Endocytic Regulation of Notch Signalling During Development

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1 Traffic 2009; 10: Review 2009 John Wiley & Sons A/S doi: /j x Endocytic Regulation of Notch Signalling During Development Maximilian Fürthauer* and Marcos González-Gaitán* Department of Biochemistry and Molecular Biology, University of Geneva, Sciences II, 30 quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland Corresponding author: Maximilian Fürthauer, and Marcos González-Gaitán, 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. Key words: asymmetric cell division, delta, development, endocytosis, multivesicular endosomes, notch, signalling Received 19 December 2008, revised and accepted for publication 12 March 2009, published online 22 April 2009 Endocytosis, the internalization of material from the cell s plasma membrane or its extracellular environment, plays a key role in the regulation of cellular physiology (1). While endocytosis is often perceived as a way to downregulate signalling, endosomal compartments can also be important for effective signal transduction. This was first shown for the EGF-receptor, which requires endocytosis to activate its full set of downstream effectors (2). The primordial role of endocytic trafficking for the transduction of biological signals is particularly evident in the case of the Delta/Notch pathway (3). Delta/Notch signalling is of major importance for embryonic development and adult life. During development, these molecules have been shown to be implicated in three different types of processes: Inductive signalling occurs at the interface between two fields of cells, one of which is presenting ligands to activate receptor molecules in neighbouring tissue. This type of interaction results in the formation of tissue boundaries: During fly wing and vertebrate limb development, Delta/Notch signalling between the dorsal and ventral compartments of the appendage is required to induce the expression of growth factors required for proper appendage outgrowth (4,5). During lateral inhibition one cell is singled out from a group of equipotent precursors. Ligand and receptor molecules are initially expressed in all cells, but stochastic variations in gene expression cause small differences in Notch activation in different cells. As Notch activation down-regulates ligand expression, Delta is progressively restricted to a single cell while Notch is activated in its neighbours. Lateral inhibition contributes notably to the equal spacing of sensory organs, be it the sensory bristles of the fly thorax or the hair cells in the vertebrate ear (4,5). Asymmetric cell divisions (ACDs) can result in unequal partitioning of regulators of Delta/Notch signalling. As a consequence, one of the daughter cells will present ligand molecules that cause Notch activation in its sibling. This type of interaction is involved for example in the specification of different cell types during the development of Drosophila mecanosensory bristles (Figure 1A) (6) as well as the maintenance of adult intestinal stem cells (7). The molecular basis of Delta/Notch signal transduction has been covered by several excellent reviews (3,8). Briefly, signalling is initiated by binding of a membranebound Delta/Serrate/Lag2 (DSL) family ligand to a transmembrane Notch receptor. This interaction triggers a first S2 cleavage in the extracellular juxtamembrane region of Notch, followed by constitutive γ -Secretasedependent intra-membrane proteolysis. This later S3 cleavage releases the Notch intracellular domain (NICD) into the cytoplasm. NICD then shuttles to the nucleus 792

2 Endocytosis and Notch Signalling in Development Figure 1: Legend on next page. Traffic 2009; 10:

3 Fürthauer and González-Gaitán to associate with transcriptional cofactors of the CBF1- Su(H)-Lag1 (CSL) family and activate target genes (8). Over 10 years ago, work carried out in Drosophila suggested that Delta/Notch signalling requires dynamin-dependent endocytosis in both signal-sending and signal-receiving cells (9). More recently, studies in flies and zebrafish showed that internalization of the ligand in signal-sending cells is required to activate Notch in signal-receiving cells (10,11). While endocytosis is crucial for Delta/Notch signalling, the basis for this requirement remains poorly understood. We review here a number of recent studies that aim at understanding the role of endocytosis mechanistically. Asymmetric Endocytosis during ACD The development of Drosophila mecanosensory bristles is a powerful model to study Delta/Notch signalling (6) (Figure 1A). In this system, differential segregation of cell fate determinants during the ACD of sensory organ precursor (SOP) cells causes the formation of two intrinsically different daughter cells, piib and piia. Neuralized, a Ubiquitin-ligase mediating Delta internalization, is segregated into piib, favouring Delta internalization in this cell (10) (Figure 1B1). Shortly after division, a Rab11-positive recycling compartment is established in the centrosomal region of piib, favouring Delta recycling in this cell (12) (Figure 1B2). In addition to the bias imposed on the endocytic regulation of Delta, ACD causes the preferential inheritance of the endocytic proteins Numb and α-adaptin by the piib daughter cell, where they retrieve Notch and its cofactor Sanpodo from the plasma membrane (13,14) (Figure 1B4). Taken together, these mechanisms predispose piib for ligand presentation and piia for signal reception. The observations mentioned above suggested that directional Delta/Notch signalling following SOP division is achieved through asymmetric segregation of factors that differentially regulate trafficking of ligand and receptor in the two daughter cells after division. But what is the behaviour of Delta and Notch themselves? This issue has previously been addressed by in vivo labelling of Delta molecules with antibodies, and following them after internalization in a chase experiment where the fate of the endocytosed cargo is assessed after fixation (10). In a recent study, endogenous Delta and Notch were followed in real time during ACD (15). Delta/Notch traffics to a population