The Ddc1/Rad17/Mec3 complex and Rad24 are DNA damage checkpoint components with limited homology ... more The Ddc1/Rad17/Mec3 complex and Rad24 are DNA damage checkpoint components with limited homology to replication factors PCNA and RF-C, respectively, suggesting that these factors promote checkpoint activation by "sensing" DNA damage directly. Mec1 kinase, however, phosphorylates the checkpoint protein Ddc2 in response to damage in the absence of all other known checkpoint proteins, suggesting instead that Mec1 and/or Ddc2 may act as the initial sensors of DNA damage. In this paper, we show that Ddc1 or Ddc2 fused to GFP localizes to a single subnuclear focus following an endonucleolytic break. Other forms of damage result in a greater number of Ddc1-GFP or Ddc2-GFP foci, in correlation with the number of damage sites generated, indicating that Ddc1 and Ddc2 are both recruited to sites of DNA damage. Interestingly, Ddc2 localization is severely abrogated in mec1 cells but requires no other known checkpoint genes, whereas Ddc1 localization requires Rad17, Mec3, and Rad24, but not Mec1. Therefore, Ddc1 and Ddc2 recognize DNA damage by independent mechanisms. These data support a model in which assembly of multiple checkpoint complexes at DNA damage sites stimulates checkpoint activation. Further, we show that although Ddc1 remains strongly localized following checkpoint adaptation, many nuclei contain only dim foci of Ddc2-GFP, suggesting that Ddc2 localization may be down-regulated during resumption of cell division. Lastly, visualization of checkpoint proteins localized to damage sites serves as a useful tool for analysis of DNA damage in living cells.
Previous work on the DNA damage checkpoint in Saccharomyces cerevisiae has shown that two complex... more Previous work on the DNA damage checkpoint in Saccharomyces cerevisiae has shown that two complexes independently sense DNA lesions: the kinase Mec1-Ddc2 and the PCNA-like 9-1-1 complex. To test whether colocalization of these components is sufficient for checkpoint activation, we fused these checkpoint proteins to the LacI repressor and artificially colocalized these fusions by expressing them in cells harboring Lac operator arrays. We observed Rad53 and Rad9 phosphorylation, Sml1 degradation, and metaphase delay, demonstrating that colocalization of these sensors is sufficient to activate the checkpoint in the absence of DNA damage. Our tethering system allowed us to establish that CDK functions in the checkpoint pathway downstream of damage processing and checkpoint protein recruitment. This CDK dependence is likely, at least in part, through Rad9, since mutation of CDK consensus sites compromised its checkpoint function.
The nematode C. elegans is attracted to nutritious bacteria and is repelled by pathogens and toxi... more The nematode C. elegans is attracted to nutritious bacteria and is repelled by pathogens and toxins. Here we show that RNAi and toxin-mediated disruption of core cellular activities, including translation, respiration, and protein turnover, stimulate behavioral avoidance of normally attractive bacteria. RNAi of these and other essential processes induces expression of detoxification and innate immune effectors, even in the absence of toxins or pathogens. Disruption of core processes in non-neuronal tissues was sufficient to stimulate aversion behavior, revealing a neuroendocrine axis of control that additionally required serotonergic and Jnk kinase signaling pathways. We propose that surveillance pathways overseeing core cellular activities allow animals to detect invading pathogens that deploy toxins and virulence factors to undermine vital host functions. Variation in cellular surveillance and endocrine pathways controlling behavior, detoxification, and immunity selected by past toxin or microbial interactions could underlie aberrant responses to foods, medicines, and microbes.
