Biology subjects

Ghosh-Roy, A.

Publications and source records attributed to Ghosh-Roy, A..

12 recordsLinked to original sources

bioRxiv2025

Deep-Pose-Tracker: a unified model for behavioural studies of Caenorhabditis elegans

Tracking and analyzing animal behaviour is a crucial step in fields such as neuroscience and developmental biology. Behavioural studies in the nematode C. elegans, for example, help in understanding how organisms respond to external cues and how the specific physiological responses link to either instantaneous or learned behaviours. Although tracking behaviour through locomotion patterns and postural dynamics is routine, it becomes laborious and time-consuming when performed manually. Automation of this process is therefore crucial for accurate and fast detection and analysis. To this end, we report Deep-Pose-Tracker (DPT), a YOLO (You Only Look Once)-based model for automated pose detection of C. elegans from videos and images. The module is further utilized for several downstream analysis algorithms to quantify essential behavioural features, including locomotion speed, orientation, forward or reverse locomotion, and complex body bends such as omega turns. In addition, it includes eigenworms decomposition to represent complex posture dynamics in a low-dimensional space. The model shows reliable performance on the validation and test datasets, with high inference speed, while being user-friendly. DPT, therefore, can be a valuable toolkit for automated behavioural quantification of C. elegans under varying experimental stimuli.

animal behavior and cognition↗

bioRxiv2025

The cytokinesis associated proteins CITK and ASPM-1 regulate neuronal microtubule dynamics and polarity in C. elegans

The polarized architecture of neurons is intricately associated with modulation of microtubule dynamics. Over the years several microtubule-associated-factors that modulate neuronal polarity have been identified. However, the precise details of how microtubule arrangement and stability is established in axon and dendrites is not clearly understood. To uncover relevant factors involved in the biological pathways governing microtubule regulation in neuron, we conducted a suppressor screen using the neuronal ectopic extension phenotype caused due to loss of kinesin-13 family microtubule depolymerizing protein KLP-7 in C. elegans. Interestingly, apart from eleven variants of (mec-12) and {beta} (mec-7) tubulins, we isolated a variant of cytokinesis associated protein, W02B8.2/citk-1, the kinase-less worm orthologue of mammalian citron-rho interacting kinase (CIT). Little is known about the role of CITK in microtubule regulation in post-mitotic neurons. In this study, we found that the kinase-less worm orthologues of CIT, citk-1 and citk-2 redundantly modulate microtubule stability in the axon-like anterior process and maintain the population of plus-end-out microtubules in the dendrite-like posterior process of the PLM mechanosensory neurons in a cell autonomous manner. In the absence of citk-1, PLM neurons exhibit variable morphological defects including defects in migration, growth, and guidance. Moreover, the neuronal and microtubule phenotypes of loss of both citk-1 and citk-2 were phenocopied by the mutant animals of aspm-1, the worm homolog of abnormal spindle-like microcephaly-associated protein (ASPM), suggesting a genetic association, similar to their association in dividing mammalian cells. These observations suggest that the cytokinesis associated citron kinase and ASPM-1 have non-mitotic roles in C. elegans mechanosensory neurons in the regulation of microtubules.

cell biology↗

bioRxiv2024

Exercise-induced differential transcriptional output of AMPK signalling improves axon regeneration and functional recovery

In adulthood, the regenerative capacity of the injured brain circuit is poor preventing functional restoration. Rehabilitative physical exercise is a promising approach to overcome such functional impairment and the metabolic sensor AMPK has emerged to be a critical mediator for this. However, the mechanistic understanding of upstream and downstream components of AMPK signalling in the physical exercise-mediated enhancement of axon regeneration is not clear. We combined swimming exercise with laser axotomy of posterior lateral microtubule (PLM) neurons of Caenorhabditis elegans to address this question. We found that direct activation of AMPK through AICAR treatment is sufficient to improve axon regeneration and functional recovery. The PAR-4/Liver kinase B1 (LKB1) acts upstream of AMPK to enhance functional recovery following swimming exercise. Using genetics, tissue-specific RNAi, and AICAR treatment, we found that the transcriptional regulators DAF-16 and MDT-15 act downstream of AMPK in mediating the positive effects of swimming. We found that MDT-15 acts in neuron to mediate the benefit of AMPK activation in axon regeneration, whereas DAF-16 acts both in neuron and muscle to promote regrowth downstream to AMPK. We also showed that swimming exercise induces nuclear localization of DAF-16 in an AMPK-dependent manner. Our results showed that neuronal and non-neuronal arms of AMPK signalling play an integrative role in response to physical exercise to promote functional recovery after axon injury. Significance statementFinding ways to promote functional recovery after accidental damage to the nervous system has been challenging as adult neurons lose the capability to regenerate. Rehabilitation therapy is the most promising approach to improve the health condition of patients with nervous system injury. Even in the roundworm C. elegans, axon regeneration could be enhanced through swimming exercise, which is mediated by the metabolic energy sensor AMP Kinase. In this study, using sensory neurons in worm, we found that PAR-4/ Liver kinase B1 acts upstream of AMPK. Whereas, the transcription factor DAF-16/ FOXO and the transcriptional co-regulator MDT-15 act as downstream signalling arms in muscle and neuron tissues. Excitingly, both of these arms could be harnessed through agonist-mediated activation of AMPK to promote functional recovery in adulthood.

neuroscience↗

bioRxiv2024

Synergistic control of axon regeneration and functional recovery by let-7 miRNA and Insulin signalling (IIs) pathways

The capability of neurons to regenerate after injury becomes poor in adulthood. Previous studies indicated that loss of either let-7 miRNA or components of Insulin signalling (IIs) can overcome the age-related decline in axon regeneration in C. elegans. In this study, we wanted to understand the relationship between these two pathways in axon regeneration. We found that the simultaneous removal of let-7 and the gene for insulin receptor daf-2 synergistically increased the functional recovery involving posterior touch sensation following axotomy of PLM neuron in adulthood. Conversely, the loss of let-7 could bypass the regeneration block due to the loss of DAF-16, a transcriptional target of DAF-2. Similarly, the loss of daf-2 could bypass the requirement of LIN-41, a transcriptional co-factor of the let-7 pathway. Our analysis revealed that these two pathways synergistically control targeting of the regenerating axon to the ventral nerve cord, which leads to functional recovery. The computational analysis of the gene expression data revealed a large number of genes, their interacting modules, and hub genes under let-7 and IIs pathway are exclusive in nature. Our study highlights a potential to promote neurite regeneration by harnessing the independent gene expression program involving the let-7 and Insulin signalling pathways.

neuroscience↗

bioRxiv2024

Activation and regulation of a p38α MAPK by its downstream MAPKAP kinase through feedback phosphorylation and LLPS-driven condensate formation.