of endosomes positive for the early endosomal marker Rab5 (16) and the transforming growth factor beta (TGFβ) signalling adapter SARA (Smad Anchor for Receptor Activation) (17,18). During ACD, SARA endosomes and Delta/Notch therein are first targeted to the contractile actin ring before being transferred to the central spindle and then directionally transported into the posterior piia daughter cell where Notch signalling will subsequently be activated (15) (Figure 1B3). SARA endosomes are a subpopulation of multivesicular endosomes (MVEs) (17). MVEs, i.e. endosomes that contain a characteristic accumulation of vesicles in their lumen (19), are formed by budding of endosomal membranes away from the cytoplasm (20). The most well-known MVEs are the so-called multivesicular bodies (MVBs), intermediates involved in the transport from early to late endosomes (19). Numerous studies have shown that the ESCRT (Endosomal Sorting Complex Required for Transport) machinery is critical to incorporate ubiquitinated cargo into the internal vesicles of MVBs in order to ensure their subsequent lysosomal degradation (20). Figure 1: Endocytic trafficking regulates the activity of Delta ligands. A) Drosophila mechanosensory bristle lineage. The SOP (also called pi) and its progeny undergo a series of ACDs to form the four cells that compose the adult sense organ (depicted on the right). Following pi division, the anterior piib cell presents Delta to activate Notch in its posterior piia sister. Similarly, directional Delta/Notch signalling is involved after the piiib and piia divisions. Asymmetric division of piib gives rise to a small glial cell that soon undergoes apoptosis. B) Endocytic regulation of Delta/Notch signalling during ACD. Images depict cells before (a), during (b,c) and after (d,e) SOP division. B1) During mitosis, Neuralized (purple) is asymmetrically recruited to the cortex of the SOP (b) and its piib daughter cell (c,d). Asymmetric Neuralized activity directs Delta internalization (broken arrows, b-e) into early endosomes (red dots) in the anterior piib daughter cell. In interphase cells before (a) and after (e) division Neuralized is detected at the nuclear envelope. B2) During mitosis Rab11-positive endosomes (yellow dots) are equally partitioned among daughter cells (c). Shortly after division, a transient Rab11-positive recycling compartment is established in the centrosomal region of the anterior piib cell (d). This enhances Delta recycling (broken arrows in d) from early endosomes (red dots) to the plasma membrane in piib. B3) In the first 10 min after internalization, Delta resides in SARA-negative Rab5-positive endosomes (red dots) that are equally partitioned during mitosis (c,d). Beyond 10 min Delta is found in endosomes that are positive for both Rab5 and SARA (green dots). SARA endosomes line up a contractile cortical actin ring (grey in b,c) in metaphase. During cytokinesis, SARA endosomes are transferred to the central spindle (black lines in c) and directionally moved into the posterior piia daughter cell (c,d). 10 min after division, de novo formation of SARA endosomes in piib occurs through recruitment of cytoplasmic SARA protein (e). B4) Before division, the Notch cofactor Sanpodo (Blue) is detected in intracellular dots (a). During division, Sanpodo is cytoplasmic (b,c). After division, asymmetric Numb and α-adaptin (orange) retrieve Sanpodo from the piib cell plasma into endosomes (d,e). In piia Sanpodo remains at the membrane, allowing it to promote Notch signalling. C) Delta endocytosis and recycling have distinct effects on ligand recycling, receptor binding and Notch activation. I) Ubiquitinated wild-type Delta-like 1 (Dll1), a Dll1 mutant that cannot be ubiquitinated (Dll1K17R) and a chimeric ligand composed of the extracellular part of Dll1 and the intracellular part of Dll3 (Dll1/3) are all endocytosed. II) Dll1K17R cannot be recycled. As Dll1K17R cannot bind Notch, this suggests that ligand recycling is a prerequisite for receptor binding. Dll1 and Dll1/3 are recycled to the plasma membrane and bind Notch. III) Only Dll1 is targeted to raft-like membrane domains allowing it to activate and trans-endocytose Notch. 794 Traffic 2009; 10:

4 Endocytosis and Notch Signalling in Development Not all proteins that accumulate in the internal membranes of MVEs are however destined for degradation (19). A ceramide-dependent mechanism has been shown to direct lipid-based sorting of specific cargo into exosomal carriers (21). Moreover, higher eucaryotic cells can retrieve proteins and lipids from the internal membranes of late endosomes through a mechanism involving the lipid Lysobisphosphatidic Acid (LBPA), its effector Alix (22) and the ESCRT component Tsg101 (23). In the case of SARA-positive MVEs, several studies have indicated a role of these endosomes in TGFβ signalling. In vertebrate cell culture, SARA is required for Smad2/3-mediated TGFβ signalling (18). During Drosophila wing development, SARA endosomes ensure the equal partitioning of Dpp signalling components among mitotic sister cells (17). The analysis of SARA endosomes during Drosophila mechanosensory bristle development similarly supports a positive role of these organelles in Delta/Notch signal transduction. Mistargeting of SARA endosomes to the wrong daughter cell causes ectopic Notch activation and cell fate transformation (15). This suggests that directional segregation of Delta/Notch-containing SARA endosomes during ACD is important for asymmetric Notch activation after division. Asymmetric partitioning of Notch has also been observed during cell division in the developing mammalian brain cortex, suggesting that this may represent a conserved mechanism for the generation of cellular diversity during development (24). SARA endosomes ensure the directional transport of Delta/Notch during ACD and affect the differential activation of Notch signalling in the two daughter cells. The asymmetric endosomes are however not dedicated exclusively to Delta/Notch signalling: SARA endosomes also mediate asymmetric segregation of Dally-like, a Glypican involved in the regulation of numerous signalling pathways (25), within a certain time-window