Caenorhabditis elegans. Reactive oxygen species (ROS) generated from dysfunctional mitochondria are required for GCN-2–dependent eIF2α phosphorylation but not ATFS-1 activation. Simultaneous deletion of ATFS-1 and GCN-2 compounds the developmental defects associated with mitochondrial stress, while stressed animals lacking GCN-2 display a greater dependence on ATFS-1 and stronger induction of mitochondrial chaperone genes. These findings are consistent with translational control and stress-dependent chaperone induction acting in complementary arms of the UPRmt.Cells respond to defects in mitochondrial function by activating signaling pathways that restore homeostasis. The mitochondrial peptide exporter HAF-1 and the bZip transcription factor ATFS-1 represent one stress response pathway that regulates the transcription of mitochondrial chaperone genes during mitochondrial dysfunction. Here, we report that GCN-2, an eIF2α kinase that modulates cytosolic protein synthesis, functions in a complementary pathway to that of HAF-1 and ATFS-1. During mitochondrial dysfunction, GCN-2–dependent eIF2α phosphorylation is required for development as well as the lifespan extension observed in  Defects in mitochondrial function are associated with numerous age-related diseases including cancer and Parkinson's. Mitochondrial function relies upon maintenance of the mitochondrial proteome, which is comprised of nuclear and mitochondrial-encoded proteins. Nuclear-encoded polypeptides are translated in the cytosol and must be transported into the mitochondrial matrix, where resident chaperones facilitate folding into their functional conformation. In order to protect against dysfunction arising from an accumulation of misfolded or unfolded mitochondrial proteins, cells employ mechanisms to maintain the folding environment. One such signaling pathway is mediated by the bZip transcription factor ATFS-1, which upregulates mitochondrial chaperones to accommodate an overwhelming misfolded protein load. Here, we describe a complementary pathway that couples the mitochondrial functional status with the rate of cytosolic protein synthesis to protect the organelle from incoming unfolded protein substrates during mitochondrial stress. This pathway is regulated by the cytosolic kinase GCN-2, which phosphorylates the translation initiation factor 2α (eIF2α) subunit to slow general translation. GCN-2 responds to ROS emitted from dysfunctional mitochondria to promote growth and extend lifespan during mitochondrial stress. Mitochondrial dysfunction and altered protein homeostasis are associated with numerous developmental and age-related diseases as well as the general process of aging ER) that combines the upregulation of molecular chaperones to accommodate the folding requirements in the organelle with a reduction of cytosolic translation and ER protein import ER, the transcription factor XBP-1 is activated and mediates the induction of ER-resident chaperones Dysfunction and accumulation of misfolded proteins in the ER triggers a multi-pronged unfolded protein response (UPRmt), couples the status of the mitochondrial protein-folding environment to the transcription of mitochondrial chaperone genes mt and upregulation of mitochondrial chaperone genes to re-establish homeostasis Maintenance of mitochondrial metabolic function depends on the efficient assembly of the mitochondrial proteome, which is comprised of nuclear-encoded as well as mitochondrial-encoded polypeptides C. elegans have identified several proteins required for signaling the response including the mitochondrial inner membrane-localized peptide transporter HAF-1 and the bZip transcription factor ZC376.7 Activating Transcription Factor associated with Stress-1). Mitochondrial dysfunction triggers the HAF-1-dependent nuclear accumulation of ATFS-1, resulting in the upregulation of mitochondrial chaperone genes including HSP-60 and mtHsp70. Activation of this pathway occurs in response to elevated levels of mitochondrial stress, which can be the result of accumulation of unfolded proteins beyond the capacity of mitochondrial molecular chaperones ER, responds to diverse insults to mitochondrial function.Our previous genetic studies in ER also mediates the attenuation of cytosolic translation to protect the ER during stress. Similarly, inhibition of cytosolic translation has been suggested to promote mitochondrial function in yeast and Drosophila models of mitochondrial stress, although a potential regulatory mechanism(s) remained to be elucidated C. elegans genetic manipulations that reduce cytosolic translation rates provide resistance to numerous stresses including heat shock and also extend lifespan In addition to chaperone induction, the UPRC. elegans, translation attenuation via GCN-2-dependent eIF2α phosphorylation acts in a responsive and adaptive protective pathway during mitochondrial stress to promote mitochondrial function. Phosphorylation levels of eIF2α are increased during mitochondrial stress, which requires ROS generated from dysfunctional mitochondria. Our data demonstrate that GCN-2-dependent translational control acts in a mitochondrial protective signaling pathway complementary to the regulation of mitochondrial chaperone gene expression mediated by HAF-1 and ATFS-1.Phosphorylation of eIF2α by four dedicated kinases  serves to attenuate cytosolic translation in response to a variety of cellular stresses including starvation, oxidative stress, viral infection and unfolded protein stress in the ER hsp-60 promoter regulates expression of GFP (pr::gfphsp-60) atfs-1(tm4525) deletion strain which lacks 432 base pairs and most of exons 2–4, and crossed it into the reporter strain. Unlike wild-type worms, atfs-1(tm4525) animals were unable to induce pr::gfphsp-60 when raised on spg-7(RNAi), a mitochondrial protease required for ETC quality control and mitochondrial ribosome biogenesis We have previously described a mitochondrial stress response pathway that upregulates mitochondrial chaperone genes in response to multiple perturbations in mitochondrial function pr::gfphsp-60 activation in strains harboring the well-characterized clk-1(qm30) or isp-1(qm150) mutations clk-1 encodes a mitochondrial protein required for ubiquinone synthesis isp-1 encodes an iron-sulfur component of complex III in the ETC. As both mutations affect respiration and display impaired development mt. Indeed, pr::gfphsp-60 expression was consistently elevated in both strains consistent with the presence of mitochondrial stress. The isp-1(qm150) mutation caused considerably stronger pr::gfphsp-60 induction suggestive of a larger impact on mitochondrial function atfs-1(RNAi) were unable to induce expression of pr::gfphsp-60 (data not shown).We next investigated ATFS-1-dependent atfs-1(RNAi) on the development of wild-type and mitochondrial stressed worms. As previously demonstrated, both clk-1(qm30) and isp-1(qm150) worms developed considerably slower than wild-type animals atfs-1(RNAi) developed at similar rates to wild-type animals (data not shown). However, feeding clk-1(qm30) and isp-1(qm150) worms atfs-1(RNAi) dramatically impaired their developmental rates  strain was chosen for the RNAi screen as it displayed mild pr::gfphsp-60 induction, potentially allowing for the identification of candidates whose knockdown by RNAi either decreased or further increased pr::gfphsp-60 expression  background.In addition to ATFS-1-regulated mitochondrial chaperone expression, we sought to identify additional components that promote mitochondrial protein homeostasis by acting in complementary pathways. To identify signaling pathways that act in parallel to ATFS-1 we generated and screened an RNAi sub-library consisting of all pression . We hypopr::gfphsp-60 expression in the clk-1(qm30) background (isp-1(qm150) strain . These findings are consistent with previous experiments demonstrating that translation attenuation is protective against mitochondrial stress in yeast and DrosophilaC. elegans. For example, knockdown of the C. elegans target of rapamycin ortholog (TOR), CeTor, which regulates mRNA translation in response to nutrient cues rsks-1, the ribosomal S6 kinase, slows development and extends lifespan in C. elegansclk-1(qm30) mitochondrial mutants have increased levels of mitochondrial stress and the stress responsive pr::gfphsp-60 reporter was specifically reduced by CeTor and rsks-1(RNAi), these findings support the hypothesis that reduced translation is beneficial to mitochondrial protein homeostasis. However, because the TOR-signaling pathway impacts many biological processes in addition to translation, other possibilities exist. Because all components identified in our RNAi screen affect protein synthesis, we sought to further characterize the role of translation attenuation in maintaining the mitochondrial protein-folding environment.Interestingly, RNAi of several kinases required for protein synthesis reduced ckground , which w) strain , suggestCeTor, rsks-1 and cel-1, we identified components that are known to regulate translation initiation by modulating the phosphorylation status of the translation initiation factor eIF2α. RNAi-knockdown of the eIF2α kinase General Control Non-derepressible-2 (GCN-2) further increased pr::gfphsp-60 expression in clk-1(qm30) animals, suggesting a role for GCN-2 in promoting mitochondrial protein homeostasis or function (gcn-2(RNAi) on pr::gfphsp-60 expression was not due to direct effects on GFP translation as gcn-2(RNAi) did not cause induction of the ER stress reporter pr::gfphsp-4 (gcn-2(RNAi) did not effect pr::gfphsp-60 expression, suggesting its primary role is during stress (gcn-2(RNAi), our RNAi screen identified gsp-1(RNAi), which reduced pr::gfphsp-60 expression in both the clk-1(qm30) and isp-1(qm150) strains ), the level of steady-state phospho-eIF2α was reduced relative to wild-type worms . Consis elegans  [35], [3 elegans . Furtherpe worms . In C. egcn-2(ok871) or pek-1(zcdf2) deletion strains fed gsp-1(RNAi) there was still an increase in steady state levels of eIF2α phosphorylation likely reflecting the ability of both kinases to constitutively phosphorylate eIF2α in the absence of exogenous stress (gsp-1(RNAi) reduces pr::gfphsp-60 induction through attenuation of cytosolic translation, thus reducing the load on the mitochondrial protein folding machinery similar to eIF2α phosphorylation and translation attenuation in the UPRERIn contrast to inhibition of GCN-2 and PEK-1, GSP-1 knockdown resulted in increased levels of phospho-eIF2α consistent with it acting as a constitutive eIF2α phosphatase . In eiths stress . As incrclk-1(qm30)mutant, which was absent in the gcn-2(ok871) mutant strain (gsp-1(RNAi) caused a further increase in phospho-eIF2α levels (isp-1(qm150) mutant, supporting the role of GCN-2 in eIF2α phosphorylation in response to stress (gcn-2(RNAi) perturbs the mitochondrial protein folding environment and GSP-1 knockdown promotes mitochondrial protein homeostasis as indicated by reduced pr::gfphsp-60 expression . Furthermore, the increase in eIF2α phosphorylation observed in the clk-1(qm30) animals was not dependent on pek-1 indicating GCN-2 is the primary eIF2α kinase involved in maintaining mitochondrial protein homeostasis  displayed a much stronger induction of the UPRER reporter upon heat exposure consistent with PEK-1 activity protecting ER protein homeostasis pek-1(RNAi), gcn-2(RNAi) had no impact on pr::gfphsp-4 during heat stress. These results indicate that the effect of gcn-2(RNAi) on pr::gfphsp-60 induction was not due to dysregulation of global translation further supporting a mitochondrial stress-specific role for GCN-2.It should be noted that deletion or knockdown of the other eostasis . In pek- reduced , however reduced . A similpression . Howevergcn-2 deletion or RNAi had no observable effect on worm development in the absence of stress (isp-1(qm150) or clk-1(qm30) mutations, gcn-2 deletion significantly slowed development (spg-7(RNAi) also significantly delayed development of gcn-2(ok871) worms relative to wild-type worms  deletion having no effect on worm development (clk-1(qm30) worms displayed a slight reduction in oxygen consumption when compared to wild-type worms consistent with mild mitochondrial dysfunction (clk-1(qm30) worms lacking gcn-2 had a much lower rate of oxygen consumption than worms harboring either the gcn-2-deletion or clk-1(qm30) alone  alone , supportclk-1(qm30) mutation causing mitochondrial dysfunction, significantly more carbonylated material was detected in lysates from clk-1(qm30) worms than lysates from wild-type worms (clk-1(qm30);gcn-2(ok871) worms displayed even more oxidative damage than worms harboring clk-1(qm30) alone. Because