Nilakshee Bhattacharya
Florida State University
Network
Latest external collaboration on country level. Dive into details by clicking on the dots.
Publication
Featured researches published by Nilakshee Bhattacharya.
Nature Structural & Molecular Biology | 2013
Alex J. Noble; Qian Zhang; Jason O'Donnell; Hanaa Hariri; Nilakshee Bhattacharya; Alan G. Marshall; Scott M. Stagg
COPII vesicles transport proteins from the endoplasmic reticulum to the Golgi apparatus. Previous COPII-cage cryo-EM structures lacked the resolution necessary to determine the residues of Sec13 and Sec31 that mediate assembly and flexibility of the COPII cage. Here we present a 12-Å structure of the human COPII cage, where the tertiary structure of Sec13 and Sec31 is clearly identifiable. We employ this structure and a homology model of the Sec13–Sec31 complex to create a reliable pseudoatomic model of the COPII cage. We combined this model with hydrogen/deuterium-exchange MS analysis to characterize four distinct contact regions at the vertices of the COPII cage. Furthermore, we found that the two-fold symmetry of the Sec31 dimeric region in Sec13–Sec31 is broken upon cage formation and that the resulting hinge is essential to form the proper edge geometry in COPII cages.
The EMBO Journal | 2015
Adam Johnson; Nilakshee Bhattacharya; Michael Hanna; Janice G. Pennington; Amber L. Schuh; Lei Wang; Marisa S. Otegui; Scott M. Stagg; Anjon Audhya
In mammalian cells, cargo‐laden secretory vesicles leave the endoplasmic reticulum (ER) en route to ER‐Golgi intermediate compartments (ERGIC) in a manner dependent on the COPII coat complex. We report here that COPII‐coated transport carriers traverse a submicron, TFG (Trk‐fused gene)‐enriched zone at the ER/ERGIC interface. The architecture of TFG complexes as determined by three‐dimensional electron microscopy reveals the formation of flexible, octameric cup‐like structures, which are able to self‐associate to generate larger polymers in vitro. In cells, loss of TFG function dramatically slows protein export from the ER and results in the accumulation of COPII‐coated carriers throughout the cytoplasm. Additionally, the tight association between ER and ERGIC membranes is lost in the absence of TFG. We propose that TFG functions at the ER/ERGIC interface to locally concentrate COPII‐coated transport carriers and link exit sites on the ER to ERGIC membranes. Our findings provide a new mechanism by which COPII‐coated carriers are retained near their site of formation to facilitate rapid fusion with neighboring ERGIC membranes upon uncoating, thereby promoting interorganellar cargo transport.
Journal of Molecular Biology | 2012
Nilakshee Bhattacharya; Jason O’Donnell; Scott M. Stagg
Structural studies have revealed some of the organizing principles and mechanisms involved in the assembly of the COPII coat including the location of the Sec23/24 adapter layer. Previous studies, however, were unable to unambiguously determine the positions of Sec23 and Sec24 in the coat. Here, we have determined a cryogenic electron microscopic structure of Sec13/31 together with Sec23. Electron tomography revealed that the binding of Sec23 induces Sec13/31 to form a variety of different geometries including a cuboctahedron, as was previously characterized for Sec13/31 alone. Single-particle reconstruction of the Sec13/31-23 cuboctahedra revealed that the binding of Sec23 induces a conformational change in Sec13/31, resulting in a more extended conformation. Docking Sec23 crystal structures into the electron microscopy map suggested that Sec24 projects its cargo binding surface out into the large open faces of the coat. These results have implications for the mechanisms by which COPII transports large cargos, cargos with large intracellular domains, and for tethering complexes that must project out of the coat in order to interact with their binding partners. Furthermore, Sec23 binds Sec13/31 at two unique sites in the coat, which suggests that each site may have unique roles in the mechanisms of COPII vesiculation.
Journal of Molecular Biology | 2014
Hanaa Hariri; Nilakshee Bhattacharya; Kerri Johnson; Alex J. Noble; Scott M. Stagg
The small GTPase protein Sar1 is known to be involved in both the initiation of COPII-coated vesicle formation and scission of the nascent vesicle from the endoplasmic reticulum. The molecular details for the mechanism of membrane remodeling by Sar1 remain unresolved. Here, we show that Sar1 transforms synthetic liposomes into structures of different morphologies including tubules and detached vesicles. We demonstrate that Sar1 alone is competent for vesicle scission in a manner that depends on the concentration of Sar1 molecules occupying the membrane. Sar1 molecules align on low-curvature membranes to form an extended lattice. The continuity of this lattice breaks down as the curvature locally increases. The smallest repeating unit constituting the ordered lattice is a Sar1 dimer. The three-dimensional structure of the Sar1 lattice was reconstructed by substituting spherical liposomes with galactoceramide lipid tubules of homogeneous diameter. These data suggest that Sar1 dimerization is responsible for the formation of constrictive membrane curvature. We propose a model whereby Sar1 dimers assemble into ordered arrays to promote membrane constriction and COPII-directed vesicle scission.
