Biology subjects

Raja, S.

Publications and source records attributed to Raja, S..

8 recordsLinked to original sources

bioRxiv2026

PBF/PTTG1IP coordinates focal adhesion formation, polarity and cell motility

Directional cell migration requires cells to sense extracellular cues and coordinate adhesion, cytoskeletal remodelling and polarity, yet the molecular regulators that integrate these events remain incompletely defined. PTTG1-binding factor (PBF/PTTG1IP) is a transmembrane glycoprotein extensively characterised in pathological overexpression and cancer models, where elevated expression promotes tumourigenic cellular phenotypes. However, its endogenous physiological function remains poorly understood. Here, separate enrichment analyses of transcriptomic and phosphoproteomic profiles from PBF-overexpressing cells collectively highlighted cell adhesion, extracellular matrix organisation, cytoskeletal regulation and cell motility as major PBF-associated programmes. Using a novel Pbf knockout mouse model, we show that primary Pbf-KO mouse embryonic fibroblasts exhibit impaired migration and invasion. PBF loss reduced fibronectin adhesion, disrupted focal adhesion number, size and distribution, attenuated FAK Tyr397 phosphorylation and delayed early adhesion formation. Live-cell imaging and Golgi orientation assays further revealed altered actin organisation and impaired front-rear polarity in Pbf-KO cells. Key phenotypes were conserved in CRISPR-Cas9 PBF-KO human thyroid cancer cells, while targeted PBF re-expression restored migration in Pbf-KO MEFs. We thus identify PBF as an endogenous regulator of cell adhesion, polarity and directional motility, providing a framework for understanding how pathological PBF overexpression may promote invasive behaviour in cancer.

cell biology↗

bioRxiv2026

Calcium triggers Cryptococcus neoformans aggregation by forming coordination bonds with capsular glucuronoxylomannan

The cryptococcal polysaccharide capsule is a unique eukaryotic virulence factor that is a target for the immune system and the development of therapeutic antibodies. Our understanding of capsular architecture is limited to a few studies suggesting that metal dications play a role. In this work we explore a mechanism of cryptococcal aggregation that depends on calcium phosphate precipitation. We describe the chemical and biophysical properties of calcium interaction with the predominant cryptococcal polysaccharide, glucuronoxylomannan (GXM). We show that cell aggregation is a pH-dependent and occurs in a calcium dose-dependent manner. Furthermore, this cellular aggregation phenomenon as well as interpolymer capsular polysaccharide interactions are unique to calcium dications and do not occur with other mono- or dications as shown by size exclusion chromatography and circular dichroism. Diffusion ordered spectroscopy nuclear magnetic resonance and ab-initio calculations support complexation of calcium with glucuronic acid (GlcA). The ab-initio calculations also suggest that calcium ions can complex up to four GlcA monomers. Not only does calcium act as a scaffold for the cryptococcal capsule, interacting with up to four glucuronic acid residues of GXM, but calcium phosphate treatment of cells reduces the anti-phagocytic properties of the capsule, promoting ingestion by macrophages and altering antibody interactions with the capsule. This work advances our understanding of the cryptococcal capsule, its biophysical properties, by providing a model for the critical role of calcium interactions with capsular polymers of Cryptococcus neoformans including important impacts at the host-cell interface.

biochemistry↗

bioRxiv2025

Classifier-guided Deep Oscillatory Neural Networks (cDONN) for capturing both Neural Dynamics and Behavior simultaneously

