Role of conserved intracellular motifs in Serrate signalling, cis-inhibition and endocytosis

Notch is the receptor in a signalling pathway that operates in a diverse spectrum of developmental processes. Its ligands (e.g. Serrate) are transmembrane proteins whose signalling competence is regulated by the endocytosis-promoting E3 ubiquitin ligases, Mindbomb1 and Neuralized. The ligands also inhibit Notch present in the same cell (cis-inhibition). Here, we identify two conserved motifs in the intracellular domain of Serrate that are required for efficient endocytosis. The first, a dileucine motif, is dispensable for trans-activation and cis-inhibition despite the endocytic defect, demonstrating that signalling can be separated from bulk endocytosis. The second, a novel motif, is necessary for interactions with Mindbomb1/Neuralized and is strictly required for Serrate to trans-activate and internalise efficiently but not for it to inhibit Notch signalling. Cis-inhibition is compromised when an ER retention signal is added to Serrate, or when the levels of Neuralized are increased, and together these data indicate that cis-inhibitory interactions occur at the cell surface. The balance of ubiquitinated/unubiquitinated ligand will thus affect the signalling capacity of the cell at several levels.     

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.     

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.     

Intracellular trafficking in Drosophila visual system development: a basis for pattern formation through simple mechanisms

Intracellular trafficking underlies cellular functions ranging from membrane remodeling to receptor activation. During multicellular organ development, these basic cell biological functions are required as both passive machinery and active signaling regulators. Exocytosis, endocytosis, and recycling of several key signaling receptors have long been known to actively regulate morphogenesis and pattern formation during Drosophila eye development. Hence, intracellular membrane trafficking not only sets the cell biological stage for receptor-mediated signaling but also actively controls signaling through spatiotemporally regulated receptor localization. In contrast to eye development, the role of intracellular trafficking for the establishment of the eye-to-brain connectivity map has only recently received more attention. It is still poorly understood how guidance receptors are spatiotemporally regulated to serve as meaningful synapse formation signals. Yet, the Drosophila visual system provides some of the most striking examples for the regulatory role of intracellular trafficking during multicellular organ development. In this review we will first highlight the experimental and conceptual advances that motivate the study of intracellular trafficking during Drosophila visual system development. We will then illuminate the development of the eye, the eye-to-brain connectivity map and the optic lobe from the perspective of cell biological dynamics. Finally, we provide a conceptual framework that seeks to explain how the interplay of simple genetically encoded intracellular trafficking events governs the seemingly complex cellular behaviors, which in turn determine the developmental product.     

The ins and outs of Wingless signaling

Signaling through the highly conserved Wingless/Wnt pathway plays a crucial role in a diverse array of developmental processes, many of which depend upon the precise regulation of Wingless/Wnt signaling levels. Recent evidence has indicated that the intracellular trafficking of Wingless/Wnt signaling components can result in significant changes in the level of signaling. Here, we examine three mechanisms through which intracellular trafficking might regulate Wingless signaling--the degradation of Wingless, its transport and the transduction of its signal. The intracellular trafficking of several Wingless/Wnt signaling components, including LRP5, LRP6, Dishevelled and Axin, as well as the functional implications of protein localization on Wingless/Wnt signaling, will be discussed.     

Integrin regulation of caveolin function

Caveolae are unique organelles that are found in the plasma membrane of many cell types. They participate in various processes such as lipid recycling, cellular signalling and endocytosis. A variety of signalling molecules localize to caveolae in response to various stimuli, providing a potential mechanism for the spatial regulation of signal transduction pathways. Caveolin-1, a constitutive protein of caveolae, has been implicated in the regulation of cell growth, lipid trafficking, endocytosis and cell migration. Phosphorylation of caveolin-1 on Tyr 14 is involved in integrin-regulated caveolae trafficking and also in signalling at focal adhesions in migrating cells. In this review, we focus on recent studies that describe the role of caveolin-1 in integrin signal transduction, and how this interplay links extracellular matrix anchorage to cell proliferation, polarity and directional migration.     

Endocytosis and mitosis: A two-way relationship

Cell division involves a vast remodelling of cellular membranes. This is most apparent for the cell surface, but it is also true for internal vesicular organelles such as the Golgi apparatus. While the contribution of endocytosis to membrane trafficking and signal processing in interphase cells is well established, the role of the endocytic system in cell division has long been neglected. A number of recent studies have however shed novel light on this issue. Here, we review findings supporting the existence of two important links between endocytosis and mitosis: First, endocytic trafficking is essential to reshape the plasma membrane during cell division. Second, cell division affects the partitioning, the trafficking and hence the activity of the signalling molecules that are contained within endocytic compartments.     

