Cargo and Dynamin Regulate Clathrin-Coated Pit Maturation

Total internal reflection fluorescence microscopy (TIR-FM) has become a powerful tool for studying clathrin-mediated endocytosis. However, due to difficulties in tracking and quantifying their heterogeneous dynamic behavior, detailed analyses have been restricted to a limited number of selected clathrin-coated pits (CCPs). To identify intermediates in the formation of clathrin-coated vesicles and factors that regulate progression through these stages, we used particle-tracking software and statistical methods to establish an unbiased and complete inventory of all visible CCP trajectories. We identified three dynamically distinct CCP subpopulations: two short-lived subpopulations corresponding to aborted intermediates, and one longer-lived productive subpopulation. In a manner dependent on AP2 adaptor complexes, increasing cargo concentration significantly enhances the maturation efficiency of productive CCPs, but has only minor effects on their lifetimes. In contrast, small interfering RNA (siRNA) depletion of dynamin-2 GTPase and reintroduction of wild-type or mutant dynamin-1 revealed dynamin's role in controlling the turnover of abortive intermediates and the rate of CCP maturation. From these data, we infer the existence of an endocytic restriction or checkpoint, responsive to cargo and regulated by dynamin

Cargo- and adaptor-specific mechanisms regulate clathrin-mediated endocytosis

Clathrin-coated pit size and dynamic behavior varies with low density lipoprotein receptor (LDLR) expression levels in a manner dependent on the LDLR-specific adaptors, Dab2 and ARH

Control of actin polymerization via the coincidence of phosphoinositides and high membrane curvature

The conditional use of actin during clathrin-mediated endocytosis in mammalian cells suggests that the cell controls whether and how actin is used. Using a combination of biochemical reconstitution and mammalian cell culture, we elucidate a mechanism by which the coincidence of PI(4,5)P2 and PI(3)P in a curved vesicle triggers actin polymerization. At clathrin-coated pits, PI(3)P is produced by the INPP4A hydrolysis of PI(3,4)P2, and this is necessary for actin-driven endocytosis. Both Cdc42⋅guanosine triphosphate and SNX9 activate N-WASP–WIP- and Arp2/3-mediated actin nucleation. Membrane curvature, PI(4,5)P2, and PI(3)P signals are needed for SNX9 assembly via its PX–BAR domain, whereas signaling through Cdc42 is activated by PI(4,5)P2 alone. INPP4A activity is stimulated by high membrane curvature and synergizes with SNX9 BAR domain binding in a process we call curvature cascade amplification. We show that the SNX9-driven actin comets that arise on human disease–associated oculocerebrorenal syndrome of Lowe (OCRL) deficiencies are reduced by inhibiting PI(3)P production, suggesting PI(3)P kinase inhibitors as a therapeutic strategy in Lowe syndrome.J.L. Gallop is supported by a Wellcome Trust Research Career Development Fellowship (grant WT095829AIA). F.  Daste, A.  Walrant, J.R. Gadsby, and J. Mason are supported by an H2020 European Research Council Starting Grant (281971) awarded to J.L. Gallop. Gurdon Institute funding is provided by the Wellcome Trust (grant 092096) and Cancer Research UK (grant C6946/A14492). The Swedish Medical Research Council and the Swedish Foundation for Strategic Research supported the work of M.R. Holst and R. Lundmark. S.F. Lee is funded by a Royal Society University Research Fellowship (grant UF120277). M. Mettlen is funded by grant MH73125 to Sandra L. Schmid (University of Texas Southwestern Medical Center)

