fission screens


SMrT templates (see Resources page for more details) are membrane nanotubes arrayed on a glass coverslip. They can be prepared from diverse lipids and in different sizes, which make them a good mimic of tubular membrane intermediates generated during vesicle formation in cells. We employ these templates to screen tissue lysates for membrane fission activity. Using such screens, we hope to expand the repertoire of fission catalysts. 

Read more in this paper:

A screen for membrane fission catalysts identifies the ATPase EHD1.

Kamerkar SC, Roy K, Bhattacharyya S, Pucadyil TJ. Biochemistry

vesicle production in recycling pathways

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. We are interested in understanding mechanistic and regulatory principles leading to vesicle formation at the ERC. 

Read more in this paper:

Cellular functions and intrinsic attributes of the ATP-binding Eps15 homology domain-containing (EHD) proteins.

Bhattacharyya S, Pucadyil TJ. Protein Science

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. Nature Communications

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. Using nanotubes that mimic the mitochondria in form and composition and use them to understand mitochondrial division. 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.

Read more in this paper:

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.  Nature Communications

fission pathways

Fission 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. Using nanotubes that mimic dimensions of necks of budded vesicles, we monitor dynamics of fission in real time using fluorescence microscopy. Together, these technological developments set the stage for dynamic and correlative analyses of conformational changes in proteins that render them capable of catalyzing membrane fission.

Read more in these papers:

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.

Journal of Physics D Applied Physics

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

Dar S, Pucadyil TJ. Molecular Biology of the Cell

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

Dar S, Kamerkar SC, Pucadyil TJ. Nature Cell Biology

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. 

Read more in these papers:

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

Andhare D, Holkar SS, Pucadyil TJ.

Methods in Molecular Biology

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

Pucadyil TJ, Holkar SS.

Molecular Biology of the Cell

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

Holkar SS, Kamerkar SC, Pucadyil TJ.

Journal of Biological Chemistry