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

RAB-5 Controls the Cortical Organization and Dynamics of PAR Proteins to Maintain C. elegans Early Embryonic Polarity

In all organisms, cell polarity is fundamental for most aspects of cell physiology. In many species and cell types, it is controlled by the evolutionarily conserved PAR-3, PAR-6 and aPKC proteins, which are asymmetrically localized at the cell cortex where they define specific domains. While PAR proteins define the antero-posterior axis of the early C. elegans embryo, the mechanism controlling their asymmetric localization is not fully understood. Here we studied the role of endocytic regulators in embryonic polarization and asymmetric division. We found that depleting the early endosome regulator RAB-5 results in polarity-related phenotypes in the early embryo. Using Total Internal Reflection Fluorescence (TIRF) microscopy, we observed that PAR-6 is localized at the cell cortex in highly dynamic puncta and depleting RAB-5 decreased PAR-6 cortical dynamics during the polarity maintenance phase. Depletion of RAB-5 also increased PAR-6 association with clathrin heavy chain (CHC-1) and this increase depended on the presence of the GTPase dynamin, an upstream regulator of endocytosis. Interestingly, further analysis indicated that loss of RAB-5 leads to a disorganization of the actin cytoskeleton and that this occurs independently of dynamin activity. Our results indicate that RAB-5 promotes C. elegans embryonic polarity in both dynamin-dependent and -independent manners, by controlling PAR-6 localization and cortical dynamics through the regulation of its association with the cell cortex and the organization of the actin cytoskeleton

Untersuchung der molekularen Dynamik von Clathrin mit Totalreflektionsmikroskopie

Das Protein Clathrin bildet dreizackförmige Moleküle (Triskelia), die aus je drei light chain- und drei heavy chain-Untereinheiten bestehen. Die Triskelia bauen sich im Zellinneren auf der Oberfläche von Membranen selbständig zu flachen sechseckigen oder zu gewölbten fünfeckigen Gittern zusammen. Clathrin spielt insbesondere für den intrazellulären Transport eine wichtige Rolle; in clathrin pits werden durch steigenden Krümmungsradius des Clathringitters kleine bläschenförmige Einstülpungen der Zellmembran gebildet - Transportvesikel - die anschließend abgeschnürt und ins Zellinnere transportiert werden. Gegenstand dieses Projekts war die Frage, wie die Zelle den selbständigen Aufbau von Clathringittern reguliert. Hierfür wurde mit Totalreflektionsmikroskopie die Kinetik des Austausches von fluoreszenten Konstrukten der beiden Clathrin-Untereinheiten untersucht.Bei der Totalreflektion eines Laserstrahls an einer dielektrischen Grenzfläche, z.B. zwischen Glas und einer Zelle in wässrigem Medium, entsteht im optisch dünneren Medium ein inhomogenes, sog. evaneszentes elektromagnetisches Nahfeld. In der Totalreflektionsmikroskopie wird dieses Feld zur Fluoreszenzanregung in der Zelle verwendet, und da seine Intensität mit steigendem Abstand von der Oberfläche exponentiell abklingt, beschränkt dies die Fluoreszenzanregung auf eine dünne Schicht unmittelbar auf der Grenzfläche. Aus diesem Grund ist diese Technik in besonderer Weise geeignet, solche zellulären Prozesse zu beobachten, die innerhalb weniger hundert Nanometer Entfernung von der Zellmembran stattfinden.Mit der FRAP-Technik (fluorescence recovery after photobleaching) wird die Geschwindigkeit untersucht, mit der einzelne Clathrin-Moleküle aus dem Gitter aus- und wieder eingebaut werden. Das sich nach einem Bleichpuls wieder erholende Fluoreszenzsignal des Gitters ist eine Exponentialfunktion, deren Zeitkonstante τder Kehrwert der Reaktionsrate koff (Abbindungsrate) ist. Das Reaktionssignal aus dem Gitter wird dabei teilweise durch ein diffusives Signal aus dem Zytosol überdeckt, weshalb die eindeutige Trennung von Reaktions- und Diffusionsignal ein wichtiger Teil dieser Arbeit war, um eine quantitative Abschätzung der Abbindungskinetik zu erlauben.Bei Zimmertemperatur betrug die Zeitkonstante für die Abbindung der Clathrin light chain τLC=18.9±1.3s. Der Austausch der light chain wurde durch Behandlung mit Sucrose, sowie durch Kalzium- und ATP-Depletion blockiert. Für Clathrin heavy chain war die Zeitkonstante der Abbindungsreaktion schneller als für light chain; außerdem waren Kalzium- und ATP-Depletion nicht ausreichend, um den Austausch vollständig zu unterbinden. Daraus schließen wir, daß für heavy chain ein von der light chain unabhängiger Pfad mit schnellerer Zeitkonstante existiert, welcher durch ATP- oder Kalziumdepletion isoliert wird und dessen Zeitkonstante 10.0±0.9s beträgt. Der jeweilige Beitrag der beiden einzelnen Austauschmechanismen wurde experimentell durch die selektive Überexpression einer Clathrin-Untereinheit moduliert.Diese Ergebnisse bestätigen, daß die Anwesenheit der Clathrin light chain die heavy chain Untereinheiten im Gittern stabilisiert, und zeigen, daß in vivo die light chain die Schaltstelle darstellt, über die die Zelle mittels Kalzium und ATP die Clathrinfunktion reguliert

