Members of the kinesin-8 subfamily are plus end-directed molecular motors that accumulate at the plus-ends of kinetochore-microtubules (kt-MTs) where they regulate MT dynamics. Loss of vertebrate kinesin-8 function induces hyperstable MTs and elongated mitotic spindles accompanied by severe chromosome congression defects. It has been reported that the motility of human kinesin-8, Kif18A, is required for its accumulation at the plus tips of kt-MTs.In vitro studies reveal that Kif18A possesses a non-motor MT binding site located within its C-proximal 121 residues. Using single molecule measurements we find that Kif18A is a highly processive motor and, furthermore, that the C-terminal tail is essential for the high processivity of Kif18A.Here, we investigate how Kif18A localizes to the plus-ends of kt-MTs. We find that Kif18A lacking its C-terminus does not accumulate on the tips of kt-MTs and fails to fulfill its mitotic function. These results show that Kif18A like its yeast orthologue is a highly processive motor. The ability of Kif18A to walk on MTs for a long distance without dissociating depends on a non-motor MT binding site located at the C-terminus of Kif18A. This C-proximal tail of Kif18A is essential for its plus-end accumulation and mitotic function. These findings advance our understanding of how Kif18A accumulates at the tips of kt-MTs to fulfill its function in mitosis. The integrity of each organism is intrinsically tied to the faithful distribution of its replicated chromosomes during mitosis. This challenging task is mediated by the mitotic spindle; a cellular machine composed of microtubules (MTs) and associated proteins kinesin superfamily proteins (Kifs) share a common 360 amino acid (aa) sequence that is highly conserved throughout the eukaryotic phyla. This conserved globular domain, called the catalytic core, contains both a catalytic pocket for the hydrolysis of ATP and the binding site for MTs. The mechanical properties of kinesins are determined by the motor-domains , whereas the stalk and tail domains can mediate dimerization and binding of cargo molecules, respectively. ATP hydrolysis mediates conformational changes in the catalytic core and neck region resulting in the movement of the motor along the MT lattice A. nidulans; Kip3p, S. cerevisiae; klp5/6+, S. pombe), and Drosophila (Klp67A), to mammals (Kif18A). Loss of kinesin-8 activity results in hyperstable MTs and elongated spindles, accompanied by severe chromosome congression defects. In vitro, kinesin-8 proteins are slow plus-end directed motors and, as shown for the budding yeast orthologue, are characterized by a remarkably high processivity The In vitro studies have shown that Kif18A like Kip3p depolymerizes MT in a length-dependent manner Upon entry into mitosis, Kif18A localizes to the lattice of spindle MTs from where it translocates to the plus-ends of kt-MTs during early metaphase In vitro studies demonstrated that the C-terminus of Kif18A can bind directly to MTs. Intriguingly, single molecule measurements revealed that Kif18A lacking this additional MT binding site displays shorter run lengths and higher velocity compared to full-length protein. Thus, the C-proximal tail of Kif18A contributes to its processivity required for its plus-end localization and, hence, its function in mitosis.In this study, we investigated how Kif18A localizes to the plus-ends of kt-MTs. We found that a non-motor binding region site located at the C-terminus of Kif18A is essential for correct plus-end localization in mitosis. In HeLa-cells, Kif18A lacking the C-proximal 121 residues decorated the lattice of spindle MTs but did not display prominent plus-end localization. In line with its inability to localize correctly, tail-less Kif18A failed to rescue spindle length and chromosome alignment in cells depleted of endogenous Kif18A. The complementary C-tail fragment accumulated at spindle poles where it partially co-localized with pericentrin. http://www.hprd.org/) revealed an N-proximal motor domain consisting of a catalytic motor and neck (aa 9-363), a centrally positioned putative coiled-coil region (aa 375-454), and a C-terminal tail (aa 453-898) that includes a functional nuclear localization signal  FL) like the endogenous protein localized to the tips of spindle microtubules  using the human protein reference database ; 14,98 µm ± 0,44 µm (GFP-Kif18A778-898)  . Consist778-898)  to 8,80 778-898) . As expe 0.18 µm . In lineectively .1-777) and tail-only Kif18A (GFP-Kif18A778-898). Our previous studies revealed that Kif18A depletion results in SAC activation and, thus, in a mitotic delay FL  and supernatant (SN) fractions were analyzed by immunoblotting. As expected, GFP-Kif18A1-777 comprising the motor MT binding domain co-sedimented with MTs in the pellet fraction  and as control the last residue of Kif18A (residue 898) fused at their amino- and carboxy-terminus to maltose-binding protein (MBP) and GFP, respectively. Recombinant proteins bound to the amylose resin were eluted by cleaving off the MBP tag using human rhinovirus 3C protease  tag from insect cells N = 782) and 199 nm/s  for His-Kif18A1-777-GFP and His-Kif18AFL-GFP, respectively  for His-Kif18AFL-GFP : 5′ primer: ATTAGGCCGGCCGATGAGCTCGAAGTGTAA-ATTACCCG; 3′ Primer: ATTAGGCGCGCCCTTAGATTTCCTTTTGAAATATTTC. The following primers were used to generate silent mutations for rendering pCS2-GFP-Kif18AFL and pCS2-GFP-Kif18A1-777 resistant to the Kif18A siRNA oligo: primer pair 1: TACCAACAACAGTGCCACAAGCAAATAGAAATGATGTG; CACATCATTTCTATTTGCTTGTGGCACTGTTGTTGGTA; Primer pair 2: TGAACTTAAATCATTC-TATCAGCAACAGTGCCACAAGCAA; TTGCTTGTGGCACTGTTGCTGATAGAATG-ATTTAAGTTCA; Primer pair 3: TAAATCATTCTATCAGCAGCAATGCCACAAGCAA-ATAGAA; TTCTATTTGCTTGTGGCATTGCTGCTGATAGAATGATTTA; Primerpair4: ATTCTATCAGCAGCAATGTCACAAGCAAATAGAAATG; CATTTCTATTTGCTTGTGACATTGCTGCTGATAGAAT. The amplified cDNAs were cloned into pCS2-GFP plasmids harboring engineered FseI and AscI restriction sites at the 5′ and 3′ end. For single molecule studies Kif18A(aa 1-898) and Kif18A (aa 1-777) were cloned into a modified FastBacM13 vector carrying an n-terminal 6xHis tag and a c-terminal GFP-Avitag. Kif18A (aa 776-898)-GFP and Kif18A (aa 898)-GFP were cloned from the FastBacM13 into a modified bacterial expression vector petM42 .All human Kif18A (NM_031217) constructs were PCR amplified from human testis cDNA (Invitrogen) with the following primer pairs: (aa 1–898): 5′ primer: ATTAGG-CCGGCCAATGTCTGTCACTGAGGAAG; 3′ primer: ATTAGGCGCGCCCTTAGATTT-CCTTTTGAAATATTTC; huKif18A (aa 1–777): 5′ primer: ATTAGGCCGGCCAATGT-CTGTCACTGAGGAAG; 3′ Primer: 776-898–GFP and Kif18A898–GFP were expressed in BL21(DE3)-T1R competent Escherichia coli (Sigma B2935) transformed with pRARE . Expression was induced at OD 0.6 with 0.5 mM IPTG over night at 18°C. Proteins were extracted with B-PER (Thermo Fischer) according to manufacturer's protocol. The extract was cleared by centrifugation at 10 min at 10,000 x g. The supernatant was incubated with Amylose resin (NEB) for 2 h at 4°C and the resin was washed four times with 3C-buffer . 3C-Protease  was added to cleave Kif18A C-terminus-GFP from its N-terminal maltose binding protein. Resin beads where pelleted and supernatant was snap frozen in liquid nitrogen.The BAC-TO-BAC expression system  was used to express 6xHis-Kif18A (aa 1-898)-GFP-Avi and 6xHis-Kif18A (aa 1-777)-GFP-Avi in insect cells SF+ (obtained from protein science). Cells were cultured in serum free SF900II Medium (Invitrogen). Protein Purification was performed as described in GST-tagged huKif18A (aa 1-367) was purified as described in The following primary antibodies were used: affinity-purified anti-huKif18A 1∶400 ; polyclonal anti-Pericentrin  1∶000 ; anti-tubulin : 1∶1000 ; CREST serum (Immunovision) 1∶3000 (IF), polyclonal anti-GFP  (1∶1000) (WB). The following secondary antibodies were used: Horseradish-peroxidase-conjugated anti-mouse or anti-rabbit (Dianova) 1∶3000 (WB); goat anti-rabbit alexa fluor 568 (Invitrogen) (1∶1000), goat anti-human alexa fluor 647  (1∶500), goat anti-mouse alexa fluor 350  (1∶50).2. For immunofluorescence experiments, we used HeLa-cells (same clone as used in All cell lines were grown in Dulbecco's modified Eagle medium (Invitrogen) with 10% fetal-bovine serum (Invitrogen) and 1 U penicillin-streptomycin (Invitrogen) at 37°C in a humid atmosphere with 5% CORNAi transfections were performed at 75 nM final concentration using Oligofectamine (invitrogen) following the manufacturer's protocol. For experiments performed on coverslips (Marienfeld) in 6-well plates, cells were plated 24h prior to transfection. The following siRNA oligonucleotides were used: Luciferase GL2 target sequence: (5′-AACGUACGC-GGAAUACUUCGA-3′) (Dharmacon Research). Kif18A target sequence: 5′-AACCAA-CAACAGUGCCAUAAA-3′ (Dharmacon Research). Cells were fixed and processed for immunofluorescence or lysed for western-blot analysis 48 hr after transfections.4 cells/ml in a six-well plate 24 hr before transfection. HeLa-cells were transfected with the following plasmids: GFP, GFP-Kif18AFL, GFP-Kif18A1-777 and GFP-Kif18A778-898 using Fugene6 in accordance with the manufacturer's protocol (Roche). After 10 hours medium was removed and the siRNA transfection-mix was dropped carefully. After 20 hours Thymidine (Sigma) was added to a final concentration of 2 mM for 18 hours. Cells were released and after 9 hours MG132 (Sigma) was added to a final concentration of 20 µM for 1 hour. Cells were then either lysed in sample buffer or processed for immunofluorescence as described. For co-immunoprecipitations, 293T cells were harvested and lysed with lysisbuffer , incubated with the cleared extract and bound proteins were precipitated.HeLa-cells were seeded on coverslips at 4*10Cell extracts were boiled in sample buffer and resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to Nitrocellulose transfer membrane (Schleicher and Schuell) and then blocked with 5% slim-milk powder in PBS + 0.1% Tween-20 solution. Blots were developed by an ECL westernblotting detection system and visualized with a CCD camera .2, 0.4% Triton X-100, and 4% formaldehyde). Samples were washed with TBS + 0.1% Triton X-100 (TBST) and incubated for 1 hr in TBST + 2% bovine-serum albumin (Sigma). Antibodies were incubated for 1 hr at RT. Washed samples were embedded in mounting media . High resolution images as shown in 2-independent media before recording on a Zeiss Observer.Z1 inverted microscope with a heating chamber at 37°C on 12-well plate using a Plan-Neofluar 40 x, N.A. 0.6, (Zeiss). Images were taken every 7 min. and analyzed with the Metamorph software.Cells were fixed for 12 min in fixation buffer . Projections were generated using the ‘quick projection’ function using the maximal intensity in the stack for