Biology subjects

Nath, S.

Publications and source records attributed to Nath, S..

36 records · Page 2Linked to original sources

bioRxiv2024

Understanding flux switching in metabolic networks through an analysis of synthetic lethals

Biological systems are extremely robust and exhibit high levels of redundancy for multiple cellular functions. Some of this redundancy manifests as alternative pathways in metabolism. Synthetic double lethals in metabolic networks comprise pairs of reactions, which, when deleted simultaneously, abrogate cell growth. However, when one reaction from such pairs is removed, the cell reroutes its metabolites through alternative pathways. Very little is known about the set of reactions through which fluxes are rerouted. Analysing this redistribution would help us to uncover the linkage between the reactions in a synthetic double lethal and also understand the complexity underlying the reroutings. Studying synthetic lethality in the context of pathogenic bacteria can offer valuable insights into therapeutic interventions. In this work, we propose a constraint-based approach to unravel these alternate pathways and complex interdependencies within and across metabolic modules. The approach involves a generic optimisation that minimises the extent of rerouting between two reaction deletions, corresponding to synthetic lethal pairs. We also include a systematic analysis of synthetic lethals by identifying the reaction classes that make up these synthetic lethals. We applied our computational workflow to several existing high-quality genome-scale models to show that these rerouted reactions span across metabolic modules, thereby illustrating the complexity and uniqueness of metabolism. Our results provide interesting insights into the organisation of metabolic networks and their redundancy. The algorithm is available at https://github.com/RamanLab/minRerouting. Contact: kraman@iitm.ac.in

systems biology↗

bioRxiv2023

Astroglia proliferate upon biogenesis of tunneling nanotubes and clearance of α-synuclein toxicities

Astrocytic cells are a subtype of glial cells that engulf pathogenic aggregates derived from degenerative neurons to facilitate its degradation. Here, we show that exposure to -SYN protofibrils caused a transient increase in biogenesis of tunneling nanotubes (TNTs) in primary astrocytes and astrocyte-origin cancer cell-lines (U-87 MG, U251). Biogenesis of nascent TNTs corresponds to -SYN protofibril-induced organelle toxicities, increased reactive oxygen species (ROS), and oxidative stress-induced premature cellular senescence. These TNTs mediate cell-to-cell transfer of -SYN protofibrils, toxic lysosomes and mitochondria. Biogenesis of TNTs precedes clearance of -SYN-induced organelle toxicities, cellular ROS levels and reversal of cellular senescence. Consequences of cellular clearance results in enhanced cell proliferation. Further, we have shown -SYN-induced senescence promotes transient localization of focal adhesion kinase (FAK) in the nucleus. FAK mediated regulation of Rho-associated kinases may have a role in the biogenesis of TNTs, successively proliferation. Our study emphasizes that TNT biogenesis may have a potential role in the clearance of -SYN toxicities and reversal of stress-induced cellular senescence, consequences of which cause enhanced proliferation in the post-recovered astroglia cells. HighlightsO_LI-SYN protofibrils treated astroglia cells proliferate upon transient biogenesis of TNTs. C_LIO_LITransient TNT biogenesis precedes clearance of -SYN toxicities and reversal of senescence. C_LIO_LIStress-induced senescence results in nuclear localization of FAK and ROCK mediated TNT biogenesis. C_LIO_LIThe rescued cells enhance proliferation through ROCK mediated ERK1/2 and NF{kappa}B signalling cascades. C_LI Synopsis O_FIG O_LINKSMALLFIG WIDTH=167 HEIGHT=200 SRC="FIGDIR/small/554645v1_figa1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@9dc5bcorg.highwire.dtl.DTLVardef@20e924org.highwire.dtl.DTLVardef@aa7920org.highwire.dtl.DTLVardef@1f6274b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO -SYN protofibrils-induced biogenesis of tunneling nanotubes (TNTs) aids to enhance cellular clearance of toxic burdens as a cellular survival strategy. -SYN protofibrils treated toxic senescence cells regulate FAK mediated modulation of ROCK signalling cascades to promote TNT biogenesis and rescue the cellular toxicities. The rescued cells eventually enhance cell proliferation. C_FIG

cell biology↗

bioRxiv2023

Targeting staphylococcal cell-wall biosynthesis protein FemX through steered molecular dynamics and drug-repurposing approach

Staphylococcus aureus-mediated infection is a serious threat in this antimicrobial-resistant world. S. aureus has become a superbug by challenging conventional as well as modern treatment strategies. Nowadays, drug repurposing has become a new trend for the discovery of new drug molecules. This study focuses on evaluating FDA-approved drugs that can be repurposed against S. aureus infection. Steered molecular dynamics (SMD) has been performed for Lumacaftor and Olaparib against staphylococcal FemX to understand their binding to the active site. A time-dependent external force or rupture has been applied to the ligands to calculate the force required to dislocate the ligand from the binding pocket. SMD analysis indicates that Lumacaftor has a high affinity for the substrate binding pocket in comparison to Olaparib. Umbrella sampling exhibits that Lumacaftor possesses a higher free energy barrier to displace it from the ligand-binding site. The bactericidal activity of Lumacaftor and Olaparib has been tested, and it shows that Lumacaftor has shown moderate activity along with biofilm inhibition potential (MIC value with conc. 128 g/mL). Pharmacokinetic and toxicology evaluations indicate that Lumacaftor has higher pharmacokinetic potential with lower toxicity. This is the first experimental report where staphylococcal FemX has been targeted for the discovery of new drugs. It is suggested that Lumacaftor may be a potential lead molecule against S. aureus.

pharmacology and toxicology↗

bioRxiv2022

Characterising replisome disassembly in human cells

To ensure faultless duplication of the entire genome, eukaryotic replication initiates from thousands of replication origins. Replication forks emanating from origins move through the chromatin until they encounter forks from neighbouring origins, at which point they terminate. In the final stages of this process the replication machinery (replisome) is unloaded from chromatin and disassembled. Work from model organisms has elucidated that during replisome unloading, the MCM7 subunit of the terminated replicative helicase is polyubiquitylated and processed by p97/VCP segregase, leading to disassembly of the helicase and the replisome, which is built around it. In higher eukaryotes (worms, frogs, mouse embryonic stem cells), MCM7 ubiquitylation is driven by a Cullin2-based ubiquitin ligase, with LRR1 as a substrate receptor. To date, most of our knowledge of replication termination comes from model organisms and embryonic systems and little is known about how this process is executed and regulated in human somatic cells. Here we thus established methods to study replisome disassembly in human model cell lines. Using flow cytometry, immunofluorescence microscopy and chromatin isolation with western blotting, we can visualise unloading of the replisome (MCM7 and CDC45) from chromatin by the end of S-phase. We observe interaction of replicative helicase (CMG complex) with CUL2LRR1 and ubiquitylation of MCM7 on chromatin, specifically in S-phase, suggesting that this is a replication-dependent modification. Importantly, we are able to show that replisome disassembly in this system also requires Cullin2, LRR1 and p97, demonstrating conservation of the mechanism. Moreover, we present evidence that the back-up mitotic replisome disassembly pathway is also recapitulated in human somatic cells. Finally, while we find that treatment with small molecule inhibitors against cullin-based ubiquitin ligases (CULi) and p97 (p97i) does lead to phenotypes of replisome disassembly defects, they also both lead to induction of replication stress in somatic cells, which limits their usefulness as tools to specifically target replisome disassembly processes in this setting.

cell biology↗

bioRxiv2022

Modified methods for bovine sperm RNA isolation for consistent quality and RNA yield

Sperm mRNA transcriptional profiling can be used to evaluate the fertility of breeding bulls. This study aimed to compare the modified RNA isolation methods for higher RNA yield and quality from freshly ejaculated sperm of cattle and buffalo bull for further transcriptome analysis. Ten fresh ejaculates from each Sahiwal (n = 10 bulls x 10 ejaculates) and Murrah bulls (n = 10 bulls x 10 ejaculates) were used for RNA isolation. Swim-up technique was used for live sperm separation and recovery. From the recovered live sperm, total sperm RNA was isolated by conventional methods (TRIzol, Double TRIzol), membrane-based methods combined with TRIzol (RNeasy + TRIzol) with the addition of {beta}-mercaptoethanol (BME) and Kit (RNeasy mini) methods in fresh semen. Among different isolation methods; the membrane-based modified methods combined with TRIzol (RNeasy + TRIzol) with the addition of {beta}-mercaptoethanol (BME) resulted significantly (P < 0.05) higher total RNA quantity (300-340 ng/L) and better purity in different concentrations of spermatozoa viz., 30-40 million, 70-80 million and 300-400 million sperm. The study concluded that the inclusion of BME to the combined membrane-based methods with somatic cell lysis buffer solution was best for constant increased yield and purity of RNA isolation from Sahiwal cattle and Murrah buffalo bull sperm. This method will help with the interpretation of data from animal models and the consistency of clinical assessments of male factor fertility employing RNA molecular biomarkers.

molecular biology↗

bioRxiv2022

Assessment of local potato cultivars found in Cis-Himalayan region of West Bengal through morphology and biochemical profiling

Potato is a major global food crop grown for tubers (underground storage stems) that are high in carbohydrates, minerals, and vitamins. The presence of anthocyanins in tuber skin and flesh can have a significant impact on customer preferences. Potatoes are also high in resistant starches, which have a variety of health benefits, including enhanced fullness, cholesterol reduction, and a reduced risk of colon cancer etc. In West Bengal, diabetes is prevalent. Similarly, according to ICMR studies, colon cancer affects 8.9% of all cancer patients in West Bengal cancer which is caused by ill eating habits or the consumption of foods that are low in antioxidants. 5-10% of colon cancers are genetically caused, while the remainder are caused by poor eating habits or the consumption of foods that are low in antioxidants. To address these issues, one strategy is to eat foods with a low glycemic index and high antioxidant content as a staple food. The tuber tissues of the potato (S. tuberosum L.) accumulate various quantities of anthocyanins, which are commonly consumed around the world. Anthocyanins are pigments that range in colour from red to purple and are found throughout the plant kingdom. Anthocyanins are powerful antioxidants that are water soluble. The red skin potato is well-known among the general public. The epidermal layer contains a high quantity of red anthocyanins, which causes the skin to be red. With this background the present study has been undertaken to address the following objectives morphological (quantitative and qualitative traits) and biochemical characterization of local potato cultivars and identification of most stable genotypes based on the anthocyanin content and tuber yield of local potato cultivars. For our recent research 14 local potato cultivar from North Bengal were collected and evaluated for DUS characterization showed wide range of variability with respect to different phenotypic variants of ten characters. Overall predominant DUS characters of local potato cultivars found in North Bengal are medium sized apical length of sprout, short stem height, compact foliage structure, open leaf structure, ovate lanceolate type leaflet shape, purple leaf sprout predominant colour, spherical leaf sprout shape, medium intensity of anthicyanin coloraton at the base, light intensity of anthicyanin coloraton at the tip and weak nature of light sprout pubescence base. The results of the present investigation suggest that local potato cultivars collected from the northern part of West Bengal showed a high level of genetic variation. The differences between genotypes were highly significant at the 1% and 5% alpha level for all characters like tuber yield, length and weight of tuber, length of sprout, chlorophyll content, canopy temperature, and number of tuber per plant, according to the analysis of variance. Three quantitative traits namely tuber yield, tuber breadth, tuber length played major role in the genetic variance. Anthocyanin content had more contribution to diversify the local potato cultures according to biochemical characterization AMMI analysis suggested that Jalpai from CoochBehar is the most stable potato cultivars in respect to anthocyanin content and tuber yield per plant.

plant biology↗

bioRxiv2022

Meiotic pairing and double strand break formation along the heteromorphic threespine stickleback sex chromosomes

Double strand break repair during meiosis is normally achieved using the homologous chromosome as a repair template. Heteromorphic sex chromosomes have reduced sequence homology with one another, presenting unique challenges to the repair of double strand breaks. Our understanding of how heteromorphic sex chromosomes behave during meiosis has been largely limited to the ancient sex chromosomes of mammals, where the X and Y differ markedly in overall structure and gene content. Consequently, pairing of the X and Y chromosomes is limited to a small pseudoautosomal region. It remains unclear how more recently evolved sex chromosomes, that share considerably more sequence homology with one another, pair and repair double strand breaks during meiosis. One possibility is barriers to pairing evolve rapidly. Alternatively, recently evolved sex chromosomes may exhibit pairing and double strand break repair that more closely resembles that of their autosomal ancestors. Here we use the recently evolved X and Y chromosomes of the threespine stickleback fish (Gasterosteus aculeatus) to study patterns of pairing and double stranded break formation using molecular cytogenetics. We found that the sex chromosomes of threespine stickleback fish do not pair exclusively in the pseudoautosomal region. Instead, the chromosomes fully pair in a non-homologous fashion. To achieve this, the X chromosome underwent synaptic adjustment during pachytene to match the axis length of the Y chromosome. Double strand break formation and repair rate also matched that of the autosomes. Our results highlight that recently evolved sex chromosomes exhibit meiotic behavior that is reminiscent of autosomes and argues for further work to identify the homologous templates that are used to repair double strand breaks on the X and Y chromosomes.

molecular biology↗

bioRxiv2020

Repression of MUC1 promotes expansion and suppressive function of myeloid-derived suppressor cells in pancreatic ductal adenocarcinoma and breast cancer murine models.

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that are responsible for immunosuppression in tumor microenvironment. Here we report the impact of mucin 1 (MUC1), a transmembrane glycoprotein, on proliferation and functional activity of MDSCs. To determine the role of MUC1 in MDSC phenotype, we analyzed MDSCs derived from wild type (WT) and MUC1-knockout (MUC1KO) mice bearing pancreatic ductal adenocarcinoma KCKO and breast cancer C57MG xenografts. We observed enhanced tumor growth in MUC1KO mice compared to WT mice in both pancreatic KCKO and breast C57MG cancer models due to increased MDSC population and enrichment of Tregs in tumor microenvironment. Our current study shows that knockdown of MUC1 in MDSCs promotes proliferation and immature suppressive phenotype indicated by increased level of iNOS, ARG1 activity and TGF-{beta} secretion under cancer conditions. Increased activity of MDSCs leads to repression of IL-2 and IFN-{gamma} production by T-cells. We were able to find that MDSCs from MUC1KO mice have higher levels of c-Myc and activated pSTAT3 as compared to MUC1 WT mice, that are signaling pathways leading to increased survival, proliferation and prevention of maturation. In summary, MUC1 regulates signaling pathways that maintain immunosuppressive properties of MDSCs. Thus, immunotherapy must target only tumor associated MUC1 on epithelial cells and not MUC1 on hematopoietic cells to avoid expansion and suppressive functions of MDSC.

cancer biology↗

bioRxiv2020

The Response of Bacterial Flagellar Motor to Stepwise Increase in NaCl Concentration

Many species of bacteria use flagella to navigate in its environment. The flagellum is a 7-10 m long helical filament with a rotary motor at its base embedded in the cell membrane and almost a dozen stator complexes. Proton motive force across the cell membrane powers the flagellar motors of E.coli and Salmonella. The motor stochastically switches between clockwise and counter-clockwise direction. A chemotaxis system causes the motor to change its direction, but the process is more complex as the switch is sensitive to load and proton motive force as well. NaCl is significant with regard to the flagellar motor as it affects the stator dynamics, proton motive force, and osmotaxis at higher concentration. Chemotaxis helps the bacteria for its growth and survival. E.colis natural habitat has high osmolarity and the organism uses use various mechanisms for osmoregulation. However, the role of flagellar motor to adapt to the changes in osmolarity, or osmotaxis, is not well studied. In this work, we dissipated the membrane potential of bacteria in pH 7 using step-wise increase in concentration of NaCl in motility buffer and studied the output of E.colis flagellar motor using tethered bead assay and swimming Salmonella enteritidis cells. We observed decrease in motor speed and switching rates with stepwise increase in NaCl concentration in the motility buffer. The mean speed of the motors decreased with NaCl concentration. The population of swimming cells tumbled more with increase in concentration of NaCl. At the single motor level, the motors biased to CCW rotation with decrease in membrane potential. In this study, we present our observations of the flagellar motor in high NaCl concentration, and explore how NaCl can be used to study various aspects of the bacterial flagellar motor. Statement of significanceSodium ion has been significant in the both the cellular energetics and the function of bacterial flagellar motor. Growing evidence show that the effect of sodium ions was not what hitherto thought it would be. It is involved in the sodium energetics, dissipate membrane potential, affect the flagellar stator dynamics of bacteria. Being an osmolyte, it influences the osmotaxis of bacteria. In this work, we studied the effect of NaCl on the response of the single bacterial flagellar motor of E.coli and swimming cells of Salmonella enteritidis. We observed that the effect of NaCl on the output of the flagellar motor was significant and it may affect the cells in various ways.

biophysics↗

bioRxiv2020

Sexual dimorphism of synaptonemal complex length in threespine stickleback fish

Crossover frequencies often differ substantially between sexes (i.e. heterochiasmy). Although this phenomenon is widespread throughout taxa, the mechanisms that lead to heterochiasmy remain unclear. One pattern that has emerged is that the overall length of the synaptonemal complex likely has a direct influence on the total number of crossovers in each sex. However, this has only been investigated in a handful of species. The threespine stickleback fish (Gasterosteus aculeatus) is an excellent species to explore whether synaptonemal complex length is associated with differences in the total number of crossovers, as females have much longer linkage maps than males. We used an immunocytological approach to quantify synaptonemal complex length in late pachytene female and male meiocytes in two different populations of threespine stickleback fish. Overall, the freshwater population had shorter synaptonemal complex lengths than the marine population. In both populations we observed sexual dimorphism, with females possessing longer axes. Our results support a model where chromosome axis length determines overall crossover frequency and establish the threespine stickleback as a useful species to explore the mechanistic basis of heterochiasmy as well as the genetic basis underlying variation in synaptonemal complex length.

genetics↗

bioRxiv2020

Improved contiguity of the threespine stickleback genome using long-read sequencing

While the cost and time for assembling a genome have drastically reduced, it still remains a challenge to assemble a highly contiguous genome. These challenges are rapidly being overcome by the integration of long-read sequencing technologies. Here, we use long sequencing reads to improve the contiguity of the threespine stickleback fish (Gasterosteus aculeatus) genome, a prominent genetic model species. Using Pacific Biosciences sequencing, we were able to fill over 76% of the gaps in the genome, improving contiguity over five-fold. Our approach was highly accurate, validated by 10X Genomics long-distance linked-reads. In addition to closing a majority of gaps, we were able to assemble segments of telomeres and centromeres throughout the genome. This highlights the power of using long sequencing reads to assemble highly repetitive and difficult to assemble regions of genomes. This latest genome build has been released through a newly designed community genome browser that aims to consolidate the growing number of genomics datasets available for the threespine stickleback fish.

genomics↗

bioRxiv2020

Monomeric amyloid beta-peptide (1-42) significantly populates compact fibril-like conformations

The aggregation of the amyloid {beta} (A{beta}) peptide is a major hallmark of Alzheimers disease. This peptide can aggregate into oligomers, proto-fibrils, and mature fibrils, which eventually assemble into amyloid plaques. The peptide monomers are the smallest assembly units, and play an important role in most of the individual processes involved in amyloid fibril formation, such as primary and secondary nucleation and elongation. The structure of the A{beta} monomer has been shown to be very dynamic and mostly disordered, both in experimental and in computational studies, similar to a random coil. This structural state of the monomer contrasts with the very stable and well defined structural core of the amyloid fibrils. An important question is whether the monomer can adopt transient fibril-like conformations in solution and what role such conformations might play in the aggregation process. Here we use enhanced and extensive molecular dynamics simulations to study the A{beta}42 monomer structural flexibility with different force fields, water models and salt concentrations. We show that the monomer behaves as a random coil under different simulation conditions. Importantly, we find a conformation with the N-terminal region structured very similarly to that of recent experimentally determined fibril models. This is to the best of our knowledge the first monomeric structural ensemble to show such a similarity with the fibril structure.

biophysics↗

bioRxiv2020

Effect of cell size and tethering geometry on rotation rate and torque of E.coli cells

The bacterial flagellum has a rotary motor embedded in its membrane surrounded by stator-units. Torque is generated by electro-steric interactions between rotor and stator-units. Chemotactic signals entail the motor to switch its direction of rotation. However, other factors such as protonmotive force and torque are involved in switching. In this work, we used peritrichously flagellated E.coli that stochastically tethers on surfaces in random geometries and studied how the cell size and position of the rotational axis affect the output of the motor. We developed a Cell Tethering Analysis Program (CTAP) to measure the length of the cells, the axis of rotation, rotational frequency of tethered cells. A D/L ratio (diameter traced by the cell body to the length of the cell body) was used to quantify the location of the rotational axis wherein, a D/L ratio of 1 and 1.9 means the axis of rotation is at the center of the cell body and near the tip of the cell body respectively. We performed experiments in controlled conditions and quantified the effect of cell size and tethering geometry on the output of the flagellar motor. The estimated torque of the tethered cells was 951 pN.nm and 1390 pN.nm for a D/L ratio of [~]1.0 and [~]1.9 respectively. As the torque increased, the motors rotated exclusively in CCW direction. We conclude that quantifying the cell size and tethering geometry is significant to characterize the output of the flagellar motors in a tethered cell assay.

biophysics↗

bioRxiv2020

Tumor -Associated MUC1 Regulates TGF-β Signaling and Function in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal human cancers. Transforming Growth Factor Beta (TGF-{beta}) is a cytokine that switches from a tumor-suppressor to a tumor promoter throughout tumor development, by a yet unknown mechanism. Tumor associated MUC1 (tMUC1) is aberrantly glycosylated and overexpressed in >80% of PDAs and is associated with poor prognosis. The cytoplasmic tail of MUC1 (MUC1-CT) interacts with other oncogenic proteins promoting tumor progression and metastasis. We hypothesize that tMUC1 levels regulate TGF-{beta} functions in PDA in vitro and in vivo. We report that high-tMUC1 expression positively correlates to TGF-{beta}RII and negatively to TGF-{beta}RI receptors. In response to TGF-{beta}1, high tMUC1 expressing PDA cells undergo c-Src phosphorylation, and activation of the Erk/MAPK pathway; while low tMUC1 expressing cells activate the Smad2/3 pathway, enhancing cell death. Correspondingly, mice bearing tMUC1-high tumors responded to TGF-{beta}1 neutralizing antibody in vivo showing significantly retarded tumor growth. Analysis of clinical data from TCGA revealed significant alterations in gene-gene correlations in the TGF-{beta} pathway in tMUC1 high versus tMUC1 low samples. This study deepens our understanding of tMUC1-regulated TGF-{beta}s paradoxical function in PDA and establishes tMUC1 as a potential biomarker to predict response to TGF-{beta}-targeted therapies.

cancer biology↗

bioRxiv2020

The bacterial quorum sensing signal DSF hijacks Arabidopsis thaliana sterol biosynthesis to suppress plant innate immunity

Quorum sensing (QS) is a recognized phenomenon that is crucial for regulating population-related behaviors in bacteria. However, the direct specific effect of QS molecules on host biology is largely under-studied. In this work, we show that the QS molecule DSF (cis-11-methyl-dodecenoic acid) produced by Xanthomonas campestris pv. campestris can suppress pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) in Arabidopsis thaliana, mediated by flagellin-induced activation of flagellin receptor FLS2. The DSF-mediated attenuation of innate immunity results from the alteration of oligomerization states and endocytic internalization of plasma membrane FLS2. DSF altered the lipid profile of Arabidopsis, with a particular increase of the phytosterol species, which impairs the general endocytosis pathway mediated by clathrin and FLS2 nano-clustering on the plasma membrane. The DSF effect on receptor dynamics and host immune responses could be entirely reversed by sterol removal. Together, our results highlighted the importance of sterol homeostasis to plasma membrane organization and demonstrate a novel mechanism by which pathogenic bacteria use their communicating molecule to manipulate PAMP-triggered host immunity. SIGNIFICANCE STATEMENTBacteria rely on small signalling molecules called quorum sensing (QS) signals to communicate and coordinate their behaviors. QS is known to regulate gene expression, production of virulence factors, and biofilm formation for pathogenic bacteria to effectively colonize their hosts and cause diseases. In this work, we found a class of QS molecule called diffusible-signal factor (DSF), produced by devastating phytopathogenic bacteria such as Xanthomonas spp. and Xylella fastidiosa, could communicate directly with plant host and subvert plant innate immunity by inducing plant sterol production and thereby, attenuating receptor signalling through hindering the receptor clustering and plant endocytosis. The results significantly enrich our understanding of the mechanisms in the tug-of-war between bacterial pathogenesis and host immunity.

plant biology↗

bioRxiv2019

Dynamic actin-mediated nano-scale clustering of CD44 regulates its meso-scale organization at the plasma membrane

Transmembrane adhesion receptors at the cell surface, such as CD44, are often equipped with modules to interact with the extracellular-matrix(ECM) and the intra-cellular cytoskeletal machinery. CD44 has been recently shown to compartmentalize the membrane into domains by acting as membrane pickets, facilitating the function of signaling receptors. While spatial organization and diffusion studies of membrane proteins are usually conducted separately, here we combine observations of organization and diffusion by using high spatio-temporal resolution imaging on living cells to reveal a hierarchical organization of CD44. CD44 is present in a meso-scale meshwork pattern where it exhibits enhanced confinement and is enriched in nano-clusters of CD44 along its boundaries. This nanoclustering is orchestrated by the underlying cortical actin dynamics. Interaction with actin is mediated by specific segments of the intracellular-domain(ICD). This influences the organization of the protein at the nano-scale, generating a selective requirement for formin over Arp2/3-based actin-nucleation machinery. The extracellular-domain(ECD) and its interaction with elements of ECM do not influence the meso-scale organization, but may serve to reposition the meshwork with respect to the ECM. Taken together, our results capture the hierarchical nature of CD44 organization at the cell surface, with active cytoskeleton-templated nano-clusters localized to a meso-scale meshwork pattern.

cell biology↗

bioRxiv2019

Amyloid-β induced membrane damage instigates tunnelling nanotubes and direct cell-to-cell transfer

Alzheimers disease (AD) pathology progresses gradually via anatomically connected brain regions. Earlier studies have shown that amyloid-{beta}1-42 oligomers (oA{beta}) can be directly transferred between connected neurons. However, the mechanism of transfer is not fully revealed. We observed formation of oA{beta} induced tunneling nanotubes (TNTs), nanoscaled f-actin containing membrane conduit, in differentially differentiated SH-SY5Y neuronal models. Time-lapse images showed that TNTs propagate oligomers from one cell to another. Preceding the TNT-formation, we detected oA{beta} induced plasma membrane (PM) damage and calcium-dependent repair through lysosomal-exocytosis and significant membrane surface expansion, followed by massive endocytosis to re-establish the PM. Massive endocytosis was monitored by an influx of the membrane-impermeable dye TMA-DPH and PM damage was quantified by propidium iodide influx in the absence of calcium. The massive endocytosis eventually caused accumulation of internalized oA{beta} in Lamp1 positive multi vesicular bodies/lysosomes via the actin cytoskeleton remodulating p21-activated kinase1 (PAK1) dependent endocytic pathway. Three dimensional quantitative and qualitative confocal imaging, structured illumination superresolution microscopy (SIM) and flowcytometry data revealed that oA{beta} induces activated phospho-PAK1, which modulates the formation of long stretched f-actin extensions between cells. Moreover, formation of TNTs can be inhibited by preventing PAK1 dependent internalization of oA{beta} using small-molecule inhibitor IPA-3, a highly selective cell permeable auto-regulatory inhibitor of PAK1. The present study gives insight that the TNTs are probably instigated as a consequence of oA{beta} induced PM damage and repair process, followed by PAK1 dependent endocytosis and actin remodeling, probably to maintain cell surface expansion and/or membrane tension in equilibrium.

cell biology↗

bioRxiv2019

Polyglutamine-expanded androgen receptor disrupts muscle triad, calcium dynamics and the excitation-contraction coupling gene expression program

Spinal and bulbar muscular atrophy (SBMA) is caused by polyglutamine (polyQ) expansions in the androgen receptor (AR) gene. Although clinical and experimental evidence highlight a primary role for skeletal muscle in the onset, progression, and outcome of disease, the pathophysiological and molecular processes underlying SBMA muscle atrophy are poorly understood. Here we show that polyQ-expanded AR alters intrinsic muscle force generation before denervation. Reduced muscle force was associated with a switch in fiber-type composition, disrupted muscle striation, altered calcium (Ca++) dynamics in response to muscle contraction, and aberrant expression of excitation-contraction coupling (ECC) machinery genes in transgenic, knock-in and inducible SBMA mice and patients. Importantly, treatment to suppress polyQ-expanded AR toxicity restored ECC gene expression back to normal. Suppression of AR activation by surgical castration elicited similar ECC gene expression changes in normal mice, suggesting that AR regulates the expression of these genes in physiological conditions. Bioinformatic analysis revealed the presence of androgen-responsive elements on several genes involved in muscle function and homeostasis, and experimental evidence showed AR-dependent regulation of expression and promoter occupancy of the most up-regulated gene from transcriptomic analysis in SBMA muscle, i.e. sarcolipin, a key ECC gene. These observations reveal an unpredicted role for AR in the regulation of expression of genes involved in muscle contraction and Ca++ dynamics, a level of muscle function regulation that is disrupted in SBMA muscle, yet restored by pharmacologic treatment.

neuroscience↗