Biology subjects

Vasudevan, D.

Publications and source records attributed to Vasudevan, D..

13 recordsLinked to original sources

bioRxiv2026

ATF4 and EcR interact to mediate both transcriptional activation and repression in the Drosophila fat body

AbstractCells with high secretory and metabolic loads such as adipocytes and hepatocytes rely on constitutive activation of stress response pathways for their homeostatic function and to cope with exogenous stressors such as nutrient deprivation or excess lipids. The evolutionarily conserved stress response factor, Activating Transcription Factor 4 (ATF4), is known to be required for both homeostatic function and exogenous burden in these tissues. However, the molecular mechanism by which ATF4 specifies distinct transcriptional targets under homeostasis versus stress conditions remains an open question. Here, we use the Drosophila larval fat tissue as a model to establish that ATF4 interacts with the steroid hormone receptor Ecdysone Receptor (EcR) to transcriptionally activate and repress genes involved in lipid metabolism. Our data show that EcR and its ligand, 20-hydroxyecdysone, are required for transcription activation of the bona fide ATF4 target 4E-BP in the fat body. We also find that ATF4 and EcR co-repress transcription of the triglyceride lipase brummer (bmm). In Forster resonance energy transfer (FRET) experiments, we find that ATF4 interacts with EcR and does so by competing with the canonical EcR-binding partner, Ultraspiracle (Usp). Using a genetic model of nutrient deprivation, we find that while EcR is required for homeostatic signaling, it is dispensable for the elevated ATF4 signaling associated with nutrient deprivation as measured by induction of 4E-BP. Together, these data provide a mechanistic starting point for understanding how changes in interaction partner allows ATF4 to engage in context-specific transcriptional programs in metabolic tissues.

developmental biology↗

bioRxiv2025

Translation regulation of ATF4 by the termination complex Hbs1-Pelo is required for visual system development and function

Inherited retinal diseases are a class of genetically heterogenous disorders characterized by mutations in genes required for retinal function, resulting in progressive loss of vision in human patients. One such deletion mutation in the translation termination factor, HBS1L, results in a suite of developmental anomalies in human patients, including progressive vision loss defects. HBS1L, and its interaction partner Pelota (Pelo), are required for recycling stalled ribosomes on mRNAs, but the specific mRNA target causative of vision defects seen with HBS1L deletion remains unknown. Further, the specific cell types in the visual system that require HBS1L for proper development and function are also unknown. Here, we discover that loss of the Drosophila HBS1L homolog, Hbs1, results in reduced expression of the stress responsive Activating Transcription Factor 4 (ATF4) which is encoded by an mRNA containing multiple upstream open reading frames (uORFs) in its 5 leader. We corroborate these results in cultured human cells, where we find that HBS1L and Pelo promote translation reinitiation at the ATF4 ORF by facilitating proper translation termination at the preceding uORFs. Like human HBS1L deficiency patients, loss of function Drosophila mutants for Hbs1, pelo, and ATF4 show vision defects as measured by electroretinograms (ERG). Depleting Hbs1 in lamina neurons replicated the ERG defects seen in Hbs1 mutants, suggesting that Hbs1-Pelo is required for proper lamina neuron function. Further confocal analysis of Hbs1 mutants revealed vacuolization defects in the lamina layer, which are indicative of defective synapse formation between lamina neurons and photoreceptors. Strikingly, restoring ATF4 expression in the lamina partially rescued ERG defects in Hbs1 mutants, indicating that ATF4 is likely a relevant mRNA target regulated by Hbs1-Pelo in these cells. Together, our data support a model wherein Hbs1-Pelo mediated translation regulation of ATF4 in lamina neurons underlies the inherited retinal disease caused by HBS1L deletion.

genetics↗

bioRxiv2025

Ring canals in the larval adipose of Drosophila buffer stress response

Cells in metabolically active tissues with high biosynthetic and secretory demands often use robust stress-responsive mechanisms to maintain endoplasmic reticulum (ER) homeostasis. Coordinating such robust stress response mechanisms requires intercellular communication and coordination. Such modalities of intercellular communication have been relatively understudied in the context of stress tolerance. Here, we use the Drosophila melanogaster third instar fat body to demonstrate that adipocytes communicate with each other through intercellular bridges called ring canals to buffer endoplasmic reticulum stress. The fat body supports the exponential growth from embryo to late larval stage over a short period of time through its energy storage and secretory functions, enduring a high basal level of ER stress in the process. We discovered that individual cells in the fat body are paired to one neighboring cell through ring canals. We further demonstrate that ring canals mediate rapid and highly specific intercellular cargo and organellar trafficking, and allow the transport of cytoplasmic, ER-bound and Golgi vesicular proteins. Disrupting fat body ring canals resulted in higher levels of ER stress response markers, aberrant cell size, as well as increased cell lethality in response to exogenous stress. We also find that animals with disrupted fat body ring canals display an overall delay in larval development, likely due to reduced secretion of larval serum proteins from the fat body. In sum, our work reveals a novel feature of intercellular communication in adipose tissue that serves to buffer ER stress across cells which is required for both homeostatic secretory function and maintaining tissue viability under exogenous stress.