each pixel. Images of fixed samples were exported as 8-bit grayscale tiff files for every channel. Figures were assembled in Adobe Photoshop CS2 and Adobe Illustrator CS2 without any further adjustments to microscopy images. Live-cell recordings were analyzed and processed with Metamorph software (Molecular Devices). Images in http://fiji.sc/wiki/index.php/Fiji) and the velocities and run lengths calculated. These criteria led to small biases in the measurements of mean run length: for the more processive full-length kinesin the mean run length was underestimated , whereas for the less processive truncated kinesin the mean run length was overestimated . These biases lead to an underestimate of the difference in processivity between the full-length and truncated kinesins. For a less biased estimate of the mean run length of full length Kif18A, all run distances in the kymographs where summed and divided by the number of dissociations. To analyze diffusion of Kif18A (aa 776-898)-GFP, the times and distances that particles travelled between landing and dissociating from the microtubule lattice were measured. Runs shorter than 1 s were excluded. The squared displacement was plotted over time and fit to a line using least-squares. Data were analyzed in Igor Pro (Wavemetrics) and Origin (Originlab).High-resolution images were acquired as 1-777 and GFPKif18A778-898 as described above. After 24 hours Thymidine (Sigma) was added to a final concentration of 2 mM for 20 hours. Afterwards cells were released and VS-83  before centrifugation at 14.000 rpm at 4°C for 5 min. The clarified supernatant was then centrifuged at 90.000 rpm  at 4°C for 40 minutes. 1 mM DTT, 1 mM GTP and 5 mM MgCl2 were added to the high speed supernatant (Input-sample) and protein concentration was determined using Bradford reagent (Biorad). The supernatants were warmed to 37°C for 5 minutes to allow microtubule polymerisation, and then taxol was added to a final concentration of 10 µM. Extracts were left at 37°C for further 10 minutes to stabilize microtubules, then transferred to RT for 30 min to allow binding of proteins to microtubules. Afterwards supernatants were centrifuged at 90.000 rpm  at 25°C for 5 min. Equal fractions of supernatant (SN) and pellet (P) were resuspended in protein sample buffer, boiled and subjected to SDS-PAGE.HeLa-cells were transfected with plasmids encoding for GFP, GFP-Kif18A2, 1 mM EGTA) supplemented with 1 mM MgCL2, 2 mM GMPCPP overnight at 37 °C. After pelleting in an Airfuge  microtubules where resuspended in BRB80. Microscopy chambers were constructed as described Porcine brain tubulin was purified and rhodamine labelled  as previously described Figure S11-367, Kif18A1-467 and Kif18A1-526 fail to properly localize to kt-MTs plus-ends. (A)Kif18A Localization of transiently GFP-Kif18A1-367 during metaphase in HeLa-cells treated with GL2 control siRNA determined by immunofluorescence. HeLa-cells were stained with CREST antisera (red), anti α-tubulin (blue), anti-pericentrin. Kif18A was visualized by GFP-tag. The scale bar is 15 µm. Images are z-projections of deconvolved 3D stacks. The merge image represents GFP, CREST and tubulin. (B) Lysates (input) were prepared from 293T cells transfected with empty GFP vector or the indicated Kif18A variants followed by immunoprecipitation of the fusion proteins with GFP antibodies. Western blots were probed with GFP and myc antibodies. (C) SDS-PAGE showing Kif18A776-898–GFP (42 kDa) and Kif18A898–GFP (28.3 kDa) as purified from bacteria. The 3C-Protease band (45 kDa) falls on top of the Kif18A776-898-GFP band. (D) SDS-PAGE showing His- Kif18A1-777-GFP (123 kDa) and His- Kif18AFL-GFP (137 kDa) as purified from insect cells. At∼75 kDa appears a typical insect-cell protein purification background band. (E) Kymographs of His-Kif18AFL-GFP molecules and His- Kif18A1-777-GFP recorded with TIRF microscopy in streaming mode under low ATP conditions. Arrows mark traces with bleaching events. Quantification states fractions observed among the traces that started and ended within one kymograph. (F) and (G) Fluorescence signal along a trace of moving His- Kif18A1-777-GFP and His- Kif18AFL-GFP molecules showing 2-step bleaching in a low ATP assay. Horizontal scale bars indicate 5 s, vertical scale bars indicate 1 µm.(TIF)Click here for additional data file.Movie S1Original movie sequence showing HeLa-cells stably expressing H2B-mRFP transfected with short interfering RNA-oligonucleotides (siRNAs) targeting luciferase (GL2) and transiently overexpressing GFP. (AVI)Click here for additional data file.Movie S2Original movie sequence showing HeLa-cells stably expressing H2B-mRFP transfected with short interfering RNA-oligonucleotides (siRNAs) targeting luciferase (GL2) and transiently overexpressing GFP. (AVI)Click here for additional data file.Movie S3Original movie sequence showing HeLa-cells stably expressing H2B-mRFP transfected with short interfering RNA-oligonucleotides (siRNAs) targeting Kif18A and transiently overexpressing GFP. (AVI)Click here for additional data file.Movie S4Original movie sequence showing HeLa-cells stably expressing H2B-mRFP transfected with short interfering RNA-oligonucleotides (siRNAs) targeting Kif18A and transiently overexpressing GFP. (AVI)Click here for additional data file.Movie S5FL. FL, respectively. Selected frames of this movie are shown in Original movie sequence showing HeLa-cells stably expressing H2B-mRFP transfected with short interfering RNA-oligonucleotides (siRNAs) targeting Kif18A and transiently overexpressing GFP-Kif18A(AVI)Click here for additional data file.Movie S6FL. FL, respectively. Selected frames of this movie are shown in Original movie sequence showing HeLa-cells stably expressing H2B-mRFP transfected with short interfering RNA-oligonucleotides (siRNAs) targeting Kif18A and transiently overexpressing GFP-Kif18A(AVI)Click here for additional data file.Movie S71-777. 1-777, respectively. Selected frames of this movie are shown in Original movie sequence showing HeLa-cells stably expressing H2B-mRFP transfected with short interfering RNA-oligonucleotides (siRNAs) targeting Kif18A and transiently overexpressing GFP-Kif18A(AVI)Click here for additional data file.Movie S81-777. 1-777, respectively. Selected frames of this movie are shown in Original movie sequence showing HeLa-cells stably expressing H2B-mRFP transfected with short interfering RNA-oligonucleotides (siRNAs) targeting Kif18A and transiently overexpressing GFP-Kif18A(AVI)Click here for additional data file.Movie S9778-898. 778-898, respectively. Selected frames of this movie are shown in Original movie sequence showing HeLa-cells stably expressing H2B-mRFP transfected with short interfering RNA-oligonucleotides (siRNAs) targeting Kif18A and transiently overexpressing GFP-Kif18A(AVI)Click here for additional data file.Movie S10778-898. 778-898, respectively. Selected frames of this movie are shown in Original movie sequence showing HeLa-cells stably expressing H2B-mRFP transfected with short interfering RNA-oligonucleotides (siRNAs) targeting Kif18A and transiently overexpressing GFP-Kif18A(AVI)Click here for additional data file.Movie S11FL-GFP moves uni-directionally along microtubules in vitro and is highly processive. TIRF microscopy pictures were taken at 1 frame per second. Video playback is 20x real-time.Subnanomolar His-Kif18A(AVI)Click here for additional data file.Movie S121-777-GFP moves uni-directionally along microtubules in vitro. TIRF microscopy pictures were taken at 1 frame per second. Video playback is 20x real-time.Subnanomolar His-Kif18A(AVI)Click here for additional data file.


This suggests that this novel Mcl1L variant efficiently protects tumor cells against extrinsic death signalling and therefore may provide a survival advantage for highly aggressive tumors.Members of the Bcl-2 protein family are frequently deregulated in tumors as they critically control cell death induction in mammalian cells. Alterations of these proteins may cause resistance to chemotherapy-induced cell death and immune responses. By serendipity we cloned a variant of the anti-apoptotic Bcl2-family member Myeloid cell leukemia-1 (Mcl1) from human neuroblastoma and leukemia cells. This Mcl1L variant lacks a 45 bp sequence that codes for 15 highly conserved amino acids ranging from Gly158 to Asp172. This region is part of the so called PEST-sequence of Mcl1L and contains two phosphorylation sites (Ser159 and Thr163) that regulate Mcl1L stability. A caspase 3/caspase 8 cleavage site at Asp157 which has been reported to be critical for death-receptor-induced apoptosis and for the conversion of Mcl1L into a pro-apoptotic protein is also missing in this novel variant. Importantly, Mcl1L FBW7 and β-TRCP regulate Mcl1 degradation [Mcl1 was originally identified in differentiating myeloid cells . The secradation , 23. Theradation , 24.Beside anti-apoptotic full-length Mcl1L, there is evidence for several pro-apoptotic Mcl1 variants. Pro-apoptotic variants are generated either by cleavage of Mcl1 by caspase-3 or 8 , 26 or bdel158-172 variant Mcl1JAM (Just Another Mcl1). PCR-products corresponding to the full length and the Mcl1LJAM variant were detected also in a CEM leukemia cell line  for up to 3 hours. In SH-EP/Ctr cells endogenous Mcl1L steady state expression is already significantly lowered within 30 minutes of CHX treatment to about 50 %  with a rapid decay after 60 min (12% versus 34%) and 90 min (7% versus 23%). However, the expression of Mcl1LJAM stabilized endogenous Mcl1L and significantly increased the half-life of endogenous Mcl1L to 93% after 30 min and still 47% after one hour . As Mclcl1 Fig. . Next weJAM cells did not contain elevated Noxa-levels, although Bortezomib-treatment increased Noxa steady state levels similar to SH-EP/Mcl1L cells  or EYFP-tagged Mcl1LJAM (EYFP- Mcl1LJAM). Treatment of SH-EP/tetNoxa-ECFP-Mcl1L cells with doxycyline (doxy) induces Noxa expression  increased steady state levels of Mcl1LJAM and Mcl1L which is in line with results from Wuillème-Toumi et al, who found that Bim and Mcl1L protect each other from degradation [JAM cells, doxy-induced Bim precipitated EYFP-Mcl1LJAM and to a lesser extend endogenous Mcl1L. Co-purification of BclxL was only observed in cells treated with both, doxy and bortezomib . To study long-term survival after treatment we performed clonogenic survival assays which demonstrated that survival of neuroblastoma cells after FAS-receptor activation by CH11 antibody was significantly increased in Mcl1LJAM -expressing cells  compared to Mcl1L-expressing cells (143%) and SH-EP/Ctr cells  including a caspase 3/8 cleavage site and two regulatory amino acids . Both, Mcl1L and Mcl1LJAM accumulate after proteasome inhibition by bortezomib treatment  