after internalization (15). This suggests that the asymmetric transport of molecules by SARA endosomes is not due to a specific sorting of the cargo that enters these carriers, but rather to the specific kinetics with which particular molecules transit through this endocytic compartment. Integrating Different Mechanisms of Asymmetric Delta Trafficking There are three mechanisms known to bias Delta trafficking during sensory bristle development: (i) Directional transport by SARA endosomes (15) (Figure 1B3); (ii) Neural ized-dependent ligand internalization (10) (Figure 1B1) and (iii) Rab11-dependent ligand recycling (12) (Figure 1B2). How do the three mechanisms relate to each other? A tantalizing possibility is that the three mechanisms allow to adapt the endocytic trafficking of Delta to a changing environment: As development proceeds, ligand trafficking changes both in time (before division versus after division) and space (in piib versus piia). Ligand molecules that have been internalized long before division (i.e. more than 45 min before the completion of cytokinesis) will already reside in late degradative compartments by the time of mitosis. As these molecules will be degraded and have no impact on signalling, there is probably no need for the cell to enforce any bias on the inheritance of late endosomes. Delta molecules that have been internalized more recently (less than 45 min but more than 10 min before cytokinesis) will reside in SARA-positive MVEs at the moment of division. As these endosomes can affect Delta/Notch signalling in the daughter cells, it seems important to transfer these organelles and/or the signalling molecules therein to the piia daughter cell in which Notch signalling will be subsequently activated (15) (Figure 1B3c,d). The transport of Delta in SARA endosomes allows to impart an asymmetric behaviour on ligand molecules that have been internalized at a stage where the SOP itself did not yet show any signs of asymmetry. Delta molecules that have been internalized in the last 10 min before cytokinesis will not have entered the asymmetrically segregating SARA compartment by the moment of division. They reside in Rab5-positive SARA-negative endosomes that partition equally among the two daughter cells (Figure 1B3c,d). After division, Neuralized, which has been segregated to the cortex of the anterior daughter cell, enhances the internalization of Delta from the surface of the piib cell, as compared with its piia sister (10) (Figure 1B1d,e). Moreover, a Rab11-positive recycling compartment is established in the centrosomal region of piib about 3 min after cytokinesis (12) and persists for about 10 min (our own unpublished observations). This compartment will likely recycle Delta molecules that have been internalized after division as well as those that resided in Rab5-positive SARA-negative carriers during division (Figure 1B2d). If Delta molecules were not recycled from pre-sara compartments in piib, they could otherwise reach SARApositive MVEs and might elicit signalling in the wrong daughter cell. Beyond a short time window (about 15 min) following cytokinesis, the molecular asymmetries that bias Delta processing in the two daughter cells decay: Neuralized is less enriched at the piib cell membrane (Figure 1B1e), the Rab11-positive compartment at the piib centrosome dissociates (Figure 1B2e) and the asymmetry of SARA endosomes diminishes (from a 15-fold enrichment in piia right after the abscission to 3-fold later; Figure 1B3e). These observations raise the question whether asymmetric processing of Delta and/or Notch activation occur only in a short time-window following division. While we can distinguish different phases in Delta behaviour, we do not know at which step (and by which Delta molecules: endocytosed in the mother or in the daughter?) Notch is Traffic 2009; 10:

5 Fürthauer and González-Gaitán activated. While temporal analysis of Notch signalling in this system has mapped receptor activation to the first hour after division (15,26), the development of dynamic reporters of Notch activity will be crucial to address this issue. Why Is Ligand Endocytosis Required for Notch Activation? The existence of different phases of ligand trafficking and/or activity is further substantiated by the group of Frédérique Logeat, which has studied the biological consequences of Delta internalization for ligand function (27). It is easily conceivable that endocytosis of an activated receptor favours its interaction with endosomal downstream effectors. But why should internalization of a ligand contribute to receptor activation? Two types of models have been considered to explain the positive effect of Delta endocytosis on Notch activation (28,29). On one hand, internalization of receptor-bound Delta could exert a physical pulling force on the Notch peptide that would change its conformation and expose the juxtamembrane cleavage S2 site to allow its access by ADAM (A Disintegrin And Metalloprotease) family metalloproteases. Alternatively, Delta internalization and recycling have been proposed to promote the formation of active ligand molecules. Activation could occur through modification of Delta in an endocytic compartment, or by preferential trafficking to a cellular environment prone to productive ligand/receptor interactions. Using mammalian cell culture, Heuss and coworkers show that, in their system, Ubiquitination of the Delta homologue Delta-like 1 (Dll1) is not required for ligand internalization (Figure 1CI). Rather, Ubiquitination acts as a sorting signal that sends Dll1 towards recycling to specific lipid raft-like areas of the plasma membrane. Only Dll1 molecules that have been recycled are able to bind Notch, providing a point in favour of the model stating that Delta endocytosis achieves ligand activation. These findings are in good agreement with previous work performed in the context of boundary formation in Drosophila wing imaginal discs (30). In this system, mono-ubiquitination and the action of the endocytic adapter protein Epsin are dispensable for bulk internalization of Delta, but required for its sorting into a specific ligand-activating compartment. The new study adds