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© 2022 The Authors. This is an open access article under the CC BY-NC-ND license http://creativecommons.org/licenses/by-nc-nd/4.0/)., Intracellular pathogens manipulate host cells to survive and thrive. Cellular sensing and signaling pathways are among the key host machineries deregulated to favor infection. In this study, we show that liver-stage Plasmodium parasites compete with the host to sequester a host endosomal-adaptor protein (APPL1) known to regulate signaling in response to endocytosis. The enrichment of APPL1 at the parasitophorous vacuole membrane (PVM) involves an atypical Plasmodium Rab5 isoform (Rab5b). Depletion of host APPL1 alters neither the infection nor parasite development; however, upon overexpression of a GTPase-deficient host Rab5 mutant (hRab5_Q79L), the parasites are smaller and their PVM is stripped of APPL1. Infection with the GTPase-deficient Plasmodium berghei Rab5b mutant (PbRab5b_Q91L) in this case rescues the PVM APPL1 signal and parasite size. In summary, we observe a robust correlation between the level of APPL1 retention at the PVM and parasite size during exoerythrocytic development., This work was supported by grants from the la Caixa Banking Foundation and Fundação para a Ciência e a Tecnologia (HR17-00264 and PTDC/SAU-PAR/30751/2017 respectively, both to M.M.M.). A.L and S.S.B were sponsored by Fundação para a Ciência e a Tecnologia fellowships (PD/BD/114036/2015, SFRH/BD/114464/2016, respectively). V.S acknowledges funding from the Science and Engineering Research Board (SERB) (EMR/2016/006810), Department of Science and Technology (DST), Government of India. V.P. and C.S. were supported by GRK2581 (P6) SPHINGOINF of the Growing Spine Foundation (DFG).

International audience; Many cellular organelles, including endosomes, show compartmentalization into distinct functional domains, which, however, cannot be resolved by diffraction-limited light microscopy. Single molecule localization microscopy (SMLM) offers nanoscale resolution but data interpretation is often inconclusive when the ultrastructural context is missing. Correlative light electron microscopy (CLEM) combining SMLM with electron microscopy (EM) enables correlation of functional subdomains of organelles in relation to their underlying ultrastructure at nanometer resolution. However, the specific demands for EM sample preparation and the requirements for fluorescent single-molecule photo-switching are opposed. Here, we developed a novel superCLEM workflow that combines triple-color SMLM (dSTORM & PALM) and electron tomography using semi-thin Tokuyasu thawed cryosections. We applied the superCLEM approach to directly visualize nanoscale compartmentalization of endosomes in HeLa cells. Internalized, fluorescently labeled Transferrin and EGF were resolved into morphologically distinct domains within the same endosome. We found that the small GTPase Rab5 is organized in nanodomains on the globular part of early endosomes. The simultaneous visualization of several proteins in functionally distinct endosomal sub-compartments demonstrates the potential of superCLEM to link the ultrastructure of organelles with their molecular organization at nanoscale resolution.

Long-distance axonal trafficking plays a critical role in neuronal function, and transport defects have been linked to neurodegenerative disorders. Various lines of evidence suggest that the small GTPase Rab5 plays a role in neuronal signaling via early endosomal transport. Here, we characterized the motility of Rab5 endosomes in primary cultures of mouse hippocampal pyramidal cells by live-cell imaging and showed that they exhibit bi-directional long-range motility in axons, with a strong bias toward retrograde transport. Characterization of key Rab5 effectors revealed that endogenous Rabankyrin-5, Rabenosyn-5 and APPL1 are all present in axons. Further analysis of APPL1-positive endosomes showed that, similar to Rab5-endosomes, they display more frequent long-range retrograde than anterograde movement, with the endosomal levels of APPL1 correlated with faster retrograde movement. Interestingly, APPL1-endosomes transport the neurotrophin receptor TrkB and mediate retrograde axonal transport of the kinase Akt1. FRET analysis revealed that APPL1 and Akt1 interact in an endocytosis-dependent manner. We conclude that Rab5-APPL1 endosomes exhibit the hallmarks of axonal signaling endosomes to transport Akt1 in hippocampal pyramidal cells.

Detection of nucleic acids within sub-cellular compartments is key to understanding their function. Determining the intracellular distribution of nucleic acids requires quantitative retention and estimation of their association with different organelles by immunofluorescence microscopy. This is important also for the delivery of nucleic acid therapeutics which depends on endocytic uptake and endosomal escape. However, the current methods fail to preserve the majority of exogenously delivered nucleic acids in the cytoplasm. To solve this problem, by monitoring Cy5-labeled mRNA delivered to primary human adipocytes via lipid nanoparticles (LNP), we optimized cell fixation, permeabilization and immuno-staining of a number of organelle markers, achieving quantitative retention of mRNA and allowing visualization of levels which escape detection using conventional procedures. Additionally, we demonstrated the protocol to be effective on exogenously delivered siRNA, miRNA, as well as endogenous miRNA. Our protocol is compatible with RNA probes of single molecule fluorescence in-situ hybridization (smFISH) and molecular beacon, thus demonstrating that it is broadly applicable to study a variety of nucleic acids.

Delivery of exogenous mRNA using lipid nanoparticles (LNP) is a promising strategy for therapeutics. However, a bottleneck remains the poor understanding of the parameters that correlate with endosomal escape vs. cytotoxicity. To address this problem, we compared the endosomal distribution of six LNP-mRNA formulations of diverse chemical composition and efficacy, similar to those employed in mRNA-based vaccines, in primary human adipocytes, fibroblasts and HeLa cells. Surprisingly, we found that total uptake is not a sufficient predictor of delivery and different LNP vary considerably in endosomal distributions. Prolonged uptake impaired endosomal acidification, a sign of cytotoxicity, and caused mRNA to accumulate in compartments defective in cargo transport and unproductive for delivery. In contrast, early endocytic/recycling compartments have the highest probability for mRNA escape. By super-resolution microscopy we could resolve single LNP-mRNA within sub-endosomal compartments and capture events of mRNA escape from endosomal recycling tubules. Our results change the view of the mechanisms of endosomal escape and define quantitative parameters to guide the development of mRNA formulations towards higher efficacy and lower cytotoxicity.

Proteins can self-organize into spatial patterns via non-linear dynamic interactions on cellular membranes. Modelling and simulations have shown that small GTPases can generate patterns by coupling guanine nucleotide exchange factors (GEF) to effector binding, generating a positive feedback of GTPase activation and membrane recruitment. Here, we reconstituted the patterning of the small GTPase Rab5 and its GEF/effector complex Rabex5/Rabaptin5 on supported lipid bilayers as a model system for membrane patterning. We show that there is a “handover” of Rab5 from Rabex5 to Rabaptin5 upon nucleotide exchange. A minimal system consisting of Rab5, RabGDI and a complex of full length Rabex5/Rabaptin5 was necessary to pattern Rab5 into membrane domains. Surprisingly, a lipid membrane composition mimicking that of the early endosome was required for Rab5 patterning. The prevalence of GEF/effector coupling in nature suggests a possible universal system for small GTPase patterning involving both protein and lipid interactions.

High-content screens (HCS) using chemical and genomic interference based on light microscopy and quantitative image analysis yielded a large amount of multi-parametric (MP) phenotypic data. Such data-sets hold great promise for the understanding of cellular mechanisms by systems biology. However, extracting functional information from data-sets, such as links between cellular processes and the functions of unknown genes, remains challenging. The limitation of HCS analysis lies in the complexity of cellular organization. Here, we assumed that cellular processes have a modular structure, and deconvolved the MP data into separate signals from different cellular modules by Blind Source Separation. We applied a combination of quantitative MP image analysis (QMPIA) and Independent Component Analysis (ICA) to an image-based HCS of endocytosis, the process whereby cells uptake molecules from the outside and distribute them to different sub-cellular organelles. We named our approach Independent Phenotypes Analysis (IPA). Phenotypic traits revealed by IPA are interpretable in terms of perturbation of specific endosomal populations (e.g. specific cargo, specific molecular markers) and of specific functional modules (early stages of endocytosis, recycling, cell cycle, etc.). The profile of perturbation of each gene in such basic phenotypic coordinates intrinsically suggest its possible mode of action.

Many cellular organelles, including endosomes, show compartmentalization into distinct functional domains, which however cannot be resolved by diffraction-limited light microscopy. Single molecule localization microscopy (SMLM) offers nanoscale resolution but data interpretation is often inconclusive when the ultrastructural context is missing. Correlative light electron microscopy (CLEM) combining SMLM with electron microscopy (EM) enables correlation of functional sub-domains of organelles in relation to their underlying ultrastructure at nanometer resolution. However, the specific demands for EM sample preparation and the requirements for fluorescent single-molecule photo-switching are opposed. Here, we developed a novel superCLEM workflow that combines triple-colour SMLM (dSTORM & PALM) and electron tomography using semi-thin Tokuyasu thawed cryosections. We applied the superCLEM approach to directly visualize nanoscale compartmentalization of endosomes in HeLa cells. Internalized, fluorescently labelled Transferrin and EGF were resolved into morphologically distinct domains within the same endosome. We found that the small GTPase Rab5 is organized in nano-domains on the globular part of early endosomes. The simultaneous visualization of several proteins in functionally distinct endosomal sub-compartments demonstrates the potential of superCLEM to link the ultrastructure of organelles with their molecular organization at nanoscale resolution.SynopsisSuborganelle compartmentalization cannot be resolved by diffraction limited light microscopy and not interpreted without knowledge of the underlying ultrastructure. This work shows a novel superCLEM workflow that combines multi-colour single-molecule localization-microscopy with electron tomography to map several functional domains on early endosomes. superCLEM reveals that the small GTPase Rab5 is organized in nano-domains largely devoid from cargo molecules Transferrin and EGF and opens new possibilities to perform structure-function analysis of organelles at the nanoscale.

Mitochondrial stress response is essential for cell survival, and damaged mitochondria are a hallmark of neurodegenerative diseases. Thus, it is fundamental to understand how mitochondria relay information within the cell. Here, by investigating mitochondrial-endosomal contact sites we made the surprising observation that the small GTPase Rab5 translocates from early endosomes to mitochondria upon oxidative stress. This process is reversible and accompanied by an increase in Rab5-positive endosomes in contact with mitochondria. Interestingly, activation of Rab5 on mitochondria depends on the Rab5-GEF ALS2/Alsin, encoded by a gene mutated in amyotrophic lateral sclerosis (ALS). Alsin-deficient human-induced pluripotent stem cell-derived spinal motor neurons are defective in relocating Rab5 to mitochondria and display increased susceptibility to oxidative stress. These findings define a novel pathway whereby Alsin catalyzes the assembly of the Rab5 endocytic machinery on mitochondria. Defects in stress-sensing by endosomes could be crucial for mitochondrial quality control during the onset of ALS., eLife digest The inside of a human cell is divided into compartments called organelles, which are surrounded by membranes. Each organelle plays a specific role in keeping the cell healthy and also has unique mix of molecular markers on its surface. These markers allow other molecules to identify the different organelles, meaning that specific organelles can communicate with each other and coordinate their activities. One way that organelles can do this is via so-called membrane contact sites, which are small areas where the compartments’ outer membranes come close together. Mitochondria are organelles that release energy inside human cells. These compartments also work to keep the levels of toxic chemicals called reactive oxygen species in the cell within a safe range. This is important because cells can die if these levels become too high – a state known as oxidative stress. Mitochondria also communicate with other organelles called endosomes, which receive materials from the cell surface, sort and direct them to different destinations throughout the cell. In many diseases affecting the nervous system, the mitochondria and endosomes in nerve cells do not work properly. These cells also have higher than normal levels of oxidative stress. Hsu et al. therefore wanted to find out if mitochondria and endosomes worked together to help cells to cope with this kind of stress. Hsu et al. triggered oxidative stress in human cancer cells by exposing them first to a dye that stained the mitochondria and then to intense light. In stressed cells, a subset of endosomes called early endosomes formed many more membrane contact sites with mitochondria than in non-stressed cells. At the same time, the protein Rab5, usually found on early endosomes, relocated to the surface of mitochondria. Human cells previously engineered to produce larger than normal amounts of Rab5 were also more likely to survive oxidative stress. These experiments suggested that early endosomes cooperate with mitochondria, via Rab5, to protect cells from oxidative stress. So, how is Rab5 relocated to mitochondria? Hsu et al. searched for activators of Rab5 and found that Alsin also migrated to mitochondria in stressed cells. The gene for Alsin is also mutated in amyotrophic lateral sclerosis (ALS), a degenerative nerve disorder that remains poorly understood. Next, Hsu et al. deleted the gene for Alsin from human stem cells growing in the laboratory and coaxed these cells into becoming nerve cells. Experiments with these cells showed that the absence of Alsin prevented Rab5 from moving to the mitochondria. Nerve cells lacking Alsin were also more susceptible to oxidative stress than normal cells. Together, these findings show that early endosomes work with mitochondria to sense and ward off oxidative stress. They also reveal an unexpected connection between this process and a gene mutated in ALS. Further experiments are now needed to explore if problems with endosomes or mitochondria, and specifically with molecules like Alsin and Rab5, are responsible for other neurodegenerative disorders, like Parkinson’s disease and Huntington’s disease.

APPL1 vesicles represent a distinct population of Rab5-positive early endosomes that undergoes fusion and fission and sorts and exchanges cargo with EEA1 endosomes, suggesting a new organization of the endocytic pathway., Endocytosis allows cargo to enter a series of specialized endosomal compartments, beginning with early endosomes harboring Rab5 and its effector EEA1. There are, however, additional structures labeled by the Rab5 effector APPL1 whose role in endocytic transport remains unclear. It has been proposed that APPL1 vesicles are transport intermediates that convert into EEA1 endosomes. Here, we tested this model by analyzing the ultrastructural morphology, kinetics of cargo transport, and stability of the APPL1 compartment over time. We found that APPL1 resides on a tubulo-vesicular compartment that is capable of sorting cargo for recycling or degradation and that displays long lifetimes, all features typical of early endosomes. Fitting mathematical models to experimental data rules out maturation of APPL1 vesicles into EEA1 endosomes as a primary mechanism for cargo transport. Our data suggest instead that APPL1 endosomes represent a distinct population of Rab5-positive sorting endosomes, thus providing important insights into the compartmental organization of the early endocytic pathway.

Most delivery systems for small interfering RNA therapeutics depend on endocytosis and release from endo-lysosomal compartments. One approach to improve delivery is to identify small molecules enhancing these steps. It is unclear to what extent such enhancers can be universally applied to different delivery systems and cell types. Here, we performed a compound library screen on two well-established siRNA delivery systems, lipid nanoparticles and cholesterol conjugated-siRNAs. We identified fifty-one enhancers improving gene silencing 2-5 fold. Strikingly, most enhancers displayed specificity for one delivery system only. By a combination of quantitative fluorescence and electron microscopy we found that the enhancers substantially differed in their mechanism of action, increasing either endocytic uptake or release of siRNAs from endosomes. Furthermore, they acted either on the delivery system itself or the cell, by modulating the endocytic system via distinct mechanisms. Interestingly, several compounds displayed activity on different cell types. As proof of principle, we showed that one compound enhanced siRNA delivery in primary endothelial cells in vitro and in the endocardium in the mouse heart. This study suggests that a pharmacological approach can improve the delivery of siRNAs in a system-specific fashion, by exploiting distinct mechanisms and acting upon multiple cell types.

Early endosomes are organized in a network of vesicles shaped by cycles of fusion, fission, and conversion to late endosomes. In yeast, endosome fusion and conversion are regulated, among others, by CORVET, a hexameric protein complex. In the mammalian endocytic system, distinct subpopulations of early endosomes labelled by the Rab5 effectors APPL1 and EEA1 are present. Here, the function of mammalian CORVET with respect to these endosomal subpopulations was investigated. Tgfbrap1 as CORVET-specific subunit and functional ortholog of Vps3p was identified, demonstrating that it is differentially distributed between APPL1 and EEA1 endosomes. Surprisingly, depletion of CORVET-specific subunits caused fragmentation of APPL1-positive endosomes but not EEA1 endosomes in vivo. These and in vitro data suggest that CORVET plays a role in endosome fusion independently of EEA1. Depletion of CORVET subunits caused accumulation of large EEA1 endosomes indicative of another role in the conversion of EEA1 endosomes into late endosomes. In addition, depletion of CORVET-specific subunits caused alterations in transport depending on both the type of cargo and the specific endosomal subpopulation. These results demonstrate that CORVET plays distinct roles at multiple stages in the mammalian endocytic pathway.

Liprin-α1 and ERC1 are interacting scaffold proteins regulating the motility of normal and tumor cells. They act as part of plasma membrane-associated platforms at the edge of motile cells to promote protrusion by largely unknown mechanisms. Here we identify an amino-terminal region of the liprin-α1 protein (liprin-N) that is sufficient and necessary for the interaction with other liprin-α1 molecules. Similar to liprin-α1 or ERC1 silencing, expression of the liprin-N negatively affects tumor cell motility and extracellular matrix invasion, acting as a dominant negative by interacting with endogenous liprin-α1 and causing the displacement of the endogenous ERC1 protein from the cell edge. Interfering with the localization of ERC1 at the cell edge inhibits the disassembly of focal adhesions, impairing protrusion. Liprin-α1 and ERC1 proteins colocalize with active integrin β1 clusters distinct from those colocalizing with cytoplasmic focal adhesion proteins and influence the localization of peripheral Rab7-positive endosomes. We propose that liprin-α1 and ERC1 promote protrusion by displacing cytoplasmic adhesion components to favour active integrin internalization into Rab7-positive endosomes.

SummaryBackgroundEndocytosis allows the import and distribution of cargo into a series of endosomes with distinct morphological and biochemical characteristics. Our current understanding of endocytic cargo trafficking is based on the kinetics of net cargo transport between endosomal compartments without considering individual endosomes. However, endosomes form a dynamic network of membranes undergoing fusion and fission, thereby continuously exchanging and redistributing cargo. The macroscopic kinetic properties, i.e., the properties of the endosomal network as a whole, result from the collective behaviors of many individual endosomes, a problem so far largely unaddressed.ResultsHere, we developed a general theoretical framework to describe the dynamics of cargo distributions in the endosomal network. We combined the theory with quantitative experiments to study how the macroscopic kinetic properties of the endosomal network emerge from microscopic processes at the level of individual endosomes. We compared our theory predictions to experimental data in which dynamic distributions of endocytosed low-density lipoprotein (LDL) were quantified.ConclusionsOur theory can quantitatively describe the observed cargo distributions as a function of time. Remarkably, the theory allows determining microscopic kinetic parameters such as the fusion rate between endosomes from still images of cargo distributions at different times of internalization. We show that this method is robust and sensitive because cargo distributions result from an average over many stochastic events in many cells. Our results provide theoretical and experimental support to the “funnel model” of endosome progression and suggest that the conversion of early to late endosomes is the major mode of LDL trafficking.

An outstanding question is how cells control the number and size of membrane organelles. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis. Here, to test this hypothesis, we developed a mathematical model of endosome dependency on Rab5 and validated it by titrating down all three Rab5 isoforms in adult mouse liver using state-of-the-art RNA interference technology. Unexpectedly, the endocytic system was resilient to depletion of Rab5 and collapsed only when Rab5 decreased to a critical level. Loss of Rab5 below this threshold caused a marked reduction in the number of early endosomes, late endosomes and lysosomes, associated with a block of low-density lipoprotein endocytosis. Loss of endosomes caused failure to deliver apical proteins to the bile canaliculi, suggesting a requirement for polarized cargo sorting. Our results demonstrate for the first time, to our knowledge, the role of Rab5 as an endosome organizer in vivo and reveal the resilience mechanisms of the endocytic system.

Contains fulltext : 70768.pdf (Publisher’s version ) (Closed access) During development of multicellular organisms, cells respond to extracellular cues through nonlinear signal transduction cascades whose principal components have been identified. Nevertheless, the molecular mechanisms underlying specificity of cellular responses remain poorly understood. Spatial distribution of signaling proteins may contribute to signaling specificity. Here, we tested this hypothesis by investigating the role of the Rab5 effector Appl1, an endosomal protein that interacts with transmembrane receptors and Akt. We show that in zebrafish, Appl1 regulates Akt activity and substrate specificity, controlling GSK-3beta but not TSC2. Consistent with this pattern, Appl1 is selectively required for cell survival, most critically in highly expressing tissues. Remarkably, Appl1 function requires its endosomal localization. Indeed, Akt and GSK-3beta, but not TSC2, dynamically associate with Appl1 endosomes upon growth factor stimulation. We propose that partitioning of Akt and selected effectors onto endosomal compartments represents a key mechanism contributing to the specificity of signal transduction in vertebrate development.

An outstanding question is how receptor tyrosine kinases (RTKs) determine different cell-fate decisions despite sharing the same signalling cascades. Here, we uncovered an unexpected mechanism of RTK trafficking in this process. By quantitative high-resolution FRET microscopy, we found that phosphorylated epidermal growth factor receptor (p-EGFR) is not randomly distributed but packaged at constant mean amounts in endosomes. Cells respond to higher EGF concentrations by increasing the number of endosomes but keeping the mean p-EGFR content per endosome almost constant. By mathematical modelling, we found that this mechanism confers both robustness and regulation to signalling output. Different growth factors caused specific changes in endosome number and size in various cell systems and changing the distribution of p-EGFR between endosomes was sufficient to reprogram cell-fate decision upon EGF stimulation. We propose that the packaging of p-RTKs in endosomes is a general mechanism to ensure the fidelity and specificity of the signalling response. DOI: http://dx.doi.org/10.7554/eLife.06156.001, eLife digest Molecules called growth factors can stimulate cells to grow, divide, or differentiate into more specialised cell types. Cells detect these molecules via proteins called receptor tyrosine kinases that span their surface membrane. The growth factor binds to the portion of the receptor outside the cell, which makes the receptor send signals to the cell's nucleus that change how the cell grows, divides, or specialises. Different growth factors and receptor tyrosine kinases affect cell development in different ways. However, it was unclear how this occurred, as the receptors all send signals via the same signalling pathways. Some researchers proposed that specific responses could be triggered if some receptor tyrosine kinases activated these pathways more strongly than other receptors, or if they activated the pathways for different lengths of time. Now, Villaseñor et al. have looked at a receptor tyrosine kinase for a growth factor called EGF. Activated EGF receptors are marked with a phosphate group (or ‘phosphorylated’) and are then removed from the surface membrane and packaged into structures within the cell called endosomes. Villaseñor et al. found that different endosomes contain the same mean amount of phosphorylated EGF receptor. When exposed to higher EGF concentrations, the cells respond by increasing the number of endosomes, and so the average number of phosphorylated EGF receptors in each endosome remains almost constant. Villaseñor et al. used mathematical modelling to show that this mechanism, which they refer to as an ‘analogue-to-digital conversion’, ensures a robust signal, and can regulate the signalling of the activated receptors in both space and time. Different growth factors either increase or decrease the number and size of endosomes in various cell types. Moreover, Villaseñor et al. found that changing how the phosphorylated EGF receptor is distributed between endosomes alters how the cells interpret the signal and differentiate when they are exposed to EGF. These findings mean that the signalling activity of a cell could be predicted from the number and size of its endosomes. Moreover, the findings also suggest that interfering with this mechanism could change cell behaviour, for example, it could stop cancer cells proliferating and force them to differentiate instead. DOI: http://dx.doi.org/10.7554/eLife.06156.002

The molecular mechanisms underlying the targeting of Huntingtin (Htt) to endosomes and its multifaceted role in endocytosis are poorly understood. In this study, we have identified Htt-associated protein 40 (HAP40) as a novel effector of the small guanosine triphosphatase Rab5, a key regulator of endocytosis. HAP40 mediates the recruitment of Htt by Rab5 onto early endosomes. HAP40 overexpression caused a drastic reduction of early endosomal motility through their displacement from microtubules and preferential association with actin filaments. Remarkably, endogenous HAP40 was up-regulated in fibroblasts and brain tissue from human patients affected by Huntington's disease (HD) as well as in STHdhQ111 striatal cells established from a HD mouse model. These cells consistently displayed altered endosome motility and endocytic activity, which was restored by the ablation of HAP40. In revealing an unexpected link between Rab5, HAP40, and Htt, we uncovered a new mechanism regulating cytoskeleton-dependent endosome dynamics and its dysfunction under pathological conditions.

The mechanisms of endosome biogenesis and maintenance are largely unknown. The small GTPases Rab 5 and Rab 7 are key determinants of early and late endosomes, organizing effector proteins into specific membrane subdomains. Whether such Rab machineries are indefinitely maintained on membranes or can disassemble in the course of cargo transport is an open question. Here, we combined novel image-analysis algorithms with fast live-cell imaging. We found that the level of Rab 5 dynamically fluctuates on individual early endosomes, linked by fusion and fission events into a network in time. Within it, degradative cargo concentrates in progressively fewer and larger endosomes that migrate from the cell periphery to the center where Rab 5 is rapidly replaced with Rab 7. The class C VPS/HOPS complex, an established GEF for Rab 7, interacts with Rab 5 and is required for Rab 5-to-Rab 7 conversion. Our results reveal unexpected dynamics of Rab domains and suggest Rab conversion as the mechanism of cargo progression between early and late endosomes.

Endocytosis comprises several routes of internalization. An outstanding question is whether the caveolar and endosomal pathways intersect. Following transport of the caveolar protein Caveolin-1 and two cargo complexes, Simian Virus 40 and Cholera toxin, in live cells, we uncovered a Rab5-dependent pathway in which caveolar vesicles are targeted to early endosomes and form distinct and stable membrane domains. In endosomes, the low pH selectively allowed the toxin to diffuse out of the caveolar domains into the surrounding membrane, while the virus remained trapped. Thus, we conclude that, unlike cyclic assembly and disassembly of coat proteins in vesicular transport, oligomeric complexes of caveolin-1 confer permanent structural stability to caveolar vesicles that transiently interact with endosomes to form subdomains and release cargo selectively by compartment-specific cues.

Recent studies indicate that endocytic organelles can play a more active role in signal propagation and amplification than was recognised before. By deciphering the interplay between endocytosis and signalling, we will be able to gain a more sophisticated level of understanding of signal transduction mechanisms.

International audience; Signals generated in response to extracellular stimuli at the plasma membrane are transmitted through cytoplasmic transduction cascades to the nucleus. We report the identification of a pathway directly linking the small GTPase Rab5, a key regulator of endocytosis, to signal transduction and mitogenesis. This pathway operates via APPL1 and APPL2, two Rab5 effectors, which reside on a subpopulation of endosomes. In response to extracellular stimuli such as EGF and oxidative stress, APPL1 translocates from the membranes to the nucleus where it interacts with the nucleosome remodeling and histone deacetylase multiprotein complex NuRD/MeCP1, an established regulator of chromatin structure and gene expression. Both APPL1 and APPL2 are essential for cell proliferation and their function requires Rab5 binding. Our findings identify an endosomal compartment bearing Rab5 and APPL proteins as an intermediate in signaling between the plasma membrane and the nucleus.

Early endosomes move bidirectionally between the cell periphery and the interior through a mechanism regulated by the low molecular weight GTPase RhoD. Here, we identify a novel splice variant of human Diaphanous, hDia2C, which specifically binds to RhoD and is recruited onto early endosomes. Expression of RhoD and hDia2C induces a striking alignment of early endosomes along actin filaments and reduces their motility. This activity depends on the membrane recruitment and activation of c-Src kinase, thus uncovering a new role in endosome function. Our results define a novel signal transduction pathway, in which hDia2C and c-Src are sequentially activated by RhoD to regulate the motility of early endosomes through interactions with the actin cytoskeleton.

Cargo transfer from trans-Golgi network (TGN)-derived transport carriers to endosomes involves a still undefined set of tethering/fusion events. Here we analyze a molecular interaction that may play a role in this process. We demonstrate that the GGAs, a family of Arf-dependent clathrin adaptors involved in selection of TGN cargo, interact with the Rabaptin-5-Rabex-5 complex, a Rab4/Rab5 effector regulating endosome fusion. These interactions are bipartite: GGA-GAE domains recognize an FGPLV sequence (residues 439-443) in a predicted random coil of Rabaptin-5 (a sequence also recognized by the gamma1- and gamma2-adaptin ears), while GGA-GAT domains bind to the C-terminal coiled-coils of Rabaptin-5. The GGA-Rabaptin-5 interaction decreases binding of clathrin to the GGA-hinge domain, and expression of green fluorescent protein (GFP)-Rabaptin-5 shifts the localization of endogenous GGA1 and associated cargo to enlarged early endosomes. These observations thus identify a binding sequence for GAE/gamma-adaptin ear domains and reveal a functional link between proteins regulating TGN cargo export and endosomal tethering/fusion events.

The three GTPases Rab5, Rab4 and Rab11 regulate sequential transport steps along the endocytic/recycling pathway, and occupy distinct membrane domains on early and recycling endosomes. To address the mechanisms that regulate communication between such domains, we searched for proteins that interact with both Rab5 and Rab4. Here, we report that Rabenosyn-5, a previously identified Rab5 effector, also binds to Rab4. Rabenosyn-5 overexpression increased the association between Rab5 and Rab4 endosomal domains and decreased the fraction of Rab4- and Rab11-positive structures. This redistribution was accompanied by a faster rate of transferrin recycling from early endosomes to the cell surface and reduced transport to Rab11-containing perinuclear recycling endosomes. These effects depend on the ability of Rabenosyn-5 to interact with Rab4. We propose that divalent Rab effectors regulate protein sorting and recycling by connecting Rab domains on early endosomes.

The small GTPase Rab5 regulates membrane docking and fusion in the early endocytic pathway. Here we reveal a new role for Rab5 in the regulation of endosome interactions with the microtubule network. Using Rab5 fused to green fluorescent protein we show that Rab5-positive endosomes move on microtubules in vivo. In vitro, Rab5 stimulates both association of early endosomes with microtubules and early-endosome motility towards the minus ends of microtubules. Moreover, similarly to endosome membrane docking and fusion, Rab5-dependent endosome movement depends on the phosphatidylinositol-3-OH kinase hVPS34. Thus, Rab5 functionally links regulation of membrane transport, motility and intracellular distribution of early endosomes.

C-Met, the receptor of hepatocyte growth factor (HGF), through overexpression or mutation, is a major protooncogene that provides an attractive molecular target for cancer therapy. HGF/c-Met–induced tumorigenesis is dependent, in part, on the transcription factor and oncogene signal transducer and activator of transcription 3 (STAT3), which is believed to be activated by the receptor at the plasma membrane and then to travel to the nucleus where it acts. We demonstrate that although the robust signal to STAT3 elicited from the cytokine oncostatin-M does indeed support this mechanism of STAT3 action, for the weaker STAT3 signal emanating from c-Met, the activated receptor itself needs to be delivered to a perinuclear endosomal compartment to sustain phosphorylated STAT3 in the nucleus. This is signal specific because c-Met–induced extracellular signal-regulated kinase nuclear accumulation does not require receptor trafficking to the perinuclear compartment. This response is triggered from peripheral endosomes. Thus, control of growth factor receptor traffic determines the nature of the signal output, providing novel opportunities for intervention.

GTPases and lipid kinases regulate membrane traffic along the endocytic pathway by mechanisms that are not completely understood. Fusion between early endosomes requires phosphatidylinositol-3-OH kinase (PI(3)K) activity as well as the small GTPase Rab5. Excess Rab5-GTP complex restores endosome fusion when PI(3)K is inhibited. Here we identify the early-endosomal autoantigen EEA1 which binds the PI(3)K product phosphatidylinositol-3-phosphate, as a new Rab5 effector that is required for endosome fusion. The association of EEA1 with the endosomal membrane requires Rab5-GTP and PI(3)K activity, and excess Rab5-GTP stabilizes the membrane association of EEA1 even when PI(3)K is inhibited. The identification of EEA1 as a direct Rab5 effector provides a molecular link between PI(3)K and Rab5, and its restricted distribution to early endosomes indicates that EEA1 may confer directionality to Rab5-dependent endocytic transport.

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains while selectively allowing transport of proteins and lipids from one pole to the opposite by transcytosis. The small GTPase, rab17, a member of the rab family of regulators of intracellular transport, is specifically induced during cell polarization in the developing kidney. We here examined its intracellular distribution and function in both nonpolarized and polarized cells. By confocal immunofluorescence microscopy, rab17 colocalized with internalized transferrin in the perinuclear recycling endosome of BHK-21 cells. In polarized Eph4 cells, rab17 associated with the apical recycling endosome that has been implicated in recycling and transcytosis. The localization of rab17, therefore, strengthens the proposed homology between this compartment and the recycling endosome of nonpolarized cells. Basolateral to apical transport of two membrane-bound markers, the transferrin receptor and the FcLR 5-27 chimeric receptor, was specifically increased in Eph4 cells expressing rab17 mutants defective in either GTP binding or hydrolysis. Furthermore, the mutant proteins stimulated apical recycling of FcLR 5-27. These results support a role for rab17 in regulating traffic through the apical recycling endosome, suggesting a function in polarized sorting in epithelial cells.

