Biology subjects

Yerramilli, V. S.

Publications and source records attributed to Yerramilli, V. S..

4 recordsLinked to original sources

bioRxiv2024

Human ESCRT-I and ALIX function as scaffolding helical filaments in vivo

The Endosomal Sorting Complex Required for Transport (ESCRT) is an evolutionarily conserved machinery that performs reverse-topology membrane scission in cells universally required from cytokinesis to budding of enveloped viruses. Upstream acting ESCRT-I and ALIX control these events and link recruitment of viral and cellular partners to late-acting ESCRT-III CHMP4 through incompletely understood mechanisms. Using structure-function analyses combined with super-resolution imaging, we show that ESCRT-I and ALIX function as distinct helical filaments in vivo. Together, they are essential for optimal structural scaffolding of HIV-1 nascent virions, the retention of viral and human genomes through defined functional interfaces, and recruitment of CHMP4 that itself assembles into corkscrew-like filaments intertwined with ESCRT-I or ALIX helices. Disruption of filament assembly or their conformationally clustered RNA binding interfaces in human cells impaired membrane abscission, resulted in major structural instability and leaked nucleic acid from nascent virions and nuclear envelopes. Thus, ESCRT-I and ALIX function as helical filaments in vivo and serve as both nucleic acid-dependent structural scaffolds as well as ESCRT-III assembly templates. Significance statementWhen cellular membranes are dissolved or breached, ESCRT is rapidly deployed to repair membranes to restore the integrity of intracellular compartments. Membrane sealing is ensured by ESCRT-III filaments assembled on the inner face of membrane; a mechanism termed inverse topology membrane scission. This mechanism, initiated by ESCRT-I and ALIX, is universally necessary for cytokinesis, wound repair, budding of enveloped viruses, and more. We show ESCRT-I and ALIX individually oligomerize into helical filaments that cluster newly discovered nucleic acid-binding interfaces and scaffold-in genomes within nascent virions and nuclear envelopes. These oligomers additionally appear to serve as ideal templates for ESCRT-III polymerization, as helical filaments of CHMP4B were found intertwined ESCRT-I or ALIX filaments in vivo. Similarly, corkscrew-like filaments of ALIX are also interwoven with ESCRT-I, supporting a model of inverse topology membrane scission that is synergistically reinforced by inward double filament scaffolding.

cell biology↗

bioRxiv2019

IQGAP1 connects phosphoinositide signaling to cytoskeletal reorganization

IQGAP1 is a multi-domain protein that acts as a scaffold for multiple signaling pathways. IQGAP1 generates the lipid messenger PI(3,4,5)P3 by scaffolding the phosphoinositide kinases PIPKIs and PI3K. The dynamics of this scaffolding protein complex in intact, living cells are unknown. Here, we delineate the role of IQGAP1 in PI(3,4,5)P3-mediated signaling in live cells under basal and stimulated conditions using fluorescence lifetime imaging microscopy. We demonstrate that IQGAP1 interacts strongly with PIPKI{gamma} at intracellular entities and on the plasma membrane, and scaffolds PI3K and PIPKI{gamma} in response to physiological changes. Additionally, we show that IQGAP1 scaffolds phosphoinositides with PI3K, PIPKI{gamma} and EGFR, and forms clusters upon cell stimulation with epidermal growth factor. Importantly, we show that IQGAP1 connects PI(3,4,5)P3-mediated signaling and cytoskeletal signaling pathways by binding PIPKI{gamma} in proximity of the cytoskeletal proteins talin and Cdc42. Our results support a model in which IQGAP1 mediates crosstalk between phosphoinositide signaling and the cytoskeleton to promote directed cell movement.

biophysics↗

bioRxiv2017

Labeling RNAs in live cells using malachite green aptamer scaffolds as fluorescent probes

RNAs mediate many different processes that are central to cellular function. The ability to quantify or image RNAs in live cells is very useful in elucidating such functions of RNA. RNA aptamerfluorogen systems have been increasingly used in labeling RNAs in live cells. Here, we use the malachite green aptamer (MGA), an RNA aptamer that can specifically bind to malachite green (MG) dye and induces it to emit far-red fluorescence signals. Previous studies on MGA showed a potential for the use of MGA for genetically tagging other RNA molecules in live cells. However, these studies also exhibited low fluorescence signals and high background noise. Here we constructed and tested RNA scaffolds containing multiple tandem repeats of MGA as a strategy to increase the brightness of the MGA aptamer-fluorogen system as well as to make the system fluoresce when tagging various RNA molecules, in live cells. We demonstrate that our MGA scaffolds can induce fluorescence signals by up to ~20 fold compared to the basal level as a genetic tag for other RNA molecules. We also show that our scaffolds function reliably as genetically-encoded fluorescent tags for mRNAs of fluorescent proteins and other RNA aptamers.

synthetic biology↗

bioRxiv2017

Context-Dependent Genetic Regulation

Cells process extra-cellular signals with multiple layers of complex biological networks. Due to the stochastic nature of the networks, the signals become significantly noisy within the cells and in addition, due to the nonlinear nature of the networks, the signals become distorted, shifted, and (de-)amplified. Such nonlinear signal processing can lead to non-trivial cellular phenotypes such as cell cycles, differentiation, cell-to-cell communication, and homeostasis. These nonlinear pheno-types, when observed at the cell population levels, can be quite different from the single-cell level observation. As one of the underlying mechanisms behind this difference, we report the interplay between nonlinearity and stochasticity in genetic regulation. Here we show that nonlinear genetic regulation, characterized at the cellular population level, can be affected by cell-to-cell variability in the regulatory factor concentrations. The observed genetic regulation at the cell population is shown to be significantly dependent on the upstream DNA sequences of the regulator, in particular, 5 untranslated region. This indicates that genetic regulation observed at the cell population level can be significantly dependent on its genetic context, and that its characterization needs a careful attention on noise propagation.\n\nOne Sentence SummaryGenetic regulation observed at the cell population level can be significantly affected by cell-to-cell variability in the regulatory factor copy numbers, indicating that the observed regulation is dependent on 5 UTR of the regulator coding gene.

synthetic biology↗