Regulation of membrane traffic by integrin signaling

Membrane trafficking pathways function to sort and transport cargoes to various intracellular compartments and to the plasma membrane. This allows precise spatiotemporal control of processes such as signal transduction, which in turn is crucial for complex cell functions such as cell division, migration and polarity. Recent studies identified cell-matrix adhesions as regulators of exocytosis, endocytosis and the recycling machinery, thus establishing a new layer of crosstalk between cell adhesion and signaling. This review discusses these findings and considers their implications for signaling events downstream of integrins and growth factor receptors.     

Endocytosis and exocytosis protect cells against severe membrane tension variations

The ability of cells to regulate their shape and volume is critical for many cell functions. How endocytosis and exocytosis, as important ways of membrane trafficking, affect cellular volume regulation is still unclear. Here, we develop a theoretical framework to study the dynamics of cell volume, endocytosis, and exocytosis in response to osmotic shocks and mechanical loadings. This model can not only explain observed dynamics of endocytosis and exocytosis during osmotic shocks but also predict the dynamics of endocytosis and exocytosis during cell compressions. We find that a hypotonic shock stimulates exocytosis, while a hypertonic shock stimulates endocytosis; and exocytosis in turn allows cells to have a dramatic change in cell volume but a small change in membrane tension during hyposmotic swelling, protecting cells from rupture under high tension. In addition, we find that cell compressions with various loading speeds induce three distinct dynamic modes of endocytosis and exocytosis. Finally, we show that increasing endocytosis and exocytosis rates reduce the changes in cell volume and membrane tension under fast cell compression, whereas they enhance the changes in cell volume and membrane tension under slow cell compression. Together, our findings reveal critical roles of endocytosis and exocytosis in regulating cell volume and membrane tension.     

Endocytosis: A Positive or a Negative Influence on Wnt Signalling?

Endocytosis, with subsequent targeting to lysosomes for degradation, is traditionally seen as a way for cells to terminate signalling. However, in a few instances, endocytosis has been demonstrated to contribute positively to signalling. Here we review recent work on the role of endocytosis in Wnt signalling. Biochemical evidence suggests that the branch of Wnt signalling that controls planar cell polarity (PCP) does require endocytosis, although how endocytosis of Frizzled receptors is translated into PCP in vivo remains unknown. With respect to the main signalling branch (called the canonical or beta-catenin pathway), the literature is divided as to whether endocytosis is required. Results of in vivo experiments are inconclusive because of the toxic side-effects of blocking endocytosis. Some results with cultured cells suggest the need for endocytosis in canonical signalling; however, it remains unclear whether the ligand-receptor complex must enter the cell by clathrin-mediated or caveolae-mediated endocytosis in order to signal. Means of specifically altering Wnt trafficking as well as of tracking the internalization route in different cell types are needed.     

Notch信号转导与调控

Notch是一个进化上十分保守的跨膜受体蛋白家族,它可以通过与表达配体的相邻细胞间的相互作用转导信号,从而决定动物系统发育过程中多种细胞的“命运”.Notch信号转导过程包括Notch受体与配体的结合、Notch受体的酶切活化、可溶性NICD转移至细胞核并与CSL DNA结合蛋白相互作用,从而调控靶基因的表达.Notch活性水平、时间和空间分布受到包括配体、蛋白质转运、泛素化降解等多水平内源性和外源性诱导因素的调节.系统介绍了Notch信号转导通路的分子组成、Notch信号激活的生化机制、Notch信号的多水平调节以及与部分相关疾病的关系.     

