Biology subjects

Mitra, A.

Publications and source records attributed to Mitra, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

DYF-5 regulates intraflagellar transport by affecting train turnaround

Intraflagellar transport (IFT) coordinates the transport of cargo in cilia and is essential for ciliary function. CILK1 has been identified as a key regulator of IFT. The mechanism by which it acts has, however, remained unclear. In this study, we use fluorescence imaging and single-molecule tracking in the phasmid cilia of live C. elegans to study the effect of the CILK1 homolog DYF-5 on the dynamics of the IFT. We show that in the absence of DYF-5, IFT components accumulate at the ciliary tip. kinesin-II is no longer restricted to the proximal segment of the cilium but is present all throughout the cilium, while its velocity is different from that of OSM-3. The frequency of IFT trains is reduced and in particular retrograde trains were rarely observed. In the absence of DYF-5, retrograde transport is vastly reduced, resulting in the accumulation of IFT components at the tip and depletion at the base. The latter results in impeded anterograde train assembly, resulting in fewer trains with irregular composition. Our results show that DYF-5 plays a key role in regulating the turnarounds of IFT trains at the ciliary tip.

molecular biology↗

bioRxiv2024

Evidencing the role of a conserved polar signaling channel in the activation mechanism of the u-opioid receptor

The activity of G protein-coupled receptors has been generally linked to dynamically interconverting structural and functional states and the process of activation was proposed to be controlled by an interconnecting network of conformational switches in the transmembrane domain. However, it is yet to be uncovered how ligands with different extent of functional effect exert their actions. According to our recent hypothesis, the transmission of the external stimulus is accompanied by the shift of macroscopic polarization in the transmembrane domain, furnished by concerted movements of conserved polar amino acids and the rearrangement of polar species. Previously, we have examined the -opioid, {beta}2-adrenergic and type 1 cannabinoid receptors by performing molecular dynamics simulations. Results revealed correlated dynamics of a polar signaling channel connecting the orthosteric binding pocket and the intracellular G protein-binding surface in all three class A receptors. In the present study, the interplay of this polar signaling channel in the activation mechanism was evidenced by systematic mutation of the channel residues of the -opioid receptor. Mutant receptors were analyzed utilizing molecular dynamics simulations and characterized in vitro by means of radioligand receptor binding and G protein stimulation assays. Apart from one exception, all mutants failed to bind the endogenous agonist endomorphin-2 and to stimulate the Gi protein complex. Furthermore, mutation results confirmed strong allosteric coupling between the binding pocket and the intracellular surface. The strong association and optimal bioactive orientation of the bound agonist was found to be crucial for the initiation of correlated motions and consequent signaling.

molecular biology↗

bioRxiv2024

Adventitial fibroblasts direct smooth muscle cell-state transition in pulmonary vascular disease

Pulmonary vascular remodeling is a progressive pathological process characterized by functional alterations within pulmonary artery smooth muscle cells (PASMC) and adventitial fibroblasts (PAAF). Mechanisms driving the transition to a diseased phenotype remain elusive. Utilizing a combination of transcriptomic and proteomic profiling, along with phenotyping of source-matched cells from healthy controls and individuals with idiopathic pulmonary arterial hypertension (IPAH), our investigation uncovered that while PASMC and PAAF retained their original cellular identities, they acquired distinct disease-associated states. Though both cell types exhibited reduced mitochondrial content and hyperpolarization, IPAH-PASMC displayed heightened glycosaminoglycan production and downregulation of contractile machinery, contrasting a hyperproliferative phenotype of IPAH-PAAF. We investigated the involvement of cellular crosstalk in regulating cell state dynamics and identified pentraxin-3 and hepatocyte growth factor as potential modulators of PASMC phenotypic transition orchestrated by PAAF. Our findings contribute to a deeper understanding of pulmonary vascular mesenchyme dynamics in disease pathogenesis.