<p>(A-D) Decreased TORC1 induces neuronal expression of HPK-1. Representative images of <... more <p>(A-D) Decreased TORC1 induces neuronal expression of HPK-1. Representative images of <i>Phpk-1</i>::<i>HPK-1</i>::<i>GFP</i> neuronal expression in day 3 adult animals after treatment with control (A), <i>daf-2</i> (B), <i>rict-1</i> (C) and <i>daf-15</i> RNAi (D). Outlines of animals are traced in white. White space was artificially filled for (D). Additional images and quantification can found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.s009" target="_blank">S9 Fig</a>. (E) Induction of endogenous <i>hpk-1</i> after <i>daf-15</i> inactivation as measured by qRT-PCR. Values are mean fold change and S.E.M. Three independent experiments were performed and normalized to <i>cdc-42</i>. * indicates a p-value <0.05 (Student’s t-test). (F, G) <i>hpk-1</i> is essential for increased lifespan of <i>daf-15(RNAi)</i> and <i>let-363(RNAi)</i>-treated animals, respectively. Tabulated lifespan is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.s012" target="_blank">S1 Table</a>.</p
<p>(A) <i>hpk-1(pk1393)</i> null mutant animals (red) are short-lived relative ... more <p>(A) <i>hpk-1(pk1393)</i> null mutant animals (red) are short-lived relative to wild-type N2 (black). <i>Phpk-1</i>::<i>hpk-1</i>::<i>GFP</i> largely restores wild-type lifespan to <i>hpk-1(pk1393)</i> mutant animals (green). Lifespan of non-transgenic <i>hpk-1(pk1393)</i> siblings (blue) is similar to <i>hpk-1(pk1393)</i>. (B) Overexpression of <i>hpk-1</i> using the heterologous <i>sur-5</i> promoter increases lifespan compared to N2 (dark red versus black). Each graph is representative of at least 3 independent lifespan experiments. Full lifespan data can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.s012" target="_blank">S1 Table</a>.</p
<p>(A) Overexpression of <i>hpk-1</i> increases lifespan (grey versus black) an... more <p>(A) Overexpression of <i>hpk-1</i> increases lifespan (grey versus black) and is <i>pha-4</i> dependent (red traces). (B) Overexpression of <i>hpk-1</i> increases lifespan dependent on <i>mxl-2</i> (blue traces). (C) Loss of <i>hlh-30</i> (green traces) partially suppresses the increased lifespan of <i>hpk-1</i>, consistent with parallel signaling or independence. (D) Loss of <i>nhr-62</i> (pink/purple traces) has a minimal negative effect on both normal and the increased lifespan conferred by <i>hpk-1</i> overexpression. In all cases, black traces are N2 and grey traces are <i>Psur-5</i>::<i>HPK-1</i>::<i>CFP</i> animals treated on control RNAi. For each panel, darker colored traces are respective RNAi-treatment of N2 animals and lighter colored traces are RNAi treatment of <i>Psur-5</i>::<i>HPK-1</i>::<i>CFP</i> animals. In some cases, experiments shown within this figure were performed simultaneously and split into multiple figures for readability. Full lifespan data can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.s012" target="_blank">S1 Table</a>.</p
<p>Model of HPK-1 functions in longevity control: HPK-1 functions as a central hub to maint... more <p>Model of HPK-1 functions in longevity control: HPK-1 functions as a central hub to maintain proteostasis by preventing sumoylation and inactivation of HSF-1 and by stimulating the expression of autophagy genes by <i>pha-4</i> and <i>mxl-2</i>. TORC1 inhibits <i>hpk-1</i> expression to limit the induction of autophagy genes under basal conditions. Under nutrient stress, TORC1 is inactivated resulting in increased <i>hpk-1</i> expression, which promotes autophagy gene expression through PHA-4/FoxA and MXL-2/Mlx. Thermal stress increases HPK-1 protein levels to reduce the threshold of activation of the heat shock response, and HPK-1 promotes longevity through modulation of HSF-1 activity under normal growth conditions.</p
<p>(A) <i>hpk-1</i> is necessary for the induction the autophagy genes <i>... more <p>(A) <i>hpk-1</i> is necessary for the induction the autophagy genes <i>atg-18</i> and <i>bec-1</i> (Beclin1) in response to inactivation of TORC1 by <i>daf-15(RNAi)</i> (** indicates p<0.01, Student’s t-test). (B) In contrast, decreased TORC1 signaling represses the expression of the translation initiation factor genes <i>ifg-1</i> and <i>iftb-1</i> independently from <i>hpk-1</i>. (C) Similarly, TORC1 inhibition mildly induces <i>hsp-16</i>.<i>2</i> and <i>hsp-70</i> independently from <i>hpk-1</i>. Columns labeled <i>hpk-1</i> indicate <i>hpk-1(pk1393)</i>. Expression levels are presented as fold change +/- S.D. normalized to <i>cdc-42</i> and averaged across four independent experiments.</p
