The Canonical Notch Signaling Pathway: Unfolding the Activation Mechanism

Endocytic mechanisms control the lipid and protein composition of the plasma membrane, thereby regulating how cells interact with their environments. Here, we review what is known about mammalian endocytic mechanisms, with focus on the cellular proteins that control these events. We discuss the well-studied clathrin-mediated endocytic mechanisms and dissect endocytic pathways that proceed independently of clathrin. These clathrin-independent pathways include the CLIC/GEEC endocytic pathway, arf6-dependent endocytosis, flotillin-dependent endocytosis, macropinocytosis, circular doral ruffles, phagocytosis, and trans-endocytosis. We also critically review the role of caveolae and caveolin1 in endocytosis. We highlight the roles of lipids, membrane curvature-modulating proteins, small G proteins, actin, and dynamin in endocytic pathways. We discuss the functional relevance of distinct endocytic pathways and emphasize the importance of studying these pathways to understand human disease processes.

Mechanisms of Endocytosis

A small number of signalling pathways are used iteratively to regulate cell fates, cell proliferation and cell death in development. Notch is the receptor in one such pathway, and is unusual in that most of its ligands are also transmembrane proteins; therefore signalling is restricted to neighbouring cells. Although the intracellular transduction of the Notch signal is remarkably simple, with no secondary messengers, this pathway functions in an enormous diversity of developmental processes and its dysfunction is implicated in many cancers.

Notch signalling: a simple pathway becomes complex

Cell signalling and endocytic membrane trafficking have traditionally been viewed as distinct processes Although our present understanding is incomplete and there are still great controversies, it is now recognized that these processes are intimately and bidirectionally linked in animal cells Indeed, many recent examples illustrate how endocytosis regulates receptor signalling (including signalling from receptor tyrosine kinases and G protein-coupled receptors) and, conversely, how signalling regulates the endocytic pathway The mechanistic and functional principles that underlie the relationship between signalling and endocytosis in cell biology are becoming increasingly evident across many systems

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Endocytosis and signalling: intertwining molecular networks

Notch signaling is evolutionarily conserved and operates in many cell types and at various stages during development. Notch signaling must therefore be able to generate appropriate signaling outputs in a variety of cellular contexts. This need for versatility in Notch signaling is in apparent contrast to the simple molecular design of the core pathway. Here, we review recent studies in nematodes, Drosophila and vertebrate systems that begin to shed light on how versatility in Notch signaling output is generated, how signal strength is modulated, and how cross-talk between the Notch pathway and other intracellular signaling systems, such as the Wnt, hypoxia and BMP pathways, contributes to signaling diversity.

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Notch signaling: simplicity in design, versatility in function

Intracellular protein transport is a key factor in epithelial cell polarity. Here we report that mutations in two core components of the vesicle trafficking machinery — a syntaxin and a Rab protein — cause an expansion of the apical membrane domain of Drosophila melanogaster epithelia; this polarity defect is coupled with overproliferation to form neoplastic tumours. Surprisingly, these proteins are associated with the endocytic, and not the exocytic, pathway. The syntaxin Avalanche (Avl) localizes to early endosomes, and loss of avl results in the cellular accumulation of specific membrane proteins, including the Notch signalling receptor and the polarity determinant Crumbs (Crb). Protein accumulation results from a failure in endosomal entry and progression towards lysosomal degradation; these and other avl phenotypes are also detected in Rab5 null mutant cells. Overexpression of Crb alone is sufficient to induce overproliferation of wild-type imaginal tissue, suggesting that polarity alterations in avl and Rab5 mutants directly contribute to tumour formation. Our findings reveal a critical and specific role for endocytic traffic in the control of both apico-basal polarity and cell proliferation.

Endocytic control of epithelial polarity and proliferation in Drosophila

Signaling through the transmembrane receptor Notch is widely used throughout animal development and is a major regulator of cell proliferation and differentiation. During canonical Notch signaling, internalization and recycling of Notch ligands controls signaling activity, but the involvement of endocytosis in activation of Notch itself is not well understood. To address this question, we systematically assessed Notch localization, processing, and signaling in a comprehensive set of Drosophila melanogaster mutants that block access of cargo to different endocytic compartments. We find that γ-secretase cleavage and signaling of endogenous Notch is reduced in mutants that impair entry into the early endosome but is enhanced in mutants that increase endosomal retention. In mutants that block endosomal entry, we also uncover an alternative, low-efficiency Notch trafficking route that can contribute to signaling. Our data show that endosomal access of the Notch receptor is critical to achieve physiological levels of signaling and further suggest that altered residence in distinct endocytic compartments could underlie pathologies involving aberrant Notch pathway activation.

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Endosomal entry regulates Notch receptor activation in Drosophila melanogaster

Scribble (Scrib) module proteins are major regulators of cell polarity, but how they influence membrane traffic is not known. Endocytosis is also a key regulator of polarity through roles that remain unclear. Here we link Scrib to a specific arm of the endocytic trafficking system. Drosophila mutants that block AP-2-dependent endocytosis share many phenotypes with Scrib module mutants, but Scrib module mutants show intact internalization and endolysosomal transport. However, defective traffic of retromer pathway cargo is seen, and retromer components show strong genetic interactions with the Scrib module. The Scrib module is required for proper retromer localization to endosomes and promotes appropriate cargo sorting into the retromer pathway via both aPKC-dependent and -independent mechanisms. We propose that the Scrib module regulates epithelial polarity by influencing endocytic itineraries of Crumbs and other retromer-dependent cargo.

The Scribble module regulates retromer-dependent endocytic trafficking during epithelial polarization

Extensins are hydroxyproline-rich glycoproteins (HRGPs) and generally play a structural role in cell wall integrity. In this study, we determined a novel role of tomato (Solanum lycopersicum) senescence-associated extensin1 (SAE1) in leaf senescence. Both gain-of-function and loss-of-function analyses suggest that SAE1 plays a positive role in leaf senescence in tomato. Transgenic tomato plants overexpressing the SAE1 gene (SAE1-OX) exhibited premature leaf senescence and enhanced dark-induced senescence, whereas SAE1 knock-out (SAE1-KO) plants displayed delayed development-dependent or dark-induced leaf senescence. Heterologous overexpression of SAE1 in Arabidopsis also led to premature leaf senescence and enhanced dark-induced senescence. In addition, the SAE1 protein interacted with the tomato ubiquitin ligase SlSINA4 and SlSINA4 promoted SAE1 degradation in a ligase-dependent manner when co-expressed in Nicotiana benthamiana leaves, suggesting SlSINA4 controls SAE1 protein levels via the ubiquitin-proteasome pathway (UPS). Consistently, introduction of the SlSINA4 overexpression construct into the SAE1-OX tomato completely eliminated accumulation of SAE1 protein and suppressed the phenotypes conferred by overexpression of SAE1. Taken together, our data suggest that the tomato extensin SAE1plays a positive role in leaf senescence and is regulated by the ubiquitin ligase SlSINA4.

Han Lu

Papers

Endocytic control of epithelial polarity and proliferation in Drosophila

Endosomal entry regulates Notch receptor activation in Drosophila melanogaster

The Scribble module regulates retromer-dependent endocytic trafficking during epithelial polarization

The extensin protein SAE1 plays a role in leaf senescence and is targeted by the ubiquitin ligase SlSINA4 in tomato.

Tomato SlSTK is involved in glucose response and regulated by the ubiquitin ligase SlSINA4.