Clathrin coated pits and vesicles mediate the major pathway for uptake of plasma membrane receptors and their ligands into the cell to regulate signal transduction, the immune response and cell-cell and cell substrate interactions. Recent advances have enabled visualization of the dynamic behavior of fluorescently-labeled clathrin coated structures (CCSs) on the plasma membrane and during endocytosis, although the driving forces for this motility are unknown. The plasma membrane is structurally supported by an underlying cortical actin filament cytoskeleton whose dynamic remodeling maintains and changes the shape of the cell. Clearly the deformation of the plasma membrane during endocytic vesicle formation must somehow require concomitant local regulation of the underlying cortical actin cytoskeleton. Indeed, we have recently shown that actin assembly and disassembly are essential for multiple diverse aspects of CCS dynamics. How are the complex dynamics of clathrin-mediated endocytosis integrated with cortical actin assembly and disassembly? What are the functional linkages between these two highly dynamic and complex cellular machineries? What is the physiological significance of these diverse, actin-dependent CCS dynamic behaviors? To address these issues, we have established an intense collaborative effort that brings together experts on endocytosis with experts on actin dynamics and combines our technical expertise in cellular biochemistry, quantitative fluorescence microscopy, computational image analysis and mathematical modeling.
In Aim 1, we propose to develop new imaging assays, methods of trajectory analysis and statistical metrics to classify the diverse kinematics of clathrin-coated structures on the cell surface, during endocytosis and after nascent vesicle formation.
In Aim 2, using total internal reflection microscopy and quantitative fluorescent speckle microscopy, we will generate the first high-resolution, cross-correlation, spatio-temporal map of cortical actin and clathrin-coated structure dynamics in each kinematic class, to quantitatively examine the functional consequences of perturbing actin dynamics on endocytosis and to probe the physiological significance of the dynamic heterogeneity in CCSs.
In Aim 3, we will establish the molecular mechanisms that coordinate cortical actin dynamics with clathrin-mediated endocytosis. The project will advance us towards our long-term objective of developing a biochemical network model of the functional linkage between endocytosis and the actin cytoskeleton based on a large data repository of integrated dynamics of clathrin coated structures and cortical actin. ? ? ?
|Xiao, Guan-Yu; Mohanakrishnan, Aparna; Schmid, Sandra L (2018) Role for ERK1/2-dependent activation of FCHSD2 in cancer cell-selective regulation of clathrin-mediated endocytosis. Proc Natl Acad Sci U S A 115:E9570-E9579|
|Kadlecova, Zuzana; Spielman, Stephanie J; Loerke, Dinah et al. (2017) Regulation of clathrin-mediated endocytosis by hierarchical allosteric activation of AP2. J Cell Biol 216:167-179|
|Schmid, Sandra L (2017) Reciprocal regulation of signaling and endocytosis: Implications for the evolving cancer cell. J Cell Biol 216:2623-2632|
|Zaritsky, Assaf; Obolski, Uri; Gan, Zhuo et al. (2017) Decoupling global biases and local interactions between cell biological variables. Elife 6:|
|Reis, Carlos R; Chen, Ping-Hung; Bendris, Nawal et al. (2017) TRAIL-death receptor endocytosis and apoptosis are selectively regulated by dynamin-1 activation. Proc Natl Acad Sci U S A 114:504-509|
|Chen, Ping-Hung; Bendris, Nawal; Hsiao, Yi-Jing et al. (2017) Crosstalk between CLCb/Dyn1-Mediated Adaptive Clathrin-Mediated Endocytosis and Epidermal Growth Factor Receptor Signaling Increases Metastasis. Dev Cell 40:278-288.e5|
|Elkin, Sarah R; Lakoduk, Ashley M; Schmid, Sandra L (2016) Endocytic pathways and endosomal trafficking: a primer. Wien Med Wochenschr 166:196-204|
|Aguet, François; Upadhyayula, Srigokul; Gaudin, Raphaël et al. (2016) Membrane dynamics of dividing cells imaged by lattice light-sheet microscopy. Mol Biol Cell 27:3418-3435|
|Dean, Kevin M; Roudot, Philippe; Reis, Carlos R et al. (2016) Diagonally Scanned Light-Sheet Microscopy for Fast Volumetric Imaging of Adherent Cells. Biophys J 110:1456-65|
|Elkin, Sarah R; Bendris, Nawal; Reis, Carlos R et al. (2015) A systematic analysis reveals heterogeneous changes in the endocytic activities of cancer cells. Cancer Res 75:4640-50|
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