Many critical cellular processes, like vesicular transport and signaling, rely on the establishment and maintenance of membrane domains. Membrane domains consist of a cluster of specific lipids and proteins that provide membranes with a distinct molecular identity. Rab GTPases are one of the main coordinators of membrane domain formation and dynamics. In this chapter, we will give a brief introduction into Rab GTPases and focus on how they create and coordinate membrane domains. This will include an in-depth look into the Rab effectors and binding partners that define membrane domains. Throughout we will highlight how these proteins are regulated such as via feedback and feed-forward loops to create cascades. Finally, we propose that signaling domains on organelles are also coordinated by Rab GTPases.

Cells undergoing apoptosis exhibit striking changes in membrane organization, including plasma membrane blebbing and invagination, vacuolation and fragmentation of organelles, and alterations in the surface expression of receptors. The underlying mechanisms for these changes are unknown, though alterations in vesicular fusion are likely to play a role. Using a cell-free system based on Xenopus laevis egg extracts we have found that endosome fusion is blocked during apoptosis. Inhibition of fusion is prevented by Bcl-2 or Bcl-xL, two negative regulators of apoptosis, or by specific inhibitors of members of the caspase family of apoptotic proteases. Selective cleavage of Rabaptin-5, an essential and rate-limiting component of endosome fusion, is responsible for the loss of fusion activity. Cleavage of Rabaptin-5 also occurs in cellular models for apoptosis. These results suggest that inactivation of Rabaptin-5 and inhibition of vesicle transport lead to fragmentation of endosomes and inhibition of the endocytic pathway during the execution phase of apoptosis. We propose that parallel changes to other membrane transport pathways would give rise to general membrane fragmentation in apoptotic cells. These changes are likely to play an important role in the generation of apoptotic bodies and their recognition by phagocytosing cells.

Delivery of short interfering RNAs (siRNAs) remains a key challenge in the development of RNA interference (RNAi) therapeutics. A better understanding of the mechanisms of siRNA cellular uptake, intracellular transport and endosomal release could critically contribute to the improvement of delivery methods. Here we monitored the uptake of lipid nanoparticles (LNPs) loaded with traceable siRNAs in different cell types in vitro and in mouse liver by quantitative fluorescence imaging and electron microscopy. We found that LNPs enter cells by both constitutive and inducible pathways in a cell type-specific manner using clathrin-mediated endocytosis as well as macropinocytosis. By directly detecting colloidal-gold particles conjugated to siRNAs, we estimated that escape of siRNAs from endosomes into the cytosol occurs at low efficiency (1-2%) and only during a limited window of time when the LNPs reside in a specific compartment sharing early and late endosomal characteristics. Our results provide insights into LNP-mediated siRNA delivery that can guide development of the next generation of delivery systems for RNAi therapeutics.

Vesicular transport is a dynamic process that requires coordinated interactions between membrane and cytoskeleton. The mechanisms and molecules integrating these interactions are unclear. A Rho protein, RhoD, might provide a molecular link between membrane traffic and the cytoskeleton. Activated RhoD causes rearrangements of the actin cytoskeleton and cell surface, and governs early endosome motility and distribution.

Small GTPases of the Rab family are key regulators of intracellular transport. They are associated with the cytoplasmic surface of distinct exocytic and endocytic organelles and with transport vesicles connecting these compartments. Rab proteins are also present in the cytosol in the GDP-bound conformation complexed to Rab GDP dissociation inhibitor (RabGDI). Upon membrane association, RabGDI is released, and the Rab protein is converted into the GTP-bound form. In this paper we have investigated whether Rab5, which regulates the clathrin-coated vesicle-mediated pathway of endocytosis, can directly associate with the membrane of clathrin-coated vesicles (CCV) purified from bovine brain in vitro. We found that RabGDI can specifically deliver Rab5 but not Rab7, which is localized to late endosomes, to CCV. Furthermore, CCV contain a heat- and trypsin-sensitive activity that stimulates the dissociation of GDP from Rab5, but not from Rab7, and the subsequent binding of GTP. The activity was found to be associated with the CCV membrane but not with the coat components. CCV weakly stimulated GDP release from either post-translationally modified or unmodified Rab5 alone. However, maximal GDP dissociation stimulation required the presence of RabGDI, suggesting that the factor(s) responsible for the membrane association and GDP/GTP exchange of Rab5 recognize the protein complexed to RabGDI. These data demonstrate that CCV are competent for acquiring Rab5 and for converting the molecule into the GTP-bound active form.

RAB4A,B,C (CCV, EE, RE)Endocytic recycling ZFYVE20- VPS45, RABEP1, RUFY1, GRIPAP1, AKAP10, RAB11FIP1, CD2APRAB5A,B,C EE fusion, EE biogenesis EEA1, ZFYVE20- VPS45, APPL1/2, ANKFY1, RABEP1, RABEP2, PIK3R4, PIK3CB, OCRL, INPP5B, INPP4A, F8A1, VPS8 (yeast)RAB13 (EE, TJ) RE- to- PM transport, GLUT4 traffi ckingMICALL2, MICALL1RAB21 (EE) Integrin endocytosis, cytokinesisN.D.RAB22A EE- to- Golgi transport TBC1D2B, EEA1RAB31/RAB22B (EE, TGN, PG)TGN- to- EE transport, PG maturationTBC1D2B, OCRLRAB23 (PM, EE) PG- LY fusion, Hedgehog signaling, ciliogenesisN.D.RAB35 (PM, CCV, EE)Endocytic recycling, cytokine-sis, actin dynamicsOCRL, ACAP2, FSCN1RAB3A,B,C,D (SG, SV)Regulated exocytosis (SV cycle)RPH3A, RPH3AL, RIMS1/2, RAB3IP, SYN1, SYTL4/5RAB6A,B,C (Golgi)Golgi- to- PM, Golgi- to-endosome, intra- Golgi and ER- to- Golgi transport, cytokinesisGOLGB1, GCC2, OCRL, KIF20A, MYH9, MYH10, DENND5A, BICD1, CCDC64, TMF1, APBA3, VPS52RAB8A,B (RE, G4V)TGN- to- PM transport, GLUT4 and E- cadherin traffi cking, ciliogenesis, AJ assemblyOPTN, RPH3A, MYO5B, SYTL1, MICALL2, ODF2RAB10 (TGN, G4V)TGN- to- PM transport, GLUT4 traffi cking, PG maturationMICALL1, MYO5A/B/C, RIMS1RAB11A,B (TGN, RE)Endocytic recycling, RE- to-PM transport, cytokinesis, ciliogenesis, autophagyRAB11FIP1/FIP2/FIP3/FIP4/FIP5, EXOC6, RAB3IP, ZFYVE27*, MYO5B, TBC1D14RAB25 (RAB11C) (RE)RE- to- PM transport, integrin recyclingRAB11FIP1/FIP2/FIP3/FIP4/FIP5, RAB3IP, MYO5BRAB14 (EE, TGN) Golgi- to- EE transport KIF16B, ZFYVE20, RUFY1RAB15 (EE, RE) Endocytic recycling RABIF*, REP15RAB17 (RE, ML) Endocytic recycling, ciliogen-esis, melanosome traffi ckingN.D.RAB26 (SG) Regulated exocytosis RIMS1RAB33B (Golgi, ER- Golgi and intra- Golgi transport, autophagyGOLGA2, ATG16L1, RABEP1RAB37 (Golgi, SG)Mast cell degranulation, Wnt signaling, TNF- alpha secretionRIMS1, UNC13BRAB39A, B (Golgi, PG)Phagosomal acidifi cation, IL-1 secretionCASP1RAB7A,B (LE, LY, EE- to- LE and LE- to- Golgi transport, LY biogenesisRILP, VPS35 (retromer), VPS41 (HOPS complex), PIK3R4, FYCO1RAB9A,B (LE) Endosome- to- TGN transport PLIN3, GCC2, RHOBTB3, RABEPK, SGSM2, HPS4 (BLOC- 3 complex)RAB12 (RE, LY) Transport to LY N.D.RAB27A (ML, SG) Dynamics of LRO and SG RPH3A , RPH3AL MYO5AMLPH, SYTL1- 5, UNC13D, MYRIP, CORO1CRAB32 (TV, ML, Mito, APG)Autophagy, distribution of mitochondria, traffi cking of melanogenic enzyme to MLPRKAR2A, ANKRD7RAB34 (Golgi, PM, PG)Macropinocytosis, LY positioningRILP, UNC13BRAB36 (Golgi) Spatial distribution of LE and LY RILPRAB38 (TV, ML) Transport of tyrosinase to immature MLANKRD7RAB24 (APG?) - GABARAP

