Heterochromatic silencing and HP1 localization in Drosophila are dependent on the RNAi machinery

Genes normally resident in euchromatic domains are silenced when packaged into heterochromatin, as exemplified in Drosophila melanogaster by position effect variegation (PEV). Loss-of-function mutations resulting in suppression of PEV have identified critical components of heterochromatin, including proteins HP1, HP2, and histone H3 lysine 9 methyltransferase. Here, we demonstrate that this silencing is dependent on the RNA interference machinery, using tandem mini-white arrays and white transgenes in heterochromatin to show loss of silencing as a result of mutations in piwi, aubergine, or spindle-E (homeless), which encode RNAi components. These mutations result in reduction of H3 Lys9 methylation and delocalization of HP1 and HP2, most dramatically in spindle-E mutants

Drosophila Activated Cdc42 Kinase Has an Anti-Apoptotic Function

Activated Cdc42 kinases (Acks) are evolutionarily conserved non-receptor tyrosine kinases. Activating somatic mutations and increased ACK1 protein levels have been found in many types of human cancers and correlate with a poor prognosis. ACK1 is activated by epidermal growth factor (EGF) receptor signaling and functions to regulate EGF receptor turnover. ACK1 has additionally been found to propagate downstream signals through the phosphorylation of cancer relevant substrates. Using Drosophila as a model organism, we have determined that Drosophila Ack possesses potent anti-apoptotic activity that is dependent on Ack kinase activity and is further activated by EGF receptor/Ras signaling. Ack anti-apoptotic signaling does not function through enhancement of EGF stimulated MAP kinase signaling, suggesting that it must function through phosphorylation of some unknown effector. We isolated several putative Drosophila Ack interacting proteins, many being orthologs of previously identified human ACK1 interacting proteins. Two of these interacting proteins, Drk and yorkie, were found to influence Ack signaling. Drk is the Drosophila homolog of GRB2, which is required to couple ACK1 binding to receptor tyrosine kinases. Drk knockdown blocks Ack survival activity, suggesting that Ack localization is important for its pro-survival activity. Yorkie is a transcriptional co-activator that is downstream of the Salvador-Hippo-Warts pathway and promotes transcription of proliferative and anti-apoptotic genes. We find that yorkie and Ack synergistically interact to produce tissue overgrowth and that yorkie loss of function interferes with Ack anti-apoptotic signaling. Our results demonstrate how increased Ack signaling could contribute to cancer when coupled to proliferative signals

The Mitochondria-Regulated Immune Pathway Activated in the C. elegans Intestine Is Neuroprotective

Immunological mediators that originate outside the nervous system can affect neuronal health. However, their roles in neurodegeneration remain largely unknown. Here, we show that the p38MAPK-mediated immune pathway activated in intestinal cells of Caenorhabditis elegans upon mitochondrial dysfunction protects neurons in a cell-non-autonomous fashion. Specifically, mitochondrial complex I dysfunction induced by rotenone activates the p38MAPK/CREB/ATF-7-dependent innate immune response pathway in intestinal cells of C. elegans. Activation of p38MAPK in the gut is neuroprotective. Enhancing the p38MAPK-mediated immune pathway in intestinal cells alone suppresses rotenone-induced dopaminergic neuron loss, while downregulating it in the intestine exacerbates neurodegeneration. The p38MAPK/ATF-7 immune pathway modulates autophagy and requires autophagy and the PTEN-induced putative kinase PINK-1 for conferring neuroprotection. Thus, mitochondrial damage induces the clearance of mitochondria by the immune pathway, protecting the organism from the toxic effects of mitochondrial dysfunction. We propose that mitochondria are subject to constant surveillance by innate immune mechanisms

Neuronal Serotonin Release Triggers the Heat Shock Response in C. elegans in the Absence of Temperature Increase