Src triggers apical endocytosis in polarized MDCK cells

L'endocytose apicale dans les cellules polarisées montre des propriétés distinctes, mais on ne connaît pas le rôle dans ce processus de la tyrosine kinase non-récepteur, Src. Ma thèse a cherché à répondre à cette question en utilisant des cellules MDCK qui expriment un mutant thermosensible de v-Src (inactif à 40°C; activé à 34°C). A 40°C, ces cellules forment sur un support poreux une monocouche étanche, et leur endocytose fluide est ~ 2 fois supérieure au pôle basolatéral qu'au pôle apical. v-Src est rapidement activé à 34°C (révélé par la phosphorylation de la Tyr416) et le domaine apical des cellules se bombe. Toutefois, l'immuno-marquage continu de ZO-1, l'arrêt abrupt du marquage ultrastructural par le rouge de ruthénium aux jonctions serrées, la résistance transépithéliale accrue, et la préservation de la restriction membranaire apicale de la gp135/podocalixine et basolatérale de la Na+/K+-ATPase, montrent que la polarité est préservée durant au moins 6 h; les jonctions serrées disparaissent après 24 h. Après 6 h à 34°C (pour étudier les effets de l'activation de v-Src tout en préservant la polarité cellulaire), v-Src est transloquée du cytoplasme basolatéral vers des membranes apicales. L'activation de cette tyrosine kinase induit à l'apex cellulaire un "ruffling" membranaire associé à la cortactine et l'apparition de grandes structures vésiculaires. De manière concomitante, l'endocytose fluide apicale est stimulée d'environ 5 fois, sans modification de l'endocytose fluide basolatérale. Tant les changements morphologiques (c.-à-d. le remodelage du cytocortex apical et l'apparition des grandes structures vésiculaires), que la stimulation de l'endocytose fluide apicale sont sélectivement abolis par des inhibiteurs de la PI3-K (wortmannine et LY294002), de la PLC (NCDC), de la PLD (1-butanol), et de la polymérisation de l'actine (latrunculine B). De plus, l'activation de v-Src accélère d'environ 2 fois l'endocytose médiée par récepteur de la transferrine aux deux pôles. Cependant, la stimulation de l'efficacité d'internalisation de la transferrine aux deux pôles est insensible aux inhibiteurs mentionnés ci-dessus, indiquant que l'accélération de l'endocytose fluide au pôle apical ne peut pas être expliquée par une formation accélérée des puits tapissés de clathrine, et soulignant le contrôle distinct de la micro- et macropinocytose apicale. Src induit la fusion microtubule-dépendante des macropinosomes avec l'endosome de recyclage apical, provoquant ainsi sa vacuolisation. Pourtant, la préservation de tubulation de ce compartiment et de la polarité apicale indique que sa fonction n'est pas affectée. L'endosome de recyclage apical est marqué par v-Src, Rab11, et rabankyrine-5, mais non par l'antigène des endosomes précoces, distinguant ainsi deux itinéraires dépendants de Rab-5. La compréhension des mécanismes cellulaires et moléculaires de la stimulation du "ruffling" membranaire et de la macropinocytose apicale par Src pourrait clarifier le mode de pénétration apicale induite par des bactéries enteropathogènes et la différentiation apicale des ostéoclastes.Apical endocytosis in polarized epithelial cells shows distinct properties but the role of Src-tyrosine kinase in this process is unknown. My thesis addresses this question using MDCK cells harboring a thermo-sensitive mutant of v-Src (inactive at 40°C; activated at 34°C). At 40°C, these cells formed tight monolayers on a permeable support, with a ~ 2-fold higher fluid-phase uptake at the basolateral compared to the apical surface. Upon shifting to 34°C, v-Src was rapidly activated (based on Tyr416 phosphorylation) and the apical domain of cells started bulging. Continuous ZO-1 immunolabeling, the abrupt arrest of ruthenium red ultrastructural staining at tight junctions, an even enhanced transepithelial resistance, and preserved membrane restriction of apical gp135/podocalyxin and basolateral Na+/K+-ATPase, all showed that polarity was maintained for at least 6 h; tight junctions disappeared after 24 h. At 6 h after transfer to 34°C (to study the effects of v-Src activation while preserving epithelial polarity), v-Src was translocated from the basolateral cytoplasm to apical membranes. Activation of Src-tyrosine kinase triggered cortactin-associated membrane ruffling at the cell apex and the appearance of large apical vesicular structures. Concomitantly, apical fluid-phase endocytosis was stimulated by 5-fold, without effect on basolateral uptake. Morphologic changes (i.e. apical cytocortex remodelling and the appearance of large vesicular structures) as well as the stimulation of apical fluid-phase endocytosis were selectively abrogated by inhibitors of PI3-K (wortmannin and LY294002), of PLC (NCDC), of PLD (1-butanol), and of actin polymerization (latrunculin B). In addition, activation of v-Src also caused a ~ 2-fold increase of receptor-mediated endocytosis of transferrin at both the apical and the basolateral domains. However, stimulation of transferrin internalization efficiency at both surfaces was insensitive to these inhibitors, indicating that acceleration of fluid-phase endocytosis at the apical domain could not be explained by a faster formation of clathrin-coated vesicles, and underscoring the distinct control of apical micro- and macropinocytosis. Src promoted microtubule-dependent fusion of macropinosomes with the apical recycling endosome, causing its strong vacuolation. However, preservation of tubulation and apical polarity indicated that the function of the apical recycling endosome was not affected. In contrast to apical endosomes, this organelle was labeled for v-Src, Rab11, and rabankyrin-5, but not for early endosome antigen-1, thus distinguishing two separate Rab5-dependent apical pathways. The mechanisms of Src-induced apical ruffling and macropinocytosis could shed light on the triggered apical entry of enteroinvasive pathogens and on the apical differentiation of osteoclasts.Thèse de doctorat en sciences biomédicales (biologie cellulaire)(SBIM 3) -- UCL, 200

v-Src accelerates spontaneous motility via phosphoinositide 3-kinase, phospholipase C and phospholipase D, but abrogates chemotaxis in Rat-1 and MDCK cells.

