Endocytic Regulation of Notch Signalling During Development

Delta/Notch signalling is of major importance for embryonic development and adult life. While endocytosis is often viewed as a way to down-regulate biological signals, ligand and receptor internalization are essential for Notch activation. The development of Drosophila mecanosensory bristles is a powerful model to study Delta/Notch signalling. Following the asymmetric division of bristle precursor cells, Delta ligands and Notch receptors traffic differently in the two daughter cells, leading to directional signal activation. Recent evidence suggests that in addition to differential ligand endocytosis after division, a subpopulation of multivesicular endosomes ensures the directional transport of Delta/Notch already during asymmetric cell division. Biochemical analysis suggests that different phases of endocytic Delta trafficking exert complementary but distinct actions required for ligand recycling, ligand/receptor interaction and ligand-mediated receptor activation, respectively. Finally, novel data suggest that different endosomal compartments may act as Delta/Notch signalling platforms. In this review, we discuss the implications of these novel findings for our cell biological understanding of Delta/Notch signalling.     

Endocytic routes to Notch activation

It is well established that Notch signalling is activated in response to ligand binding through a series of proteolytic cleavages that release the Notch intracellular domain, allowing it to translocate to the nucleus to regulate downstream target gene expression. However there is still much to learn about the mechanisms that can bring about these proteolytic events in the numerous physiological contexts in which signal activation occurs. A number of studies have suggested that endocytosis of Notch contributes to the signal activation process, but the molecular details are unclear and controversial. There is conflicting data as to whether endocytosis of the receptor is essential for ligand-induced signalling or supplements it. Other studies have revealed that Notch can be activated in the endosomal pathway, independently of its ligands, through the activity of Deltex, a Ring-domain Ubiquitin ligase that binds to the Notch intracellular domain. However, it is unclear how the Deltex-activation mechanism relates to that of ligand-induced signalling, or to ectopic Notch signalling brought about by disruption of ESCRT complexes that affect multivesicular body formation. This review will address these issues and argue that the data are best reconciled by proposing distinct activation mechanisms in different cellular locations that contribute to the cellular pool of the soluble Notch intracellular domain. The resulting signalling network may provide developmental robustness to environmental and genetic variation.     

Endocytosis and control of Notch signaling

The Notch signaling pathway controls patterning and cell fate decisions during development in metazoans, and is associated with human diseases such as cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and certain cancers. Studies over the last several years have revealed sophisticated regulation of both the membrane-bound Notch receptor and its ligands by vesicle trafficking. This is perhaps most evident in neural progenitor cells in Drosophila, which divide asymmetrically to segregate Numb, an endocytic adaptor protein that acts as a Notch pathway inhibitor, to one daughter cell. Here, we discuss recent findings addressing how receptor and ligand trafficking to specific membrane compartments control activation of the Notch pathway in asymmetrically dividing cells and other tissues.     

Multiple levels of Notch signal regulation (review)

Notch is a vitally important signalling receptor controlling cell fate determination and pattern formation in numerous ways during development of both invertebrate and vertebrate species. An intriguing pathway for the Notch signal has emerged where, after ligand-dependent proteolysis, an intracellular fragment of the receptor itself translocates to the nucleus to regulate gene expression. The nuclear activity of the Notch intracellular domain is linked to complexes regulating chromatin organization through histone deacetylation and acetylation. To allow the Notch signal to be deployed in numerous contexts, many different mechanisms have evolved to regulate the level, duration and spatial distribution of Notch activity. Regulation occurs at multiple levels including patterns of ligand and receptor expression, Notch-ligand interactions, trafficking of the receptor and ligands, and covalent modifications including glycosylation, phosphorylation and ubiquitination. Several Notch regulatory proteins have conserved domains that link them to the ubiquitination pathway, and ubiquitination of the Notch intracellular domain has recently been linked to its degradation. Different proteolytically derived isoforms of Notch have also been identified that may be involved in alternative Notch-dependent signals or regulatory mechanisms, and differences between the four mammalian Notch homologues are beginning to be appreciated.     