cell biology↗

bioRxiv2024

Sorting at ciliary base and ciliary entry of BBSome, IFT-B and IFT-A

Summary/AbstractAnterograde intraflagellar transport (IFT) trains, composed of IFT-B, IFT-A and BBSome subcomplexes, are responsible for transporting ciliary proteins into the cilium. How IFT subcomplexes reach the ciliary base and assemble into IFT trains is poorly understood. Here, we perform quantitative single-molecule imaging in C. elegans chemosensory cilia to uncover how IFT subcomplexes arrive at the base, organize in IFT trains, and enter the cilium. We find that BBSomes reach the base via diffusion where they either associate with assembling IFT trains or with the membrane surrounding the base. In contrast, IFT-B and IFT-A reach the base via directed transport on vesicles that stop at distinct locations near the base. Individual subcomplexes detach from the vesicles into a diffusive pool and associate to assembling trains. Our results indicate that the assembly of IFT trains is a step-wise process involving the subsequent incorporation of first IFT-B, then IFT-A and finally BBSomes.

molecular biology↗

bioRxiv2024

Direct modulation of TRPM8 ion channels by rapamycin and analog macrolide immunosuppressants

Rapamycin (sirolimus), a macrolide compound isolated from the bacterium Streptomyces hygroscopicus, is widely used as oral medication for the prevention of transplant rejection and the treatment of lymphangioleiomyomatosis. It is also incorporated in coronary stent coatings to prevent restenosis and in topical preparations for the treatment of skin disorders. Rapamycins in vivo activities are generally ascribed to its binding to the protein FKBP12, leading to potent inhibition of the mechanistic target of rapamycin kinase (mTOR) by the FKBP12-rapamycin complex. The specific rapamycin-induced interaction between domains from mTOR and FKBP12 is also frequently employed in cell biological research, for rapid chemically-induced protein dimerization strategies. Here we show that rapamycin activates TRPM8, a cation channel expressed in sensory nerve endings that serves as the primary cold sensor in mammals. Using a combination of electrophysiology, Saturation Transfer Triple-Difference (STTD) NMR spectroscopy and molecular docking-based targeted mutagenesis, we demonstrate that rapamycin directly binds to TRPM8. We identify a rapamycin-binding site in the groove between voltage sensor-like domain and the pore domain, distinct from the interaction sites of cooling agents and known TRPM8 agonists menthol and icilin. Related macrolide immunosuppressants act as partial TRPM8 agonists, competing with rapamycin for the same binding site. These findings identify a novel molecular target for rapamycin and provide new insights into the mechanisms of TRPM8 activation, which may assist in the development of therapies targeting this ion channel. Moreover, our findings also indicate that caution is needed when using molecular approaches based on rapamycin-induced dimerization to study ion channel regulation.

neuroscience↗

bioRxiv2023

Human kinesin-5 KIF11 drives the helical motion of anti-parallel and parallel microtubules around each other

During mitosis, motor proteins and microtubule-associated protein organize the spindle apparatus by cross-linking and sliding microtubules. Kinesin-5 plays a vital role in spindle formation and maintenance, potentially inducing twist in the spindle fibers. The off-axis power stroke of kinesin-5 could generate this twist, but its implications in microtubule organization remain unclear. Here, we investigate 3D microtubule-microtubule sliding mediated by the human kinesin-5, KIF11, and found that the motor caused right-handed rotation of anti-parallel microtubules around each other. The effective sidestepping probability of KIF11 increased with reduced ATP concentration, indicating that forward and sideways stepping of the motor are not strictly coupled. Further, the microtubule-microtubule distance (motor extension) during sliding decreased with increasing sliding velocity. Intriguingly, parallel microtubules cross-linked by KIF11 orbit without forward motion, with nearly full extension. Altering the length of the neck linker increased the forward velocity and pitch of microtubules in anti-parallel overlaps. Taken together, we suggest that helical motion and orbiting of microtubules, driven by KIF11, enable flexible and context-dependent filament organization, as well as torque regulation within the mitotic spindle.

