Hongying Shen

Coordinated actions of actin and BAR proteins upstream of dynamin at endocytic clathrin-coated pits.

The GTPase dynamin, a key player in endocytic membrane fission, interacts with numerous proteins that regulate actin dynamics and generate/sense membrane curvature. To determine the functional relationship between these proteins and dynamin, we have analyzed endocytic intermediates that accumulate in cells that lack dynamin (derived from dynamin 1 and 2 double conditional knockout mice). In these cells, actin-nucleating proteins, actin, and BAR domain proteins accumulate at the base of arrested endocytic clathrin-coated pits, where they support the growth of dynamic long tubular necks. These results, which we show reflect the sequence of events in wild-type cells, demonstrate a concerted action of these proteins prior to, and independent of, dynamin and emphasize similarities between clathrin-mediated endocytosis in yeast and higher eukaryotes. Our data also demonstrate that the relationship between dynamin and actin is intimately connected to dynamins endocytic role and that dynamin terminates a powerful actin- and BAR protein-dependent tubulating activity.

Recruitment of Endophilin to Clathrin-Coated Pit Necks Is Required for Efficient Vesicle Uncoating after Fission

Endophilin is a membrane-binding protein with curvature-generating and -sensing properties that participates in clathrin-dependent endocytosis of synaptic vesicle membranes. Endophilin also binds the GTPase dynamin and the phosphoinositide phosphatase synaptojanin and is thought to coordinate constriction of coated pits with membrane fission (via dynamin) and subsequent uncoating (via synaptojanin). We show that although synaptojanin is recruited by endophilin at bud necks before fission, the knockout of all three mouse endophilins results in the accumulation of clathrin-coated vesicles, but not of clathrin-coated pits, at synapses. The absence of endophilin impairs but does not abolish synaptic transmission and results in perinatal lethality, whereas partial endophilin absence causes severe neurological defects, including epilepsy and neurodegeneration. Our data support a model in which endophilin recruitment to coated pit necks, because of its curvature-sensing properties, primes vesicle buds for subsequent uncoating after membrane fission, without being critically required for the fission reaction itself.

Dynamin triple knockout cells reveal off target effects of commonly used dynamin inhibitors.

Summary Dynamin, which is encoded by three genes in mammals, is a GTPase implicated in endocytic membrane fission. Dynamin 1 and 3 are predominantly expressed in brain, whereas dynamin 2 is ubiquitously expressed. With the goal of assessing the impact of the lack of dynamin on cell physiology, we previously generated and characterized dynamin 1 and 2 double knockout (DKO) fibroblasts. These DKO cells were unexpectedly viable in spite of a severe impairment of clathrin-mediated endocytosis. As low-level expression of the dynamin 3 gene in these cells could not be excluded, we have now engineered dynamin 1, 2 and 3 triple KO (TKO) fibroblasts. These cells did not reveal any additional defects beyond what was previously observed in DKO fibroblasts. Surprisingly, although fluid-phase endocytosis and peripheral membrane ruffling were not impaired by the lack of all three dynamins, two structurally similar, widely used dynamin inhibitors, dynasore and Dyngo-4a, robustly inhibited these two processes both in wild-type and TKO cells. Dynamin TKO cells will be useful tools for the further exploration of dynamin-dependent processes and the development of more specific dynamin inhibitors.

Suppression of EGFR endocytosis by dynamin depletion reveals that EGFR signaling occurs primarily at the plasma membrane

The role of endocytosis in the control of EGF receptor (EGFR) activation and cell signaling was explored by using mouse fibroblasts in which dynamin was conditionally depleted. Dynamin is a GTPase shown to play an important role in the control clathrin mediated endocytosis of EGFR and other cell surface receptors. In this report, we demonstrate that EGF binding activity and the display of high and low affinity EGFRs on the cell surface are not affected by dynamin depletion. By contrast, dynamin depletion leads to a strong inhibition of EGFR endocytosis, robust enhancement of EGFR autophosphorylation and ubiquitination, and slower kinetics of EGFR degradation. Surprisingly, MAPK stimulation induced by either low or high EGF concentrations is not affected by dynamin depletion. While a similar initial Akt response is detected in control or dynamin depleted fibroblasts, a somewhat more sustained Akt stimulation is detected in the dynamin depleted cells. These experiments demonstrate that dynamin-mediated endocytosis leads to attenuation of EGFR activation and degradation and that stimulation of the MAPK response and Akt activation are primarily mediated by activated EGFR located in the plasma membrane.

Coupling between endocytosis and sphingosine kinase 1 recruitment

Genetic studies have suggested a functional link between cholesterol/sphingolipid metabolism and endocytic membrane traffic. Here we show that perturbing the cholesterol/sphingomyelin balance in the plasma membrane results in the massive formation of clusters of narrow endocytic tubular invaginations positive for N-BAR proteins. These tubules are intensely positive for sphingosine kinase 1 (SPHK1). SPHK1 is also targeted to physiologically occurring early endocytic intermediates, and is highly enriched in nerve terminals, which are cellular compartments specialized for exo/endocytosis. Membrane recruitment of SPHK1 involves a direct, curvature-sensitive interaction with the lipid bilayer mediated by a hydrophobic patch on the enzyme’s surface. The knockdown of SPHKs results in endocytic recycling defects, and a mutation that disrupts the hydrophobic patch of Caenorhabditis elegans SPHK fails to rescue the neurotransmission defects in loss-of-function mutants of this enzyme. Our studies support a role for sphingosine phosphorylation in endocytic membrane trafficking beyond the established function of sphingosine-1-phosphate in intercellular signalling.

