Rab-domain dynamics in endocytic membrane trafficking

Eukaryotic cells depend on cargo uptake into the endocytic membrane system, which comprises a functionally interconnected network of endosomal compartments. The establishment and maintenance of such diverse compartments in face of the high rates of exchange between them, poses a major challenge for obtaining a molecular understanding of the endocytic system. Rab-GTPases have emerged as architectural key element thereof: Individual family members localize selectively to endosomal compartments, where they recruit a multitude of cytoplasmic effector proteins and coordinate them into membrane sub-domains. Such "Rab-domains" constitute modules of molecular membrane identity, which pattern the endocytic membrane system into a mosaic of Rab-domains. The main objective of this thesis research was to link such "static" mosaic-view with the highly dynamic nature of the endosomal system. The following questions were addressed: How are neighbouring Rab-domains coordinated? Are Rab-domains stable or can they undergo assembly and disassembly? Are the dynamics of Rab-domains utilized in cargo transport? The first part of this thesis research focused on the organization of Rab-domains in the recycling pathway. Utilizing Total Internal Reflection (TIRF) microscopy, Rab11-, but neither Rab4- nor Rab5-positive vesicles were observed to fuse with the plasma membrane. Rab4-positive membranes, however, could be induced to fuse in presence of Brefeldin A. Thus, these experiments complete the view of the recycling pathway by the following steps: a) Rab11-carriers likely mediate the return of recycling cargo to the surface; b) such carriers are presumably generated in an Arf-dependent fission reaction from Rab4-positive compartments. Rab11-chromatography was subsequently carried out in the hope of identifying Rab11-effectors functioning at the Rab4-Rab11 domain interface. An as yet uncharacterized ubiquitin ligase was identified, which selectively interacts with both Rab4 and Rab11. Contrary to expectations, however, the protein (termed RUL for *R*ab interacting *U*biquitin *L*igase) does not function in recycling,but appears to mediate trafficking between Golgi/TGN and endosomes instead.In order to address the dynamics of Rab-domains, fluorescently tagged Rab-GTPases were imaged during cargo transport reactions in living cells. Herefore high-speed/long-term imaging procedures and novel computational image analysis tools were developed. The application of such methodology to the analysis of Rab5-positive early endosomes showed that a) The amount of Rab5 associated with individual endosomes fluctuates strongly over time; b) such fluctuations can lead to the "catastrophic" loss of the Rab5-machinery from membranes; c) Rab5 catastrophe is part of a functional cycle of early endosomes, involving net centripetal motility, continuous growth and increase in Rab5 density. Next, the relevance of Rab5 catastrophe with respect to cargo transfer into either the recycling- or degradative pathway was examined. Recycling cargo (transferrin) could be observed to exit Rab5-positive early endosomes via the frequent budding of tubular exit carriers. Exit of degradative cargo (LDL) from Rab5-positive endosomes did not involve budding, but the rapid loss of Rab5 from the limiting membrane.Rab5-loss was further coordinated with the concomitant acquisition of Rab7, suggesting "Rab conversion" as mechanism of transport between early- and late endosomes.Altogether, this thesis research has shown that first, Rab-machineries can be acquired and lost from membranes. Second, such dynamics provide a molecular mechanism for cargo exchange between endosomal compartments. Jointly, these findings lead to the concept of Rab-domain dynamics modulation in /trans/ between neighbouring domains as mechanistic principle behind the dynamic organization of membrane trafficking pathways

Intracellular membrane trafficking in Osteoclasts The role of direct Rab protein – Rac 1 interactions in the targeting of intracellular vesicles

Osteoclasts are cells responsible for bone resorption. These cells undergo extensive membrane re-organization during their polarization for bone resorption and form four distinct membrane domains, namely the ruffled border, the basolateral membrane, the sealing zone and the functional secretory domain. The endocytic/biosynthetic pathway and transcytotic route(s) are important for the resorption process, since the endocytic/biosynthetic pathway brings the specific vesicles to the ruffled border whereas the transcytotic flow is believed to transport the degraded bone matrix away from the resorption lacuna to the functional secretory domain. In the present study, we found a new transcytotic route from the functional secretory domain to the ruffled border, which may compensate membrane loss from the ruffled border during the resorption process. We also found that lipid rafts are essential for the ruffled border-targeted late endosomal pathways. A small GTP-binding protein, Rab7, has earlier been shown to regulate the late steps of the endocytic pathway. In bone-resorbing osteoclasts it is involved in the formation of the ruffled border, which displays several features of late endosomal membranes. Here we discovered a new Rab7-interacting protein, Rac1, which is another small GTP-binding protein and binds to the GTP-form of Rab7 in vitro. We demonstrated further that Rab7 colocalizes with Rac1 at the fusion zone of the ruffled border in bone-resorbing osteoclasts. In other cell types, such as fibroblast-like cells, this colocalization is mainly perinuclear. Because Rac1 is known to control the actin cytoskeleton through its effectors, we suggest that the Rab7-Rac1 interaction may mediate late endosomal transport between microtubules and microfilaments, thus enabling endosomal vesicles to switch tracks from microtubules to microfilaments before their fusion to the ruffled border. We then studied the role of Rab-Rac1 interaction in the slow recycling pathway. We revealed that Rac1 also binds directly to Rab11 and to some other but not all Rab-proteins, suggesting that Rab-Rac1 interaction could be a general regulatory mechanism to direct the intracellular vesicles from microtubule mediated transport to actin filament mediated transport and vice versa. On the basis of our results we thus propose a new hypothesis for these GTPases in the regulation of intracellular membrane flow.Siirretty Doriast

