Caenorhabditis elegans myotubularin MTM-1 as a negative regulator of cell corpse engulfment. Myotubularins (MTMs) constitute a large, highly conserved family of lipid phosphatases. MTM gene mutations are associated with various human diseases, but the cellular functions of MTM proteins are not clearly defined. We found that inactivation of MTM-1 caused significant reduction in cell corpses in strong loss-of-function mutants of ced-1, ced-6, ced-7, and ced-2, but not in animals deficient in the ced-5, ced-12, or ced-10 genes. In contrast, overexpression of MTM-1 resulted in accumulation of cell corpses. This effect is dependent on the lipid phosphatase activity of MTM-1. We show that loss of mtm-1 function accelerates the clearance of cell corpses by promoting their internalization. Importantly, the reduction of cell corpses caused by mtm-1 RNAi not only requires the activities of CED-5, CED-12, and CED-10, but also needs the functions of the phosphatidylinositol 3-kinases (PI3Ks) VPS-34 and PIKI-1. We found that MTM-1 localizes to the plasma membrane in several known engulfing cell types and may modulate the level of phosphatidylinositol 3-phosphate (PtdIns(3)P) in vivo. We propose that MTM-1 negatively regulates cell corpse engulfment through the CED-5/CED-12/CED-10 module by dephosphorylating PtdIns(3)P on the plasma membrane.During programmed cell death, apoptotic cells are recognized and rapidly engulfed by phagocytes. Although a number of genes have been identified that promote cell corpse engulfment, it is not well understood how phagocytosis of apoptotic cells is negatively regulated. Here we have identified  Caenorhabditis elegans for negative regulators of cell corpse engulfment and identified myotubularin MTM-1. Myotubularins (MTMs) are a family of highly conserved enzymes that remove phosphate groups from membrane lipids. Mutations in human MTM genes are associated with various severe diseases including X-linked myotubular myopathy and Charcot-Marie-Tooth disease, but the normal functions of MTMs are unknown. In this study, we found that MTM-1 inhibits cell corpse engulfment through a series of evolutionarily conserved signaling molecules (the bipartite GEF (CED-5/DOCK180-CED-12/ELMO) and the GTPase CED-10/Rac). The negative regulatory effect of MTM-1 requires both its lipid phosphatase activity and the function of another group of enzymes called PI3-kinases. We propose that MTM-1 acts through Rac GTPase CED-10 by dephosphorylating the lipid PtdIns(3)P on the plasma membrane. We have identified a negative regulatory mechanism of cell corpse engulfment and a previously unknown cellular function of MTM-1, which may provide further insights into the basis of human MTM-related diseases.Clearance of dead cells is crucial for normal animal development. Cell corpses are recognized, engulfed, and removed by phagocytic cells. However, the mechanisms that regulate phagocytosis are still not well understood, especially the ways in which the process is inhibited (negatively regulated). We screened the nematode worm  C. elegans, phagocytosis of apoptotic cells is controlled by two partially redundant signaling pathways. In one pathway, three genes, ced-1, ced-7 and ced-6, are involved in recognizing and transducing the engulfment signal(s), while dyn-1 acts downstream of them to promote vesicle delivery for cell corpse internalization C. elegans homologue of the human phosphatidylserine receptor. These signaling molecules mediate activation of the small GTPase CED-10/Rac, leading to rearrangement of the actin cytoskeleton which is needed for cell corpse engulfment mig-2 or unc-73 or both, indicating that more complex regulatory mechanisms are involved in CED-10/Rac activation. In addition, as earlier studies mainly focused only on positive regulation of engulfment, it is less well understood whether any negative regulatory mechanism is involved.Phagocytosis of apoptotic cells is essential for animal development, tissue homeostasis and regulation of immune responses. Defects in this process contribute to the development of various human diseases including persistent inflammatory diseases and autoimmune disorders MTM1, the founder member of this family, cause X-linked myotubular myopathy (XLMTM), a severe congenital muscular disorder. Mutations in MTMR2 and MTMR13 are associated with Charcot-Marie-Tooth disease (CMT4B1) 5R). Intriguingly, nearly half of the known MTM1-related proteins (MTMRs) carry sequence variations in this motif and are predicted to be catalytically inactive Myotubularin phosphatases belong to the tyrosine/dual-specificity phosphatase super-family (PTP/DSP) whose members have been found in almost all eukaryotes. Mutations in myotubularin genes are associated with several human diseases 2) 22 are key regulators of the endocytic pathway 2 as substrates suggests that they may regulate endocytosis and/or other class III PI3-kinase-mediated cellular events. This idea is supported by evidence from a variety of experiments in yeast and C. elegans. Overexpression of human MTM1 decreased the level of PtdIns(3)P in S. pombe and induced a vacuolar phenotype similar to that of mutants defective in VPS34C. elegans mtm-6 activity by RNAi rescued the larval lethality of vps-34(lf) mutants, while inactivation of mtm-1 rescued the endocytosis defect in vps-34(lf) coelomocytes , indicating that myotubularins likely modulate VPS-34-mediated cellular processes in worms C. elegans MTM-1 regulates endocytosis or whether it functions in other cellular processes by regulating PtdIns(3)P.Surprisingly, instead of acting as protein tyrosine phosphatases, MTM1 and its related proteins were later found to function primarily as lipid phosphatases with specificity for phosphatidylinositol 3-phosphate (PtdIns(3)P) and its metabolite phosphatidylinositol 3,5-bisphosphate P2On the other hand, the cellular function of MTM1 in mammals appears to be more obscure. In one study, it was reported that MTM1 translocates to late endosomes after EGF stimulation and negatively regulates EGFR degradation and vesicular formation at the late stage of endosomal trafficking C. elegans MTM-1 as a negative regulator of cell corpse engulfment. We found that inactivation of MTM-1 by RNAi promotes cell corpse engulfment, whereas overexpression of MTM-1 results in accumulation of cell corpses in a manner dependent on its lipid phosphatase activity. We show that the reduction of cell corpses caused by mtm-1 RNAi requires the functions of CED-5, CED-12 and CED-10 and the activities of PI3-kinases VPS-34 and PIKI-1. MTM-1 is widely expressed in many cell types, localizes to the plasma membrane and negatively regulates the vesicular accumulation of PtdIns(3)P in C. elegans. Our data suggest that MTM-1 acts as a lipid phosphatase to negatively regulate cell corpse engulfment through the CED-5/CED-12/CED-10 module, which is likely achieved by dephosphorylating PtdIns(3)P on the plasma membrane.In the present study, we have identified ced-1(e1735) mutants (mtm-1 was one of the candidate genes identified from this screen because the accumulation of cell corpses in ced-1(e1735);rrf-3(pk1426) double mutants was significantly reduced when treated with mtm-1 RNAi  mutants ) and found that it was indistinguishable from that in wild-type embryos (mtm-1(ok742) deletion mutants caused significant reduction in cell corpses in ced-2(n1994) mutants, but not in ced-5(n1812) or ced-10(n3246) mutants ;ced-2(n1994) double mutants, but not in ced-1(e1735);ced-5(n1812), ced-1(e1735)ced-12(n3261) or ced-1(e1735);ced-10(n3246) double mutants  led to accumulation of cell corpses at every embryonic stage ;mig-2(mu28) and ced-2(n1994);mig-2(mu28) double mutants, suggesting that an additional input of the CED-10/Rac pathway in parallel to mig-2 and ced-2 may exist and mtm-1 probably functions downstream of them to inhibit the CED-5/CED-12/CED-10 module , in which ced-10 activity is only partially blocked  animals treated with control RNAi, most cell corpses persisted from 30 to 70 min, with an average duration of 63 min (ced-6(qx17) embryos treated with mtm-1 RNAi, most cell corpses persisted from 20 to 40 min, with an average duration of 34 min, which is 46% shorter than in control animals  animals treated with either control or mtm-1 RNAi (data not shown). Therefore, inactivation of mtm-1 accelerates the clearance of cell corpses.To further investigate whether the reduction in cell corpses caused by inactivation of reatment . In ced-f 63 min . In cont animals . This sull death . Consistced-1 encodes a transmembrane phagocytic receptor that acts specifically in engulfing cells to mediate the recognition and internalization of cell corpses hspannexin v::mrfp) (ced-6(qx17) mutants (at least 15 embryos were scored in each strain). We therefore used CED-1::GFP as a marker to examine whether the acceleration of cell corpse clearance caused by mtm-1 RNAi is achieved by facilitating the internalization of apoptotic cells. We found that more cell corpses were surrounded by CED-1::GFP in ced-6(qx17) animals treated with mtm-1 RNAi, suggesting that more apoptotic cells might be internalized  animals treated with mtm-1 RNAi, the CED-1::GFP ring formed rapidly around dying cells, in an average of 4.5 min (ced-6(qx17) embryos, however, many cell corpses were not fully surrounded by CED-1::GFP even after 10 min and the average formation time of a full CED-1::GFP ring was 7.4 min, which is 64% longer than that in mtm-1 RNAi-treated embryos (ced-6(qx17);mtm-1(RNAi) embryos  mutants.v::mrfp) . Annexinrnalized . A similot ced-5 . Since C 4.5 min . In cont embryos . Moreove embryos . To exclic cells . Collectmtm-1 RNAi promotes the engulfment of cell corpses, we examined the internalization of apoptotic cells by using a GFP fusion of a cytosolic actin isoform, ACT-5, which clusters around cell corpses during early stages of engulfment and disappears after they are fully engulfed ced-6(qx17) embryos treated with mtm-1 RNAi than in control animals (ced-6(qx17);mtm-1(RNAi) embryos when two different phagosomal markers, GFP::RAB-7 and LMP-1::GFP, were monitored , ced-6(n2095) and ced-2(n1994) mutants but not ced-5(n1812) mutants after mtm-1 RNAi treatment , which are located at the tips of the two gonad arms and guide the formation of gonads during larval development ced-2, ced-5, ced-12 and ced-10 mutants, the DTCs often make extra turns, which causes abnormally shaped gonads mtm-1 negatively regulates cell corpse engulfment through the CED-5/CED-12/CED-10 complex and both MTM-1 and CED-10 are highly expressed in DTCs  mutants (which are hypersensitive to RNAi treatment). Moreover, inactivation of mtm-1 by RNAi significantly enhanced the defect of DTC migration in ced-10(n1993) weak loss-of-function mutants  and found that it efficiently rescued the reduced cell corpse phenotype in mtm-1(ok742);ced-2(n1994) mutants  super-family and which are evolutionarily conserved in yeast, worms and humans  mutants . This suC. elegans, 5 MTMs have been identified based on sequence homology C. elegans MTMs are also involved in cell corpse clearance, we analyzed the cell corpse phenotype of mtm(lf) in the background of ced-1(e1735), which has a cell corpse phenotype that can be attenuated by mtm-1 RNAi. We found that the persistent cell corpse phenotype of ced-1(e1735) mutants was not affected when mtm-9, mtm-6, or mtm-5 was inactivated either individually or in combination (mtm-3 was inactivated by RNAi in ced-1(e1735) mutants, we observed a slight enhancement of cell corpse numbers which was further enhanced by mtm-6(ok330) but not mtm-5(ok469) mutants  but not mtm-5(ok469) mutants  mutants, these MTMs appear not to act redundantly with MTM-1 in cell corpse engulfment.In bination . Interes mutants . In wild mutants . This su5R active site motifs characteristic of the protein tyrosine phosphatase super-family, they primarily function as lipid phosphatases to dephosphorylate phosphatidylinositol 3-phosphate (PtdIns(3)P) or phosphatidylinositiol 3, 5-bisphosphate P2) C. elegans heat-shock promoters (PhspMTM-1(C378S)) failed to cause accumulation of cell corpses and was unable to rescue the reduced cell corpse phenotype in mtm-1(ok742);ced-2(n1994) double mutants (Although myotubularins contain CX mutants . This inmtm-1 RNAi. Three classes of phosphatidylinositol 3-kinases (PI3K) are responsible for generating 3-phosphoinositides, among which class I PI3Ks are mainly responsible for generating PtdInsP3, while the synthesis of PtdIns(3)P in vivo is mostly carried out by class III PI3Ks C. elegans contains a single homolog of each class: AGE-1, a class I PI3K essential