Biology subjects

Sundaram, V. K.

Publications and source records attributed to Sundaram, V. K..

5 recordsLinked to original sources

bioRxiv2026

SCN- induces prestin contraction through reduced intermonomer angles and a modified transporter like movement of each monomer.

Prestin is the membrane based molecular motor responsible for outer hair cell electromotility that expands and contracts in response to transmembrane voltage. How voltage brings these about is unknown but cryoEM structures in the presence of ligands has led to the extrapolation of structures with the transporter cycle and its expansion contraction coupling. We find prestin to adopt novel increasingly contracted states that are brought about by a reduced intermonomer angles in the obligate dimers and a transmembrane contraction of each monomer at its cytoplasmic half in the presence of SCN-. These changes are associated with a loss in the intermonomer beta sheet at its N terminus. There is a hyperpolarizing shift in Vh along with increased affinity for SCN-. SCN- binding at the internal binding site likely cause the structural changes since they are reversed by SCN- displacement by salicylate.

physiology↗

bioRxiv2021

RNA-Seq is not required to determine stable reference genes for qPCR normalization

Assessment of differential gene expression by qPCR is heavily influenced by the choice of reference genes. Although numerous statistical approaches have been proposed to determine the best reference genes, they can give rise to conflicting results depending on experimental conditions. Hence, recent studies propose the use of RNA-Seq to identify stable genes followed by the application of different statistical approaches to determine the best set of reference genes for qPCR data normalization. In this study, we demonstrate that the statistical approach to determine the best reference genes from randomly selected candidates is more important than the preselection of stable candidates from RNA-Seq data. Using a qPCR data normalization workflow that we have previously established; we show that qPCR data normalization using randomly chosen conventional reference genes renders the same results as stable reference genes selected from RNA-Seq data. We validated these observations in two distinct cross-sectional experimental conditions involving human iPSC derived microglial cells and mouse sciatic nerves. These results taken together show that given a robust statistical approach for reference gene selection, stable genes selected from RNA-Seq data do not offer any significant advantage over commonly used reference genes for normalizing qPCR assays.

molecular biology↗

bioRxiv2020

Vesicle capture by discrete self-assembled clusters of membrane-bound Munc13

Munc13 is a large banana-shaped soluble protein that is involved in the regulation of synaptic vesicle docking and fusion. Recent studies suggested that multiple copies of Munc13 form nanoassemblies in active zones of neurons. However, it is not known if such clustering is an inherent self-assembly property of Munc13 or whether Munc13 clusters indirectly by multivalent binding to synaptic vesicles or specific plasma membrane domains at docking sites in the active zone. The functional significance of putative Munc13 clustering is also unknown. Here we report that nano-clustering is an inherent property of Munc13, and is indeed required for vesicle binding to bilayers containing Munc13. Pure Munc13 reconstituted onto supported lipid bilayers assembled into clusters containing from 2 to [~]20 copies as revealed by a combination of quantitative TIRF microscopy and step-wise photobleaching. Surprisingly, only clusters a minimum of 6 copies of Munc13 were capable of efficiently capturing and retaining small unilamellar vesicles. The C-terminal C2C domain of Munc13 is not required for Munc13 clustering, but is required for efficient vesicle capture.

biochemistry↗

bioRxiv2020

Retracing Schwann cell developmental transitions in embryonic dissociated DRG cultures

Embryonic Dissociated Dorsal Root Ganglia cultures are often used to investigate the role of novel molecular pathways or drugs in Schwann cell development and myelination. These cultures largely recapitulate the order of cellular and molecular events that occur in Schwann cells of embryonic nerves. However, the timing of Schwann cell developmental transitions, notably the transition from Schwann Cell Precursors to immature Schwann cells and then to myelinating Schwann cells, has not been estimated so far in this culture system. In this study, we determined the expression profiles of Schwann cell developmental genes during the first week of culture and then compared our data to the expression profiles of these genes in developing spinal nerves. This helped in identifying that Schwann Cell Precursors transition into immature Schwann Cells between the 5th and 7th day in vitro. Furthermore, we also investigated the transition of immature cells into pro-myelinating and myelinating Schwann cells upon the induction of myelination in vitro. Our results suggest that Schwann cell differentiation beyond the immature stage can be observed as early as 4 days post the induction of myelination in cocultures. Finally, we compared the myelinating potential of coculture-derived Schwann cell monocultures to cultures established from neonatal sciatic nerves and found that both these cultures system exhibit similar myelinating phenotypes. In effect, our results allow for a better understanding and interpretation of coculture experiments especially in studies that aim to elucidate the role of a novel actor in Schwann Cell development and myelination.

developmental biology↗

bioRxiv2019

Optimal use of statistical methods to validate reference gene stability in longitudinal studies

Multiple statistical approaches have been proposed to validate reference genes in qPCR assays. However, conflicting results from these statistical methods pose a major hurdle in the choice of the best reference genes. Indeed, as their respective approaches to calculating reference gene stability is different, their suitability has to be tested for a given experimental setting. In this study, the stability of 10 candidate reference genes (Actb, Gapdh, Tbp, Sdha, Pgk1, Ppia, Rpl13a, Hsp60, Mrpl10, Rps26) was assessed using four common statistical approaches (GeNorm, NormFinder, Coefficient of Variation analysis and Pairwise {Delta}Ct method) in a longitudinal setting. We used the development of the cerebellum and the spinal cord of mice as a model to assess the suitability of these statistical methods for reference gene validation. GeNorm and the Pairwise {Delta}Ct were found to be ill suited due to a fundamental assumption in their stability calculations. Whereas, NormFinder and Coefficient of Variation analysis fare better provided they are used complementarily. We therefore devised a workflow combining these two methods for validating reference genes in developmental studies. This workflow proves to be more robust than any of the methods used individually.

molecular biology↗