<p>(A-C) <i>hpk-1</i> activity affects the accumulation of Q35::YFP foci in mus... more <p>(A-C) <i>hpk-1</i> activity affects the accumulation of Q35::YFP foci in muscle cells. Shown are representative images of <i>Punc-54</i>::<i>polyQ</i>::<i>YFP</i> animals treated with (A) control RNAi or (B) <i>hpk-1</i> RNAi, and (C) transgenic animals overexpressing <i>hpk-1</i> (<i>Psur-5</i>::<i>HPK-1</i>::<i>CFP</i>). (D) Time course of polyQ::YFP foci accumulation in conjunction with: treatment with control RNAi (black circles), <i>hpk-1</i> RNAi (white circles), <i>hpk-1(pk1393)</i> (white squares), or <i>hpk-1</i> overexpression (open triangles). Data points display the mean +/- standard deviation (S.D.) of at least 15 animals per biological replicate; at least 5 independent experiments were performed. P-values are provided in Results and in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.s014" target="_blank">S3 Table</a>. (E) Time course of paralysis of <i>Punc-54</i>::<i>polyQ</i>::<i>YFP</i> animals in conjunction with: treatment with control RNAi (black circles), <i>hpk-1</i> RNAi (white circles), <i>hpk-1(pk1393)</i> (white squares), or <i>hpk-1</i> overexpression (open triangles). Plotted data display the results for a single biological replicate. P-values and data from all trials are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.s014" target="_blank">S3 Table</a>.</p
<p>(A-D) DIC and GFP overlay for <i>Phsp-16</i>.<i>2</i>::<i>... more <p>(A-D) DIC and GFP overlay for <i>Phsp-16</i>.<i>2</i>::<i>GFP</i> worms on empty vector (A, B) or <i>smo-1(RNAi</i>) (C, D) with (+HS) and without (-HS) heat shock. Scale bar = 100μm. (E) Western blot for HSP-16.2, GFP and β-actin from <i>hsp-16</i>.<i>2p</i>::<i>GFP</i> worms grown on empty vector (EV) without heat shock (no HS) or with heat shock (EV), <i>GFP(RNAi)</i>, <i>hsf-1(RNAi)</i> or <i>smo-1(RNAi)</i>. Fold-increases for HSP-16.2/actin on <i>smo-1(RNAi)</i> relative to EV in three independent replicates were 2.4, 4.7, and 1.8.</p
<p>(A) Expression of HPK-1 protein (<i>Phpk-1</i>::<i>HPK-1</i>::&l... more <p>(A) Expression of HPK-1 protein (<i>Phpk-1</i>::<i>HPK-1</i>::<i>GFP</i>) under basal conditions is primarily restricted to neurons. Fluorescent intestinal speckles are non-specific gut granules[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.ref111" target="_blank">111</a>]. (B-D) Heat shock induces HPK-1 protein (<i>Phpk-1</i>::<i>HPK-1</i>::<i>GFP</i>) levels most strongly within hypodermal seam cells (indicated by arrows) independent of <i>hsf-1</i> (C) and transcription (D). <i>Phpk-1</i>::<i>HPK-1</i>::<i>GFP</i> animals after heat shock with (B) empty vector HT115, (C) <i>hsf-1(RNAi)</i>, and (D) α-amanitin treatment. Increased HPK-1 expression within neurons and hypodermal seam cells is specific to heat stress as neither oxidative stress (E) or UV damage (F) altered expression. White space was artificially filled for some images and animals are outlined. GFP quantification and further analysis can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.s006" target="_blank">S6 Fig</a>.</p
<p>(A-D) Representative images of at least 30 <i>Phsp-16</i>.<i>2</i&g... more <p>(A-D) Representative images of at least 30 <i>Phsp-16</i>.<i>2</i>::<i>GFP</i> animals in wild type (A, C) and <i>hpk-1(pk1393)</i> animals (B, D) under basal conditions (A, B) or after heat shock (C, D). Animals are outlined in panels A-B. (E-F) Induction of endogenous <i>hsp-16</i>.<i>2</i> (E) and <i>hsp-70</i> (C12C8.1) (F), in N2 and <i>hpk-1(pk1393)</i> animals as measured by qRT-PCR. (G) Loss of <i>hpk-1</i> did not alter endogenous expression of <i>hsp-16</i>.<i>2</i> (blue) or <i>hsp-70</i> (orange). Values in (E-G) are normalized to expression of <i>act-1</i> and the mean fold change relative to wild-type animals, and the S.E.M. between technical replicates is shown. In total three independent experiments were performed with similar results. P-values for (E) and (F) are <0.05 and <0.01, respectively (Student’s t-test).</p
<p>(A-B) Loss of <i>pha-4</i> (red traces) does not increase foci formation (A)... more <p>(A-B) Loss of <i>pha-4</i> (red traces) does not increase foci formation (A) or the onset of paralysis (B) in the absence of <i>hpk-1</i> (open circles/squares). (C-D) Loss of <i>mxl-2</i> (blue traces) does not increase foci formation (C) or the onset of paralysis (D) in the absence of <i>hpk-1</i> (open circles/squares). (E-F) Loss of <i>hlh-30</i> (green traces) does not increase foci formation (E) but delays the onset of paralysis in the absence of <i>hpk-1</i> (F) (open circles/squares). For foci formation, data are the mean and standard error of the mean (S.E.M.) of at least 15 animals from one representative trial; three independent experiments were performed. ***, **, and * indicate p-values of <0.001, <0.01, and <0.05, respectively. For paralysis, data is representative of one of two trials performed with the same conditions. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.s014" target="_blank">S3 Table</a> for additional details.</p
<p>(A-C) HPK-1 and HSF-1 colocalize in neurons under basal conditions. Representative image... more <p>(A-C) HPK-1 and HSF-1 colocalize in neurons under basal conditions. Representative image of transgenic animal co-expressing <i>Phpk-1</i>::<i>HPK-1</i>::<i>tdtomato</i>,<i>Phsf-1</i>::<i>HSF-1</i>::<i>GFP</i>: (A) red fluorescence, (B) green fluorescence, and (C) overlay.</p
