endocytic and recycling pathways

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A screen for membrane fission catalysts identifies the ATPase EHD1.

Kamerkar SC, Roy K, Bhattacharyya S, Pucadyil TJ. Biochemistry. 2019 PMID: 30403133

ATP-dependent membrane remodeling links EHD1 functions to endocytic recycling.

Deo R, Kushwah MS, Kamerkar SC, Kadam NY, Dar S, Babu K, Srivastava A, Pucadyil TJ. Nat Commun. 2018 PMID: 30518883

Endocytosis and recycling pathways are vital to cellular physiology as it regulates nutrient uptake and display of adhesion molecules, ion-channels, and antigen-presenting receptors. Recycling is managed by the endocytic recycling compartment (ERC) or the tubular recycling endosome (TRE), which is a dynamic organelle composed of a network of membrane tubules and vesicles concentrated in the perinuclear region. The ERC receives a high density of soluble and membrane-bound cargo from endocytic vesicles, which are then sorted and released for recycling in transport vesicles from this compartment. The mechanisms by which such vesicles are released from the ERC remain ill defined. Using an unbiased screen to identify proteins that catalyze fission, we discovered an ATP-binding Eps15-homology domain-containing protein1 (EHD1), a member of the dynamin superfamily of proteins. Addition of purified EHD1 with ATP to membrane tubes that mimic the tubular ERC causes fission thus establishing it as a fission catalyst and the first one reported to utilize ATP hydrolysis. 

mitochondrial division

Mitochondria are double-membrane tubular organelles and their fusion and division dynamics is linked to ATP production, quality control, segregation of mitochondrial DNA, mitophagy, and apoptosis. Mitochondrial division is regulated by a plethora of cellular factors including the metabolic state of the cell, reactive oxygen species, calcium signaling, and contacts with other organelles. We prepare artificial membrane templates that mimic the mitochondria in its size and tubular architecture as well as its lipid and protein composition and use these as substrates for fission. Insights gained from such in vitro assays are then tested in cell culture and animal models to arrive at a comprehensive mechanistic understanding of mitochondrial division.

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Dynamin-related protein 1 has membrane constricting and severing abilities sufficient for mitochondrial and peroxisomal fission.

Kamerkar SC, Kraus F, Sharpe AJ, Pucadyil TJ, Ryan MT.  Nat Commun. 2018 PMID: 30531964

mechanism of membrane fission

Fission or the splitting of a vesicular compartment is central to diverse cellular processes such as cytokinesis, organelle inheritance and vesicular transport. This process requires the enclosing lipid bilayer to be brought to close proximity, which, from theory, represents a distance of separation of 5 nm. Since cellular compartments are of much larger dimensions, fission follows a topological transformation of the limiting membrane into a highly curved tube-like intermediate. The seminal discovery of reversible paralysis in flies harbouring a temperature-sensitive allele of the GTPase dynamin, which causes defects in the fission-induced birth of synaptic vesicles, prompted a surge in researching form and function of this molecule. Using nanotubes engineered to mimic the dimensions of necks of these vesicles, we monitor dynamics of a tube-severing reaction in real time using fluorescence microscopy. Together, these technological developments set the stage for dynamic and correlative analyses of conformational changes in dynamin that  render it capable of catalyzing membrane fission.

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The 2018 biomembrane curvature and remodeling roadmap.

Bassereau P, Jin R, Baumgart T, Deserno M, Dimova R, Frolov VA, Bashkirov PV, Grubmüller H, Jahn R, Risselada HJ, Johannes L, Kozlov MM, Lipowsky R, Pucadyil TJ, Zeno WF, Stachowiak JC, Stamou D, Breuer A, Lauritsen L, Simon C, Sykes C, Voth GA, Weikl TR.

J Phys D Appl Phys. 2018 PMID: 30655651

The pleckstrin-homology domain of dynamin is dispensable for membrane constriction and fission.

Dar S, Pucadyil TJ. Mol Biol Cell. 2016 PMID: 28035046

A high-throughput platform for real-time analysis of membrane fission reactions reveals dynamin function.

Dar S, Kamerkar SC, Pucadyil TJ. Nat Cell Biol. 2015 PMID: 26479317

prelude to fission - membrane budding

Clathrin-mediated endocytosis (CME) manages the sorting and uptake of the bulk of membrane proteins from the plasma membrane in cells. CME is initiated by the formation of clathrin-coated pits (CCPs), in which adaptors nucleate clathrin assembly and in turn get clustered. Clathrin adaptors display diversity in both the type and number of evolutionarily conserved clathrin-binding boxes. How this diversity relates to the process of adaptor clustering as clathrin assembles around a growing pit remains unclear. Using real-time, fluorescence microscopy-based assays, we reconstitute formation of clathrin assemblies on planar lipid bilayers that display various adaptor proteins. 

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SMrT assay for real-time visualization and analysis of clathrin assembly reactions.

Andhare D, Holkar SS, Pucadyil TJ.

Methods Mol Biol. 2018 PMID: 30129016

Comparative analysis of adaptor-mediated clathrin assembly reveals general principles for adaptor clustering.

Pucadyil TJ, Holkar SS.

Mol Biol Cell. 2016 PMID: 27559129

Spatial control of epsin-induced clathrin assembly by membrane curvature.

Holkar SS, Kamerkar SC, Pucadyil TJ.

J Biol Chem. 2015 PMID: 25837255