The hepatitis C virus non-structural protein NS5A alters the trafficking profile of the epidermal growth factor receptor

Hepatitis C virus (HCV) frequently establishes a persistent infection, leading to chronic liver disease. The NS5A protein has been implicated in this process as it modulates a variety of intracellular signalling pathways that control cell survival and proliferation. In particular, NS5A associates with several proteins involved in the endocytosis of the epidermal growth factor receptor (EGFR) and has been previously shown to inhibit epidermal growth factor (EGF)-stimulated activation of the Ras–Erk pathway by a mechanism that remains unclear. As EGFR signalling involves trafficking to late endosomes, we investigated whether NS5A perturbs EGFR signalling by altering receptor endocytosis. We demonstrate that NS5A partially localizes to early endosomes and, although it has no effect on EGF internalization, it colocalizes with the EGFR and alters its distribution. This redistribution correlates with a decrease in the amount of active EGF–EGFR ligand–receptor complexes present in the late endosomal signalling compartment and also results in a concomitant increase in the total levels of EGFR. These observations suggest that NS5A controls EGFR signalling by diverting the receptor away from late endosomes. This represents a novel mechanism by which a viral protein attenuates cell signalling and suggests that NS5A may perturb trafficking pathways to maintain an optimal environment for HCV persistence.     

Internal affairs: investigating the Brucella intracellular lifestyle

Bacteria of the genus Brucella are Gram-negative pathogens of several animal species that cause a zoonotic disease in humans known as brucellosis or Malta fever. Within their hosts, brucellae reside within different cell types where they establish a replicative niche and remain protected from the immune response. The aim of this article is to discuss recent advances in the field in the specific context of the Brucella intracellular 'lifestyle'. We initially discuss the different host cell targets and their relevance during infection. As it represents the key to intracellular replication, the focus is then set on the maturation of the Brucella phagosome, with particular emphasis on the Brucella factors that are directly implicated in intracellular trafficking and modulation of host cell signalling pathways. Recent data on the role of the type IV secretion system are discussed, novel effector molecules identified and how some of them impact on trafficking events. Current knowledge on Brucella gene regulation and control of host cell death are summarized, as they directly affect intracellular persistence. Understanding how Brucella molecules interplay with their host cell targets to modulate cellular functions and establish the intracellular niche will help unravel how this pathogen causes disease.     

Emerging concepts of receptor endocytosis and concurrent intracellular signaling: Mechanisms of guanylyl cyclase/natriuretic peptide receptor-A activation and trafficking

Endocytosis is a prominent clathrin-mediated mechanism for concentrated uptake and internalization of ligand-receptor complexes, also known as cargo. Internalization of cargo is the fundamental mechanism for receptor-dependent regulation of cell membrane function, intracellular signal transduction, and neurotransmission, as well as other biological and physiological activities. However, the intrinsic mechanisms of receptor endocytosis and contemporaneous intracellular signaling are not well understood. We review emerging concepts of receptor endocytosis with concurrent intracellular signaling, using a typical example of guanylyl cyclase/natriuretic peptide receptor-A (NPRA) internalization, subcellular trafficking, and simultaneous generation of second-messenger cGMP and signaling in intact cells. We highlight the role of short-signal motifs located in the carboxyl-terminal regions of membrane receptors during their internalization and subsequent receptor trafficking in organelles that are not traditionally studied in this context, including nuclei and mitochondria. This review sheds light on the importance of future investigations of receptor endocytosis and trafficking in live cells and intact animals in vivo in physiological context.     

EphA2 Signaling Following Endocytosis: Role of Tiam1

Eph receptors and their membrane‐bound ligands, the ephrins, represent a complex subfamily of receptor tyrosine kinases (RTKs). Eph/ephrin binding can lead to various and opposite cellular behaviors such as adhesion versus repulsion, or cell migration versus cell‐adhesion. Recently, Eph endocytosis has been identified as one of the critical steps responsible for such diversity. Eph receptors, as many RTKs, are rapidly endocytosed following ligand‐mediated activation and traffic through endocytic compartments prior to degradation. However, it is becoming obvious that endocytosis controls signaling in many different manners. Here we showed that activated EphA2 are degraded in the lysosomes and that about 35% of internalized receptors are recycled back to the plasma membrane. Our study is also the first to demonstrate that EphA2 retains the capacity to signal in endosomes. In particular, activated EphA2 interacted with the Rho family GEF Tiam1 in endosomes. This association led to Tiam1 activation, which in turn increased Rac1 activity and facilitated Eph/ephrin endocytosis. Disrupting Tiam1 function with RNA interference impaired both ephrinA1‐dependent Rac1 activation and ephrinA1‐induced EphA2 endocytosis. In summary, our findings shed new light on the regulation of EphA2 endocytosis, intracellular trafficking and signal termination and establish Tiam1 as an important modulator of EphA2 signaling .     

The range of spalt-activating Dpp signalling is reduced in endocytosis-defective Drosophila wing discs.