Parameter estimation with bio-inspired meta-heuristic optimization: modeling the dynamics of endocytosis

<p>Abstract</p> <p>Background</p> <p>We address the task of parameter estimation in models of the dynamics of biological systems based on ordinary differential equations (ODEs) from measured data, where the models are typically non-linear and have many parameters, the measurements are imperfect due to noise, and the studied system can often be only partially observed. A representative task is to estimate the parameters in a model of the dynamics of endocytosis, i.e., endosome maturation, reflected in a cut-out switch transition between the Rab5 and Rab7 domain protein concentrations, from experimental measurements of these concentrations. The general parameter estimation task and the specific instance considered here are challenging optimization problems, calling for the use of advanced meta-heuristic optimization methods, such as evolutionary or swarm-based methods.</p> <p>Results</p> <p>We apply three global-search meta-heuristic algorithms for numerical optimization, i.e., differential ant-stigmergy algorithm (DASA), particle-swarm optimization (PSO), and differential evolution (DE), as well as a local-search derivative-based algorithm 717 (A717) to the task of estimating parameters in ODEs. We evaluate their performance on the considered representative task along a number of metrics, including the quality of reconstructing the system output and the complete dynamics, as well as the speed of convergence, both on real-experimental data and on artificial pseudo-experimental data with varying amounts of noise. We compare the four optimization methods under a range of observation scenarios, where data of different completeness and accuracy of interpretation are given as input.</p> <p>Conclusions</p> <p>Overall, the global meta-heuristic methods (DASA, PSO, and DE) clearly and significantly outperform the local derivative-based method (A717). Among the three meta-heuristics, differential evolution (DE) performs best in terms of the objective function, i.e., reconstructing the output, and in terms of convergence. These results hold for both real and artificial data, for all observability scenarios considered, and for all amounts of noise added to the artificial data. In sum, the meta-heuristic methods considered are suitable for estimating the parameters in the ODE model of the dynamics of endocytosis under a range of conditions: With the model and conditions being representative of parameter estimation tasks in ODE models of biochemical systems, our results clearly highlight the promise of bio-inspired meta-heuristic methods for parameter estimation in dynamic system models within system biology.</p

Small GTPases of the Rab and Arf Families: Key Regulators of Intracellular Trafficking in Neurodegeneration

Small guanosine triphosphatases (GTPases) of the Rab and Arf families are key regulators of vesicle formation and membrane trafficking. Membrane transport plays an important role in the central nervous system. In this regard, neurons require a constant flow of membranes for the correct distribution of receptors, for the precise composition of proteins and organelles in dendrites and axons, for the continuous exocytosis/endocytosis of synaptic vesicles and for the elimination of dysfunctional proteins. Thus, it is not surprising that Rab and Arf GTPases have been associated with neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Both pathologies share characteristics such as the presence of protein aggregates and/or the fragmentation of the Golgi apparatus, hallmarks that have been related to both Rab and Arf GTPases functions. Despite their relationship with neurodegenerative disorders, very few studies have focused on the role of these GTPases in the pathogenesis of neurodegeneration. In this review, we summarize their importance in the onset and progression of Alzheimer’s and Parkinson’s diseases, as well as their emergence as potential therapeutical targets for neurodegeneration.A.A.S. is a recipient of a predoctoral fellowship (PRE_2017_1_0016) from the Basque Government. M.L.M. is a recipient of a fellowship from Foundation “Jesús de Gangoiti y Barrera”. J.L.Z. was supported by the Instituto de Salud Carlos III (PI18/00207) and the University of Basque Country Grant (US19/04)