An activity of Notch regulates JNK signalling and affects dorsal closure in Drosophila.

BACKGROUND: The Drosophila Notch protein is a receptor that controls cell fate during embryonic development, particularly in lateral inhibition, a process that acts on groups of cells that share a particular developmental potential to restrict the number of cells that will adopt that cell fate. The process of lateral inhibition is implemented by the nuclear protein Suppressor of Hairless (Su(H)) and is triggered by the ligand Delta. Recent results have shown that the interaction between Delta and Notch triggers the cleavage of the intracellular domain of Notch which then translocates to the nucleus and binds to Su(H). RESULTS: We find that Notch plays a role in the patterning of the dorsal epidermis of the Drosophila embryo and that this function of Notch is independent of Su(H), requires Notch at the plasma membrane and targets the c-Jun N-terminal kinase (JNK) signalling pathway. Notch mutants show high levels of JNK activity and can rescue the effects of lowered JNK signalling resulting from mutations in the hemipterous and basket genes. Two regions of the intracellular domain of Notch are involved: the Cdc10/ankyrin repeats, which downregulate signalling through the JNK pathway, and a region carboxy-terminal to these repeats, which regulates this negative function. CONCLUSIONS: Our results reveal a novel signalling activity of Notch that does not require its cleavage and acts by modulating signalling through the JNK pathway. In the Drosophila embryo, this activity plays an important role in the morphogenetic movements that drive dorsal closure.     

Notch信号途径及其调控

Notch是一个进化上十分保守的跨膜受体蛋白家族,对无脊椎动物和脊椎动物发育过程中的细胞命运决定起重要作用。一条重要的Notch信号途径涉及Notch的“三步蛋白质水解”活化。许多相关分子和体内生化过程参与Notch信号途径调控。调控发生在不同水平,包括Notch-配体互作、受体和配体的运输、泛素化降解等。现就Notch受体、Notch信号途径及其所受的不同水平的调控进行综述。     

Role of Cre-loxP-mediated conditional gene targeting in understanding the function of Notch receptors

The Notch family of cell surface receptors and their ligands constitute an evolutionarily conserved signaling pathway that is used by invertebrates and vertebrates to regulate a broad spectrum of cell specification events through local cell interactions. After ligand binding Notch receptor undergoes proteolytic processing ultimately liberating the cytoplasmic domain of the Notch receptor which translocates to the nucleus and activates target genes. In all animal models tested, mutations in Notch genes invariably resulted in developmental abnormalities. In mammals, Notch signaling controls key stages of lymphocyte differentiation as well as activation and several abnormalities in Notch pathway have been suggested to cause human leukemias. Cre-loxP mediated conditional gene targeting significantly contributed to our current understanding of the physiological roles of different Notch family members in hematopoietic compartment. This technique helped to overcome embryonic lethality of Notch mutants providing the opportunity to inactivate specific Notch gene in adulthood.     

Notch pathway: making sense of suppressor of hairless

Suppressor of Hairless (Su(H)) is a DNA-binding protein component of the Notch signalling pathway, thought to be required, with a fragment of the Notch receptor, for target gene activation. Recent studies show that this is only one side of the story: target gene enhancers may be regulated by Su(H) in a variety of different ways.     

Endosomal entry regulates Notch receptor activation in Drosophila melanogaster

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 gamma-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.     

Modulation of developmental signals by endocytosis: different means and many ends

Recent advances have highlighted the importance of endocytic processes in regulating the activity and distribution of developmental signals. Classically, signalling is downregulated by endocytosis and subsequent trafficking to lysosomes (e.g. Notch, Hedgehog, Roundabout). However, endocytosis can also have a positive role in signalling. For example, endocytosis of Delta, the ligand of Notch, is needed for activation of the signal. In the case of signalling by Hedgehog, endocytic trafficking segregates an inhibitory receptor (Patched) from the positive effector (Smoothened). Endosomes could also be the site where signalling is activated (e.g. transforming growth factor beta). Finally, endocytosis could power the transport of morphogens along epithelia.