Signaling is regulated by endocytosis at multiple levels along endocytic routes. Endocytic control of signaling starts already at the plasma membrane, where cells employ different mechanisms to finely tune the type and strength of signals emanating from the cell surface. Here, we will review some of the most recently described endocytic mechanisms controlling signaling at the plasma membrane, through the regulation of internalization dynamics and through the integration of different internalization pathways triggered by canonical chemical stimuli or physical forces.
Membrane contact sites (MCSs) are hubs allowing various cell organelles to coordinate their activities. The dynamic nature of these sites and their small size hinder analysis by current imaging techniques. To overcome these limitations, we here design a series of reversible chemogenetic reporters incorporating improved, low-affinity variants of splitFAST, and study the dynamics of different MCSs at high spatiotemporal resolution, both in vitro and in vivo. We demonstrate that these versatile reporters suit different experimental setups well, allowing one to address challenging biological questions. Using these probes, we identify a pathway in which calcium (Ca2+) signalling dynamically regulates endoplasmic reticulum-mitochondria juxtaposition, characterizing the underlying mechanism. Finally, by integrating Ca2+-sensing capabilities into the splitFAST technology, we introduce PRINCESS (PRobe for INterorganelle Ca2+-Exchange Sites based on SplitFAST), a class of reporters to simultaneously detect MCSs and measure the associated Ca2+ dynamics using a single biosensor.
The endocytic protein NUMB has been implicated in the control of various polarized cellular processes, including the acquisition of mesenchymal migratory traits through molecular mechanisms that have only been partially defined. Here, we report that NUMB is a negative regulator of a specialized set of understudied, apically restricted, actin-based protrusions, the circular dorsal ruffles (CDRs), induced by either PDGF or HGF stimulation. Through its PTB domain, NUMB binds directly to an N-terminal NPLF motif of the ARF6 guanine nucleotide exchange factor, EFA6B, and promotes its exchange activity in vitro. In cells, a NUMB-EFA6B-ARF6 axis regulates the recycling of the actin regulatory cargo RAC1 and is critical for the formation of CDRs that mark the acquisition of a mesenchymal mode of motility. Consistently, loss of NUMB promotes HGF-induced cell migration and invasion. Thus, NUMB negatively controls membrane protrusions and the acquisition of mesenchymal migratory traits by modulating EFA6B-ARF6 activity.
Abstract During wound repair, branching morphogenesis and carcinoma dissemination, cellular rearrangements are fostered by a solid-to-liquid transition known as unjamming. The biomolecular machinery behind unjamming, its physiological and clinical relevance remain, however, a mystery. Here, we combine biophysical and biochemical analysis to study unjamming in a variety of epithelial 2D and 3D collectives: monolayers, differentiated normal mammary cysts, spheroid models of breast ductal carcinoma in situ (DCIS), and ex vivo slices of orthotopically-implanted DCIS. In all cases, elevation of the small GTPase RAB5A sparks unjamming by promoting non-clathrin-dependent internalization of epidermal growth factor receptor that leads to hyper-activation of endosomally-confined ERK1/2 and phosphorylation of the actin nucleator WAVE2. Physically, activation of this pathway causes highly coordinated flocking of the cells, with striking rotational motion in 3D that eventually leads to matrix remodelling and collective invasiveness of otherwise jammed carcinoma. The identified endo-ERK1/2 pathway provides an effective switch for unjamming through flocking to promote epithelial tissues morphogenesis and carcinoma invasion and dissemination.
Abstract One open question in the biology of growth factor receptors is how a quantitative input (i.e., ligand concentration) is decoded by the cell to produce specific response(s). Here, we show that an EGFR endocytic mechanism, non-clathrin endocytosis (NCE), which is activated only at high ligand concentrations and targets receptor to degradation, requires a tripartite organelle platform involving the plasma membrane (PM), endoplasmic reticulum (ER) and mitochondria. At these contact sites, EGFR-dependent, ER-generated Ca 2+ oscillations are sensed by mitochondria, leading to increased metabolism and ATP production. Locally released ATP is required for cortical actin remodeling and EGFR-NCE vesicle fission. The same biochemical circuitry is also needed for an effector function of EGFR, i.e., collective motility. The multiorganelle signaling platform herein described mediates direct communication between EGFR signaling and mitochondrial metabolism, and is predicted to have a broad impact on cell physiology as it is activated by another growth factor receptor, HGFR/MET.
Adaptor protein 2 (AP2) is a major constituent of clathrin-coated pits (CCPs). Whether it is essential for all forms of clathrin-mediated endocytosis (CME) in mammalian cells is an open issue. Here, we demonstrate, by live TIRF microscopy, the existence of a subclass of relatively short-lived CCPs lacking AP2 under physiological, unperturbed conditions. This subclass is retained in AP2-knockout cells and is able to support the internalization of epidermal growth factor receptor (EGFR) but not of transferrin receptor (TfR). The AP2-independent internalization mechanism relies on the endocytic adaptors eps15, eps15L1, and epsin1. The absence of AP2 impairs the recycling of the EGFR to the cell surface, thereby augmenting its degradation. Accordingly, under conditions of AP2 ablation, we detected dampening of EGFR-dependent AKT signaling and cell migration, arguing that distinct classes of CCPs could provide specialized functions in regulating EGFR recycling and signaling.
ER-PM contacts in nonclathrin endocytosis The epidermal growth factor receptor (EGFR) is internalized through both clathrin-mediated endocytosis and nonclathrin endocytosis (NCE). The two pathways act in concert to sustain EGFR signaling or its long-term attenuation. The mechanistic underpinnings of EGFR-NCE are unclear. Caldieri et al. used a variety of cell and molecular biology approaches to identify nine regulators of EGFR-NCE (see the Perspective by Tan and Anderson). They also identified an additional cargo of the pathway (CD147). One of the regulators of the pathway was the endoplasmic reticulum (ER)-resident protein reticulon 3 (RTN3). Unexpectedly, EGFR-NCE required the formation of specific contacts between the plasma membrane (PM) and the cortical ER, mediated by RTN3. ER-PM contact sites were required in the very early steps of the internalization process for the maturation of NCE tubular intermediates. Science , this issue p. 617 ; see also p. 584
Abstract One open question in the biology of growth factor receptors is how a quantitative input ( i.e., ligand concentration) is decoded by the cell to produce specific response(s). Here, we show that an EGFR endocytic mechanism, non-clathrin endocytosis (NCE), which is activated only at high ligand concentrations and targets receptor to degradation, requires a tripartite organelle platform involving the plasma membrane (PM), endoplasmic reticulum (ER) and mitochondria. At these contact sites, EGFR-dependent, ER-generated Ca 2+ oscillations are sensed by mitochondria, leading to increased metabolism and ATP production. Locally released ATP is required for cortical actin remodeling and EGFR-NCE vesicle fission. The same biochemical circuitry is also needed for an effector function of EGFR, i.e., collective motility. The multiorganelle signaling platform herein described mediates direct communication between EGFR signaling and mitochondrial metabolism, and is predicted to have a broad impact on cell physiology as it is activated by another growth factor receptor, HGFR/MET.