Characterisation of Novel Rab5 Effector Proteins in the Endocytic Pathway

Endocytosis, a process of plasma membrane invaginations, is a fundamental cellular mechanism, ensuring uptake of nutrients, enhanced communication between cells, protective functions against invasive pathogens and remodelling of the plasma membrane composition. In turn, endocytic mechanisms are exploited by pathogens to enter their host cells. Endocytosis comprises multiple forms of which our molecular understanding has mostly advanced with respect to clathrin-mediated endocytosis and phagocytosis. Studies on the small GTPase Rab5 have provided important insights into the molecular mechanism of endocytosis and transport in the early stages of the endocytic pathways. Rab5 is a key regulator of clathrin-mediated endocytosis, but in addition, localises to several distinct endocytic carriers including phagosomes and pinocytic vesicles. On early endosomes, Rab5 coordinates within a spatially restricted domain enriched in phosphatidylinositol-3 phosphate PI(3)P a complex network of effectors, including PI3-Kinase (PI3-K), the FYVE-finger proteins EEA1 and Rabenosyn-5 that functionally cooperate in membrane transport. Moreover, Rab5 regulates endocytosis from the apical and basolateral plasma membrane in polarised epithelial cells. During my PhD thesis, I investigated the molecular mechanisms of endocytosis both in polarised and non-polarised cells. I obtained new insights into the molecular mechanisms of endocytosis and their coordination through the functional characterization of a novel Rab5 effector, termed Rabankyrin-5. I could demonstrated that Rabankyrin-5 is a novel PI(3)P-binding Rab5 effector that localises to early endosomes and stimulates their fusion activity in vitro. The latter activity depends on the oligomerisation of Rabankyrin-5 on the endosomal membrane via the N-terminal BTB/POZ domain. In addition to early endosomes, however, Rabankyrin-5 localises to large vacuolar structures that correspond to macropinosomes in epithelial cells and fibroblasts. Overexpression of Rabankyrin-5 increases the number of macropinosomes and stimulates fluid phase uptake whereas its downregulation through RNA interference inhibits these processes. In polarised epithelial cells, the function of Rabankyrin-5 is primarily restricted to the apical membrane. It localises to large pinocytic structures underneath the apical surface of kidney proximal tubule cells and its overexpression in polarised MDCK cells specifically stimulates apical but not basolateral, non-clathrin mediated pinocytosis. In demonstrating a regulatory role in endosome fusion and (macro)-pinocytosis, my studies suggest that Rab5 regulates and coordinates different endocytic mechanisms through its effector Rabankyrin-5. Furthermore, the active role in apical pinocytosis in epithelial cells suggests an important function of Rabankyrin-5 in the physiology of polarised cells. The results obtained in this thesis are central not only for our understanding of the basic principles underlying the regulation of multiple endocytic mechanisms. They are also relevant for the biomedical field, since actin-dependent (macro)-pinocytosis is an important mechanism for the physiology of cells and organisms and is upregulated under certain pathological conditions (e.g. cancer)

Integrin traffic and signalling – from plasma membrane to endosomes

Integrins are the main cell surface receptors by which cells adhere to the surrounding extracellular matrix (ECM). Cells regulate integrin-mediated adhesions by integrin endo/exocytic trafficking or by altering the integrin activation status. Integrin binding to ECM-components induces several intracellular signalling cascades, which regulate almost every aspect of cell behaviour from cell motility to survival, and dysregulation of integrin traffic or signalling is associated with cancer progression. Upon detachment, normal cells undergo a specialised form of programmed cell death namely anoikis and the ECM-integrin -mediated activation of focal adhesion kinase (FAK) signalling at the cell surface has been considered critical for anoikis suppression. Integrins are also constantly endocytosed and recycled back to the plasma membrane, and so far the role of integrin traffic in cancer has been linked to increased adhesion site turnover and cell migration. However, different growth factor receptors are known to signal also from endosomes, but the ability of integrins to signal from endosomes has not been previously studied. In this thesis, I demonstrate for the first time that integrins are signalling also from endosomes. In contrast to previous believes, integrin-induced focal adhesion kinase (FAK) signalling occurs also on endosomes, and the endosomal FAK signalling is critical for anoikis suppression and for cancer related processes such as anchorage-independent growth and metastasis. Moreover, we have set up a new integrin trafficking assay and demonstrate for the first time in a comprehensive manner that active and inactive integrins undergo distinct trafficking routes. Together these results open up new horizons in our understanding of integrins and highlight the fundamental connection between integrin traffic and signalling.Siirretty Doriast