<p>(A-F) Inactivation of <i>hpk-1</i> but not <i>hsf-1</i> disrupts... more <p>(A-F) Inactivation of <i>hpk-1</i> but not <i>hsf-1</i> disrupts autophagosome formation after bacterial deprivation (BD) as visualized by puncta formation for the autophagosomal reporter <i>Plgg-1</i>::<i>LGG-1</i>::<i>GFP</i> (Atg8p/MAP-LC3) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.ref018" target="_blank">18</a>]. (G) Quantification of LGG-1:GFP foci in L3 stage animals under <i>ad libitum</i> (AL) and bacterial deprivation (BD) conditions. BD was imposed by removal from bacterial food for 6 hours prior to scoring puncta formation. Plotted are the mean number of LGG-1::GFP puncta/seam cell visualized +/-S.D. *** indicates a p-value of <0.001 (Student’s t-test). Summary data provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.s015" target="_blank">S4 Table</a>.</p
Recent investigation of the DNA-damage checkpoint in several organisms has highlighted the conser... more Recent investigation of the DNA-damage checkpoint in several organisms has highlighted the conservation of this pathway. The checkpoint's signal transduction pathway consists of four conserved classes of molecules: two large protein kinases having homology to phosphatidylinositol 3-kinases, three 'sensor' proteins with homology to proliferating cell nuclear antigen, two serine/threonine (S/T) kinases, and two adaptors for the S/T kinases. This review compares the role of these four classes of checkpoint proteins in humans and model organisms.
An extensive proteostatic network comprised of molecular chaperones and protein clearance mechani... more An extensive proteostatic network comprised of molecular chaperones and protein clearance mechanisms functions collectively to preserve the integrity and resiliency of the proteome. The efficacy of this network deteriorates during aging, coinciding with many clinical manifestations, including protein aggregation diseases of the nervous system. A decline in proteostasis can be delayed through the activation of cytoprotective transcriptional responses, which are sensitive to environmental stress and internal metabolic and physiological cues. The homeodomain-interacting protein kinase (hipk) family members are conserved transcriptional co-factors that have been implicated in both genotoxic and metabolic stress responses from yeast to mammals. We demonstrate that constitutive expression of the sole Caenorhabditis elegans Hipk homolog, hpk-1, is sufficient to delay aging, preserve proteostasis, and promote stress resistance, while loss of hpk-1 is deleterious to these phenotypes. We show...
Rictor is a component of the target of rapamycin complex 2 (TORC2). While TORC2 has been implicat... more Rictor is a component of the target of rapamycin complex 2 (TORC2). While TORC2 has been implicated in insulin and other growth factor signaling pathways, the key inputs and outputs of this kinase complex remain unknown. We identified mutations in the Caenorhabditis elegans homolog of rictor in a forward genetic screen for increased body fat. Despite high body fat, rictor mutants are developmentally delayed, small in body size, lay an attenuated brood, and are short-lived, indicating that Rictor plays a critical role in appropriately partitioning calories between long-term energy stores and vital organismal processes. Rictor is also necessary to maintain normal feeding on nutrient-rich food sources. In contrast to wild-type animals, which grow more rapidly on nutrient-rich bacterial strains, rictor mutants display even slower growth, a further reduced body size, decreased energy expenditure, and a dramatically extended life span, apparently through inappropriate, decreased consumption of nutrient-rich food. Rictor acts directly in the intestine to regulate fat mass and whole-animal growth. Further, the high-fat phenotype of rictor mutants is genetically dependent on akt-1, akt-2, and serum and glucocorticoid-induced kinase-1 (sgk-1). Alternatively, the life span, growth, and reproductive phenotypes of rictor mutants are mediated predominantly by sgk-1. These data indicate that Rictor/TORC2 is a nutrient-sensitive complex with outputs to AKT and SGK to modulate the assessment of food quality and signal to fat metabolism, growth, feeding behavior, reproduction, and life span.
We report on the observation of sex-restricted, non-Mendelian inheritance over a region of mouse ... more We report on the observation of sex-restricted, non-Mendelian inheritance over a region of mouse Chromosome (Chr) 11, occurring in the offspring of crosses between two commonly used Mus musculus-derived inbred strains, C57BL/6J and DBA/2J. In the surviving backcross progeny of reciprocal matings between (C57BL/6J × DBA/2J)F 1 hybrids and the C57BL/6J parental strain, we observed the preferential appearance of C57BL/6J alleles along a region of Chr 11. The deviation from Mendelian predictions was observed only in female offspring from both reciprocal backcrosses, and not in males from either cross. The sexspecificity of the observed non-Mendelian inheritance points to an explanation based on embryonic or neonatal lethality. Our data add to previously obtained evidence for a Chr 11 locus or loci with sex-specific and allele-specific effects on viability.
