Regulated delivery of molecular cargo to invasive tumour-derived microvesicles - PubMed (original) (raw)
Regulated delivery of molecular cargo to invasive tumour-derived microvesicles
James W Clancy et al. Nat Commun. 2015.
Abstract
Cells release multiple, distinct forms of extracellular vesicles including structures known as microvesicles, which are known to alter the extracellular environment. Despite growing understanding of microvesicle biogenesis, function and contents, mechanisms regulating cargo delivery and enrichment remain largely unknown. Here we demonstrate that in amoeboid-like invasive tumour cell lines, the v-SNARE, VAMP3, regulates delivery of microvesicle cargo such as the membrane-type 1 matrix metalloprotease (MT1-MMP) to shedding microvesicles. MT1-MMP delivery to nascent microvesicles depends on the association of VAMP3 with the tetraspanin CD9 and facilitates the maintenance of amoeboid cell invasion. VAMP3-shRNA expression depletes shed vesicles of MT1-MMP and decreases cell invasiveness when embedded in cross-linked collagen matrices. Finally, we describe functionally similar microvesicles isolated from bodily fluids of ovarian cancer patients. Together these studies demonstrate the importance of microvesicle cargo sorting in matrix degradation and disease progression.
Figures
Figure 1. VAMP3 and MT1-MMP co-traffic to sites of active membrane blebbing
(A) TMVs are shed from areas of high blebbing activity. Sequential frames captured during imaging of unlabeled cells plated on gelatin-coated dish. Movie frames acquired 1.5 seconds apart. Vesicles shed from regions of membrane blebbing (arrow). Scale bar = 20 μm. LOX cells alone or co-transfected with VAMP3-GFP and MT1-MMP-mCherry or VAMP7-GFP and MT1-MMP-mCherry, were plated on unlabeled gelatin. Cells were visualized using phase contrast or live-cell spinning disk confocal microscopy. (B) VAMP3-GTP and MT1-MMP-mCherry colocalize on intracellular vesicles (arrows) in amoeboid cells. Scale bar = 25 μm. (C) Small, highly mobile vesicles (arrow) containing MT1-MMP and VAMP3 travel outward from the vicinity of larger vesicular structures in an anterograde fashion. Individual frames captured from Supplementary Movie 3 were captured 1.5 seconds apart, total time 18 seconds. Scale bar = 25 μm. (D) VAMP3-MT1-MMP intracellular vesicles traffic into newly formed membrane blebs. This montage is composed of stills from Supplementary Movie 1 to highlight trafficking into membrane blebs. Scale bar = 25 μm. (E) LOX cells with flattened, mesenchymal, morphology exhibit less colocalization between VAMP3 and MT1-MMP. Sequential frames approximately 40 seconds apart. Scale bar = 25 μm. (F) A pool of MT1-MMP localizes to the center of VAMP7 rosettes. Sequential frames approximately 40 seconds apart. Scale bar = 25 μm. (G) LOX cells that have adopted an amoeboid morphology when plated on gelatin show a high level of colocalization between VAMP3 and MT1-MMP as measured by the Pearson's coefficient (upper) while cells that adopt a flattened, mesenchymal morphology show demonstrably less colocalization between VAMP3 and MT1-MMP (lower) Images are representative. (H) Increasing bleb index* in LOX cells correlates with an increase in the colocalization between VAMP3 and MT1-MMP as measured by the Pearson's coefficient. n= 30 randomly selected cells. *Bleb index = blebs per second/cell perimeter.
Figure 2. VAMP3 knockdown disrupts trafficking and delivery of MT1-MMP; and alters in vitro invasive capacity
TMVs were isolated from LOX cells expressing VAMP3-shRNA (A) or scrambled-shRNA (B). TMVs were plated on FITC-gelatin coated coverslips and allowed to degrade substrate. Slides were fixed and stained as indicated to show vesicle-associated matrix degradation (dark areas). Scale bar = 10 μm. TMV-associated matrix degradation quantified in (C). Data presented as mean ± 1 standard deviation, n = 120 per condition across 3 independent experiments, p < 0.02 determined by student's t test. (D) TMVs from VAMP3 or scrambled-shRNA cells were stained to examine endogenous MT1-MMP. Scale bar = 5 μm. (E) TMVs were isolated from VAMP3 or scrambled-shRNA electroporated LOX cells, lysed, and equal protein (determined by BCA) separated by SDS-PAGE, blotted and probed as indicated. Immunoblots are representative of at least 3 independent experiments. (F) VAMP3-shRNA or scrambled-shRNA transfected LOX cells were plated on unlabeled gelatin substrate. Cells were fixed and stained to identify endogenous MT1-MMP. Cells carrying scrambled-shRNA show intracellular MT1-MMP identifiable in surface blebs. This staining, and the presence of MT1-MMP in surface blebs are absent in VAMP3-shRNA cells. Scale bar = 5 μm. Scrambled-shRNA (G) or VAMP3-shRNA (H) transfected LOX cells were plated on FITC-conjugated gelatin coated coverslips. Cells were fixed and stained as indicated to examine the matrix degradation. Higher magnification image of VAMP3-shRNA invasion in inset. (I) Linear displacement of the cell's center of mass at time t=0 and time t=14 hours. Results from minimum 15 cells per condition from 3 independent experiments. Data presented as mean ± 1 standard deviation, p < 0.01 determined by students t test. (J) The approximate center of mass in scrambled-shRNA or VAMP3-shRNA transfected cells embedded in PurCol collagen matrix was tracked over time by live cell imaging. Positions plotted relative to the origin (time t=0). (K) Linear displacement of invading cells was quantified over 14 hours. Results represent at least 15 cells per condition over independent experiments. Data presented as mean ± 1 standard deviation, p < 0.01 determined by students t test.