whereas ectopic Mcl1L showed a similar decay as endogenous Mcl1L during CHX treatment. This is in line with a recent report from Thomas et al, who demonstrated that mutation at Ser162 reduces the stability of Mcl1 [JAM enhances the half-life of endogenous Mcl1L suggesting that both proteins compete for the same binding partners and Mcl1LJAM is preferentially targeted for degradation and therefore more instable than the wild type. Since no ubiquitin residues are affected by the deletion in Mcl1JAM  [JAM, which might affect binding to BH3-only proteins, for example Puma which was shown to protect Mcl1L from MULE induced degradation by binding to its BH1 domain, [JAM -expressing cancer cells. We also observed this enhanced stability of Mcl1L when coexpressed with Mcl1LJAM after treatment with CH11 or TRAIL. Mcl1LJAM was degraded after CH11 and TRAIL treatment but delayed phosphorlation and degradation of Mcl1L .The lines SH-EP , CEM-C7HJAM was amplified from human cDNA and inserted into the BamH1-EcoRI sites of pLIB-MCS2-iresPuro. For live-cell imaging, the coding sequence of ECFP or EYFP were amplified from pECFP-C1 and pEYFP-C1  and inserted into the EcoR1 site of pLIB-Mcl1L-iresPuro (resulting in pLIB-ECFP-Mcl1L-iresPuro) or pLIB-Mcl1LJAM-iresPuro (resulting in pLIB-EYFP-Mcl1LJAM-iresPuro). The production of retrovirus supernatants was described previously [JAM, SH-EP/ECFP-Mcl1L or SH-EP/EYFP-Mcl1LJAM cells. The tetracyclin-regulated cell lines SH-EP/tetNoxa and SH-EP/tetBim have been described previously [JAM-iresPuro, generating SH-EP/tetNoxa-ECFP-Mcl1L, SH-EP/tetNoxa-EYFP-Mcl1LJAM, SH-EP/tetBim-EYFP- Mcl1LJAM and SH-EP/tetBim-ECFP-Mcl1L cells.pLIB-MCS2-iresPuro, pLIB-MCL1-iresPuro pLIB-rtTA-M2-iresTRSID-iresPuro, pQ-tetCMV-Bim-SV40Neo, pQ-tetCMV-Noxa-SV40Neo, pQ-tetH1-SV40-Puro have been described before , 43, 44.eviously , 44. TheJAM expression was analysed via RT-PCR. For PCR detection the coding sequence of MCL1 was amplified from cDNA and a nested PCR was performed using 5' AAGAGGAGCTGGACGGGTAC and 3' TGGCTTTGTGTCCTTGGC which amplifies part of the PEST region. PCR products were analysed on 2% agarose gels.Mcl1L7 cells were lysed in PBS containing 1% IGEPAL, phosphatase- and protease-inhibitors. For immunoprecipitation 1 μg of anti-rabbit Bim antibody , 2.5 μg of anti-mouse Noxa , or mouse or rabbit immunoglobulin, as a negative control, were covalently coupled to Tachisorb™ Immunoadsorbent  or Affi-Prep Protein A Support  using dimethylpimelidate dihydrochloride/Borax buffer. Antibody-bead complexes were added to 500 μg lysate and incubated at 4°C for 6 hours. Tachisorb™-/Protein A-immunocomplexes were washed four times in PBS/IGEPAL-buffer, resuspended in SDS-sample buffer and subjected to SDS-PAGE and blotting. Equal amounts of total-protein and cleared supernatants were loaded as controls. Immunoblot analysis was performed as previously described [For immuoprecipitation 1×10escribed  using pr3 cells were seeded into a 6well and cultured up to 7 days. For chemotherapeutic treatment 4×104 cells were seeded into 6wells and treated for 72 hours with chemotherapeutic agents . Afterwards medium was removed and cells were fixed and stained with 0.2% crystal violet in 50% methanol. Cell density was measured photometrically after discoloration with 0.5% SDS in 50% ethanol. Untreated/ mock-infected cells were set as 100%.To determine the ability of SH-EP cells to form colony units, 2×105 cells were harvested and resuspended in hypotonic PI solution for 2-4 hours at 4°C. Stained nuclei in the sub-G1 marker window were considered to represent apoptotic cells. Statistical analysis was performed using GraphPad Prism 4.0 software.Apoptosis was assessed by staining the cells with propidium-iodide (PI) using a CytomicsFC-500 Beckman Coulter as previously described . In shorFor live cell analyses cells were grown on LabTek Chamber Slides™  coated with 0.1 mg/ml collagen. Images were collected with an Axiovert200M microscope with a 63x-oil objective . Mitochondria staining was performed using 300 nM CMXRos .Statistical significance of differences between controls and treated cells were calculated using unpaired t-test. All statistical analyses were performed using Graph Pad Prism 4.0 software.


AML1/RUNX1. Our analysis of 13 BC cases revealed that three cases had AML1 mutations and the transcript levels of wild-type (wt.) AML1 were elevated in BC compared with CP. Functional analysis of representative AML1 mutants using mouse hematopoietic cells revealed the possible contribution of some, but not all, mutants for the BC-phenotype. Specifically, K83Q and R139G, but neither R80C nor D171N mutants, conferred upon BCR-ABL-expressing cells a growth advantage over BCR-ABL-alone control cells in cytokine-free culture, and the cells thus grown killed mice upon intravenous transfer. Unexpectedly, wt.AML1 behaved similarly to K83Q and R139G mutants. In a bone marrow transplantation assay, K83Q and wt.AML1s induced the emergence of blast-like cells. The overall findings suggest the roles of altered functions of AML1 imposed by some, but not all, mutants, and the elevated expression of wt.AML1 for the disease progression of CML.Patients in the chronic phase (CP) of chronic myelogenous leukemia (CML) have been treated successfully following the advent of ABL kinase inhibitors, but once they progress to the blast crisis (BC) phase the prognosis becomes dismal. Although mechanisms underlying the progression are largely unknown, recent studies revealed the presence of alterations of key molecules for hematopoiesis, such as  BCR-ABL generated by the chromosomal translocation t in hematopoietic stem cells constitutively activates tyrosine kinase on its own and leads to CML Although BCR-ABL plays a central role in the pathogenesis of CML-CP, the unrestrained expression and continuous activity of BCR-ABL kinase itself are thought to accelerate the disease AML1, a founding member of the RUNX family AML1 is the most frequent target gene of chromosomal translocation associated with human leukemia AML1 point mutations have been often identified in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and CML-BC AML1 deletion in established hematopoietic stem cells leads to an expansion of hematopoietic progenitor cells. These findings imply that impaired AML1 functions may enhance self-renewal of progenitor cells and block their differentiation, thus priming the cells for leukemic transformation AML1-MDS1-EVI1 inhibits differentiation of 32Dcl3 AML1-MDS1-EVI1 to elicit leukemia RUNX1, ASXL1, WT1, NRAS, KRAS, TET2, CBL, TP53, IDH and IKZF) were reported to occur in 76.9% of CML-BC patients. Significantly, AML1 mutations account for 33.3% of CML-BC patients, while no such mutation was detected in CML-CP samples AML1 mutants contribute to the transition from CML-CP to CML-BC. Although somewhat paradoxically, experimental expression of wt., but not mutant, AML1 protects BCR-ABL-transformed cells from imatinib-induced apoptosis, indicating the role of up-regulated wt.AML1 in imatinib resistance and disease progression of CML Recently, mutations of genes crucial for hematopoiesis  to the BC phenotype using mouse cells in vitro and in vivo.We hypothesized that the deregulated activity of SCIDPrkdc Il2rgtm1Wjl/SzJ) mice were purchased from Charles River and Jackson Laboratory , respectively. All animal experiments were performed according to protocols (Permit Number: 22-7) approved by the Institutional Animal Care and Use Committee at the Aichi Cancer Center.C57BL/6N mice were purchased from Charles River  and SLC . NOD/SCID and NSG  and bone marrow (BM) cells were obtained from healthy volunteers. CML cell lines used were as follows: K562 (ATCC-CLL-243), Nalm1 (ATCC-CRL-1567), BV-173(DSMZ ACC-20), MegO1 BCR/ABL-IRES-GFP plasmid AML1bAML1R139GNUP98-HOXA9Bmi1Hes1R (Clontech) and MSCV-IRES-human(h)CD8 MSCV-p210Anti-Gr-1 (RB6-8C5), anti-Mac-1(M1/70), anti-B220 (RA3-6B2), anti-CD3 (145-2C11), anti-TER-119 (TER-119), anti-CD41 (eBioMWReg30), anti-c-kit (2B8), anti-Sca-1 (D7)  and anti-Fc receptor  antibodies were used. Flow cytometry was performed on FACSCalibar (BD Bioscience) and JSAN  devices. Data were analyzed using Flow Jo software ver.7.6.2 . Lineage marker-negative (Lin-)/Sca-1-negative (Sca-1-) e14 C57BL/6N mouse fetal liver cells were isolated using antibodies for Gr-1/Mac-1/CD3/B220/Ter-119/Sca-1 and MACS columns . Cells were spin-infected with retrovirus, as described 5) were grown in Iscove’s Modified Dulbecco Medium (IMDM) supplemented with 10% fetal calf serum (FCS) without cytokines, in triplicate, and counted using the trypan blue exclusion method. For the colony-forming assay, 1×104 cells were plated in triplicate, as described Cells .Target sequences for shRNA were AML1 and 5′-GCGGGAAATCGTGCGTGACATT-3′/5′-GATGGAGTTGAAGGTAGTTTCGTG-3′ for human β-actin.Quantitative PCR was performed as described Anti-AML1  and anti-tubulin  antibodies were used.AML1 were amplified using RT-PCR and subjected to direct sequencing as described cDNAs for A nuclear extract of 293T cells transfected with Myc-tagged CBFβ was mixed with that of cells transfected with either FLAG-tagged wt. or mutant AML1s, and then incubated with annealed 5′-biotinylated oligo-DNAs for the binding site of AML1. The DNA was captured by streptavidin-agarose beads, and co-purified proteins were analyzed by immunoblotting with anti-FLAG antibody, as described M-CSFR) promoter (M-CSF-R-luc) 293T cells were transfected with plasmids for the luciferase reporter containing macrophage colony-stimulating factor receptor  4 days earlier, and then cultured in the presence of SCF (stem cell factor), IL-3, IL-6 and 1×10−5 M hydrocortisone, and infected with retrovirus, as described in the literature BCR-ABL-IRES-GFP and either MSCV-wt.AML1-IRES-hCD8, MSCV-AML1K83Q-IRES-hCD8 or MSCV-AML1R80C-IRES-hCD8 were transplanted intravenously into 2.5 Gy-irradiated NOD-SCID or NSG mice. Probabilities of event-free survival were estimated using the Kaplan-Meier method, and statistical analysis was performed using the log-rank test with post hoc analysis using Holm’s method.Cultured cells were intravenously transferred into lethally irradiated syngeneic mice, along with 0.5×10AML1 and its mutations, in clinical samples of 13 CML-BC   of 12 evaluable CML-BC patients: G190R, R135EfsX42 and A297LfsX7. AML1 mutations were not found in any of the 5 CML-BC cell lines examined (exons 3–5 of MegA2 were excluded because of the consistent failure of RT-PCR-mediated amplification). Interestingly, the four outlier CML-BC cases had no mutation in the AML1 gene, raising the possibility that some CML-BC might be causally related to elevated expression of wt.AML1. To gain insights into this possibility, we chose three cell lines MegA2, MegO1 (megakaryocytic) and Nalm1 (B lymphoid) that exhibited higher AML1 transcript levels compared with those of normal and CML-CP samples, and examined the effects of shRNA-mediated silencing of AML1 (AML1 transcript levels obtained following infection with shRNA virus in these cell lines were comparable to those of CML-CP patients (We first examined transcript levels of 3 CML-BC  and 5 CM samples . Investip<0.001) . Direct  of AML1 . Two shR of AML1  and inhi of AML1 . Cell depatients . These fWe next investigated the DNA-binding and transactivation abilities of BC-related AML1 mutants. For this purpose, we chose AML1K83Q and AML1R80C mutants  because NUP98-HOXA9Bmi1Hes1BCR-ABL. Cells were then cultured in a cytokine-free condition. BCR-ABL-alone-transduced cells grew initially, but ceased to do so after a week  and either mutant or wt.AML1 (that co-expresses the puromycin-resistant gene) or vector-only control were selected for puromycin-resistance, and cultured without cytokines. GFP-positive cells in this culture therefore represent BCR-ABL- and mutant/wt.AML1-doubly transduced cells, while GFP-negative cells represent mutant/wt.AML1-alone-transduced cells. Results showed that while BCR-ABL/AML1K83Q and BCR-ABL/AML1R139G cells continued to grow for at least 11 days, BCR-ABL/AML1R80C and BCR-ABL/AML1D171N cells lacked such an ability  ratio  ratio . While tC) ratio . In controphages . These din vivo, we intravenously transferred cells grown in cytokine-free culture (shown in BCR-ABL-only-transduced cells (n = 2) survived without any signs of disease for at least 4 months (BCR-ABL and AML1K83Q (n = 5) or wt.AML1 (n = 5) became moribund or died within 30 days after the transfer. BCR-ABL/AML1R139G-co-transduced cells were less potent in the lethality   . All disAML1K83Q and wt.AML1 to investigate their co-operability with BCR-ABL using a conventional BMT assay. To this end, retroviruses for wt. and mutant AML1s that co-express an extracellular domain of human CD8 (hCD8) by virtue of an IRES element were used to facilitate identification of infected cells. AML1R80C was also included in the analysis, because this mutant showed, unlike AML1K83Q, dominant-negative activity against wt.AML1 in the transactivation assay conducted in AML1K83Q and wt.AML1 in culture  within 23 days following transplantation. The MPN was characterized by expansion of mature myeloid cells (AML1R80C did not appreciably affect the matured myeloid-dominant phenotype (n = 3)   , which w (n = 3) . In cont (n = 3) . The GFP1+ cells , while t1+ cells . These f 3 cases  comparedantation . These fcipients  comparedcipients , and hiscipients . Flow cy B cells , and thove cells . These recipient , are notAML1 mutations and quantitated AML1 transcripts in BC samples. We found AML1 mutations in three of 12 evaluable BC cases (25%) and elevated levels of transcripts of wt.AML1 in BC when compared with CP and normal bone marrow samples. The latter finding is consistent with the results of our analysis of published data of a larger number of cases AML1 and/or elevated expression of AML1 with the disease progression of CML.Molecular mechanisms underlying the disease progression of CML are largely unknown, but comparison of BC with CP following deep sequencing and gene expression analysis may provide clues. Several reports have revealed the presence of recurrent somatic mutations specific to BC BCR-ABL-alone control cells in culture. However, AML1R139G was much less potent regarding colony forming ability than AML1K83Q, suggesting a lower clonogenic potential. In contrast, AML1R80C and AML1D171N did not confer a growth advantage. Rather unexpectedly, wt.AML1 behaved similarly to AML1K83Q. Although AML1G190R and AML1A297LfsX7 mutants found in our clinical samples co-operated with BCR-ABL to confer growth advantage upon cells in culture , it is important to investigate AML1 mutants individually for their capacity amid BCR-ABL expression to induce BC-like disease in mouse models. In addition, a difference in cooperative genes may impose additional factors for consideration AML1 mutation displayed karyotypic abnormalities and/or mutations in ASXL1, IKZF1, WT, TET2, IHD1 or CBL genes, thus pointing to the presence of additional genetic and epigenetic abnormalities. Here, the search for mutations focused on 11 genes , and therefore leaves open the possibility that more genes are mutated.A recent report by Zhao AML1 transcripts in BC compared with CP, and the presence of outlier patients in BC regarding AML1 transcript levels. Elevated expression of wt.AML1 is known to confer upon BCR-ABL-expressing cells resistance to imatinib Our examination of clinical samples revealed elevated levels of wt.Figure S1AML1 mRNA levels of CML patient samples. Data published by Radich et al. https://www.oncomine.org/resource/login.html) and analyzed for statistical differences. The bottom side of the box represents the first quartile, and the third quartile is represented by the top side. The vertical width of the box represents the inter-quartile deviation. The horizontal line inside the box is the median. The vertical lines protruding from the box extend to the minimum and maximum values of the data set. The values plotted above the top whiskers are outliers. The difference in mRNA levels among CP, AP and BC cases were statistically significant (p = 0.00014) . Pairwise comparisons were made using the Mann-Whitney U-test and post hoc analysis with P-value adjustment using Holm’s method.(TIF)Click here for additional data file.Figure S2Western blot analysis of BC cell lines infected with shRNA viruses for AML1. MegA2, Nalm1 and MegO1 cells were infected with the indicated shRNA for AML1(shRNA1 and shRNA2) and luciferase (control), and subjected to Western analysis with anti-AML1 antibody. Anti-tubulin blot was included to allow comparison of amounts of loaded protein. Relative expression levels of AML1 are also presented.(TIF)Click here for additional data file.Figure S3BCR-ABL (B/A) and either NUP98-HOXA9, Bmi1 or Hes1 in the liquid culture without cytokine supplementation. Cells were counted every other day with the use of trypan blue.Sustained growth of cells cotransduced with (TIF)Click here for additional data file.Figure S4Cytokine-free liquid culture of mouse hematopoietic progenitor cells transduced with wt.AML1 and AML1 mutants. Lin-negative, Sca-1-negative progenitor cells were transduced with the indicated AML1, selected for puromycin-resistance and cultured without cytokines. Note that cell numbers rapidly decreased due to cell death.(TIF)Click here for additional data file.Figure S5Kaplan-Meier survival curves of recipient mice of progenitor cells that were transduced with the indicated retroviral constructs and grown in cytokine-free culture.(TIF)Click here for additional data file.Figure S6BCR-ABL and hCD8-only control.Flow cytometric analysis of bone marrow (BM) cells from a mouse receiving BM cells that were transduced with (TIF)Click here for additional data file.Figure S7BCR-ABL and AML1R80C.Flow cytometric analysis of spleen (SP) cells from 2 mice transplanted with BM cells that were transduced with (TIF)Click here for additional data file.Figure S8BCR-ABL and AML1K83Q.Flow cytometric analysis of spleen (SP) cells from 2 mice transplanted with BM cells that were transduced with (TIF)Click here for additional data file.Figure S9BCR-ABL and wt.AML1.Flow cytometric analysis of spleen (SP) cells from 2 mice transplanted with BM cells that were transduced with (TIF)Click here for additional data file.Figure S10Compositions of cell fractions transduced with BCR-ABL (GFP) and/or AML1 (hCD8). Indicated fractions in spleen cells were analyzed for cell compositions  following primary and secondary transplantations. * indicates fractions with insufficient number of cells to evaluate.(TIF)Click here for additional data file.Figure S11BCR-ABL/AML1K83Q- and BCR-ABL/wt.AML1-transduced cells.(A) Photographs showing hepatosplenomegaly of representative secondary recipient mice transplanted with (TIF)Click here for additional data file.Figure S12Analysis of functions of AML1R135EfsX42, AML1A297LfsX7 and AML1G190R. (A) Transactivation potentials of the indicated AML1 mutants. Assay was performed as described in the legend to (TIF)Click here for additional data file.Figure S13Analysis of sensitivity of cells to imatinib. (A)(B) MegA2, MegO1 and Nalm1 cells were infected with shRNA virus for AML1 (AML1–1 and AML1–2 used in (TIF)Click here for additional data file.Table S1Summary of BC samples used for quantification of AML1 transcript in (TIF)Click here for additional data file.Table S2AML1 in CML-BC patients.Reported mutations of (TIF)Click here for additional data file.


Programmed death-1 (PD-1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) receptors are immune checkpoint receptors involved in tumor immune evasion. Nivolumab and ipilimumab are fully human monoclonal PD-1 and CTLA-4 blocking antibodies, respectively, and have shown clinical antitumor activity as monotherapy. In a multicohort, phase 1 study with melanoma patients (pts), nivolumab/ipilimumab combination produced objective response rates (ORRs) of up to 53% (NCT01024231). We evaluated putative predictive biomarkers for nivolumab and ipilimumab monotherapy in the combination setting, including tumor PD-L1 expression, absolute lymphocyte count (ALC), peripheral blood (PB) myeloid-derived suppressor cells (MDSCs), and pharmacodynamic changes in T-cell phenotype.+ and CD8+ T cells and MDSC.An automated immunohistochemistry assay (developed by Dako) using the 28-8 PD-L1 antibody was used to evaluate tumor PD-L1 membrane expression in archival FFPE specimens. ALC was measured in serial PB samples. Flow cytometry was used to characterize changes in percentage, number, and phenotype of activated CD4P<0.05).Using a cut-off of 5% tumor cell membrane staining, 37% of pts (13/35) exhibited PD-L1 expression. Nine of 22 pts (41%) with PD-L1 negative tumors and 6/13 pts (46%) with PD-L1 positive tumors achieved an OR. ALC increase vs baseline was not observed, but phenotypic changes in PB T-cell subsets  were evident. Six of 14 pts (43%) with low ALC (<1.0 at wk 6-7) achieved an OR, vs 15/38 pts (39%) with ALC ≥1.0 at wk 6-7. Lower pretreatment percent MDSC correlated with higher ORR (ORRs with nivolumab/ipilimumab combination in this small pt subset were not associated with PD-L1 expression or ALC, in contrast to prior observations with nivolumab or ipilimumab monotherapy, respectively. Compared with either therapy alone, the combination may induce an immune response with distinct characteristics. PB and tumor biomarkers, and their relationship to clinical activity patterns, will be further investigated in this study and in phase 2/3 randomized studies.