The Ddc1/Rad17/Mec3 complex and Rad24 are DNA damage checkpoint components with limited homology ... more The Ddc1/Rad17/Mec3 complex and Rad24 are DNA damage checkpoint components with limited homology to replication factors PCNA and RF-C, respectively, suggesting that these factors promote checkpoint activation by "sensing" DNA damage directly. Mec1 kinase, however, phosphorylates the checkpoint protein Ddc2 in response to damage in the absence of all other known checkpoint proteins, suggesting instead that Mec1 and/or Ddc2 may act as the initial sensors of DNA damage. In this paper, we show that Ddc1 or Ddc2 fused to GFP localizes to a single subnuclear focus following an endonucleolytic break. Other forms of damage result in a greater number of Ddc1-GFP or Ddc2-GFP foci, in correlation with the number of damage sites generated, indicating that Ddc1 and Ddc2 are both recruited to sites of DNA damage. Interestingly, Ddc2 localization is severely abrogated in mec1 cells but requires no other known checkpoint genes, whereas Ddc1 localization requires Rad17, Mec3, and Rad24, but not Mec1. Therefore, Ddc1 and Ddc2 recognize DNA damage by independent mechanisms. These data support a model in which assembly of multiple checkpoint complexes at DNA damage sites stimulates checkpoint activation. Further, we show that although Ddc1 remains strongly localized following checkpoint adaptation, many nuclei contain only dim foci of Ddc2-GFP, suggesting that Ddc2 localization may be down-regulated during resumption of cell division. Lastly, visualization of checkpoint proteins localized to damage sites serves as a useful tool for analysis of DNA damage in living cells.
Previous work on the DNA damage checkpoint in Saccharomyces cerevisiae has shown that two complex... more Previous work on the DNA damage checkpoint in Saccharomyces cerevisiae has shown that two complexes independently sense DNA lesions: the kinase Mec1-Ddc2 and the PCNA-like 9-1-1 complex. To test whether colocalization of these components is sufficient for checkpoint activation, we fused these checkpoint proteins to the LacI repressor and artificially colocalized these fusions by expressing them in cells harboring Lac operator arrays. We observed Rad53 and Rad9 phosphorylation, Sml1 degradation, and metaphase delay, demonstrating that colocalization of these sensors is sufficient to activate the checkpoint in the absence of DNA damage. Our tethering system allowed us to establish that CDK functions in the checkpoint pathway downstream of damage processing and checkpoint protein recruitment. This CDK dependence is likely, at least in part, through Rad9, since mutation of CDK consensus sites compromised its checkpoint function.
The nematode C. elegans is attracted to nutritious bacteria and is repelled by pathogens and toxi... more The nematode C. elegans is attracted to nutritious bacteria and is repelled by pathogens and toxins. Here we show that RNAi and toxin-mediated disruption of core cellular activities, including translation, respiration, and protein turnover, stimulate behavioral avoidance of normally attractive bacteria. RNAi of these and other essential processes induces expression of detoxification and innate immune effectors, even in the absence of toxins or pathogens. Disruption of core processes in non-neuronal tissues was sufficient to stimulate aversion behavior, revealing a neuroendocrine axis of control that additionally required serotonergic and Jnk kinase signaling pathways. We propose that surveillance pathways overseeing core cellular activities allow animals to detect invading pathogens that deploy toxins and virulence factors to undermine vital host functions. Variation in cellular surveillance and endocrine pathways controlling behavior, detoxification, and immunity selected by past toxin or microbial interactions could underlie aberrant responses to foods, medicines, and microbes.