Assessment of functional characteristics of small GTPases using small molecules

Rab and Rho subfamilies of GTPases are functionally linked to intracellular trafficking and organization of the cytoskeleton respectively. Despite their roles, use of small molecule inhibitors or activators to map the functionality of these GTPases remains largely underexplored due to lack of suitable compounds. In this dissertation, we report on the functional characterization of Rab7 and Rac1 GTPases using small molecules. 2-(benzoylcarbamothioylamino)-5,5-dimethyl-4,7- dihydrothieno[2,3-c]pyran-3-carboxylic acid (PubChem #: CID1067700) has been used to characterize Rab7. Using bead based flow cytometry, CID1067700 was found to have significant inhibitory potency on Rab7 nucleotide binding with a respective inhibitory efficacy of 80% for BODIPY-GTP and 60% for BODIPY-GDP binding. Rac1 has been functionally characterized by non-steroidal anti-inflammatory drug (NSAID), R-Naproxen in the context of ovarian cancer. R-Naproxen isoform functionally inhibited Rac1 in the cell lines assayed relative to S-Naproxen and structurally similar 6-methoxy-2-naphthylacetic Acid (6-MNA). Inhibition is based on interference with membrane distribution of Rac1 rather than overall protein levels. Taken together, this study has identified the first competitive GTPase inhibitor (CID1067700) and also demonstrated the potential utility of the compound for dissecting GTPase enzymology. The study has also shown that R-Naproxen blocks activation of Rac1 small GTPase in ovarian cancer cells with implications for the inhibition of ovarian cancer cell proliferation, migration, and invasion

Macropinosome Maturation is a Clathrin Dependent Process in Bone Marrow Macrophages

Macrophages nonspecifically take up extracellular fluids, solutes and macromolecules by macropinocytosis. Understanding the mechanisms of macropinosome maturation will inform the study of lipid uptake, viral entry, antigen processing and presentation, as well as regulation of cell growth. Colony stimulating factor-1 receptor (CSF-1R) is internalized by small vesicle endocytosis, trafficked to nascent macropinosomes and degraded. These CSF-1R positive macropinosomes mature through a sequence similar to endosomes, progressing from EEA1 and Rab5 to Rab7 positive vesicles before fusing with lysosomes. Here we report the assembly of clathrin on internalized macropinosomes shown both by live-cell microscopy of cells expressing clathrin light chain-yellow fluorescent protein (CLTA-YFP) and by immuno-staining of endogenous clathrin heavy chain (CHC). Partial depletion of the clathrin heavy chain by siRNA prevented macropinosome-lysosome fusion and impaired degradation of the CSF- 1R, with only minimal effects on the delivery of the CSF-1R to the macropinosome. Expression of fluorescent protein fusions demonstrates that clathrin assembled on macropinosomes co-localizes with dynamin and possibly the clathrin adaptor, clathrinassembly lymphoid myeloid leukaemia protein (CALM). These data indicate a novel role for clathrin in mediating macropinosome maturation, cargo trafficking and macropinosomes-lysosome fusion in macrophages. We hypothesize that clathrin is involved in an outward budding event during the Rab5 to Rab7 stage of macropinosome maturation. Protein and membrane sorting at this time may predispose the macropinosome to further maturation processes such as attachment to motor proteins and lysosome fusion

Intracellular regulation of Wnt and FGF signal transduction by the late endosomal compartment in Xenopus laevis