more detail to this picture by showing that ligand recycling and acquisition of Notchbinding affinity are not sufficient for receptor activation: A chimeric ligand containing the extracellular part of Dll1 and the intracellular part of Delta-like 3 (Dll3, which is not ubiquitinated) can be internalized, recycled and bind Notch (Figure 1CII). Despite this, chimeric Dll1/3 is unable to activate Notch and induce subsequent T- cell differentiation. Moreover, only wild-type Dll1 but not Dll1/3 is able to elicit trans-endocytosis of the extracellular part of Notch upon receptor binding, an event that occurs concomitant with receptor activation (31,32) (Figure 1CIII). Interestingly, Dll1/3 differs from wild-type Dll1 by its inability to be sorted to lipid-ordered membrane domains. These observations suggest that lipid rafts may represent a membrane environment that is required for Dll1-mediated Notch activation. Taken together, the data in this study suggest that, to activate Notch, Dll1 has to undergo two rounds of internalization: a first one to acquire affinity for the receptor and be sorted to a specific membrane environment and a second one that leads to receptor trans-endocytosis and activation. It is clear that the second event conforms well to the model according to which Delta endocytosis physically pulls on Notch to activate it. This work therefore suggests that the activation versus pulling functions that have until now been assigned to Delta endocytosis may not be mutually exclusive but rather provide complementary inputs into the regulation of this signalling molecule. What Are the Factors that Direct Ligand Internalization? Several studies have shown that the activity of the E3 Ubiquitin-ligase Neuralized is required for Delta internalization (10,11). As the work of Heuss and coworkers shows that Delta ubiquitination is dispensable for ligand internalization (27), it is tempting to speculate that Neuralized may promote Delta internalization through a novel, ubiquitination-independent mechanism. The group of Gabrielle Boulianne has shown that, in addition to previously identified protein domains, Neuralized contains an N-terminal phosphoinositide-binding motif (33). Interestingly, deletion analysis reveals that this motif does not seem to affect Delta ubiquitination while being required for ligand internalization during embryonic neurogenesis and wing development in Drosophila. It will be interesting to see how phosphoinositide binding by Neuralized contributes to the regulation of Delta internalization. The function of Delta ligands is to bind Notch. Is this interaction required for Delta internalization? While it is clear that the E3-Ubiquitin ligases Neuralized and Mindbomb direct Delta internalization in flies and vertebrates (10,11,34), it is not clear whether endocytosed ligand molecules are bound to receptors. Work by the group of Ajay Chitnis has revealed that during zebrafish neurogenesis Notch1a and Notch3 are required for the internalization of DeltaD and DeltaA (35). Interestingly knock-down of Notch has no effect on DeltaC, suggesting once again that different Delta homologues follow different endocytic routes. While the loss of Notch inhibits Delta endocytosis, no such effect is observed upon inactivation of signalling components that act downstream of the receptor. This suggests that it is 796 Traffic 2009; 10:

6 Endocytosis and Notch Signalling in Development A. Intercellular Delta/Notch signalling I. Delta/Notch binding II. S2 cleavage Delta III.a. S3 cleavage at the plasma membrane γ-sec Notch III.b. Endosomal S3 cleavage nucleus γ-sec B. Intraendosomal Delta/Notch signalling Delta Intralumenal vesicle NICD Notch MVE limiting membrane nucleus 1 2' 3' 4' 5' C. Ligand-independent Notch signalling from late endosomes Notch Dx Rab5 HOPS AP3 Rab7 Su(Dx) Dx Complete degradation Partial degradation nucleus γ-sec Figure 2: Legend on next page. not the signal transduced by Notch, but rather the presence of the Notch protein itself that is required for Delta internalization. One possible interpretation of these results is that direct ligand/receptor interactions may be required for Delta internalization. The presence of Notch in a cell can promote the internalization of Delta molecules both in the same cell (i.e. in cis) and in neighbouring cells (i.e. in trans). It is tempting to speculate that the later interaction may correspond to productive ligand/receptor binding, while the former could be inhibitory. Overexpression of high levels of Delta ligands in Drosophila has indeed been shown to exert a cis-inhibitory effect on Notch activation (36,37). In flies as well as in vertebrates, the physiological relevance of this type of interaction remains however to be established. Traffic 2009; 10:

7 Fürthauer and González-Gaitán Notch Activation from Endosomal Compartments Until now, we have discussed the role of endocytosis for the formation of active ligand molecules. But what about the importance of Notch internalization for receptor activation? According to the current textbook model, Notch signalling is initiated when a DSL ligand on the surface of one cell binds a receptor molecule presented by a neighbouring cell. This ligand/receptor interaction triggers the metalloprotease-mediated S2 cleavage and subsequent shedding of the Notch extracellular domain (NECD). In a second step, γ -Secretase causes the intramembrane S3 proteolyis of the remaining fragment, releasing hence the NICD in the cytoplasm from where it shuttles to the nucleus to activate target genes (8). While the different steps to Notch activation have been identified, the cellular compartment in which Notch is cleaved and activated remains controversial. Some studies have suggested that the entire cascade of activating cleavages can occur at the plasma membrane (Figure 2A, (38,39)). However, the increasing number of studies reporting effects of endocytic trafficking on Notch activation prompts the question whether transit through endocytic compartments may actually be required for receptor activation! One of the first indications that endocytosis may be important for Notch activation came from work of Gupta Rossi and coworkers who showed