Pattern formation along the anterior-posterior (A/P) axis of the developing Drosophila wing depends on Decapentaplegic (Dpp), a member of the conserved transforming growth factor beta (TGFbeta) family of secreted proteins. Dpp is expressed in a stripe along the A/P compartment boundary of the wing imaginal disc and forms a long-range concentration gradient with morphogen-like properties which generates distinct cell fates along the A/P axis. We have monitored Dpp expression and Dpp signalling in endocytosis-mutant wing imaginal discs which develop severe pattern defects specifically along the A/P wing axis. The results show that the size of the Dpp expression domain is expanded in endocytosis-mutant wing discs. However, this expansion did not result in a concomitant expansion of the functional range of Dpp activity but rather its reduction as indicated by the reduced expression domain of the Dpp target gene spalt. The data suggest that clathrin-mediated endocytosis, a cellular process necessary for membrane recycling and vesicular trafficking, participates in Dpp action during wing development. Genetic interaction studies suggest a link between the Dpp receptors and clathrin. Impaired endocytosis does not interfere with the reception of the Dpp signal or the intracellular processing of the mediation of the signal in the responder cells, but rather affects the secretion and/or the distribution of Dpp in the developing wing cells.     

Ligand endocytosis drives receptor dissociation and activation in the Notch pathway

Endocytosis of the ligand delta; is required for activation of the receptor Notch during Drosophila development. The Notch extracellular domain (NotchECD) dissociates from the Notch intracellular domain (NotchICD) and is trans-endocytosed into delta;-expressing cells in wild-type imaginal discs. Reduction of dynamin-mediated endocytosis in developing eye and wing imaginal discs reduces Notch dissociation and Notch signalling. Furthermore, dynamin-mediated delta endocytosis is required for Notch trans-endocytosis in Drosophila cultured cell lines. Endocytosis-defective delta proteins fail to mediate trans-endocytosis of Notch in cultured cells, and exhibit aberrant subcellular trafficking and reduced signalling capacity in Drosophila. We suggest that endocytosis into delta-expressing cells of NotchECD bound to delta plays a critical role during activation of the Notch receptor and is required to achieve processing and dissociation of the Notch protein.     

The role of palmitoylation in signalling, cellular trafficking and plasma membrane localization of protease-activated receptor-2

Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. This irreversible activation mechanism leads to rapid receptor desensitization by internalisation and degradation. We have explored the role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Experiments using the palmitoylation inhibitor 2-bromopalmitate indicated that palmitate addition is important in trafficking of PAR2 endogenously expressed by prostate cancer cell lines. This was supported by palmitate labelling using two approaches, which showed that PAR2 stably expressed by CHO-K1 cells is palmitoylated and that palmitoylation occurs on cysteine 361. Palmitoylation is required for optimal PAR2 signalling as Ca²⁺ flux assays indicated that in response to trypsin agonism, palmitoylation deficient PAR2 is ∼9 fold less potent than wildtype receptor with a reduction of about 33% in the maximum signal induced via the mutant receptor. Confocal microscopy, flow cytometry and cell surface biotinylation analyses demonstrated that palmitoylation is required for efficient cell surface expression of PAR2. We also show that receptor palmitoylation occurs within the Golgi apparatus and is required for efficient agonist-induced rab11a-mediated trafficking of PAR2 to the cell surface. Palmitoylation is also required for receptor desensitization, as agonist-induced β-arrestin recruitment and receptor endocytosis and degradation were markedly reduced in CHO-PAR2-C361A cells compared with CHO-PAR2 cells. These data provide new insights on the life cycle of PAR2 and demonstrate that palmitoylation is critical for efficient signalling, trafficking, cell surface localization and degradation of this receptor.     

Microtubule motors at the intersection of trafficking and transport

Molecular motors drive the transport of vesicles and organelles within the cell. Traditionally, these transport processes have been considered separately from membrane trafficking events, such as regulated budding and fusion. However, recent progress has revealed mechanistic links that integrate these processes within the cell. Rab proteins, which function as key regulators of intracellular trafficking, have now been shown to recruit specific motors to organelle membranes. Rab-independent recruitment of motors by adaptor or scaffolding proteins is also a key mechanism. Once recruited to vesicles and organelles, these motors can then drive directed transport; this directed transport could in turn affect the efficiency of trafficking events. Here, we discuss this coordinated regulation of trafficking and transport, which provides a powerful mechanism for temporal and spatial control of cellular dynamics.