Apically enriched Rab11-positive recycling endosomes (Rab11-REs) are important for establishing and maintaining epithelial polarity. Yet, little is known about the molecules controlling trafficking of Rab11-REs in an epithelium in vivo. Here, we report a genome-wide, image-based RNA interference screen for regulators of Rab11-RE positioning and transport of an apical membrane protein (PEPT-1) in C. elegans intestine. Among the 356 screen hits was the 14-3-3 and partitioning defective protein PAR-5, which we found to be specifically required for Rab11-RE positioning and apicobasal polarity maintenance. Depletion of PAR-5 induced abnormal clustering of Rab11-REs to ectopic sites at the basolateral cortex containing F-actin and other apical domain components. This phenotype required key regulators of F-actin dynamics and polarity, such as Rho GTPases (RHO-1 and the Rac1 orthologue CED-10) and apical PAR proteins. Our data suggest that PAR-5 acts as a regulatory hub for a polarity-maintaining network required for apicobasal asymmetry of F-actin and proper Rab11-RE positioning.

Rab5a is a small GTPase that regulates fusion of endocytic vesicles to early endosomes. We investigated whether Rab5a is involved in early endocytic traffic in both the axonal and the somatodendritic domains of polarized neurons. Using immunofluorescence, endogenous Rab5a was detected in axons and dendrites. Its localization in axons strongly overlapped that of the synaptic vesicle protein synaptophysin. Indeed, Rab5a co-immunoisolated with synaptophysin-containing vesicles, and antibodies against Rab5a labeled synaptic vesicle-like structures in nerve terminals. The functional association of Rab5a with dendritic and axonal early endosomes was assayed by electron microscopy after overexpression of wild-type and mutant Rab5a in cultured hippocampal neurons. This induced the formation of abnormal endosomes in both the somatodendritic and the axonal domains. These results show a role for Rab5a in axonal and dendritic endocytosis, and the presence of Rab5a on synaptic vesicles indicates that the axonal endosomes participate in the biogenesis of these vesicles.

In nonpolarized cells, the small GTPase Rab5a is localized to the plasma membrane, clathrin-coated vesicles, and early endosomes. Rab5a is required for early endosome fusion in vitro and regulates transport between the plasma membrane and early endosomes, in vivo. In polarized epithelial cells endocytosis occurs from separate apical and basolateral plasma membrane domains. Internalized molecules are initially delivered to distinct apical or basolateral early endosomes. In vitro, apical early endosomes can readily fuse with one another but not with the basolateral endosomes and vice versa, thereby indicating that the apical and basolateral early endocytic pathways are controlled by distinct machineries. Here, we have investigated the localization and function of Rab5a in polarized epithelial cells. Confocal immunofluorescence microscopy on mouse kidney sections revealed association of the protein with the apical and basolateral plasma membrane domains and underlying structures. In polarized Madin-Darby canine kidney I cells, endogenous and overexpressed Rab5a have the same distribution. Moreover, overexpression of the protein causes a 2-fold increase in fluid-phase uptake from both domains of the cell, thus showing that Rab5a functions in apical and basolateral endocytosis. Our data indicate that the apical and basolateral endocytic machineries of epithelial cells share common regulatory components and that Rab5a per se is not sufficient to target endocytic vesicles to apical or basolateral early endosomes.

Small GTPases of the rab family control distinct steps of intracellular transport. The function of their GTPase activity is not completely understood. To investigate the role of the nucleotide state of rab5 in the early endocytic pathway, the effects of two mutants with opposing biochemical properties were tested. The Q79L mutant of rab5, analogous with the activating Q61L mutant of p21-ras, was found to have a strongly decreased intrinsic GTPase activity and was, unlike wild-type rab5, found mainly in the GTP-bound form in vivo. Expression of this protein in BHK and HeLa cells led to a dramatic change in cell morphology, with the appearance of unusually large early endocytic structures, considerably larger than those formed upon overexpression of wild-type rab5. An increased rate of transferrin internalization was observed in these cells, whereas recycling was inhibited. Cytosol containing rab5 Q79L stimulated homotypic early endosome fusion in vitro, even though it contained only a small amount of the isoprenylated protein. A different mutant, rab5 S34N, was found, like the inhibitory p21-ras S17N mutant, to have a preferential affinity for GDP. Overexpression of rab5 S34N induced the accumulation of very small endocytic profile and inhibited transferrin endocytosis. This protein inhibited fusion between early endosomes in vitro. The opposite effects of the rab5 Q79L and S34N mutants suggest that rab5:GTP is required prior to membrane fusion, whereas GTP hydrolysis by rab5 occurs after membrane fusion and functions to inactivate the protein.