MAP kinases (MAPKs) represent a class of evolutionarily conserved stress and extracellular stimuli responsive signaling molecules. p38 group of MAPKs known to be activated by dual-specific upstream MAPK kinases and also by autophosphorylation. They activate MAPK activated protein kinases (MAPKAPKs) in a context dependent manner by specific phosphorylation, and together, they play crucial biological roles. One such pair is PMK3, p38-MAPK and its cognate MAPKAPK, MAK2, downstream of DLK1 (MAPK kinase kinase) and MKK4 (MAPK kinase) in C. elegans. They are implicated in axonal regeneration, degeneration and synaptic pruning in response to neuronal injury. Here, we report that PMK3 and MAK2 engage in a feedback relationship, leading to phosphorylation-mediated activation of both kinases. Interaction of PMK3 with MAK2 through the canonical docking site leads to negligible de novo autophosphorylation of PMK3. Phosphorylation of both Thr and Tyr residues on the TxY-motif necessary for the full activation of PMK3 requires catalytic activity of MAK2 as confirmed by mass spectrometry. Distribution of phosphorylation sites on either kinase when incubated together, and presence of long intrinsically disordered regions in each kinase indicate that PMK3:MAK2 complex is conformationally plastic in nature. MAK2 increases bioavailability of aggregation-prone PMK3 by virtue of its ability to form LLPS-driven condensates in vitro, wherein they retain enzymatic activities and phosphorylate each other. Furthermore, experiments with transgenic C. elegans reveal that MAK2 controls stability of PMK3 and localization of PMK3 puncta in touch neuron. Our observations offer an unreported activation mechanism of a p38-MAPK by its downstream MAPKAPK. Significance StatementMAPK kinases are evolutionarily conserved, and are key players in stress response, cell survival, differentiation, metabolic processes and neuronal response to injury. MAPK pathways are primarily activated through unidirectional flow of phosphorylation-signal MAP3K to MAP2K to MAPK, and in some cases to a downstream MAPKAPK. We found that a C. elegans MAPK (PMK3) can also be activated by its downstream MAPKAPK (MAK2) through feedback phosphorylation that ensures robust activation of PMK3 by MAK2 without requiring the MAP2K. Furthermore, MAK2 increases bioavailability of activation-competent and active PMK3 by preventing its aggregation through LLPS-driven condensate formation in vitro and in the worm neuron. This feedback relationship might ensure rapid activation of such MAPK pathways in response to nervous system injury or stress.

biochemistry↗

bioRxiv2023

Optimization of RNAi efficiency in PVD neuron of C. elegans

PVD neuron of C. elegans has become an attractive model for the study of dendrite development and regeneration due to the elaborate and stereotype dendrite morphology in this neuron. The molecular basis for dendrite maintenance and regeneration is poorly understood. RNA interference (RNAi) by feeding E. coli expressing dsRNA has been the basis of several genome wide screens performed using C. elegans. However, the feeding method often fails when it comes to nervous system. Using an optimal induction condition for the dsRNA expression in E coli, we fed the worm strains with HT115 bacteria expressing dsRNA against genes like mec-3, hpo-30, and tiam-1, whose loss of function are known to show dendrite morphology defects in PVD neuron. We found that RNAi of these genes in the strains such as nre-1(-) lin-15b(-), lin-15b(-) and sid-1(-); lin-15b(-); Punc-119::sid-1[+] resulted in significant reduction of dendrite branching. However, the phenotypes were significantly modest compared to the respective loss of function mutants. To obtain stronger phenotype for PVD specific genes, we have made a strain, which strongly expresses sid-1 under mec-3 promoter specific for PVD. When Pmec-3::sid-1 is expressed in either nre-1(-);lin-15b(-) or lin-15b(-) background, the higher order branching phenotype after RNAi of mec-3, hpo-30, and tiam-1 was significantly enhanced as compared to nre-1(-);lin-15b(-) and lin-15b(-) background alone. Next we tested the nre-1(-) lin-15b(-), Pmec-3-sid-1[+] strain for the knockdown of genes playing role in dendrite regeneration process. We found that when aff-1 and ced-10 genes were knocked down in the nre-1(-) lin-15b(-), Pmec-3-sid-1[+] background, the dendrite regeneration was significantly reduced and the extent of reduction was comparable to that of the mutants of aff-1 and ced-10. Essentially, our strain expressing sid-1 in PVD neuron optimizes the condition for RNAi for high throughput screening for PVD development, maintenance and regeneration.

genetics↗

bioRxiv2023

KLP-7/Kinesin-13 orchestrates axon-dendrite checkpoints for polarized trafficking in neurons

Neurons are compartmentalized cells with spatiotemporal distinction of anatomy and molecular repertoire. Microtubule organization in the neuron is crucial for its polarized structure and composition. Microtubule dynamics are differentially optimized in the axons and dendrites by the interplay between the microtubule-stabilizing and destabilizing factors. It is unclear how the destabilizing factors are important for developing and maintaining neuronal polarity. We investigated the function of KLP-7, a microtubule depolymerizing motor from the Kinesin-13 family, in the compartmentalization of axons and dendrites using the PVD neurons in Caenorhabditis elegans. In the absence of KLP-7, axonal proteins such as RAB-3 and SAD-1 were mislocalized to dendrites, suggesting a disruption in axon-dendrite compartmentalization. Notably, this phenomenon was independent of other depolymerizing factors like EFA-6, highlighting the specific role of KLP-7 in this process. We observed a reduced rate of microtubule polymerization and an altered polarity of microtubules in the PVD major dendrite due to the loss of klp-7. Additionally, the deletion of klp-7 led to the formation of ectopic neurites from the cell body and the ectopic localization of UNC-44/Ankyrin-G, a protein associated with the axon initial segment (AIS), to the dendrites. Additionally, live imaging of GFP::KLP-7 revealed that KLP-7 is more dynamic in the dendrites as compared to the axon. These observations indicate that the precise dynamics of KLP-7 in neurites are crucial for maintaining distinct microtubule polymerization in the axons and dendrites, thereby influencing neuronal polarity. Our findings shed light on the pivotal role of KLP-7/Kinesin-13 in the establishment of axon-dendrite checkpoints, which in turn impact the polarized trafficking of cellular components within neurons.

cell biology↗

bioRxiv2023

Functional recovery associated with dendrite regeneration in PVD neuron of C. elegans

PVD neuron of C. elegans is a highly polarized cell with well-defined axonal, and dendritic compartments. PVD neuron operates in multiple sensory modalities controlling both nociceptive touch sensation and body posture. Although both axon and dendrites of this neuron show regeneration response following laser-assisted injury, it is rather unclear how the behavior associated with this neuron is affected by the loss of these structures. It is also unclear whether neurite regrowth would lead to functional restoration in these neurons. Upon axotomy, using a femtosecond laser, we saw that harsh touch response was specifically affected leaving the body posture unperturbed. Subsequently, recovery in the touch response is highly correlated to the axon regrowth, which was dependent on DLK-1 MAP Kinase. Dendrotomy of both major and minor primary dendrites affected the wavelength and amplitude of sinusoidal movement without any apparent effect on harsh touch response. We further correlated the recovery in posture behavior to the type of dendrite regeneration events. We found that dendrite regeneration with the fusion and reconnection between the proximal and distal branches of the injured dendrite corresponded to improved recovery of posture. Our data revealed that the axons and dendrites differentially regulate the functions of PVD neurons. It also revealed that dendrite and axon regeneration are both functionally and molecularly distinct.

neuroscience↗

bioRxiv2022

Musleblind-1 regulates microtubule cytoskeleton in C. elegans mechanosensory neuron through tubulin mRNAs

Regulation of microtubule cytoskeleton is fundamental for the development and maintenance of neuronal architecture. Recent studies have shown that regulated RNA processing is also critical for the establishment and maintenance of neural circuits. In a genetic screen using mechanosensory neurons of C. elegans, we identified a mutation in muscleblind-1 as a suppressor of loss of kinesin-13 family microtubule destabilizing factor klp-7. Muscleblind-1(MBL-1) is an RNA-binding protein that regulates the splicing, localization, and stability of RNA. We found that mbl-1 is required cell-autonomously for axon growth and synapse formation in the posterior lateral microtubule (PLM) neuron. Loss of mbl-1 affects stability and plus-end-out organization of microtubules in the anterior process of PLM. These defects are also accompanied by abnormal axonal transport of the synaptic protein RAB-3 and loss of gentle touch sensation in mbl-1 mutant. Our data showed that mbl-1 is genetically epistatic to mec-7 ({beta} tubulin) and mec-12 (a tubulin) for axon growth. The immunoprecipitation of MBL-1 pulls down the mec-7, mec-12, and sad-1 mRNAs. Additionally, the mbl-1 mutants show a reduction in the level and stability of mec-7 and mec-12 transcripts. Independently, mbl-1 is epistatic to sad-1 for synapse formation. Our work elucidated a previously unknown link between RNA binding protein and cytoskeletal machinery for the development and maintenance of the nervous system.

neuroscience↗

bioRxiv2021

Dendrite regeneration in C. elegans is controlled by the RAC GTPase CED-10 and the RhoGEF TIAM-1

Neurons are vulnerable to physical insults which compromise the integrity of both dendrites and axons. Although several molecular pathways of axon regeneration are identified, our knowledge of dendrite regeneration is limited. To understand the mechanisms of dendrite regeneration, we used PVD neurons in C. elegans having stereotyped branched dendrites. Using femtosecond laser, we severed the primary dendrites and axon of this neuron. After severing the primary dendrites near the cell body, we observed sprouting of new branches from the proximal site within 6 hours, which regrew further with timein an unstereotyped manner.This was accompanied by reconnection between the proximal and distal dendrites as well as the fusion among the higher-order branches as reported before. We quantified the regeneration pattern in threeaspects -territory length, number of branchesand fusion phenomena.Axonal injury causes a retraction of the severed end followed by a Dual leucine zipper kinase-1 (DLK-1) dependent regrowth from the severed end.We tested the roles of the major axon regenerationsignaling hubs such as DLK-1-RPM-1, cAMP elevation, let-7 miRNA, AKT-1, Phosphatidyl serine exposure/PS in dendrite regeneration. We found that neither regrowth nor fusionis affected by the axon injury pathway molecules. Surprisingly, we found that the RAC GTPase CED-10and its upstream GEF TIAM-1 play a cell-autonomous role in dendrite regeneration. Additionally, function of CED-10 in epidermal cell is critical for post-dendrotomy fusion phenomena. This work describes a novel regulatory mechanism of dendrite regeneration andprovides a framework for understanding the cellular mechanism of dendrite regeneration using PVD neuron as a model system.

neuroscience↗

bioRxiv2020

The G-protein coupled receptor SRX-97 is required for concentration dependent sensing of Benzaldehyde in Caenorhabditis elegans

The G-protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors in the olfactory system function to sense the surroundings and respond to various odorants. The genes encoding for the olfactory receptors in C. elegans are larger in number in comparison to those in mammals, suggesting complexity in the receptor- odorant relationships. Recent studies have shown that the same odorant in different concentration could act on multiple receptors in different neurons to induce attractive or repulsive responses. The ASH neuron is known to be responsible for responding to high concentrations of volatile odorants. Here we characterize a new GPCR, SRX-97. We found that the srx-97 promoter shows expression specifically in the head ASH and tail PHB chemosensory neurons of C. elegans. Further, the SRX-97 protein localizes to the ciliary ends of the ASH neurons. Analysis of CRISPR/based deletion mutants of the srx-97 gene suggest that this gene is involved in the recognition of high concentrations of benzaldehyde. This was further confirmed through rescue and neuronal ablation experiments. Our work gives insight into concentration dependent receptor function in the olfactory system and provides details of an additional molecule that could help the animal navigate its surroundings.

neuroscience↗

bioRxiv2019

Loss of dop-2 causes increased dopamine release and locomotory defects in the presence of ethanol

Ethanol is a widely used drug, excessive consumption of which could lead to medical conditions with diverse symptoms. Ethanol abuse causes disinhibition of memory, attention, speech and locomotion across species. Dopamine signaling plays an essential role in ethanol dependent behaviors in animals ranging from C. elegans to humans. We devised an ethanol dependent assay in which mutants in the dopamine autoreceptor, dop-2, displayed a unique sedative locomotory behavior causing the animals to move in circles while dragging the posterior half of their body. We identify the posterior dopaminergic sensory neuron as being essential to modulate this behavior. We further demonstrate that in dop-2 mutants, ethanol exposure increases dopamine secretion and results in enhanced function of the DVA interneuron. DVA releases the neuropeptide NLP-12 and leads to the excitation of cholinergic motor neurons that affect movement. Thus, DOP-2 modulates dopamine levels at the synapse and regulates alcohol induced movement through NLP-12.

neuroscience↗