MicroRNAs are exported from malignant cells in customized particles.

MicroRNAs (miRNAs) are released from cells in association with proteins or microvesicles. We previously reported that malignant transformation changes the assortment of released miRNAs by affecting whether a particular miRNA species is released or retained by the cell. How this selectivity occurs is unclear. Here we report that selectively exported miRNAs, whose release is increased in malignant cells, are packaged in structures that are different from those that carry neutrally released miRNAs (n-miRNAs), whose release is not affected by malignancy. By separating breast cancer cell microvesicles, we find that selectively released miRNAs associate with exosomes and nucleosomes. However, n-miRNAs of breast cancer cells associate with unconventional exosomes, which are larger than conventional exosomes and enriched in CD44, a protein relevant to breast cancer metastasis. Based on their large size, we call these vesicles L-exosomes. Contrary to the distribution of miRNAs among different microvesicles of breast cancer cells, normal cells release all measured miRNAs in a single type of vesicle. Our results suggest that malignant transformation alters the pathways through which specific miRNAs are exported from cells. These changes in the particles and their miRNA cargo could be used to detect the presence of malignant cells in the body.

Novel function for AP-1B during cell migration.

The epithelial cell-specific clathrin adaptor protein (AP)-1B has a well-established role in polarized sorting of cargos to the basolateral membrane. Here we show that ß1 integrin was dependent on AP-1B and its coadaptor, autosomal recessive hypercholesterolemia protein (ARH), for sorting to the basolateral membrane. We further demonstrate an unprecedented role for AP-1B at the basal plasma membrane during collective cell migration of epithelial sheets. During wound healing, expression of AP-1B (and ARH in AP-1B-positive cells) slowed epithelial-cell migration. We show that AP-1B colocalized with ß1 integrin in focal adhesions during cell migration using confocal microscopy and total internal reflection fluorescence microscopy on fixed specimens. Further, AP-1B labeling in cell protrusions was distinct from labeling for the endocytic adaptor complex AP-2. Using stochastic optical reconstruction microscopy we identified numerous AP-1B-coated structures at or close to the basal plasma membrane in cell protrusions. In addition, immunoelectron microscopy showed AP-1B in coated pits and vesicles at the plasma membrane during cell migration. Lastly, quantitative real-time reverse transcription PCR analysis of human epithelial-derived cell lines revealed a loss of AP-1B expression in highly migratory metastatic cancer cells suggesting that AP-1B's novel role at the basal plasma membrane during cell migration might be an anticancer mechanism.

Using replication defective viruses to analyze membrane trafficking in polarized epithelial cells.

Epithelial cells in culture, especially once they are polarized, are extremely hard to manipulate by transient transfection methods. The use of replication defective adenoviruses for gene expression or replication defective retroviruses or lentiviruses to express shRNA for gene knockdown provides efficient tools to manipulate gene expression patterns even in hard-to-transfect cell lines. One of the advantages of using defective adenoviruses for gene expression is that once the virus has been generated, it can easily be applied to a wide variety of cells. In addition, replication defective retro- and lentiviruses are used to stably deplete proteins from cell lines, which subsequently may be used for analyzing the polarized surface delivery of receptors that may be expressed using defective adenoviruses. The latter approach is especially useful if the expressed shRNA also encodes GFP for easy assessment of shRNA-expressing cells. Thus the use of defective viruses in epithelial cell research is convenient. This makes a detailed infection protocol a research tool that would be valuable to many laboratories. Here we describe in detail how cells are infected with defective retro- or lentiviruses and subsequently selected for stable gene knockdown. We then describe how these cells may be used for infection with defective adenoviruses and the subsequent analyses.

Arf6 regulates AP-1B-dependent sorting in polarized epithelial cells.

The epithelial cell-specific clathrin adaptor complex AP-1B facilitates the sorting of various transmembrane proteins from recycling endosomes (REs) to the basolateral plasma membrane. Despite AP-1B's clear importance in polarized epithelial cells, we still do not fully understand how AP-1B orchestrates basolateral targeting. Here we identify the ADP-ribosylation factor 6 (Arf6) as an important regulator of AP-1B. We show that activated Arf6 pulled down AP-1B in vitro. Furthermore, interfering with Arf6 function through overexpression of dominant-active Arf6Q67L or dominant-negative Arf6D125N, as well as depletion of Arf6 with short hairpin RNA (shRNA), led to apical missorting of AP-1B-dependent cargos. In agreement with these data, we found that Arf6 colocalized with AP-1B and transferrin receptor (TfnR) in REs. In addition, we observed specific recruitment of AP-1B into Arf6-induced membrane ruffles in nonpolarized cells. We conclude that activated Arf6 directs membrane recruitment of AP-1B, thus regulating AP-1B's functions in polarized epithelial cells.

Selective release of microRNA species from normal and malignant mammary epithelial cells.

MicroRNAs (miRNAs) in body fluids are candidate diagnostics for a variety of conditions and diseases, including breast cancer. One premise for using extracellular miRNAs to diagnose disease is the notion that the abundance of the miRNAs in body fluids reflects their abundance in the abnormal cells causing the disease. As a result, the search for such diagnostics in body fluids has focused on miRNAs that are abundant in the cells of origin. Here we report that released miRNAs do not necessarily reflect the abundance of miRNA in the cell of origin. We find that release of miRNAs from cells into blood, milk and ductal fluids is selective and that the selection of released miRNAs may correlate with malignancy. In particular, the bulk of miR-451 and miR-1246 produced by malignant mammary epithelial cells was released, but the majority of these miRNAs produced by non-malignant mammary epithelial cells was retained. Our findings suggest the existence of a cellular selection mechanism for miRNA release and indicate that the extracellular and cellular miRNA profiles differ. This selective release of miRNAs is an important consideration for the identification of circulating miRNAs as biomarkers of disease.