SUMO modifies GßL and mediates mTOR signaling.

The mechanistic target of rapamycin (mTOR) signaling is influenced by multiple regulatory proteins and post-translational modifications; however, underlying mechanisms remain unclear. Here, we report a novel role of small ubiquitin-like modifier (SUMO) in mTOR complex assembly and activity. By investigating the SUMOylation status of core mTOR components, we observed that the regulatory subunit, GßL (G protein ß-subunit-like protein, also known as mLST8), is modified by SUMO1, 2, and 3 isoforms. Using mutagenesis and mass spectrometry, we identified that GßL is SUMOylated at lysine sites K86, K215, K245, K261, and K305. We found that SUMO depletion reduces mTOR-Raptor (regulatory protein associated with mTOR) and mTOR-Rictor (rapamycin-insensitive companion of mTOR) complex formation and diminishes nutrient-induced mTOR signaling. Reconstitution with WT GßL but not SUMOylation-defective KR mutant GßL promotes mTOR signaling in GßL-depleted cells. Taken together, we report for the very first time that SUMO modifies GßL, influences the assembly of mTOR protein complexes, and regulates mTOR activity.

Tunneling nanotubes and related structures: molecular mechanisms of formation and function.

Tunneling nanotubes (TNTs) are F-actin-based, membrane-enclosed tubular connections between animal cells that transport a variety of cellular cargo. Over the last 15 years since their discovery, TNTs have come to be recognized as key players in normal cell communication and organism development, and are also exploited for the spread of various microbial pathogens and major diseases like cancer and neurodegenerative disorders. TNTs have also been proposed as modalities for disseminating therapeutic drugs between cells. Despite the rapidly expanding and wide-ranging relevance of these structures in both health and disease, there is a glaring dearth of molecular mechanistic knowledge regarding the formation and function of these important but enigmatic structures. A series of fundamental steps are essential for the formation of functional nanotubes. The spatiotemporally controlled and directed modulation of cortical actin dynamics would be required to ensure outward F-actin polymerization. Local plasma membrane deformation to impart negative curvature and membrane addition at a rate commensurate with F-actin polymerization would enable outward TNT elongation. Extrinsic tactic cues, along with cognate intrinsic signaling, would be required to guide and stabilize the elongating TNT towards its intended target, followed by membrane fusion to create a functional TNT. Selected cargoes must be transported between connected cells through the action of molecular motors, before the TNT is retracted or destroyed. This review summarizes the current understanding of the molecular mechanisms regulating these steps, also highlighting areas that deserve future attention.

Nucleolin regulates 14-3-3ζ mRNA and promotes cofilin phosphorylation to induce tunneling nanotube formation.

Tunneling nanotubes (TNTs) mediate intercellular communication between animal cells in health and disease, but the mechanisms of their biogenesis and function are poorly understood. Here we report that the RNA-binding protein (RBP) nucleolin, which interacts with the known TNT-inducing protein MSec, is essential for TNT formation in mammalian cells. Nucleolin, through its RNA-binding domains (RBDs), binds to and maintains the cytosolic levels of 14-3-3ζ mRNA, and is, therefore, required for TNT formation. A specific region of the 3'-untranslated region (UTR) of the 14-3-3ζ mRNA is likely to be involved in its regulation by nucleolin. Functional complementation experiments suggest that nucleolin and 14-3-3ζ form a linear signaling axis that promotes the phosphorylation and inactivation of the F-actin depolymerization factor cofilin to induce TNT formation. MSec also similarly inactivates cofilin, but potentiates TNT formation independent of the nucleolin-14-3-3ζ axis, despite biochemically interacting with both proteins. We show that 14-3-3ζ and nucleolin are required for the formation of TNTs between primary mouse neurons and astrocytes and in multiple other mammalian cell types. We also report that the Caenorhabditis elegans orthologs of 14-3-3ζ and MSec regulate the size and architecture of the TNT-like cellular protrusions of the distal tip cell (DTC), the germline stem cell niche in the gonad. Our study demonstrates a novel and potentially conserved mRNA-guided mechanism of TNT formation through the maintenance of cellular 14-3-3ζ mRNA levels by the RBP nucleolin.

Transgelin-2 and phosphoregulation of the LIC2 subunit of dynein govern mitotic spindle orientation.

The molecular motor dynein is essential for mitotic spindle orientation, which defines the axis of cell division. The light intermediate chain subunits, LIC1 and LIC2, define biochemically and functionally distinct vertebrate dynein complexes, with LIC2-dynein playing a crucial role in ensuring spindle orientation. We reveal a novel, mitosis-specific interaction of LIC2-dynein with the cortical actin-bundling protein transgelin-2. Transgelin-2 is required for maintaining proper spindle length, equatorial metaphase chromosome alignment, spindle orientation and timely anaphase onset. We show that transgelin-2 stabilizes the cortical recruitment of LGN-NuMA, which together with dynein is required for spindle orientation. The opposing actions of transgelin-2 and LIC2-dynein maintain optimal cortical levels of LGN-NuMA. In addition, we show that the highly conserved serine 194 phosphorylation of LIC2 is required for proper spindle orientation, by maintaining mitotic centrosome integrity to ensure optimal astral microtubule nucleation. The work reveals two specific mechanisms through which LIC2-dynein regulates mitotic spindle orientation; namely, through a new interactor transgelin-2, which is required for engagement of LGN-NuMA with the actin cortex, and through mitotic phosphoregulation of LIC2 to control microtubule nucleation from the poles.This article has an associated First Person interview with the first author of the paper.

The chaperone ERp29 is required for tunneling nanotube formation by stabilizing MSec.

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.