Sandra L. Schmid

Regulated portals of entry into the cell

The plasma membrane is the interface between cells and their harsh environment. Uptake of nutrients and all communication among cells and between cells and their environment occurs through this interface. ‘Endocytosis’ encompasses several diverse mechanisms by which cells internalize macromolecules and particles into transport vesicles derived from the plasma membrane. It controls entry into the cell and has a crucial role in development, the immune response, neurotransmission, intercellular communication, signal transduction, and cellular and organismal homeostasis. As the complexity of molecular interactions governing endocytosis are revealed, it has become increasingly clear that it is tightly coordinated and coupled with overall cell physiology and thus, must be viewed in a broader context than simple vesicular trafficking.

Control of EGF Receptor Signaling by Clathrin-Mediated Endocytosis

Epidermal growth factor receptor (EGFR) signaling was analyzed in mammalian cells conditionally defective for receptor-mediated endocytosis. EGF-dependent cell proliferation was enhanced in endocytosis-defective cells. However, early EGF-dependent signaling events were not uniformly up-regulated. A subset of signal transducers required the normal endocytic trafficking of EGFR for full activation. Thus, endocytic trafficking of activated EGFR plays a critical role not only in attenuating EGFR signaling but also in establishing and controlling specific signaling pathways.

Robust single particle tracking in live cell time-lapse sequences

Single-particle tracking (SPT) is often the rate-limiting step in live-cell imaging studies of subcellular dynamics. Here we present a tracking algorithm that addresses the principal challenges of SPT, namely high particle density, particle motion heterogeneity, temporary particle disappearance, and particle merging and splitting. The algorithm first links particles between consecutive frames and then links the resulting track segments into complete trajectories. Both steps are formulated as global combinatorial optimization problems whose solution identifies the overall most likely set of particle trajectories throughout a movie. Using this approach, we show that the GTPase dynamin differentially affects the kinetics of long- and short-lived endocytic structures and that the motion of CD36 receptors along cytoskeleton-mediated linear tracks increases their aggregation probability. Both applications indicate the requirement for robust and complete tracking of dense particle fields to dissect the mechanisms of receptor organization at the level of the plasma membrane.

Tubular membrane invaginations coated by dynamin rings are induced by GTP-γS in nerve terminals

THE mechanisms through which synaptic vesicle membranes are reinternalized after exocytosis remain a matter of debate1–5. Because several vesicular transport steps require GTP hydrolysis6–9, GTP-γS may help identify intermediates in synaptic vesicle recycling. In GTP-γS-treated nerve terminals, we observed tubular invaginations of the plasmalemma that were often, but not always, capped by a clathrin-coated bud. Strikingly, the walls of these tubules were decorated by transverse electron-dense rings that were morphologically similar to structures formed by dynamin around tubular templates10,11. Dynamin is a GTPase implicated in synaptic vesicle endocytosis12–14 and here we show that the walls of these membranous tubules, but not their distal ends, were positive for dynamin immunoreactivity. These findings demonstrate that dynamin and clathrin act at different sites in the formation of endocytic vesicles. They strongly support a role for dynamin in the fission reaction and suggest that stabilization of the GTP-bound conformation of dynamin leads to tubule formation by progressive elongation of the vesicle stalk.

Dynamin and its partners: A progress report

Dynamins role in clathrin-mediated endocytosis is now well established. Here we review new evidence from the past two years for the function of dynamin and related GTPases in other Intracellular trafficking events. We then summarize current information on the domain structure and function of this multidomain GTPase. Finally, we describe dynamin partners and their function in the context of clathrin-mediated endocytosis.

Impairment of dynamin's GAP domain stimulates receptor-mediated endocytosis.

Dynamin is a GTP-hydrolysing protein that is an essential participant in clathrin-mediated endocytosis by cells. It self-assembles into ‘collars’ in vitro which also form in vivo at the necks of invaginated coated pits. This self-assembly stimulates dynamins GTPase activity and it has been proposed that dynamin hydrolyses GTP in order to generate the force needed to sever vesicles from the plasma membrane. A mechanism is now described in which self-assembly of dynamin is coordinated by a domain of dynamin with a GTPase-activating function. Unexpectedly, when dynamin mutants defective in self-assembly-stimulated GTPase activity are overexpressed, receptor-mediated endocytosis is accelerated. The results indicate that dynamin, like other members of the GTPase superfamily, functions as a molecular regulator in receptor-mediated endocytosis, rather than as a force-generating GTPase.

Actin assembly plays a variable, but not obligatory role in receptor-mediated endocytosis in mammalian cells.

Three cell‐permeant compounds, cytochalasin D, latrunculin A and jasplakinolide, which perturb intracellular actin dynamics by distinct mechanisms, were used to probe the role of filamentous actin and actin assembly in clathrin‐mediated endocytosis in mammalian cells. These compounds had variable effects on receptor‐mediated endocytosis of transferrin that depended on both the cell line and the experimental protocol employed. Endocytosis in A431 cells assayed in suspension was inhibited by latrunculin A and jasplakinolide, but resistant to cytochalasin D, whereas neither compound inhibited endocytosis in adherent A431 cells. In contrast, endocytosis in adherent CHO cells was more sensitive to disruption of the actin cytoskeleton than endocytosis in CHO cells grown or assayed in suspension. Endocytosis in other cell types, including nonadherent K562 human erythroleukemic cells or adherent Cos‐7 cells was unaffected by disruption of the actin cytoskeleton. While it remains possible that actin filaments can play an accessory role in receptor‐mediated endocytosis, these discordant results indicate that actin assembly does not play an obligatory role in endocytic coated vesicle formation in cultured mammalian cells.

Two distinct subpopulations of endosomes involved in membrane recycling and transport to lysosomes

Functionally distinct subpopulations of endosomes involved in targeting internalized material to specific intracellular destinations were resolved as two distinct peaks by free-flow electrophoresis. The less anodally shifted peak contained a population of early endosomes selectively labeled by brief exposures to endocytic tracers or by receptor-bound transferrin. Late endosomes, labeled only after longer periods of internalization, migrated more toward the anode. While both fluid phase and certain receptor-bound markers could be rapidly chased from early to late endosomes, transferrin remained in vesicles comigrating with early endosomes even after prolonged uptake. Thus early and late endosomes are kinetically related but functionally distinct: early endosomes serve as the major site of recycling of membrane and surface receptors and late endosomes are involved in delivery to lysosomes. The subpopulations each contain unique polypeptides not found on the plasma membrane. Thus, endosomes cannot be derived entirely from internalized cell surface components, and may have at least partly independent biosynthetic origins.

Regulation of signal transduction by endocytosis.

Endocytosis of ligand-activated receptors has generally been considered a mechanism to attenuate signaling. There is now a growing body of evidence suggesting that this process is much more sophisticated and that endocytic membrane trafficking regulates both the intensity of signaling and the co-localization of activated receptors with downstream signaling molecules.

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 dynamins 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.