oxidative damage can perturb protein folding, these data support a role for GCN-2 in protecting the folding environment as well as mitochondrial function.Elevated ROS produced by dysfunctional mitochondria can damage proteins through the formation of irreversible carobonyl modifications on lysine, cysteine, proline and threonine residues pe worms . clk-1(qmyo-3). High-level expression of mitochondria-targeted GFP challenges the organelle's protein folding environment by increasing the load of unfolded proteins pr::gfpmtmyo-3 worms raised on gcn-2(RNAi) displayed severely perturbed mitochondrial morphology consistent with a loss of protein homeostasis and mitochondrial function To further assess the contribution of GCN-2 in maintaining mitochondrial protein homeostasis we targeted GFP to the mitochondrial matrix via the strong muscle-specific myosin promoter (clk-1(qm30) and isp-1(qm150) animals, which activate ATFS-1-dependent pr::gfphsp-60 expression and GCN-2-dependent eIF2α phosphorylation, are among the numerous C. elegans mitochondrial mutants that exhibit lifespan extension ubl-5, a small ubiquitin-like protein required for UPRmt signaling ubl-5 prevented pr::gfphsp-60 induction in the long-lived clk-1(qm30) worms (atfs-1(RNAi).0) worms  similar clk-1(qm30) animals reduced their lifespan to that of wild-type worms (gcn-2(RNAi) was not generally toxic, as it did not affect lifespan or development in the absence of stress (gcn-2(RNAi) also shortened the lifespan of isp-1(qm150) animals, but because the animals were very sick with considerable developmental defects, we were unable to determine a role for GCN-2 in longevity of these animals (data not shown). These data are consistent with GCN-2 and increased eIF2α phosphorylation contributing to the lifespan extension observed in mitochondrial mutants and further emphasizes the importance of protein homeostasis in aging.Additionally, cytosolic translation attenuation also contributes to longevity in several animal models pe worms  consistef stress . gcn-2(Rgsp-1(RNAi) caused an increase in eIF2α phosphorylation in the absence or presence of mitochondrial stress (gsp-1(RNAi) would promote lifespan extension. However, the lifespan of wild-type or clk-1(qm30) worms on gsp-1(RNAi) were severely shortened (gsp-1(RNAi) also prevents C. elegans germline formation (data not shown) and is required for a variety of cellular processes including mitosis Because l stress , we hypohortened . Interprclk-1(qm30) and isp-1(qm150) worms produce increased levels of ROS  animals with the ROS scavenger ascorbate resulted in increased pr::gfphsp-60 activation, similar to gcn-2(RNAi)  as observed with gcn-2(RNAi) (clk-1(qm30) and isp-1(qm150) animals. Ascorbate treatment, like GCN-2 inhibition, caused a reduction of eIF2α phosphorylation in both mutants supporting a role for ROS in GCN-2 signaling during mitochondrial stress  . Ascorba-2(RNAi) . We nextl stress .clk-1(qm30) and isp-1(qm150) mutants. In addition to these two mutants which generate ROS, the herbicide paraquat is known to generate excessive ROS and extend C. elegans lifespan clk-1(qm30) and isp-1(qm150) mutants, exposure of wild-type worms to paraquat increased eIF2α phosphorylation in a GCN-2-dependent manner  animals lacking HAF-1 or ATFS-1. clk-1(qm30) animals displayed an increase in eIF2α phosphorylation which was further increased in combination with the haf-1(ok705) deletion or when fed atfs-1(RNAi)  , consistgcn-2(ok871);atfs-1(tm4525) animals or the gcn-2(ok871) strain fed atfs-1(RNAi) developed somewhat slower in the absence of exogenous stress  or the clk-1(qm30) mutation, development of worms lacking gcn-2 and atfs-1 was severely compromised. When raised on spg-7(RNAi), most worms arrested at the L1 or L2 larval stage and no animals were able to reach adulthood (clk-1(qm30) (isp-1(qm150) (data not shown) animals lacking both GCN-2 and ATFS-1 developed more slowly than worms lacking either individual gene. No synthetic interactions were observed between ATFS-1 and PEK-1 as atfs-1(tm4525) animals raised on pek-1(RNAi) developed at similar rates to atfs-1(tm4525) animals raised on a control RNAi in the absence or presence of stress (data not shown). Despite the developmental defect observed in animals lacking both ATFS-1 and GCN-2 (In the presence of stress caused by dulthood . Further-1(qm30)  or isp-1nd GCN-2 , animalsnd GCN-2  suggestind GCN-2 .gcn-2(RNAi) or deletion inhibited eIF2α phosphorylation during mitochondrial stress. These results, along with recent experiments in yeast and flies The experiments described here implicate the eIF2α kinase GCN-2 in the maintenance of mitochondrial function and protein homeostasis. Development of worms lacking GCN-2 was impaired in the presence of mitochondrial stress which caused further induction of ATFS-1-dependent mitochondrial chaperone genes consistent with perturbed mitochondrial protein homeostasis. Furthermore, simultaneous deletion or knockdown of GCN-2 and ATFS-1 has a negative synergistic effect on animal development suggesting that GCN-2-dependent translation attenuation and ATFS-1-dependent mitochondrial chaperone gene induction act in parallel pathways to maintain mitochondrial protein homeostasis. Additionally, GCN-2 was required for development and lifespan extension in the presence of mitochondrial stress suggesting it is responsive to and protective against mitochondrial dysfunction. gcn-2(RNAi) further exacerbating mitochondrial chaperone induction in the presence of stress (clk-1(qm30) and isp-1(1qm150) mutant worms, suggesting that ROS could participate in GCN-2 activation either through a direct interaction with the GCN-2 tRNA synthetase domain or through effects on amino acid metabolism Our results demonstrate that ROS generated from stressed or dysfunctional mitochondria f stress  suggestiAttenuation of cytosolic translation slows mitochondrial import, thus reducing the folding load on mitochondrial chaperones. However, continued translation of proteins encoded by the mitochondrial genome could become detrimental when the expression of cytosolic components required for ETC complex formation is reduced. Interestingly, mitochondrial translation is tightly linked to the accumulation of imported ETC subunits and complex assembly. In their absence, mitochondrial translation is also attenuated ER, which regulates expression of ER chaperones and additional protein handling machinery Protection of mitochondrial protein homeostasis and function appears to be a novel role for GCN-2 in addition to its established role during starvation clk-1(qm30) animals (clk-1(qm30) animals display elevated levels of ROS that have also been shown to contribute to longevity C. elegansIn addition to a protective role during development, GCN-2 also contributes to the lifespan extension of  animals . These m animals , consistS. cerevisiaeC. elegans, and this avenue was not further pursued. Our RNAi screen identified components that when knocked down slow cytosolic translation as suppressors of pr::gfphsp-60 activation in stressed animals supporting a role for translation attenuation in promoting mitochondrial protein homeostasis.An additional eIF2α-dependent protective activity not addressed here involves the preferential translation of mRNAs with small upstream open reading frames (uORFs). A number of uORF containing transcripts have been identified in Our finding that GCN-2-dependent eIF2α phosphorylation protects mitochondrial protein homeostasis raises the possibility that manipulation of phospho-eIF2α status may be a therapeutic entry point for the diverse number of degenerative diseases associated with mitochondrial dysfunction pr::gfp(zcIs9)Vhsp-60, pr::gfpmt(zcIs14)myo-3 and pr::gfp(zcIs4)Vhsp-4 have been described previously pr::gfp(zcIs9)Vhsp-60 transgene was crossed into individual mutant strains of interest, with the exception of atfs-1(tm4525)V, which was backcrossed with N2 three times prior to crossing into the pr::gfphsp-60 background. The clk-1(qm30), isp-1(qm150) and gcn-2(ok871) strains were obtained from the Caenorhabditis Genetics Center  and the atfs-1(tm4525) strain was obtained from the National BioResource Project . RNAi feeding experiments were performed as described Reporter strains Worms were synchronized via bleaching and allowed to develop on the described RNAi plate or condition. For development in the presence of oxidative stress, rotenone was applied to vector(RNAi) plates and allowed to soak in prior to seeding eggs. At the time points indicated, the numbers of L1, L2, L3, L4, young adult (non-gravid) or gravid adult worms were counted on each plate and quantified as a percent of the total number of animals. For each plate, the worms in 6–8 individual fields of view were counted, and the total number combined.For lifespan analysis, worms were synchronized as eggs and allowed to develop under the described condition for two days. At that point, 100 L4 animals were transferred to new RNAi plates and subsequently transferred to fresh plates every day for the next 5–6 days and every two days thereafter. The numbers of dead and censored worms were counted every second day for the duration of the assay clk-1(qm30) animals.Worms were grown under the described conditions and collected at the L4 stage for analysis as previously described For the eIF2α dephosphorylation assay, 150 µg of worm lysate was treated with calf intestinal phosphatase (CIP) for 30 minutes at 30°C prior to SDS-PAGE analysis. For ascorbate treatment, synchronized worms were grown to adulthood in liquid medium, when 25 mM ascorbate was added for 16 hours prior to western analysis.eif2α using iQ sybr green supermix and MyiQ2 Two-Color Real-Time PCR Detection System (Bio-Rad). Actin was used as a control. Fold changes in gene expression were calculated using the comparative CtΔΔCt method.Total RNA was isolated using RNA STAT . RNA samples were prepared from the described worms at the L4 stage. cDNA was then synthesized from total RNA using a iScript cDNA Synthesis Kit . Following mRNA isolation and cDNA synthesis, qPCR was used to determine the expression level of Fluorescent photomicrographs were obtained using a Zeiss AxioCam MRm mounted on a Zeiss Imager.Z2 microscope or Zeiss M2 Bio stereo microscope .clk-1(qm30) and clk-1(qm30);gcn-2(ok871) worms were harvested once they reached the L4 stage. Worm lysates were separated by SDS-Page and treated according to the Oxyblot manufacturer .Oxygen consumption assays were performed as described Figure S1isp-1(qm150) worms fed vector(RNAi) or atfs-1(RNAi) Animals were synchronized as in Photomicrographs of (TIF)Click here for additional data file.Figure S2gcn-2(RNAi), CeTor(RNAi) and gsp-1(RNAi) effect pr::gfphsp-60 expression in isp-1(qm150) worms similar to clk-1(qm30) worms. (A) Photomicrographs of pr::gfpisp-1(qm150);hsp-60 worms raised on vector, gcn-2 or CeTor(RNAi). (B) Photomicrographs of pr::gfpisp-1(qm150);hsp-60 worms raised on vector or gsp-1(RNAi).(TIF)Click here for additional data file.Figure S3gcn-2 knockdown is specific for mitochondrial dysfunction and does not effect eif2α mRNA levels. (A) Phase contrast images of a pr::gfphsp-4 transgenic animals raised at 20°C in top panels of eif2α mRNA in wild-type, gcn-2(ok871) and gcn-2(ok871);pek-1(zcdf2) worms raised on vector(RNAi). Displayed is the mean +/− SEM, n = 3. In support of eIF2α protein levels in (TIF)Click here for additional data file.Figure S4gcn-2(ok871) worms develop at similar rates in the absence of stress. Quantification of developmental rates of wild-type and gcn-2(ok871) animals. Synchronized worms were raised from eggs and animals of different developmental stages were scored and plotted as percent of total animals on day 3.Wild-type and (TIF)Click here for additional data file.Figure S5ubl-5(RNAi) inhibits pr::gfphsp-60 expression in clk-1(qm30) worms. Fluorescent photomicrographs of pr::gfpclk-1(qm30);hsp-60 worms raised on vector or ubl-5(RNAi).(TIF)Click here for additional data file.Figure S6gsp-1(RNAi) dramatically shortens the lifespan of unstressed or stressed worms. Wild-type, clk-1(qm30) or isp-1(qm150) worms were synchronized on vector or gsp-1(RNAi). The number of worms alive was recorded on day 9 after hatching.(TIF)Click here for additional data file.Figure S7atfs-1(tm4525) and atfs-1(tm4525);gcn-2(ok871) raised on vector or spg-7(RNAi) as described in clk-1(qm30) and clk-1(qm30);gcn-2(ok871) animals raised on vector(RNAi) or atfs-1(RNAi). Synchronized worms were raised from eggs on the described RNAi plate and imaged on day 8.GCN-2 and ATFS-1 act in parallel to protect mitochondria during stress. (A) Photomicrographs of (TIF)Click here for additional data file.


