Biology subjects

Mukherjee, A.

Publications and source records attributed to Mukherjee, A..

At least 91 records · Page 5Linked to original sources

bioRxiv2023

Increase in primary cilia number and length upon VDAC1 depletion contributes to attenuated proliferation of cancer cells

Primary cilia (PCs) that are present in most human cells and perform sensory function or signal transduction are lost in many solid tumors. Previously, we identified VDAC1, best known to regulate mitochondrial bioenergetics, to negatively regulate ciliogenesis. Here, we show that downregulation of VDAC1 in pancreatic cancer-derived Panc1 and glioblastoma-derived U-87 cells significantly increased ciliation. Those PCs were remarkably longer than the control cells. Such increased ciliation inhibited cell cycle, which contributed to reduced proliferation of these cells. VDAC1-depletion also led to longer PCs in quiescent RPE1 cells. Therefore, serum-induced PC disassembly was slower in VDAC1-depleted RPE1 cells. Overall, this study reiterates the importance of VDAC1 in modulating tumorigenesis, due to its novel role in regulating PC length and disassembly.

cell biology↗

bioRxiv2023

mRNA capping enzyme exports to cytoplasm, localizes to stress granules and maintains cap homeostasis of the target mRNAs

mRNA decapping is believed to trigger RNA degradation until the identification of cytoplasmic capping that has changed the epitome of RNA stability. Unlike nuclear capping machinery that includes RNA polymerase II bound mRNA Capping Enzyme (CE), N-7 RNA methyl transferase and RNMT activating protein RAM, cytoplasmic capping complex consist of cytoplasmic pool of CE (cCE) and N-7 RNA methyl transferase-RAM along with a few cytoplasmic proteins of various functions. Cytoplasmic capping has been shown to recap selective uncapped mRNAs and maintains cap homeostasis by a cyclic process of decapping and recapping. Thus, it acts as post-transcriptional nexus for the target transcripts. Our data show nuclear export of mammalian CE is regulated by Exportin1 (XPO1) pathway via a conserved Nuclear Export Signal sequence. In order to examine biological function of cCE, we show cCE forms granules during stress and majority of these granules co-localize with SGs. In order to identify how cCE regulates cap homeostasis during stress and recovery, we measured the cap status of specific cCE targeted mRNA transcripts along with non-targeted transcripts during non-stress, stress and recovery phase using Xrm1 susceptibility assay. Our data show cCE targeted mRNA transcripts lost their caps in stress condition when cCE is sequestered in granules. After removal of stress, when cCE is released, the cap status has been restored for these transcripts pointing towards the role of cCE in altering cap homeostasis and thus promoting cellular recovery from stress.

molecular biology↗

bioRxiv2022

Multiparameter stimulation mapping of signaling states in single pediatric immune cells reveals heightened tonic activation during puberty

Cellular stimulation via factors such as cytokines followed by multiparameter single-cell measurements is a powerful approach to interrogate cellular functions. However, transforming such high-dimensional data into biological insights presents unique challenges, particularly given the extensive response heterogeneity among single cells, such as the presence of bimodal responding versus non-responding subpopulations upon stimulation. Here we present an unsupervised high-dimensional approach for analyzing stimulation responses at the single cell level (HDStIM) and apply it to evaluate how pediatric development may shape peripheral immune cell signaling states and responsiveness to stimulations in 42 subjects (age: 2 - 16). We show that in comparison to the conventional approach of assessing one marker at a time by averaging across single cells, HDStIM can effectively learn, in an unsupervised fashion, the multi-parameter signature of responding versus non-responding cells to accurately quantify responses within cell populations. HDStIM reveals that the extent of pre-stimulation/baseline activation of interferon-related and TCR signaling molecules in myeloid and T cells, respectively, increases during puberty. This suggests that puberty is marked by a heightened "tonic" activation state in these cells, perhaps to strengthen defense against pathogens during this period of human development.

immunology↗

bioRxiv2022

Activity-regulated growth of motoneurons at the neuromuscular junction is mediated by NADPH oxidases

Neurons respond to changes in the levels of activity they experience in a variety of ways, including structural changes at pre- and postsynaptic terminals. An essential plasticity signal required for such activity-regulated structural adjustments are reactive oxygen species (ROS). To identify sources of activity-regulated ROS required for structural plasticity in vivo we used the Drosophila larval neuromuscular junction as a highly tractable experimental model system. For adjustments of presynaptic motor terminals, we found a requirement for both NADPH oxidases, Nox and Dual Oxidase (Duox), that are encoded in the Drosophila genome. This contrasts with the postsynaptic dendrites from which Nox is excluded. NADPH oxidases generate ROS to the extracellular space. Here, we show that two aquaporins, Bib and Drip, are necessary ROS conduits in the presynaptic motoneuron for activity regulated, NADPH oxidase dependent changes in presynaptic motoneuron terminal growth. Our data further suggest that different aspects of neuronal activity-regulated structural changes might be regulated by different ROS sources: changes in bouton number require both NADPH oxidases, while activity-regulated changes in the number of active zones might be modulated by other sources of ROS. Overall, our results show NADPH oxidases as important enzymes for mediating activity-regulated plasticity adjustments in neurons.

neuroscience↗

bioRxiv2022

Adipocytes reprogram glucose metabolism in cancer cells promoting metastasis

In the tumor microenvironment, adipocytes function as an alternate fuel source for cancer cells. However, whether adipocytes influence macromolecular biosynthesis in cancer cells is unknown. Here, we systematically characterized the bi-directional interaction between primary human adipocytes and ovarian cancer (OvCa) cells using multi-platform metabolomics, imaging mass spectrometry, [13C]-glucose isotope tracing, and gene expression analysis. We report that omental tumor explants and OvCa cells co-cultured with adipocytes divert part of the glucose from glycolysis and TCA cycle towards glycerol-3-phosphate (G3P) synthesis. Normoxic HIF1 protein, stabilized by adipokines, regulate this altered flow of glucose-derived carbons in cancer cells, resulting in increased synthesis of glycerophospholipids (GPL) and triacylglycerols. Blocking adipocyte-induced HIF1 expression increases lipid peroxidation levels in cancer cells and sensitizes them to ferroptosis-mediated cell death. Subsequently, the knockdown of HIF1 or G3P acyltransferase 3 (a regulatory enzyme of GPL synthesis) reduced metastasis in xenograft models of OvCa. In summary, we show that in an adipose-rich tumor microenvironment, cancer cells generate G3P as a precursor for critical membrane and signaling components, thereby promoting metastasis. Targeting biosynthetic processes specific to adipose-rich tumor microenvironments might be an effective strategy against metastasis.

cancer biology↗

bioRxiv2022

Triggered functional dynamics of AsLOV2 by time-resolved electron paramagnetic resonance at high magnetic fields

We present time-resolved Gd-Gd electron paramagnetic resonance (TiGGER) at 240 GHz for tracking inter-residue distances during a proteins mechanical cycle in the solution state. TiGGER makes use of Gd-sTPATCN as spin labels, whose favorable qualities include a spin-7/2 EPR-active center, short linker, narrow intrinsic linewidth, and virtually no anisotropy at high fields (8.6 T) when compared to nitroxide spin labels. Using TiGGER, we determined that upon light activation, the C-terminus and N-terminus of AsLOV2 separate in less than 1 s and relax back to equilibrium with a time constant of approximately 60 s. TiGGER revealed that the light-activated long-range mechanical motion is slowed in the Q513A variant of AsLOV2 and is correlated to the similarly slowed relaxation of the optically excited chromophore as described in recent literature. TiGGER has the potential to valuably complement existing methods for the study of triggered functional dynamics in proteins.

biophysics↗

bioRxiv2022

Escalation of alcohol intake is associated with regionally decreased insular cortex activity but not associated with changes in taste quality

BackgroundIntermittent access to ethanol (EtOH) drives persistent escalation of intake and rapid transition from moderate to compulsive-like drinking. Intermittent EtOH drinking may facilitate escalation in part by altering aversion-sensitive neural substrates, such as the insular cortex (IC), thus driving greater approach toward stimuli previously treated as aversive. MethodsWe conducted a series of experiments in rats to examine behavioral and neural responses associated with escalation of EtOH intake. First, taste reactivity analyses quantified the degree that intermittent brief-access ethanol exposure (BAEE) alters sensitivity to the aversive properties of EtOH. Next, we determined whether pharmacological IC inhibition facilitated EtOH escalation. Finally, given that IC is primary gustatory cortex, we employed psychophysical paradigms to assess whether escalation of EtOH intake induced changes in EtOH taste. These paradigms measured changes in sensitivity to the intensity of EtOH taste and whether escalation shifts the salient taste quality of EtOH by measuring the degree that the taste of EtOH generalized to a sucrose-like ( sweet) or quinine-like ( bitter) percept. ResultsWe found a near complete loss of aversive oromotor responses in EtOH-exposed relative to -naive rats. Additionally, we observed significantly reduced expression of EtOH-induced c-Fos expression in the posterior IC in exposed rats relative to naive rats. Inhibition of the IC resulted in a modest, but statistically reliable increase in acceptance of higher EtOH concentrations in naive rats. Finally, we found no evidence of changes in the psychophysical assessment of the taste of EtOH in exposed, relative to naive, rats. ConclusionsOur results demonstrate that neural activity within the IC adapts following escalation of EtOH intake in a manner that correlates with reduced sensitivity to the aversive hedonic properties of EtOH. These data further establish that IC may be driving exposure-induced escalations in EtOH intake and directly contributing to development of compulsive-like EtOH drinking.

neuroscience↗

bioRxiv2022

An Engineered T7 RNA Polymerase for efficient co-transcriptional capping with reduced dsRNA byproducts in mRNA synthesis

Messenger RNA (mRNA) therapies have recently gained tremendous traction with the approval of mRNA vaccines for the prevention of SARS-CoV-2 infection. However, manufacturing challenges have complicated large scale mRNA production, which is necessary for the clinical viability of these therapies. Not only can the incorporation of the required 5 7-methylguanosine cap analog be inefficient and costly, in vitro transcription (IVT) using wild-type T7 RNA polymerase generates undesirable double-stranded RNA (dsRNA) byproducts that elicit adverse host immune responses and are difficult to remove at large scale. To overcome these challenges, we have engineered a novel RNA polymerase, T7-68, that co-transcriptionally incorporates both di- and tri-nucleotide cap analogs with high efficiency, even at reduced cap analog concentrations. We also demonstrate that IVT products generated with T7-68 have reduced dsRNA content.

molecular biology↗

bioRxiv2022

Identification of Long noncoding RNAs as substrate of cytoplasmic Capping Enzyme

Cytoplasmic capping returns a cap to specific mRNAs thus protecting uncapped RNAs from decay. Prior to the identification of cytoplasmic capping, uncapped mRNAs were thought to be degraded. Here, we test whether long non-coding RNAs (lncRNAs) are cytoplasmic capping enzyme (cCE) substrates. The subcellular localization of fourteen lncRNAs associated with sarcomas were examined in U2OS osteosarcoma cells. We used 5 rapid amplification of cDNA ends (RACE) to assay uncapped forms of these lncRNAs. Inhibiting cytoplasmic capping elevated uncapped forms of selected lncRNAs indicating a plausible role of cCE in targeting them. Analysis of published cap analysis of gene expression (CAGE) data shows increased prevalence of certain 5-RACE cloned sequences suggesting that these uncapped lncRNAs are cytoplasmic capping targets.

molecular biology↗

bioRxiv2022

A genetically engineered phage-based nanomaterial for detecting bacteria with magnetic resonance imaging

The ability to noninvasively detect bacteria at any depth inside opaque tissues has important applications ranging from infection diagnostics to tracking therapeutic microbes in their mammalian host. Current examples of probes for detecting bacteria with strain-type specificity are largely based on optical dyes, which cannot be used to examine bacteria in deep tissues due to the physical limitation of light scattering. Here, we describe a new biomolecular probe for visualizing bacteria in a cell-type specific fashion using magnetic resonance imaging (MRI). The probe is based on a peptide that selectively binds manganese and is attached in high numbers to the capsid of filamentous phage. By genetically engineering phage particles to display this peptide, we are able to bring manganese ions to specific bacterial cells targeted by the phage, thereby producing MRI contrast. We show that this approach allows MRI-based detection of targeted E. coli strains while discriminating against non-target bacteria as well as mammalian cells. By engineering the phage coat to display a protein that targets cell surface receptors in V. cholerae, we further show that this approach can be applied to image other bacterial targets with MRI. Finally, as a preliminary example of in vivo applicability, we demonstrate MR imaging of phage-labeled V. cholerae cells implanted subcutaneously in mice. The nanomaterial developed here thus represents a path towards noninvasive detection and tracking of bacteria by combining the programmability of phage architecture with the ability to produce three- dimensional images of biological structures at any arbitrary depth with MRI.

bioengineering↗

bioRxiv2022

Actin filaments couple the protrusive tips to the nucleus through the I-BAR domain protein IRSp53 for migration of elongated cells on 1D fibers

The cell migration cycle proceeds with shaping the membrane to form new protrusive structures and redistribution of contractile machinery. The molecular mechanisms of cell migration are well-studied in 2D, but membrane shape-driven molecular migratory landscape in 3D fibrous matrices remains poorly described. 1D fibers recapitulate 3D migration, and here, we examined the role of membrane curvature regulator IRSp53 as a coupler between actin filaments and plasma membrane during cell migration on suspended 1D fibers. Cells attached, elongated, and migrated on the 1D fibers with the coiling of their leading-edge protrusions. IRSp53 depletion reduced cell-length spanning actin stress fibers, reduced protrusive activity, and contractility, leading to uncoupling of the nucleus from cellular movements. Using a theoretical model, the observed transition of IRSp53 depleted cells from rapid stick-slip migration to smooth, and slower migration was predicted to arise from reduced actin polymerization at the cell edges, which was verified by direct measurements of retrograde actin flow using speckle microscopy. Overall, we trace the effects of IRSp53 deep inside the cell from its actin-related activity at the cellular tips, thus demonstrating a unique role of IRSp53 in controlling cell migration in 3D.

cell biology↗

bioRxiv2022

Non-cancer stem cell-derived Fibromodulin activates Integrin-dependent Notch signaling in endothelial cells to promote tumor angiogenesis and growth

Cancer stem cells alone can initiate and maintain tumors, but the function of non-cancer stem cells that form the tumor bulk remains poorly understood. Proteomic analysis showed a higher abundance of the extracellular matrix small leucine-rich proteoglycan Fibromodulin (FMOD) in the conditioned medium of non-cancer stem cells (DGCs; differentiated glioma cells) of glioma compared to that of glioma stem-like cells (GSCs). DGCs silenced for FMOD fail to cooperate with co-implanted GSCs to promote tumor growth. FMOD downregulation neither affects GSC growth and differentiation nor DGC growth and reprogramming in vitro. DGC-secreted FMOD promotes angiogenesis by activating Integrin-dependent Notch signaling in endothelial cells. Furthermore, conditional silencing of FMOD in newly generated DGCs in vivo inhibits the growth of GSC-initiated tumors due to poorly developed vasculature and increases mouse survival. Collectively, these findings demonstrate that DGC-secreted FMOD promotes glioma tumor angiogenesis and growth through paracrine signaling in endothelial cells and identifies a DGC-produced protein as a potential therapeutic target in glioma.

cancer biology↗

bioRxiv2021

The energy requirements of ion homeostasis determine the lifespan of starving bacteria

The majority of microbes on earth, whether they live in the ocean, the soil or in animals, are not growing, but instead struggling to survive starvation1-6. Some genes and environmental conditions affecting starvation survival have been identified7-13, but despite almost a century of study14-16, we do not know which processes lead to irreversible loss of viability, which maintenance processes counteract them and how lifespan is determined from the balance of these opposing processes. Here, we used time-lapse microscopy to capture and characterize the cell death process of E. coli during carbon starvation for the first time. We found that a lack of nutrients results in the collapse of ion homeostasis, triggering a positive-feedback cascade of osmotic swelling and membrane permeabilization that ultimately results in lysis. Based on these findings, we hypothesized that ion transport is the major energetic requirement for starving cells and the primary determinant of the timing of lysis. We therefore developed a mathematical model that integrates ion homeostasis and cannibalistic nutrient recycling from perished cells16,17 to predict lifespan changes under diverse conditions, such as changes of cell size, medium composition, and prior growth conditions. Guided by model predictions, we found that cell death during starvation could be dramatically slowed by replacing inorganic ions from the medium with a non-permeating osmoprotectant, removing the cost of ion homeostasis and preventing lysis. Our quantitative and predictive model explains how survival kinetics are determined in starvation and elucidates the mechanistic underpinnings of starvation survival.

microbiology↗

bioRxiv2021

CDK1 and HSP90AA1 appears as novel regulatory gene in Non-Small Cell Lung Cancer: A Bioinformatics Approach

Lung cancer is one of the most invasive cancer affecting over a million of population. Non-small cell lung cancer constitutes up to 85% of all lung cancer cases. Therefore, it is important to identify prognostic biomarkers of NSCLC for therapeutic purpose. The complex behaviour of the NSCLC gene-regulatory network interaction is investigated using a network theoretical approach. We used eight NSCLC microarray datasets GSE19188, GSE118370, GSE10072, GSE101929, GSE7670, GSE33532, GSE31547, GSE31210 and meta analyse them to find differentially expressed genes (DEGs), construct protein-protein interaction (PPI) network, analysed its topological properties, significant modules using network analyser with MCODE, construct a PPI-MCODE network using the genes of the significant modules. We used topological properties such as Maximal Clique Centrality (MCC) and bottleneck from the PPI-MCODE network. We compare them with hub genes (those with highest degrees) to find key regulator (KR) gene. This result is also validated by finding of common genes among top twenty hub genes, genes with highest betweenness, closeness centrality and eigenvector values. It was found that two genes, CDK1 and HSP90AA1 were common in PPI-MCODE combined analysis, and it was also found that CDK1, HSP90AA1 and HSPA8 were common among hub and bottle neck properties and suggesting significant regulatory role of CDK1 in non-small cell lung cancer. After validation, the common genes among top twenty hubs and centrality values like Betweenness Centrality, Closeness Centrality and eigen vector properties, CDK1 again appeared as the common gene. Our study as a summary suggested CDK1 as key regulator gene in complex NSCLC network interaction using network theoretical approach and described the complex topological properties of the network.

systems biology↗

bioRxiv2021

γ-TuRCs are required for asymmetric microtubule nucleation from the somatic Golgi of Drosophila neurons

Microtubules are polarised polymers nucleated by multi-protein {gamma}-tubulin ring complexes ({gamma}-TuRCs). Within neurons, microtubule polarity is plus-end-out in axons and mixed or minus-end-out in dendrites. Previously we showed that within the soma of Drosophila sensory neurons {gamma}-tubulin localises asymmetrically to Golgi stacks, Golgi-derived microtubules grow asymmetrically towards the axon, and growing microtubule plus-ends are guided towards the axon and restricted from entering dendrite in a Kinesin-2-dependent manner (Mukerjee et al., 2020). Here we show that depleting {gamma}-TuRCs perturbs the direction of microtubule growth from the Golgi stacks, consistent with a model for asymmetric microtubule nucleation involving {gamma}-TuRCs and other nucleation-promoting factors. We also directly observe microtubule turning along microtubule bundles and show that depleting APC, proposed to link Kinesin-2 to plus ends, reduces microtubule turning and increases plus end growth into dendrites. These results support a model of asymmetric nucleation and guidance within the neuronal soma that helps establish and maintain overall microtubule polarity.

cell biology↗

bioRxiv2021

Topological morphogenesis of neuroepithelial organoids

Animal organs exhibit complex topologies involving cavities and tubular networks, which underlie their form and function. However, how topology emerges during organ morphogenesis remains elusive. Here, we combine tissue reconstitution and quantitative microscopy to show that trans and cis epithelial fusion govern tissue topology and shape. These two modes of topological transitions can be regulated in neuroepithelial organoids, leading to divergent topologies. The morphological space can be captured by a single control parameter which is analogous to the reduced Gaussian rigidity of an epithelial surface. Finally, we identify a pharmacologically accessible pathway that regulates the frequency of trans and cis fusion, and demonstrate the control of organoid topology and shape. The physical principles uncovered here provide fundamental insights into the self-organization of complex tissues.

biophysics↗

bioRxiv2021

Evolutionary flexibility in routes to mat formation by Pseudomonas

Many bacteria form mats at the air-liquid interface of static microcosms. These structures typically involve the secretion of exopolysaccharide(s), the production of which is often controlled by the secondary messenger c-di-GMP. Mechanisms of mat formation have been particularly well characterized in Pseudomonas fluorescens SBW25; mutations that lead to an increase in c-di-GMP production by diguanylate cyclases (WspR, AwsR, or MwsR) result in the secretion of cellulose, and mat formation. Here, we characterize and compare mat formation in two close relatives of SBW25: Pseudomonas simiae PICF7 and Pseudomonas fluorescens A506. We find that PICF7 - the strain more closely related to SBW25 - can form mats through mutations affecting the activity of the same three diguanylate cyclases as SBW25. However, instead of cellulose, these mutations activate the production of the Pel exopolysaccharide. We also provide evidence for at least two further - as yet uncharacterized - routes to PICF7 mat formation. P. fluorescens A506, while retaining the same mutational routes to mat formation as SBW25 and PICF7, forms mats by a semi-heritable mechanism that likely culminates in Pga and/or Psl production. Overall, our results demonstrate a high level of evolutionary flexibility in the molecular and structural routes to mat formation, even among close relatives.

evolutionary biology↗

bioRxiv2021

Transcriptomic analysis of ribosome biogenesis and pre-rRNA processing during growth stress in Entamoeba histolytica

Ribosome biogenesis, a multi-step process involving the transcription, modification, folding and processing of rRNA is the major consumer of cellular energy. It involves the sequential assembly of ribosomal proteins (RP)s via more than 200 ribogenesis factors. Unlike model organisms where transcription of rRNA and RP genes slows down during stress, in Entamoeba histolytica, pre-rRNA synthesis continues, and unprocessed pre-rRNA accumulates. To gain insight into the vast repertoire of ribosome biogenesis factors and understand the major components playing role during stress we computationally identified the ribosome biogenesis factors in E. histolytica. Of the total [~]279 S. cerevisiae proteins, we could only find 188 proteins in E. histolytica. Some of the proteins missing in E. histolytica were also missing in humans. A number of proteins represented by multiple genes in S. cerevisiae had only a single copy in E. histolytica. It was interesting to note that E. histolytica lacked mitochondrial ribosome biogenesis factors and had far less RNase components as compared to S. cerevisiae. Northern hybridization using probes from different spacer regions depicted the accumulation of unprocessed intermediates during stress. Transcriptomic studies revealed the differential regulation of a number of ribosomal factors both in serum-starved and RRP6KD conditions. The ARB1 protein involved at multiple steps of ribosome biogenesis and NEP1 and TSR3 involved in chemical modification of 18S rRNA previously shown to accumulate pre-rRNA precursors upon downregulation in S. cerevisiae and humans were included. The data reveals the importance of some of the major factors required for regulating pre-rRNA processing during stress. This is the first report on the complete repertoire of ribosome biogenesis factors in E. histolytica.

microbiology↗