for the C. elegans insulin-like signaling pathway vps-34 function when mtm-6 is inactivated by RNAi ok2346 of the class II PI3-kinase F39B1.1, which we named piki-1 (phosphatidylinositol 3 -kinase), resulted in accumulation of cell corpses at several embryonic stages similar to vps-34(h797), a strong loss-of-function mutant of vps-34 (vps-34(h797);piki-1(ok2346) double mutants than in either single mutant alone (vps-34(h797);piki-1(ok2346) double mutants is likely due to a defect in apoptotic cell clearance because cell corpses persist on average 1.6 times longer in embryos lacking both vps-34 and piki-1 activities than in wild type .Since human MTM1 mainly uses PtdIns(3)P as a substrate both in vitro and in vivo f vps-34 . Interesnt alone . The incild type . This inmtm-1 RNAi is able to reduce cell corpse numbers in ced-1(e1735) mutants when PI3K activity is blocked. Although a reduction in cell corpses was still observed in vps-34(h797)ced-1(e1735) or ced-1(e1735);piki-1(ok2346) double mutants treated with mtm-1 RNAi, no obvious difference in the number of cell corpses was seen in vps-34ced-1;piki-1 triple mutants after mtm-1 RNAi treatment (ced-1(e1735) mutants by mtm-1 RNAi requires the activity of both class III and class II PI3Ks. Similar effects were also observed when mtm-1 was inactivated in vps-34;piki-1 double mutants or in ced-6 or ced-7 mutants lacking both vps-34 and piki-1 functions (We next examined whether reatment . This inunctions . Therefomtm-1 promoter (Pmtm-1mtm-1::gfp), which fully rescued the cell corpse phenotype of mtm-1(ok742);ced-2(n1994) mutants (mtm-1 RNAi rescues the coelomocyte uptake defect in vps-34(lf) mutants (ced-1 promoter (Pced-1mtm-1), which drives gene expression specifically in engulfing cells, fully rescued the reduced cell corpse phenotype in mtm-1(ok742);ced-2(n1994) mutants. This rescuing activity was not observed when MTM-1 expression was controlled by the egl-1 promoter (Pegl-1mtm-1), which drives gene expression specifically in dying cells  also localizes to plasma membranes and overlaps with CED-1::mCHERRY, a cell surface phagocytic receptor  that contains the catalytic activity, and a C-terminal coiled-coil domain (CC) which might be involved in protein-protein interactions ced-1 promoter and examined their localizations. All of the MTM-1 truncations were expressed at the expected size in C. elegans (mtm-1(ok742);ced-2(n1994) mutants, suggesting that the coiled-coil motif is dispensable for MTM-1 function in cell corpse engulfment  mutants in which the production of PtdIns(3)P is largely blocked vps-34(h797) mutants (piki-1(ok2346) mutants or vps-34(h797);piki-1(ok2346) double mutants P is required for locating MTM-1 to the cell membrane by examining the MTM-1::GFP expression pattern in  mutants . Moreove mutants . Therefomtm-1 can negatively regulate PtdIns(3)P levels in C. elegans, we monitored the localization and accumulation of PtdIns(3)P in both wild type and mtm-1(ok742) mutants using a YFP::2xFYVE probe which specifically binds PtdIns(3)P on both endosomes and phagosomes mtm-1(ok742) larvae, and the number of positive vesicles was greatly reduced when vps-34 activity was inhibited (vps-34 RNAi (vps-34 is responsible for generating PtdIns(3)P on these vesicles. In contrast, over 66% of mtm-1(ok742) worms contained more than 110 vesicles positive for YFP::2xFYVE. The average number of YFP-2xFYVE-positive vesicles in mtm-1(ok742) animals was 128, which is 62% more than in wild type, suggesting that MTM-1 negatively regulates the vesicular accumulation of PtdIns(3)P P. To test this hypothesis and assess whether nhibited . This in-34 RNAi . This indIns(3)P .C. elegans showed that blocking engulfment enhances the survival of cells triggered to initiate programmed cell death The rapid and efficient phagocytosis of apoptotic cells is crucial for maintaining homeostasis as well as regulating the immune responses. Therefore, it seems to be counterintuitive to think that engulfment needs to be down-regulated. In fact, phagocytosis of apoptotic cell not only clears dead cells but promotes cell killing. For example, macrophages taking up apoptotic cells will release FasL and promote Fas-mediated apoptosis of monocytes and neutrophils for quick resolution of inflammation C. elegans ABL-1 kinase was reported to inhibit cell corpse engulfment and DTC migration through its interacting protein ABI-1 Although many genes are known be involved in apoptotic cell clearance, very few of them play a negative role. In mammalian macrophages, the small GTPase RhoA and its effector Rho-kinase were shown to negatively regulate the engulfment of apoptotic cells C. elegans myotubularin MTM-1, which was previously reported to play a role in endocytosis. Our genetic and cell biological analyses indicate that inactivation of MTM-1 promotes cell corpse engulfment, while overexpression of MTM-1 results in accumulation of cell corpses, suggesting that MTM-1 negatively regulates the engulfment of apoptotic cells. Given that inactivation of mtm-1 reduced the number of cell corpses in strong loss-of-function mutants of ced-1, ced-6, ced-7 and ced-2 but not ced-5, ced-12 or ced-10, mtm-1 likely functions through the CED-5/CED-12/CED-10 complex. We still observed a significant reduction in cell corpses when mtm-1 was inhibited in ced-2(n1994);mig-2(mu28) double mutants, which suggests that MIG-2 and CED-2 do not mediate all the inputs into the CED-10 pathway and an additional branch may exist in parallel to both of them. Since overexpression of MTM-1 failed to enhance the engulfment phenotype in ced-2, ced-5, ced-12 or ced-10 mutants, we prefer a model in which mtm-1 functions downstream of mig-2 and ced-2 to negatively regulate the CED-5/CED-12/CED-10 complex rather than a model where mtm-1 acts in a parallel pathway which requires CED-10 function for engulfment. Recently, ABL-1 kinase was reported to inhibit both cell corpse engulfment and DTC migration by acting in a parallel pathway to the two known engulfment pathways. Moreover, the functions of CED-5, CED-12 and CED-10 were required for its inhibition of engulfment but not DTC migration In order to gain more insight into the negative regulation of phagocytosis, we performed an RNAi screen to search for negative regulators of cell corpse engulfment and identified C. elegans, 5 MTMs  have been identified based on sequence homology. They belong to 5 different subgroups with mtm-5 and mtm-9 encoding catalytically inactive phosphatases and probably perform non-redundant functions like mammalian MTMs mtm-3(lf);mtm-6(lf) double mutants contain significantly higher numbers of cell corpses than wild type and they also enhance the cell corpse phenotype of ced-1(e1735) mutants, suggesting that mtm-3 and mtm-6 may play a redundant role to either promote cell corpse clearance or affect cell death activation. Since mtm-6 has only been implicated in regulating an ARF-6- and RME-1-dependent endocytic pathway together with mtm-9, whereas the functions of mtm-3 are mostly unknown, it will be important to determine whether these MTMs are directly involved in cell death processes and if so, which specific steps they regulate and how their activities are coordinated.In 2vps-34 and the class II PI3K piki-1 are required for reducing cell corpses by mtm-1 RNAi and that loss of mtm-1 function increases the vesicular accumulation of PtdIns(3)P in vivo, PtdIns(3)P likely serves as a substrate of MTM-1 during engulfment. Although PtdIns(3)P is enriched in endocytic compartments vps-34(lf), piki-1(lf) or vps-34(lf);piki-1(lf) double mutants, in which PtdIns(3)P generation is probably blocked 2 are concentrated on early and late endocytic compartments, respectively, which leads to the hypothesis that myotubularins may act to prevent accumulation of PtdIns(3)P or PtdInsP2 at inappropriate compartments Myotubularins are lipid phosphatases that specifically dephosphorylate PtdIns(3)P or its metabolite PtdInsPmtm-1 antagonizes cell corpse internalization, which requires both its lipid phosphatase activity and the functions of PI3Ks VPS-34 and PIKI-1, (ii) MTM-1 localizes to the plasma membrane, functions in engulfing cells and clusters around cell corpses during engulfment, and (iii) MTM-1 negatively regulates the vesicular accumulation of PtdIns(3)P in vivo. Interestingly, DOCK180, the mammalian homolog of CED-5, binds directly to PtdInsP3 through its DHR1 domain in vitro and translocates to the cell membrane in response to PtdInsP3 production in NIH3T3 cells 3 appears not to be required for cell corpse engulfment in C. elegans, it is possible that CED-5/CED-12 GEF is recruited to the plasma membrane by PtdIns(3)P to activate CED-10/Rac, and that MTM-1 acts antagonistically to terminate the signal and release the complex after engulfment. Alternatively, PtdIns(3)P may bind and facilitate the nucleotide exchange of CED-10/Rac during engulfment, similar to the way in which PtdInsP2 promotes Rho and Rac activation under certain conditions Although PtdIns(3)P is a proven critical regulator of early endosomal traffic and phagosome maturation, its involvement in cell corpse engulfment has not been reported. We propose that PtdIns(3)P acts as a positive signal for engulfment based on our findings that (i) mtm-1(lf) mutants suggests that the role of MTM-1 in cell corpse engulfment is likely conserved from worms to humans and that similar mechanisms may also be used in mammals to regulate the removal of apoptotic cells.Myotubularin family phosphatases are conserved amongst all eukaryotic organisms, but their cellular functions are not well understood. Our finding that overexpression of human MTM1 can efficiently rescue the cell corpse phenotype of mtm-1 seems to play a positive instead of a negative role in the migration of DTCs, a process which also requires the activity of CED-5, CED-12 and CED-10. MTM-1 may therefore play distinct roles in different CED-10/Rac-mediated processes. However, we currently do not know whether the function of MTM-1 in DTC migration is also mediated by PtdIns(3)P and, if so, whether the opposite roles of MTM-1 in cell corpse engulfment and DTC migration are caused by distinct effects of PtdIns(3)P on CED-10/Rac in these two processes. On the other hand, loss of myotubularin has been found to cause muscle defects in human, mouse and zebrafish We found that MTM-1 acts as a lipid phosphatase to negatively regulate the engulfment of apoptotic cells through CED-10/Rac, which raises the interesting question of whether MTM-1 is similarly involved in other CED-10/Rac-mediated cellular processes by regulating PtdIns(3)P. Intriguingly, we found that C. elegans were cultured at 20°C using standard procedures C. elegans II ced-1(e1735), vps-34(h797), dpy-5(e61), unc-13(e450am), mtm-1(ok742) (this study), ced-12(n3261)hT2(bli-4(e937);let-?