Caenorhabditis elegans axon pathfinding, yet UNC-73 does not control CED-10/Rac downstream of UNC-6/Netrin in attractive axon guidance. Here we show that C. elegans TIAM-1 is a Rac-specific GEF that links CDC-42 and Rac signaling in lamellipodia and filopodia formation downstream of UNC-40/DCC. We also show that TIAM-1 acts with UNC-40/DCC in axon guidance. Our results indicate that a CDC-42/TIAM-1/Rac GTPase signaling pathway drives lamellipodia and filopodia formation downstream of the UNC-40/DCC guidance receptor, a novel set of interactions between these molecules. Furthermore, we show that TIAM-1 acts with UNC-40/DCC in axon guidance, suggesting that TIAM-1 might regulate growth cone protrusion via Rac GTPases in response to UNC-40/DCC. Our results also suggest that Rac GTPase activity is controlled by different GEFs in distinct axon guidance contexts, explaining how Rac GTPases can specifically control multiple cellular functions.The mechanisms linking guidance receptors to cytoskeletal dynamics in the growth cone during axon extension remain mysterious. The Rho-family GTPases Rac and CDC-42 are key regulators of growth cone lamellipodia and filopodia formation, yet little is understood about how these molecules interact in growth cone outgrowth or how the activities of these molecules are regulated in distinct contexts. UNC-73/Trio is a well-characterized Rac GTP exchange factor in  Axons extend great distances to make precise synaptic connections in the developing nervous system. Axons are guided to their targets by the growth cone, a dynamic structure at the axon distal tip that senses extracellular cues telling the axon where to go. In response to guidance cues, growth cones alter their shape and motility resulting in outgrowth and turning. The cytoskeleton (actin and microtubules) underlies growth cone motility and guidance. The signaling mechanisms linking guidance receptors to cytoskeletal change remain mysterious. Here, we define a new signaling mechanism downstream of the guidance receptor UNC-40/DCC involving the GTPases CDC-42 and Rac, which have long been known to control growth cone protrusion. We show that CDC-42 and Rac act in a linear pathway in axon guidance; CDC-42 acts upstream of the GTPase regulatory molecule TIAM-1, which is a GTP exchange factor specific for Rac and which activates Rac signaling. We also show that TIAM-1 acts with UNC-40/DCC signaling in protrusion and axon guidance. Our results imply that Rac GTPase function in axon guidance is complex and that distinct GEFs (TIAM-1 and UNC-73/Trio) might control Rac GTPases in different aspects of axon guidance. In a developing nervous system, axonal growth happens via extension and migration of the growth cone, an actin-rich neuronal structure from which filopodial and lamellipodial structures emanate in order to sense the surrounding environment for appropriate guidance cues Drosophila melanogaster and the nematode Caenorhabditis elegansC. elegans growth cone development and axon pathfinding, the two redundant Rac GTPases CED-10/Rac1 and MIG-2/RhoG act via two distinct pathways to activate the Arp2/3 complex: CED-10/Rac1 acts with WVE-1/Wave, while MIG-2/RhoG acts with WSP-1/Wasp The intracellular signaling mechanisms linking guidance receptors to cytoskeletal alterations are an intense area of study, and many remain to be elucidated. Rac GTPases, members of the Rho subfamily of small GTPases , have been well established as modulators of the actin cytoskeleton C. elegans genome encodes 19 predicted Dbl-homology (DH) GEF proteins (Wormbase). UNC-73/Trio is a well-characterized GEF for MIG-2/RhoG and CED-10/Rac1 in C. elegans axon pathfinding rh40) disrupts axon pathfinding mig-2; ced-10 rac double mutants are more severe than unc-73(rh40)Although much is known about the downstream effectors of the Rac GTPases in axon development, less is known about pathways upstream of Rac GTPases that control their activity in vivo. Rac GTPases switch between an inactive, GDP-bound state, and an active, GTP-bound state. Rac GTPase activation can be reached with the aid of a Guanine-nucleotide Exchange Factor (GEF), which catalyzes the exchange of GTP for GDP Here we show that TIAM-1 is a Rac GEF similar to mammalian T-cell lymphoma Invasion and Metastasis Factor 1 (Tiam1) C11D9.1 gene is predicted to encode a molecule with DH and Pleckstrin homology (PH) domains most similar to mammalian T-cell lymphoma Invasion and Metastasis Factor 1 (Tiam1) and Drosophila Still Life (Sif), but lacks an apparent EVH1 domain and PDZ domain found in Tiam1 and Sif C11D9.1 were obtained and sequenced (courtesy of Yuji Kohara). One of the cDNAs contained sequences complementary not only to C11D9.1, but also to the predicted upstream gene T21E12.2  T21E12.2/C11D9.1 locus collectively as tiam-1.A search of the additional predicted sequence encoded by  and Sif . While ttiam-1 cDNAs are shown in tiam-1 used here (ok772 and tm1556) remove at least part of the DH domain , but when in a double mutant combination (mig-2(mu28); ced-10(n1993M+); M+ indicates that the genotype has wild-type maternally-supplied gene function), most PDE neurons were affected  substitution in the Rac-specific DH1 domain) mig-2(mu28); ced-10(n1993M+) (p<0.001). mig-2(mu28); unc-73(rh40) and ced-10(n1993); unc-73(rh40) doubles display pathfinding defects equivalent to mig-2(mu28); ced-10(n1993M+)The ted 84%, . UNC-73/C. elegans genome encodes 19 predicted DH-containing GEFs including UNC-73/Trio . We screened mutant alleles or RNAi of the other 18 DH-GEFs alone or in combination with ced-10(n1993) and mig-2(mu28) for PDE axon pathfinding defects . The two deletion alleles of tiam-1 described above, ok772 and tm1556, caused low-penetrance defects in PDE axon pathfinding  (mig-2(mu28) and ced-10(n1993), these defects were synergistically enhanced (tiam-1(tm1556); mig-2(mu28) displayed 15% misrouted PDEs (p = 0.001), and tiam-1(tm1556); ced-10(n1993) 12% (p = 0.008). tiam-1 mutations slightly but significantly enhanced unc-73(rh40) (tiam-1 gene rescued the PDE axon pathfinding defects of tiam-1(tm1556); ced-10(n1993)  (tiam-1(+) expression in a wild-type background caused a low level of PDE axon pathfinding defects ; ced-10(n1993) rescued animals. Thus, TIAM-1 transgenic expression caused defects in axon pathfinding.The ctively) . In doubenhanced . For exa= 0.028) . We noteects 5%; , equivaltiam-1(ok772); mig-2(mu28) was significantly weaker than that of tiam-1(tm1556); mig-2(mu28) (p = 0.02) and other tiam-1 doubles with mig-2 and ced-10 ; mig-2(mu28) is still significant compared to tiam-1(ok772) alone (p = 0.046).The phenotype of d ced-10 . This mitiam-1 mutants alone also displayed weak defects in other neurons. In the AVM neurons, tiam-1(ok772) displayed ventral axon pathfinding defects and 6% ectopic neurite formation, defects not seen in wild-type (tiam-1(ok772) and tiam-1(tm1556) also displayed weak defects in VD/DD commissural motor neuron pathfinding  but not significant for tiam-1(ok772)) ((ok772)) .tiam-1 enhances mig-2 and ced-10 could indicate that TIAM-1 acts in parallel to these GTPases or that TIAM-1 acts in both of the redundant MIG-2 and CED-10 pathways in pathfinding. Studies below indicate that TIAM-1 acts with both MIG-2 and CED-10. However, the mig-2; ced-10 double mutant phenotype is much stronger than tiam-1, suggesting that another molecule might regulate MIG-2 and CED-1 in parallel to TIAM-1, possibly another GEF.These studies indicate that TIAM-1 displays a genetic interaction with the Rac GTPases CED-10 and MIG-2 and the UNC-73/Trio GEF in PDE axon pathfinding. That tiam-1 gene might by expressed, we studied expression of a tiam-1 transcriptional reporter transgene. We generated by PCR a 4793-bp region upstream of T21E12.2  (tiam-1 promoter region was placed upstream of the cyan fluorescent protein (cfp) coding region. Expression of the tiam-1promoter::cfp construct was primarily in neurons  , and thi neurons . Most ifC. elegans TIAM-1 is similar to the Rac GEFs Tiam1 and Sif. We tested if TIAM-1 from C. elegans acts as a GTP exchange factor in vitro using the mant-GTP RhoGEF exchange assay  S1216F missense mutation in the Rac-specific DH GEF domain of UNC-73 specifically abolishes Rac GEF activity unc-73(rh40) mutation S1216F. This mutation also abolished the ability of TIAM-1[DHPH] to induce ectopic protrusions in neurons as described below.The in vivo, we constructed transgenes that express constitutively-activated forms of the TIAM-1[DHPH] and UNC-73[DHPH1] Rac GEF domains in the PDE neurons. We based these experiments on previous results with Drosophila Trio (UNC-73 in C. elegans), in which a myristoylation sequence was added to the Rac-specific DHPH domain of Trio . The MYR::DHPH::GFP fusion proteins displayed a punctate, peripheral accumulation pattern consistent with localization to cellular membranes as seen in other MYR::GFP fusion proteins Based on these results, we created transgenes consisting of the TIAM-1 DHPH domain and both UNC-73 DHPH domains  fused inIn a wild-type background, the Rac-specific