The accurate diagnosis of acute myocarditis on cardiovascular magnetic resonance imaging (CMR) often requires multiple modalities, including T2-weighted (T2W), early and late gadolinium imaging. Novel CMR techniques are now available, including bright-blood T2W-CMR, and T1-mapping which is also sensitive to changes in free water content. We hypothesized that these emerging methods can serve as new diagnostic criteria for myocarditis.We studied 34 patients with suspected acute myocarditis and 45 healthy controls. All patients presented with chest pain, troponin I > 0.04 ug/L and had unobstructed coronary arteries on angiogram or ruled out clinically (e.g. young age < 35 years). CMR at 1.5T within 12 days of presentation included (1) dark-blood T2 (STIR); (2) bright-blood T2 (ACUT2E); (3) T1-mapping (ShMOLLI); and (4) late gadolinium enhancement (LGE) , bright-blood T2W-CMR  and mean myocardial T1 . Receiver operator characteristic analysis showed good diagnostic performance for all methods, with T1-mapping having a significantly larger area-under-the-curve (0.95) compared to dark-blood T2W (0.79) and bright-blood T2W imaging  Clinical Fellowship and the University of Oxford Clarendon Fund Scholarship. Dr. Robin Choudhury is a Wellcome Trust Senior Research Fellow in Clinical Science. Stefan Neubauer and Robin Choudhury acknowledge support from the British Heart Foundation Centre of Research Excellence, Oxford


Highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) has caused large economic losses in swine industry in recent years. However, current antiviral strategy could not effectively prevent and control this disease. In this research, five artificial microRNAs (amiRNAs) respectively targeted towards ORF5  and ORF6  were designed and incorporated into a miRNA-based vector that mimics the backbone of murine miR-155 and permits high expression of amiRNAs in a GFP fused form mediated by RNA Pol II promoter CMV.It was found that amirGP5-370 could effectively inhibit H-PRRSV replication. The amirM-263-M-263, which was a dual pre-amiRNA expression cassette where two amirM-263s were chained, showed stronger virus inhibitory effects than single amirM-263. H-PRRSV replication was inhibited up to 120 hours in the MARC-145 cells which were stably transduced by recombinant lentiviruses . Additionally, efficacious dose of amirGP5-370 and amirM-263 expression did not trigger the innate interferon response.Our study is the first attempt to suppress H-PRRSV replication in MARC-145 cells through vector-based and lentiviral mediated amiRNAs targeting GP5 or M proteins coding sequences of PRRSV, which indicated that artificial microRNAs and recombinant lentiviruses might be applied to be a new potent anti-PRRSV strategy. Nidovirales, family Arteriviridae[Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant viral diseases in swine and it threats global swine industries. It has riviridae. The PRRriviridae. Both ofriviridae-9, for eriviridae. In receriviridae,12. HoweRNA interference (RNAi) is a conserved natural mechanism by which homologous small interference RNA (siRNA) duplexes induce potent and sequence-specific posttranscriptional inhibition of gene expression via degradation of complementary messenger RNA (mRNA),14. SincIn this research, two recombinant plasmids expressing amiRNAs, which are designed to be perfectly homologous to PRRSV GP5 and M proteins coding sequences respectively, were found to be capable of inhibiting H-PRRSV replication without inducing innate interferon response. Additionally, it was found that H-PRRSV replication could be effectively inhibited in MARC-145 cells up to 120 h at least by stably transduced with recombinant lentiviruses expressing verified amiRNAs.Renilla luciferase signals were normalized to the firefly luciferase signals in order to correct the differences of transfection efficiencies. Relative luciferase expression in the presence of psiCHECK2 alone was set at 100%. We observed that inhibitory effects of all the amiRNAs were more than 70%. The relative renilla luminescence (RLU) activities in amiRNAs transfected cells were significantly lower than that in the cells transfected with psiCHECK2 alone (p < 0.05). Negative amiRNA transfected cells did not show significantly reduction in RLU activity compare to psiCHECK2 alone transfected cells , which were designed using online tools (Invitrogen), target PRRSV GP5 and M proteins coding sequences, as shown in Figure s Figure  and 2D. s Figure  and 2D.6 TU/ml (Lenti-amirGP5-370), 3.33 × 106 TU/ml (Lenti-amirM-263-M-263), 1.81 × 106 TU/ml (Lenti-negative amiRNA).Six amiRNAs expressing plasmids , two luciferase report plasmids psiCHECK2-GP5 and psiCHECK2-M, and three recombinant lentiviral expression plasmids  were constructed. All the nucleotides sequences inserted into vectors were confirmed by sequencing. The titers of the three recombinant lentiviruses were 3.35 × 10To determine whether amiRNAs could inhibit CPE induced by H-PRRSV and protect MARC-145 cells, MARC-145 cells were transfected with plasmids expressing amiRNAs that target PRRSV GP5 and M protein coding sequence. In parallel, a nonspecific amiRNA that does not target any known vertebrate genes was transfected. Transfection efficiency was determined by GFP fluorescence intensity of transfected cells on an inverted fluorescence microscope. At six hours after transfection, MARC-145 cells were infected with H-PRRSV. Sixty-hour post-infection, we found that cells pre-transfected with pcDNA™6.2-GW/EmGFP-amirGP5-370 or pcDNA™6.2-GW/EmGFP-amirM-263-M-263 showed less CPE compared to others. MARC-145 cells treated with pcDNA™6.2-GW/EmGFP-neagtive-amiRNA demonstrated the same typical PRRSV-induced CPE as cells that infected with H-PRRSV alone (positive control), which became reticulated and detached from the monolayer, as shown in Figure To determine whether the viral suppression was due to specific viral gene expression inhibition, the cells were infected with H-PRRSV at six hours post-transfected with amiRNAs expressing plasmids. At sixty-hour post-infection, the cells were harvested to detect H-PRRSV GP5 or M protein coding genes and β-actin gene in each experimental group by real-time PCR. β-actin gene was served as an internal reference. AmirGP5-370 showed highest inhibition of expression of its target gene (GP5 protein coding gene) in H-PRRSV infected MARC-145 cells (p < 0.05)  were carried out in the experiment groups that were transfected efficacious dose amiRNAs expressing plasmids. It was observed that amplifications of IFN-β and OAS were absence in the ethidium bromide stained agarose gels   may indus Figure . Meanwhis Figure .50. We found that H-PRRSV titers were at least approximately 1000-fold lower in MARC-145 cells expressing amirGP5-370 or amirM-263-M-263 compared to those infected with H-PRRSV alone or expressing negative amiRNA  demonstrated that adenovirus-mediated FMDV-specific shRNA could significantly reduce the susceptibility of swine to FMDV infection[et al. (2006) demonstrated that anti-HBx shRNA could effectively inhibited HBV replication[Vaccination is the principal means used to control and treat PRRSV infection. An array of PRRS vaccines have been developed, but these all could not provide sustainable disease control because they suffer both from the immune evasion strategies of the virus and the antigenic heterogeneity of field strains. Hence, infection. Carmonaplication.However, shRNAs are not the optimal substrate for RNAi process in that shRNAs will not be processed by Drosha that could create cleavage sites for further cleave by Dicer which is critical for efficient RNAi effect. TherefoRecently, pre-artificial microRNAs (pre-amiRNA) driven by Polymerase II promoters that via naturally existing endogenous microRNA pathway to become mature amiRNA have shown to be more potent antiviral RNAi inducers but with less dangerous of toxicities compared to conventional shRNAs-25. RNA Renilla luciferase signal at least 70% via cleavage their target sequences. Except for amirM-82, inhibitory effects of all the other amiRNAs were found to be in dose dependent  targeting PRRSV GP5 or M proteins coding sequences were designed via online tool. Then recombinant plasmids expressing theses amiRNAs were constructed. In the duel luciferase signal assay experiment, we observed that all amiRNAs could inhibit t Figure  and 2D. To investigate whether amiRNAs could inhibit accumulation of viral mRNAs or proteins of target genes, we carried out CPE analysis, real-time PCR, IFA and Western blotting assays. Among five amiRNAs tested in this study, amirGP5-370 showed the highest inhibitory effect on H-PRRSV replication and ORF5 gene expression in MARC-145 cells infected with H-PRRSV  may induce type I interferon (IFN-I) activation, which will result in off-target effects of RNAi in mammalian cells. HoweverWe designed five artificial amiRNAs respectively targeting PRRSV GP5  or M  proteins coding sequences and constructed recombinant plasmids expressing theses amiRNA. The results showed that amirGP5-370 and amirM-263-M-263 could effectively suppress H-PRRSV replication without inducing innate interferon response. And H-PRRSV replication could be effectively inhibited in MARC-145 cells up to 120 h at least by stably transduced with recombinant lentiviruses expressing verified amiRNAs. To our knowledge, the study presented here is the first attempt to suppress H-PRRSV replication in MARC-145 cells through vector-based and lentiviral mediated amiRNAs targeting GP5 or M proteins coding sequences of PRRSV. The study indicated that artificial microRNAs and recombinant lentiviruses might be applied to be a new potent anti-PRRSV strategy.Five precursor miRNAs (pre-miRNA) sequences respectively targeting on PRRSV GP5 and M protein coding genes were designed through an internet application system (Invitrogen). Double-stranded oligonucleotide encoding pre-miRNA sequence were annealed and inserted into pcDNA™6.2-GW/EmGFP-miR expression vector (Invitrogen) containing cytomegalovirus (CMV) promoter and herpes simplex virus thymidine kinase polyadenylation signal. All recombinant plasmids have been sequenced to confirm the sequences inserted.African green monkey kidney cell line MARC-145 were applied to grow PRRSV and to determine virus titers. Human embryo kidney cells (293FT) were applied to produce lentivirus. MARC-145 cells and 293FT cells were both cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 0.1 mM MEM Non-Essential Amino Acids, 1% penicillin/streptomycin, and 1 mM MEM Sodium Pyruvate. Cells were typsinized and seeded in 96-well, 12-well or 6-well plate nearly 24 hours before tansfection. For transfection, lipofectamine 2000 (Invitrogen) was used according to manufacturer's instruction. Six hours post-transfection, MARC-145 cells were infected with Highly pathogenic PRRSV GD isolate  at a multiplicity of infection of 0.01 as described in previous study.Renilla luciferase activities were evaluated using the Dual-Luciferase Reporter Assay system (Promega), and Renilla luciferase activity was normalized to firefly luciferase activity.Luciferase reporter assays were performed using the psiCHECK2-GP5 and psiCHECK2-M. 293FT cells were grown to approximately 80% confluence in 24-well plates and cotransfected with psiCHECK2-GP5, psiCHECK2-M or psiCHECK2 empty vector plus 0.2 μg, 0.5 μg or 0.8 μg pcDNA™6.2-GW/EmGFP-miR. Cells were incubated with transfection reagent lipofectamine 2000/DNA complex for 6 hours and then refreshed with fresh DMEM containing 1% FBS for 48 hours. Firefly and 50) was calculated by the Reed-Muench method.MARC-145 cells were typsinized and seeded in 96-well plate 24 hours before virus infection. Virus supernatants were 10-fold serially diluted and added 100 μl to each well in eight repeated. Six days after infection, the 50% cell culture infection dose (CCID18 primers (TaKaRa). 1 μl cDNA was submitted to real-time PCR analysis using specific primers for β-actin, PRRSV GP5 and M protein coding genes. β-actin-F:5'TGACTGACTACCTCATGAAGATCC3';β-actin-R:5'TCTCCTTAATGTCACGCACGATT3'[® Premix Ex Taq™ (TaKaRa). Real-time PCR was performed in a LightCycler® 480 Real-Time PCR System and analyzed with LightCycler® 480software (Roche). Amplification was carried out in a 10 μl reaction mixture containing 5 μl SYBR® Premix Ex Taq™ (TaKaRa) 2×, 0.2 μM concentration of each primer, 1 μl cDNA. The reaction procedure was 95°C 10s, followed by 40 cycles at 95°C for 5s and 60°C for 40s. β-actin gene was served as an internal reference. To confirm specific amplification, melting curve analysis was performed.Total RNA was isolated from MARC-145 cells at 60 h after H-PRRSV infection as described above using RNApreppure total cell RNA isolation kit . Genomic DNA was removed using DNase I (NEB). 2 μg RNA was reverse-transcribed into first strand cDNA with M-MLV transcriptase (Promega) and oligo d(T)CACGATT3', GP5-F:5MARC-145 cells seeded in 96-well plate were transfected with pcDNA™6.2-GW/EmGFP-amiR-GP5-370, pcDNA™6.2-GW/EmGFP-amiR-M-263-M-263 or pcDNA™6.2-GW/EmGFP-amiR-negative, and then were infected with H-PRRSV. At 60 hours after virus infection, MARC-145 cells were fixed with pre-cooled methanol for 20 minutes on ice. Following three washes by phosphate-buffer saline , the fixed MAR-145 cells were incubated with PRRSV positive serum for 2 hours at 37°C. Unbound antibodies were washed three times with TBST. Then, FITC labeled goat anti-pig IgG antibody (KPL) was added and incubated for 1 hour at 37°C. After 3 washes by TBST, fluorescence was analyzed using a fluorescence microscopy (Nikon).Western blotting was processed as described previously. The PVD293FT cells were respectively transfected with 1 μg amiRNA construct in 24-well plate (pcDNA™6.2-GW/EmGFP-amiR-GP5-370 or pcDNA™6.2-GW/EmGFP-amiR-M-263-M-263) using Lipofectamine 2000 (Invitrogen). The 2 μg poly I:C (Sigma) that was transfected into 293FT cells in 24-well plate served as a positive control for innate response induction as described previously and natiPCR amplification was performed on 1 μl RT product with interferon-β(IFN-β), 2',5'-oligoadenylate synthetase (OAS) and β-actin(endogenous control) specific primers. IFN-β-F: 5' GATTCATCTAGCACTGGCTGG 3'; IFN-β-R: 5' CTTCAGGTAATGCAGAATCC 3'(186 bp), OAS-F: The pDONR™ 211 vector was used as intermediate to transfer the pre-amiRNA expression cassette constructed before into the lentiviral expression plasmid (pLenti6/V5-DEST) using Gateway technology (Invitrogen) to generate pLenti6/V5-GW/EmGFP-miR. The pLenti6/V5-GW/EmGFP-miR then was co-transfected with packaging vectors (Invitrogen) into 293FT cells using lipofectamine 2000 (Invitrogen). The cell supernatants were collected at 72 h post-transfection and used as a virus stock. All lentiviruses expressed enhanced green fluorescence protein (GFP), allowing for titering and measuring their infection efficiency in transfected cells. The viral titers were determined by counting GFP-positive cells after transduced in the presence of polybrene (6 μg).Statistical significance was determined by student's t-test. A P-value<0.05 was considered statistical significant. N values represent the number independent experiments.HP-PRRS: Highly pathogenic porcine reproductive and respiratory syndrome; amiRNAs: artificial microRNAs; PRRSV: porcine reproductive and respiratory syndrome virus; H-PRRSV: highly pathogenic porcine reproductive and respiratory syndrome virus; RNAi: RNA interference; siRNA: small interference RNA; shRNA: short hairpin RNA; CPE: cytopathic effect; pre-amiRNA: pre-artificial microRNAs; CCID50: 50% cell culture infection dose; GFP: green fluorescence protein.The authors declare that they have no competing interests.SX and QW conceived and designed the study. SX and QW performed the experiments, analyzed data, and wrote the manuscript. JG, LW, ZH and DM coordinated the study. YC and XL contributed to the interpretation of the results and took part to the critical revision of the manuscript. All authors read and approved the final manuscript.


Caenorhabditis elegans, depletion of the clathrin heavy chain CHC-1 and individual components of AP2 led to a significant accumulation of germ cell corpses, which resulted from defects in both cell corpse engulfment and phagosome maturation required for corpse removal. CHC-1 and AP2 components associate with phagosomes in an inter-dependent manner. Importantly, we found that the phagocytic receptor CED-1 interacts with the α subunit of AP2, while the CED-6/Gulp adaptor forms a complex with both CHC-1 and the AP2 complex, which likely mediates the rearrangement of the actin cytoskeleton required for cell corpse engulfment triggered by the CED-1 signaling pathway. In addition, CHC-1 and AP2 promote the phagosomal association of LST-4/Snx9/18/33 and DYN-1/dynamin by forming a complex with them, thereby facilitating the maturation of phagosomes necessary for corpse degradation. These findings reveal a non-classical role of clathrin and AP2 and establish them as indispensable regulators in phagocytic receptor-mediated apoptotic cell clearance.Clathrin and the multi-subunit adaptor protein complex AP2 are central players in clathrin-mediated endocytosis by which the cell selectively internalizes surface materials. Here, we report the essential role of clathrin and AP2 in phagocytosis of apoptotic cells. In  Phagocytosis of apoptotic cells is an indispensable part of the cell death program. During phagocytosis, the evolutionarily conserved CED-1 family phagocytic receptors recognize cell corpses and transduce engulfment signals to induce the formation and maturation of phagosomes. However, it remains largely unknown how the CED-1 signaling pathway induces the cytoskeletal reorganization required for corpse internalization and initiates phagosome maturation. Interestingly, cell corpse phagocytosis appears to resemble clathrin-mediated endocytosis (CME) of cell surface molecules in that both events cause receptor-dependent internalization of extracellular cargoes differing only in size. In CME, the recognition of plasma membrane receptors by adaptor proteins such as the AP2 complex triggers the formation of clathrin-coated vesicles (CCVs) with diameters ranging from 10–200 nm. Nevertheless, it is not known whether clathrin and AP2 also play a role in phagocytosis of apoptotic cells that are much larger than CCVs. Here we provide genetic, cell biological, and biochemical experimental findings to demonstrate that clathrin and AP2 act downstream of CED-1 to regulate the actin cytoskeleton rearrangement required for cell corpse internalization and cell corpse degradation. Clathrin and AP2 form two types of complex with factors required for engulfment and phagosome maturation. These findings establish clathrin and AP2 as essential players in phagocytic receptor-mediated apoptotic cell clearance. C. elegans hermaphrodite, 131 somatic cells and about half the germ cells undergo apoptosis and the resulting cell corpses are quickly removed by neighboring cells in the soma or by sheath cells encasing the germ line. The engulfment of cell corpses is essentially controlled by two partially redundant signaling pathways that induce the cytoskeletal reorganization of engulfing cells Drosophila Draper and Six-microns-under (SIMU) Phagocytosis of apoptotic cells is critical to tissue remodeling, suppression of inflammation and control of immune responses C. elegans, DYN-1/dynamin regulates the initiation of phagosome maturation The phagocytic receptor CED-1 plays a leading role in apoptotic cell clearance by recognizing cell corpses and transducing signals for engulfment and phagosome maturation. Nevertheless, it remains largely unknown how the CED-1-mediated signaling pathway triggers the cytoskeletal reorganization required for corpse internalization. In addition, the mechanisms governing the transition from corpse internalization to phagosome maturation are poorly understood. Interestingly, CED-1-mediated phagocytosis of cell corpses appears to resemble clathrin-mediated endocytosis (CME) of cell surface molecules in that both events cause receptor-dependent internalization of extracellular cargoes differing only in size C. elegans. We found that clathrin and AP2 act downstream of CED-1 and CED-6 by forming a complex with them, which mediates the rearrangement of the actin cytoskeleton required for cell corpse engulfment. In addition, we demonstrated that LST-4, the C. elegans homolog of Snx9/18/33, functions at an early step of phagosome maturation by promoting phagosomal association of DYN-1/dynamin. Furthermore, we demonstrated that clathrin and AP2 also interact with LST-4 and DYN-1 to regulate the initiation of phagosome maturation required for cell corpse degradation. These findings suggest that clathrin and AP2 play essential roles in the phagocytic receptor CED-1-mediated apoptotic cell clearance pathway by regulating cytoskeletal reorganization and facilitating phagosome maturation.In this study, we investigated the mechanisms underlying CED-1-mediated cytoskeleton reorganization for phagocytosis and uncovered the role of CME regulators in apoptotic cell clearance. Our findings revealed that clathrin and the AP2 complex, the central players in CME, are critical to apoptotic cell removal in ced-1(e1735) and ced-6(n1813) strong loss-of-function mutants, and analyzed proteins in the precipitates using liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS). Interestingly, multiple peptides of the heavy chain of clathrin (CHC-1) were identified from proteins co-precipitated with CED-1 and CED-6 in the N2 lysate but not in lysates of ced-1(e1735) or ced-6(n1813) mutants (chc-1 and examined the persistence of cell corpses in C. elegans germ lines. We found that germ cell corpses accumulated significantly in an age-dependent manner in chc-1(RNAi) animals. A similar increase was observed at 25°C in a chc-1 temperature-sensitive mutant, b1025tschc-1(RNAi) animals   mutants . We ther animals . These rC. elegans homologs of individual mammalian proteins involved in CME and examined the persistence of germ cell corpses. Compared to animals with control RNAi, a significant increase in germ cell corpses was observed in animals treated with RNAi of apa-2, apb-1 and dpy-23, which encode the α, β2 and μ2 subunits of the AP2 complex, respectively  and apb-1(RNAi) animals persisted longer than 120 min, and no cell corpses existed less than 60 min (chc-1(RNAi) animals, 28 out of 50 germ cell corpses examined (56%) from 5 gonad arms appeared not to be fully engulfed by sheath cells (apb-1(RNAi) animals were found not to be internalized . In contrast, the majority of germ cell corpses in n 60 min , suggestth cells . Similarrnalized . In contth cells  [22], [3C. elegans, the ced-1/6/7 and ced-2/5/12/10 signaling pathways act redundantly to mediate cell corpse engulfment chc-1 could exert an additive effect on defective corpse engulfment in mutants deficient in either engulfment pathway. In ced-1(e1735) and ced-6(n2095) strong loss-of-function mutants in the cell corpse recognition pathway, RNAi of apb-1, dpy-23, or chc-1 did not obviously change the numbers of germ cell corpses at all adult ages examined (ced-2(n1994) and ced-5(n1812) strong loss-of-function mutants affecting the cytoskeletal reorganization pathway  and mCherry::CHC-1 driven by the ced-1 promoter (yqIs98) rescued the cell corpse phenotype in chc-1(b1025ts) mutants (apa-2 promoter (yqEx481) or the ced-1 promoter (yqIs99) rescued the accumulation of germ cell corpses in animals treated with RNAi complementary to the 3′ untranslated region (3′ UTR) of apa-2 , ced-1(RNAi) and ced-6(RNAi) animals, few germ cell corpses were labeled by APA-2::GFP or mCherry::CHC-1, suggesting that CED-1 and CED-6 are required for phagosomal association of AP2 and clathrin  and ced-6(n2095) mutants (apb-1(RNAi) and chc-1(RNAi) animals was similar to that in wild type and in lst-4(tm2423) mutants affecting phagosome maturation (see below) -tagged CHC-1, APA-2 and DPY-23 under the control of their own promoters. We found that these GFP-tagged proteins associated with phagosomes in the germ line . Similaragosomes . The fus mutants . Likewisof apa-2 . Interesclathrin . On the  mutants . The sure below) , indicatDrosophila Moesin (GFP::Moesin) ced-1 promoter. In wild-type animals, about 50% of germ cell corpses were surrounded by GFP::ACT-1. In ced-1(e1735) or ced-6(RNAi) animals, however, the labeling of germ cell corpses by GFP::ACT-1 was strongly reduced (chc-1(RNAi) and apb-1(RNAi) animals  and apb-1(RNAi) animals. RNAi of chc-1 or apb-1 caused a strong decrease in labeling of CED-1::GFP-positive phagosomes by mCherry::ACT-1 from 77% to 35–40%  did not show a detectable interaction with CED-1C, suggesting that CED-1 likely interacts with the AP2 complex through its α subunit (smIs110) could rescue the defective phagosomal recruitment of mCherry::CHC-1 in ced-1(e1735) mutants. CED-1ΔC::GFP failed to rescue the cell corpse phenotype (ced-1(e1735) mutants; however, barely any mCherry::CHC-1 was found to co-localize with CED-1ΔC::GFP (smIs34) fully rescued the cell corpse phenotype in ced-1(e1735) animals , but not GST, directly interacted with nslation . However subunit . To detehenotype  but labe1ΔC::GFP . In cont animals  and abou35S-labeled APA-2, APB-1 and DPY-23, and His6-tagged CHC-1C  and apb-1(RNAi) animals, we wondered whether maturation of phagosomes containing cell corpses was affected in these animals. To assess this, we examined the acidification of phagosomes with LysoSensor Green DND-189, an indicator of normal progression of phagosome maturation. We found that germ cell corpses were mostly negative for LysoSensor Green DND-189 staining in chc-1(RNAi) and apb-1(RNAi) animals