biophysics↗

bioRxiv2023

IFT cargo and motors associate sequentially with IFT trains to enter cilia

Intraflagellar transport (IFT) orchestrates entry of proteins into primary cilia. At the ciliary base, assembled IFT trains, driven by kinesin-2 motors, can transport cargo proteins into the cilium, across the crowded transition zone (TZ). How trains assemble at the base and how proteins associate with them is far from understood. Here, we use single-molecule imaging in the cilia of C. elegans chemosensory neurons to directly visualize the entry of kinesin-2 motors kinesin-II and OSM-3, as well as anterograde cargo proteins IFT dynein and tubulin. Single-particle tracking shows that IFT components associate with trains sequentially, both in time and space. Super-resolution maps of IFT components in wild-type and mutant worms reveal ciliary ultrastructure and show that kinesin-II is essential for axonemal organization. Finally, imaging cilia lacking kinesin-II and/or TZ function uncovers the interplay of kinesin-II and OSM-3 in driving efficient transport of IFT trains across the TZ.

biophysics↗

bioRxiv2023

Molecular insight into the mechanism of action of some beneficial flavonoids for the treatment of Parkinson's Disease

Parkinsons Disease (PD) is a severe neurodegenerative disorder. It is characterized by the declination of dopaminergic (DA) neurons of substantia nigra pars compacta in the mid brain. Decrease in dopamine level in substantia nigra is a major event in PD. Mutation in Parkin protein is responsible for the onset of Autosomal Recessive Juvenile Parkinsons (ARJP) Disease. Till date, there is no available treatment of PD without any side-effects. Therefore, the focus has been shifted to identifying natural product inhibitors for the treatment of the disorder. Flavonoids, a class of natural products, have proven neuroprotective effects. Some of the flavonoids have their abilities to influence the activities of central nervous system. Therefore, many studies are being conducted to analyze their activities to lower the progression of neurodegenerative diseases. This study was conducted to identify the flavonoids to be used as potential drugs for PD. From the literature, we picked up the flavonoids active on the nervous systems of human beings. We employed a literature mining approach to build a Structure Activity Relationship (SAR) to measure their efficacies. We performed molecular docking simulations using the flavonoids as the ligands and computed their binding free energy values. Our study would therefore point towards future drug- designing endeavours to come up with plausible therapeutics against PD onset.

bioinformatics↗

bioRxiv2023

Integrated spatiotemporal transcriptomic resolution of embryonic palate osteogenesis

The differentiation of osteoblasts and the subsequent formation of bone marks an important terminal phase in palate formation that leads to the separation of the oral and nasal cavities. While the developmental events that precede palatal osteogenesis are well explored, major gaps remain in our understanding of the molecular mechanisms that lead to the bony union of fusing palatal shelves. Herein, the timeline of osteogenic transcriptional programming is unveiled in the embryonic palate by way of integrated bulk, single-cell, and spatially resolved RNA-seq analyses. We define spatially restricted expression patterns of key marker genes, both regulatory and structural, that are differentially expressed during palatal fusion, including the identification of several novel genes (Deup1, Dynlrb2, Lrrc23) spatially restricted in expression to the palate, providing a relevant framework for future studies that identify new candidate genes for cleft palate anomalies in humans as well as the timing of mammalian embryonic palatal osteogenesis.

developmental biology↗

bioRxiv2023

SIN3 gene regulatory activity is linked to RNA polymerase II pausing

The chromatin environment has a significant impact on gene expression. Chromatin structure is highly regulated by histone modifications and RNA polymerase II binding dynamics. The SIN3 histone modifying complex regulates the chromatin environment leading to changes in gene expression. In Drosophila melanogaster, the Sin3A gene is alternatively spliced to produce different protein isoforms, two of which include SIN3 220 and SIN3 187. Both SIN3 isoforms are scaffolding proteins that interact with several other factors to regulate the chromatin landscape. The mechanism through which the SIN3 isoforms regulate chromatin is not well understood. Here, we analyze publicly available data sets to allow us to ask specific questions on how SIN3 isoforms regulate chromatin and gene activity. We determined that genes repressed by the SIN3 isoforms exhibited enrichment in histone H3K4me2 and H3K4me3 near the transcription start site. We observed an increase in the amount of paused RNA polymerase II on the promoter of genes repressed by the isoforms as compared to genes that require SIN3 for maximum activation. Furthermore, we analyzed a subset of genes regulated by SIN3 187 that suggest a mechanism in which SIN3 187 might exhibit hard regulation as well as soft regulation. Data presented here expand our knowledge of how the SIN3 isoforms regulate the chromatin environment and RNA polymerase II binding dynamics. Summary statementSIN3 cofactors can activate or repress genes. Using bioinformatic analysis, we find that histone methylation and RNA polymerase II binding profiles differ at SIN3-regulated genes with distinct transcriptional outcomes.