MYR::UNC-73[DHPH1] induced ectopic protrusions that emanated from the cell body and axons of PDEs . MYR::TIunc-73(rh40) S1216F mutation tiam-1(tm1556) background and observed ectopic protrusions similar to the wild-type background (data not shown), consistent with the idea that MYR::TIAM-1DHPH represents a constitutively-activated rather than a dominant negative molecule.To test the requirement of Rac GEF activity in ectopic protrusion, we introduced point mutations into the transgenes that abolish the Rac GEF activities of UNC-73 and TIAM-1  and ced-10(n1993) significantly suppressed the ectopic projections caused by MYR::TIAM-1[DHPH] and MYR::UNC-73[DHPH1] (mig-2(mu28) suppressed the ectopic lamellipodia and filopodia caused by MYR::UNC-73[DHPH1]  and MYR::TIAM-1[DHPH] . ced-10(n1993) suppressed MYR::TIAM-1[DHPH] strongly  and MYR::UNC-73[DHPH1] more weakly but significantly . The same integrated MYR::TIAM-1[DHPH] transgene (lqIs165) was used in both the mig-2 and ced-10 double mutants, yet upon outcrossing, the transgene showed significantly different effects (p<0.01). We do not know the nature of this variability, but it might be due to effects of different genetic backgrounds after outcrossing. Regardless, in both cases the effects increased significantly upon outcrossing from ced-10 and mig-2. We also scored integrated lines of the GEF dead constructs and the Rho-specific UNC-73 DHPH2 domain  and the osm-6 promoter (for PDE expression). The yk730h9 cDNA contained two errors relative to the reference gene models that led to frame shifts. These were repaired before proceeding  expression in In the PDE and VD/DD neurons, TIAM-1::mCherry localized to puncta primarily at the periphery of the cell bodies, consistent with plasma membrane localization, as well as in the axons . Consistmig-2/RhoG and ced-10/Rac1 transgenes tagged with gfp driven by the unc-25 promoter. MIG-2 contains an N-terminal myristoylation site and a C-terminal CAAX prenylation site. We placed gfp immediately after the Myr sequence in frame with mig-2, such that both myristoylated and prenylated forms of MIG-2 could be tagged with GFP. CED-10 was N-terminally tagged with GFP. GFP::CED-10 and GFP::MIG-2 accumulated at the cell periphery as previously described We constructed We analyzed the localization of N-terminally tagged mCherry::TIAM-1 and GFP::CED-10 in cultured NIH-3T3 fibroblasts. As expected, expression of GFP::CED-10 in fibroblasts caused marginal lamellipodial ruffling, a phenotype canonically associated with expression of vertebrate Rac1 in these cells . GFP::CEC. elegans, these data indicate that TIAM-1 and CED-10/Rac1 might interact in specific regions of the cell. In 3T3 cells, they co-localize where CED-10/Rac1 induces lamellipodial ruffles at the cell periphery   displayed ectopic lamellipodial and filopodial protrusions (44%) (cdc-42(G12V) transgenic lines, and each induced ectopic protrusions. We used one representative line for further studies.Evidence from cell and neuronal culture studies -1(DHPH)  [41]. Wens (44%) , as wellcdc-42(G12V) in tiam-1(ok772) and tiam-1(tm1556) mutants. In both cases, the ectopic lamellipodia and filopodia induced by CDC-42(G12V) were significantly suppressed  (unc-73(rh40) did not suppress CDC-42(G12V) (47% ectopic lamellipodia and filopodia in unc-73(rh40); cdc-42(G12V) compared to 44% in cdc-42(G12V) alone; p = 0.07)). These data suggest that TIAM-1, but not UNC-73, is required for the effects of CDC-42(G12V).To test if TIAM-1 was required for the effects of CDC-42(G12V), we assayed the effects of <0.0001) . These dosm-6p::tiam-1::mCherry, which produces full-length TIAM-1 tagged with mCherry in the PDE neurons but not surrounding cells, restored the effects of CDC-42(G12V) in tiam-1(ok772) and tiam-1(tm1556) mutants -induced ectopic lamellipodia and filopodia  were consistently enhanced compared to CDC-42(G12V) alone . This was also the case with fosmid rescue . Possibly, the excess TIAM-1 expressed from the transgene is activated by CDC-42(G12V), leading to increased defects. However, TIAM-1 expression alone causes ectopic protrusions  deletion removes the initiator codon and a large portion of the coding region and is likely a null allele (Wormbase). cdc-42(gk388M+) homozygotes, with wild-type maternal gene function, survived until late larval/early adulthood and were sterile. Alone, cdc-42(gk388M+) animals display 15% PDE axon pathfinding defects (tiam-1(ok772); cdc-42(gk388M+) animals showed no increase in pathfinding defects and resembled cdc-42(gk388M+) alone. However, unc-73(rh40); cdc-42(gk388M+) mutants displayed a synergistic increase in the number of PDE pathfinding defects (90%) compared to cdc-42(gk388M+) and unc-73(rh40) alone (p<0.001) (These findings were corroborated with loss-of-function studies. The  defects . tiam-1(p<0.001) . These lmig-2(mu28) and ced-10(n1993) suppressed the effects of CDC-42(G12V) (ced-10(tm597M+) deletion allele also suppressed CDC-42(G12V) (p<0.001). Suppression by ced-10(n1993) was significantly weaker than that of mig-2(mu28)  and ced-10(tm597M+) . ced-10(n1993) is a mutation in the C-terminal prenylation sequence and is a hypomorph, consistent with weaker suppression ced-10(n1993M+); mig-2(mu28) double mutant suppressed  slightly better than mig-2(mu28) or ced-10(tm597M+) alone . These data indicate that CED-10/Rac1 and MIG-2/RhoG are required for the ectopic protrusions caused by CDC-42(G12V).Our results suggest that TIAM-1 acts downstream of CDC-42 in ectopic lamellipodial protrusion. Given that TIAM-1 can act as a Rac GEF, we expect that MIG-2/RhoG and CED-10/Rac1 might also be required for the effects of CDC-42(G12V). Indeed, 42(G12V) ; p<0.001C. elegans. UNC-6/Netrin is a secreted ligand expressed in the ventral side of the animal that attracts axons that express its receptor UNC-40/DCC C. elegans Ephrin receptor We have shown evidence that the Rac GEF TIAM-1 regulates the Rac GTPases CED-10 and MIG-2 in response to CDC-42 signaling. We next sought to determine which dorsal-ventral axon guidance ligand/receptor pathway might be upstream of TIAM-1. Distinct parallel ventral guidance pathways control dorsal ventral guidance in tiam-1 with the loss-of-function alleles unc-6(ev400), slt-1(eh15), and vab-1(dx31), and scored defects in PDE axon ventral guidance (tiam-1(ok772) significantly increased the defects of vab-1(dx31) , and tiam-1(tm1556) enhanced slt-1(eh15) , suggesting that TIAM-1 might act in parallel to VAB-1/Eph and SLT-1/Slit. In contrast, neither tiam-1(ok772) nor tiam-1(tm1556) increased the amount of PDE pathfinding defects of unc-6(ev400) . slt-1(eh15) is a small deletion and is likely a null vab-1(dx31) is a complex rearrangement that deletes exons 1–4 and is also likely a null tiam-1 enhanced slt-1 and vab-1 but did not enhance unc-6 is consistent with TIAM-1 acting in the UNC-6/Netrin pathway in parallel to VAB-1 and SLT-1 in PDE ventral guidance.We constructed double mutants of guidance . tiam-1 did not suppress (tiam-1(ok772) significantly suppressed the ectopic lamellipodia incidence in these neurons . Furthermore, cdc-42(gk388M+) suppressed MYR::UNC-40 , suggesting that CDC-42 is also downstream of UNC-40. Consistent with this idea, cdc-42(gk388M+) did not enhance unc-6(ev400) in PDE ventral axon guidance (cdc-42(gk388M+) slightly suppressed unc-6(ev400) (p = 0.027). The nature of this possible suppression is unclear, but it is possible that cdc-42(gk388M+) might also normally attenuate UNC-6/Netrin signaling. While tiam-1 loss of function suppressed the ectopic protrusions driven by MYR::UNC-40, the axon guidance and branching defects were not suppressed significantly (data not shown), suggesting that MYR::UNC-40 perturbation of axon guidance and branching are not dependent upon TIAM-1 and might be due to effects distinct from ectopic lamellipodial protrusion.We tested if TIAM-1 might act downstream of MYR::UNC-40 in AVM. When expressed in the PDE neurons, MYR::UNC-40 did not result in ectopic lamellipodial protrusions. We do not know why this is the case, but possibly the PDE neuron is less sensitive to the effects of MYR::UNC-40 than is AVM, or the suppress ; 58%, coguidance . In factunc-6, unc-40, and tiam-1 (unc-6(ev400) and unc-40(n324) both displayed defective AVM ventral guidance as expected, but unc-6(ev400) was significantly stronger than unc-40(n324) . Both are predicted null mutations, suggesting that UNC-40 might not be the only receptor for UNC-6 in AVM ventral guidance. AVM defects in unc-6(ev400) mutants were scored previously by another group unc-6(ev400) (data not shown), similar to those previously described We assayed AVM axon guidance in single and double loss-of-function mutants involving d tiam-1 . unc-6(etiam-1(tm1556), tiam-1(ok772), and tiam-1(RNAi) significantly enhanced the AVM ventral guidance defects of unc-6(ev400)  caused defects in AVM guidance similar to unc-40(n324) that were not enhanced by tiam-1 mutations; and tiam-1(RNAi), which enhanced unc-6(ev400), did not enhance unc-40(n324) (unc-40(n324) and unc-40(RNAi) enhanced