The endosomal network depicts a vast playground of multiple processing capabilities in terms of signaling. Distinct compartments of the endosomal machinery exert specific functions and thus contribute in signal termination, transduction, attenuation or amplification. Initially, these functions were attributed to early endosomes but recent research likewise considers late endosomes to be just as relevant in mediating such processes. Functionality as well as the molecular identity of these intracellular membranous platforms are orchestrated by a large superfamily of small Ras like GTPases. The collected data of this study particularly highlight the involvement of late endosomes and its associated regulator Rab7 in the early development of the African clawed frog Xenopus laevis. In particular, the first two chapters address the Rab7-dependent specification of the mesodermal germ layer by regulating intracellular pathway activity of Wnt and FGF/MAPK signaling. After fertilization formation of the germ layers is one of the first processes to be initiated. An essential part of mesoderm development comprises subdivision into different mesodermal regions, thus clustering it into ventrolateral and dorsal mesoderm. This patterning is crucial to promote further differentiation into various tissues arising from the mesodermal germ layer. It turned out, Rab7 regulates ventrolateral fates in a Wnt-dependent manner. The small GTPase exerts its function upstream of the Wnt co-transcription factor Ctnnb1 to ensure its nuclear relocalization. In addition to that, Rab7-positive endosomes are likewise required to mediate intracellular FGF/MAPK signal transduction in order to specify dorsal mesoderm. Here, Rab7 regulates proper signaling at the level or downstream of Ras and upstream of Erk/Mapk1. The last chapter then elicits further regulative properties of the late endosomal platform, concerning Cd63 function. The tetraspanin Cd63, which constitutes a transmembrane protein, associates with late endolysosomal compartments and exhibits a similar expression pattern like the small GTPase Rab7 in Xenopus laevis. Contrary to Rab7, function of Cd63 seems to be dispensable whilst gastrulation. However, the presented studies in this chapter suggest a vital function of the tetraspanin Cd63 during axial elongation and correct eye development. Therefore, these investigations regarding Cd63 demonstrated an involvement of the regulative function of late endosomes as signaling platforms for embryonic development beyond mesoderm specification and gastrulation. Overall, the summarized data of this study provides further insights into the determining capacity of Rab7-positive endosomal platforms in intracellular signal transduction of different pathways during early embryonic development.Das endosomale Netzwerk stellt einen enormen Schauplatz für mannigfaltige Prozessierungsmöglichkeiten in Bezug auf Signalübertragung dar. Die verschiedenen Kompartimente der endosomalen Maschinerie üben spezifische Funktionen aus und tragen somit zur Signaltermination, -übertragung, -abschwächung oder -verstärkung bei. Anfänglich wurden diese Funktionen frühen Endosomen zugeschrieben, neuere Forschungen erachten jedoch auch späte Endosomen als gleichermaßen relevant für die Vermittlung solcher Prozesse. Sowohl die Funktionalität als auch die molekulare Identität dieser intrazellulären membranösen Plattformen werden durch eine große Superfamilie kleiner Ras ähnlicher GTPasen gesteuert. Die zusammengestellten Daten dieser Studie heben besonders die Beteiligung später Endosomen und des mit ihnen assoziierten Regulators Rab7 in der frühen Entwicklung des Afrikanischen Krallenfroschs Xenopus laevis hervor. Die ersten zwei Kapitel thematisieren jeweils die Rab7-abhängige Spezifizierung des mesodermalen Keimblatts durch Regulation der intrazellulären Signalwegsaktivität von Wnt und FGF/MAPK Signalen. Nach der Befruchtung ist die Entstehung der Keimblätter einer der ersten initiierten Prozesse. Ein wesentlicher Teil der Mesodermentwicklung umfasst die Unterteilung in unterschiedliche mesodermale Bereiche, wodurch dieses in ventrolaterales und dorsales Mesoderm gegliedert wird. Diese Strukturierung ist essentiell um die weitere Differenzierung in die verschiedenen Gewebe, welche aus dem mesodermalen Keimblatt entstehen, voranzutreiben. Es stellte sich heraus, dass Rab7 ventrolaterales Schicksal in einer Wnt-abhängigen Weise reguliert. Die kleine GTPase vermittelt ihre Funktion oberhalb des Wnt Ko Transkriptionsfaktors Ctnnb1 um wiederum dessen nukleare Relokalisierung zu gewährleisten. Darüber hinaus werden Rab7-positive Endosomen ebenfalls für die intrazelluläre Vermittlung der FGF/MAPK Signaltransduktion benötigt um dorsales Mesoderm zu spezifizieren. Hier reguliert Rab7 die korrekte Signalgebung auf Ebene oder unterhalb von Ras beziehungsweise oberhalb von Erk/Mapk1. Im letzten Kapitel werden die weiteren regulativen Eigenschaften später endosomaler Plattformen in Bezug auf die Funktion von Cd63, eruiert. Das Tetraspanin Cd63, welches ein Transmembranprotein darstellt, ist mit späten endolysosomalen Kompartimenten assoziiert und weist in Xenopus laevis ein ähnliches Expressionsmuster wie die kleine GTPase Rab7 auf. Im Gegensatz zu Rab7, scheint die Funktion von Cd63 während der Gastrulation entbehrlich zu sein. Allerdings lassen die in diesem Kapitel aufgeführten Studien eine entscheidende Funktion des Tetraspanins Cd63 während der axialen Elongation und der korrekten Augenentwicklung vermuten. Daher legen die Untersuchungen hinsichtlich Cd63 eine Beteiligung an der regulativen Funktion von späten Endosomen als Signalplattformen in der embryonalen Entwicklung dar, welche über die Mesodermspezifizierung und Gastrulation hinausgehen. Insgesamt bieten die gesammelten Daten dieser Studie weitere Erkenntnisse über die entscheidende Kapazität Rab7-positiver endosomaler Plattformen in der intrazellulären Signaltransduktion verschiedener Signalwege während der frühen embryonalen Entwicklung