bioinformatics↗

bioRxiv2022

A small excitation window allows long-duration imaging of OCR-2-associated vesicles in C. elegans sensory neurons

Single-particle imaging using laser-illuminated widefield epi-fluorescence microscopy is a powerful tool to investigate molecular processes in vivo. Performing high-quality single-molecule imaging in such biological systems, however, remains a challenge due to difficulties in controlling the number of fluorescing molecules, photobleaching, and the autofluorescence background. Here, we show that by exciting only a small, 5-15 {micro}m wide region in chemosensory neurons in live C. elegans, we can significantly improve the duration and quality of single-molecule imaging. Small-window illumination microscopy (SWIM) allows long-duration single-particle imaging since fluorescently labelled proteins are only excited upon entering the small excited area, limiting their photobleaching. Remarkably, we also find that using a small excitation window significantly improves the signal-to-background ratio of individual particles. With the help of theoretical calculations, we explain that the improved signal-to-background ratio is due to reduced background, mostly caused by out-of-focus autofluorescence. We demonstrate the potential of this approach by studying the dendritic transport of a ciliary calcium channel protein, OCR-2, in the chemosensory neurons of C. elegans. We reveal that OCR-2-associated vesicles are continuously transported back and forth along the length of the dendrite and can switch between directed and diffusive states. Furthermore, we perform single-particle tracking of OCR-2-associated vesicles to quantitatively characterize the transport dynamics. SWIM can be readily applied to other in vivo systems where intracellular transport or cytoskeletal dynamics occur in elongated protrusions, such as axons, dendrites, cilia, microvilli and extensions of fibroblasts.

biophysics↗

bioRxiv2022

Acute hyper- and hypoglycemia uncouples the metabolic cooperation between glucose and lactate to disrupt sleep

The sleep-wake cycle is a master regulator of metabolic and neuronal activity and when altered, can have profound effects on metabolic health and disease. Although consideration is given to how fluctuations in blood glucose affect peripheral physiology and metabolism, less is known about how glucose dysregulation impacts the intrinsic cooperation between brain metabolism and neuronal activity to regulate sleep. To understand the effect of peripheral hyper- and hypoglycemia on these relationships, we paired biosensors measuring hippocampal interstitial fluid (ISF) levels of glucose and lactate with cortical EEG/EMG recordings to produce simultaneous subsecond recordings of ISF glucose, lactate, and sleep-wake states. First, we describe a conserved temporal relationships between ISF glucose and lactate based on their intrinsic oscillations, diurnal rhythms, and sleep/wake cycles. ISF glucose and lactate oscillations are largely anti-correlated but the frequency of their oscillations dictate their power, coherence, and phase. While ISF glucose and lactate both have diurnal fluctuations, only ISF lactate is consistently elevated during wake. During wake, fluctuations in ISF lactate are associated with changes in the EEG power spectrum, suggesting wake-related activity is more closely associated with ISF lactate. Modulation of glucose availability via both hyper- or hypoglycemia disrupts the relationship between peripheral metabolism, brain metabolism, and sleep. Hyper- and hypo-glycemia increase ISF lactate, decrease NREM, and alter EEG spectral activity, again demonstrating ISF lactate drives wake-associated behaviors and disrupts sleep. Taken together, these studies demonstrate that peripheral glucose homeostasis is necessary for maintaining the relationships between brain metabolism, neuronal activity, and sleep-wake patterns and deviations in blood glucose levels are sufficient to disrupt the metabolic signature of sleep-wake states, putting the brain at risk in diseases like type-2-diabetes and Alzheimers disease. Graphical Abstract. Peripheral glucose homeostasis directly modifies sleep/wake patterns O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/507967v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@9102eborg.highwire.dtl.DTLVardef@5699c5org.highwire.dtl.DTLVardef@1897ebeorg.highwire.dtl.DTLVardef@1e00661_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2022