SummaryBackgroundCellular mechanisms aimed at repairing protein damage and maintaining homeostasis, widely understood to be triggered by the damage itself, have recently been shown to be under cell nonautonomous control in the metazoan C. elegans. The heat shock response (HSR) is one such conserved mechanism, activated by cells upon exposure to proteotoxic conditions such as heat. Previously, we had shown that this conserved cytoprotective response is regulated by the thermosensory neuronal circuitry of C. elegans. Here, we investigate the mechanisms and physiological relevance of neuronal control.ResultsBy combining optogenetic methods with live visualization of the dynamics of the heat shock transcription factor (HSF1), we show that excitation of the AFD thermosensory neurons is sufficient to activate HSF1 in another cell, even in the absence of temperature increase. Excitation of the AFD thermosensory neurons enhances serotonin release. Serotonin release elicited by direct optogenetic stimulation of serotonergic neurons activates HSF1 and upregulates molecular chaperones through the metabotropic serotonin receptor SER-1. Consequently, excitation of serotonergic neurons alone can suppress protein misfolding in C. elegans peripheral tissue.ConclusionsThese studies imply that thermosensory activity coupled to serotonergic signaling is sufficient to activate the protective HSR prior to frank proteotoxic damage. The ability of neurosensory release of serotonin to control cellular stress responses and activate HSF1 has powerful implications for the treatment of protein conformation diseases

Ack and Yorkie genetically interact.

<p>The eye size assay was employed to assess the involvement of Yorkie (yki) in Ack suppression of apoptosis. Genotypes are indicated on each panel. (A) <i>GMR-Gal4</i> control eye. (B) Ack overexpression does not modify <i>GMR-Gal4</i> roughness, but does result in a slightly increased eye size. (C) Yki overexpression results in a disorganized eye that is overgrown. (D) Ack and yki overexpression yields a massively overgrown eye. (E) Hid small eye control phenotype. (F) Ack overexpression suppresses hid induced PCD. The eye is outlined with a dashed oval that has been reproduced in panels G and J–L to aid in size comparisons. (G) Yki overexpression in the hid background not only results in hid PCD suppression, but also shows a reduction in pigmentation. (H) Yki and Ack overexpression in a hid background results in further PCD suppression than either transgene alone, but still shows loss of pigmentation. (I) <i>Yki</i> single copy loss results in an eye that is a similar size as the hid control phenotype, shown by a dotted oval. (J) Single copy loss of <i>yki</i> reduces the ability of Ack to suppress hid induced PCD. (K) Single copy loss of <i>Ack</i> does not modify yki suppression of PCD. (L) Knockdown of yki by RNAi further interferes with the ability of Ack to suppress hid induced apoptosis compared to <i>yki</i> single copy loss. (M) Exogenous expression of human YAP^S94A/S127A does not affect eye size. (N) Combined expression of YAP^S94A/S127A and Ack results in an increased eye size.</p

Drk is required for Ack anti-apoptotic activity.

<p>The influence of Drk loss of function on PCD was assessed using the eye size assay. Genotypes are indicated in each panel. Hid (A) and hid^Ala5 (B) backgrounds were combined with RNAi mediated knockdown of Drk (D and E). Hid and hid^Ala5 unmodified eye sizes have been outlined with dotted and dashed ovals that have been reproduced in panels D and E respectively. Drk knockdown in the hid (D) or hid^Ala5 (E) backgrounds results in no reduction in eye size but a loss of pigmentation, which is more pronounced with hid^Ala5. (C and F) The requirement of Drk for Ack anti-apoptotic function is assessed. (C) Hid^Ala5, Ack overexpression baseline eye. (F) RNAi mediated knockdown of Drk inhibits Ack's ability to suppress programmed cell death. The dashed oval from panels B and E is reproduced here to show that the eye size is similar to the hid^Ala5 baseline. (G and H) Western analysis of Drk immunoprecipitation. (G) Drk was immunoprecipitated with anti-Drk 12201 antiserum and blotted with Ack (top panel) and Drk (bottom panel) antibodies. Ack co-precipitates with Drk from extracts prepared from S2 cells expressing endogenous Ack (S2 lane) as well as Ack overexpressing S2 cells (Ack OE lane). Ack knockdown by RNAi eliminates the immunoreactive band, confirming that it is Ack (Ack RNAi lane). (H) Drk was immunoprecipitated from Ack overexpressing S2 cells using two different Drk antisera, 12201 (01) and 12203 (03), and blotted with an anti-phosphotyrosine antibody (4G10). An immunoreactive band corresponding to phospho-Ack is indicated (P-Ack). No immunoreactivity is detected where Drk is predicted to run (P-Drk).</p

Ack is required for mature sperm production.