compared to animals with gla-3 RNAi that induces an elevation in apoptosis without affecting cell corpse clearance apb-1(RNAi) and chc-1(RNAi) germ lines, including GFP-fused RAB-5 (GFP::RAB-5) and mCherry-fused RAB-14 (mCherry::RAB-14), two small GTPases required for phagosomal progression from early to late stages, and mCherry-fused NUC-1 (NUC-1::mCherry), a lysosomal DNase that indicates the formation of phagolysosomes apb-1(RNAi) and chc-1(RNAi) animals was greatly reduced compared to that in wild type  germ cell corpses were found to be encircled by GFP::Moesin, compared with 60% in wild type, indicating that loss of lst-4 did not affect cell corpse internalization (lst-4(tm2423) animals, indicating that loss of lst-4 inhibited the recruitment of factors required for phagosome maturation  single mutants or double mutants of lst-4(tm2423) with vps-18(tm1125) that was previously shown to cause defective phagolysosome formation but not phagosome acidification gla-3(RNAi) animals, in which cell corpses are normally removed, and in vps-18(tm1125) single mutants (lst-4(tm2423) mutants, even though they appeared cytoplasmic in several tissues  mutants, both proteins were found to colocalize on phagosomes (C. elegans (lst-4(tm2423) animals, and phagosomal association of LST-4::GFP in wild-type and dyn-1(RNAi) germ lines. In the wild type, DYN-1 was initially localized to the periphery of the phagosome and then quickly became enriched to form a large patch-like structure (lst-4(tm2423) mutant germ lines  and control RNAi animals (lst-4(tm2423) mutants expressing DYN-1::GFP (qxIs139) displayed an obvious reduction in germ cell corpses compared with the same mutants without DYN-1::GFP expression (lst-4(tm2423) animals expressing DYN-1::GFP (qxIs139) contained 6.3±0.6 (mean±SEM) and 7.6±0.6 corpses per gonad arm at adult ages of 36 and 48 h post L4, respectively, compared with 29.9±0.6 and 55.2±1.1 in lst-4(tm2423) mutants   . Taken tchc-1 and AP2 had an additive role in cell corpse accumulation in lst-4(tm2423) animals, and found that RNAi of chc-1, apb-1, and dpy-23 did not affect the numbers of germ cell corpses in lst-4(tm2423) mutants (chc-1 and the AP2 complex. In chc-1(RNAi) and apb-1(RNAi) germ lines, the labeling of germ cell corpses by LST-4::GFP and DYN-1::GFP was strongly reduced compared to that in wild type (lst-4(tm2423), dyn-1(ky51), or dyn-1(RNAi) animals were similar to that in wild type  but failed to detect an obvious accumulation of germ cell corpses (data not shown). Thus it is possible that multiple factors may function redundantly to mediate the recruitment of actin by the CED-1-CED-6-AP2-clathrin complex. Further studies will be necessary to unveil the underlying mechanism. In addition, unlike clathrin recruitment during bacterial phagocytosis by mammalian cells, phagosomal recruitment of clathrin requires the AP2 complex in C. elegans. The requirement for different adaptors may be attributed to the use of different receptors for engulfment of apoptotic cells and bacteria. Besides, as the sizes of cell corpses in C. elegans are normally ≥1 µm, which is much larger than endocytic CCVs (<200 nm), it also remains to be determined how clathrin is assembled  when it associates with phagosomes. Moreover, the engulfment of cell corpses in C. elegans appears to involve fewer CME regulators compared with mammalian phagocytosis of pathogens. In our unbiased RNAi screen of C. elegans CME regulators, we found that only CHC-1 and AP2 components obviously affected cell corpse engulfment and degradation while LST-4/Snx9/18/33 and DYN-1/dynamin were essential for phagosome maturation; in contrast, RNAi inactivation of several major CME regulators has been shown to inhibit bacterial infection of mammalian cells. Thus, whereas both cell corpse engulfment in C. elegans and pathogen invasion in mammals make use of clathrin for actin rearrangement, other factors may differ owing to the requirement of distinct signaling mechanisms.The recruitment of actin around germ cell corpses mediated by the complex of CED-1, CED-6, AP2 and clathrin may resemble the pathway used by mammalian cells to phagocytose pathogens. In mammalian cells, clathrin and some other regulators of CME are found to be essential for invasion of pathogenic bacteria, fungi and large viruses c-1 RNAi . We alsoDrosophila Ced-6 was identified as a clathrin-associated sorting protein (CLASP) as it binds to clathrin and AP2 via the C-terminal region Drosophila Ced-6 specifically recognizes a noncanonical sorting signal in the vitellogenin receptor Yolkless. Thus Ced-6 participates in clathrin-mediated yolk uptake in Drosophila egg chambers C. elegans, Drosophila and mammals, our discovery that clathrin and AP2 play an essential role in removal of apoptotic cells suggests that the non-classical function of clathrin and its adaptor proteins in phagocytosis is likely conserved across diverse species.Remarkably, MEGF10, the mammalian ortholog of CED-1, was reported to interact with the μ2 subunit of AP2 in a yeast 2-hybrid screen, and the existence of a protein complex containing MEGF10 and AP2 subunits was further confirmed by a protein purification assay lst-4(tm2423) deletion mutants were provided by Dr. Shohei Mitani . lst-4(qx159) mutants were isolated in Dr. Xiaochen Wang's lab . The lst-4(qx159) mutation is a deletion of 4573 bp including 1962 bp of the lst-4 gene (from exon 4 to the stop codon) and 2611 bp downstream of the lst-4 open reading frame (ORF), which also affects the gene Y37A1B.4. The flanking sequences of the deletion region are 5′-TGCCCAGAAATTTTATTTTT-3′ and 5′-ATGTTCTTGTTGACCTTATT-3′. Other mutant alleles used in this study are listed by linkage groups: LG I: ced-1(e1735), ced-12(n3261). LG III: ced-6(n1813), ced-6(n2095), chc-1(b1025ts). LG IV: ced-2(n1994), ced-5(n1812). LG V: unc-76(e911). LG X: dyn-1(ky51). The integrated arrays qxIs405(Pced-1gfp::act-1), qxIs105 (Prab-14 mcherry::rab-14), qxIs139 (derived from qxEx957) (Pced-1dyn-1::gfp), qxIs408 (Pced-1gfp::rab-5), qxIs66 (Pced-1gfp::rab-7), qxIs257 (Pced-1 nuc-1::mcherry) were provided by Dr. Xiaochen Wang. The integrated arrays smIs34 (Pced-1 ced-1::gfp) and smIs110 (Pced-1 ced-1ΔC::gfp) were provided by Dr. Ding Xue . The integrated array opIs334 (Pced-1yfp::2×fyve) was provided by Dr. K. S. Ravichandran  and Dr. M. O. Hengartner . The integrated array yqIs120 (Pdpy-23dpy-23::gfp) was generated by integrating an extrachromosomal transgene harboring the pMG4 (Pdpy-23dpy-23::gfp) plasmid kindly provided by Dr. Erik M. Jorgensen  and the unc-76 rescuing plasmid in an unc-76(e911) background. Other strains used in this study carrying integrated or extrachromosomal arrays are as follows: yqIs98 (Pced-1mCherry::chc-1), yqIs99 (Pced-1apa-2::gfp), yqIs100 (Pced-1mCherry::act-1), yqIs101 (Pced-1gfp::ced-6), yqIs112 (Pced-1gfp::chc-1), yqIs114 (Plst-4lst-4(cDNA)::gfp), yqIs119 (Plst-4lst-4(cDNA)::mCherry), yqIs121(Pced-1gfp::Moesin), yqEx368 (Plst-4::lst-4(cDNA)::gfp), yqEx376 (Plst-4::lst-4(gDNA)::gfp), yqEx480 (chc-1chc-1::gfpP), yqEx481 (apa-2apa-2::gfpP). Animals carrying the stably integrated array were outcrossed with the N2 strain 4 times. C. elegans cultures and genetic crosses were performed essentially according to standard procedures C. elegans transformation was carried out essentially as described before The Bristol strain N2 was used as wild type. chc-1chc-1::gfp construct was generated by cloning a genomic DNA fragment containing a promoter region of 3 kb and the open reading frame (ORF) of the chc-1 gene in frame with GFP into the pPD95.77 vector. The Papa-2apa-2::gfp construct was similarly generated by cloning a genomic fragment containing a promoter region of 2 kb and the ORF of apa-2. Genomic DNA containing the ORF of chc-1 was amplified and inserted into Pced-1mCherry1 or Pced-1gfp1 via the KpnI site to generate the Pced-1mCherry::chc-1 and Pced-1gfp::chc-1 constructs. To generate Pced-1apa-2::gfp, a genomic fragment containing the apa-2 ORF was amplified and inserted into Pced-1gfp3 via the KpnI site. To construct Pced-1gfp::Moesin, the C-terminal of Moesin was amplified from the plasmid pJWZ6 ced-1gfp1 via the KpnI site. To generate Plst-4lst-4(cDNA)::gfp, a DNA fragment containing a 2 kb promoter region and the first intron followed by the remaining cDNA sequence of the lst-4 isoform c was inserted between the HindIII and KpnI sites of the vector pPD95.77. Plst-4lst-4(cDNA)::mCherry was derived from Plst-4lst-4(cDNA)::gfp by replacing gfp with mCherry. To generate Plst-4lst-4(gDNA)::gfp, a genomic fragment containing a 2 kb promoter region and the lst-4 genomic ORF were amplified and inserted between the XbaI and XmaI sites of the vector pPD95.77.The Papa-2 with its 3′ UTR was performed by feeding animals with bacteria expressing dsRNA corresponding to the 3′UTR of 516 bp. Germ cell corpses and other phenotypes were observed in adults of the progeny. For RNAi of chc-1, apb-1 and dyn-1, which may cause embryonic lethality in the progeny, L3- to L4-stage animals were transferred to plates seeded with bacteria expressing dsRNA of individual genes and phenotypes were observed in adults of the same generation.RNAi experiments were performed by using bacterial feeding assays as described previously t-tests. For cell corpse analysis of chc-1(b1025ts) mutants, animals were grown to L4 at 20°C and then shifted to 25°C. Germ cell corpses were scored at 12, 24, 36 and 48 h after the shift.Cell corpses in synchronized animals were scored under Nomarski optics. To quantify germ cell corpses, cell corpses in the germline meiotic region of one gonad arm in each of at least 15 animals were scored at various adult ages . The average numbers of germ cell corpses from one gonad arm were calculated for each adult age. Data derived from different genetic backgrounds were compared using unpaired 2PO4, 6 g Na2HPO4, 5 g NaCl, 1 mM MgSO4) with 2 mM levamisole and then examined by fluorescence microscopy. To analyze the labeling of germ cell corpse by phagosomal markers in dyn-1(ky51ts) mutants, animals were grown to L4 stage at 20°C and then shifted to 25°C; cell corpses were analyzed 24 h after the shift. To view germ cell corpses in dyn-1 RNAi-treated animals, L4 larvae were cultured on RNAi plates and germ cell corpses were analyzed 24 h after the L4 molt.To quantify the percentage of germ cell corpses labeled by various phagosomal markers, adult animals at 36 h after the L4 molt were mounted on agar pads in M9 buffer  containing 1 µM LysoSensor Green DND-189 (Invitrogen) and examined by fluorescence microscopy.Adult animals (36 h after the L4 molt) were dissected in gonad dissection buffer , and recorded under Nomarski optics at 20°C. The gonadal region was recorded every 1 min at 1 µm/section for 20 chc-1 or apb-1. 