Cell-cell communication is essential for the development and homeostasis of multicellular organisms. Recently, a new type of cell-cell communication was discovered that is based on the formation of thin membranous nanotubes between remote cells. These long membrane tethers, termed tunneling nanotubes (TNTs), form an intercellular conduit and have been shown to enable the transport of various cellular components and signals. However, the molecular basis for TNT formation remains to be elucidated. Here we report that a mammalian protein, M-Sec, induces de novo formation of numerous membrane protrusions extending from the plasma membrane, some of which tether onto adjacent cells and subsequently form TNT-like structures. Depletion of M-Sec by RNA interference (RNAi) greatly reduced endogenous TNT formation as well as intercellular propagation of a calcium flux in a macrophage cell line. Furthermore, blockage of the interaction of M-Sec with Ral and the exocyst complex, which serves as a downstream effector of Ral, attenuated the formation of membrane nanotubes. Our results reveal that M-Sec functions as a key regulator of membrane nanotube formation through interaction with the Ral-exocyst pathway.

The study of macroautophagy in mammalian cells has described induction, vesicle nucleation, and membrane elongation complexes as key signaling intermediates driving autophagosome biogenesis. How these components are recruited to nascent autophagosomes is poorly understood, and although much is known about signaling mechanisms that restrain autophagy, the nature of positive inductive signals that can promote autophagy remain cryptic. We find that the Ras-like small G-protein, RalB, is localized to nascent autophagosomes and is activated upon nutrient deprivation. RalB and its effector Exo84 are required for nutrient starvation-induced autophagocytosis, and RalB activation is sufficient to promote autophagosome formation. Through direct binding to Exo84, RalB induces the assembly of catalytically active ULK1 and Beclin1-VPS34 complexes on the exocyst, which are required for isolation membrane formation and maturation. Thus, RalB signaling is a primary adaptive response to nutrient limitation that directly engages autophagocytosis through mobilization of the core vesicle nucleation machinery.

The closely related GTPases RalA and RalB are required for assembly of tight junction gate, but not fence, function. These activities depend on direct binding to the exocyst complex. Whereas RalA–exocyst complexes are required for exocytosis of junction proteins, RalB–exocyst complexes are required for endocytosis of these components.

Metastasis is a complex process during which several gross cellular changes occur. Cells must dissociate from the tumor mass and gain the ability to degrade extracellular matrix and migrate in order to ultimately attach and form a satellite tumor. Regulation of the actin cytoskeleton is an indispensible aspect of cell migration, and many different factors have been implicated in this process. We identified interactions between RalA and its effectors in the Exocyst complex as directly necessary for migration and invasion of prostate cancer tumor cells. Blocking RalA-Exocyst binding caused significant morphological changes and defects in single and coordinated cell migration.

Epithelial tissues can be polarized along two axes, in addition to apical-basal polarity they are often also polarized within the plane of the epithelium, known as planar cell polarity (PCP). PCP depends upon the conserved Wnt/Frizzled (Fz) signaling factors, including Fz itself and Van Gogh (Vang/Vangl). Here, taking advantage of the complementary features of Drosophila wing and mouse skin PCP establishment, we dissect how Vang phosphorylation on a specific conserved tyrosine residue affects its interaction with two cytoplasmic core PCP factors, Dsh/Dvl and Pk. We demonstrate that Pk and Dsh/Dvl bind to Vang/Vangl in an overlapping region centered around this tyrosine. Strikingly, Vang/Vangl2 phosphorylation promotes its binding to Pk, a key effector of the Vang/Vangl complex, and inhibits its interaction with Dsh/Dvl, and thus phosphorylation of this tyrosine appears to promote the formation of the mature and stable Vang/Vangl-Pk complex during PCP establishment. Interestingly, as our single point mutations allow selective binding inhibition of either Dsh or Pk, we can demonstrate for the first time that Dsh interaction with Vang is physiologically important, as all single point mutations fail to rescue the Vang null mutant wing phenotype.