Biology subjects

Shekhar, S.

Publications and source records attributed to Shekhar, S..

25 records · Page 2Linked to original sources

bioRxiv2023

Trem2 Agonist Reprograms Foamy Macrophages to Promote Atherosclerotic Plaque Stability

ObjectiveTrem2, a surface lipid receptor, is expressed on foamy macrophages within atherosclerotic lesions and regulates cell survival, proliferation, and anti-inflammatory responses. Studies examining the role of Trem2 in atherosclerosis have shown that deletion of Trem2 leads to impaired foamy macrophage lipid uptake, proliferation, survival, and cholesterol efflux. Thus, we tested the hypothesis that administration of a validated Trem2 agonist antibody (AL002a) to atherogenic mice could drive macrophage survival and decrease necrotic core formation to improve plaque stability. Approach and ResultsTo model a therapeutic intervention approach, atherosclerosis-prone mice (Ldlr-/-) were fed a high fat diet (HFD) for 8 weeks, then transitioned to treatment with AL002a or isotype control for an additional 8 weeks while continuing on an HFD. AL002a-treated mice had increased lesion size in both the aortic sinus and whole mount aorta, which correlated with an expansion of plaque macrophage area. This expansion was due to increased macrophage survival and proliferation in plaques. Importantly, plaques from AL002a-treated mice showed improved features of plaque stability, including smaller necrotic cores, increased fibrous caps, and greater collagen deposition. Single cell RNA sequencing of whole aorta suspensions from isotype and AL002a-treated atherosclerotic mice revealed that Trem2 agonism dramatically altered foamy macrophage transcriptome. This included upregulation of oxidative phosphorylation and increased expression of collagen genes. In vitro studies validated that Trem2-agonism with AL002a promoted foamy macrophage oxLDL uptake, survival, and cholesterol efflux in culture. ConclusionsTrem2 agonist expands plaque macrophages by promoting cell survival and proliferation but improves features of plaque stability by rewiring foamy macrophage function to enhance collagen deposition.

immunology↗

bioRxiv2023

Multicomponent regulation of actin barbed end assembly by twinfilin, formin and capping protein

Living cells assemble their actin networks by regulating reactions at the barbed end of actin filaments. Formins accelerate elongation, capping protein (CP) arrests growth and twinfilin promotes depolymerization at barbed ends. How cells integrate these disparate activities within a shared cytoplasm to produce diverse actin networks, each with distinct morphologies and finely tuned assembly kinetics, is unclear. We used microfluidics-assisted TIRF microscopy to investigate how formin mDia1, CP and twinfilin influence the elongation of actin filament barbed ends. We discovered that the three proteins can simultaneously bind a barbed end in a multiprotein complex. Three-color single molecule experiments showed that twinfilin cannot bind actin filament ends occupied by formin mDia1 unless CP is present. The trimeric complex is short-lived ([~]1s) and results in rapid dissociation of CP by twinfilin causing resumption of rapid formin- based elongation. Thus, the depolymerase twinfilin acts as a pro-formin factor that promotes polymerization when both CP and formin are present. While a single twinfilin binding event is sufficient to displace CP from the trimeric complex, it takes about 30 independent twinfilin binding events to remove capping protein from CP-bound barbed end. Our findings establish a new paradigm in which polymerases, depolymerases and cappers work in concert to tune cellular actin assembly.

biophysics↗

bioRxiv2023

Cooperative hydrodynamics accompany multicellular-like colonial organization in the unicellular ciliate Stentor

Evolution of multicellularity from early unicellular ancestors is arguably one of the most important transitions since the origin of life1,2. Multicellularity is often associated with higher nutrient uptake3, better defense against predation, cell specialization and better division of labor4. While many single-celled organisms exhibit both solitary and colonial existence3,5,6, the organizing principles governing the transition and the benefits endowed are less clear. Using the suspension-feeding unicellular protist Stentor coeruleus, we show that hydrodynamic coupling between proximal neighbors results in faster feeding flows that depend on the separation between individuals. Moreover, we find that the accrued benefits in feeding current enhancement are typically asymmetric- individuals with slower solitary currents gain more from partnering than those with faster currents. We find that colony-formation is ephemeral in Stentor and individuals in colonies are highly dynamic unlike other colony-forming organisms like Volvox carteri 3. Our results demonstrate benefits endowed by the colonial organization in a simple unicellular organism and can potentially provide fundamental insights into the selective forces favoring early evolution of multicellular organization.