Cellular Microbiology | 2015
Antonella Gianfelice; Phuong H.B. Le; Luciano A. Rigano; Susan Saila; Georgina C. Dowd; Tina McDivitt; Nilakshee Bhattacharya; Wanjin Hong; Scott M. Stagg; Keith Ireton
Listeria monocytogenes is a food‐borne pathogen that uses actin‐dependent motility to spread between human cells. Cell‐to‐cell spread involves the formation by motile bacteria of plasma membrane‐derived structures termed ‘protrusions’. In cultured enterocytes, the secreted Listeria protein InlC promotes protrusion formation by binding and inhibiting the human scaffolding protein Tuba. Here we demonstrate that protrusions are controlled by human COPII components that direct trafficking from the endoplasmic reticulum. Co‐precipitation experiments indicated that the COPII proteins Sec31A and Sec13 interact directly with a Src homology 3 domain in Tuba. This interaction was antagonized by InlC. Depletion of Sec31A or Sec13 restored normal protrusion formation to a Listeria mutant lacking inlC, without affecting spread of wild‐type bacteria. Genetic impairment of the COPII component Sar1 or treatment of cells with brefeldin A affected protrusions similarly to Sec31A or Sec13 depletion. These findings indicated that InlC relieves a host‐mediated restriction of Listeria spread otherwise imposed by COPII. Inhibition of Sec31A, Sec13 or Sar1 or brefeldin A treatment also perturbed the structure of cell–cell junctions. Collectively, these findings demonstrate an important role for COPII in controlling Listeria spread. We propose that COPII may act by delivering host proteins that generate tension at cell junctions.
Journal of Structural Biology | 2018
Mohammadreza Paraan; Nilakshee Bhattacharya; Vladimir N. Uversky; Scott M. Stagg
In COPII mediated vesicle formation, Sec13/Sec31 heterotetramers play a role in organizing the membranes into a spherical vesicle. There they oligomerize into a cage that interacts with the other COPII proteins to direct vesicle formation and concentrate cargo into a bud. In this role they must be flexible to accommodate different sizes and shapes of cargo, but also have elements that provide rigidity to help deform the membrane. Here we characterize the influence the C-terminal disordered region of Sec31 has on cage flexibility and rigidity. After deleting this region (residues 820-1220), we characterized Sec13/Sec31ΔC heterotetramers biophysically and structurally through cryo-EM. Our results show that Sec13/31ΔC self-assembles into canonical cuboctahedral cages in vitro at buffer conditions similar to wild type. The distribution of cage sizes indicated that unlike the wild type, Sec13/31ΔC cages have a more homogeneous geometry. However, the structure of cuboctahedrons exhibited more conformational heterogeneity than wild type. Through localized reconstruction of cage vertices and molecular dynamics flexible fitting we found a new hinge for the flexing of Sec31 β-propeller domain and more flexibility of the previously known hinge. Together, these results show that the C-terminal region of Sec31 regulates the flexing of other domains such that flexibility and rigidity are not compromised during transport of large and/or asymmetric cargo.
Cell Reports | 2018
Erin L. Slosarek; Amber L. Schuh; Iryna Pustova; Adam Johnson; Jennifer Bird; Matthew C. Johnson; E.B. Frankel; Nilakshee Bhattacharya; Michael Hanna; Jordan E. Burke; David A. Ruhl; Kyle Quinney; Samuel Block; Jennifer L. Peotter; Edwin R. Chapman; Michael D. Sheets; Samuel E. Butcher; Scott M. Stagg; Anjon Audhya
SUMMARY Length-dependent axonopathy of the corticospinal tract causes lower limb spasticity and is characteristic of several neurological disorders, including hereditary spastic paraplegia (HSP) and amyotrophic lateral sclerosis. Mutations in Trk-fused gene (TFG) have been implicated in both diseases, but the pathomechanisms by which these alterations cause neuropathy remain unclear. Here, we biochemically and genetically define the impact of a mutation within the TFG coiled-coil domain, which underlies earlyonset forms of HSP. We find that the TFG (p.R106C) mutation alters compaction of TFG ring complexes, which play a critical role in the export of cargoes from the endoplasmic reticulum (ER). Using CRISPR-mediated genome editing, we engineered human stem cells that express the mutant form of TFG at endogenous levels and identified specific defects in secretion from the ER and axon fasciculation following neuronal differentiation. Together, our data highlight a key role for TFG-mediated protein transport in the pathogenesis of HSP.
Biophysical Journal | 2011
Nilakshee Bhattacharya; Jason O’Donnell; Abbas Razvi; William E. Balch; Scott M. Stagg
COPII coated vesicles are responsible for packaging and transporting newly synthesized proteins from the endoplasmic reticulum to the Golgi apparatus. The COPII coat consists of Sec13/31, Sec23/24, and Sar1. Mutation in these coat protein cause medical conditions like Anderson disease, chylomicron retention disease and cranio-lenticulo-sutural dysplasia, which highlights the biological relevance of the coat proteins. Previously we solved two different COPII structures (Stagg et. Al., Nature 2006 and Stagg et. Al., Cell 2008) that suggest that the hinge region formed by the four heterotetramer can direct cage expansion to accommodate cargo of various sizes. Recently a tubular structure of Sec 13/31 solved where the tubules were formed by the concatenation of individual sec13/31 cage (O’Donell et. Al, J. STruc. Biol.). Earlier, we hypothesized that the distribution of Sec23/24 dictates the geometry of the COPII coat. We now show that Sec23 by itself influences the outer geometry of the cage. We have reconstructed a structure of a COPII coat cage assembled from Sec13/31 and Sec23. The assemblies form at least two geometries, and the most common size is 600 A, similar to what has been observed for Sec13/31. We will discuss how the orientation of Sec23 may dictate cage geometry and orient Sar1 to participate in the fission of COPII coated vesicles in the cell.
Biochemistry | 2005
Gregory P. Wylie; Vijayaraghavan Rangachari; Ewa A. Bienkiewicz; Vedrana Marin; Nilakshee Bhattacharya; John F. Love; John R. Murphy; Timothy M. Logan
Cell Reports | 2014
Nancy F. Ramia; Michael Spilman; Li Tang; Yaming Shao; Joshua R. Elmore; Caryn R. Hale; Alexis I. Cocozaki; Nilakshee Bhattacharya; Rebecca M. Terns; Michael P. Terns; Hong Li; Scott M. Stagg