Identification of potential natural compound inhibitors and drug like molecules against human METTL3 by docking and molecular dynamics simulation

Nucleotide level chemical modification in transcriptome is critical in regulating different cellular processes, including cancer. The most investigated epitranscriptomic modification is methylation at the N6-position of adenosine (m6A). This dynamic modification process is carried out by: writer, reader and eraser proteins. Writers are methyltransferases, METTL3 is the major writer that works in association with METTL14, an accessory protein. Extensive study revealed that cancer progression for acute myeloid leukaemia, gastric cancer, colorectal cancer, hepatocellular carcinoma, and lung cancer is directly contributed by irregular expression of METTL3. Targeting METTL3, has opened a new window in the development of new inhibitors/drugs. In this study, 80 commercially available compounds were found from an unbiased screening by molecular docking, showing better score when compared with the existing substrate/substrate-analogue and the inhibitor bound crystal structures in terms of docking score and binding energy calculation. Among this pool of compounds, the best seven small molecules, AMF, RAD, JNJ, MEH, ECP, MHN, SGI, have been selected and further validated by different computational tools like binding energy calculation, molecular dynamics simulation etc. The novel hits found in this study can function as lead compounds which can be developed into inhibitors as well as drugs, specific against METTL3.

bioinformatics↗

bioRxiv2022

Decreasing Wapl dosage partially corrects transcriptome phenotypes in Nipbl-/+ embryonic mouse brain

Cohesin rings interact with DNA and modulate expression of thousands of genes. NIPBL loads cohesin onto chromosomes and WAPL takes it off. Heterozygous mutations in NIPBL lead to a developmental disorder called Cornelia de Lange syndrome. Nipbl heterozygous mice are a good model for this disease but mutations in WAPL were not known to cause disease or gene expression changes in mammals. Here we show dysregulation of >1000 genes in Wapl{Delta}/+ embryonic mouse brains. The patterns of dysregulation are highly similar to Nipbl heterozygotes, suggesting that Wapl mutations may also cause disease in humans. Since WAPL and NIPBL have opposite effects on cohesins association with DNA, we asked whether a heterozygous Wapl mutation could correct phenotypes seen in Nipbl heterozygous mice. In fact, both gene expression and embryonic growth are partially corrected. Our data are consistent with the view that cohesin dynamics play a key role in regulating gene expression.

genetics↗

bioRxiv2022

In silico identification of novel peptides as potential modulators of Aβ42 Amyloidogenesis

Alzheimers Disease is a neurodegenerative disease for which no cure is available at present. The presence of amyloid plaques in the extracellular space of neural cells is the key feature of this fatal disease. Amyloid-Beta (A{beta}) is a 40-42 amino acid peptide and the main component of amyloid plaques. This peptide is produced by the proteolysis of Amyloid Precursor Protein by presenilin. Deposition of 42 residual A{beta} peptides forms fibrils structure, leading to disruption of neuron synaptic transmission, inducing neural cell toxicity, ultimately leading to neuron death. To modulate the amyloidosis of A{beta} peptides, various novel peptides have been investigated via molecular docking and molecular dynamic simulation studies. The sequence-based peptides were designed and investigated for their interaction with A{beta}42 monomer and fibril using the molecular docking method, and their influence on the structural stability of target proteins was studied using molecular simulations. According to the docking results, amongst all the synthetic peptides, the peptide YRIGY (P6) has the highest binding affinity with A{beta}42 fibril, and the peptide DKAPFF (P12) shows better binding with A{beta}42 monomer. Moreover, simulation results also suggest that the higher the binding affinity, the better the inhibitory action. From these findings, it is suggested that both the peptides can modulate the amyloidogenesis, but peptide (P6) has better potential for the disaggregation of the fibrils, whereas peptide P12 stabilizes the native structure of the A{beta}42 monomer more effectively and hence can serve as a potential amyloid inhibitor. Thus, these peptides can be explored as therapeutic agents against Alzheimers Disease.