<p>(A) A schematic representation of Ack family members is shown. Ack family members can be grouped into three categories depending on their domain structure. All family members contain an N-terminal tyrosine kinase domain flanked by sterile alpha motif (SAM) and Src homology 3 (SH3) domains. The C-terminal half of these proteins also contains multiple proline-rich motifs. <i>Drosophila</i> Ack and human TNK1 do not contain a Cdc42/Rac interactive binding (CRIB) domain. All Ack family members except for Ark-1 in <i>C. elegans</i> contain an ubiquitin binding associated domain (UBA). (B–G) drICE activity (anti-cleaved caspase-3, green), actin rich investment cones (phalloidin, red) and DNA (DAPI, blue) are visualized in wild type (Oregon R) and Ack null mutant backgrounds (<i>Ack⁸⁶/Ack⁸⁶</i>) during sperm cell individualization. (B and C) The rostral or coiled region of the testis containing compacted nuclei is shown. The phalloidin staining individualization complexes (*) in wild type testis travel along the outer edge of the testis coil (B), while in Ack mutant testis they are positioned on the inside of the coil (C). (D–E) Higher magnification of individualization complexes assembling on condensed DNA (arrowheads) or beginning to migrate (arrows). (F–G) Higher magnification of more caudal migrating individualization complexes. (H–I) Visualization of dj-GFP fluorescence (green) shows the presence of elongated spermatids in the testes (arrows) and mature sperm in the seminal vesicle (arrowheads) of Ack⁸⁶ hetrozygous males (H). (I) Elongated spermatids are present in the testes of Ack⁸⁶ homozygous males (arrows) but no mature sperm are present in the seminal vesicle (arrowheads).</p

Ack expression produces fewer TUNEL positive cells.

<p>Proliferation and apoptosis in <i>Drosophila</i> third instar eye discs (posterior is up) were analyzed by BrdU incorporation (red) and TUNEL (green), respectively. Genotypes are indicated in each panel; the morphogenetic furrow (MF) is labeled in panels A and B. (A) <i>GMR-Gal4</i> eye discs have TUNEL positive cells posterior to the second mitotic wave (SMW). (B) Hid expression results in TUNEL staining posterior to the SMW as well as in the posterior region of the eye disc within the zone of compensatory proliferation (ZCP). (C) Ack overexpression results in fewer TUNEL positive cells and no observable change in BrdU incorporation patterns. (D) TUNEL staining is also decreased when Ack is expressed in a hid background. The Ack mediated decrease in apoptosis also decreases the number of BrdU positive cells in the location of the ZCP as expected. (E and F) Ack kinase dead mutant shows an enhancement of cell death post furrow. (G) Quantification of TUNEL assay from 4 different eye discs per genotype. P-values using a paired t-test verifies that Ack expression is statistically significant compared to control (* = 0.004, # = 0.0393), whereas, Ack kinase inactive mutant is not statistically significant (* = 0.8007, # = 0.765)(GraphPad Software, QuickCalcs online).</p

Ack expression genetically interacts with <i>argos</i> and <i>Gap1</i> loss of function.

<p>Genotypes are shown in each panel. (A–G) 42% pupa retina dissections stained with anti-armadillo and false colored to indicate primary (blue) and secondary or tertiary (red) pigment cells. (A) Oregon R, wildtype phenotype contains nine interommatidial pigment cells and three bristle cells per ommatidial unit. (B) Overexpression of Ack produces a duplication of bristles and slight disorganization; however, there is still an average of 9 pigment cells present surrounding each core unit. (C) Expression of kinase inactive Ack results in fewer but larger pigment cells. (D–E) <i>Gap1</i> single copy loss (D) and <i>argos</i> single copy loss (E) do not affect eye patterning. (F–G) Single copy loss of <i>argos</i> (F) or <i>Gap1</i> (G) in combination with Ack overexpression results in increased interommatidial pigment cells. (H) Quantification of pigment cell number from all dissections. P-values using a paired t-test verify that the increased number of pigment cells when Ack is combined with modifiers of the EGF receptor pathway is statistically significant compared to control (* = 0.0001). Loss of pigment cells in the Ack kinase inactive mutant is also statistically significant compared to the Oregon R control (⧫ = 0.0001)(GraphPad Software, QuickCalcs online).</p

Top ten Ack interacting proteins based on their Mascot score.

a<p>Mascot score = −10log(P) where P is the probability that the match is incorrect.</p