In Rat-1 fibroblasts, v-Src causes a profound remodelling of cortical actin cytoskeleton. This transformation includes membrane ruffling, a hallmark of the leading edge in migrating cells, and results from activation of phosphoinositide 3-kinase (PI 3-kinase), phospholipase C (PLC) and phospholipase D (PLD). We therefore reexamined whether motility is constitutively triggered by v-Src and studied whether this response is controlled by the same signalling pathway. The study was performed using Rat-1/tsLA29 and MDCK/tsLA31 cells, each harbouring a different thermosensitive v-Src kinase, active at 34 degrees C but inactivated at 40 degrees C. In both cell lines, overnight v-Src activation induced transformation and accelerated spontaneous motility by approximately twofold, as evidenced by wound-healing assay and by single-cell track, time-lapse recording in Dunn chambers. Inhibitors of PI 3-kinase, PLC and PLD selectively abrogated acceleration of motility by v-Src. Since mechanisms that co-ordinate spontaneous, as distinct from oriented, cell migration are separable, we further analysed in Dunn chambers chemotactic response of Rat-1/tsLA29 cells to PDGF and of MDCK/tsLA31 cells to EGF. In both cases, v-Src decreased the steady-state level of growth factor receptors at the cell surface twofold, and abrogated movement directionality at comparable level of occupancy as in non-transformed cells. The burst of pinocytosis in response to growth factors was also abolished by v-Src. Altogether, these results indicate that v-Src triggers motility in a PI 3-kinase-, PLC- and PLD-dependent manner, but abrogates directionality by suppressing polarised signalling downstream of growth factor receptors.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Regulation of matrix metalloproteinase (MMP) activity by the low-density lipoprotein receptor-related protein (LRP). A new function for an "old friend".

Matrix metalloproteinases (MMPs) are essential contributors to a microenvironment that promotes tumour progression. During the two last decades, inhibition of MMPs has become the focus of considerable interest for cancer therapy, and numerous synthetic metalloproteinase inhibitors have been developed by the pharmaceutical industry. However, clinical trials have shown disappointing efficacy or unexpected toxicity and new targets are thus eagerly awaited. The identification of endocytic clearance of several MMPs by the low-density lipoprotein receptor-related protein (LRP) might provide insight into novel strategies for controlling MMP level during malignant processes. This review attempts to summarize recent aspects on the cellular and molecular basis of LRP-mediated endocytic disposal of MMPs

Constitutive diffuse activation of phosphoinositide 3-kinase at the plasma membrane by v-Src suppresses the chemotactic response to PDGF by abrogating the polarity of PDGF receptor signalling.

Cancer cells depend on chemotaxis for invasion and frequently overexpress and/or activate Src. We previously reported that v-Src accelerates motility by promoting phosphoinositide 3-kinase (PI3-K) signalling but abrogates chemotaxis. We here addressed the mechanism of the loss of chemotactic response to platelet-derived growth factor (PDGF) gradients in fibroblasts harbouring a thermosensitive v-Src kinase. At non-permissive temperature, PDGF receptor (PDGFR) signalling, assessed by phosphoY(751)-specific antibodies (a docking site for PI3-K), was not detected without PDGF and showed a concentration-dependent PDGF response. Both immunolabeling of PI3-K (p110) and live cell imaging of its product (phosphatidylinositol 3,4,5 tris-phosphate) showed PI3-K recruitment and activation at lamellipodia polarized towards a PDGF gradient. Centrosomes and PDGFR- and Src-bearing endosomes were also oriented towards this gradient. Upon v-Src thermoactivation, (i) Y(751) phosphorylation was moderately induced without PDGF and synergistically increased with PDGF; (ii) PI3-K was recruited and activated all along the plasma membrane without PDGF and did not polarize in response to a PDGF gradient; and (iii) polarization of centrosomes and of PDGFR-bearing endosomes were also abrogated. Thus, PDGF can further increase PDGFR auto-phosphorylation despite strong Src kinase activity, but diffuse downstream activation of PI3-K by Src abrogates cell polarization and chemotaxis: "signalling requires silence"