in vertebrate cell culture that mono-ubiquitination and internalization of Notch are required for the final γ -Secretase cleavage (40). More recently, similar results have been obtained through the use of mutants that perturb endosomal entry in Drosophila (41). Evidence for an important role for the endocytic machinery has further come from studies of the fly ESCRT machinery which directs the incorporation of endocytic cargo into the intra-lumenal vesicles of MVEs. Interestingly, inactivation of Hrs, an upstream component involved in the initial step of cargo selection, causes accumulation of Notch protein but has no effect on Notch signalling (42,43). In contrast, inactivation of more downstream components of the ESCRTI or ESCRTII subcomplexes causes ectopic Notch activation and tumorous overproliferation of eye and wing imaginal discs (44 46). This has led to the proposal that ESCRTI/II mutants accumulate Notch in a compartment that is naturally prone for receptor activation. SARA Endosomes and Notch Activation SARA endosomes ensure the asymmetric segregation of internalized Delta/Notch into piia during ACD (15) (Figure 1B3). Following division of wild-type SOPs, Notch is activated in piia but not in its piib sister. If SARA endosomes are however mistargeted to piib, they elicit ectopic Notch signalling and transform the fate of this daughter cell. These observations suggest that the SARA endosome itself or the Delta/Notch molecules therein affect the signalling levels of the cell that inherits them. The first of these two (not mutually exclusive) possibilities would be consistent with previous reports that suggested a preferential localization and activity of the γ -Secretase complex in the acidic environment of the endosomal system (Figure 2AIIIb) (47). In this scenario asymmetric segregation of Sara endosomes in piia would concentrate the organelle in which the final activating γ -Secretase cleavage of Notch could be performed, hence contributing to a signalling bias between sister cells. Alternatively, it could be envisaged that Delta and Notch contained in SARA endosomes interact within this organelle to elicit signalling. In this situation, Notch pathway activation should by accompanied by a change in endosomal cargo content. This is indeed the case. In the absence of Notch activation, Delta as well as the extra- and intracellular moieties of Notch are detected in SARA endosomes. This combined presence of NECD and NICD is commonly thought to indicate the presence of intact full-length receptor molecules. It is however Figure 2: Different modes of Notch activation. A) Receptor activation during intercellular Delta/Notch signalling. I) Delta on the membrane of the signal-sending cell binds Notch on the surface of the adjacent signal-receiving cell. II) Delta internalization elicits juxtamembrane S2 cleavage and trans-endocytosis of the NECD. III) Intra-membrane S3 proteolysis of the remaining membrane-bound Notch fragment is carried out by γ -Secretase (γ -Sec) at the plasma membrane (IIIa) or in endosomes (IIIb). In both cases, the Notch intracelular domain is released into the cytoplasm to shuttle to the nucleus and activate target genes. B) Proposed model for intra-endosomal Delta/Notch signalling. Depicted is the behaviour of Delta/Notch within a single multivesicular endosome (MVE). 1) After internalization Delta and Notch are found at the limiting membrane of the endosome. Invagination of the limiting membrane (2,2 ) can translocate Delta (3) ornotch(3 ) to intra-lumenal vesicles. This allows Delta/Notch to adopt an anti-parallel configuration required for productive ligand/receptor interaction. Notch activation releases the Notch intracellular domain (NICD) into the cytoplasm (4) orthe lumen of internal vesicles (4 ). In the later case maturation of MVEs into late endosomes could cause back-fusion of intra-lumenal vesicles with the endosomal limiting membrane, releasing hence NICD into the cytoplasm (5 ). C) Ligand-independent Notch signalling from late endosomes. The ubiquitin-ligase Deltex (Dx) promotes ligand-independent internalization of intact Notch into Rab5-positive early endosomes (red). The HOPS and AP3 complexes promote cargo transport to Rab7-positive late endosomes (blue) where Notch can follow two different routes: The ubiquitin-ligase Suppressor of Deltex (Su(Dx)) directs the incorporation of Notch into the internal vesicles (black ring) of late endosomes, followed by the complete degradation of the protein. Deltex promotes retention of Notch at the limiting membrane of late endosomes. As a consequence, only the extracellular part of Notch is degraded in the late endosomal lumen, giving rise to a fragment that can be further processed by γ -Secretase to release active NICD. 798 Traffic 2009; 10:

8 Endocytosis and Notch Signalling in Development still possible that even if NECD and NICD colocalize in the same compartment they are already separated after Notch cleavage. In contrast, in cells undergoing active Notch signalling, NICD is no more detected in SARA endosomes while Delta and NECD are still present (15). The disappearance of NICD from SARA endosomes requires the activity of both Delta and γ - Secretase, hence confirming that it can be considered as a bona fide readout of Notch activation. These data are compatible with the hypothesis that Delta and Notch have interacted productively within SARA endosomes to elicit the release of active NICD from the endocytic compartment into the cytoplasm (Figure 2B). Alternatively, Delta binding and γ -Secretase cleavage of Notch may occur at the plasma membrane, but NICD would not be released from NECD until it reaches Sara endosomes. One of the main problems for a model in which ligand and receptor are proposed to interact within the same organelle is to bring the two partners into a proper anti-parallel orientation to allow productive binding. While this is trivial if ligand and receptor are present on the membrane of two different cells that contact each other, a mechanism has to be invoked in order to achieve a similar topology if both are inside the same endosome. The fact that SARA endosomes are MVEs containing intra-lumenal vesicles