cell biology↗

bioRxiv2024

Sexually dimorphic ATF4 expression in the fat confers female stress tolerance in Drosophila melanogaster

Metabolic differences between males and females have been well documented across many species. However, the molecular basis of these differences and how they impact tolerance to nutrient deprivation is still under investigation. In this work, we use Drosophila melanogaster to demonstrate that sex-specific differences in fat tissue metabolism are driven, in part, by dimorphic expression of the Integrated Stress Response (ISR) transcription factor, ATF4. We found that female fat tissues have higher ATF4 activity than their male counter parts under homeostatic conditions. This dimorphism was partly due to a female bias in transcript abundance of specific ATF4 splice isoforms. We found that the canonical sex determinants transformer (tra) and doublesex (dsx) drive such dimorphic ATF4 transcript abundance. These differences persist in a genetic model of nutrient deprivation, where female animals showed greater resistance to lethality than males in an ATF4-dependent manner. These results suggest that higher ATF4 activity confers higher tolerance to stress in females. Together, our data describe a previously unknown facet of ISR signaling wherein sexual identity of adipose tissue confers differential stress tolerance in males and females. Since energy storage mechanisms are known to be dimorphic and have been linked to ATF4 regulation, our studies provide a mechanistic starting point for understanding how sexual identity influences metabolic disease outcomes.

cell biology↗

bioRxiv2024

Temporal cohort number, neural composition, and connectivity vary along the Drosophila larval body axis

Neural stem cells are inherently flexible, producing different lineages of neurons in different contexts; yet in vivo, development must be highly regulated because aberrant stem cell development leads to microcephalies, tumors, and neurodevelopmental disorders. However, the full complement of in vivo flexibility in neural stem cell development remains poorly understood. Within the Drosophila CNS, 30 types of neural stem cells or neuroblasts repeat in each segment with segment-specific differences in development. We studied NB3-3 neuroblast in each of the 14 segments where it is present. In different segments, NB3-3 produces different types of neurons in different numbers but always produces Even-skipped Lateral (EL) neurons. Prior work showed that in abdominal segments, ELs comprise two types of temporal cohorts. Temporal cohorts are small sets of neurons within a lineage that are born within a tight time window. In the abdomen, EL neurons in the early-born temporal cohort all receive mechanosensory input, whereas EL neurons in the late-born temporal cohort all receive proprioceptive input. Herein, we find that NB3-3 generates an early-born EL temporal cohort in all segments. In specific regions, NB3-3 neuroblasts produce additional types of temporal cohorts, including but not limited to the late-born EL temporal cohort. We show that variations in NB3-3 division pattern determine the number and type of temporal cohorts. Later in development, the number of neurons within a temporal cohort is refined by a variety of post-mitotic mechanisms: regulation of molecular identity markers, cell death, and differentiation status, acting in complex combinations in different segments. Post-mitotic refinement of neuron numbers occurs most often for neurons of the early-born EL temporal cohort. Further, neurons of the early-born EL temporal cohort are incorporated into different circuits, depending on segment. Together, our results show how a single neural stem cell type can flexibly generate segment-specific lineages and contribute to various circuits. We offer the following framework for understanding how a given type of neural stem cell generates diverse temporal cohorts: a stem cell type invariably generates one type of temporal cohort, regardless of location or pattern of division. For neurons in this invariant temporal cohort, post-mitotic mechanisms introduce diversity in number. The stem cell, on a segment-by-segment basis, can also produce additional types of temporal cohorts simply by altering its division pattern. This framework is valuable for motor circuit development, the development of sexual dimorphisms, nervous system evolution, and in vitro stem cell applications.

neuroscience↗

bioRxiv2024

Transcriptomic analysis identifies injury-responsive fibroblast populations as potential mediators of Wnt-dependent spinal cord regeneration

BackgroundIn humans and other mammals, spinal cord injury (SCI) can lead to a permanent loss of sensory and motor function, due to the inability of damaged neurons and axons to regenerate. However, other vertebrate species including zebrafish exhibit complete spinal cord regeneration and functional recovery after SCI. Wnt signaling is required for neurogenesis and axon regrowth in a larval zebrafish SCI model, but the genes regulated by this pathway and the cell types that express them remain largely unknown. ResultsIn this study, we used bulk RNA-sequencing (RNAseq) to identify candidate genes regulated by Wnt signaling that are expressed after SCI. Using this unbiased screen, we identified multiple genes previously unassociated with SCI in larval zebrafish, and confirmed by in situ hybridization that their expression is injury-responsive, Wnt-dependent, and localized to fibroblast-like cells surrounding the spinal cord. ConclusionsTogether, our data reveal potential novel gene targets and cell populations that may play important roles in spinal cord regeneration.

developmental biology↗

bioRxiv2023

Integrated Stress Response signaling acts as a metabolic sensor in fat tissues to regulate oocyte maturation and ovulation

Reproduction is an energy-intensive process requiring systemic coordination. However, the inter-organ signaling mechanisms that relay nutrient status to modulate reproductive output are poorly understood. Here, we use Drosophila melanogaster as a model to establish the Integrated Stress response (ISR) transcription factor, Atf4, as a fat tissue metabolic sensor which instructs oogenesis. We demonstrate that Atf4 regulates the lipase Brummer to mediate yolk lipoprotein synthesis in the fat body. Depletion of Atf4 in the fat body also blunts oogenesis recovery after amino acid deprivation and re-feeding, suggestive of a nutrient sensing role for Atf4. We also discovered that Atf4 promotes secretion of a fat body-derived neuropeptide, CNMamide, which modulates neural circuits that promote egg-laying behavior (ovulation). Thus, we posit that ISR signaling in fat tissue acts as a "metabolic sensor" that instructs female reproduction: directly, by impacting yolk lipoprotein production and follicle maturation, and systemically, by regulating ovulation.

genetics↗

bioRxiv2022

The plant nucleoplasmin AtFKBP43 needs its extended arms for histone interaction

The nucleoplasmin family of histone chaperones is a key player in governing the dynamic architecture of chromatin, thereby regulating various DNA-templated processes. The crystal structure of the N-terminal domain of Arabidopsis thaliana FKBP43 (AtFKBP43), an FK506-binding immunophilin protein, revealed a characteristic nucleoplasmin fold, thus confirming it to be a member of the FKBP nucleoplasmin class. Small-Angle X-ray Scattering (SAXS) analyses confirmed its pentameric nature in solution, and additional studies confirmed the nucleoplasmin fold to be highly stable. The AtFKBP43 nucleoplasmin core domain could not interact with histones and required the acidic arms, C-terminal to the core, for histone association. Furthermore, SAXS generated low-resolution envelope structure, ITC, and AUC results revealed that an AtFKBP43 pentamer with C-terminal extensions interacts with H2A/H2B dimer and H3/H4 tetramer in an equimolar ratio. Put together, AtFKBP43 belongs to a hitherto unreported subclass of FKBP nucleoplasmins that requires the C-terminal acidic stretches emanating from the core domain for histone interaction.

biophysics↗

bioRxiv2021

The Transcription Factor Xrp1 is Required for PERK-Mediated Antioxidant Gene Induction in Drosophila

PERK is an endoplasmic reticulum (ER) transmembrane sensor that phosphorylates eIF2 to initiate the Unfolded Protein Response (UPR). eIF2 phosphorylation promotes stress-responsive gene expression most notably through the transcription factor ATF4 that contains a regulatory 5 leader. Possible PERK effectors other than ATF4 remain poorly understood. Here, we report that the bZIP transcription factor Xrp1 is required for ATF4-independent PERK signaling. Cell type-specific gene expression profiling in Drosophila indicated that delta-family glutathione-S-transferases (gstD) are prominently induced by the UPR-activating transgene Rh1G69D. Perk was necessary and sufficient for such gstD induction, but ATF4 was not required. Instead, Perk and other regulators of eIF2 phosphorylation regulated Xrp1 protein levels to induce gstDs. The Xrp1 5 leader has a conserved upstream Open Reading Frame (uORF) analogous to those that regulate ATF4 translation. The gstD-GFP reporter induction required putative Xrp1 binding sites. These results indicate that antioxidant genes are highly induced by a previously unrecognized UPR signaling axis consisting of PERK and Xrp1.

cell biology↗

bioRxiv2021

A protein-trap allele reveals roles for Drosophila ATF4 in photoreceptor degeneration, oocyte maturation and wing development

Metazoans have evolved various stress response mechanisms to cope with cellular stress inflicted by external and physiological conditions. The Integrated Stress Response (ISR) is an evolutionarily conserved pathway that mediates adaptation to cellular stress via the transcription factor, ATF4. Loss of function of Drosophila ATF4, encoded by the gene cryptocephal (crc), results in lethality during pupal development. The roles of crc in Drosophila disease models and adult tissue homeostasis thus remain poorly understood. Here, we report that a protein-trap MiMIC insertion in the crc locus generates a crc-GFP fusion protein that allows visualization of crc activity in vivo, and acts as a hypomorphic mutant that uncovers previously unknown roles for crc. Specifically, the crc protein-trap line shows crc-GFP induction in a Drosophila model for Retinitis Pigmentosa (RP). This crc allele renders photoreceptors more vulnerable to age-dependent retinal degeneration. crc mutant adult animals also show greater susceptibility to amino acid deprivation and reduced levels of known crc transcriptional targets. Furthermore, this mutant allele shows defects in wing veins and oocyte maturation, uncovering previously unknown roles for crc in the development of these tissues. Together, our data establish physiological and pathological functions of crc-mediated ISR in adult Drosophila tissues.

genetics↗

bioRxiv2020

Regenerated Interneurons Integrate Into Locomotor Circuitry Following Spinal Cord Injury

Whereas humans and other adult mammals lack the ability to regain locomotor function after spinal cord injury, zebrafish are able to recover swimming behavior even after complete spinal cord transection. We have previously shown that zebrafish larvae regenerate lost neurons within 9 days post-injury (dpi), but the functional contribution of these neurons to motor recovery is unknown. Here we show that multiple interneuron subtypes known to play a role in locomotor circuitry are regenerated in injured spinal cord segments during the period of functional recovery. Further, we show that one subtype of newly-generated interneurons receives excitatory input and fires synchronously with motor output by 9 dpi. Taken together, our data show that regenerative neurogenesis in the zebrafish spinal cord produces interneurons with the physiological capacity to participate in the recovery of locomotor function.

neuroscience↗

bioRxiv2018

Macrophage migration inhibitory factor (MIF) of Syrian golden hamster (Mesocricetus auratus) has similar structure and function as human MIF and promotes pancreatic tumor growth in vivo

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that increasingly is being studied in cancers and inflammatory diseases. Though murine models have been instrumental in understanding the functional role of MIF in different pathological conditions, the information obtained from these models is biased towards a specific species. In experimental science, results obtained from multiple clinically relevant animal models always provide convincing data that might recapitulate in humans. Syrian golden hamster (Mesocricetus auratus), is a clinically relevant animal model for multiple human diseases. Hence, the major objectives of this study were to characterize structure and function of hamster MIF, and finally evaluate its effect on pancreatic tumor growth in vivo. Initially, the recombinant hamster MIF (rha-MIF) was cloned, expressed and purified in bacterial expression system. The rha-MIF primary sequence, biochemical properties and crystal structure analysis showed a greater similarity with human MIF. The crystal structure of hamster MIF illustrates that it forms a homotrimer as known in human and mouse. However, hamster MIF exhibits some minor structural variations when compared to human and mouse MIF. The in vitro functional studies show that rha-MIF has tautomerase activity and enhances activation and migration of hamster peripheral blood mononuclear cells (PBMCs). Interestingly, injection of rha-MIF into HapT1 pancreatic tumor bearing hamsters significantly enhanced the tumor growth and tumor associated angiogenesis. Together, the current study shows a structural and functional similarity between hamster and human MIF. Moreover, it has demonstrated that a high-level of circulating MIF originating from non-tumor cells might also promote pancreatic tumor growth in vivo.

biochemistry↗

bioRxiv2017

The integrated stress response pathway activates 4E-BP to bias mRNA translation and boost antimicrobial peptide synthesis in response to bacterial infection

Pathogenic bacterial infection imposes considerable cellular stress on the host and often leads to attenuation of mRNA translation. In this translation-suppressive environment, it is unclear how the host synthesizes various antimicrobial peptides (AMPs) to mount innate immune response. Here, we use Drosophila as a model to demonstrate that AMP production during infection relies on a translation bias mechanism mediated by the inhibitor of cap-dependent translation 4E-BP (Drosophila Thor), and the AMP 5UTRs that can undergo cap-independent translation. We found that 4E-BP is induced upon infection with the pathogenic bacteria Ecc15 by the stress-responsive transcription factor ATF4, and its upstream kinase GCN2. Moreover, loss of gcn2, atf4 or 4e-bp compromised immunity against Ecc15. In 4E-BP mutants, the transcriptional induction of AMPs after infection was unaffected, while the protein levels of AMPs were substantially reduced in their hemolymph. Analysis of the 5UTRs of AMPs using cell-based bicistronic reporters and in vitro translation analysis indicated that AMPs are translated in a cap-independent mechanism. Analysis of bicistronic reporters in the presence of 4E-BP indicate that infection enhances cap-independent translational activity associated with AMP 5UTRs, accounting for enhanced AMP translation during infection.\n\nHighlights O_LI4E-BP is transcriptionally induced by GCN2/ATF signaling in response to bacterial infection\nC_LIO_LI4E-BP mutants show unaltered antimicrobial peptide (AMP) transcript levels, but have reduced AMP translation\nC_LIO_LIAMP 5UTRs are translated cap-independently\nC_LIO_LITranslation bias by 4E-BP drives cap-independent AMP translation\nC_LI

immunology↗