Analyzing the functional, mechanistic and regulatory aspects of membrane tubulation, fission and fusion during vesicular transport
Cells are compartmentalised into various membrane-bound organelles, which are highly dynamic in shape and composition. They are formed and maintained by the constant exchange of material between donor and acceptor compartments via vesicular transport carriers. These carriers are formed by membrane tubulation of the donor membrane. Membrane fission releases them from the donor compartment, and their fusion with the acceptor compartment completes the transport cycle.
The throughput and fidelity of vesicular transport pathways relies on a diverse and complex set of proteins, as well as an intricate coordination between the various step of the transport cycle. Remarkably, many of the participant proteins are conserved from yeast to humans, thus highlighting their importance to sustain life. Furthermore, mutations in many of the participant proteins are linked to various developmental disorders and several pathogens subvert the intrinsic coordination in vesicular transport pathways during infection.
Movie credit - Soumya Bhattacharyya
At the Pucadyil lab, we focus on discovering proteins that orchestrated membrane tubulation, fission and fusion and understand their mechanism.
Evolution of membrane intermediates during vesicular transport
Membrane tubulation, fission and fusion are extremely dynamic processes that involve a myriad of proteins in cells.
But at the core of these processes lies the ability of specific proteins to have evolved to overcome the hydrophobic effect, which otherwise ensures that the membrane retains its planar topology. In other words, these proteins manage to bend membrane out of shape - a process collectively referred to as membrane remodelling.
Original image credit - Himani Khurana
Reconstituting membrane remodelling ex vivo
Cellular complexity, while fascinating, frequently comes in the way of assign specific functions to proteins and building testable models. To get around these problems, we use a bottom-up reconstitution approach that allows recreating membrane tubulation, fission and fusion with specific proteins or their combinations on a define membrane template outside of the cell - a process that is referred to as reconstitution.
Reconstituted tubulation and fission of Supported Membrane Templates
For our reconstitution efforts, we utilise Supported Membrane Templates (SMrT) that represent a versatile assay system displaying a wide range of topologies, from planar bilayers to highly curved membrane tubes, resting on a passive glass surface (see schematic above and Swaminathan and Pucadyil (2024) Biochem. Soc. Transac.). Our experimental workflow is simple and typically involves the real-time monitoring of membrane tubulation, fission and fusion using fluorescence microscopy.
Read more here
Dynamics of membrane tubulation coupled with fission by a two-component module
Bhattacharyya and Pucadyil (2024) PNAS
"Gearing" up for dynamin-catalyzed membrane fission
Khurana and Pucadyil (2023) Current Opinions in Cell Biology
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