Browsing by Author "Madhu, Bhoomi"
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Item Caenorhabditis elegans egg-laying and brood-size changes upon exposure to Serratia marcescens and Staphylococcus epidermidis are independent of DBL-1 signaling(Caltech Library, 2019-04) Madhu, Bhoomi; Salazar, Aileen E.; Gumienny, Tina L.Effects of S. marcescens and S. epidermidis on egg laying and brood size in wild-type and dbl-1(nk3) populations. (A) Comparison of mean eggs laid between wild-type (WT) and dbl-1(nk3) populations fed on E. coli OP50, S. marcescens, or S. epidermidis. The counts of number of eggs laid were reported only for plates with live animals. Error bars represent SEM. n = 7-10 surviving animals each. (B) Comparison of total brood size between wild-type and dbl-1(nk3) populations fed on E. coli OP50, S. marcescens, or S. epidermidis. Total brood sizes of animals that desiccated were censored. Error bars represent SEM. n = 7-10 surviving animals each. Within each genetic background, significant differences between the E. coli OP50-fed control and the pathogen-fed populations are marked with asterisks (*, p< 0.05, **, p< 0.001).Item Genetic interactions between the DBL-1/BMP-like pathway and dpy body size–associated genes in Caenorhabditis elegans(American Society for Cell Biology, 2019-12-12) Lakdawala, Mohammed Farhan; Madhu, Bhoomi; Faure, Lionel; Vora, Mehul; Padgett, Richard W.; Gumienny, Tina L.Bone morphogenetic protein (BMP) signaling pathways control many developmental and homeostatic processes, including cell size and extracellular matrix remodeling. An understanding of how this pathway itself is controlled remains incomplete. To identify novel regulators of BMP signaling, we performed a forward genetic screen in Caenorhabditis elegans for genes involved in body size regulation, a trait under the control of BMP member DBL-1. We isolated mutations that suppress the long phenotype of lon-2, a gene that encodes a negative regulator that sequesters DBL-1. This screen was effective because we isolated alleles of several core components of the DBL-1 pathway, demonstrating the efficacy of the screen. We found additional alleles of previously identified but uncloned body size genes. Our screen also identified widespread involvement of extracellular matrix proteins in DBL-1 regulation of body size. We characterized interactions between the DBL-1 pathway and extracellular matrix and other genes that affect body morphology. We discovered that loss of some of these genes affects the DBL-1 pathway, and we provide evidence that DBL-1 signaling affects many molecular and cellular processes associated with body size. We propose a model in which multiple body size factors are controlled by signaling through the DBL-1 pathway and by DBL-1–independent processes.Item Reduced bone morphogenic protein signaling along the gut–neuron axis by heat shock factor promotes longevity(Wiley, 2022) Arneaud, Sonja L.B.; McClendon, Jacob; Tatge, Lexus; Watterson, Abigail; Zuurbier, Kielen R.; Madhu, Bhoomi; Gumienny, Tina L.; Douglas, Peter M.Aging is a complex and highly regulated process of interwoven signaling mechanisms. As an ancient transcriptional regulator of thermal adaptation and protein homeostasis, the Heat Shock Factor, HSF-1, has evolved functions within the nervous system to control age progression; however, the molecular details and signaling dynamics by which HSF-1 modulates age across tissues remain unclear. Herein, we report a nonautonomous mode of age regulation by HSF-1 in the Caenorhabditis elegans nervous system that works through the bone morphogenic protein, BMP, signaling pathway to modulate membrane trafficking in peripheral tissues. In particular, HSF-1 represses the expression of the neuron-specific BMP ligand, DBL-1, and initiates a complementary negative feedback loop within the intestine. By reducing receipt of DBL-1 in the periphery, the SMAD transcriptional coactivator, SMA-3, represses the expression of critical membrane trafficking regulators including Rab GTPases involved in early (RAB-5), late (RAB-7), and recycling (RAB-11.1) endosomal dynamics and the BMP receptor binding protein, SMA-10. This reduces cell surface residency and steady-state levels of the type I BMP receptor, SMA-6, in the intestine and further dampens signal transmission to the periphery. Thus, the ability of HSF-1 to coordinate BMP signaling along the gut–brain axis is an important determinate in age progression.Item Small-scale extraction of Caenorhabditis elegans genomic DNA(JOVE, 2022) Madhu, Bhoomi; Lakdawala, Mohammed Farhan; Gumienny, Tina L.Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCRbased applications.Item The DBL-1/TGF-β signaling pathway regulates an array of behavioral, molecular, and physiological defenses in Caenorhabditis elegans(8/18/2021) Madhu, Bhoomi; Gumienny, Tina LOrganisms possess mechanisms to protect themselves from environmental threats. Animals have innate immune responses that include behavioral, molecular, and physiological components. A conserved cell-cell signaling pathway, transforming growth factor-beta (TGF-β) signaling pathway, is a major regulator of innate immune responses in animals. However, the requirement for this pathway in generating specific, robust responses to different bacterial challenges has not previously been well characterized. We used the roundworm Caenorhabditis elegans, which has conserved innate immune responses and TGF-β signaling pathways, to address how organisms use TGF-β signaling to tailor immune responses to different environmental threats. We tested the requirement for DBL-1/TGF-β signaling in a diverse array of immune responses to a selected panel of Gram-negative and Gram-positive bacteria. We showed that robust, protective, and specific responses to different bacteria require functional DBL-1 signaling. Animals lacking DBL-1 were more susceptible to all test bacteria. We discovered that canonical DBL-1 signaling is required to suppress avoidance to Gram-negative bacteria, but non-canonical DBL-1 signaling represses the avoidance to Gram-positive bacteria. Furthermore, this work identified a novel role for SMA-4/co-Smad that is independent of the DBL-1 signaling pathway. This indicates that the DBL-1/TGF-β signaling pathway plays an important role in tailoring the animals’ innate immune responses to the bacterial threat. Additionally, to investigate the role of saposin-like antimicrobial proteins in response to different environmental threats, we characterized the regulation of spp-9 in response to the bacterial panel. We identified that spp-9 is affected by both bacterial exposure and by starvation. We reported that the pathogen-specific regulation of spp-9 expression was dependent on not only DBL-1/TGF-β signaling but also other innate immune signaling pathways including insulin-like and p38/MAP kinase. Lastly, to determine the role of DBL-1 signaling in regulating the epicuticle, a secreted lipid-rich barrier layer, we developed a method to isolate epicuticular lipids and characterized their composition. We showed that DBL-1 signaling regulates the composition and levels of both epicuticle and internal lipids. DBL-1 signaling also regulates expression of lipid metabolism genes. Collectively, these findings demonstrate bacteria-specific host immune responses regulated by the DBL-1/TGF-β signaling pathway.Item The DBL-1/TGF-β signaling pathway tailors behavioral and molecular host responses to a variety of bacteria in Caenorhabditis elegans(2023-09-26) Madhu, Bhoomi; Lakdawala, Mohammed Farhan; Gumienny, Tina L.Generating specific, robust protective responses to different bacteria is vital for animal survival. Here, we address the role of transforming growth factor β (TGF-β) member DBL-1 in regulating signature host defense responses in Caenorhabditis elegans to human opportunistic Gram-negative and Gram-positive pathogens. Canonical DBL-1 signaling is required to suppress avoidance behavior in response to Gram-negative, but not Gram-positive bacteria. We propose that in the absence of DBL-1, animals perceive some bacteria as more harmful. Animals activate DBL-1 pathway activity in response to Gram-negative bacteria and strongly repress it in response to select Gram-positive bacteria, demonstrating bacteria-responsive regulation of DBL-1 signaling. DBL-1 signaling differentially regulates expression of target innate immunity genes depending on the bacterial exposure. These findings highlight a central role for TGF-β in tailoring a suite of bacteria-specific host defenses.