Biology subjects

Kalra, S.

Publications and source records attributed to Kalra, S..

11 recordsLinked to original sources

bioRxiv2026

Environmental Regulation and Gene-by-Environment Interaction Influence RAP1 Activity and its Impact on Gene Expression

Gene-by-environment (GxE) interactions play a major role in shaping both phenotypic and molecular variation, with important implications for human health and disease. In this study, we used the Doxycycline (Dox) regulated, tetracycline-responsive (Tet-Off) promoter system to sequentially reduce or titrate gene expression levels of the essential yeast transcription factor Repressor Activator Protein 1 (RAP1) similar to a hypomorph allele series, across three distinct environments: Yeast Peptone Dextrose (YPD) media, YPD media with Heat Shock (HS), and Yeast Peptone Acetate (YPAC) media. We then performed RNA sequencing (RNA Seq) to assess global transcriptional responses to RAP1 reduction in these different growth environments. Our analysis first focused on the independent effects of varying RAP1 expression levels within and across environments. We then explored GxE interactions, revealing a subset of genes with significant consequences of reduced levels of RAP1 and environment-specific expression patterns. Notably, many genes exhibited opposite effects of RAP1 titration on gene expression when yeast were grown in YPAC media compared to YPD media and/or HS, suggesting environment-dependent regulatory architecture. This design reveals how cells integrate internal transcriptional and regulatory changes with external environmental cues, providing a deeper view of GxE architecture. Using Weighted Gene Co-expression Network Analysis (WGCNA), we identified co-regulated gene modules, and by combining this with transcription factor motif enrichment tests, our study identified candidate regulators driving their dynamics. Our findings demonstrate that gene regulatory networks can vary dramatically depending on the environmental context an organism experiences, which can then influence the specific phenotypes produced by a particular genetic perturbation. This illustrates the complexity of genotype-environment interactions and the importance of studying gene function in multiple environments to gain a truly comprehensive understanding of a genes sometimes numerous and diverse functions.

genomics↗

bioRxiv2025

Respiratory vocal coordination increases as zebra finches prepare to sing

Complex vocalizations like human speech and bird song require precise coordination between respiration and vocalizations with most vocalization being produced during the exhalation phase of respiration. However, when this coordination is established each time animals vocalize remains poorly understood. Here, we addressed this question by recording respiratory pressure in adult, male, zebra finches during singing. Zebra finches begin their song bouts by repeating a short vocalization called an introductory note (IN), before producing song and these INs have been hypothesized to reflect motor preparation for song. We found that each IN is associated with a large amplitude expiratory pulse. Birds that did not produce INs had a few silent, large amplitude, IN-like, expiratory pulses just before starting song. Expiratory pressure and inspiratory pressure increased with successive repeats of the IN (silent or vocalized). Further, for birds with INs, coordination between respiration and vocalization increased with vocalizations beginning earlier in the exhalation and ending later in the exhalation, such that more of the expiratory pulse was filled with the vocalizaton. Overall, these results show that respiratory vocal coordination improves with each repetition of the IN and suggest that INs reflect preparation that involves coordination of respiratory and vocal neural circuitry.

neuroscience↗

bioRxiv2025

Decoding RAP1's Role in Yeast mRNA Splicing

Messenger RNA (mRNA) splicing is a fundamental and tightly regulated process in eukaryotes, where the spliceosome removes non-coding sequences from pre-mRNA to produce mature mRNA for protein translation. Alternative splicing enables the generation of multiple RNA isoforms and protein products from a single gene, regulating both isoform diversity and abundance. While splicing is widespread in eukaryotes, only [~]3% of genes in Saccharomyces cerevisiae undergo splicing, with most containing a single intron. However, intron-containing genes, primarily ribosomal protein genes, are highly expressed and constitute about one-third of the total mRNA pool. These genes are transcriptionally regulated by Repressor Activator Protein 1 (RAP1), prompting us to investigate whether RAP1 influences mRNA splicing. Using RNA sequencing, we identified a novel role for RAP1 in alternative splicing, particularly in intron retention (IR) while minor effects were observed on alternative 3 and 5 splice site usage. Many IR-containing transcripts introduced premature termination codons, likely leading to degradation via nonsense-mediated decay (NMD). Consistent with previous literature, genes with predicted NMD in our study also had reduced overall expression levels suggesting that RAP1 plays an important role in this understudied mechanism of gene expression regulation.

genomics↗

bioRxiv2025

Zebra finch introductory notes do not influence female response to song and juvenile song learning accuracy

Many songbirds begin their song bouts with introductory notes (INs). In some songbird species like rufous-sided towhees and white-crowned sparrows, INs are believed to function in communication, to alert conspecifics about song onset or as a signal for species identification, but it is unclear whether this function is conserved in other species. Here, we addressed this question using the zebra finch, a well-studied songbird. Male zebra finches begin their song bouts with several INs and the number of INs increases when males sing to females or juveniles, suggesting a possible role of INs in female song preference and/or juvenile song learning. To test these hypotheses, we first recorded and analyzed call responses of female zebra finches to playbacks of different song stimuli. Female zebra finches responded equally to songs with and without INs, even when songs were degraded or when background noise was present. Second, we examined song learning accuracy of juveniles and found that juveniles copied songs accurately from their fathers independent of the fathers number of INs. Overall, these results show that zebra finch INs do not influence female response to song or juvenile song learning and suggest that INs might have different functions in different songbird species.

animal behavior and cognition↗

bioRxiv2025

Giant KASH proteins and ribosomes synergistically establish cytoplasmic biophysical properties in vivo

Understanding how cells control their biophysical properties during development remains a fundamental challenge. While cytoplasmic macromolecular crowding affects multiple cellular processes in single cells, its regulation in living animals remains poorly understood. Using genetically encoded multimeric nanoparticles for in vivo rheology, we discovered that C. elegans tissues maintain distinct cytoplasmic biophysical properties that differ from those observed across diverse systems, including bacteria, yeast species, and cultured mammalian cells. We identified two conserved mechanisms controlling cytoplasmic macromolecular diffusion: ribosome concentration, a known regulator of cytoplasmic crowding, works in concert with a previously unknown function for the giant KASH protein ANC-1 scaffolding the endoplasmic reticulum. These findings reveal mechanisms by which tissues establish and maintain distinct cytoplasmic biophysical properties, with implications for understanding cellular organization across species. One-Sentence SummaryLiving tissues maintain unique intracellular biophysical properties under the control of cytoplasmic constraints and crowding.

biophysics↗

bioRxiv2023

High quality, high throughput, and low-cost simultaneous video recording of 60 animals in operant chambers using PiRATeMC

BackgroundThe development of Raspberry Pi-based recording devices for video analyses of drug self-administration studies has shown to be promising in terms of affordability, customizability, and capacity to extract in-depth behavioral patterns. Yet, most video recording systems are limited to a few cameras making them incompatible with large-scale studies. New MethodWe expanded the PiRATeMC (Pi-based Remote Acquisition Technology for Motion Capture) recording system by increasing its scale, modifying its code, and adding equipment to accommodate large-scale video acquisition, accompanied by data on the throughput capabilities, video fidelity, synchronicity of devices, and comparisons between the Raspberry Pi 3B+ and 4B models. ResultsUsing PiRATeMC default recording parameters resulted in minimal storage ([~]350MB/h), high throughput (< [~]120 seconds/Pi), high video fidelity, and synchronicity within [~]0.02 seconds, affording the ability to simultaneously record 60 animals in individual self-administration chambers at a fraction of current commercial costs. No consequential differences were found between Raspberry Pi 3B+ and 4B models. Comparison with Existing Method(s)This system allows greater acquisition of video data simultaneously than other video recording systems by an order of magnitude with less storage needs and lower costs. Additionally, we report in-depth quantitative assessments of throughput, fidelity, and synchronicity, displaying real-time system capabilities. ConclusionsThe system presented is able to be fully installed in a months time by a single technician and provides a scalable, low cost, and quality-assured procedure with a high-degree of customization and synchronicity between recording devices, capable of recording a large number of subjects with high turnover in a variety of species and settings.

neuroscience↗

bioRxiv2023

Caenorhabditis elegans models for striated muscle disorders caused by missense variants of human LMNA

Striated muscle laminopathies caused by missense mutations in the nuclear lamin gene LMNA are characterized by cardiac dysfunction and often skeletal muscle defects. Attempts to predict which LMNA variants are pathogenic and to understand their physiological effects lags behind variant discovery. We created Caenorhabditis elegans models for striated muscle laminopathies by introducing pathogenic human LMNA variants and variants of unknown significance at conserved residues within the lmn-1 gene. Severe missense variants reduced fertility and/or motility in C. elegans. Nuclear morphology defects were evident in the hypodermal nuclei of many lamin variant strains, indicating a loss of nuclear envelope integrity. Phenotypic severity varied within the two classes of missense mutations involved in striated muscle disease, but overall, variants associated with both skeletal and cardiac muscle defects in humans lead to more severe phenotypes in our model than variants predicted to disrupt cardiac function alone. We also identified a separation of function allele, lmn-1(R204W), that exhibited normal viability and swimming behavior but had a severe nuclear migration defect. Thus, we established C. elegans avatars for striated muscle laminopathies and identified LMNA variants that offer insight into lamin mechanisms during normal development. Author summaryMuscular dystrophy is a progressive muscle-wasting disorder that eventually leads to cardiac disease. Mutations in the LMNA gene, which encodes an intermediate filament protein involved in the structure and organization of the nucleus, is a common but poorly understood cause of this disease. How variants across the breadth of LMNA contribute to mechanistic cellular defects that lead to disease is poorly understood, leading to hurdles in diagnosing disease and developing treatments. We found that by introducing amino acid substitutions found in patients with striated muscle disorders caused by LMNA into the conserved lmn-1 gene of the nematode C. elegans, we could rapidly test the function of these variants to better understand their roles. We found that variants modeling diseases that involve both skeletal and cardiac muscle in humans were the most pathogenic in C. elegans, typically affecting both viability and movement, while those that modeled cardiac disease alone had less deleterious effects in C. elegans. Furthermore, we uncovered molecular mechanisms for how lamins interact with other nuclear envelope proteins to carry out their cellular functions. Thus, our new C. elegans models can be used to diagnose and predict the severity of new variants of human LMNA as well as better understanding the molecular mechanisms of lamins in normal development.

cell biology↗

bioRxiv2022

CineFinch: An animated female zebra finch for studying courtship interactions

Dummies, videos and computer animations have been used extensively in animal behaviour to study simple social interactions. These methods allow complete control of one interacting animal, making it possible to test hypotheses about the significance and relevance of different elements of animal displays. Recent studies have demonstrated the potential of videos and interactive displays for studying more complex courtship interactions in the zebra finch, a well-studied songbird. Here, we extended these techniques by developing an animated female zebra finch and showed that ~40% of male zebra finches (n=5/12) sing to this animation. To study real-time social interactions, we developed two possible methods for closed loop control of animations; (1) an arduino based system to initiate videos/animations based on perch hops and (2) a video game engine based system to change animations. Overall, our results provide an important tool for understanding the dynamics of complex social interactions during courtship. SUMMARY STATEMENTWe develop and test an animation of a female zebra finch to study song and courtship interactions in the male zebra finch.

animal behavior and cognition↗

bioRxiv2020

The Nesprin-1/-2 ortholog ANC-1 regulates organelle positioning in C. elegans without its KASH or actin-binding domains

KASH proteins in the outer nuclear membrane comprise the cytoplasmic half of LINC complexes that connect nuclei to the cytoskeleton. Caenorhabditis elegans ANC-1, an ortholog of Nesprin-1/2, contains actin-binding and KASH domains at opposite ends of a long spectrin-like region. Deletion of either the KASH or calponin homology (CH) domains does not completely disrupt nuclear positioning, suggesting neither KASH nor CH domains are essential. Deletions in the spectrin-like region of ANC-1 led to significant defects, but only recapitulated the null phenotype in combination with mutations in the trans-membrane span. In anc-1 mutants, the ER was unanchored, moving throughout the cytoplasm, and often fragmented. The data presented here support a cytoplasmic integrity model where ANC-1 localizes to the ER membrane and extends into the cytoplasm to position nuclei, ER, mitochondria, and likely other organelles in place.

cell biology↗

bioRxiv2020

Introductory gestures before songbird vocal displays are shaped by learning and biological predispositions

Introductory gestures are present at the beginning of many animal displays. For example, lizards start their head-bobbing displays with introductory push-ups and animal vocal displays begin with introductory notes. Songbirds also begin their vocal displays by repeating introductory notes (INs) before producing their learned song and these INs are thought to reflect motor preparation. Between individuals of a given species, the acoustic structure of INs and the number of times INs are repeated before song varies considerably. While similar variation in songs between individuals is known to be a result of learning, whether INs are also learned remains poorly understood. Here, using natural and experimental tutoring with male zebra finches, we show that mean IN number and IN acoustic structure are learned from a tutor, independent of song learning. We also reveal biological predispositions in IN production; birds artificially tutored with songs lacking INs still repeated a short-duration syllable, thrice on average, before their songs. Overall, these results show that INs, just like elements in song, are shaped both by learning and biological predispositions and suggest multiple, independent, learning processes underlying the acquisition of complex vocal displays.

animal behavior and cognition↗

bioRxiv2020

Cerebral Atrophy in Amyotrophic Lateral Sclerosis Parallels the Pathological Distribution of TDP43

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a preferential involvement of both upper and lower motor neurons. Evidence from neuroimaging and post-mortem studies confirms additional involvement of brain regions extending beyond the motor cortex. The aim of this study was to assess the extent of cerebral disease in ALS cross-sectionally and longitudinally, and to compare the findings with a recently proposed disease-staging model of ALS pathology. Deformation-based morphometry (DBM) was used to identify the patterns of brain atrophy associated with ALS and to assess their relationship with clinical symptoms. Longitudinal T1-weighted MRI data and clinical measures were acquired at baseline, 4 months, and 8 months, from 66 ALS patients and 43 age-matched controls who participated in the Canadian ALS Neuroimaging Consortium (CALSNIC) study. Whole brain voxel-wise mixed-effects modelling analysis showed extensive atrophy patterns differentiating ALS patients from the normal controls. Cerebral atrophy was present in the motor cortex and corticospinal tract, involving both GM and WM, and to a lesser extent in non-motor regions. More specifically, the results showed significant bilateral atrophy in the motor cortex, the corticospinal tract including the internal capsule and brainstem, with an overall pattern of ventricular enlargement; along with significant progressive longitudinal atrophy in the precentral gyrus, frontal and parietal white matter, accompanied by ventricular and sulcal enlargement. Atrophy in the precentral gyrus was significantly associated with greater disability as quantified with the ALS Functional Rating Scale-Revised (ALSFRS-R) (p<0.0001). The pattern of atrophy observed using DBM was consistent with the Brettschneiders four stage pathological model of the disease. Deformation based morphometry provides a sensitive indicator of atrophy in ALS, and has potential as a biomarker of disease burden, in both gray and white matter.

neuroscience↗