Discovering and analyzing functions of membrane fission proteins
Formation of organelles requires the limiting membrane to be constricted to nanometer dimensions before undergoing fission. Since organelles are of much larger dimensions, fission necessitates a topological transformation of organelles into a highly curved tube-like intermediate. Using Supported Membrane Templates (SMrT) that represent an array membrane nanotubes (see schematic below and Dar, Kamerkar and Pucadyil (2017) Nature Protocols for more details) and display a wide range of curvatures, mimicking intermediates in the process of fission, we screen tissue lysates for fission activity and identify the sets of proteins that contribute to such activity. We then purify and reconstitute their functions to better understand how they work. The hits that emerge from our screen are tested for cellular function by making knock-out models and analysing morphological changes to various membrane compartments.
Schematic of formation of SMrTs and a movie showing fission of nanotubes seen upon flowing a fission protein.
Workflow of the discovery process.
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Membrane contacts, lipid flux, and fission
Pucadyil (2023) Molecular Cell
Mechanistic analysis of a novel membrane-interacting variable loop in the pleckstrin-homology domain critical for dynamin function
Khurana et al. (2023) PNAS
Function and regulation of the divisome for mitochondrial fission
Kraus et al. (2021) Nature
Cellular functions and intrinsic attributes of the ATP-binding Eps15 homology domain-containing (EHD) proteins
Bhattacharyya and Pucadyil (2020) Protein Science
A Screen for Membrane Fission Catalysts Identifies the ATPase EHD1
Kamerkar et al. (2019) Biochemistry
Dynamin-related protein 1 has membrane constricting and severing abilities sufficient for mitochondrial and peroxisomal fission
Kamerkar and Kraus et al. (2018) Nature Communications
ATP-dependent membrane remodeling links EHD1 functions to endocytic recycling
Deo and Kushwah et al. (2018) Nature Communications
The pleckstrin-homology domain of dynamin is dispensable for membrane constriction and fission
Dar and Pucadyil (2017) Molecular Biology of the Cell
A high-throughput platform for real-time analysis of membrane fission reactions reveals dynamin function
Dar et al. (2015) Nature Cell Biology