unc-6(ev400) in a manner similar to tiam-1. Enhancement of unc-6(ev400) by tiam-1 and unc-40 was also significant when only failure to reach the ventral nerve cord was considered (data not shown). Previous results showed that unc-40(e1430); unc-6(ev400) doubles had only slightly increased AVM guidance defects (∼25% to ∼33%) unc-40(n324) and unc-40(RNAi) and the previous study used unc-40(e1430)unc-6(ev400) that we report is due to different unc-40 alleles used.We found that 6(ev400) . This rece unc-6 , suggest40(n324) . These dunc-40 has a more severe phenotype than tiam-1 suggests that additional molecules might act in parallel to TIAM-1 downstream of UNC-40.In sum, these genetic interactions suggest that UNC-6 and UNC-40 act partially in parallel and also suggest that UNC-6 might not be the only ligand for UNC-40/DCC in AVM ventral guidance. They are also consistent with TIAM-1 acting in the UNC-40 pathway in AVM ventral axon guidance. That C. elegans molecule TIAM-1, which acts specifically as a GEF for Rac GTPases and interacts with them in axon guidance and neuronal protrusion. We show that TIAM-1 regulates neuronal protrusion downstream of the CDC-42 GTPase and upstream of the two redundant Rac GTPases MIG-2/RhoG and CED-10/Rac1. Genetic interactions of these molecules suggest that that they act in a common pathway in axon pathfinding. Furthermore, we provide evidence that TIAM-1 acts with the UNC-6/Netrin receptor UNC-40/DCC in axon guidance and downstream of UNC-40/DCC in neuronal protrusion. Previous studies have implicated Rac GTPases and the Rac GEF Trio as players in Netrin signaling and interaction with the Netrin receptor DCC In recent years, attention has focused on how guidance receptors engage cytoplasmic signaling mechanisms to control growth cone outgrowth and turning during axon guidance. In this report we present evidence of a signaling pathway  involvinGTP exchange assays indicated that TIAM-1 is a Rac-specific GEF . The actin vivo, whether or not their normal role is in protrusion. However, we found that transgenic expression of full-length TIAM-1::mCherry also induced ectopic protrusions in the PDE neurons similar to but at a much lower level than the MYR::DHPH construct, suggesting that regulation of protrusion is a normal cellular role of TIAM-1. This is consistent with tiam-1 suppression of protrusion caused by CDC-42(G12V) and MYR::UNC-40. In contrast, unc-73 suppressed neither of these, suggesting that UNC-73/Trio might not normally regulate protrusive events downstream of UNC-40. We speculate that UNC-73 might control Rac activity upstream of guidance receptors to control their cell surface trafficking  Our results and those of others How might our results fit with these known roles of Trio in Netrin signaling? Cell-specific and context-specific differences in Trio function downstream of guidance receptors are possible explanations. In other words, Trio might act with UNC-40/DCC in some neurons and not in others, or might display distinct, cell-specific interactions with guidance receptors.C. elegans, Rac GTPases engage the actin-nucleating Arp2/3 complex and the actin-binding protein UNC-115/abLIM to promote filopodial extension mig-2; ced-10 doubles are more severe than tiam-1 alone. Rac GTPases might affect multiple aspects of axon guidance, whereas TIAM-1 might specifically regulate them in downstream cytoskeletal effects.An alternative but not mutually exclusive explanation, consistent with previous results, is that Trio and TIAM-1 affect distinct aspects of Rac function in axon guidance. It is becoming apparent that the role of Rac GTPases in axon pathfinding is complex. Rac GTPases clearly regulate cytoskeletal events in the growth cone in response to guidance cues. For example, in C. elegansunc-73 did not suppress MYR::UNC-40. Thus, suppression of MYR::UNC-40 might target those molecules that act downstream of UNC-40/DCC but not those involved in it's trafficking . Our results provide experimental evidence for this idea, as we have shown that the TIAM-1 GEF but not the UNC-73/Trio GEF controls Rac function downstream of UNC-6/Netrin signaling in protrusion. UNC-73/Trio might act upstream of UNC-40/DCC and regulate its trafficking Rho-family GTPases control many cellular functions, and it has been assumed that upstream regulators confer specificity to Rho GTPase activity. Indeed, genomes encode many GTP exchange factors for Rho GTPases (e.g. the C. elegans handling and culture were performed using standard techniques tiam-1(tm1556) allele was provided by the National Bioresource Project for the Experimental Animal “Nematode C. elegans” (S. Mitani), and the tiam-1(ok772) allele was provided to us by the C. elegans Gene Knockout Consortium via the C. elegans Genetic Center (CGC). Polymerase chain reaction (PCR) was used to verify the homozygosity of tiam-1(ok772) and (tm1556) in strains. The following mutations and genetic constructs were used: LGI: unc-73(rh40), tiam-1(ok772) and (tm1556), lqIs37[osm-6::cdc-42(G12V)]; LGII: cdc-42(gk388), mIn1,vab-1(dx31), juIs76[unc-25::gfp]; LGIV: ced-10(n1993), zdIs4[mec-4::gfp], lqIs3[osm-6::gfp]; LGV: lqIs123[osm-6::myr::unc-73(DHPH1)]; LGX: mig-2(mu28), unc-6(ev400), slt-1(eh15), lqIs2[osm-6::gfp]; LG?: lqIs125[osm-6::myr::unc-73(DHPH2)], lqIs131[mec-7::myr::unc-40(cyto)::gfp], lqIs134[osm-6::myr::unc-73(DHPH1 S1216F)], lqIs165[osm-6::myr::tiam-1(DHPH)], lqIs216[osm-6::myr::tiam-1(DHPH T516F)]. Extrachromosomal arrays: lqEx504-506, osm-6::myr::unc-73(DHPH1); lqEx509-510, osm-6::myr::unc-73(DHPH)2; lqEx517-518, osm-6::myr::unc-73(DHPH1 S1216F); lqEx559-561, osm-6::myr::tiam-1(DHPH); lqEx571-572, lqEx584, osm-6::myr::tiam-1(DHPH T516F).C. elegans transformation was performed with standard methods by microinjection of DNA into the hermaphroditic syncytial germline All micrographs were obtained on a Leica DMRE microscope with a Qimaging Rolera MGi EMCCD camera or a Qimaging Retiga CCD camera. Openlab and IPlab software were used to obtain images.lqIs2 X and lqIs3 IV [osm-6 promoter::gfp] were used juIs76 II [unc-25 promoter::gfp] was used zdIs4 IV [mec-4 promoter::gfp] was used.Fluorescence microscopy was used to score axon pathfinding defects of fourth larval stage (L4) or young adult hermaphrodite animals expressing a green fluorescent protein transgene for specific cells. To score and visualize PDE axons, the integrated transgenes The cell bodies of the PDE neurons (PDEL and PDER) are situated in the posterior lateral post-deirid ganglion. PDEs extend an axon ventrally to the ventral nerve cord, which then bifurcates and extends anteriorly and posteriorly in the ventral nerve cord. If the axon failed to reach the ventral nerve cord or wandered beyond a 45° angle from a straight line ventrally from the cell body, it was considered mutant.A VD/DD commissural axon that failed to reach the dorsal nerve cord or that wandered laterally before reaching the dorsal nerve cord was considered mutant. The percent of animals with pathfinding defects was noted, and the percentage of defective axons was noted.The AVM cell body is situated laterally on the right side, in the anterior half of the body of the animal. The axon extends ventrally towards the ventral nerve cord, which then extends anteriorly until the first pharyngeal bulb. Similarly to PDE axons, if the axon failed to reach the ventral nerve cord or wandered beyond a 45° angle from a straight line ventrally, it was considered mutant. p-value significance was determined using Fisher Exact Analysis.Ectopic lamellipodial and filopodial protrusions were identified as any thin ruffle-like or finger-like protrusion from the cell body, which is normally smooth and round, or axon of the neurons.C. elegans expression plasmids utilized the standard Fire expression vectors with the unc-54 3′ UTR . Primer sequences used to amplify C. elegans genomic and cDNA sequences to construct these plasmids are shown in tiam-1 transcriptional promoter fusion plasmid was generated by amplifying by PCR the tiam-1 upstream promoter region, a 4793-bp region upstream of T21E12.2  (cyan fluorescent protein (cfp) coding region. For cell specific expression of full length TIAM-1, the open reading frame of the yk370h9 tiam-1 cDNA was amplified by PCR and fused to mCherry at the 3′ end to create a C-terminally tagged TIAM-1::mCherry molecule (tiam-1::mCherry expression plasmids contained the osm-6 promoter (to drive expression in the PDEs) unc-25 promoter (VD/DDs) All 21E12.3) , and plamolecule . tiam-1:gfp::mig-2 fusion plasmid was constructed by placing gfp in frame after the N-terminal myristoylation sequence of mig-2 and before the remaining genomic coding region, allowing for GFP tagging of both myristoylated and prenylated forms of MIG-2 (gfp::ced-10 was constructed by placing gfp at the N-terminus as previously described gfp::mig-2 and gfp::ced-10 expression was driven by the osm-6 or unc-25 promoter.A of MIG-2 . gfp::cecdc-42(G12V) genomic coding region under the control of the osm-6 promoter was constructed using site directed mutagenesis as described