(q782)qIs48/sep-1(e2406) (Wormbase:www.wormbase.org). LGII: rrf-3(pk1426)ced-6(n2095), ced-6(qx17) (this study and see below), ced-7(n2094), mtm-6(ok330) . LGIV: ced-2(n1994), ced-5(n1812), ced-10(n3246), ced-10(n1993). LGX: mig-2(mu28)piki-1(ok2346) and mtm-5(ok469) . smIs34 (Pced-1ced-1::gfp) and smIs95 (Phspannexin v::mrfp) were provided by Dr. Ding Xue . opIs334 (Pced-1YFP::2xFYVE) was a gift from Dr. K. S. Ravichandran  and Dr. M. O. Hengartner  Strains of Other strains carrying integrated arrays used in this study are listed below:qxIs156 (PhspMTM-1), qxIs210 (PhspMTM-1(C378S)), qxIs197 (Pmtm-1MTM-1::GFP), qxIs66 (Pced-1GFP::RAB-7), qxIs40 (Pced-1ACT-5::GFP), qxIs60 (Pced-1LMP-1::GFP). The vps-34-deficient strain, vps-34(h797), is maintained as dpy-5(e61)vps-34(h797); qxEx . Non-green embryos (dpy-5(e61)vps-34(h797)) were scored as vps-34(lf) and green embryos that carry the vps-34(+) transgene were scored as wild type for vps-34.qx17 is a recessive mutation isolated from a forward genetic screen for additional regulators of cell corpse engulfment and was mapped to linkage group III very close to ced-6. A complementation test between qx17 and ced-6(n2095) was performed and a similar engulfment phenotype was observed in the double heterozygote, indicating that qx17 is an allele of ced-6. We determined the sequence of ced-6 in qx17 mutants and identified a T to A transition that results in a premature stop codon after Glu 475 and generates a truncated CED-6 protein lacking the last 17 amino acids at the C-terminus.C. elegans embryos were mounted on agar pads in M9 and viewed using a 100× Plan-Neofluar DIC objective on an Axioimager M1 microscope . The number of cell corpses was quantified in the head region of living embryos either at the six different embryonic stages  for a time-course analysis or at the 4-fold embryonic stage as described before vps-34(h797);piki-1(ok2346) mutants vps-34(lf) causes embryonic lethality and most vps-34(h797) mutant embryos die before the 4-fold embryonic stage, it is maintained as dpy-5(e61)vps-34(h797); qxEx . To monitor the cell corpse duration in vps-34(h797);piki-1(ok2346) mutants, only non-green embryos which developed normally until the 2.5- or 3-fold stage were followed and quantified. To examine the internalization of cell corpses, apoptotic cells were first identified by their refractile disk-like morphology using Nomarski DIC microscopy and then different fluorescent markers that associate with cell corpses at different stages of engulfment were examined. To monitor the occurrence of embryonic cell death, embryos at the 2–4 cell stage were mounted on agar pads and images in a 30 µm z series (0.75 µm/section) were captured every 1.5 minutes for 8 h using a Zeiss Axioimager M1 microscope . Images were processed and viewed using Axiovision Rel 4.5 software .Somatic cell corpses were directly visualized by differential interference contrast (DIC) microscopy as highly refractile button-like objects distinct from normal living cells ced-1(e1735);rrf-3(pk1426) were bleached on NGM plates without OP50. The hatched larvae were washed off with M9 and added to individual RNAi plates with 30 larvae per plate . Cell corpses were scored in the progeny at the 4-fold embryonic stage. For mtm-1 RNAi by feeding, gravid adults of the indicated strains (P0) were picked and bleached on either control (pPD129.36-gfp or pPD129.36 for examining MTM-1::GFP expression) or mtm-1 RNAi plates (I-6C09) and the number of embryonic cell corpses or the expression of MTM-1::GFP was scored in the F2 generation. To quantify the DTC migration defect, L4 larvae of the F2 generation were aged 24 h before examination. mtm-1 RNAi caused 14% and 40% embryonic lethality in wild type and the RNAi hypersensitive mutant rrf-3(pk1426), respectively, while 7% of wild-type and 29% of rrf-3(pk1426) embryos died when treated with gfp RNAi. For mtm-1 RNAi by injection, a double-stranded RNA (dsRNA) of mtm-1 (436–2489 bp of Y110A7A.5) was synthesized in vitro and injected into ced-1(e1735);qxIs197 animals, which carry an integrated array of MTM-1::GFP controlled by the mtm-1 promoter (Pmtm-1MTM-1::GFP). Embryonic cell corpses and expression of MTM-1::GFP were both examined 24 h post injection. For mtm-3 and mtm-9 RNAi, double-stranded RNAs (dsRNA) were synthesized  and injected individually or in combination. Embryonic cell corpses were quantified 24 h post injection.The genome-wide RNAi screen was performed as described before with some modifications Young adults were moved to fresh nematode growth medium (NGM) plates and cultured at 20°C for 12 h before they were incubated at 33°C for 1 h (+HS) or left at 20°C without heat-shock treatment (−HS), followed by recovery at 20°C for 1.5 h. Adult worms were removed and embryos were incubated at 20°C and scored for the number of cell corpses 5 to 10 h after treatment.C. elegans embryos at the pre-comma or comma stage were mounted on agar pads and images in a 20–25 z series (1.0 µm/section) were captured every 1 min, 1.5 min or 2 min for 120 min using a Zeiss LSM 510 Pascal inverted confocal microscope . Images were processed and viewed using LSM Image Browser software.A Zeiss LSM 510 Pascal inverted confocal microscope with 488, 514, 633 lasers (Carl Zeiss Inc.) was used to capture fluorescent images which were processed and viewed using LSM Image Browser software. Time-lapse imaging of CED-1::GFP, GFP::MTM-1 and CED-1::mCHERRY was performed as described mtm-1(ok742) mutants derived from hT2/mtm-1(ok742) worms, and vps-34 RNAi-treated wild type or mtm-1(ok742) mutants were mounted on agar pads. Nomarski and fluorescent images of the midbody region of L2 larvae in a 20 z series (1.0 µm/section) were captured using a fixed exposure time with a Zeiss Axioimager A1 equipped with epifluorescence and an Axiocam monochrome digital camera. The exposure time was 550 ms for wild type and mtm-1(ok742) mutants, but 1500 ms for animals treated with vps-34 RNAi because only very faint YFP::2xFYVE signal can be observed at 550 ms in these worms due to significant reduction of PtdIns(3)P accumulation caused by loss of vps-34 function. Serial optical sections were analyzed and the numbers of YFP::2xFYVE-positive vesicles were quantified in hypodermal cells in a region of approximately 88 µm×18 µm. At least 30 animals were quantified in each strain.To examine the level of PtdIns(3)P using the YFP::2xFYVE probe, L2 larvae of wild type, surviving mtm-1mtm-1::gfp , a 3.9 kb genomic fragment of mtm-1 including a 0.8 kb promoter region was amplified by PCR from WRM0617dG02 using primers PQL121/120 and cloned into the pPD95.77 vector through its Bam HI and Sma I sites. A 3.1 kb fragment containing the full-length genomic sequence of the mtm-1 gene was PCR-amplified from WRM0617dG02 using primers PWDL108/109 and cloned into both pPD49.78 and pPD49.83 through the Nhe I-Kpn I sites to generate Phspmtm-1. The same mtm-1 genomic fragment was also amplified by primers PWZ215/PWDL109 and cloned into Pced-1gfp vector through the Kpn I site to generate Pced-1gfp::mtm-1. The C378S mutation was introduced into pPD49.83-mtm-1 by site-directed mutagenesis using primers PQL148/149  and re-cloned into both pPD49.78 and pPD49.83 via their Nhe I and Kpn I sites. To construct Pced-1mtm-1 and Pegl-1mtm-1, the full-length cDNA of mtm-1 was amplified from a C. elegans cDNA library  by PWZ215/PWDL109 and cloned into both Pced-1 and Pegl-1 vector through the Kpn I site. To generate Pced-1GFP::MTM-1(ΔGRAM), Pced-1GFP::MTM-1(ΔPTP) and Pced-1GFP::MTM-1(Δcoiled-coil), a 2.1 kb genomic fragment of mtm-1 (ΔGRAM: 975–3072 bp) or a 1.7 kb mtm-1 genomic sequence (1–1739 bp) (ΔPTP) or a 3 kb mtm-1 fragment (Δcoiled-coil: 1–3000 bp) were PCR-amplified from WRM0617dG02 using primers PWZ291/PWDL109, PWZ215/PWZ151 and PWZ215/PWZ322, respectively, and cloned into the Pced-1gfp vector via its Kpn I site. To construct PhsphMTM1, the full-length cDNA of human MTM1 was amplified from a human cDNA library  with primers PWZ384/385 and cloned into both pPD48.78 and pPD49.83 through the Nhe I-Kpn I sites. To generate Phspmyri::mcherry, mcherry was amplified from pAA65 PCR primer sequences are shown in Figure S1mtm-1 RNAi treatments specifically inhibit the expression of mtm-1. (A) mtm-1 RNAi treatments (either feeding with bacteria expressing mtm-1 dsRNA or injecting in vitro-synthesized mtm-1 dsRNA) result in reduction of cell corpse numbers and inhibition of MTM-1::GFP expression. RNAi experiments were performed as described in mtm-1 RNAi treatment are also shown. The exposure time of both pictures was 2000 ms.  The gene structures of mtm-1 and piki-1 are shown, with filled boxes representing the exons and thin lines indicating the introns. The arrows show the direction of the transcript. The gray bars below the genes indicate the position and size of the deletions in the ok742 and ok2346 mutant.(1.05 MB TIF)Click here for additional data file.Figure S2mtm-1 RNAi accelerates internalization of the apoptotic cell C3. (A) DIC and fluorescence images of a wild-type embryo co-expressing CED-1::GFP (Pced-1ced-1::gfp) and a secreted Annexin V::mRFP under the control of heat-shock promoters (Phspannexin v::mrfp) are shown. The apoptotic cell C3 (white arrow) and a posterior apoptotic cell (blue arrow) were labeled by both CED-1::GFP and Annexin V::mRFP. The ventral hypodermal cell that engulfs C3 is indicated by the arrowhead. Bars: 5 µm.  The formation and duration of the CED-1::GFP ring around C3 (arrowed) were followed in ced-6(qx17) mutants treated with either control (i–iv in B and i–v in C) or mtm-1 RNAi (v–viii in B and vi–x in C). To monitor the formation of CED-1 rings, the “0 min” time point was set immediately prior to the appearance of trace amounts of CED-1::GFP around C3. To monitoring the duration of CED-1 rings, the “0 min” time point was set when a full CED-1::GFP ring was just visible. 13 C3 corpses were monitored and quantified for either formation or duration of CED-1::GFP rings (ix in B and xi in C). The numbers in parenthesis indicate average formation or duration times of CED-1::GFP rings (±s.e.m). Bars: 5 µm.(2.95 MB TIF)Click here for additional data file.Figure S3t tests were performed to compare the data derived from mtm-1 RNAi-treated embryos with that from control animals. **P<0.0001, *P<0.01; all other points had P value>0.01.Inactivation of MTM-1 promotes cell corpse internalization. Clustering of CED-1::GFP around cell corpses (A) or the phagosomal association of GFP::RAB-7 (B) and LMP-1::GFP (C) was quantified in 1.5-fold stage embryos in the indicated strains. At least 15 embryos were scored in each strain. Error bars indicate s.e.m. Unpaired (0.13 MB TIF)Click here for additional data file.Figure S4C. elegans MTM-1 (c.eMTM-1), human myotubularin (hMTM1) and yeast myotubularin (Ymr1p) are shown. Identical residues are in black and similar ones are in gray. Conserved motifs are boxed. The signature CX5R active site motif for the protein tyrosine phosphatase super-family is circled in red. The critical cysteine residue, which is changed to serine in the C. elegans MTM-1(C378S) mutant, is marked by a red arrowhead.Myotubularin is conserved in yeast, worms and humans. Protein sequence alignments of (0.50 MB TIF)Click here for additional data file.Figure S5mtm-1mtm-1::gfp are shown. MTM-1::GFP was seen in distal tip cells , coelomocytes  and vulva cells . Bars: 10 µm. (B) Both full-length and truncated GFP::MTM-1 were stably expressed in C. elegans. Lysates were prepared from 200 adult transgenic worms carrying Pced-1GFP::MTM-1, Pced-1GFP::MTM-1(δGRAM), Pced-1GFP::MTM-1(δPTP) or Pced-1GFP::MTM-1(δCC) and western blot analysis was performed using an anti-GFP antibody. Full-length GFP::MTM-1 (93 Kd) and the three GFP::MTM-1 truncations  were all expressed at the expected size. (C) DIC and fluorescence images of MTM-1::GFP in wild-type , vps-34(h797) , piki-1(ok2346)  and vps-34(h797);piki-1(ok2346)  embryos are shown. The plasma membrane localization of MTM-1::GFP is not affected in the loss-of-function mutants of PI3-kinases. Bars: 5 µm.Plasma membrane localization of MTM-1 does not require the activities of PI3-kinases. (A) MTM-1::GFP is expressed in many different cell types. DIC and fluorescence images of wild-type animals expressing P(2.85 MB TIF)Click here for additional data file.Table S1Inactivation of MTM-1 affects the migration of DTCs.(0.04 MB DOC)Click here for additional data file.Table S2The lipid phosphatase activity and conserved domains of MTM-1 are important for its function in cell corpse engulfment.(0.04 MB DOC)Click here for additional data file.Table S3Other MTMs do not play redundant roles with MTM-1 in cell corpse engulfment.(0.04 MB DOC)Click here for additional data file.Table S4Primers used for plasmid construction.(0.04 MB DOC)Click here for additional data file.Video S1ced-6(qx17) embryo treated with mtm-1 RNAi. The formation of a CED-1::GFP ring around a dying cell in a mtm-1 RNAi-treated ced-6(qx17) embryo is shown. The cell corpse followed is indicated by an arrow. The frames were collected every 1 min and displayed every 1 sec. Selected images are shown in The clustering of CED-1::GFP in a (0.35 MB AVI)Click here for additional data file.Video S2ced-6(qx17) embryo treated with control RNAi.The formation of a CED-1::GFP ring around a dying cell in a control RNAi-treated ced-6(qx17) embryo is shown. The cell corpse followed is indicated by an arrow. The frames were collected every 1.5 min and displayed every 1 sec. Selected images are shown in The clustering of CED-1::GFP in a (0.33 MB AVI)Click here for additional data file.Video S3ced-6(qx17) embryo treated with control RNAi.The duration of the CED-1::GFP ring around a cell corpse in a control RNAi-treated ced-6(qx17) embryo is shown. The cell corpse followed is indicated by an arrow. The frames were collected every 1 min and displayed every 1 sec. Selected images are shown in The duration of CED-1::GFP around dying cell in a (0.81 MB AVI)Click here for additional data file.Video S4ced-6(qx17) embryo treated with mtm-1 RNAi.The duration of CED-1::GFP around a cell corpse in a mtm-1 RNAi-treated ced-6(qx17) embryo is shown. The cell corpse followed is indicated by an arrow. The frames were collected every 1 min and displayed every 1 sec. Selected images are shown in The duration of CED-1::GFP around dying cell in a (0.52 MB AVI)Click here for additional data file.