could however provide a relatively simple answer to this question. After internalization Delta and Notch would first be found at the limiting membrane of SARA endosomes. At this stage Notch and Delta would be in a parallel orientation in the membrane, precluding their positive interaction and preventing premature Notch activation (Figure 2B1). Interestingly, the morphology of SARA endosomes changes as cells enter division, i.e. before the activation of Notch signalling (15). While these endosomes appear as hollow spheres before division, progression through mitosis appears to correlate with increased formation of intra-lumenal vesicles. Concomitantly, Delta and Notch which are detected mostly at the MVE limiting membrane before division are detected in the MVE lumen after mitosis, in structures that may correspond to intralumenal vesicles. It is tempting to speculate that these changes play a causative role in the activation of Notch signalling following cell division. If ligand or receptor molecules are incorporated into these internal vesicles, they would then be in an anti-parallel configuration with respect to their binding partners remaining on the limiting membrane. At this point, two topologies are conceivable (Figure 2B2). If Delta is present on the internal vesicles, it may interact now with Notch on the limiting membrane in order to elicit release of NICD into the cytoplasm (Figure 2B3). Conversely, if Delta remains on the limiting membrane to interact with Notch on intra-lumenal vesicles, productive interaction between these two molecules would result in the release of NICD into the lumen of the internal vesicles (Figure 2B4). Interestingly, acidification and maturation of MVEs into late endosomes have been shown to be accompanied by a back-fusion process in which the internal vesicles merge again with the limiting membrane (22). As a result, the NICD fragments that have been initially released into the lumen of the intra-lumenal vesicles would now be expelled into the cytoplasm and able to access the nucleus (Figure 2B5). ph-dependent Regulation of Delta/Notch Signalling Interestingly, intra-endosomal Delta/Notch signalling would be compatible with a recent report concerning the ph-dependency of ligand/receptor interaction (48). Using a biochemical approach, Pei and Baker analyse the interactions between Delta, the ligand-binding domain of Notch as well as the Notch Abruptex domain. Mutations in the Abruptex domain have long been known to give rise to Notch gain-of-function phenotypes in Drosophila (49). Pei and Baker find that under the neutral ph conditions encountered in the extracellular space, Delta and the Notch Abruptex domain bind the ligand-binding domain of Notch with comparable affinities. As a consequence the Abruptex domain may successfully compete with Delta for interaction with the Notch ligand-binding domain and prevent productive Delta/Notch binding at the cell surface. If the ph is however lowered to a more acidic state compatible with an endosomal environment, the affinity of Delta for the ligand-binding domain of Notch increases substantially compared to the one of the Abruptex domain. This in vitro study therefore suggests that productive Delta/Notch interactions would not only be possible, but may actually be favoured in an endosomal environment. A study of the fly ESCRT complex has similarly suggested that the spectacular tumorigenic overgrowth which is observed during the wing and eye development of vps25 mutant flies could be due to the combined accumulation of Delta and Notch in the same endosomal compartment (46). As these tumours represent clearly a pathological condition, this raises the question as to whether intra-endosomal Delta/Notch signalling is something that can be observed under mutant conditions or something that should also contribute to normal development. A similar point can be made for the directional segregation of SARA endosomes during ACD: it is unclear whether the primary function of the asymmetric segregation of Delta/Notch into piia is to enforce signalling in this daughter cell or to prevent signalling in its piib sister (15). The importance of endosomal ph regulation is also highlighted by a recent study of the Drosophila Aquaporin mutant big brain by Kanwar and Fortini (50). Big brain mutations impair Notch signalling during boundary formation in wing imaginal discs as well as during lateral inhibition (51). Under wild-type conditions, Delta/Notch Traffic 2009; 10:

9 Fürthauer and González-Gaitán binding triggers a proteolytic cleavage of the NECD. The remaining Notch extracellular truncation (NEXT) fragment is then constitutively processed by γ -secretase to release the NICD into the cytoplasm from where it shuttles to the nucleus to activate target genes. While misexpression of NICD can restore Notch signalling in big brain mutants, a NEXT construct fails to elicit a similar rescue. This suggests that although a functional γ -Secretase complex is present in Aquaporin mutants, these animals fail to process NEXT to generate NICD. Loss of Aquaporin function impairs endosomal acidification and maturation so that early endosomes form abnormal apical clusters and progression towards late endocytic compartments is inhibited. Taken together, these data suggest that the transit of Notch through different endocytic compartments is essential for the final stages of receptor activation. Notch Activation from Late MVEs The model of intra-endosomal Delta/Notch signalling presented above proposes that translocation of signalling molecules from the limiting membrane of the MVE to its internal vesicles may be important for receptor activation. Interestingly a recent report by the lab of Martin Baron suggest that this type of internalversus-limiting sorting is important for the regulation of ligand-independent Notch signalling from late endosomes during inductive boundary formation in Drosophila wing discs (52) (Figure 2C). Through a study of the HOPS (Homotypic fusion and vacuole protein sorting) and AP-3 (Adaptor protein 3) complexes involved in late endosomal trafficking, Wilkin and coworkers show that the E3-Ubiquitin-ligase Deltex directs the internalization of