In addition to apicobasal polarization, some epithelia also display polarity within the plane of the epithelium. To what extent polarized endocytosis plays a role in the establishment and maintenance of planar cell polarity (PCP) is at present unclear. Here, we investigated the role of Rabenosyn-5 (Rbsn-5), an evolutionarily conserved effector of the small GTPase Rab5, in the development of Drosophila wing epithelium. We found that Rbsn-5 regulates endocytosis at the apical side of the wing epithelium and, surprisingly, further uncovered a novel function of this protein in PCP. At early stages of pupal wing development, the PCP protein Fmi redistributes between the cortex and Rab5- and Rbsn-5-positive early endosomes. During planar polarization, Rbsn-5 is recruited at the apical cell boundaries and redistributes along the proximodistal axis in an Fmi-dependent manner. At pre-hair formation, Rbsn-5 accumulates at the bottom of emerging hairs. Loss of Rbsn-5 causes intracellular accumulation of Fmi and typical PCP alterations such as defects in cell packing, in the polarized distribution of PCP proteins, and in hair orientation and formation. Our results suggest that establishment of planar polarity requires the activity of Rbsn-5 in regulating both the endocytic trafficking of Fmi at the apical cell boundaries and hair morphology.

We have investigated the in vivo functional role of rab5, a small GTPase associated with the plasma membrane and early endosomes. Wild-type rab5 or rab5ile 133, a mutant protein defective in GTP binding, was overexpressed in baby hamster kidney cells. In cells expressing the rab5ile 133 protein, the rate of endocytosis was decreased by 50% compared with normal, while the rate of recycling was not significantly affected. The morphology of early endosomes was also drastically changed by the mutant protein, which induced accumulation of small tubules and vesicles at the periphery of the cell. Surprisingly, overexpression of wild-type rab5 accelerated the uptake of endocytic markers and led to the appearance of atypically large early endosomes. We conclude that rab5 is a ratelimiting component of the machinery regulating the kinetics of membrane traffic in the early endocytic pathway.

SummarySequential transport from early to late endosomes requires the coordinated activities of the small GTPases Rab5 and Rab7. The transition between early and late endosomes could be mediated either through transport carriers or by Rab conversion, a process in which the loss of Rab5 from an endosome occurs concomitantly to the acquisition of Rab7. We demonstrate that Rab conversion is the mechanism by which proteins pass from early to late endosomes in Caenorhabditis elegans coelomocytes. Moreover, we identified SAND-1/Mon1 as the critical switch for Rab conversion in metazoa. SAND-1 serves a dual role in this process. First, it interrupts the positive feedback loop of RAB-5 activation by displacing RABX-5 from endosomal membranes; second, it times the recruitment of RAB-7, probably through interaction with the HOPS complex to the same membranes. SAND-1/Mon1 thus acts as a switch by controlling the localization of RAB-5 and RAB-7 GEFs.

The small GTP-binding protein rab5 was previously localized on early endosomes and on the cytoplasmic face of the plasma membrane. Using a cell-free assay, we have now tested whether rab5 is involved in controlling an early endocytic fusion event. Fusion could be inhibited by cytosol containing the overexpressed mutant rab5lle133, which does not bind GTP on blots, and by antibodies against rab5, but not against rab2 or rab7. In contrast, fusion was stimulated with cytosol containing overexpressed wild-type rab5. Cytosols containing high levels of rab2 or mutant rab5 with the 9 carboxy-terminal amino acids deleted, which bind GTP on blots, had no effects. Finally, the inhibition mediated by anti-rab5 antibodies could be overcome by complementing the assay with the cytosol containing wild-type rab5, but not with the same cytosol depleted of rab5, nor with cytosol containing the rab5 mutants or rab2. These in vitro findings strongly suggest that rab5 is involved in the process of early endosome fusion.

Endocytosis is a complex process fulfilling many cellular and developmental functions. Understanding how it is regulated and integrated with other cellular processes requires a comprehensive analysis of its molecular constituents and general design principles. Here, we developed a new strategy to phenotypically profile the human genome with respect to transferrin (TF) and epidermal growth factor (EGF) endocytosis by combining RNA interference, automated high-resolution confocal microscopy, quantitative multiparametric image analysis and high-performance computing. We identified several novel components of endocytic trafficking, including genes implicated in human diseases. We found that signalling pathways such as Wnt, integrin/cell adhesion, transforming growth factor (TGF)-beta and Notch regulate the endocytic system, and identified new genes involved in cargo sorting to a subset of signalling endosomes. A systems analysis by Bayesian networks further showed that the number, size, concentration of cargo and intracellular position of endosomes are not determined randomly but are subject to specific regulation, thus uncovering novel properties of the endocytic system.

Small GTPases of the Rab family have been long recognized to be key regulators of membrane trafficking. However, recent studies have uncovered their more fundamental role as determinants of organelle biogenesis and maintenance in all cells. Rab proteins acting in the endocytic pathway were shown to occupy nonoverlapping, morphologically and biochemically distinct domains on membranes of endosomes. Molecular characterization of Rab5 and its effectors revealed basic principles by which this GTPase mediates local changes in membrane structure and function, thus organizing a specific domain on early endosomes. Rab domains on endosomes appear to coordinate multiple functions related to membrane trafficking, organelle motility and signal transduction and are dynamically linked through the activity of bivalent Rab effectors. The concept of Rab proteins acting as membrane organizers provides a framework explaining the biogenesis of endocytic organelles composed of separate but functionally coupled domains which are arranged in a dynamic fashion.

Vesicular transport of signaling molecules, specifically neurotrophins, in neurons is essential for their differentiation, survival, and plasticity. Neurotrophins such as neuron growth factor (NGF) and brain‐derived neurotrophic factor (BDNF) are internalized by receptor‐mediated endocytosis at synaptic terminals and loaded into endosomes for microtubule‐based transport along axons to the cell body where they exert their signaling function in the nucleus. The molecular mechanisms underlying this intracellular transport are not only relevant from a basic knowledge viewpoint, but have also important implications for neurodegenerative diseases. Defects in trafficking are increasingly implicated in the pathology of Huntington's disease (HD) and other neurodegenerative disorders. The small GTPases Rab5 and Rab7 play important roles in the endocytic trafficking of neurotrophins. We have recently identified Huntingtin (Htt) and Huntingtin associated protein of 40 kDa (HAP40) as a novel Rab5 effector complex that regulates endosome motility. In HD, we detected higher HAP40 protein levels compared with normal cells. Such increase causes an augmented recruitment of Htt onto Rab5‐positive early endosomes that drastically reduces their motility by “switching” these organelles from microtubules to F‐actin. These findings suggest a mechanism by which impaired Rab5‐mediated trafficking of neurotrophic factors may be a key event of the pathogenetic process leading to neurodegeneration in HD. To dissect the mechanisms by which Htt, HAP40, and Rab5 function in early endosome interactions with the cytoskeleton, we developed assays to investigate endosome–cytoskeleton interactions that can be applied to normal and pathological conditions. We provide here detailed protocols for, first, an assay that measures binding of early endosomes to microtubules and F‐actin. Second, we describe an improved protocol for a cell‐free assay that recapitulates the motility of early endosomes along microtubules in vitro. These assays provide mechanistic insights into the dysfunction of endosome motility occurring in HD as well as other neurodegenerative disorders.