<p>(A-D) Decreased TORC1 induces neuronal expression of HPK-1. Representative images of <... more <p>(A-D) Decreased TORC1 induces neuronal expression of HPK-1. Representative images of <i>Phpk-1</i>::<i>HPK-1</i>::<i>GFP</i> neuronal expression in day 3 adult animals after treatment with control (A), <i>daf-2</i> (B), <i>rict-1</i> (C) and <i>daf-15</i> RNAi (D). Outlines of animals are traced in white. White space was artificially filled for (D). Additional images and quantification can found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.s009" target="_blank">S9 Fig</a>. (E) Induction of endogenous <i>hpk-1</i> after <i>daf-15</i> inactivation as measured by qRT-PCR. Values are mean fold change and S.E.M. Three independent experiments were performed and normalized to <i>cdc-42</i>. * indicates a p-value <0.05 (Student’s t-test). (F, G) <i>hpk-1</i> is essential for increased lifespan of <i>daf-15(RNAi)</i> and <i>let-363(RNAi)</i>-treated animals, respectively. Tabulated lifespan is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.s012" target="_blank">S1 Table</a>.</p
<p>(A) <i>hpk-1(pk1393)</i> null mutant animals (red) are short-lived relative ... more <p>(A) <i>hpk-1(pk1393)</i> null mutant animals (red) are short-lived relative to wild-type N2 (black). <i>Phpk-1</i>::<i>hpk-1</i>::<i>GFP</i> largely restores wild-type lifespan to <i>hpk-1(pk1393)</i> mutant animals (green). Lifespan of non-transgenic <i>hpk-1(pk1393)</i> siblings (blue) is similar to <i>hpk-1(pk1393)</i>. (B) Overexpression of <i>hpk-1</i> using the heterologous <i>sur-5</i> promoter increases lifespan compared to N2 (dark red versus black). Each graph is representative of at least 3 independent lifespan experiments. Full lifespan data can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.s012" target="_blank">S1 Table</a>.</p
<p>(A) Overexpression of <i>hpk-1</i> increases lifespan (grey versus black) an... more <p>(A) Overexpression of <i>hpk-1</i> increases lifespan (grey versus black) and is <i>pha-4</i> dependent (red traces). (B) Overexpression of <i>hpk-1</i> increases lifespan dependent on <i>mxl-2</i> (blue traces). (C) Loss of <i>hlh-30</i> (green traces) partially suppresses the increased lifespan of <i>hpk-1</i>, consistent with parallel signaling or independence. (D) Loss of <i>nhr-62</i> (pink/purple traces) has a minimal negative effect on both normal and the increased lifespan conferred by <i>hpk-1</i> overexpression. In all cases, black traces are N2 and grey traces are <i>Psur-5</i>::<i>HPK-1</i>::<i>CFP</i> animals treated on control RNAi. For each panel, darker colored traces are respective RNAi-treatment of N2 animals and lighter colored traces are RNAi treatment of <i>Psur-5</i>::<i>HPK-1</i>::<i>CFP</i> animals. In some cases, experiments shown within this figure were performed simultaneously and split into multiple figures for readability. Full lifespan data can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.s012" target="_blank">S1 Table</a>.</p
<p>Model of HPK-1 functions in longevity control: HPK-1 functions as a central hub to maint... more <p>Model of HPK-1 functions in longevity control: HPK-1 functions as a central hub to maintain proteostasis by preventing sumoylation and inactivation of HSF-1 and by stimulating the expression of autophagy genes by <i>pha-4</i> and <i>mxl-2</i>. TORC1 inhibits <i>hpk-1</i> expression to limit the induction of autophagy genes under basal conditions. Under nutrient stress, TORC1 is inactivated resulting in increased <i>hpk-1</i> expression, which promotes autophagy gene expression through PHA-4/FoxA and MXL-2/Mlx. Thermal stress increases HPK-1 protein levels to reduce the threshold of activation of the heat shock response, and HPK-1 promotes longevity through modulation of HSF-1 activity under normal growth conditions.</p
<p>(A) <i>hpk-1</i> is necessary for the induction the autophagy genes <i>... more <p>(A) <i>hpk-1</i> is necessary for the induction the autophagy genes <i>atg-18</i> and <i>bec-1</i> (Beclin1) in response to inactivation of TORC1 by <i>daf-15(RNAi)</i> (** indicates p<0.01, Student’s t-test). (B) In contrast, decreased TORC1 signaling represses the expression of the translation initiation factor genes <i>ifg-1</i> and <i>iftb-1</i> independently from <i>hpk-1</i>. (C) Similarly, TORC1 inhibition mildly induces <i>hsp-16</i>.<i>2</i> and <i>hsp-70</i> independently from <i>hpk-1</i>. Columns labeled <i>hpk-1</i> indicate <i>hpk-1(pk1393)</i>. Expression levels are presented as fold change +/- S.D. normalized to <i>cdc-42</i> and averaged across four independent experiments.</p