previously for ced-10(G12V)myr::unc-40(cyto) plasmid under the control of the mec-7 promoter was constructed as described in myr::GEF(DHPH) plasmids with the osm-6 promoter were used as means of overactivating the GEFs as previously described A plasmid containing an activated cmv::mCherry::tiam-1 N-terminal fusion construct was generated by placing the yk37h9 tiam-1 cDNA open reading frame, fused N-terminally to mCherry, behind the CMV promoter in the pTriEx-mCherry vector . cmv::gfp::ced-10 was constructed using the ced-10 cDNA open reading frame For expression in NIH 3T3 cells, a 2 at 37°C. Cells were plated onto coverslips the day before transfection at a density of 200,000 cells per well in a 6-well plate. For co-localization assays, pEGFP-C1 , pEGFP-C1 expressing CED-10 (“GFP::CED-10”), pTriEx-mCherry  and pTriEx-mCherry expressing TIAM-1 (“mCherry::TIAM-1”) were transiently transfected into NIH3T3 cells using GeneExpresso Transfection reagent  according to the manufacturer's specifications. To visualize subcellular protein localizations at high magnification, the NIH 3T3 cells grown on coverslips and transiently transfected were fixed in 3.7% formaldehyde for 1 h at room temperature. Coverslips were then washed three times in 1× Phosphate Buffered Saline (PBS), one time in ddH2O, and then mounted onto a glass slide using ProLong Gold antifade (Invitrogen/Molecular Probes) mounting medium. The cells were then visualized using compound fluorescence microscopy .NIH 3T3 mouse fibroblasts were grown in DMEM (Cellgro) supplemented with 10% Cosmic Calf serum (HyClone) and 1% penicillin-streptomycin (P/S) and maintained in 5% COWe amplified the DHPH domains of TIAM-1 (residues 488–856 relative to isoform A), placed it into the pET-28b vector (Novagen), which has a T7 IPTG-inducible promoter and a C-terminal 6HIS-tag, and transformed this construct into BL21(DE3) Gold competent cells (Agilent Technologies). For the TIAM-1[DH(T516F)PH] construct, we used site directed mutagenesis to make a point mutation in the wild-type construct using the Quikchange kit (Stratagene). We purified TIAM-1[DHPH]::6HIS using Ni-column based chromatography (Qiagen). For the mant-GTP assay, we used the RhoGEF Exchange Assay Biochem Kit  and followed instructions in the kit. We used 12.5 µM of TIAM-1[DHPH] in these experiments.N- methylanthraniloyl-GTP (mant-GTP) is a fluorescently labeled nucleotide analog that can be incorporated by small GTPases. Using a Varian Cary Eclipse fluorescent spectrophotometer, we measured the amount of mant-GTP incorporated by the GTPase due to the spectroscopic difference between bound and free mant-GTP . The GTPase  was incubated in buffer and mant-GTP for five minutes, after which time solvent (control) or the GEF (TIAM-1[DHPH]) was added. Experiments were recorded for one half hour.Figure S1tiam-1 locus. (A) Black boxes represent exons with coding region open reading frame, white boxes represent non-coding exons, SL1 indicates that an SL1 trans-spliced leader sequence was present in the cDNA sequence, and AAAAA indicates the presence of a poly-A tail in the cDNA sequence. The regions removed by the ok772 and tm1556 deletions are indicated. ok772 is an 838-bp deletion a coupled to an 18-bp insertion (CTGTAACTTAACTGTAAC), and tm1556 is an 851-bp deletion. The yk730h9 cDNA spanned the predicted gene models C11D9.1 and T21E12.2, indicating that these regions are part of the same transcription unit and thus represent a single locus, the tiam-1 locus. The dashed lines are an expansion of the 3′ end of the locus showing the structures of three cDNAs sequenced. yk730h9 is the longest cDNA and spans the entire locus. An arrow points to an intron that is spliced out specifically in yk730h9. An SL1 trans-spliced leader sequence is present at the 5′ end of yk730h9, indicating that it is a full-length cDNA. yk1684a10 is a shorter cDNA with splice variation. An in-frame stop codon is present in the 5′ region of the cDNA sequence, indicating that this cDNA contains the entire coding potential of this isoform. yk762f12 also encodes a shorter cDNA with splice variation and contains an SL1 trans-spliced leader, indicating that it contains the complete coding potential for this isoform. The sequences of these cDNAs have been deposited in Genbank. (B) The predicted molecules encoded by the three cDNAs. yk730h9 encodes isoform A, which contains a predicted EVH1-like domain and PDZ-like domain in addition to the DH and PH domains. yk762f12 encodes isoform B, which contains the PDZ-like domain and the DH/PH domains. yk1684a10 encodes isoform C which contains a truncated PDZ-like domain and the DH/PH domains. The regions removed by the deletions ok772 and tm1556 are indicated.A model of the (TIF)Click here for additional data file.Figure S2tiam-1 A. Open reading frame of tiam-1 A encoded by a repaired version of the yk730h9 cDNA is shown, with the amino acid sequence below. The yk730h9 cDNA contained errors relative to the reference gene models that resulted in frame shifts. These were repaired in the yk730h9 cDNA by site-directed mutagenesis. An extra G residue after base 1039 relative to the start codon in ok772 and tm1556 are shaded black. These do not represent the precise breakpoints of the deletions, as three of four breakpoints are within introns.The nucleotide and amino acid sequence of (PDF)Click here for additional data file.Figure S3C. elegans TIAM-1. (Ce) (this work), Still life/Tiam1 from Ascaris suum (As) (Genbank: ADY42014), and Still Life/Tiam1 isoform E from Drosophila melanogaster (Dm) (Genbank NP_001097519). While BLASTP and CCD did not recognize the EVH1 domain of Ce TIAM-1, they did recognize a conserved region in As TIAM-1 as an EVH1 domain. Identities are indicated. (B) An analysis of the putative PDZ-like domain as described for the EVH1 domain in (A). (C) ClustalW alignments of the predicted EVH1 and PDZ domains of the same molecules in (A and B). Conserved identical residues are indicated in red and by asterisks (*), and conserved similar residues are indicated by colons (:).BLASTP and ClustalW analysis of the TIAM-1 molecule. (A) BLAST alignments showing of the predicted EVH1-like regions of (PDF)Click here for additional data file.Figure S4tiam-1 mutants. In all micrographs, anterior is to the left and dorsal is up. (A) A fluorescent micrograph of an AVM neuron of a wild-type animal visualized with mec-7::gfp. (B) An AVM neuron from a tiam-1(ok772) mutant with an ectopic neurite. (C) A VD or DD motor neuron visualized with unc-25::gfp branched prematurely in a tiam-1(tm1556) mutant (arrow). The scale bars represent 2 µm.VD/DD and AVM axon defects in (TIF)Click here for additional data file.Figure S5in vitro, represented as “(DEAD)” on the X axis Click here for additional data file.Figure S6osm-6::gfp (arrows). TIAM-1::mCherry is excluded from the nucleus and localizes to the periphery of the cell body. (B) TIAM-1::mCherry accumulation at the periphery of VD/DD motor neuron cell bodies (arrows), driven by the unc-25 promoter. The outlines of cell bodies were traced (dashed line) to indicate peripheral localization of TIAM-1::mCherry. The scale bar represents 2 µm for all micrographs.TIAM-1::mCherry localized to the periphery of neuronal cell bodies. In all micrographs, anterior is to the left and dorsal is up. (A) two PDE neurons with TIAM-1::mCherry expression driven by (TIF)Click here for additional data file.Figure S7GFP::CED-10 and TIAM-1::mCherry accumulate to specific sites in NIH-3T3 cells. GFP::CED-10 and TIAM-1 localize to discrete regions of the peripheral plasma membrane (arrowheads in (A) and (B). GFP::CED-10 induced lamellipodial ruffles but TIAM-1::mCherry did not. Neither GFP nor mCherry alone localized to discrete regions of the cell ((C) and (D)) as did the tagged CED-10 and TIAM-1 molecules. The scale bar in (A) represents 10 µm for all micrographs.(TIF)Click here for additional data file.Figure S8tiam-1 genomic fosmid clone rescued suppression of CDC-42(G12V). (B) A graph charting the percentage of PDE axons with ectopic lamellipodial and filopodial protrusions (X axis) in different genotypes (Y axis). [cdc-42(G12V)] represents animals harboring a transgene that expresses activated cdc-42(G12V) driven by the osm-6 promoter. [tiam-1(+)] represents a transgene composed of the genomic fosmid clone WRM0633ch01 that harbors a wild-type copy of tiam-1. At least 100 PDE neurons were scored for each genotype, and p value significance was determined using Fisher's Exact analysis. Error bars represent 2× standard error of the proportion in both directions.A (TIF)Click here for additional data file.Figure S9C. elegans genomic or cDNA fragments to construct expression plasmids. The fragment of genomic DNA or cDNA amplified is underlined. (+) indicates the sense strand primer relative to the gene, and (−) indicates the anti-sense strand primer relative to the gene. Spacers and restriction enzyme sites added to the ends of the primers to aid in cloning are in italics.Primer sequences used to amplify (PDF)Click here for additional data file.