Constitutive activated Cdc42-associated kinase (Ack) phosphorylation at arrested endocytic clathrin-coated pits of cells that lack dynamin

In cells in which clathrin-mediated endocytosis is arrested before fission by depleting dynamin, the major change in tyrosine phosphorylation is the increased phosphorylation/activation of Ack, a tyrosine kinase. Our finding reveals a link between the progression of clathrin-coated pits to endocytic vesicles and an activation-deactivation cycle of Ack.

SnapShot: membrane curvature sensors and generators.

Cells and intracellular organelles are enclosed by bilayer lipid membranes, whose curvatures define their shape. Generation of bilayer curvature is critical for organelle biogenesis and to mediate vectorial transport from one membrane to another via vesicular or tubular intermediates. Membrane curvature can be driven by lipid perturbations that create asymmetry in the two leaflets of the bilayer, by bilayer binding and/or penetrating proteins, or by forces applied to membranes by the cytoskeleton. This SnapShot provides an overview of the mechanisms through which these proteins generate and/or sense curvature. These two properties are tightly interconnected. Proteins that initiate curvature by inserting a wedge in the bilayer (for example, amphipathic helices and hydrophobic loops) will bind preferentially to a precurved bilayer, where bending has created a gap in lipid packing. Likewise, proteins that function primarily as curved scaffolds generally assemble into polymers that propagate curvature, and their binding to the bilayer and their polymerization are both facilitated by a precurved surface. In some proteins, both scaffold mechanisms and wedge-based mechanisms cooperate. Major mechanisms of protein-driven membrane deformation are listed below. Examples given refer to structures and schematic cartoons shown in the figure.

A Novel Multiple Hypothesis Based Particle Tracking Method for Clathrin Mediated Endocytosis Analysis Using Fluorescence Microscopy

In order to quantitatively analyze biological images and study underlying mechanisms of the cellular and subcellular processes, it is often required to track a large number of particles involved in these processes. Manual tracking can be performed by the biologists, but the workload is very heavy. In this paper, we present an automatic particle tracking method for analyzing an essential subcellular process, namely clathrin mediated endocytosis. The framework of the tracking method is an extension of the classical multiple hypothesis tracking (MHT), and it is designed to manage trajectories, solve data association problems, and handle pseudo-splitting/merging events. In the extended MHT framework, particle tracking becomes evaluating two types of hypotheses. The first one is the trajectory-related hypothesis, to test whether a recovered trajectory is correct, and the second one is the observation-related hypothesis, to test whether an observation from an image belongs to a real particle. Here, an observation refers to a detected particle and its feature vector. To detect the particles in 2D fluorescence images taken using total internal reflection microscopy, the images are segmented into regions, and the features of the particles are obtained by fitting Gaussian mixture models into each of the image regions. Specific models are developed according to the properties of the particles. The proposed tracking method is demonstrated on synthetic data under different scenarios and applied to real data.

Tracking clathrin coated pits with a multiple hypothesis based method

Cellular processes are crucial for cells to survive and function properly. To study their underlying mechanisms quantitatively with fluorescent live cell microscopy, it is necessary to track a large number of particles involved in these processes. In this paper, we present a method to automatically track particles, called clathrin coated pits (CCPs), which are formed in clathrin mediated endocytosis (CME). The tracking method is developed based on a MAP framework, and it consists of particle detection and trajectory estimation. To detect particles in 2D images and take account of Poisson noise, a Gaussian mixture model is fitted to image data, for which initial parameters are provided by a combination of image filtering and histogram based thresholding methods. A multiple hypothesis based algorithm is developed to estimate the trajectories based on detection data. To use the current knowledge about CCPs, their properties of motion and intensity are considered in our models. The tracking method is evaluated on synthetic data and real data, and experimental results show that it has high accuracy and is in good agreement with manual tracking.

A Bayesian method for 3D estimation of subcellular particle features in multi-angle TIRF microscopy

Multi-angle total internal reflection fluorescence microscopy (MA-TIRFM) is a relatively new and powerful tool to study subcellular particles near cell membrane due to its unique illumination mechanism. We present a MAP-Bayesian method to automatically estimate features of individual particles in MA-TIRF images, including 3D positions, relative sizes, and relative amount of fluorophores. Using the MAP criterion, the optimal values of the features can be obtained by maximizing a nonlinear functional. Initial feature values are estimated by using image filters and clustering algorithms. The method is evaluated on synthetic data and results show that it has high accuracy. The result on real data from our initial experiments is also presented.