Tunneling nanotubes (TNTs) are membrane conduits that mediate long-distance intercellular cross-talk in several organisms and play vital roles during development, pathogenic transmission, and cancer metastasis. However, the molecular mechanisms of TNT formation and function remain poorly understood. The protein MSec (also known as TNFα-induced protein 2 (TNFAIP2) and B94) is essential for TNT formation in multiple cell types. Here, using affinity protein purification, mass spectrometric identification, and confocal immunofluorescence microscopy assays, we found that MSec interacts with the endoplasmic reticulum (ER) chaperone ERp29. siRNA-mediated ERp29 depletion in mammalian cells significantly reduces TNT formation, whereas its overexpression induces TNT formation, but in a strictly MSec-dependent manner. ERp29 stabilized MSec protein levels, but not its mRNA levels, and the chaperone activity of ERp29 was required for maintaining MSec protein stability. Subcellular ER fractionation and subsequent limited proteolytic treatment suggested that MSec is associated with the outer surface of the ER. The ERp29–MSec interaction appeared to require the presence of other bridging protein(s), perhaps triggered by post-translational modification of ERp29. Our study implicates MSec as a target of ERp29 and reveals an indispensable role for the ER in TNT formation, suggesting new modalities for regulating TNT numbers in cells and tissues.

Cytokinesis is the final step of cell division following chromosome segregation that generates two daughter cells. The conserved exocyst complex is required for scission of the intercellular cytokinetic bridge, although the molecular mechanisms it employs in this process are unclear. We identify and validate the early endocytic GTPase Rab5 as interacting with the exocyst complex in mammalian cells. Rab5 localizes in the cytokinetic bridge and on the midbody ring in a manner similar to the exocyst complex. Depletion of Rab5 led to delayed abscission. Caenorhabditis elegans orthologs of both exocyst complex subunits and Rab5 localize along the cleavage furrow and are required for cytokinesis in early embryos. Cytokinetic cells depleted of either Rab5 or the exocyst subunits Exoc3 and Exoc4 showed impaired deposition of the endosomal sorting complexes required for transport (ESCRT) III subunits CHMP2B and/or CHMP4B near the midbody ring. The study reveals an evolutionarily conserved role for the early endocytic marker Rab5 in cytokinetic abscission. In addition, it uncovers a key requirement of the exocyst and Rab5 for the delivery of components of the membrane-severing ESCRT III machinery to complete cytokinesis.

Colorectal cancer is the third major cause of cancer-related mortality worldwide. The upward trend in incidence and mortality rates, poor sensitivity to conventional therapies and a dearth of early diagnostic parameters pose a huge challenge in the management of colorectal cancer in India. Due to the high level of genetic diversity present in the Indian population, unraveling the genetic contributions toward pathogenesis is key for understanding the etiology of colorectal cancer and in reversing this trend. We have established a novel cell line, MBC02, from an Indian colorectal cancer patient and have carried out extensive molecular characterization to unravel the pathological alterations in this cell line. In-depth molecular analysis of MBC02 revealed suppression of E-cadherin expression, concomitant with overexpression of EMT related molecules, which manifested in the form of highly migratory and invasive cells. Loss of membrane-tethered E-cadherin released β-catenin from the adherens junction resulting in its cytoplasmic and nuclear accumulation and consequently, upregulation of c-Myc. MBC02 also showed dramatic transcriptional upregulation of β-catenin. Remarkably, we observed significantly elevated proteasome activity that perhaps co-evolved to compensate for the unnaturally high mRNA level of β-catenin to regulate the increased protein load. In addition, there was substantial misregulation of other clinically relevant signaling pathways that have clinical relevance in the pathogenesis of colorectal cancer. Our findings pave the way toward understanding the molecular differences that could define pathogenesis in cancers originating in the Indian population.

The conserved exocyst complex regulates plasma membrane-directed vesicle fusion in eukaryotes. However, its role in stem cell proliferation has not been reported. Germline stem cell (GSC) proliferation in the nematode Caenorhabditis elegans is regulated by conserved Notch signaling. Here, we reveal that the exocyst complex regulates C. elegans GSC proliferation by modulating Notch signaling cell autonomously. Notch membrane density is asymmetrically maintained on GSCs. Knockdown of exocyst complex subunits or of the exocyst-interacting GTPases Rab5 and Rab11 leads to Notch redistribution from the GSC-niche interface to the cytoplasm, suggesting defects in plasma membrane Notch deposition. The anterior polarity (aPar) protein Par6 is required for GSC proliferation, and for maintaining niche-facing membrane levels of Notch and the exocyst complex. The exocyst complex biochemically interacts with the aPar regulator Par5 (14-3-3ζ) and Notch in C. elegans and human cells. Exocyst components are required for Notch plasma membrane localization and signaling in mammalian cells. Our study uncovers a possibly conserved requirement of the exocyst complex in regulating GSC proliferation and in maintaining optimal membrane Notch levels.