<p>(A-C) <i>hpk-1</i> activity affects the accumulation of Q35::YFP foci in mus... more <p>(A-C) <i>hpk-1</i> activity affects the accumulation of Q35::YFP foci in muscle cells. Shown are representative images of <i>Punc-54</i>::<i>polyQ</i>::<i>YFP</i> animals treated with (A) control RNAi or (B) <i>hpk-1</i> RNAi, and (C) transgenic animals overexpressing <i>hpk-1</i> (<i>Psur-5</i>::<i>HPK-1</i>::<i>CFP</i>). (D) Time course of polyQ::YFP foci accumulation in conjunction with: treatment with control RNAi (black circles), <i>hpk-1</i> RNAi (white circles), <i>hpk-1(pk1393)</i> (white squares), or <i>hpk-1</i> overexpression (open triangles). Data points display the mean +/- standard deviation (S.D.) of at least 15 animals per biological replicate; at least 5 independent experiments were performed. P-values are provided in Results and in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.s014" target="_blank">S3 Table</a>. (E) Time course of paralysis of <i>Punc-54</i>::<i>polyQ</i>::<i>YFP</i> animals in conjunction with: treatment with control RNAi (black circles), <i>hpk-1</i> RNAi (white circles), <i>hpk-1(pk1393)</i> (white squares), or <i>hpk-1</i> overexpression (open triangles). Plotted data display the results for a single biological replicate. P-values and data from all trials are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.s014" target="_blank">S3 Table</a>.</p
<p>(A-D) DIC and GFP overlay for <i>Phsp-16</i>.<i>2</i>::<i>... more <p>(A-D) DIC and GFP overlay for <i>Phsp-16</i>.<i>2</i>::<i>GFP</i> worms on empty vector (A, B) or <i>smo-1(RNAi</i>) (C, D) with (+HS) and without (-HS) heat shock. Scale bar = 100μm. (E) Western blot for HSP-16.2, GFP and β-actin from <i>hsp-16</i>.<i>2p</i>::<i>GFP</i> worms grown on empty vector (EV) without heat shock (no HS) or with heat shock (EV), <i>GFP(RNAi)</i>, <i>hsf-1(RNAi)</i> or <i>smo-1(RNAi)</i>. Fold-increases for HSP-16.2/actin on <i>smo-1(RNAi)</i> relative to EV in three independent replicates were 2.4, 4.7, and 1.8.</p
<p>(A) Expression of HPK-1 protein (<i>Phpk-1</i>::<i>HPK-1</i>::&l... more <p>(A) Expression of HPK-1 protein (<i>Phpk-1</i>::<i>HPK-1</i>::<i>GFP</i>) under basal conditions is primarily restricted to neurons. Fluorescent intestinal speckles are non-specific gut granules[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.ref111" target="_blank">111</a>]. (B-D) Heat shock induces HPK-1 protein (<i>Phpk-1</i>::<i>HPK-1</i>::<i>GFP</i>) levels most strongly within hypodermal seam cells (indicated by arrows) independent of <i>hsf-1</i> (C) and transcription (D). <i>Phpk-1</i>::<i>HPK-1</i>::<i>GFP</i> animals after heat shock with (B) empty vector HT115, (C) <i>hsf-1(RNAi)</i>, and (D) α-amanitin treatment. Increased HPK-1 expression within neurons and hypodermal seam cells is specific to heat stress as neither oxidative stress (E) or UV damage (F) altered expression. White space was artificially filled for some images and animals are outlined. GFP quantification and further analysis can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.s006" target="_blank">S6 Fig</a>.</p
<p>(A-D) Representative images of at least 30 <i>Phsp-16</i>.<i>2</i&g... more <p>(A-D) Representative images of at least 30 <i>Phsp-16</i>.<i>2</i>::<i>GFP</i> animals in wild type (A, C) and <i>hpk-1(pk1393)</i> animals (B, D) under basal conditions (A, B) or after heat shock (C, D). Animals are outlined in panels A-B. (E-F) Induction of endogenous <i>hsp-16</i>.<i>2</i> (E) and <i>hsp-70</i> (C12C8.1) (F), in N2 and <i>hpk-1(pk1393)</i> animals as measured by qRT-PCR. (G) Loss of <i>hpk-1</i> did not alter endogenous expression of <i>hsp-16</i>.<i>2</i> (blue) or <i>hsp-70</i> (orange). Values in (E-G) are normalized to expression of <i>act-1</i> and the mean fold change relative to wild-type animals, and the S.E.M. between technical replicates is shown. In total three independent experiments were performed with similar results. P-values for (E) and (F) are <0.05 and <0.01, respectively (Student’s t-test).</p
<p>(A-B) Loss of <i>pha-4</i> (red traces) does not increase foci formation (A)... more <p>(A-B) Loss of <i>pha-4</i> (red traces) does not increase foci formation (A) or the onset of paralysis (B) in the absence of <i>hpk-1</i> (open circles/squares). (C-D) Loss of <i>mxl-2</i> (blue traces) does not increase foci formation (C) or the onset of paralysis (D) in the absence of <i>hpk-1</i> (open circles/squares). (E-F) Loss of <i>hlh-30</i> (green traces) does not increase foci formation (E) but delays the onset of paralysis in the absence of <i>hpk-1</i> (F) (open circles/squares). For foci formation, data are the mean and standard error of the mean (S.E.M.) of at least 15 animals from one representative trial; three independent experiments were performed. ***, **, and * indicate p-values of <0.001, <0.01, and <0.05, respectively. For paralysis, data is representative of one of two trials performed with the same conditions. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.s014" target="_blank">S3 Table</a> for additional details.</p
<p>(A-C) HPK-1 and HSF-1 colocalize in neurons under basal conditions. Representative image... more <p>(A-C) HPK-1 and HSF-1 colocalize in neurons under basal conditions. Representative image of transgenic animal co-expressing <i>Phpk-1</i>::<i>HPK-1</i>::<i>tdtomato</i>,<i>Phsf-1</i>::<i>HSF-1</i>::<i>GFP</i>: (A) red fluorescence, (B) green fluorescence, and (C) overlay.</p
