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APA

Dozynkiewicz, M. A., Jamieson, N. B., MacPherson, I., Grindlay, J., vandenBerghe, P. V. E., vonThun, A., … Norman, J. C. (2012). Rab25 and CLIC3 Collaborate to Promote Integrin Recycling from Late Endosomes/Lysosomes and Drive Cancer Progression. Developmental Cell, 22(1), 131–145. https://doi.org/10.1016/j.devcel.2011.11.008

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