A new human extrahepatic bile duct carcinoma cell line, designated OCUCh-LM1, was established from a liver metastatic lesion in a 61-year-old male. OCUCh-LM1 cells proliferated in a monolayered sheet with a population doubling time of 31 h. OCUCh-LM1 cells had an aneuploid pattern with a DNA index of 1.76, and chromosome counts showed a unimode of 63. OCUCh-LM1 cells expressed various carbohydrate antigens, including sialyl Lewis X, carbohydrate antigen 19-9 and SPan-1 antigen. Subcutaneous injections of OCUCh-LM1 cells induced tumour formation in nude mice. The reconstituted tumours were classified as well-differentiated adenocarcinoma. A daughter line, designated OCUCh-LM1-H1, was also established from liver metastatic colonies induced by splenic injection of OCUCh-LM1. OCUCh-LM1-H1 cells showed a higher potential for liver metastasis than OCUCh-LM1 (100% vs 20%). Since OCUCh-LM1 retains the initial characteristics (as of 93 passages at present) and expresses various carbohydrate antigens, the OCUCh-LM1 cell line and liver metastatic model established here will be useful for the study of the biological nature of extrahepatic bile duct carcinoma and the relationship between the expression of carbohydrate antigens and metastatic potential.


Proliferation, apoptosis, migration, and angiogenesis by endothelial cells are modulated, but may involve both stimulation and inhibition depending on the specific circumstances. PPAR-γ-receptor activation stimulates the production of nitric oxide, C-type natriuretic peptide, and superoxide dismutase, while endothelin-1 production is inhibited. Together, they augment endothelial function, resulting in blood pressure lowering and direct renoprotective effects. The presentation of adhesion molecules and release of cytokines recruiting inflammatory cells are inhibited by PPAR-γ-agonism. Finally, PPAR-γ-receptors are also found on endothelial progenitor cells and PPAR-γ-agonists stimulate progenitor-mediated endothelial repair. Together, the stimulatory effects of PPAR-γ-agonism on endothelium make an important contribution to the beneficial actions of PPAR-γ-agonists on renal disease.Renal endothelial damage is pivotal in the initiation and progression of renal disease. Damaged renal endothelium may be regenerated through proliferation of local endothelium and circulation-derived endothelial progenitor cells. Activation of the PPAR- Currently, the clinicallymost used PPAR-γ-agonistic drugs are the thiazolidinediones (TZDs). PPAR-γ-agonists may favorably affect the course of renaldisease in both diabetic and nondiabetic conditions [γ receptors, which are expressed on glomerularendothelium [γ-receptors may beincreased, for example, as transiently occurs after ischemia- reperfusion [γ-agonists also increases the expression of PPAR-γ-receptorson renal endothelium in both the glomerulus and the capillary endothelium ofthe medullary vasa recta [γ-receptors on renal endotheliumis becoming increasingly clear and is the focus of this review.PPAR-nditions , 2. Innnditions ,passivenditions ,the devnditions ,renal inditions , and antnditions . Onepotothelium , 9. Inrerfusion , 9. Treasa recta . TherelRenal microvascular endothelial injury is a pivotal pathogenic factor forvarious renal diseases. Renal disease conditions involving prominentendothelial damage include ischemic nephropathy, glomerulonephritis,interstitial nephritis, and allograft rejection , 12. EndProgression of renaldisease does not only depend on the degree of microvascular endothelial injury,but also on the effectiveness of endothelial repair. Impaired glomerular capillary repair was foundto be associated with the development of glomerulosclerosis and renal failure . During Enhancing renal endothelialrepair offers therapeutic potential. Stimulating angiogenesis withVEGF-treatment augments capillary repair and renal recovery afterglomerulonephritis . Enhanciγ-receptorimply a role in the regulation of vascular function and remodeling. In humanswith dominant-negative heterozygous mutations in the PPAR-γ-receptor causing animpaired capacity for transcriptional activation, diabetes and alsohypertension occur at an unusually young age [γ-null mice areembryonically lethal due to placental dysfunction, characterized by defectivetrophoblast differentiation and markedly impaired placental vascularization [γ-knockoutmouse that was supplementedwith a wild-type placenta developed an apparently normal vascular system duringfurther embryogenesis, but died some days after birth due to a combination ofpathologies, including severe lipodystrophic changes and hemorrhages [γ-knockout mice from embryoniclethality by preserving PPAR-γ-function in the trophoblast marked lipodystrophywas also observed, together with insulin resistance. Surprisingly, these micewere hypotensive and showed increased endothelium-dependent relaxation inresponse to acetylcholine [γ-functionwas deficient in all cell types. Mice in which PPAR-γ-function was selectivelyknocked out in endothelial cells only using again a cre-lox system were foundto be hypertensive when fed a high-fat diet [γ-receptor, endothelium-dependent blood vessel dilatationis impaired, while the endothelium is more sensitive to endothelin-1-inducedvasoconstriction [γ-mutations were found not to be fully selective, therefore, it cannot beexcluded that some of the effects observed in the knockout systems areattributable to PPAR-γ-receptor-independent signaling, in particular throughthe alternative PPAR-receptors [Phenotypicalstudies in humans and animals with genetic mutations in the PPAR-oung age . Heterozoung age  and decroung age , but do oung age . Howeverrization . A surviorrhages . In a colcholine . In the fat diet . Also, itriction . Superoxtriction . Cerebratriction . Of noteeceptors .γ-agonist treatment on proliferation, migration, and angiogenicnetwork formation of cultured endothelial cells showed variable results. Fukunagaet al. demonstrated increased proliferation with both troglitazone andpioglitazone in four different endothelial cell lines [Reports on the directeffects of PPAR-ll lines . Incontll lines  and humall lines . Rosiglill lines , whileall lines .γ-agonists troglitazone andciglitazone, which was mediated by inhibition of Akt [γ-agonism observed by Xin et al [γactivation using the GW1929 compound, mainly through a VEGF-dependent mechanism [VEGF-induced endothelialmigration was found to be inhibited by PPAR-n of Akt . Thisisin et al . Howeverin et al . Also,Bechanism .γ-agonismon endothelial cell apoptosis have been similarly variable. Both spontaneousand TNF-alpha-induced endothelial apoptosis were shown to be inhibited byvarious thiazolidinedione PPAR-γ-agonists [γ-receptor overexpression [γ ligand 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2)-induced endothelialcell apoptosis [γ-receptor activation [In vitro effects of PPAR-agonists  and trogagonists , while apression . In sevpoptosis , 48, 49,tivation .γ-agonists may modulate endothelial proliferation, migration,angiogenesis, and apoptosis in vitro, but that this may result in bothstimulation and inhibition. The factors that determine whether treatment with a PPAR-γ-agonist results in a stimulatory or inhibitory effect on endothelial cells remain largely unidentified. This may involve PPAR-γ-receptor-independent effects asreported for 15d-PGJ2 [γ-agonistdosages and inhibition at higher dosages [Taken together, it is clearthat PPAR-15d-PGJ2 , 50 and 15d-PGJ2 . Fukuna dosages . Howeverγ-agonist pioglitazoneaugmented the angiogenic response and increased blood flow recovery [γ-agonist treatment stimulated local angiogenesisand improved functional neurological recovery [γ-agonisttreatment inhibited pathological choroidial and retinal neovascularization [γ-agonism may differentially affectneovascularization with inhibition of pathological neovascularization andaugmentation of physiological neovascularization. This is in line with the invitro observations of both stimulatory and inhibitory actions.Invivo studies in diabetic animals with impaired angiogenesis in response toperipheral ischemia showed that treatment with PPAR-recovery . In ratsrecovery . In contrization  and supprization , 55. Theγ-activation have consistently shown astimulating effect on NO production by endothelial cells [γ blocked the increase inNO-production confirmed that this effectis PPAR-γ-receptor mediated [γ-activation has beenshown to augment systemic NO-production and endothelial function. In diabeticrats, PPAR-γ-agonist treatment restoredimpaired endothelium-dependent arterial relaxation by increasing NO-productionwhile reducing oxidative stress [γ-agonisttreatment enhanced endothelial function [Endothelial integrity doesnot only rely on the number of cells, but also on their function. NO-productionis a key component of endothelial function. NO stimulates vasodilation, inhibitsinflammation, prevents platelet activation, and scavenges radicals . Invitral cells –60. The mediated . Inbothe stress . Inheale stress . Innonde stress . Intypee stress , 65 and e stress . Rosiglie stress . Long-tee stress . In renafunction .γ-agonisttreatment lowered blood pressure and ameliorated abnormal pressure natriuresis in association withincreased renal NO-metabolite nitrite/nitrateproduction [γ-agonist treatment also reduced bloodpressure, increased NO-metabolite nitrite/nitrate excretion, and reducedexcretion of oxidative-stress associated urinary isoprostanes and lipidperoxides [Importantly, the observedbeneficial effects on systemic endothelial function were found to extend to improving intrarenalNO-production. In human type 2 diabetic patients, rosiglitazone treatmentincreased intrarenal NO levels, which was associated with improvement of renalhemodynamics and reduction of proteinuria . This isoduction . Inobeseroxides . Inthe eroxides .γ-activating compounds in the signaling levelat which NO-production is stimulated. The NO producing enzyme endothelialnitric oxide synthase (eNOS) is not only regulated at the level oftranscription and translation, but also has multiple phosphorylation sites foractivation and deactivation of the enzyme and requires translocation to thecaveolae to associate with its cofactors and effectively produce NO [γ pathways as NO has been shown to rapidly and dose-dependentlyincrease PPAR-γ-binding, mediated by p38 MAPK activation [Differences have beenobserved between the various PPAR-oduce NO , 74. Trooduce NO ,while noduce NO , 58, piooduce NO . 15d-PGJoduce NO . Interestivation .γ-agonists rosiglitazone and pioglitazoneon endothelial NO-production was associated with increasedeNOS-phosphorylation, while eNOS mRNA and total protein levels were notaffected [Invivo, the stimulatory effect of PPAR-affected , 75. Rosaffected . This isaffected –79.γ-agonism favorablyaffects other endothelium-derived factors that act in conjunction with NO to maintainendothelial homeostasis. The release of C-type natriuretic peptide, anothervasodilatory peptide, by endothelial cells is increased by PPAR-γ-agonisttreatment [γ-agonist treatment increased Cu2+ and Zn2+-superoxidedismutase expression in cultured endothelial cells, thereby increasing their potentialfor oxygen radical scavenging [γ-agonist treatment decreasesthe production of radical oxygen species in endothelial cells [PPAR-reatment . In addiavenging , 81. Alsal cells , inpartal cells , 81.γ-agonist treatment. Pioglitazone treatment prevented hypertension and renal oxidative stressboth by reducing free-radical production and by increasing nitric oxideproduction [γ-agonist treatment in rats alsoreceiving NO-inhibitor L-NAME [γ-agonisttreatment, such as effects on the contractility and proliferation of smoothmuscle cells [Improving NO-availabilityis thought to be a major mechanism mediating the blood pressure lowering effectof PPAR-oduction . No bloor L-NAME . Howeverle cells .γ-activation inhibits the production ofendothelin-1 from endothelial cells in vitro [γ-activation on endothelin-1 secretion takes place atthe transcriptional level [γ-activation negatively interferes with the activator protein-1 signalingcascade, resulting in inhibition of thrombin-induced transcription ofendothelin-1 [A naturally occurringantagonist of NO is the vasoconstrictor endothelin-1, which is involved inatherosclerosis and hypertension . PPAR-γ-in vitro , 86–88. in vitro . Interesin vitro . In DOCAin vitro . Severalal level  andit wal level . Delerivγ signaling pathways. Infusionof angiotensin-II downregulates PPAR-γ-receptor expression in the vascular wall [γ-agonistsabrogates many of the angiotensin-II pathophysiological effects [γ-agonists [Inhypertension, increased production and secondary effects of angiotensin-II playa major role. In endothelial cells, angiotensin-II is a strong inducer ofNADPH-oxidase, resulting in the production of radical oxygen species . Intereslar wall  and trea effects . Of noteagonists .γ-agonist treatmentinhibits the increased expression of adhesion molecules such as VCAM-1, ICAM-1,and E-selectin and release of inflammatory cytokines upon stimulation ofendothelial cells with PMA [α [γ [β [γ-activation resulted in a marked reductionof monocyte adhesion and in vitro transendothelial migration, mediated byinhibition of Rac1 and RhoA GTPases [Endothelial cells form animportant barrier between the blood and peripheral tissues and regulate homing,adhesion, and transmigration of inflammatory cells. Upon activation,endothelial cells may further release inflammatory cytokines and expressadhesion molecules to attract inflammatory cells. PPAR-with PMA , 96, TNFh PMA [α , 97, IFNMA [α [γ , IL-1β [[α [γ [β , LPS [96[α [γ [β , and hig[α [γ [β . In vitr[α [γ [β , 102. In GTPases .γ-agonists is the capacity to augment the level and function of endothelialprogenitor cells. PPAR-γ-agonist treatment increases endothelialprogenitor cell levels in mice [γ-agonist treatment ex vivo [γ-agonist treatment largely prevented apoptosis of endothelial progenitorcells induced by CRP [2O2[A recently uncovered effectof PPAR- in mice , 105 and in mice , 107. A  ex vivo , 109, suCRP [2O2, 108, whCRP [2O2. PioglitCRP [2O2.γ-agonist treatment was shown to stimulate angiogenic network formation byendothelial progenitor cells in vitro [γ-agonist treated animals [γ-agonist treatment [Functionally,PPAR-in vitro  and in vin vitro . The adhin vitro , 109. In animals . Asendo animals ,we invereatment . Howeverγ-receptors present on renal endothelial cellsaffects their cellular behavior. Proliferation, apoptosis, migration, andangiogenesis by endothelial cells are modulated, but may involve both stimulation andinhibition depending on the specific circumstances. It remains unclear how thisbimodal potential of PPAR-γ-agonists is regulated. An important consequence of PPAR-γ-receptoractivation is the enhanced production of nitric oxide and C-type natriureticpeptide and superoxide dismutase, while endothelin-1 production is inhibited. Together, this improves the capacity of the endothelium to exert vasodilatory,anti-inflammatory, and antioxidative actions, resulting in blood pressurelowering and direct renoprotective effects. In addition, the presentation of adhesionmolecules and release of cytokines aimed at recruiting inflammatory cells toactivated endothelium is inhibited by PPAR-γ-agonism. This may also in partaccount for the anti-inflammatory effects of PPAR-γ-agonists, supplementary todirect effects on the inflammatory cells themselves. Finally, PPAR-γ-receptorsare also found on endothelial progenitor cells and PPAR-γ-agonist stimulateprogenitor-mediated endothelial repair, although definitive evidence that thisoccurs in the kidney is currently lacking.Activation of the PPAR-γ may also havebeneficial effects on the endothelium. Recently, selective agonists for PPAR-β/δwere developed [β/δ receptors are present on endothelium and stimulation with apharmacological PPAR-β/δ-agonist was found to increase endothelial cellproliferation and angiogenesis in vitro and in vivo, mainly mediated throughVEGF [γ-agonists, PPAR-β/δ-agonists increase circulating endothelialprogenitor cell levels, augment endothelial progenitor cell function in vitro,and improve endothelial progenitor cell-mediated neovascularization of ischemictissue [γ-agonists, PPAR-β/δ-agonist treatment reduced theexpression of adhesion molecules and monocyte binding to activated endothelialcells [Activation of other PPAR-receptors besides PPAR-eveloped . PPAR-β/ughVEGF . Like PPctissue . Also, malcells .γ-agonist treatment is not standard clinical practice innondiabetic renal patients. As shown in this review, experimental studiesindicate that stimulatory effects of PPAR-γ-agonism on endothelium may provideadditional benefit in nondiabetic renal disease. Together with otherpotentially therapeutic effects independent of the insulin-sensitizing actionsuch as the anti-inflammatory actions, this provides a rationale for furtherclinical evaluation in nondiabetic renal patients. For patients with diabetickidney disease, a pressing question is whether glycemic control with a TZD issuperior to other antidiabetic drugs for preventing the decline of renalfunction. To date, only retrospective studies, post hoc analyses, and pilot studies are availableto help answer this question. Trials designed to specifically evaluate thisquestion have yet to be performed.Currently, PPAR-


The equatorial plane is defined by four O atoms from two 4-hydroxypyridine-2,6-dicarboxylate ligands. The axial positions are occupied by the N atoms of the same ligands. There is an extensive three-dimensional hydrogen-bond network reinforcing crystal cohesion.In the title compound, (C DOI: 10.1107/S160053680802566X/bt2763Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


TSP-1 and TSP-2 gene expression by reverse transcription polymerase chain reaction (RT-PCR) analysis in 61 colon cancers. Thirty-eight of these 61 colon cancers were positive for TSP-2 expression and showed hepatic metastasis at a significantly lower incidence than those without TSP-2 expression (P = 0.02). TSP-2 expression was significantly associated with M0 stage in these colon cancers (P = 0.03), whereas TSP-1 expression showed no apparent correlation with these factors. The colon cancer patients with TSP-2 expression showed a significantly low frequency of liver metastasis correlated with the cell-associated isoform of vascular endothelial growth factor (VEGF-189) (P = 0.0006). Vascularity was estimated by CD34 staining, and TSP-2(–)/VEGF-189(+) colon cancers showed significantly increased vessel counts and density in the stroma (P < 0.0001). TSP-2(–)/VEGF-189(+) colon cancer patients also showed significantly poorer prognosis compared with those with TSP-2(+) / VEGF-189(–) (P = 0.0014). These results suggest that colon cancer metastasis is critically determined by angiogenesis resulting from the balance between the angioinhibitory factor TSP-2 and angiogenic factor VEGF-189. © 1999 Cancer Research CampaignTwo subtypes of thrombospondin (TSP-1 and TSP-2) have inhibitory roles in angiogenesis in vitro, although the biological significance of these TSP isoforms has not been determined in vivo. We examined


C. elegans germ line, syncytial germ line nuclei are arranged at the cortex of the germ line as they exit mitosis and enter meiosis, forming a nucleus-free core of germ line cytoplasm called the rachis. Molecular mechanisms of rachis formation and germ line organization are not well understood.In the fli-1 gene disrupt rachis organization without affecting meiotic differentiation, a phenotype in C. elegans referred to here as the germ line morphogenesis (Glm) phenotype. In fli-1 mutants, chains of meiotic germ nuclei spanned the rachis and were partially enveloped by invaginations of germ line plasma membrane, similar to nuclei at the cortex. Extensions of the somatic sheath cells that surround the germ line protruded deep inside the rachis and were associated with displaced nuclei in fli-1 mutants. fli-1 encodes a molecule with leucine-rich repeats and gelsolin repeats similar to Drosophila flightless 1 and human Fliih, which have been shown to act as cytoplasmic actin regulators as well as nuclear transcriptional regulators. Mutations in let-60 Ras, previously implicated in germ line development, were found to cause the Glm phenotype. Constitutively-active LET-60 partially rescued the fli-1 Glm phenotype, suggesting that LET-60 Ras and FLI-1 might act together to control germ line morphogenesis.Mutations in the FLI-1 controls germ line morphogenesis and rachis organization, a process about which little is known at the molecular level. The LET-60 Ras GTPase might act with FLI-1 to control germ line morphogenesis. C. elegans gonad is a bi-lobed organ composed of the germ line and somatic distal tip cells and sheath cells that partially envelop the germ line ..fli-1 mifli-1(ky535), including misplaced nuclei, were of roughly the same size and shape as those in wild-type  and 3.0 ± 0.6 μm diameter for wild-type). The misplaced nuclei in fli-1(ky535) had a meiotic appearance  mutant germ nuclei.In transmission electron microscopic (TEM) cross-sections, nuclei in the meiotic zone in fli-1ky55, includfli-1ky55, includfli-1(bp130) allele caused defects in oocyte production and brood size , dpy-17(e164), unc-32(e189), mpk-1(ku1), eT1. LGIV: let-60(n2021), let-60(s1124), let-60(s1155), let-60(s59), let-60(sy93), let-60(sy92), let-60(sy99), let-60(n1046), let-60(n1700), lin-3(e1417), lin-3(n1058), lin-1(n431). LGV: sos-1(s1031), lin-25(e1446), qIs56 [lag-2::gfp].chniques ,37. All C. elegans were produced by germ line microinjection of DNA solutions using standard techniques octane (DABCO) antifade reagent.The gonads of 12-hour-old adult hermaphrodite animals were dissected and fixed with 3% paraformaldehyde containing 0.1 M KEscherichia coli strain MG1693 (a thymidine-deficient E. coli strain kindly provided by the E. coli stock center) were grown minimal medium (M9) with 0.4% glucose, 1 mM MgSO4, 1.25 μg/ml vitamin B1, 0.5 μM thymidine, and 10 μM bromodeoxyuridine (BrdU) overnight at 37°C [E. coli were then plated on nematode growth medium (NGM) plates containing 100 μg/ml ampicillin. 12-hour-old adult hermaphrodite animals were placed on seeded plates and allowed to eat the BrdU-labeled E. coli for varying times depending on the experiment . Gonads were dissected immediately and fixed in methanol at -20°C for 1 hour followed by 1% paraformaldehyde for 15 minutes at room temperature.Fixed gonads were placed in 1 mg/ml BSA in PBT for 15 minutes, 2N HCl to denature DNA for 30 minutes at room temperature, and 0.1 M sodium borate to neutralize for 15 minutes at room temperature. The specimens were blocked in 1 mg/ml BSA in PBT for 15 minutes and stained with a 1:2.5 dilution in PBT of anti-BrdU antibody  at 4°C overnight. On the next day, the specimens were washed three times by 1 mg/ml BSA in PBT for 10 minutes each. A 1:500 dilution of Alexa 488-conjugated goat-anti-mouse antibody was incubated with the specimens at room temperature for 2 hours in PBT. The specimens were washed three times with 1 mg/ml BSA in PBT for 10 minutes each with DAPI in the last wash to stain DNA (see above). Gonads were mounted for microscopy as described above.Gonads of adult hermaphrodite animals (12 hours after the L4/adult molt) were fixed as described for DAPI staining. Fixed gonads were blocked for 1 hour in 1 mg/ml BSA in PBT at room temperature and then were incubated overnight with 1:50 diluted monoclonal anti-GFP  antibody at 4°C. The specimens were washed three times with PBT for 10 minutes each and incubated with 1:500 diluted Alexa 488-conjugated goat-anti-mouse antibody  for 2 hours at room temperature. DAPI was included to stain DNA. Stained gonads were rinsed three times with PBT for 10 minutes each. Gonads were mounted for microscopy as described above.JL conducted all of the experiments described in the manuscript and participated in figure construction and manuscript writing. WLD assisted with transmission electron microscopy and discussion of results. EAL oversaw all aspects of the project and contributed the bulk of manuscript writing.


Sequencing of multiple clones of three separate DNA fragments spanning 40% of the genome also indicated that DEN-4 propagated in Vero cells contained a higher number of mutations than the virus growing in MRC-5 cells. Although Vero cells yielded a peak virus titer approximately 1 to 17 folds higher than MRC-5 cells, cloned DEN-4 from MRC-5 cells maintained a greater stability than the virus from Vero cells. Serum-free microcarrier cultures of MRC-5 cells offer a potentially valuable system for the large-scale production of live-attenuated DEN vaccines.This work investigated the replication kinetics of the four dengue virus serotypes (DEN-1 to DEN-4), including dengue virus type 4 (DEN-4) recovered from an infectious cDNA clone, in Vero cells and in MRC-5 cells grown on Cytodex 1 microcarriers. DEN-1 strain Hawaii, DEN-2 strain NGC, DEN-3 strain H-87, and DEN-4 strain H-241 , and DEN-4 strain 814669 derived from cloned DNA, were used to infect Vero cells and MRC-5 cells grown in serum-free or serum-containing microcarrier cultures. Serum-free and serum-containing cultures were found to yield comparable titers of these viruses. The cloned DNA-derived DEN-4 started genetically more homogeneous was used to investigate the genetic stability of the virus propagated in Vero cells and MRC-5 cells. Sequence analysis revealed that the DEN-4 propagated in MRC-5 cells maintained a high genetic stability, compared to the virus propagated in Vero cells. Amino acid substitutions of Gly The RNA genome contain sequences coding for three structural protein genes , seven non-structural protein genes , and the flanking non-translating regions. DEN infection in humans causes a spectrum of illnesses, ranging from dengue fever (DF) to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) The four dengue serotype viruses (DEN-1 to DEN-4) .Solid microcarriers (Cytodex 1) have been shown to support the growth of Vero cells in either serum-containing or serum-free medium opically . The cel3- to 104-fold lower viral yield from Vero cells or a 102- to 103-fold lower yield from MRC-5 cells in serum-containing medium. The serotype difference was probably not responsible for the low viral yields, as the DEN-4 strain derived from cDNA replicated as efficiently as DEN-2. A plausible reason for the unexpected low yields is that these viruses have acquired mutations during passages in cultured cells or in animals. This probability was supported from the analysis of viral yields in similar infections with the wild type DEN-4 derived from a cDNA clone (see next section). Importantly, higher viral yields from Vero cells or MRC-5 cells were consistently observed at 0.3- to 2.6-fold higher for the four DEN serotypes in serum-free medium than in serum-containing media.Production of DEN-1 strain Hawaii, DEN-2 strain NGC, DEN-3 strain H-87 and DEN-4 strain H-241 in Vero cells and in MRC-5 cells grown in serum-containing and serum-free media was studied. DEN-4 strain 814669 recovered from cloned DNA was propagated in microcarrier cultures of both cell lines. Infection with the virus was carried out at the same multiplicity as described earlier. The density of Vero cells or MRC-5 cells decreased steadily during the subsequent 10-day period, indicating that both cells were readily infected . The virDEN-4 derived from cloned DNA was propagated in the microcarrier cultures of Vero cells or MRC-5 cells in serum-free or serum-containing medium for 4 days. The nucleotide sequence of progeny DEN-4 was determined from a single cDNA clone representing each of the four overlapping regions, designated as W01/W02R, W03/W04R, W05/W06R, W07/W08R, spanning the entire DEN genome. Each progeny DEN-4 sequence was compared with the parent DEN-4 DNA sequence (GenBank accession number: AF375822). The results in in vitro RNA transfection . P1 and P2 viruses were harvested from cells grown in T flask cultures and the P3 passage virus was from a microcarrier culture. Multiple clone sequencings were conducted on the three genomic fragments  because most mutations identified earlier were localized in these fragments. These fragments are: (i) WE1/W02R, consisting of the full-length E gene and the NS1 gene portion encoding the first 152 amino acids; (ii) W28/W29R, consisting of part of NS3 gene encoding the amino acids 42–455; and (iii) W21/W23R, consisting of the entire NS4B gene and the NS5 gene portion encoding the first 180 amino acids. A total of 180 independent clones were obtained for sequence determination in this study.Since the previous DEN-4 sequence determination was made from one DNA clone representing each of the four genome segments, to further investigate the genome stability of DEN-4 during passages in Vero cells and in MRC-5 cells, sequences of multiple clones were determined. The DNA-derived DEN-4 viruses were obtained in Vero cells or in MRC-5 cells at three consecutively passages after 104C, F108I, G427R, V439F, and V463L from Vero cells after three passages. Likewise, there were 3 nucleotide differences including 3 amino acid mutations in E protein at E345K, N362K, and G427R from MRC-5 cells  fragments, the P3 viruses from microcarrier cultures revealed 16 mutations resulting in 11 amino acid changes in Vero cells and 3 mutations resulting in 2 amino acid changes in MRC-5 cells. Each of these mutations in the NS3 protein occurred at a frequency of 10% except E341G at a frequency of 20%  fragments of P1 and P2 passages from Vero or MRC-5 cells . However-5 cells . The muty of 20% . In the rs at P3 .These results were then analyzed on the basis of amino acid mutations detected in each of the three fragments sequenced. The mean diversity and mean p-distance of amino acid mutations in each genomic fragment of 10 independent clones were calculated . The numThis work has investigated the replication kinetics of four-serotype and cloned DNA-derived DEN viruses in Vero and MRC-5 cells. Vero (African green monkey kidney) cells are polyploidy (recently certified as cell hosts for producing DEN vaccines) and MRC-5  are diploid. Both cell lines have been already used to develop several human vaccines 6 pfu/ml) of cloned DEN-4 (81466 strain) in both Vero cells and MRC-5 cells. The viral yield of cloned DEN-4 (81466 strain) produced in MRC-5 cells on microcarriers (at P3) was approximately 10 folds lower and had only one amino acid change compared to those in Vero cells with thirteen and six amino acid mutations in serum-free and serum-containing cultures, respectively (High titers of DEN-1 (HAWAII) and DEN-2 (NGC) but relatively low titers of DEN-3 (H-87) and DEN-4 (H-241) were obtained in this study in either Vero cells or MRC-5 cells, although both cells were grown to a similar density on microcarrieres prior to virus infection. A possible explanation is that the DEN-4 (H-241) virus strain used was the non mouse-adapted strain rather than the prototype mouse-adapted strain since the non mouse-adapted strain was associated with two E protein mutations  ectively . Almost ectively ∼5. The 104C, E-F108I in Vero cells and the E-E345K in MRC-5 cells had mutation rates exceeding 50% and the NS4B-L112F and NS4B-A240V in Vero cells had mutation rates of 30%. These results conflict with the high rate of NS3 gene mutation reported in a recent study of the full-genomic sequences of several DEN-3 clinical isolates 112F and NS4B-A240V mutations occur in Vero cells at a frequency of only 30% , DEN-2 (NGC), DEN-3 (H-87), and DEN-4 (H-241) strains used for the virus yield studies in only 30% , which pThe DEN2-PDK-53-based chimeric viruses have recently been characterized by multiple plaque selection and spot/genomic sequencing Vero cells, MRC-5 cells and BHK-21 cells were obtained from the Bioresource Collection and Research Center (BCRC), Food Industrial Research and Development Institute, Hsinchu, Taiwan. Vero cells and MRC-5 cells were grown in Dulbecco's Modified Essential Medium (DMEM) (Invitrogen) and BHK-21 cells were grown in Eagle's Minimum Essential Medium (EMEM) (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 U/ml of penicillin G sodium-Streptomycin (Invitrogen). Vero cells and MRC-5 cells were also cultured in serum-free medium using M-VSFM serum free medium (Biogro) Kpn I and then added to a transcription reaction mixture (Promega kit) containing m7G(5′)ppp(5′)G (Merck) for cap addition at the RNA 5′-end. After incubation at 37°C for 1.5 hour, the RNA product was purified with TRIzol reagent (Invitrogen) according to manufacturer's instructions. Prior to RNA transfection, subconfluent Vero cells and MRC-5 cells in a 24-well plate were rinsed once with serum-free medium and then covered with 0.3 ml of DMEM medium per well. The transfection mixture was prepared by adding 4 µl of DMRIE-C reagent (Invitrogen) to 1 ml of DMEM, then mixing with 10 µg of the RNA product. The transfection mixture was added directly to cell monolayers. After 18 hours incubation at 37°C, either DMEM+10% FBS or M-VSFM medium were added to the well. Eight days after transfection, culture supernatants were collected. All virus stocks were stored at −80°C freezer for further analysis.DEN-1 virus (Hawaii), DEN-2 virus (NGC), DEN-3 virus (H-87) and DEN-4 virus (H-241) were provided by the Center of Disease Control, Department of Health, Taiwan. Stock viruses were prepared from supernatants of infected C6/36 cells grown in Hank's MEM medium (GIBCO) plus supplements 6 days post-infection at 28°C 2 at 37°C. Cells were detached from tissue culture flasks using trypsin-EDTA, and then transferred to a spinner flask containing 2g/l Cytodex 1 microcarriers. Starting cell densities were 3×105 cells/ml in serum-containing cultures and 6×105 cells/ml in serum-free cultures. The cell cultures were infected after 3 day inoculation and by replacing 70% of the medium with fresh medium containing the virus inoculum at a multiplicity of infection (MOI) of 0.01. Inoculation continued without further replacement with fresh medium or addition of supplements.Cytodex 1 microcarriers (Amersham Biosciences) were prepared according to manufacturer's instructions. Briefly, the Cytodex 1 microcarriers were immersed in phosphate buffer saline (PBS) for at least three hours and autoclaved for 15 min before each experiment. The autoclaved microcarriers were washed twice with the culture medium. Bellco spinner flasks (100-ml) were used in the experiments for a working volume of 50 ml. The spinner flasks were stirred at 60 rpm and placed inside an incubator with 5% COg for 5 min to remove the supernatant. The pellets were treated with 1 ml 0.1 M citric acid [containing 0.1% (w/v) crystal violet] and incubated at 37°C for 1 hour. The released nuclei were counted in a hemocytometer. The virus titer was measured by 10-fold serial dilutions of the culture supernatant in duplicate infections of BHK-21 cell monolayers in a 6-well plate. After 1 hour incubation at 37°C, 4 ml of medium containing 10% FBS and 1.1% methylcellulose (4 ml/well) was added to each well. Virus plaques were stained with naphthol blue black dye six days after incubation. The infectivity titer in plaque forming units (pfu) per ml was determined.The number of cells attached to the microcarriers was determined by nuclei staining. Briefly, a 1-ml sample of the microcarrier culture was taken and centrifuged at 200 Pfx DNA polymerase (Invitrogen). Four overlapping fragments that span the entire DEN genome were produced using 4 sets of forward and reverse primers: (i) W01/W02R, (ii) W03/W04R, (iii) W05/W06R, and (iii) W07/W08R as listed in Viral RNA was extracted from culture supernatants using TRIzol reagent (Invitrogen), and the cDNA synthesized by reverse transcription using Superscript II RTase (Invitrogen). The double-stranded DNA was then generated by polymerase chain reaction (PCR) using Platinum® Pfx DNA polymerase with their respective forward and reverse primer sets: (i) WE1/W02R, (ii) W28/W29R, and (iii) W21/W23R (see Three DNA fragments were synthesized from DEN-4 RNA by RT-PCR using Platinum® Table S1Primers used in the genomic sequencing of cloned DNA-derived DEN-4 viruses in cell cultures.(0.18 MB DOC)Click here for additional data file.Figure S1Production of DEN-1 virus (HAWAII strain) from Vero and MRC-5 cells grown on 2g/l Cytodex 1 microcarriers: (A) Vero cell growth curves; (B) MRC-5 cell growth curves; (C) virus replication curves in Vero cells; (D) virus replication curves produced in MRC-5 cells.(0.06 MB TIF)Click here for additional data file.Figure S2Production of DEN-2 virus (NGC strain) from Vero and MRC-5 cells grown on 2g/l Cytodex 1 microcarriers. Symbols are the same as (0.06 MB TIF)Click here for additional data file.Figure S3Production of DEN-3 virus (H-87 strain) from Vero and MRC-5 cells grown on 2g/l Cytodex 1 microcarriers. Symbols are the same as (0.06 MB TIF)Click here for additional data file.Figure S4Production of DEN-4 virus (H-241 strain) from Vero and MRC-5 cells grown on 2g/l Cytodex 1 microcarriers. Symbols are the same as (0.06 MB TIF)Click here for additional data file.Figure S5Production of cloned DNA-derived DEN4 from Vero and MRC-5 cells grown on 2g/l Cytodex 1 microcarriers. Symbols are the same as (0.06 MB TIF)Click here for additional data file.


Dominantly inherited mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are the most common cause of familial Parkinson's disease (PD) and have also been identified in individuals with sporadic PD. Although the exact cellular function of LRRK2 remains unknown, most PD-linked mutations appear to be toxic to cells in culture via mechanisms that depend on the kinase activity of LRRK2 or on the formation of cytoplasmic inclusions. Here we show that the E3 ubiquitin ligase CHIP physically associates with LRRK2 and regulates the cellular abundance of LRRK2. We further show that LRRK2 forms a complex with overexpressed and endogenous CHIP and Hsp90. Our data indicates that the destabilization of LRRK2 by CHIP is due to ubiquitination and proteasome-dependent degradation. Hsp90 can attenuate CHIP-mediated degradation and this can be blocked by the Hsp90 inhibitor geldanamycin. These findings provide important insight into the cellular regulation of LRRK2 stability and may lead to the development of therapeutics to treat PD based on controlling LRRK2 stability. Parkinson's disease (PD) is the most common neurodegenerative movement disorder and affects 1–2% of the population over 60 years old. The primary clinical symptoms have classically been defined as bradykinesia, resting tremor, cogwheel rigidity and abnormal gait. Despite intensive research, the cause of PD remains unknown. Epidemiological studies have failed to definitively identify a single cause for PD, however, age is the greatest risk factor and many studies have implicated cumulative oxidative stress, mitochondrial dysfunction, protein aggregation and neuroinflammation in PD pathogenesis or progression In vitro studies have confirmed that LRRK2 possesses both GTPase and kinase activities, including autophosphorylation activity and phosphorylation activity towards generic substrates and potential physiologic substrates Of all the mutations linked to familial PD to date, the G2019S missense mutation in the leucine-rich repeat kinase 2 gene (LRRK2) is the most common and can account for a significant fraction of apparently sporadic PD cases in certain populations Dominantly inherited point mutations that segregate with PD have been identified in the leucine-rich repeat, ROC GTPase, COR and kinase domains of LRRK2 In an effort to better define the cellular function of LRRK2, we conducted yeast two-hybrid screens to identify potential LRRK2 interacting proteins. We identified a robust protein-protein interaction between LRRK2 and CHIP (C-terminus of Hsp70-Interacting Protein), which is an E3 ubiquitin ligase crucial for the ubiquitination of several heat shock protein (HSP)70/90 client proteins that are involved in neurodegenerative disease Full-length mouse LRRK2 cDNA was cloned by PCR from mouse brain cDNA prepared in our lab. Seven putative LRRK2 domains comprising amino acids 1–690 (ARM), 691–1009 (Ankyrin), 1010–1312 (LRR), 1331–1512 (ROC), 1514–1866 (COR), 1869–2128 (kinase) and 2141–2432 (WD40) were subcloned into pGBKT7 (Clontech) as baits for yeast two-hybrid analyses and into pMyc-CMV (Clontech) for mammalian cell transfections, along with full-length LRRK2. Point mutations  were generated using the QuickChange Site-Directed Mutagenesis kit (Stratagene). Wild-type human LRRK2 and LRRK2(R1441C) constructs plus SMARTpool CHIP siRNA (L-007201-00) and non-targeting control siRNA (D-001810-02-05) were purchased from Dharmacon .The following antibodies and other reagents were used in these studies: Mouse monoclonal anti-HA ; Mouse monoclonal anti-myc ; Rabbit anti-HA ; Rabbit anti-myc ; Rat monoclonal anti-Hsp90 ; Mouse anti-β-actin ; Rabbit anti-CHIP ; Mouse anti-Flag (#F3165 Sigma). Mouse anti-ubiquitin was kindly provided by Dr. Amyn Habib. Rabbit IgG, Peroxidase-conjugated goat anti-mouse IgG(H+L) and anti-rabbit IgG(H+L) and anti-rat IgG(H+L) were purchased from Jackson ImmunoResearch Laboratories. Lipofectamine 2000 (11668–027) was purchased from Invitrogen  and complete protease inhibitor cocktail (11873580001) was purchased from Roche Molecular Biochemicals . Geldanamycin was purchased from Alexis Biochemicals . Lactacystin (70980) was purchased from Cayman Chemical . ImmunoPure Immobilized Protein A/G beads (20421) were purchased from Pierce . X-α-Gal (MESP-1900) was purchased from Growcells . X-β-Gal (X1001-5) was purchased from Zymo Research . Yeast nitrogen base w/o amino acids (291940) and amino acid Drop-out supplements  were from Clontech. 3-AT  was purchased from Sigma. ON-TARGETMATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4Δ, gal80Δ, UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2, URA3::MEL1UAS-MEL1TATA-lacZLYS2::GAL1) (Clontech). Yeast clones harboring the bait plasmids were selected on SD/-Trp medium. Bait autoactivation was tested on SD/-Trp-His medium. The baits without or with slight autoactivation were used to screen a mouse brain cDNA library  pre-transformed in yeast strain Y187  (Clontech). Yeast two-hybrid screens were performed as previously described 6) were plated on triple selective medium (SD/-Leu-Trp-His plus 5 mM 3-AT). The prey plasmids from yeast colonies growing on the triple selective medium were rescued and sequenced. To further verify the interactions in yeast cells, the obtained prey plamsids and their bait plasmid were co-transformed into AH109 yeast cells. After growing up on double selective medium (SD/-Trp-Leu), the yeast cells were re-streaked onto quadruple selective medium (SD/-Leu-Trp-His-Ade plus 2 mM 3-AT) and were used to perform X-α-Gal assays (MEL1 reporter) and X-β-Gal colony-lift assays (LacZ reporter) according to the Yeast Protocols Handbook (Clontech).Bait plasmids consisting of each individual LRRK2 domain in pGBKT7 (Clontech) were transformed into yeast strain AH109  medium with 10% FBS and 50 U penicillin, 50 µg/ml streptomycin. For HEK293 cell transfection, cells (80% confluence) were transfected with an equal amount of total plasmid DNA (adjusted with the corresponding empty vector) by the calcium phosphate method. For siRNA transfection into SH-SY5Y cells, the cells (40–50% confluence) were transfected with 50 ng of pMyc-LRRK2 and 0–40 picomoles of CHIP siRNA or control siRNA using Lipofectamine 2000 cell transfection reagent according to the manufacturer's instructions.Twenty-four to sixty hours after cell transfection, the cells were lysed in 500 µl ice-cold buffer . The cell lysates were incubated on ice for 30 min and centrifuged at 12,000g for 10 min at 4°C. The supernatants were pre-cleared at 4°C for 1 h with 10 µl of ImmunoPure Immobilized Protein A/G beads. Rabbit polyclonal antibody (anti-myc or anti-HA) was added to the pre-cleared supernatants at 1∶500 dilution and incubated at 4°C for 2 h. 20 µl of ImmunoPure Immobilized Protein A/G beads was added and incubated for another 1 h. The beads were washed five times in ice-cold lysis buffer with 1× protease inhibitor cocktail. Proteins were eluted from the beads by heating at 95°C in 1× Laemmli buffer, resolved on 4–20% gradient gels (BioRad), transferred to nitrocellulose and detected by western blot using mouse monoclonal anti-HA or anti-myc antibodies, HRP-conjugated goat anti-mouse secondary antibodies (Jackson Immunoresearch) and chemiluminescent detection (Pierce).pMyc-LRRK2, pcDNA-CHIP and pMT-HA-Ub were co-transfected into HEK293 cells. After incubation for 24 h, immunoprecipitation was performed using a rabbit polyclonal anti-myc antibody. Mouse monoclonal anti-ubiquitin antibody was used to detect the ubiquitination of LRRK2. Monoclonal anti-myc antibody was used to probe LRRK2 expression.248-stop) and the interactor of the ROC domain corresponded to the charged domain of the E3 ubiquitin ligase CHIP from amino acid 150 to amino acid 200 (designated here as CHIP150–200). To confirm these protein-protein interactions in mammalian cells, the LRRK2 ARM and ROC domains and their interactors (Hsp90248–stop and CHIP150–200) were cloned into mammalian cell expression vectors with N-terminal myc or HA epitope tags (pMyc-CMV and pHA-CMV). Full-length LRRK2 and CHIP were also cloned into these vectors for mammalian cell expression. Co-transfection of HEK293 cells followed by immunoprecipitation and western blotting showed that the ROC domain of LRRK2 robustly co-immunoprecipated with CHIP150–200  from amino acid 248 to amino acid 704  domain, a C-terminal U-box domain and an intervening charged domain The N-terminal TPR domain of CHIP has been shown to specifically bind to several chaperones such as Hsp70 and Hsp90 These results, together with our data showing that Hsp90 binds to the ARM domain of LRRK2 , led us The above data shows that the TPR domain of CHIP is required for CHIP to bind to the N-terminal portion of LRRK2. To test whether isolated TPR domain is sufficient for binding to LRRK2, we co-transfected HEK293 cells with HA-tagged TPR domain and Myc-tagged N-terminal or C-terminal portions of LRRK2. Co-immunoprecipitation assays showed that isolated CHIP TPR domain binds to the ARM-ANK-LRR domains of LRRK2 but not to the ROC-COR-kinase-WD40 domains of LRRK2 , and many additional LRRK2 variants are being evaluated for conclusive pathogenicity or increased risk for PD To date, several missense mutations in To confirm that none of these LRRK2 mutations disrupt CHIP binding to LRRK2, we conducted co-immunoprecipitation assays using HEK293 cells co-transfected with CHIP and wild-type or mutant LRRK2. Wild-type, D1994A, R1441C and G2019S LRRK2 all co-immunoprecipitated with CHIP  domain of CHIP and the C-terminal U-box domain of CHIP are not essential for binding to LRRK2 , we examTo determine whether CHIP can ubiquitinate LRRK2, mammalian expression vectors for myc-tagged LRRK2, CHIP and HA-tagged ubiquitin (HA-Ub), were co-transfected into HEK293 cells and incubated for 24 hours. Cell lysates were immunoprecipitated with anti-myc antibody and ubiquitinated LRRK2 was detected by immunoblotting with anti-ubiquitin antibody Click here for additional data file.Figure S26 HEK293 cells in 60 mm dishes. HEK293 cell lysates were subjected to immunoprecipitation using polyclonal anti-myc or anti-HA antibodies, then immunoblotted using monoclonal anti-HA or anti-myc antibodies, respectively. Cell lysates were also directly immunoblotted