biophysics↗

bioRxiv2021

Single-molecule imaging of IQGAP1 regulating actin filament dynamics in real time

IQGAP is a conserved family of actin-binding proteins with essential roles in cell motility, cytokinesis, and cell adhesion, yet it has remained poorly understood how IQGAP proteins directly regulate actin filament dynamics. To close this gap, we used single-molecule and single-filament TIRF microscopy to directly visualize IQGAP regulating actin dynamics in real time. To our knowledge, this is the first study to do so. Our results show that full-length human IQGAP1 forms dimers that stably bind to filament sides and transiently cap barbed ends. These interactions organize actin filaments into thin bundles, suppress barbed end growth, and inhibit filament disassembly. Surprisingly, each activity depends on distinct combinations of IQGAP1 domains and/or dimerization, suggesting that different mechanisms underlie each functional effect on actin. These observations have important implications for how IQGAP functions as a direct actin regulator in vivo, and how it is deployed and regulated in different biological settings.

biochemistry↗

bioRxiv2019

Mouse IgG2a antibodies specific for the commensal Streptococcus mitis show stronger cross-reactivity with Streptococcus pneumoniae than IgG1 antibodies

Here we show that mouse IgG2a and IgG1 antibodies specific for the commensal Streptococcus mitis cross-react with the pathogen Streptococcus pneumoniae, although the cross-reactivity conferred by IgG2a is stronger than IgG1 antibodies. These findings may have implications for designing S. mitis-based vaccines against pneumococcal infections.

immunology↗

bioRxiv2019

Induction of p73, Δ133p53, Δ160p53, pAKT leads to neuroprotection via DNA repair by 5-LOX inhibition

Lipooxygenase-5 (5-LOX), protein is involved in the pathologic phenotype of AD which includes amyloid-plague and tau hyperphosphorylation. This study aims to identify the mechanistic role in neuroprotection by peptide YWCS, the 5-LOX inhibitor in neurotoxic SH-SY5Y cell line developed by the treatment of A{beta}25-35. The cells were treated with A{beta}25-35 and with different doses of YWCS. The effect on cell survival pathways were determined by western blot using polyclonal anti body of p53, anti-Akt and anti-phosphorylated-Akt. Immunoprecipitation and mass spectroscopic studies were done to identify the altered proteins. Over expression of phosphorylated-Akt and 3 bands of p53 isoforms were observed which correspond to p73, {Delta}133p53 and {Delta}160p53 in the cells treated only with 80{micro}M of YWCS compare to untreated cells. However, no alteration of total p53 and Akt were observed. The results exposed the novel mechanistic pathway of neuroprotection by 5-LOX inhibition, which is likely to be mediated by DNA DSB repair through p53 isoforms and PI3K/Akt pathway. Our finding has opened a new window in the therapeutic approach for the prevention of AD.

neuroscience↗

bioRxiv2017

Direct visualization of how Actin Depolymerizing Factor’s filament severing and depolymerization synergizes with Capping Protein's "monomer funneling" to promote rapid polarized growth of actin filaments

A living cells ability to assemble actin filaments in intracellular motile processes is directly dependent on the availability of polymerizable actin monomers which feed polarized filament growth. Continued generation of the monomer pool by filament disassembly is therefore crucial. Disassemblers like ADF/cofilin and filament cappers like Capping Protein (CP) are essential agonists of motility, but the exact molecular mechanisms by which they accelerate actin polymerization at the leading edge and filament turnover has been debated for over two decades. While filament fragmentation by ADF/cofilin has long been demonstrated by TIRF, filament depolymerization was only inferred from bulk solution assays. Using microfluidics-assisted TIRF microscopy, we provide the first direct visual evidence of ADF's simultaneous severing and rapid depolymerization of individual filaments. We have also built a conceptually novel assay to directly visualize ADFs effect on a filament population. We demonstrate that ADFs enhanced pointed-end depolymerization leads to an increase in polymerizable actin monomers co-existing with filaments, thus promoting faster barbed-end growth. We further reveal how ADF-enhanced filament depolymerization synergizes with CPs long-predicted \"monomer funneling\" and leads to skyrocketing of filament growth rates, close to estimated rates in the lamellipodia. The \"Funneling model\" hypothesized, on thermodynamic grounds, that at high enough extent of capping, the few noncapped filaments transiently grow much faster, an effect proposed to be very important for motility. We provide the first direct microscopic evidence of monomer funneling by CP at the scale of individual filaments. We believe that these results enlighten our understanding of the turnover of cellular actin networks.

biophysics↗