Tunneling NanoTubes (TNTs) are membrane conduits that mediate long distance intercellular crosstalk 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 is essential for TNT formation in multiple cell types. We identified a novel interaction of MSec with the endoplasmic reticulum chaperone ERp29. ERp29 depletion in mammalian cells led to a significant reduction in TNT formation, while its over-expression induced TNT formation, but strictly in an 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. Our study implicates MSec as a new target of ERp29 and reveals an indispensable role for the endoplasmic reticulum in the biogenesis of TNTs, thus suggesting new modalities for regulating TNT numbers.
Sample Processing Protocol
The stably expressing (MSec-MTAP) U2OS cells were cultured in large scale in selection media for affinity purification. Cultured cells were harvested in resuspension buffer containing 50 mM Tris-HCl, pH 7.5, 125 mM NaCl, 1 mM EDTA, 5% glycerol, Igepal CA-630 (0.025% - 0.2%) and a protease inhibitor cocktail (Roche). Harvested cells were lysed cryogenically by grinding them (using a mortar and pestle under liquid nitrogen) to preserve the integrity of protein complexes. The total cell lysate was centrifuged at 14,000 x g for 30 min at 40C to separate insoluble material. The supernatant was subjected to streptavidin binding peptide (SBP)-based affinity purification using a StrepTrap HP 1 ml column (GE Healthcare Life Sciences). The affinity column was equilibrated with resuspension buffer at a flow rate of 0.5 ml/ min and the supernatant applied to it at a flow rate of 0.3 ml/ min. The supernatant was incubated for 30 min, drained and the column subsequently washed with wash buffer containing 50 mM Tris-HCl, pH 7.5, 250 mM NaCl, Igepal CA-630 (0.05%) and protease inhibitor cocktail. Bound protein was eluted in elution buffer containing 25 mM Tris-HCl, pH 7.5, 125 mM NaCl, 2.5 mM desthio-biotin and protease inhibitor cocktail. Four biological experiments were performed at varying Igepal CA-630 concentrations from 0.025% to 0.2% to identify protein interactions consistently observed over this range of detergent concentrations. Mass spectrometry After affinity elution, the protein solution (eluate) was concentrated using Amicon-Ultra centrifugal filters (Millipore). Concentrated protein was estimated by using the bicinchoninic acid kit (BCA, Thermo Scientific) and subsequently processed by the filter-assisted sample preparation (FASP protocol) followed by digestion with trypsin using Trypsin-Gold (Promega) at 370C for 14-16 hours. Trypsinized peptides were desalted using ZipTips (Millipore) and analysed by electrospray ionization liquid chromatography mass spectrometry (ESI-LC-MS) by injecting a total of 1μg peptides on to a nano-reverse phase high performance liquid chromatography (RP-HPLC) C18 column (Thermo Scientific) equilibrated with buffer-A (2% acetonitrile + 0.1% formic acid in water). Peptides were eluted using a gradient of buffer-B (98% acetonitrile + 0.1% formic acid in water) applied at a flow rate of 200nl/min over 80 min.
Data Processing Protocol
The results obtained from the mass spectrometry experiments (peptide and fragmentation spectra) were analyzed through the Mascot search engine (protein database search program) with optimal parameters. The positive hits present in the bait (MSec-MTAP) experiments with a significant score from each of the four experimental conditions were overlapped using the Venny 2.1 software (http://bioinfogp.cnb.csic.es/tools/venny/index.html) and selected for further classification into various functional categories by using the PANTHER database (http://www.pantherdb.org/).
Sivaram Mylavarapu, Regional Centre for Biotechnology, Faridabad, India
Sivaram V S Mylavarapu, Laboratory of Cellular Dynamics, Regional Centre for Biotechnology, NCR Biotech Science Cluster, 3rd Milestone Faridabad-Gurgaon Expressway, Faridabad Haryana 121001, India. ( lab head )