using monoclonal anti-HA and anti-myc antibodies to verify similar protein expression levels in experimental and empty expression vector (CMV) control transfections. (A) Co-immunoprecipitation of isolated CHIP TPR domain and the ARM-Ankyrin-LRR (AAL) portion of LRRK2; (B) Absence of co-immunoprecipitation of isolated CHIP TPR domain and the ROC-COR-Kinase-WD40 (RCKW) portion of LRRK2; (C) Absence of co-immunoprecipitation of K30A point mutant CHIP TPR domain and the ARM-Ankyrin-LRR (AAL) portion of LRRK2; (D) Absence of co-immunoprecipitation of K30A point mutant CHIP TPR domain and the ROC-COR-Kinase-WD40 (RCKW) portion of LRRK2; (E) Co-immunoprecipitation of isolated CHIP TPR domain and full length LRRK2; (F) Absence of co-immunoprecipitation of K30A point mutant full length CHIP and full length LRRK2; (G) Absence of co-immunoprecipitation of K30A point mutant full length CHIP and the ARM-Ankyrin-LRR (AAL) portion of LRRK2; (H) Co-immunoprecipitation of K30A point mutant full length CHIP and the ROC-COR-Kinase-WD40 (RCKW) portion of LRRK2.The TPR domain of CHIP is sufficient to bind to the N-terminal portion of LRRK2 and the K30A point mutation that disrupts CHIP binding to Hsp90 disrupts CHIP binding to the N-terminal portion of LRRK2 but not to the C-terminal portion of LRRK2. The indicated constructs (0.5 µg for each) were co-transfected into 4.5×10(2.31 MB EPS)Click here for additional data file.Figure S36 HEK293 cells in 60 mm dishes. Geldanamycin was added into the medium to 1 µM final concentration and incubated for 1 hour prior to cell collection. HEK293 cell lysates were subjected to immunoprecipitation using polyclonal anti-myc or anti-HA antibodies, then immunoblotted using monoclonal anti-HA or anti-myc antibodies, respectively. Cell lysates were also directly immunoblotted using monoclonal anti-HA and anti-myc antibodies to verify similar protein expression levels in experimental and empty expression vector (CMV) control transfections.Geldanamycin does not impair the association of full length CHIP with full length LRRK2. The indicated constructs (0.5 µg for each) were co-transfected into 4.5×10(0.49 MB EPS)Click here for additional data file.Figure S46 HEK293 cells in 60 mm dishes. HEK293 cell lysates were subjected to immunoprecipitation using polyclonal anti-myc or anti-HA antibodies, then immunoblotted using monoclonal anti-HA or anti-myc antibodies, respectively. Cell lysates were also directly immunoblotted using monoclonal anti-HA and anti-myc antibodies to verify similar protein expression levels in experimental and empty expression vector (CMV) control transfections. (A) Co-immunoprecipitation of full-length CHIP and full length wild-type LRRK2; (B) Co-immunoprecipitation of CHIP and G2019S mutant LRRK2. (C) Co-immunoprecipitation of CHIP and R1441C mutant LRRK2. (D) Co-immunoprecipitation of CHIP and D1994A variant LRRK2.CHIP binds to wild-type, G2019S, R1441C and D1994A LRRK2. The indicated constructs (0.5 µg for each) were co-transfected into 4.5×10(1.00 MB EPS)Click here for additional data file.Figure S55 neuroblastoma SH-SY5Y cells in 24-well format. After 48-hour incubation, the cells were harvested and the proteins blotted with antibodies to Myc, CHIP and β-actin. (B). Parallel cells were similarly co-transfected using 0.05 µg of pMyc-LRRK2 and 0–40 picomoles of non-targeting siRNA in place of CHIP siRNA and western blotted to confirm no effects on the levels of endogenous CHIP or transfected Myc-LRRK2.CHIP siRNA stabilizes LRRK2. (A). 0.05 µg of pMyc-LRRK2 was co-transfected with 0-40 picomoles of CHIP siRNA into 1.5×10(0.71 MB EPS)Click here for additional data file.


C57BL/6 (B6)-derived embryonic stem (ES) cells are not widely used to generate knockout mice despite the advantage of a well-defined genetic background because of poor developmental potential. We newly established serum- and feeder-free B6 ES cells with full developmental potential by using leukemia inhibitory factor (LIF) and 6-bromoindirubin-3′-oxime (BIO), a glycogen synthase kinase-3 (GSK3) inhibitor. BIO treatment significantly increased the expression levels of 364 genes including pluripotency markers such as Nanog and Klf family. Unexpectedly, by aggregating or microinjecting those ES cells to each eight-cell-stage diploid embryo, we stably generated germline-competent ES-derived mice. Furthermore, founder mice completely derived from female XO, heterozygous, or homozygous mutant B6 ES cells were directly available for intercross breeding and phenotypic analysis. We hereby propose that serum- and feeder-free B6 ES cells stimulated with LIF plus GSK3 inhibitor are valuable for generating mouse models on B6 background. genesis 47:414–422, 2009. © 2009 Wiley-Liss, Inc. The technology to generate knockout (KO) mice has been one of the most important advances in understanding the function of genes of interest . The estLeukemia inhibitory factor (LIF)/STAT3 signaling is known to suppress lineage commitment  in Wnt/β-catenin signaling, which maintains self-renewal and pluripotency in both mouse and human ES cells cultured in the presence of serum  whether LIF/STAT3 signaling plus GSK3 inhibition maintain self-renewal and pluripotency of ES cells cultured in the absence of serum and feeder layers, and (2) whether germline-competent founder mice are generated from ES cells, which were maintained in such culture conditions. In a previous report, GSK3β deficient embryos were pale and non-viable between E 13.5 and E 14.5 (C57/BL6J ES cells (TB6-2), which were originally established by culturing the inner cell mass (ICM) of blastocyst-stage embryos, were maintained on a feeder layer in 1000 U/ml LIF plus 15% serum-containing medium as a standard culture method. However, these cells showed very low developmental potency despite normal karyotypes (38 + XY) when aggregated with tetraploid embryos to generate founder mice  alone, we performed DNA microarray analyses . In thisBecause BIO treatment enhanced self-renewal and pluripotency markers in serum- and feeder-free B6 ES cells in vitro, we tested their developmental potential by generating founder mice . To inveThe inhibition of the MEK pathway through the FGF receptor was reported to suppress lineage commitment even in the LIF absent condition . TherefoES-derived coat-color founder mice (100%) were also efficiently generated from B6/129 F1 hybrid ES (V6.5) cells under the serum- and feeder-free condition  B6 ES cells with ICR eight-cell-stage diploid embryos. These female mice with 100% black coat-color were fertile  and alsoFinally, to confirm that 100% black coat-color founder mice were fully derived from B6 ES cells treated with LIF plus BIO, we assessed the contamination of host ICR cells by quantitative PCR. For this analysis, we established homozygotes (−/−) of leptin receptor targeting in B6 ES cells cultured in 1000 U/ml LIF plus BIO under the serum- and feeder-free condition , and theThe treatment of LIF plus BIO dramatically changed the morphology of serum- and feeder-free B6 ES cells and significantly increased the expressions of self-renewal and pluripotency regulators. The aggregation or microinjection of serum- and feeder-free B6 ES cells cultured in LIF plus BIO to each eight-cell-stage diploid embryo stably provided germline-competent B6 ES-derived mice. Previously, it was reported that laser-assisted microinjection of ES cells into an eight-cell-stage diploid embryo efficiently provides founder generation mice that are available for immediate phenotypic analyses  for FGF receptor and GSK3  were originally established by a previously published procedure . The first strand cDNA synthesis by reverse transcription was performed with 0.25 μg of total RNA at 37°C for 2 h using a high capacity cDNA archive kit (Applied Biosystems) and subsequently diluted 10 times. Endogenous mRNA levels of pluripotency markers  were measured with inventoried TaqMan probes using an Applied Biosystems 7900HT (Applied Biosystems). PCR primers and TaqMan probes ; 5′-FAM- or 5′-VIC-Minor Groove Binder Non-Fluorescent Quencher  were used as follows: leptin receptor forward, 5′-cggagaccacgcaactt-3′; leptin receptor reverse, 5′-ccccgggcagtttcca-3′; leptin receptor probe, 5′-FAM-caggcctctgactact-MGBNFQ-3′; β actin forward, 5′-aggtcatcactattggcaacga-3′; β actin reverse, 5′-cacaggattccatacccaagaag-3′; β actin probe, 5′-VIC-atgccctgaggctcttttccagcctt-MGBNFQ-3′. GAPDH expression was examined by using Rodent GAPDH control regents (Applied Biosystems). All results were normalized to β actin or GAPDH expression. DNA microarrays (Agilent) were performed with total RNAs from ES cells treated with or without 2 μM of BIO for five days.ES cells were fixed with 4% paraformaldehyde/PBS for 30 min at room temperature and permeabilized with 0.1% Triton X-100/PBS at room temperature for 30 min. Primary and secondary antibodies were diluted to final working concentrations in 1% BSA, 0.1% TritonX-100/ PBS. Specimens were mounted with ProLong gold anti-fade reagent containing DAPI (Molecular Probe). Images of immunofluorescent samples were obtained with a Zeiss LSM510 confocal microscope. The final concentrations of antibodies were as follows: anti-β catenin-FITC, 1:250 (BD Bioscience); anti-Nanog, 1:250 ; goat anti-rabbit IgG-FITC, 1/100 (Invitrogen).2 in Hepes-buffered potassium simplex optimized medium (KSOM), zona pellucidae of eight-cell-stage embryos were removed with an acid-tyrode solution. The diploid embryos were transferred into each KSOM drop. For aggregation, a number of clumps of loosely connected ES cells were selected under a dissecting microscope, and then one of the ES cell clumps was put on the side of one embryo. After overnight incubation at 37°C/5% CO2 in KSOM, the aggregated embryos were developed to the late morula/ early blastocyst stage, and then transplanted into the uterine horns of pseudo-pregnant ICR mice by the standard method. Conventional or laser-assisted microinjection was performed according to a previous report (2 in KSOM before embryo transplantation. All animal procedures were carried out according to the Merck Worldwide Policy on Animal Care and Use.Two-cell-stage embryos were collected from oviducts of plugged females at 1.5 dpc. After overnight incubation of the embryos at 37°C/5% CO