Cell Ratcheting through the Sbf RabGEF Directs Force Balancing and Stepped Apical Constriction

During Drosophila melanogaster gastrulation, the invagination of the prospective mesoderm is driven by the pulsed constriction of apical surfaces. Here, we address the mechanisms by which the irreversibility of pulsed events is achieved while also permitting uniform epithelial behaviors to emerge. We use MSD-based analyses to identify contractile steps and find that when a trafficking pathway initiated by Sbf is disrupted, contractile steps become reversible. Sbf localizes to tubular, apical surfaces and associates with Rab35, where it promotes Rab GTP exchange. Interestingly, when Sbf/Rab35 function is compromised, the apical plasma membrane becomes deeply convoluted, and nonuniform cell behaviors begin to emerge. Consistent with this, Sbf/Rab35 appears to prefigure and organize the apical surface for efficient Myosin function. Finally, we show that Sbf/Rab35/CME directs the plasma membrane to Rab11 endosomes through a dynamic interaction with Rab5 endosomes. These results suggest that periodic ratcheting events shift excess membrane from cell apices into endosomal pathways to permit reshaping of actomyosin networks and the apical surface

Regulation of Clathrin-mediated Endocytosis by Hierarchical Allosteric Activation of AP2

The critical initiation phase of clathrin-mediated endocytosis (CME) determines where and when endocytosis occurs. Heterotetrameric adaptor protein 2 (AP2) complexes, which initiate clathrin-coated pit (CCP) assembly, are activated by conformational changes in response to phosphatidylinositol-4,5-bisphosphate (PIP2) and cargo binding at multiple sites. However, the functional hierarchy of interactions and how these conformational changes relate to distinct steps in CCP formation in living cells remains unknown. We used quantitative live-cell analyses to measure discrete early stages of CME and show how sequential, allosterically regulated conformational changes activate AP2 to drive both nucleation and subsequent stabilization of nascent CCPs. Our data establish that cargoes containing Yxxφ motif, but not dileucine motif, play a critical role in the earliest stages of AP2 activation and CCP nucleation. Interestingly, these cargo and PIP2 interactions are not conserved in yeast. Thus, we speculate that AP2 has evolved as a key regulatory node to coordinate CCP formation and cargo sorting and ensure high spatial and temporal regulation of CME

Nanoscale Coupling of Endocytic Pit Growth and Stability

Clathrin-mediated endocytosis, an essential process for plasma membrane homeostasis and cell signaling, is characterized by stunning heterogeneity in the size and lifetime of clathrin-coated endocytic pits (CCPs). If and how CCP growth and lifetime are coupled and how this relates to their physiological function are unknown. We combine computational modeling, automated tracking of CCP dynamics, electron microscopy, and functional rescue experiments to demonstrate that CCP growth and lifetime are closely correlated and mechanistically linked by the early-acting endocytic F-BAR protein FCHo2. FCHo2 assembles at the rim of CCPs to control CCP growth and lifetime by coupling the invagination of early endocytic intermediates to clathrin lattice assembly. Our data suggest a mechanism for the nanoscale control of CCP growth and stability that may similarly apply to other metastable structures in cells

Rab8 Directs Furrow Ingression and Membrane Addition during Epithelial Formation inDrosophila Melanogaster

One of the most fundamental changes in cell morphology is the ingression of a plasma membrane furrow. The Drosophila embryo undergoes several cycles of rapid furrow ingression during early development that culminate in the formation of an epithelial sheet. Previous studies have demonstrated the requirement for intracellular trafficking pathways in furrow ingression; however, the pathways that link compartmental behaviors with cortical furrow ingression events are unclear. Here, we show that Rab8 has striking dynamic behaviors in vivo. As furrows ingress, cytoplasmic Rab8 puncta are depleted and Rab8 accumulates at the plasma membrane in a location that coincides with known regions of directed membrane addition. We additionally use CRISPR/Cas9 technology to N-terminally tag Rab8, which is then used to address endogenous localization and function. Endogenous Rab8 displays partial coincidence with Rab11 and the Golgi, and this colocalization is enriched during the fast phase of cellularization. When Rab8 function is disrupted, furrow formation in the early embryo is completely abolished. We also demonstrate that Rab8 behaviors require the function of the exocyst complex subunit Sec5 as well as the recycling endosome protein Rab11. Active, GTP-locked Rab8 is primarily associated with dynamic membrane compartments and the plasma membrane, whereas GDP-locked Rab8 forms large cytoplasmic aggregates. These studies suggest a model in which active Rab8 populations direct furrow ingression by guiding the targeted delivery of cytoplasmic membrane stores to the cell surface through interactions with the exocyst tethering complex