Generating EEG signals alongside behavioural actions introduces substantial biological complexity, akin to an abstract model that mimics rich oscillatory brain dynamics by simultaneously producing dynamic neural activity and corresponding behavioural responses. In this work, we propose a novel class of bio-inspired classifier guided Deep Oscillatory Neural Network (cDONN), designed to simultaneously model and capture both neural signal dynamics and behavioural patterns. The cDONN integrates a feedforward neural network with Hopf oscillator neurons, specialized in handling sequential data generation tasks. The model features a Y-shaped structure with two output branches: one for classifying the input image that signifies behaviour, and the other for generating corresponding EEG signals that represent neural dynamics. We evaluate the performance of the behaviour classification branch using the standard ramp classification accuracy used for the cDONN network. For the EEG signal generation branch, we benchmark the generated signals using classification accuracy via a pre-trained DONN signal classifier, alongside comparison with real EEG signals using metrics such as Inception Score, Frechet Inception Distance (FID), power spectrum analysis and topoplot analysis. Our analysis reveals a strong correspondence between the generated and real EEG data, particularly in the midline parietal region within the alpha frequency band, and in the left hemisphere within the delta band, indicating high fidelity and neurophysiological realism in the synthesized signals. Our approach offers a novel perspective on modelling the simultaneous relationship between neural activity and behaviour, pushing the boundaries of EEG signal generation and behavioural analysis.

bioengineering↗

bioRxiv2025

Loss of direct binding of Leishmania profilin with actin adversely affected its functions and interactions with other cellular proteins

Profilins are actin binding proteins that play a central role in regulation of actin remodeling in all eukaryotic organisms. Leishmania, a family of protozoan parasites that cause leishmaniasis, express a single homolog of profilin, which differs from other eukaryotic profilins in that it contains an extra stretch of 20 amino acids (actin binding domain) through which it directly binds actin. We therefore considered it of interest to analyze the role of direct binding of profilin with actin in interactions and functions of profilin in L. donovani promastigotes. For this, we deleted the actin binding domain in L. donovani profilin (LdPfn), and then carried out comparative analysis of LdPfn and truncated LdPfn ({delta}LdPfn) interactomes by affinity pull-down and mass spectrometry. To assess the effect of deletion of the actin binding domain on the LdPfn functions, we expressed GFP conjugates of LdPfn and {delta}LdPfn in the wild type Leishmania cells and then carried out comparative analysis of their growth, cell division cycle and intracellular vesicle trafficking activity. Results revealed that expression of GFP-{delta}LdPfn in the wild type cells adversely affected their cell growth, intracellular vesicle trafficking and G1-to-S and S-to-G2/M phase transitions during their cell division cycle. Also, there was a complete loss of LdPfn binding to several cellular proteins including actin and mitochondrial outer membrane protein, porin (LdPorin) after deleting its actin binding domain. Further, to assess the effect of lack of LdPorin binding to {delta}LdPfn on the mitochondrial functions, we measured the cellular ATP levels in wild-type and transgenic promastigotes. The ATP levels were significantly increased by expressing GFP-{delta}LdPfn in wild-type cells. These results taken together strongly indicate that LdPfn-driven actin remodeling besides playing a pivotal role in regulation of intracellular vesicle trafficking and cell division cycle, it also plays an important role in regulation of Leishmania mitochondrial activity.

cell biology↗

bioRxiv2023

Nonequilibrium bending fluctuations reveal microtubule mechanics in-vivo and their regulation by glutamylation

Cells can be described as active composite materials. The mechanical properties of cells are controlled by complex polymer networks and are dynamically tuned for diverse cellular processes driven by force-generating motor proteins. Microtubules are the most rigid protein polymers in the cytoskeleton, and their material properties have been measured in vitro by active bending or by analyzing thermal bending fluctuations. Microtubule mechanics in living cells are extremely difficult to probe directly, while fluctuations are difficult to interpret because they are generated by active forces in a surrounding cytoplasm with poorly understood material properties. Here we introduce a method to measure the elastic properties of microtubules in living cells by making use of motor-generated forces that drive bending fluctuations. Bending dynamics are governed by three main factors: microtubule material properties, cytoskeletal active forces, and the response characteristics of the surrounding cytoplasm. We show theoretically that, when one factor can be independently determined, the other two can be derived from observed fluctuations. Using this method we discovered that polyglutamylation, a post-translational modification enriched on microtubule arrays that need to withstand large mechanical forces such as those in axons or cilia, increases microtubule stiffness in living cells. Our work provides a theoretical and experimental framework to study microtubule mechanics and their regulation by tubulin modifications and microtubule effectors in complex cellular environments. The approach can be extended to other large aspect ratio cellular structures such as the endoplasmic reticulum or the mitochondrial network.

biophysics↗

bioRxiv2022

Z-REX: Shepherding Reactive Electrophiles to Specific Proteins Expressed either Tissue-Specifically or Ubiquitously, and Recording the Resultant Functional Electrophile-Induced Redox Responses in Larval Fish

Summary of the Protocol ExtensionThis Protocol Extension describes the adaptation of an existing Nature Protocol detailing the use of T-REX (targetable reactive electrophiles and oxidants)--an on-demand redox targeting toolset in cultured cells. The adaptation described here is for use of REX technologies in live zebrafish embryos (Z-REX). Zebrafish embryos expressing a Halo-tagged protein of interest (POI)--either ubiquitously or tissue-specifically--are treated with a HaloTag-specific small-molecule probe housing a photocaged reactive electrophile (either natural electrophiles or synthetic electrophilic drug-like fragments). The reactive electrophile is then photouncaged at a user-defined time, enabling proximity-assisted electrophile-modification of a POI. Functional and phenotypic ramifications of POI-specific modification can then be monitored, by coupling to standard downstream assays, such as, Click chemistry-based POI-labeling and target-occupancy quantification; immunofluorescence or live imaging; RNA-Seq and qRT-PCR analyses of downstream-transcript modulations. Transient expression of requisite Halo-POI in zebrafish embryos is achieved by mRNA injection. Procedures associated with generation of transgenic zebrafish expressing a tissue-specific Halo-POI are also described. The Z-REX experiments can be completed in <1-week using standard techniques. To successfully execute Z-REX, researchers should have basic skills in fish husbandry, imaging, and pathway analysis. Experience with protein or proteome manipulation is useful. This protocol extension is aimed at helping chemical biologists study precision redox events in a model organism and fish biologists perform redox chemical biology.

biochemistry↗

bioRxiv2019

Cortico-cortical feedback from V2 exerts a powerful influence over the visually evoked local field potential and associated spike timing in V1

The local field potential (LFP) is generally thought to be dominated by synaptic activity within a few hundred microns of the recording electrode. The sudden onset of a visual stimulus causes a large downward deflection of the LFP recorded in primary visual cortex, known as a visually evoked potential (VEP), followed by rhythmic oscillations in the gamma range (30-80 Hz) that are often in phase with action potentials of nearby neurons. By inactivating higher visual areas that send feedback projections to V1, we produced a large decrease in amplitude of the VEP, and a strong attenuation of gamma rhythms in both the LFP and multi-unit activity, despite an overall increase in neuronal spike rates. Our results argue that much of the recurrent, rhythmic activity measured in V1 is strongly gated by feed-back from higher areas, consistent with models of coincidence detection that result in burst firing by layer 5 pyramidal neurons.

neuroscience↗

bioRxiv2017

Control of mechanical pain hypersensitivity through ligand-targeted photoablation of TrkB positive sensory neurons

SummaryMechanical allodynia is a major symptom of neuropathic pain whereby innocuous touch evokes severe pain. Here we identify a population of peripheral sensory neurons expressing TrkB that are both necessary and sufficient for producing pain from light touch after nerve injury. Mice in which TrkB-Cre expressing neurons are ablated are less sensitive to the lightest touch under basal conditions, and fail to develop mechanical allodynia in a model of neuropathic pain. Moreover, selective optogenetic activation of these neurons after nerve injury evokes marked nociceptive behavior. Using a phototherapeutic approach based upon BDNF, the ligand for TrkB, we perform molecule-guided laser ablation of these neurons and achieve long-term retraction of TrkB positive neurons from the skin and pronounced reversal of mechanical allodynia across multiple types of neuropathic pain. Thus we identify the peripheral neurons which transmit pain from light touch and uncover a novel pharmacological strategy for its treatment.\n\nHighlightsO_LITrkB+ neurons detect light touch under basal conditions\nC_LIO_LITrkB+ neurons convey mechanical allodynia in neuropathic pain states\nC_LIO_LIA photosensitizing derivative of BDNF allows for photoablation of TrkB+ neurons\nC_LIO_LIBDNF-guided photoablation reverses allodynia in multiple types of neuropathic pain\nC_LI

neuroscience↗