bioinformatics↗

bioRxiv2022

Cannabinoids regulate an insula circuit controlling water intake

The insular cortex, or insula, is a large brain region involved in the detection of thirst and the control of water intake. However our understanding of the topographical, circuit and molecular mechanisms the controlling water intake within the insula remains parcellated. We found that type-1 cannabinoid receptors (CB1) within the insular cortex participate to the regulation of water intake, and deconstructed circuit mechanisms of this control. Topographically, we revealed that the activity of excitatory neurons in both anterior (aIC) and posterior (pIC) insula increases in response to water intake, yet removal of CB1 receptors only in the pIC decreases water intake. Interestingly, we found that CB1 receptors are highly expressed in insula projections to the basolateral amygdala (BLA), while undetectable in the neighboring central part of the amygdala. Thus, we imaged the neurons of the anterior or posterior insula targeting the BLA (aIC-BLA and pIC-BLA), and found they oppositely respond to water intake, respectively decreasing and increasing their activity upon water drinking. Consistently, chemogenetic activation of pIC-BLA neurons decreased water intake. Finally, we uncovered CB1-dependent short term synaptic plasticity (depolarization-induced suppression of excitation, DSE) selectively in pIC-BLA, compared to aIC-BLA synapses. Altogether, our results support a model where CB1 signaling in the pIC-BLA pathway exerts a positive control on water intake.

neuroscience↗

bioRxiv2022

Guiding Irregular Nuclear Morphology on Nanopillar Array for Malignancy Differentiation in Tumor cells

For more than a century, abnormal nuclei in tumor cells, presenting subnuclear invaginations and folds on the nuclear envelope, have been known to be associated with high malignancy and poor prognosis. However, current nuclear morphology analysis focuses on the features of the entire nucleus, overlooking the malignancy-related subnuclear features in nanometer scale. The main technical challenge is to probe such tiny and randomly distributed features inside cells. We here employ nanopillar arrays to guide subnuclear features into ordered patterns enabling their quantification as a strong indicator of cell malignancy. Both breast and liver cancer cells were validated, as well as the quantification of nuclear abnormality heterogeneity. The alterations of subnuclear patterns were also explored as effective readouts for drug treatment. We envision this nanopillar-enabled quantification of subnuclear abnormal features in tumor cells opens a new angle in characterizing malignant cells and studying the unique nuclear biology in cancer. TeaserA nanopillar-based assay quantifying the abnormal nuclear morphology in tumor cells at single-cell level.

bioengineering↗

bioRxiv2022

Isoforms of the transcriptional cofactor SIN3 differentially regulate genes necessary for energy metabolism and cell survival

The SIN3 scaffolding protein is a conserved transcriptional regulator known to fine-tune gene expression. In Drosophila, there are two major isoforms of SIN3, SIN3 220 and SIN3 187, which each assemble into multi-subunit histone modifying complexes. The isoforms have distinct developmental expression patterns and non-redundant functions. Gene regulatory network analyses indicate that both isoforms affect genes encoding proteins in pathways such as the cell cycle and cell morphogenesis. Interestingly, the SIN3 187 isoform uniquely regulates a subset of pathways including post-embryonic development, phosphate metabolism and apoptosis. Target genes in the phosphate metabolism pathway include nuclear-encoded mitochondrial genes coding for proteins responsible for oxidative phosphorylation. Here, we investigate the physiological effects of SIN3 isoforms on energy metabolism and cell survival. We find that ectopic expression of SIN3 187 represses expression of several nuclear-encoded mitochondrial genes affecting production of ATP and generation of reactive oxygen species (ROS). Forced expression of SIN3 187 also activates several pro-apoptotic and represses a few anti-apoptotic genes. In the SIN3 187 expressing cells, these gene expression patterns are accompanied with an increased sensitivity to paraquat-mediated oxidative stress. These findings indicate that SIN3 187 influences the regulation of mitochondrial function, apoptosis and oxidative stress response in ways that are dissimilar from SIN3 220. The data suggest that the distinct SIN3 histone modifying complexes are deployed in different cellular contexts to maintain homeostasis.

molecular biology↗