A combination therapy of interferon-α (IFN-α) and zidovudin (AZT) shows therapeutic effects in ATL patients, although its mechanism is also obscure. We previously found that viral gene expression in IL-2-dependent HTLV-1-infected T-cells (ILTs) derived from ATL patients was markedly suppressed by stromal cells through a type I IFN response. Here, we investigated the effects of IFN-α with or without AZT on viral gene expression and cell growth in ILTs.Human T-cell leukemia virus type-1 (HTLV-1) is the causative retrovirus of adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 gene expression is maintained at low levels ILTs expressed variable but lower amounts of HTLV-1 Tax protein than HTLV-1-transformed HUT102 cells. Following the addition of IFN-α, the amounts of HTLV-1 p19 in the supernatants of these cells decreased in three days, while HTLV-1 gene expression decreased only in ILTs but not HUT102 cells. IFN-α also suppressed the spontaneous HTLV-1 induction in primary ATL cells cultured for 24 h. A time course study using ILTs revealed that the levels of intracellular Tax proteins decreased in the first 24 h after addition of IFN-α, before the reduction in HTLV-1 mRNA levels. The initial decreases of Tax protein following IFN-α treatment were observed in 6 of 7 ILT lines tested, although the reduction rates varied among ILT lines. An RNA-dependent protein kinase (PKR)-inhibitor reversed IFN-mediated suppression of Tax in ILTs. IFN-α also induced cell cycle arrest at the G0/G1 phase and suppressed NF-κB activities in these cells. AZT alone did not affect HTLV-1 gene expression, cell viability or NF-κB activities. AZT combined with IFN-α markedly induced cell apoptosis associated with phosphorylation of p53 and induction of p53-responsive genes in ILTs.IFN-α suppressed HTLV-1 gene expression at least through a PKR-mediated mechanism, and also induced cell cycle arrest in ILTs. In combination with AZT, IFN-α further induced p53 signaling and cell apoptosis in these cells. These findings suggest that HTLV-1-infected cells at an IL-2-dependent stage retain susceptibility to type I IFN-mediated regulation of viral expression, and partly explain how AZT/IFN-α produces therapeutic effects in ATL. HTLV-1 mRNA, but not proteins, are detectable in peripheral blood mononuclear cells (PBMCs) of HTLV-1-infected individuals [in vivo, as HTLV-1-infected individuals maintain antibodies against HTLV-1 structural proteins and Tax protein-specific cytotoxic T lymphocytes.Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATL) , a maligividuals . AlthougEx vivo studies have indicated that graft-versus-tumor responses including anti-Tax cytotoxic T-cells were potentially involved in the therapeutic mechanisms of allo-HSCT [in vitro[in vitro[in vivo.Recent therapeutic approaches, such as allogeneic hematopoietic stem cell transplantation  ,8, a humllo-HSCT , and thallo-HSCT . However[in vitro, and the[in vitro. The com[in vitro,19. As Iin vitro. For example, IFN-α reduced the virus release but not viral protein synthesis in HTLV-1-transformed HUT102 or MT-2 cells [in vitro, while conflicting with the clinical effects of AZT/IFN-α therapy in ATL patients. This discrepancy between in vivo and in vitro systems can be partially attributed to differences in status of HTLV-1-infected cells between the two systems.It is well established that HTLV-1-infected cells are resistant to type I IFNs -2 cells . The mec-2 cells , Tax-med-2 cells , Tax-med-2 cells ,24, and -2 cells , all of in vitro. Our experimental system differed from previous studies in two ways. First, we used IL-2-dependent HTLV-1-infected T-cells (ILTs) derived from ATL patients, while previous studies used IL-2-independent HTLV-1-transformed cell lines such as HUT102. Second, we used stromal cells as effectors; these mediated the type I IFN response, but could have also produced multiple factors other than IFNs.We previously found that HTLV-1-infected cells could induce type I IFN responses in co-cultured stromal cells . We alsoIn the present study, we investigated whether purified type I-IFNs can affect viral expression and cell growth of HTLV-1-infected cells by using various ILTs. Here we report a novel finding that IFN-α suppresses intracellular Tax expression at a translational level at least through PKR. We further demonstrate that IFN-α activates p53 pathways in cooperation with AZT, partly explaining the mechanisms of the therapeutic effects of AZT/IFN-α in ATL.We evaluated the baseline levels of HTLV-1 gene expression in HUT102, ILT-Hod and ILT-#29 cell lines  on HUT102, ILT-Hod, and ILT-#29 cells  or HAM/TSP patients  were maintained in RPMI 1640 medium  containing 10% fetal calf serum , Antibiotic Antimycotic Solution (Sigma Aldrich) and 30–100 IU/ml of recombinant human IL-2 . The IL-2-independent HTLV-1-infected T-cell line, HUT102, derived from a patient with mycosis fungoides  and MOLTAlexa Fluor 488-conjugated Lt-4 , a mouse13H8N4OS; Calbiochem) and its negative control inhibitor  were dissolved in DMSO and added at 500 nM in culture 2 h before IFN-α treatment and carried over through the culture. Pifithrin-α p-nitro cyclic, a chemical p53-inhibitor , was used at 1 μM.Natural type human IFN-α  was added to cell cultures at various concentrations. Zidovudin (AZT)  was used at 10 μM, a concentration inhibiting reverse transcription without cell toxicity . When cuAliquots (0.5 μg) of total RNA extracted from cells using Isogen  were treated with DNase  and subjected to reverse transcription (RT) with oligo(dT)20 primers followed by PCR using THUNDERBIRD qPCR Mix . To quantify HTLV-1 mRNAs, three primer sets were used; Gag primers  located at Gag region, pX primers  located at pX region, and RPX primers  located at upstream and downstream of the second splice junction site of tax/rex mRNA . The priFor intracellular staining of HTLV-1 antigens, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 100% methanol for 10 min on ice. To detect the Gag protein, cells were serially incubated with GIN-7 or control ascites and FITC-conjugated goat anti-mouse IgG + IgM. To detect the Tax protein, cells were incubated with Alexa Fluor 488-labelled Lt-4 or isotype control antibody. To stain intracellular Ki-67, cells were fixed with 4% paraformaldehyde and permeabilized with a BD Perm/Wash™ Buffer Kit (BD Pharmingen), then incubated with an R-PE-conjugated anti-human Ki-67 mouse mAb or isotype control mouse IgG1, κ antibody. For staining intracellular phosphorylated p53, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 100% methanol for 10 min on ice prior to incubation with antibody. Stained cells were analyzed with a flow cytometer  using FlowJo software (Tree Star).HTLV-1-infected or uninfected cells were dissolved in Cell Culture Lysis Reagent  containing 25 mM Tris-phosphate (pH 7.8), 2 mM DTT, 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, and 1% Triton X-100, with protease inhibitor cocktail , and were incubated on ice for 1 hour. The cell lysates were cleared by centrifugation, denatured with SDS sample buffer  and 2.5% 2-mercaptethanol  at 70°C for 15 min, and 17 μg of proteins were electrophoresed on polyacrylamide gel , and then transferred to PVDF membrane . The membranes was blocked with Block Ace  overnight, and reacted with mouse anti-Tax and anti-α-Tubulin  antibodies as primary antibodies overnight, followed by exposure to horseradish peroxidase-conjugated sheep anti-mouse IgG whole antibody  as a second antibody. The reacted bands were visualized by enhanced chemiluminescence using Novel® ECL  and analyzed on Image Quant mini LAS 4000 .Cells were cultured in the presence of 10 μM bromodeoxyuridine (BrdU) for 24 h, fixed and then permeabilized, followed by incubation with a mouse anti-BrdU mAb and 7AAD from BrdU flow Kits (BD Pharmingen), according to the manufacturers’ instructions. Stained cells were analyzed with a flow cytometer using CellQuest software (Becton Dickinson). To evaluate cell growth, Trypan blue exclusion test and a colorimetric assay using Cell Counting Kit-8  based on formazan color development were used.The concentration of HTLV-1 p19 in the supernatants from ILT-Hod, ILT-#29 or HUT102 cultures were measured using a RETRO-tek HTLV-1/II p19 antigen ELISA  according to the manufacturer’s instructions.Reporter cell lines (ILT-Hod/NF-κB-Luc and ILT-#29/NF-κB-Luc) were established by using a Cignal Lenti-NF-κB reporter Luc Kit  and Cignal Lenti thymidine kinase (TK)-Renilla control (Qiagen). Luciferase assays were conducted with Luciferase or Renilla luciferase assay systems  on cell lysates in Renilla luciferase lysis buffer (Promega). Relative NF-κB activity was calculated as the ratio of firefly luciferase to renilla luciferase activities in the same sample.t-test was performed for statistical significance, and P values less than 0.05 were considered significant.The unpaired ATL: Adult T-cell leukemia/lymphoma; AZT: Zidovudin (3'-Azido-3'-deoxythymidine); BrdU: Bromodeoxyuridine; ELISA: Enzyme-linked immunosorbent assay; FCS: Fetal calf serum; FITC: Fluorescein-isothiocyanate; HAM/TSP: HTLV-1-associated myelopathy/tropical spastic paraparesis; HSCT: Hematopoietic stem cell transplantation; HTLV-1: Human T-cell leukemia virus type-1; IFN-α: Interferon-α; IgG: Immunoglobulin G; ILT: IL-2-dependent HTLV-1-infected T-cell; mAb: Monoclonal antibody; MFI: Mean fluorescence intensity; PBMC: Peripheral blood mononuclear cell; PKR: RNA-dependent protein kinase; PMA: Phorbol 12-myristate 13-acetate; RT: Reverse transcription; TK: Thymidine kinase; VEGF: Vascular endothelial growth factorThe authors declare that they have no competing interests.SK carried out most of the experiments, analyzed data, and wrote the manuscript; MKi and AT carried out certain aspects of the experiments; AH advised on flow cytometry analysis; AS advised on signaling analysis; TM advised on RT-PCR analysis; YT provided HTLV-1-specific monoclonal antibodies; AU provided clinical samples; MKa designed the study, analyzed data, and wrote the manuscript; all authors reviewed and approved the final manuscript.


