Cytolocalization, molecular function, and the relative abundance of proteins identified in tumor-derived exosomes.

<p>(A) Cellular localization of exosome proteins. Relative subcellular distribution of proteins identified in the exosomes isolated from the breast cancer cell line, MCF-7. Classification of the subcellular location of the proteins was based on the information provided by the UniProtKB/SwissProt database. (B) GO annotation of tumor-derived proteins. Proteins identified were allocated to different molecular function categories defined by the GO consortium. Relative protein levels of exosomes isolated from MCF-7 are indicated by the bars. Two independent biological replicates from the breast cancer cells type, MCF-7 were used for MS analysis. (C) iTRAQ analysis: Differential expression profile for metastatic tumor-derived exosome proteins in MDA-MB-231 and Rab27b-transformed MCF-7 cells compared to non-invasive breast cancer cells (MCF-7). Filled and open bars represent fold change in protein expression in Rab27b and MDA-MB-231 compared to MCF-7, respectively. Two independent biological replicates from the 3 cells types (MCF-7; Rab27b; MDA-MB-231) were used for iTRAQ analysis. The raw data give absolute expression levels of the various proteins (identity) and proteins levels (abundance) in all 3 cells lines. The average of proteins from the two independent samples from Rab27b and MDA-MB-231 lines was determined and compared to that the average of two samples from MCF-7 and the ratio for differential expression was determined.</p

Characterization of exosomes.

<p>(A) Large-field view of electron micrograph of exosomes released from the human breast cancer cell line MCF-7 and stained with uranyl acetate. Scale bar, 100nm; (B) Magnified view of electron micrograph of exosomes released from the human breast cancer cell line MCF-7. Scale bars are 100nm. (C) Western blot analysis of exosomal proteins extracted from MCF-7, MCF-7/Rab27b, and MDA-MB-231.</p

Tumor-derived exosomes increase breast cell motility.

<p>(A) Time course of cell migration wound-healing assays using MCF-7 breast cancer cells as the “recipient” cell line with the three “donor” exosome preparations. Serum-free media was used as a control. Each point on the assay represents three independent experiments at 21 hours. (B) Representative images from the wound healing experiments. Quantification (C) and images (D) of wound-healing assay for MCF7/Rab27b cells in the presence of the three exosome preparations and serum-free media after 19 hours. Quantification (C) and images (D) of wound-healing assay for MDA-MB-231 cells in the presence of the three exosome preparations and serum-free media after 15 hours. Errors were calculated from wound closure at each time point and normalized to the wound closure at the initial time point (0 hour). Experiments were repeated two additional times to verify results.</p

Endocytosis of exosomes in breast cancer cells.

<p>(A) Time-course curve of exosome uptake (endocytosis) by determining fluorescent intensity of TAMRA-labeled exosomes from “donor” cancer cells at specific times. Inset shows a MCF-7 “recipient” cells incubated with TAMRA-labeled MCF-7 “donor” exosomes at 8 hours. Errors were calculated from fluorescence intensity of cells (n = 50), at each time point, normalized to the intensity at the final time point (24 hours). (B) Colocalization of TAMRA-labeled exosomes added to MCF-7 cells transiently transfected with LAMP1-GFP. Scale bar is 10μm.</p

Proteomic Analysis of Proteins Surrounding Occludin and Claudin-4 Reveals Their Proximity to Signaling and Trafficking Networks

<div><p>Tight junctions are complex membrane structures that regulate paracellular movement of material across epithelia and play a role in cell polarity, signaling and cytoskeletal organization. In order to expand knowledge of the tight junction proteome, we used biotin ligase (BioID) fused to occludin and claudin-4 to biotinylate their proximal proteins in cultured MDCK II epithelial cells. We then purified the biotinylated proteins on streptavidin resin and identified them by mass spectrometry. Proteins were ranked by relative abundance of recovery by mass spectrometry, placed in functional categories, and compared not only among the N- and C- termini of occludin and the N-terminus of claudin-4, but also with our published inventory of proteins proximal to the adherens junction protein E-cadherin and the tight junction protein ZO-1. When proteomic results were analyzed, the relative distribution among functional categories was similar between occludin and claudin-4 proximal proteins. Apart from already known tight junction- proteins, occludin and claudin-4 proximal proteins were enriched in signaling and trafficking proteins, especially endocytic trafficking proteins. However there were significant differences in the specific proteins comprising the functional categories near each of the tagging proteins, revealing spatial compartmentalization within the junction complex. Taken together, these results expand the inventory of known and unknown proteins at the tight junction to inform future studies of the organization and physiology of this complex structure.</p></div

Characterization of invasiveness associated with non-metastatic and metastatic breast cancer cell lines.

<p>(A) Punctate expression pattern and cellular extensions of MCF-7 cells stably transfected with GFP-Rab27b-expressing plasmids. (B) Cells overexpression GFP-Rab27b formed cellular extensions with peripherally-localized GFP-Rab27b, and a spreading morphology on collagen-coated coverslips. (C) Measurement of cell proliferation of MCF-7 cells stably expression GFP or GFP-Rab27b. The same number of cells was plated in triplicate into 10 cm dishes on day 1 and the total number of cells was counted on day 8. (D) Matrigel invasion assay with MCF-7 GFP, MCF-7 Rab27b GFP, and MDA-MB-231cells. 10⁵ cells were seeded in serum free media on a Matrigel-coated filter and their migration toward medium containing serum was quantified by microscopic evaluation. MCF-7 cells stably overexpressing GFP-Rab27b showed significant increased invasion into matrigel compared to MCF-7 GFP cells (mean = 55.7 vs. 14.7 cells, <i>P<0</i>.<i>0001</i>). The well-characterized invasive MDA-MB-231 cell line showed massive invasion into matrigel, which has been described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117495#pone.0117495.ref062" target="_blank">62</a>]. (E) The mean total number of invading cells from 10 different fields is shown <i>P</i> values were calculated using two-sided Student <i>t</i> tests. Statistically significant <i>P</i> values are indicated (n = 3); (F) Western blot analysis of protein extracts from MCF-7, MDA-MB-231, MCF-7 /GFP, MCF-7/GFP-RAB27a, and MCF-7/GFP-RAB27b probed with antibodies to GFP, RAB27a, and RAB27b. An anti-HRP conjugated β-actin antibody was used as a protein loading control. The established MCF-7 RAB27b GFP cell line showed over-expression of GFP-RAB27b demonstrated with an anti-GFP antibody, RAB27b antibody, and RAB27a antibody. Lysates of previously described cell lines (MCF-7 GFP, MCF-7 RAB27a GFP) were included as controls to show specificity of the antibodies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117495#pone.0117495.ref037" target="_blank">37</a>].</p

Enriched clathrin-dependent endocytosis and exocytosis/transcytosis trafficking proteins tagged by biotin ligase fused to occludin and claudin-4.

<p>Enriched clathrin-dependent endocytosis and exocytosis/transcytosis trafficking proteins tagged by biotin ligase fused to occludin and claudin-4.</p

Enriched kinases and phosphatases tagged by biotin ligase fused to occludin and claudin-4.

<p>Enriched kinases and phosphatases tagged by biotin ligase fused to occludin and claudin-4.</p

Identification and functional analysis of SOX10 phosphorylation sites in melanoma

<div><p>The transcription factor SOX10 plays an important role in vertebrate neural crest development, including the establishment and maintenance of the melanocyte lineage. SOX10 is also highly expressed in melanoma tumors, and SOX10 expression increases with tumor progression. The suppression of SOX10 in melanoma cells activates TGF-β signaling and can promote resistance to BRAF and MEK inhibitors. Since resistance to BRAF/MEK inhibitors is seen in the majority of melanoma patients, there is an immediate need to assess the underlying biology that mediates resistance and to identify new targets for combinatorial therapeutic approaches. Previously, we demonstrated that SOX10 protein is required for tumor initiation, maintenance and survival. Here, we present data that support phosphorylation as a mechanism employed by melanoma cells to tightly regulate SOX10 expression. Mass spectrometry identified eight phosphorylation sites contained within SOX10, three of which (S24, S45 and T240) were selected for further analysis based on their location within predicted MAPK/CDK binding motifs. SOX10 mutations were generated at these phosphorylation sites to assess their impact on SOX10 protein function in melanoma cells, including transcriptional activation on target promoters, subcellular localization, and stability. These data further our understanding of SOX10 protein regulation and provide critical information for identification of molecular pathways that modulate SOX10 protein levels in melanoma, with the ultimate goal of discovering novel targets for more effective combinatorial therapeutic approaches for melanoma patients.</p></div

Coomassie-stained SDS-PAGE gels reveal that streptavidin-purified biotinylated proteins from different transgenes show differing protein patterns.

<p><b>A</b>. Shown are proteins purified from cells expressing biotin ligase alone (<i>BL</i>), biotin ligase fused to the N terminus of Ocln (<i>BL-Ocln</i>). <b>B</b>. Proteins purified from the N- and C- terminus of Ocln (BL-Ocln and <i>Ocln-BL) with added</i> biotin. <b>C</b>. Shown are proteins purified from cells expressing biotin ligase fused to the N terminus of Cldn4 (<i>BL-Cldn4</i>), <i>with or without added</i> biotin. The positions of the transgenes are marked with <i>arrowheads</i>. Triplicate samples gave very similar protein patterns.</p