Notch molecules to a late endosomal compartment. Internalized Notch molecules can follow two routes. If Notch is incorporated into the intra-lumenal vesicles of late MVEs it will be degraded. Alternatively, Deltex can inhibit incorporation of Notch into internal vesicles and promote its retention at the limiting membrane. In this second scenario, only the NECD domain of Notch would be inside the lysosomal lumen where it would be progressively degraded. This process would ultimately yield a molecule that resembles the one normally generated through ligand-induced S2 cleavage and ectodomain shedding. Lysosomal γ -Secretase would then carry out the S3 cleavage and release the NICD fragment from the limiting membrane of the endosome into the cytoplasm. Conclusion As research progresses, it becomes apparent that the endocytic requirements for Delta/Notch signalling depend on the biological context. For example, Numb and Sanpodo are essential modulators of Notch activity during ACD, but do not appear to play a role during other signalling events (53). Conversely, loss of function of the Drosophila Epsin liquid facets impairs Notch activation during inductive boundary formation and lateral inhibition, but not in the context of ACD (30). For many other endocytic regulators including the ESCRT-, AP3- or HOPS complexes, their importance for Delta/Notch signalling has presently only been studied in a single biological context. An additional level of complexity is added by the observation that different vertebrate Delta homologues are internalized and recycled through different mechanisms (27,35). These observations clearly suggest that there may not be a simple, unique answer to explain the role of endocytosis for Delta/Notch signalling. It may well be that, depending on the biological context, Notch activation occurs by different mechanisms: While endocytosis may be dispensable for receptor activation at the plasma membrane, it may favour intra-endosomal ligand receptor interactions and be essential for ligand-independent signalling from late endosomes. It will be an interesting challenge for the future to find out why different Delta/Notchmediated signalling events require different endocytic regulators. Recent studies have significantly improved our understanding of the contribution of endocytic trafficking to the regulation of Delta/Notch signalling. However, the novel insights also raise a number of major issues. First, it is presently clear that it is essential to distinguish different phases of Delta trafficking. This is obvious notably during ACD where ligand molecules that have been internalized in the mother cell or in either of its daughters traffic differently (10,12,15). This raises two obvious questions. Which Delta molecules (from the mother? from the daughter?) activate Notch and when/where (at the plasma membrane? in the endosome?) do they do it? Simultaneous monitoring of ligand trafficking and receptor activation will be required to address this issue. Second, the observation that Delta-like1 activates Notch only when incorporated into raft-like domains (27) highlights the importance of cellular microenvironments for signalling. How can we identify potential signalling domains within cellular compartments in living tissue? An increasing number of studies indicate that domains of different lipid composition play an important, but presently poorly understood, role in the functional compartmentalization of cellular signalling pathways (21,54). Finally, the asymmetric segregation of endosomes and endocytic regulators contributes to the development of Drosophila mecanosensory bristles. Are these mechanisms also important for the regulation of Notch signalling in other biological contexts? It has been reported that asymmetric endosome segregation also occurs in dividing larval brain stem cells (15). It will therefore be interesting to analyse the importance of asymmetric endocytic trafficking during stem cell self-renewal. 800 Traffic 2009; 10:

10 Endocytosis and Notch Signalling in Development References 1. Conner SD, Schmid SL. Regulated portals of entry into the cell. Nature 2003;422: Vieira AV, Lamaze C, Schmid SL. Control of EGF receptor signaling by clathrin-mediated endocytosis. Science 1996;274: Hurlbut GD, Kankel MW, Lake RJ, Artavanis-Tsakonas S. Crossing paths with Notch in the hyper-network. Curr Opin Cell Biol 2007;19: Lewis J. Notch signalling and the control of cell fate choices in vertebrates. Semin Cell Dev Biol 1998;9: Bray S. Notch signalling in Drosophila: three ways to use a pathway. Semin Cell Dev Biol 1998;9: Le Borgne R, Bardin A, Schweisguth F. The roles of receptor and ligand endocytosis in regulating Notch signaling. Development 2005;132: Ohlstein B, Spradling A. The adult Drosophila posterior midgut is maintained by pluripotent stem cells. Nature 2006;439: Bray SJ. Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol 2006;7: Seugnet L, Simpson P, Haenlin M. Requirement for dynamin during Notch signaling in Drosophila neurogenesis. Dev Biol 1997;192: Le Borgne R, Schweisguth F. Unequal segregation of Neuralized biases Notch activation during asymmetric cell division. Dev Cell 2003;5: Itoh M, Kim CH, Palardy G, Oda T, Jiang YJ, Maust D, Yeo SY, Lorick K, Wright GJ, Ariza-McNaughton L, Weissman AM, Lewis J, Chandrasekharappa SC, Chitnis AB. Mind bomb is a ubiquitin ligase that is essential for efficient activation of Notch signaling by Delta. Dev Cell 2003;4: Emery G, Hutterer A, Berdnik D, Mayer B, Wirtz-Peitz F, Gaitan MG, Knoblich JA. Asymmetric Rab 11 endosomes regulate delta recycling and specify cell fate in the Drosophila nervous system. Cell 2005;122: Berdnik D, Torok T, Gonzalez-Gaitan M, Knoblich JA. The endocytic protein alpha-adaptin is required for numb-mediated asymmetric cell division in Drosophila. Dev Cell 2002;3: Hutterer A, Knoblich JA. Numb and alpha-adaptin regulate Sanpodo endocytosis to specify cell fate in Drosophila external sensory organs. EMBO Rep 2005;6: Coumailleau F, Fürthauer M, Knoblich JA, Gonzalez-Gaitan M. Directional delta/notch trafficking in asymmetric Sara endosomes during asymmetric cell division. Nature doi: /nature Wucherpfennig T, Wilsch-Brauninger M, Gonzalez-Gaitan M. Role of Drosophila Rab5 during endosomal trafficking at the synapse and evoked neurotransmitter release. J Cell Biol 2003;161: Bokel C, Schwabedissen A, Entchev E, Renaud O, Gonzalez-Gaitan M. Sara endosomes and the maintenance of Dpp signaling levels across mitosis. Science 2006;314: Tsukazaki T, Chiang TA, Davison AF, Attisano L, Wrana JL. SARA, a FYVE domain protein that recruits Smad2 to the TGFbeta receptor. Cell 1998;95: Gruenberg J, Stenmark H. The biogenesis of multivesicular endosomes. Nat Rev Mol Cell Biol 2004;5: Williams RL, Urbe S. The emerging shape of the ESCRT machinery. Nat Rev Mol Cell Biol 2007;8: Trajkovic K, Hsu C, Chiantia S, Rajendran L, Wenzel D, Wieland F, Schwille P, Brügger B, Simons M. Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science 2008;319: Le Blanc I, Luyet PP, Pons V, Ferguson C, Emans N, Petiot A, Demaurex N, Mayran N, Fauré J, Sadoul R, Parton RG, Gruenberg J. Endosome-to-cytosol transport of viral nucleocapsids. Nat Cell Biol 2005;7: Luyet PP, Falguieres T, Pons V, Pattnaik AK, Gruenberg J. The ESCRT- I subunit TSG101 controls endosome-to-cytosol release of viral RNA. Traffic 2008;9: Chenn A, McConnell SK. Cleavage orientation and the asymmetric inheritance of Notch1 immunoreactivity in mammalian neurogenesis. Cell 1995;82: Perrimon N, Hacker U. Wingless, hedgehog and heparan sulfate proteoglycans. Development 2004;131: ; author reply Remaud S, Audibert A, Gho M. S-phase favours Notch cell responsiveness in the Drosophila bristle lineage. PLoS ONE 2008;3:e Heuss SF, Ndiaye-Lobry D, Six EM, Israel A, Logeat F. The intracellular region of Notch ligands Dll1 and Dll3 regulates their trafficking and signaling activity. Proc Natl Acad Sci USA 2008;105: Le Borgne R. Regulation of Notch signalling by endocytosis and endosomal sorting. Curr Opin Cell Biol 2006;18: Chitnis A. Why is delta endocytosis required for effective activation of Notch? Dev Dyn 2006;235: Wang W, Struhl G. Drosophila Epsin mediates a select endocytic pathway that DSL ligands must enter to activate Notch. Development 2004;131: Parks AL, Klueg KM, Stout JR, Muskavitch MA. Ligand endocytosis drives receptor dissociation and activation in the Notch pathway. Development 2000;127: Nichols JT, Miyamoto A, Olsen SL, D Souza B, Yao C, Weinmaster G. DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur. J Cell Biol 2007;176: Skwarek LC, Garroni MK, Commisso C, Boulianne GL. Neuralized contains a phosphoinositide-binding motif required downstream of ubiquitination for delta endocytosis and Notch signaling. Dev Cell 2007;13: Le Borgne R, Remaud S, Hamel S, Schweisguth F. Two distinct E3 ubiquitin ligases have complementary functions in the regulation of delta and serrate signaling in Drosophila. PLoS Biol 2005;3:e Matsuda M, Chitnis AB. Interaction with Notch determines endocytosis of specific Delta ligands in zebrafish neural tissue. Development 2009;136: Micchelli CA, Rulifson EJ, Blair SS. The function and regulation of cut expression on the wing margin of Drosophila: Notch, wingless and a dominant negative role for delta and serrate. Development 1997;124: Jacobsen TL, Brennan K, Arias AM, Muskavitch MA. Cis-interactions between delta and Notch modulate neurogenic signalling in Drosophila. Development 1998;125: Struhl G, Adachi A. Requirements for presenilin-dependent cleavage of Notch and other transmembrane proteins. Mol Cell 2000;6: Lopez-Schier H, St Johnston D. Drosophila nicastrin is essential for the intramembranous cleavage of Notch. Dev Cell 2002;2: Gupta-Rossi N, Six E, LeBail O, Logeat F, Chastagner P, Olry A, Israël A, Brou C. Monoubiquitination and endocytosis direct gamma-secretase cleavage of activated Notch receptor. J Cell Biol 2004;166: Vaccari T, Lu H, Kanwar R, Fortini ME, Bilder D. Endosomal entry regulates Notch receptor activation in Drosophila melanogaster. J Cell Biol 2008;180: Lloyd TE, Atkinson R, Wu MN, Zhou Y, Pennetta G, Bellen HJ. Hrs regulates endosome membrane invagination and tyrosine kinase receptor signaling in Drosophila. Cell 2002;108: Jekely G, Rorth P. Hrs mediates downregulation of multiple signalling receptors in Drosophila. EMBO Rep 2003;4: Vaccari T, Bilder D. The Drosophila tumor suppressor vps25 prevents nonautonomous overproliferation by regulating Notch trafficking. Dev Cell 2005;9: Traffic 2009; 10:

11 Fürthauer and González-Gaitán 45. Moberg KH, Schelble S, Burdick SK, Hariharan IK. Mutations in erupted, the Drosophila ortholog of mammalian tumor susceptibility gene 101, elicit non-cell-autonomous overgrowth. Dev Cell 2005;9: Thompson BJ, Mathieu J, Sung HH, Loeser E, Rorth P, Cohen SM. Tumor suppressor properties of the ESCRT-II complex component Vps25 in Drosophila. Dev Cell 2005;9: Pasternak SH, Bagshaw RD, Guiral M, Zhang S, Ackerley CA, Pak BJ, Callahan JW, Mahuran DJ. Presenilin-1, nicastrin, amyloid precursor protein, and gamma-secretase activity are co-localized in the lysosomal membrane. J Biol Chem 2003;278: Pei Z, Baker NE. Competition between Delta and the Abruptex domain of Notch. BMC Dev Biol 2008;8: de Celis JF, Garcia-Bellido A. Modifications of the Notch function by Abruptex mutations in Drosophila melanogaster. Genetics 1994;136: Kanwar R, Fortini ME. The big brain aquaporin is required for endosome maturation and Notch receptor trafficking. Cell 2008;133: Rao Y, Jan LY, Jan YN. Similarity of the product of the Drosophila neurogenic gene big brain to transmembrane channel proteins. Nature 1990;345: Wilkin M, Tongngok P, Gensch N, Clemence S, Motoki M, Yamada K, Hori K, Taniguchi-Kanai M, Franklin E, Matsuno K, Baron M. Drosophila HOPS and AP-3 complex genes are required for a Deltex-regulated activation of Notch in the endosomal trafficking pathway. Dev Cell 2008;15: O Connor-Giles KM, Skeath JB. Numb inhibits membrane localization of Sanpodo, a four-pass transmembrane protein, to promote asymmetric divisions in Drosophila. Dev Cell 2003;5: Rajendran L, Schneider A, Schlechtingen G, Weidlich S, Ries J, Braxmeier T, Schwille P, Schulz JB, Schroeder C, Simons M, Jennings G, Knölker HJ, Simons K. Efficient inhibition of the Alzheimer s disease beta-secretase by membrane targeting. Science 2008;320: Traffic 2009; 10: