Higuchi-Sanabria R et al. (2018) Mol Biol Cell "Spatial regulation of the actin cytoskeleton by HSF-1 during aging."

Tronnes SU, Durieux J, Higuchi-Sanabria R, Daniele JR, Dillin A, Garcia G, Frankino PA, Paul JW, Monshietehadi S, Benitez C

[Mol Biol Cell, 2018]

There are many studies suggesting an age-associated decline in the actin cytoskeleton, and this has been adopted as common knowledge in the field of aging biology. However, a direct identification of this phenomenon in aging, multi-cellular organisms have not been performed. Here, we express LifeAct::mRuby in a tissue-specific manner to interrogate cytoskeletal organization as a function of age. We show for the first time in C. elegans that the organization and morphology of the actin cytoskeleton deteriorates during advanced age in the muscle, intestine, and hypodermids. Moreover, hsf-1 is essential for regulating cytoskeletal integrity during aging, such that knockdown of hsf-1 results in premature aging of actin and its overexpression protects actin cytoskeletal integrity in muscle, intestine, and the hypodermis. Finally, hsf-1 overexpression in neurons alone is sufficient to protect cytoskeletal integrity in non-neuronal cells.

Garcia G et al. (2022) Methods Mol Biol "Imaging of Actin Cytoskeletal Integrity During Aging in C. elegans."

Homentcovschi S, Garcia G, Higuchi-Sanabria R, Kelet N

[Methods Mol Biol, 2022]

The actin cytoskeleton plays a fundamental role in the regulation of multiple cellular pathways, including trafficking and locomotion. The functional integrity of the cytoskeleton is important during aging, as the decline of cytoskeletal integrity contributes to the physiological consequence of aging. Moreover, improving cytoskeletal form and function throughout aging is sufficient to drive life span extension and promote organismal health in multiple model systems. For these reasons, optimized protocols for visualization of the actin cytoskeleton and its downstream consequences on health span and life span are critical for understanding the aging process. In C. elegans, the actin cytoskeleton shows diverse morphologies across tissues, potentially due to the significantly different functions of each cell type. This chapter describes an imaging platform utilizing LifeAct to visualize the actin cytoskeleton in live, whole nematodes throughout the aging process and methods to perform follow-up studies on the life span and health span of these organisms.

Garcia G et al. (2023) Elife "Lipid homeostasis is essential for a maximal ER stress response."

Moreno S, Dillin A, Webster BM, Higuchi-Sanabria R, Garcia G, Tsui CK, Zhang H

[Elife, 2023]

Changes in lipid metabolism are associated with aging and age-related diseases, including proteopathies. The endoplasmic reticulum (ER) is uniquely a major hub for protein and lipid synthesis, making its function essential for both protein and lipid homeostasis. However, it is less clear how lipid metabolism and protein quality may impact each other. Here, we identified <i>let-767</i>, a putative hydroxysteroid dehydrogenase in <i>Caenorhabditis elegans</i>, as an essential gene for both lipid and ER protein homeostasis. Knockdown of <i>let-767</i> reduces lipid stores, alters ER morphology in a lipid-dependent manner, and blocks induction of the Unfolded Protein Response of the ER (UPR^ER). Interestingly, a global reduction in lipogenic pathways restores UPR^ER induction in animals with reduced <i>let-767</i>. Specifically, we find that supplementation of 3-oxoacyl, the predicted metabolite directly upstream of <i>let-767</i>, is sufficient to block induction of the UPR^ER. This study highlights a novel interaction through which changes in lipid metabolism can alter a cell's response to protein-induced stress.

Castro Torres T et al. (2022) J Vis Exp "Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans."

Moaddeli D, Coakley AJ, Higuchi-Sanabria R, Garcia G, Dutta N, Averbukh M, Castro Torres T

[J Vis Exp, 2022]

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

Galindo-Malagn XA et al. (2022) Zootaxa "New species, synonymies and records in the genus Rhagovelia Mayr, 1865 (Hemiptera: Heteroptera: Veliidae) from Colombia."

Moreira FFF, Morales I, Mondragn-F SP, Galindo-Malagn XA

[Zootaxa, 2022]

Rhagovelia medinae sp. nov., of the hambletoni group (angustipes complex), and R. utria sp. nov., of the hirtipes group (robusta complex), are described, illustrated, and compared with similar congeners. Based on the examination of type specimens, six new synonymies are proposed: R. elegans Uhler, 1894 = R. pediformis Padilla-Gil, 2010, syn. nov.; R. cauca Polhemus, 1997 = R. azulita Padilla-Gil, 2009, syn. nov., R. huila Padilla-Gil, 2009, syn. nov., R. oporapa Padilla-Gil, 2009, syn. nov, R. quilichaensis Padilla-Gil, 2011, syn. nov.; and R. gaigei, Drake Hussey, 1947 = R. victoria Padilla-Gil, 2012 syn. nov. The first record from Colombia is presented for R. trailii (White, 1879), and the distributions of the following species are extended in the country: R. cali Polhemus, 1997, R. castanea Gould, 1931, R. cauca Polhemus, 1997, R. gaigei Drake Hussey, 1957, R. elegans Uhler, 1894, R. femoralis Champion, 1898, R. malkini Polhemus, 1997, R. perija Polhemus, 1997, R. sinuata Gould, 1931, R. venezuelana Polhemus, 1997, R. williamsi Gould, 1931, and R. zeteki Drake, 1953.

Bar-Ziv R et al. (2020) J Vis Exp "Measurements of Physiological Stress Responses in C. Elegans."

Frankino PA, Higuchi-Sanabria R, Gildea HK, Bolas T, Frakes AE, Dillin A, Metcalf MG, Bar-Ziv R

[J Vis Exp, 2020]

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPR^ER), the unfolded protein response of the mitochondria (UPR^MT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Frankino PA et al. (2022) iScience "SKN-1 regulates stress resistance downstream of amino catabolism pathways."

Bolas T, Higuchi-Sanabria R, Dillin A, Siddiqi TF, Gildea HK, Bar-Ziv R, Frankino PA, Zhang H

[iScience, 2022]

The deleterious potential to generate oxidative stress is a fundamental challenge to metabolism. The oxidative stress response transcription factor, SKN-1/NRF2, can sense and respond to changes in metabolic state, although the mechanism and consequences of this remain unknown. Here, we performed a genetic screen in C. elegans targeting amino acid catabolism and identified multiple metabolic pathways as regulators of SKN-1 activity. We found that knockdown of the conserved amidohydrolase T12A2.1/amdh-1 activates a unique subset of SKN-1 regulated genes. Interestingly, this transcriptional program is independent of canonical P38-MAPK signaling components but requires ELT-3, NHR-49 and MDT-15. This activation of SKN-1 is dependent on upstream histidine catabolism genes HALY-1 and Y51H4A.7/UROC-1 and may occur through accumulation of a catabolite, 4-imidazolone-5-propanoate. Activating SKN-1 results in increased oxidative stress resistance but decreased survival to heat stress. Together, our data suggest that SKN-1 acts downstream of key catabolic pathways to influence physiology and stress resistance.

Anderson EC et al. (2019) Dev Cell "X Chromosome Domain Architecture Regulates Caenorhabditis elegans Lifespan but Not Dosage Compensation."

Yang Q, Meyer BJ, Bian Q, Podshivalova K, Anderson EC, Higuchi-Sanabria R, Frankino PA, Kenyon C, Shin A, Dillin A

[Dev Cell, 2019]

Mechanisms establishing higher-order chromosome structures and their roles in gene regulation are elusive. We analyzed chromosome architecture during nematode X chromosome dosage compensation, which represses transcription via a dosage-compensation condensin complex (DCC) that binds hermaphrodite Xs and establishes megabase-sized topologically associating domains (TADs). We show that DCC binding at high-occupancy sites (rex sites) defines eight TAD boundaries. Single rex deletions disrupted boundaries, and single insertions created new boundaries, demonstrating that a rex site is necessary and sufficient to define DCC-dependent boundary locations. Deleting eight rex sites (8rex) recapitulated TAD structure of DCC mutants, permitting analysis when chromosome-wide domain architecture was disrupted but most DCC binding remained. 8rex animals exhibited no changes inXexpression and lacked dosage-compensation mutant phenotypes. Hence, TAD boundaries are neither the cause nor the consequence of DCC-mediated gene repression. Abrogating TAD structure did, however, reduce thermotolerance, accelerate aging, and shorten lifespan, implicating chromosome architecture in stress responses and aging.

Anderson, E. C. et al. (2019) International Worm Meeting "X-Chromosome Domain Achitecture Regulates Caenorhabditis elegans Lifespan but Not Dosage Compensation."

Dillin, A., Shin, A., Kenyon, C., Frankino, P. A., Bian, Q., Meyer, B. J., Yang, Q, Anderson, E. C., Podshivalova, K., Higuchi-Sanabria, R.

[International Worm Meeting, 2019]

Interphase chromosomes are organized into a series of structures ranging from kilobase-scale chromatin loops, to megabase-scale topologically associating domains (TADs), to territories comprising hundreds of megabases. Loci within a TAD interact predominantly with each other while being insulated from interactions with loci in neighboring TADs. Mechanisms that define TAD boundaries and the biological functions of TADs have been elusive. We analyzed chromosome architecture and its impact on gene expression during C. elegans X-chromosome dosage compensation. A condensin dosage compensation complex (DCC) binds to both hermaphrodite X chromosomes via rex sites (recruitment elements on X) to reduce transcription by half, while also establishing an X structure composed of TADs. Without DCC binding, eight TAD boundaries are lost, causing X structure to resemble that of autosomes. We defined the requirements for creating a DCC-dependent TAD boundary by making a series of rex deletions and insertions and measuring chromosome structure. Each rex deletion eliminated the associated DCC-dependent TAD boundary, revealing that a high-occupancy rex site is necessary for boundary formation. Inserting a rex site at a new location on X defined a new boundary, indicating that DCC binding at a high-occupancy rex site is sufficient to define the boundary. Deleting all eight rex sites at the eight DCC-dependent boundaries recapitulated the TAD structure of a DCC mutant. These 8rex? animals allowed us to measure transcription when TAD structure was grossly disrupted across an entire metazoan chromosome, but DCC binding was largely intact, and X chromosomes were properly compacted. The 8rex? worms lacked canonical dosage compensation phenotypes, and embryos did not show statistically significant changes in X-chromosome expression, indicating that TAD structure does not underlie the mechanism of dosage compensation. The absence of TADs allowed us to identify additional DCC-mediated X-chromosome structure that may underlie X compaction and facilitate transcriptional repression. Strikingly, though TADs do not mediate dosage compensation, abrogating TAD structure in hermaphrodites did reduce thermotolerance, accelerate aging, and shorten lifespan, implicating higher-order chromosome structure in regulating stress responses and aging. Our discoveries offer new directions for understanding the regulation of lifespan and the connection between chromosome organization and gene regulation.

Lu WX et al. (1990) J Biol Chem "Cloning, structure, and expression of the gene for a novel regulatory subunit of cAMP-dependent protein kinase in Caenorhabditis elegans."

Bagchi S, Lu WX, Rubin CS, Gross RE

[J Biol Chem, 1990]

The nematode Caenorhabditis elegans (C. elegans) expresses the regulatory subunit (R) of cAMP-dependent protein kinase at a level similar to the levels determined for R subunits in mammalian tissues. Approximately 60% of the C. elegans cAMP-binding protein is tightly associated with particulate structures by noncovalent interactions. Ionic detergents or 7 M urea solubilize particulate R. Solubilized and cytosolic R subunits have apparent Mr values of 52,000 and pI values of 5.5. cDNA and genomic DNA encoding a unique C. elegans R subunit were cloned and sequenced. The derived amino acid sequence contains 375 residues; carboxyl-terminal residues 145-375 are 69% identical with mammalian RI. However, residues 44-145 are markedly divergent from the corresponding regions of all other R sequences. This region might provide sufficient structural diversity to adapt a single R subunit for multiple functional roles in C. elegans. Antibodies directed against two epitopes in the deduced amino acid sequence of C. elegans R avidly bound nematode cytosolic and particulate R subunits on Western blots and precipitated dissociated R subunits and R2C2 complexes from solution. Immunofluorescence analysis revealed that the tip of the head, which contains chemosensory and mechanosensory neurons, and the pharyngeal nerve ring were enriched in R. The R subunit concentration is low during early embryogenesis in C. elegans. A sharp increase (approximately 6-fold) in R content begins several hours before the nematodes hatch and peaks during the first larval stage. Developmental regulation of R expression occurs at translational and/or post-translational levels. The 8-kilobase pair C. elegans R gene is divided into 8 exons by introns ranging from 46 to 4300 base pairs. The 5'-flanking region has no TATA box and contains preferred and minor transcription start sites.