![]() PCR products and the GeneRuler 100 bp or 1 kb DNA ladder (Thermo Fisher Scientific) were diluted in DNA Gel Loading Dye (6X Thermo Fisher Scientific) and separated by electrophoresis on 2% agarose gels, prepared in 40 mM Tris, 20 mM acetate and 1 mM EDTA (TAE buffer) with 0.01% GelRed fluorescent DNA stain (Biotium, Hayward, CA, USA). The PCR was performed on a MiniOpticon PCR System (BioRad Laboratories) or a 2720 Thermal Cycler (Applied Biosystems, Thermo Fisher Scientific) with the following protocol: one cycle of 95☌ for 3 min 39 cycles of 95☌ for 30 sec 60☌ for 30 sec 72☌ for 1 min and one cycle of 72☌ for 7 min. Taq polymerase and Buffer II were added as per the manufacturers instructions additionally, one PCR volume contained 0.5 mM MgCl 2, 5% dimethyl sulfoxide (DMSO), 0.1 μM dNTPs, 0.5 μM forward primer, 0.5 μM reverse primer, and 1.5 μl of sample DNA (1 μg/μl). PCR was carried out with AmpliTaq Gold ® DNA polymerase with Buffer II and MgCl 2 (Thermo Fisher Scientific). The existence of this splice variant of Exoc3l2 expands the potential for EXOC3L2 functional diversity, as an integrated component of the exocyst or as an independent agent, and will need to be accounted for in future studies of EXOC3L2. Here we report an alternative splice variant of murine Exoc3l2 and using a structural homology model based on M-Sec we compare the properties of the canonical isoform of EXOC3L2 with this alternative isoform. Together these findings indicate that EXOCs and their paralogs serve central membrane-bending functions, including the formation of TNTs. ![]() Furthermore, EXOCs have also been identified as effectors of the HIV Nef-1 protein, mediating the formation of nanotubes that facilitate intercellular virus transfer. Studies of the membrane-bending capacity of the exocyst have largely focused on EXOC7, where its ability to dimerize and generate membrane protrusions has lead to its comparison with the inverse BAR (I-BAR) proteins, which induce similar membrane deformations and like EXOC7 and EXOC3L2 are required for directional cell migration. Importantly, M-Sec’s ability to induce TNTs relies on its interaction with the GTPase Ral and the exocyst. A crystal structure for M-Sec was recently published and highlights its structural similarities with yeast EXOC3 (Sec6) and mouse EXOC7 (Exo70). TNTs are fine intercellular membrane connections that are implicated in the transport of organelles and protein complexes. M-Sec plays a key role in the formation of tunneling nanotubes (TNTs). M-Sec is another paralog of Exoc3 and is also known as tumor necrosis factor α-inducible protein 2 (TNFαIP2) and EXOC3元. It remains to be determined whether these alternative EXOC isoforms operate independently of the exocyst furthermore, if recruited to the exocyst, it is unclear if they replace their canonical EXOC counterparts or can be integrated in addition to them. Alternative splicing of EXOC7 permits isoform switching during epithelial to mesenchymal transition (EMT) in breast cancer cells, such that the mesenchymal EXOC7 isoform promotes a migratory phenotype through its ability to recruit remodelers of the actin cytoskeleton. We have previously reported on the EXOC3 paralog EXOC3L2 and established that it is required for directional migration of endothelial cells and can associate with other EXOCs. Isoform variants exist for several of the EXOCs and these are derived from alternative splicing of the canonical EXOC transcripts or paralogous gene expression. The exocyst complex is composed of eight distinct components (EXOC1-8) and tethers secretory vesicles to the plasma membrane prior to SNARE-mediated fusion and exocytosis. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: All relevant data are within the paper.įunding: This study was supported by grants received by Paul O'Callaghan from Alzheimerfonden (AF-552581, AF-649251 ) and Demensförbundet ( ), and by Johan Kreuger from Cancerfonden (CAN 2014/820 ) and Uppsala University. Received: MaAccepted: JPublished: August 7, 2018Ĭopyright: © 2018 O’Callaghan et al. ![]() Citation: O’Callaghan P, Zarb Y, Noborn F, Kreuger J (2018) Modeling the structural implications of an alternatively spliced Exoc3l2, a paralog of the tunneling nanotube-forming M-Sec.
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