<p>(A-F) Inactivation of <i>hpk-1</i> but not <i>hsf-1</i> disrupts... more <p>(A-F) Inactivation of <i>hpk-1</i> but not <i>hsf-1</i> disrupts autophagosome formation after bacterial deprivation (BD) as visualized by puncta formation for the autophagosomal reporter <i>Plgg-1</i>::<i>LGG-1</i>::<i>GFP</i> (Atg8p/MAP-LC3) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.ref018" target="_blank">18</a>]. (G) Quantification of LGG-1:GFP foci in L3 stage animals under <i>ad libitum</i> (AL) and bacterial deprivation (BD) conditions. BD was imposed by removal from bacterial food for 6 hours prior to scoring puncta formation. Plotted are the mean number of LGG-1::GFP puncta/seam cell visualized +/-S.D. *** indicates a p-value of <0.001 (Student’s t-test). Summary data provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.s015" target="_blank">S4 Table</a>.</p
Recent investigation of the DNA-damage checkpoint in several organisms has highlighted the conser... more Recent investigation of the DNA-damage checkpoint in several organisms has highlighted the conservation of this pathway. The checkpoint's signal transduction pathway consists of four conserved classes of molecules: two large protein kinases having homology to phosphatidylinositol 3-kinases, three 'sensor' proteins with homology to proliferating cell nuclear antigen, two serine/threonine (S/T) kinases, and two adaptors for the S/T kinases. This review compares the role of these four classes of checkpoint proteins in humans and model organisms.
An extensive proteostatic network comprised of molecular chaperones and protein clearance mechani... more An extensive proteostatic network comprised of molecular chaperones and protein clearance mechanisms functions collectively to preserve the integrity and resiliency of the proteome. The efficacy of this network deteriorates during aging, coinciding with many clinical manifestations, including protein aggregation diseases of the nervous system. A decline in proteostasis can be delayed through the activation of cytoprotective transcriptional responses, which are sensitive to environmental stress and internal metabolic and physiological cues. The homeodomain-interacting protein kinase (hipk) family members are conserved transcriptional co-factors that have been implicated in both genotoxic and metabolic stress responses from yeast to mammals. We demonstrate that constitutive expression of the sole Caenorhabditis elegans Hipk homolog, hpk-1, is sufficient to delay aging, preserve proteostasis, and promote stress resistance, while loss of hpk-1 is deleterious to these phenotypes. We show...
Rictor is a component of the target of rapamycin complex 2 (TORC2). While TORC2 has been implicat... more Rictor is a component of the target of rapamycin complex 2 (TORC2). While TORC2 has been implicated in insulin and other growth factor signaling pathways, the key inputs and outputs of this kinase complex remain unknown. We identified mutations in the Caenorhabditis elegans homolog of rictor in a forward genetic screen for increased body fat. Despite high body fat, rictor mutants are developmentally delayed, small in body size, lay an attenuated brood, and are short-lived, indicating that Rictor plays a critical role in appropriately partitioning calories between long-term energy stores and vital organismal processes. Rictor is also necessary to maintain normal feeding on nutrient-rich food sources. In contrast to wild-type animals, which grow more rapidly on nutrient-rich bacterial strains, rictor mutants display even slower growth, a further reduced body size, decreased energy expenditure, and a dramatically extended life span, apparently through inappropriate, decreased consumption of nutrient-rich food. Rictor acts directly in the intestine to regulate fat mass and whole-animal growth. Further, the high-fat phenotype of rictor mutants is genetically dependent on akt-1, akt-2, and serum and glucocorticoid-induced kinase-1 (sgk-1). Alternatively, the life span, growth, and reproductive phenotypes of rictor mutants are mediated predominantly by sgk-1. These data indicate that Rictor/TORC2 is a nutrient-sensitive complex with outputs to AKT and SGK to modulate the assessment of food quality and signal to fat metabolism, growth, feeding behavior, reproduction, and life span.
We report on the observation of sex-restricted, non-Mendelian inheritance over a region of mouse ... more We report on the observation of sex-restricted, non-Mendelian inheritance over a region of mouse Chromosome (Chr) 11, occurring in the offspring of crosses between two commonly used Mus musculus-derived inbred strains, C57BL/6J and DBA/2J. In the surviving backcross progeny of reciprocal matings between (C57BL/6J × DBA/2J)F 1 hybrids and the C57BL/6J parental strain, we observed the preferential appearance of C57BL/6J alleles along a region of Chr 11. The deviation from Mendelian predictions was observed only in female offspring from both reciprocal backcrosses, and not in males from either cross. The sexspecificity of the observed non-Mendelian inheritance points to an explanation based on embryonic or neonatal lethality. Our data add to previously obtained evidence for a Chr 11 locus or loci with sex-specific and allele-specific effects on viability.
Uploads
Papers by Justine Melo