Figure 3. VAMP3 depletion disrupts association between MT1-MMP and CD9
(A) Lysates from control-shRNA or VAMP3-shRNA electoporated cells were immunoprecipitated using polyclonal anti-CD9 antibody. Total cell lysates (left) and immunoprecipitates (right) were resolved by SDS-PAGE, transferred to PVDF membrane and probed as indicated. Blots shown are representative of more than 4 independent immunoprecipitation replicates. (B) LOX cells and those expressing shRNA as indicated, were incubated with mouse anti-MT1-MMP (lanes 1, 4, and 5) or control mouse IgG (lane 3) as described in Methods. These cells were incubated for 16 hours in pre-cleared complete culture media before shed microvesicles were isolated. Microvesicle lysate was then resolved by SDS-PAGE and analyzed by western blotting as indicated. (C) LOX cells expressing either scrambled-shRNA or VAMP3-shRNA were embedded in BD Rat Tail collagen I matrix and imaged 24 hours post embedding to allow for shRNA expression. Frames shown are representative stills captured approximately every 20 minutes. Scale bar = 25 μm. Control (D) or NSC405020 treated (E) LOX cells were plated on FITC- gelatin and allowed to invade overnight. Coverslips were fixed and stained as indicated to examine the extent of cell invasion. Scale bar = 25 μm. (F) Cells expressing scrambled siRNA (top) or MT1-MMP siRNA (bottom) were allowed to invade TRITC conjugated matrix overnight. The cells were then fixed, stained as indicated and analyzed by confocal microscopy to examine invasive capacity. Scale bar = 25 μm. (G) LOX cells transfected to express MT1-MMP-siRNA (top) or scrambled siRNA (bottom) were embedded in a layer of rat tail collagen-I and allowed to invade. Cell invasion was monitored by phase contrast microscopy over the course of 12 hours. Stills represent sequential images every 90 minutes. Scale bar = 25 μm.
Figure 4. Invasive microvesicles can be isolated from peripheral bodily fluids of patients with diagnosed abdomino-pelvic mass
(A) Unfractionated ascites or isolated TMVs were resuspended in sterile, filtered 1X PBS and subjected to nano particle tracking analysis using a NanoSight LM10 as per the manufacturers protocol. Measurements of concentration (upper panel) and percentage undersized (lower panel) vs. particle diameter (nm) shown represent the mean of 10 individual acquisitions for each sample type. The curves presented are representative of the patient population studied. (B) Microvesicles from patients with Stage IIB serous adenocarcinoma were fixed and examined by whole mount transmission electron microscopy as described in Methods. Scale bar = 500 nm. (C) Equal amounts of unfractionated ascites fluid and isolated microvesicles (determined using BCA assay) from patient samples, were probed by western blotting as indicated. Note, protein is equal within but not between patients. Data shown is from patients later diagnosed with serous cystadenocarcinoma of the ovary (28), high-grade serous ovarian carcinoma (32), poorly-differentiated ovarian carcinoma (33). Unfractionated fluid in parallel with isolated TMVs, were resuspended in sterile, filtered, 1X PBS (D) or 1X PBS +NSC405020 (E) prior to mixing with TMV-free complete cell culture media. The mixture was overlaid onto FITC-gelatin coated coverslips and allowed to degrade matrix for a period of 14 hours. TMVs were fixed, stained as indicated, and subjected to confocal microscopy to examine levels of matrix degradation. Scale bar = 50 μm. (F) Equal amounts of protein from unfractionated serum and isolated TMVs (determined using BCA assay) were probed by western blot as indicated. In parallel, microvesicles were mixed with 1X PBS prior to addition to TMV-free complete cell culture media and incubation with FITC-conjugated gelatin-coated coverslips for a period of 14 hours. Scale bar = 10 μm. Data shown is from a patient later diagnosed with serous ovarian carcinoma.
Figure 5. Working model for VAMP3-mediated trafficking of MT1-MMP into nascent tumor microvesicles
VAMP3 facilitates interactions between MT1-MMP and CD9, which are required for the routing of newly synthesized MT1-MMP to the cell surface. VAMP3 also regulates the delivery of endosomal MT1-MMP to the cell surface. ARF6 and VAMP3 positive intracellular vesicles may serve as holding and sorting stations for MT1-MMP being delivered to surface TMVs. Whether CD9 interacts with the protease on endosomes or at the cell surface requires further investigation.
References
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources