Biology subjects

Mittal, N.

Publications and source records attributed to Mittal, N..

29 records · Page 2Linked to original sources

bioRxiv2021

Dual roles of mTORC1-dependent activation of the ubiquitin-proteasome system in muscle proteostasis

Muscle size is controlled by the PI3K-PKB/Akt-mTORC1-FoxO pathway, which integrates signals from growth factors, energy and amino acids to activate protein synthesis and inhibit protein breakdown. While mTORC1 activity is necessary for PKB/Akt-induced muscle hypertrophy, its constant activation alone induces muscle atrophy. Here we show that this paradox is based on mTORC1 activity promoting protein breakdown through the ubiquitin-proteasome system (UPS) by simultaneously inducing ubiquitin E3 ligase expression via feedback inhibition of PKB/Akt and proteasome biogenesis via Nuclear Factor Erythroid 2-Like 1 (Nrf1). Muscle growth was restored by reactivation of PKB/Akt, but not by Nrf1 knockdown, implicating ubiquitination as the limiting step. However, both PKB/Akt activation and proteasome depletion by Nrf1 knockdown led to an immediate disruption of proteome integrity with rapid accumulation of damaged material. These data highlight the physiological importance of mTORC1-mediated PKB/Akt inhibition and point to juxtaposed roles of the UPS in atrophy and proteome integrity.

physiology↗

bioRxiv2021

Nascent adhesions differentially regulate lamellipodium velocity and persistence

Cell migration is essential to physiological and pathological biology. Migration is driven by the motion of a leading edge, in which actin polymerization pushes against the edge and adhesions transmit traction to the substrate while membrane tension increases. How the actin and adhesions synergistically control edge protrusion remains elusive. We addressed this question by developing a computational model in which the Brownian ratchet mechanism governs actin filament polymerization against the membrane and the molecular clutch mechanism governs adhesion to the substrate (BR-MC model). Our model predicted that actin polymerization is the most significant driver of protrusion, as actin had a greater effect on protrusion than adhesion assembly. Increasing the lifetime of nascent adhesions also enhanced velocity, but decreased the protrusions motional persistence, because filaments maintained against the cell edge ceased polymerizing as membrane tension increased. We confirmed the model predictions with measurement of adhesion lifetime and edge motion in migrating cells. Adhesions with longer lifetime were associated with faster protrusion velocity and shorter persistence. Experimentally increasing adhesion lifetime increased velocity but decreased persistence. We propose a mechanism for actin polymerization-driven, adhesion-dependent protrusion in which balanced nascent adhesion assembly and lifetime generates protrusions with the power and persistence to drive migration.

cell biology↗

bioRxiv2021

Distinct and additive effects of calorie restriction and rapamycin in aging skeletal muscle

As global life expectancy continues to climb, maintaining skeletal muscle function is increasingly essential to ensure a good life quality for aging populations. Calorie restriction (CR) is the most potent and reproducible intervention to extend health and lifespan, but is largely unachievable in humans. Therefore, identification of "CR mimetics" has received much attention. CR targets nutrient-sensing pathways centering on mTORC1. The mTORC1 inhibitor, rapamycin, has been proposed as a potential CR mimetic and is proven to counteract age-related muscle loss. Therefore, we tested whether rapamycin acts via similar mechanisms as CR to slow muscle aging. Contrary to our expectation, long-term CR and rapamycin-treated geriatric mice display distinct skeletal muscle gene expression profiles despite both conferring benefits to aging skeletal muscle. Furthermore, CR improved muscle integrity in a mouse with nutrient-insensitive sustained muscle mTORC1 activity and rapamycin provided additive benefits to CR in aging mouse muscles. Therefore, RM and CR exert distinct, compounding effects in aging skeletal muscle, opening the possibility of parallel interventions to counteract muscle aging.

physiology↗

bioRxiv2020

A Small Molecule Fluorogenic Probe for the Detection of Sphingosine in Living Cells

ABSTRACTThe single-chained sphingolipid sphingosine is an essential structural lipid and signaling molecule. Abnormal sphingosine metabolism is observed in several diseases, including cancer, diabetes, and Alzheimer’s. Despite its biological importance, there are a lack of tools for detecting sphingosine in living cells. This is likely due to the broader challenge of developing highly selective and live-cell compatible affinity probes for hydrophobic lipid species. In this work, we have developed a small molecule fluorescent turn-on probe for labeling sphingosine in living cells. This probe utilizes a selective reaction between sphingosine and salicylaldehyde esters to fluorescently label sphingosine molecules. We demonstrate that this probe exhibits a dose-dependent response to sphingosine and is able to detect endogenous pools of sphingosine. Using our probe, we successfully detected sphingosine accumulation in live Niemann-Pick type C1 (NPC1) patient cells, a lipid transport disorder in which increased sphingosine mediates disease progression. This work provides a simple and accessible method for the detection of sphingosine and should facilitate study of this critical signaling lipid in biology and disease.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2020

Multistage and transmission-blocking targeted antimalarials discovered from the open-source MMV Pandemic Response Box

New chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium. Here, we report the parallel screening of the Medicines for Malaria Venture Pandemic Response Box to identify multistage-active and stage-specific compounds against various life cycle stages of Plasmodium parasites (asexual parasites, stage IV/V gametocytes, gametes, oocysts and liver stages) and for endectocidal activity. Hits displayed unique chemotypes and included two multistage-active compounds, 16 asexual-targeted, six with prophylactic potential and ten gametocyte-targeted compounds. Notably, four structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity were identified: the JmjC inhibitor ML324, two azole antifungals including eberconazole, and the antitubercular clinical candidate SQ109. Besides ML324, none of these have previously attributed antiplasmodial activity, emphasizing the success of de novo parallel screening against different Plasmodium stages to deliver leads with novel modes-of-action. Importantly, the discovery of such transmission-blocking targeted compounds covers a previously unexplored base for delivery of compounds required for malaria elimination strategies.

microbiology↗

bioRxiv2019

Direct Arp2/3-vinculin binding is essential for cell spreading, but only on compliant substrates and in 3D

Cells modify their shape in response to the extracellular environment through dynamic remodeling of the actin cytoskeleton by actin-binding proteins (ABPs) 1-4. The relation between actin dynamics and spreading is well-understood for cells on flat glass coverslips; much less is known about cell morphogenesis in compliant three-dimensional environments, and, in particular, how ABPs contribute to this process 5. Here, we knocked-out a diverse set of ABPs, and evaluated the effect of each on cell spreading on planar glass surfaces (2D) and in reconstituted collagen gels (3D). Our morphometric analyses identify the Arp2/3 complex and its associated regulatory genes among the ABPs that contribute most strongly to cell spreading in 3D, but marginally in 2D. Cells lacking Arp3 have reduced spreading specifically in 3D, and display stiffness-dependent cell-matrix adhesion defects. Through manipulation of vinculin activity, we determine that the Arp3 knock-out phenotype largely arises from the lack of direct interaction between vinculin and Arp2/3 complex. This interaction is dispensable for cell spreading in 2D. These data highlight that actin architectural features necessary for adhesion formation and cell spreading in 3D are efficiently compensated on flat and stiff surfaces.

cell biology↗

bioRxiv2019

Perturbation of the titin/MURF1 signaling complex is associated with hypertrophic cardiomyopathy in a fish model and in human patients.

Hypertrophic cardiomyopathy (HCM) is a hereditary disease characterized by cardiac hypertrophy with diastolic dysfunction. Gene mutations causing HCM have been found in about half of the patients, while the genetic etiology and pathogenesis remain unknown for many cases of HCM. To identify novel mechanisms underlying HCM pathogenesis, we generated a cardiovascular-mutant medaka fish non-spring heart (nsh), which showed diastolic dysfunction and hypertrophic myocardium. The nsh homozygotes had fewer myofibrils, disrupted sarcomeres and expressed pathologically stiffer titin isoforms. In addition, the nsh heterozygotes showed M-line disassembly that is similar to the pathological changes found in HCM. Positional cloning revealed a missense mutation in an immunoglobulin (Ig) domain located in the M-line-A-band transition zone of titin. Screening of mutations in 96 unrelated patients with familial HCM, who had no previously implicated mutations in known sarcomeric gene candidates, identified two mutations in Ig domains close to the M-line region of titin. In vitro studies revealed that the mutations found in both medaka fish and in familial HCM increased binding of titin to muscle-specific ring finger protein 1 (MURF1) and enhanced titin degradation by ubiquitination. These findings implicate an impaired interaction between titin and MURF1 as a novel mechanism underlying the pathogenesis of HCM.

developmental biology↗

bioRxiv2019

Local translation of yeast ERG4 mRNA at the endoplasmic reticulum requires the brefeldin A resistance protein Bfr1

Brefeldin A resistance factor 1 (Bfr1p) is a non-essential RNA-binding protein and multi-copy suppressor of brefeldin A sensitivity in Saccharomyces cerevisiae. Deletion of BFR1 leads to multiple defects, including altered cell shape and size, change in ploidy, induction of P-bodies and chromosomal mis-segregation. Bfr1p has been shown to associate with polysomes, binds to several hundred mRNAs, and can target some of them to P-bodies. Although this implies a role of Bfr1p in translational control of mRNAs, its molecular function remains elusive. In the present study, we show that mutations in RNA-binding residues of Bfr1p impede its RNA-dependent co-localization with ER, yet do not mimic the known cellular defects seen upon BFR1 deletion. However, a Bfr1 RNA-binding mutant is impaired in binding to ERG4 mRNA which encodes an enzyme required for the final step of ergosterol biosynthesis. Consistently, bfr1{Delta} strains show a strong reduction in Erg4p protein levels. Polysome profiling of bfr1{Delta} or bfr1 mutant strains reveals a strong shift of ERG4 mRNA to polysomes, consistent with a function of Bfr1p in elongation. Collectively, our data reveal that Bfr1 has at least two separable functions: one in RNA-binding and elongation during translation, in particular at the ER membrane, and one in ploidy control or chromosome segregation.

cell biology↗

bioRxiv2018

Protein synthesis rates and ribosome occupancies reveal determinants of translation elongation rates

Although protein synthesis dynamics has been studied both with theoretical models and by profiling ribosome footprints, the determinants of ribosome flux along open reading frames (ORFs) are not fully understood. Combining measurements of protein synthesis rate with ribosome footprinting data, we here inferred translation initiation and elongation rates for over a thousand ORFs in exponentially-growing wildtype yeast cells. We found that the amino acid composition of synthesized proteins is as important a determinant of translation elongation rate as parameters related to codon and tRNA adaptation. We did not find evidence of ribosome collisions curbing the protein output of yeast transcripts, either in high translation conditions associated with exponential growth, or in strains in which deletion of individual ribosomal protein genes leads to globally increased or decreased translation. Slow translation elongation is characteristic of RP-encoding transcripts, which have markedly lower protein output than other transcripts with equally high ribosome densities.\n\nSignificance StatementAlthough sequencing of ribosome footprints has uncovered new aspects of mRNA translation, the determinants of ribosome flux remain incompletely understood. Combining ribosome footprint data with measurements of protein synthesis rates, we here inferred translation initiation and elongation rates for over a thousand ORFs in yeast strains with varying translation capacity. We found that the translation elongation rate varies up to ~20-fold among yeast transcripts, and is significantly correlated with the rate of translation initiation. Furthermore, the amino acid composition of synthesized proteins impacts the rate of translation elongation to the same extent as measures of codon and tRNA adaptation. Transcripts encoding ribosomal proteins are translated especially slow, having markedly lower protein output than other transcripts with equally high ribosome densities.

biophysics↗

bioRxiv2018

Complex Interplay between Serum and Fibroblasts in 3D Hepatocyte Co-culture

Previous studies have suggested that primary hepatocytes cultured in vitro undergo a rapid loss of function. On the other hand, in the clinic, drug induced liver injury typically manifests 5 days to 3 months after starting a medication. Thus, novel approaches that can maintain the function of primary human hepatocytes for longer durations of time may enable the development of improved in vitro assays for detecting hepatotoxicity. Previous studies have demonstrated that two-dimensional micro-patterning of hepatocytes with fibroblasts leads to improved maintenance of the hepatocyte phenotype relative to hepatocyte monocultures, in serum containing medium. Additionally, we, and others, have shown that three-dimensional culture of hepatocytes leads to enhanced function (in serum-free medium). In this study we wanted to (i) examine the effect of combining the above two approaches on hepatocyte function, and (ii) to further delineate the effect of serum on hepatocyte function. We developed a user-friendly and inexpensive approach for constructing layered spheroids. Similar to previous results in two-dimensional (2d) culture, we observed that 3d culture of hepatocytes alone (i.e. monoculture) in serum-containing medium led to an increase in the urea production rate, but near-complete loss of cytochrome activity in both lots of primary human hepatocytes (PHH) tested. In serum-free sandwich culture, cytochrome activity was maintained at the level observed in freshly thawed PHH for one lot, but almost completely lost in another lot. Spheroid culture of both lots of PHH in serum-free medium led to maintenance of CYP3A4 and CYP1A2 activity at the fresh thaw level, though CYP2B6 activity was reduced. In contrast to PHH monoculture, co-cultures of PHH with NIH 3T3 fibroblast cells benefitted from the presence of serum, and led to 3-5-fold increases in CYP activity relative to even serum-free spheroid monocultures. Layering of the fibroblasts did not result in improvements over mixed co-cultures. These results indicate the importance of appropriate serum-free monoculture control experiments in the evaluation of novel biomaterials and techniques for hepatocyte co-culture. Further, urea production and cytochrome production are decoupled; therefore, urea production is an insufficient readout when developing models for pharmaceutical applications.

cell biology↗

bioRxiv2017

Sub-Confluent Culture of Mouse Embryonic Stem Cell-derived Ventricular Cardiomyocytes On & In Gels - Enhancement of Maturation Phenotype Relative to Tissue Culture Polystyrene via Enabling of Auxotonic Contraction

Cardiac myocytes (CMs) obtained by differentiating embryonic stem cells (ES-CMs) have an immature phenotype and promoting the maturation of such PSC-derived cardiomyocytes remains a major limitation in the development of stem cell models of human cardiovascular disease. We cultured murine ES-CMs in a collagen gel (3D) at a low density, or on collagen-coated polystyrene (2D) and found that 3D culture results in dramatic improvement of the maturation rate and end-state gene expression of ES-CMs. There are two main differences between CMs cultured in 3D versus 2D; in 3D the mechanical stiffness of the environment is lower, enabling auxotonic instead of isometric contraction; and, in 3D the amount of cell-cell interaction is higher. To isolate the contributions, we first cultured ES-CMs on gels (2D substrates) that are softer than tissue culture plastic, enabling auxotonic contraction, while controlling for dimensionality and cell interaction. This indeed promoted a mature gene expression profile, while also enabling the maintenance of sarcomeres. Next, we determined that increased cell-cell interaction inhibits the mature gene expression of ES-CMs. Thus, auxotonic contraction is the likely mechanism for improved gene expression in sub-confluent 3D culture. However, 2D auxotonic contraction may offer a suitable compromise between obtaining enhanced gene expression and morphology. After 6 weeks of culture on gels, via Di-8-ANEPPs and WGA staining we also detected CMs forming a t-tubule network. Collectively these results demonstrate that 3D and 2D cultures that enable auxotonic contraction enhance aspects of the maturation of ES-CMs.

biophysics↗