Bladder transitional cell carcinoma greatly threatens human health all overthe world. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)shows a strong apoptosis-inducing effect on a variety of cancer cellsincluding bladder cancer. However, adenovirus-mediated TRAIL expressionstill showed cytotoxicity to normal cells mainly due to lack of tumorspecificity.miR-1, miR-133 andmiR-218 to confer TRAIL expression with specificity tobladder cancer cells.To solve the problem, we applied miRNA response elements (MREs) ofmiR-1, miR-133 andmiR-218 was greatly decreased in bladder cancer thannormal bladder tissue. Luciferase assay showed that application of the 3MREs was able to restrain exogenous gene expression to within bladder cancercells. Subsequently, we constructed a recombinant adenovirus with TRAILexpression regulated by MREs of miR-1,miR-133 and miR-218, namelyAd-TRAIL-MRE-1-133-218. qPCR, immunoblotting and ELISA assays demonstratedthat Ad-TRAIL-MRE-1-133-218 expressed in bladder cancer cells, rather thannormal bladder cells. The differential TRAIL expression also led toselective apoptosis-inducing and growth-inhibiting effect ofAd-TRAIL-MRE-1-133-218 on bladder cancers. Finally, bladder cancer xenograftin mouse models further confirmed that Ad-TRAIL-MRE-1-133-218 effectivelysuppressed the growth of bladder cancers.Expression of Collectively, we demonstrated that MREs-based TRAIL delivery into bladdercancer cells was feasible and efficient for cancer gene therapy. Among the most common malignant cancers, bladder transitional cell carcinoma severelyrisks health of the people on the earth . DownregTumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted muchattention due to its specific induction of apoptosis in various types of cancercells by binding death receptors and activating mitochondria-independent signaltransduction pathway ,6. Like Cytotoxicity to normal cells, however, seriously hurdles the clinical application ofadenoviral vector for cancer gene therapy, since adenoviral vector lacks the abilityto discriminate cancer and normal cells. To confer adenovirus with bladder cancerspecificity, researchers developed many strategies including employingcancer-specific promoter. Although UP II promoter has been used to specificallydrive TRAIL expression in bladder cancer cells, more novel strategies are needed toprevent the cytotoxicity of adenovirus-based gene therapy to normal cells -16.Differential expression profile of miRNAs has been widely reported between bladdercancer and normal cells . DecreasHere, we intended to identify suitable MREs for bladder cancer specificadenovirus-mediated TRAIL expression from the miRNAs with downregulated expressionin bladder cancer, including miR-1 -21, miR-We employed primary cultures derived from bladder transitional carcinoma andnormal bladder mucosal cells (BMC) in this study. For the culturing of bladdercancer, the samples were obtained with written informed consent from allpatients according to protocols approved by Ethical Review Board in GeneralHospital of Chengdu Military Area Command of Chinese PLA . Allpatients underwent surgical resection of bladder carcinoma at Department ofUrology, General Hospital of Chengdu Military Area Command of Chinese PLA. Bladder cancer samples were sheared into small pieces,followed by mechanical manipulation to obtain single cell suspension. Theprimary cultures were maintained in DMEM supplemented with 15% FBS.For primary BMC culture, the samples were obtained from 8 patients that underwentcystoscopic examination of asymptomatic haematuria . The previously describedprocedures that have been approved by Ethical Review Board in General Hospitalof Chengdu Military Area Command of Chinese PLA  was followed toestablish the primary BMC culture . The BMCACATTCCAACAAACACCACATTCC.Ad-EGFP and Ad-TRAIL were preserved in our laboratory. We constructedAd-TRAIL-MRE-1-133-218 as follows. A DNA fragment was synthesized(5′-ACAAACACCAACAAACACCGGACCAAAACAAACACCGGACCAAAACAAACACCAAGCACAAACAAACACCAAGCACAA-3′),which contained two copies of miR-1 MREs, two copies of miR-133 MREs and twocopies of miR-218 MREs. This fragment was released from the temporary vector byEcoRV and then inserted into pShuttle-CMV-TRAIL at the same site,generating pShuttle-CMV-TRAIL-MRE-1-133-218. This plasmid was subsequentlycotransfected into HEK-293 cells with pAdEasy. After plague purification forthree times and PCR-based identification, adenoviruses were harvested and thenpurified with the CsCl gradient centrifugation. The involved adenoviruses weretitrated with TCID50 method on HEK-293 cells and represented asplaque-forming units per milliliter (pfu/ml) [(pfu/ml) . The ade2 and humidified atmosphere at 37°C.Human bladder transitional cell carcinoma cell line T24 and RT-4 were bothpurchased from the American Type Culture Collection  and weregrown in McCoy’s 5a Medium Modified supplemented with 10% (v/v) fetal bovine serum . Human endothelial cells HUV-EC-C and normal liver cells L-02 were obtainedfrom Shanghai Cell Collection . HUV-EC-C and L-02 cells werecultured using DMEM media supplemented with 10% (v/v) fetal bovine serum. Allmedia was supplemented with 4 mM glutamine, 100 units/mL penicillin and 100μg/ml streptomycin. All cells in this experiment were cultured under a 5%COTotal RNA was extracted from 14 bladder cancer samples with Trizol solution and pooled as one group for subsequent experiments. Anotherpool of RNA was also obtained from 8 normal bladder mucosal tissues according tothe same protocol. Also, T24, RT-4, HUV-EC-C and L-02 cells were processed forextracting RNA with Trizol solution. Reverse transcription reaction wassubsequently performed with TaqMan® MicroRNA Reverse Transcription Kit(Applied Biosystems) according to the manufacturer’s instructions. qPCRwas finally performed with TaqMan® 2 × Universal PCR Master Mix(Applied Biosystems) on CFX96™ Real-Time PCR Detection System  supplied with analytical software.4 cells were cultured in each well of 6-well plates. TRAILmRNA abundance was determined in Ad-TRAIL-MRE-1-133-218-infected cells aftertreated with 10 MOI of adenoviruses. After 48h, cells were lysed for RNAextraction and then inversely transcribed into cDNAs with Rever Tra Ace qPCR RTKit  according to the manufacturer’s instructions. qPCR wasperformed with SYBR premix Ex Taq (TaKaRa) on CFX96™ Real-Time PCRDetection System  supplied with analyticalsoftware.4 × 105 cells were cultured in each well of 6-well plates. 10MOI of adenoviruses were added to cell cultures. Proteins were lyzed withM-PER® Mammalian Protein Extraction Reagent  after48 h, separated using polyacrylamide gel electrophoresis and transferred onto0.45 μm nitrocellulose membranes. 5% fat-free dry milk was used forblocking. The membrane was then incubated with specific primary antibodies for6h. The membrane was incubated with corresponding secondary antibody and thenwith SuperSignal West Dura Extended Duration Substrate .Protein in adenovirus-infected cells was quantified with immunoblotting assay.3.5 × 105 cells were cultured in each well of 6-wellplates. 10 MOI of adenoviruses were added to cell media. After 48h, two-antibodysandwich ELISA was applied to determine human TRAIL expression level in thesupernatant of cells. The involved antibodies are monoclonal mouse anti-humanTRAIL antibody (R&D Systems), peroxidase-conjugated rabbit anti-goat IgG(H&L) and goat anti-human TRAIL antibody (R&D Systems). The absorbancewas assessed at a 450 nm wavelength.We performed ELISA assay to evaluate the secreted TRAIL protein in media.Briefly, 3.5 × 10miR-1, miR-133, miR-218and control mimics were synthesized by GenePharma . T24 andRT-4 cells were transfected with 300 nM control mimic or the mixture of 100 nMmiR-1, 100 nM miR-133 and 100 nMmiR-218.5 cells were cultured in each well of 6-well plates.After 24h, the cells were infected with adenoviruses of 10 MOI. After 48h, thecells were stained with Annexin V-PE Apoptosis Detection Kit based on the manufacturer’s instructions. The percentages of apoptoticcells were examined with FACS analysis.3.5 × 10XhoI and NotI sites of psiCheck2 vectors to construct recombinant luciferase reporter (psiCheck2-*). Theinvolved MREs sequences in our study were described in detail inTable The synthesized DNA constructs, which contains two copies of indicated MREs, wereinserted into the 4 cells were cultured in each well of 24-well plates. Aftertransfecting T24, RT-4 and BMCs with the above plasmids, cells were processedwith lysis buffer, and subsequently, luciferase activities were assessed withthe Dual-Luciferase reporter system  according to themanufacturers’ instructions.4 × 104 T24 and RT-4 cells, 1.5 × 104 primarybladder cancer cells or 2 × 104 BMCs were cultured in each wellof 96-well plates. Adenoviruses of indicated MOIs were added to cell cultures.After 6d, 50 μl of MTT (1 mg/ml) was added, and 4 h later, MTT-containingmedia was replaced with 150 μl of DMSO. The spectrophotometric absorbancewas assessed on a model 550 microplate reader  at 570 nm with a reference wavelength of 655 nm. Cell viability = absorbancevalue of infected cells / absorbance value of uninfected control cells.1 × 10Procedures for animal experiments were all approved by the Committee on the Useand Care on Animals in Qingdao Municipal Hospital .6 T24 cells were inoculated at the left flanks of 5-week-oldfemale BALB/c nude mice . When tumors reached 7–9 mm in diameter, 24mice were equally assigned into 4 groups (n=6). 100 μL of PBS with orwithout 2×108 pfu of Ad-EGFP, Ad-TRAIL andAd-TRAIL-MRE-1-133-218 was directly administrated into tumors by injection,respectively. The administrations were performed every other day for five timeswith a total dosage of 1×109 pfu of adenoviruses.2×106 cellsat the right flanks of 5-week-old female BALB/c nude mice. 24 mice were equallydivided into 4 groups (n=6). The doses of used adenoviruses and injectionprocedures were the same as those on T24 tumor xenograft.T-24 cancer xenograft was established by incubating 1.5×103) = maximal length (mm) × perpendicular width (mm)2 / 2.We periodically measured tumor diameter using calipers. Tumor volume was harvested by cardiac puncture, followed by being incubated with 12U of heparin. Alanine aminotransferase (ALT) levels in blood were detected atthe Clinical Laboratory, Qingdao Manucipal Hospital .To evaluate the hepatoxicity induced by adenovirus treatment, BALB/c mice (n=5)were intravenously injected with 1×10On day 7 after adenovirus injection, one mouse was sacrificed from each group andits tumor, brain and liver were collected and fixed according to the routineprocedures. Histological staining was then performed on formalin-fixed,paraffin-embedded tumor, brain and liver tissue sections using thestreptavidinbiotin peroxidase complex method. Anti-TRAIL antibody  was used to specifically recognize TRAIL protein. Thesections were finally counterstained with hematoxylin.The statistical tests in this manuscript were two-tailed student’s t-test.Differences were considered as statistically significant (*) when P < 0.05and statistically very significant (**) when P < 0.01.miR-1, miR-99a,miR-101, miR-133a,miR-218, miR-490-5p,miR-493 and miR-517a had reduction of greaterthan 90% in their expression level (P<0.01)  Etoptic miRNA expression profile of T24 and RT-4 cells. Expression ofClick here for filemiR-1,miR-133a and miR-218 betweennormal cells and bladder cancer Expression of miR-1,miR-133a and miR-218 weredetected in HUV-EC-C and L-02 cells. miRNA level in HUV-EC-C cells wasregarded as standard and U6 was selected as endogenous reference. Means± SEM of three independent experiments were shown. (PPT 115 kb)Differential expression levels of Click here for file


Gentamicin (GM) is an aminoglycoside frequently used in the neonatal ICU to treat infections. Despite low resistance and costs, GM is also nephrotoxic and may cause acute kidney injury (AKI). Serum creatinine appears to be an insensitive and unreliable marker in this setting. The objective of this study was to determine whether urine biomarkers are useful for early detection of gentamicin-induced AKI in neonates in the neonatal ICU.n = 20) and a reference group (n = 13).Thirty-three neonates  with a bladder catheter without pre-existent kidney disease were divided into a GM group (S-transferase A1-1 (GSTA1-1), GSTP1-1, kidney injury marker-1 (KIM-1), N-acetyl-β-D-glucosaminidase (NAG) and neutrophil gelatinase-associated lipocalin (NGAL) were determined. Residual blood samples were used to measure serum creatinine (sCr).A prospective, clinical observational trial with non-invasive procedures. Demographics, vital signs and clinical conditions were recorded. Every 2 hours, during the period of bladder catheter, urine samples were collected and renal injury biomarkers glutathione-P < 0.04), which disappeared after the first day of the study. No significant differences were found in baseline kidney function, hemodynamics, ventilation support and reason for admission. Treatment with GM resulted in higher levels of sCr compared with the reference group (58.5 (44.8 to 78.5) vs. 34 (28.3 to 58.8) mmol/l; P < 0.05). The average time until the highest peak was shorter for both GSTA1-1 and GSTP1-1 compared with sCr (P < 0.05). Furthermore, higher levels of sCr corresponded with higher levels of urine biomarkers and KIM-1, NAG and GSTP1-1 could differentiate between the GM and reference group.Demographics were similar between both groups expect for baseline BUN (Higher sCr levels correspond with higher urinary excretion of all biomarkers, especially after GM use. In addition, the urinary biomarker GSTP1-1 might be useful for early detection of AKI in the neonatal ICU.