30 h later, animals were collected for fixation, embedding and sectioning following a procedure essentially as described by Gumienny et al. L3- or L4-stage animals were fed with bacteria expressing dsRNA of 35S-labeled APA-2, APB-1 and DPY-23 were prepared by in vitro translation. Purified GST, GST-CED-9, GST-CED-1C or GST-CED-6 proteins (3 µg of each) immobilized on glutathione-Sepharose beads was incubated with 35S-labeled APA-2, APB-1, DPY-23 or CHC-1CHis6, LST-4His6 and DYN-1His6 at 4°C for ≥4 h and washed extensively. Bound proteins were resolved on sodium dodecyl sulfate (SDS) polyacrylamide gels (SDS-PAGE) and visualized by autoradiography or immunoblotting with anti-His6 antibody.Recombinant GST-CED-1C, GST-CED-6, GST-DYN-1, GST-LST-4, GST-CED-9 proteins were expressed in bacterial BL21(DE3) cells and purified with glutathione-Sepharose beads (Amersham) according to the instructions provided by the supplier. His6-tagged CHC-1C (amino acids 825–1682), LST-4, DYN-1 and mCherry were purified with Ni-NTA beads. CED-1C antibody was generated previously Adult animals (24 h post L4 molt) were anesthetized with 0.1 mM levamisole in M9 buffer, mounted on 2% agar pads, and maintained at 22°C. Time-lapse images of DYN-1::GFP, LST-4::mCherry and LST-4::GFP were captured every 2 min by using an imaging system consisting of an Axiovert 200M microscope  equipped with a 100×, 1.45 N.A. objective, an EM CCD camera , and the 488 nm and 561 nm lines of an Argon and Krypton laser attached to a spinning disk confocal scan head (Yokogawa CSU10 obtained from Solamere Inc.).Figure S1ced-1(e1735), and ced-6(n1813) animals. The precipitated proteins were resolved on SDS-PAGEs and visualized with silver staining. Gel regions indicated by the brackets were cut off and subjected to mass spectrometry analysis by using a TripleTOF5600 mass spectrometer . Protein identification was performed by searching the C. elegans proteome sequence database (SwissProt) using ProteinPilot software 4.2, with a mass tolerance of 0.05 Da and a false discovery rate of 1%. The unique peptides identified for CHC-1, CED-1, or CED-6 are shown on the right. Asterisks indicate the precipitated CED-1 (A) or CED-6 (B). Arrowheads indicate the IgG heavy chain.Detection of CHC-1 in proteins co-immunoprecipitated with CED-1 (A) and CED-6 (B). Immunopreciptations were performed with CED-1C (A) and CED-6 (B) antibodies on lysates of N2, (JPG)Click here for additional data file.Figure S2chc-1chc-1::gfp, Papa-2apa-2::gfp, and Pdpy-23dpy-23::gfp, respectively. Arrows indicate cell corpses. Bars, 10 µm. (B) Rescue of the cell corpse phenotype in chc-1(b1025ts) mutants by Pchc-1chc-1::gfp (yqEx480) and Pced-1mCherry::chc-1 (yqIs98). (C) Rescue of the cell corpse phenotype in animals with RNAi of the 3′UTR of apa-2 by Papa-2apa-2::gfp (yqEx481) and Pced-1apa-2::gfp (yqIs99). Germ cell corpses in one gonad arm were scored in each animal of the indicated strains at 36 h and 48 h post L4 stage. Error bars represent SEM. Comparisons were performed using unpaired t-tests. ** p<0.001. (D) Rescue of the cell corpse phenotype in ced-1(e1735) mutants by Pced-1ced-1::gfp (smIs34) and Pced-1ced-1ΔC::gfp (smIs110). (E) Rescue of the cell corpse phenotype in ced-6(n2095) mutants by Pced-1gfp::ced-6 (yqIs101). In (D) and (E), the average number of cell corpses per embryo in 4-fold embryos  and the average number of germ cell corpses per gonad arm  are shown for each genotype. Comparisons were performed with unpaired t-tests. ** p<0.001.Clathrin and AP2 associate with phagosomes containing cell corpses. (A) Representative DIC and fluorescence images of germ cell corpses surrounded by CHC-1::GFP, APA-2::GFP, and DPY-23::GFP in animals expressing P(JPG)Click here for additional data file.Figure S3lst-4 does not affect the encircling of germ cell corpses by CED-1::GFP and GFP::CED-6. (A and B) Representative images of germ cell corpses labeled by CED-1::GFP (A) or GFP::CED-6 (B) in N2, apb-1(RNAi), chc-1(RNAi) and lst-4(tm2423) animals. Arrows point to cell corpses labeled by CED-1::GFP or GFP::CED-6; arrowheads indicate unlabeled corpses. Bars, 10 µm. (C) Quantification of cell corpse labeling by CED-1::GFP and GFP::CED-6 in the animals indicated. ≥100 corpses were analyzed for each genotype.Loss of clathrin, AP2 and (JPG)Click here for additional data file.Figure S4Ctrl(RNAi), ced-1(RNAi), ced-6(RNAi), chc-1(RNAi) and apb-1(RNAi) germ lines. Bars, 10 µm. (B) Quantification of the labeling of germ cell corpses by GFP::Moesin as shown in (A). ≥100 corpses were scored for each genotype.CHC-1 and AP2 are required for the rearrangement of the actin cytoskeleton. (A) Representative images of cell corpse labeling by GFP::Moesin in (JPG)Click here for additional data file.Figure S5lst-4(tm2423) and lst-4(qx159) deletion mutation. Solid boxes indicate exons and thin lines indicate introns. Deleted regions are indicated by the bars above and below the gene. (B) Quantification of germ cell corpses in N2, lst-4(tm2423) and lst-4(qx159). Error bars represent SEM. N2 and lst-4 mutants were compared using unpaired t-tests. ** p<0.001. (C–G) Representative DIC and fluorescence images of germ cell corpse labeling by GFP:Moesin (C), YFP::2XFYVE (D), GFP::RAB-5 (E), mCherry::RAB-14 (F) and GFP::RAB-7 (G) in N2 and lst-4(tm2423) mutants. Arrows indicate cell corpses labeled by phagosomal markers and arrowheads indicate unlabeled corpses. Bars, 10 µm. (H) Quantification of germ cell corpse labeling as shown in (C–G). The data represent average numbers of 3 independent experiments. ≥100 corpses were scored for each phagosomal marker at each time. Error bars represent SEM.LST-4 affects phagosomal recruitment of factors required for phagosome maturation. (A) Schematic representation of the (JPG)Click here for additional data file.Figure S6gla-3(RNAi), lst-4(tm2423), vps-18(tm1125) and vps-18(tm1125);lst-4(tm2423) germ lines. Arrows point to germ cell corpses positive for LysoSensor Green DND-189; arrowheads indicate unstained corpses. Bars, 10 µm. (B) Quantification of cell corpse staining as shown in (A). ≥100 corpses were scored for each genotype. (C) Expression and localization of LST-4::GFP driven by the lst-4 promoter. The transgenic array used is yqEx376 (Plst-4::lst-4(gDNA)::gfp). LST-4::GFP is observed in ∼100-cell stage embryos and in adult gonadal sheath cells. Bars, 10 µm. (D) Rescue of the cell corpse phenotype in lst-4(tm2423) mutants by Plst-4::lst-4(gDNA)::gfp (yqEx376), Plst-4::lst-4(cDNA)::gfp (yqEx368), and Plst-4::lst-4(cDNA)::mCherry (yqIs119). Germ cell corpses in one gonad arm were scored in each animal of the indicated strains at 36 h post L4 stage. For transgenes, the number of cell corpses in one transgenic line from a total of three lines exhibiting similar rescuing activity is shown. Error bars represent SEM. Comparisons were performed between lst-4(tm2423) and transgenic animals by unpaired t-tests. ** p<0.001. (E and F) Representative DIC and fluorescence images of germ cell corpse labeling by LST-4::GFP (yqIs114) in mutants affecting engulfment (E) and animals with RNAi of genes required for phagosome maturation (F). Arrows indicate cell corpses labeled by LST-4::GFP and arrowheads indicate unlabeled corpses. Bars, 10 µm.Characterization of LST-4-mediated phagosome acidification. (A) Representative DIC and fluorescence images of cell corpse staining by LysoSensor Green DND-189 in (JPG)Click here for additional data file.Figure S7dyn-1(ky51) mutants by Pced-1dyn-1::gfp (qxEx957). Germ cell corpse were scored at 24 and 36 h post L4 stage and analyzed as in Ctrl(RNAi), apb-1(RNAi) and chc-1(RNAi) animals as shown in lst-4(tm2423) and dyn-1(ky51)(25°C) germ lines (E) and phagosomal association of mCherry::CHC-1 in gla-3(RNAi), lst-4(tm2423), and dyn-1(RNAi) germ lines (F). Adult animals (24 h after the L4 molt) were analyzed. Arrows indicate cell corpses labeled by APA-2::GFP or mCherry::CHC-1. Bars, 10 µm. (G) Quantification of phagosomal association of APA-2::GFP as shown in (E) and chc-1(RNAi) germ lines (left) and phagosomal association of mCherry::CHC-1 as shown in (F) and apb-1(RNAi) germ lines (right). ≥100 corpses were analyzed for each genotype.LST-4 interacts with DYN-1 and acts downstream of clathrin and AP2. (A) DYN-1::GFP and LST-4::mCherry colocalize on the surface of germ cell corpses. DIC, GFP, mCherry and merged GFP and mCherry images are shown. Cell corpses are indicated by arrows. Bar, 10 µm. (B) Rescue of the cell corpse phenotype in (JPG)Click here for additional data file.Table S1C. elegans genes involved in clathrin-mediated endocytosis. C. elegans genes involved in clathrin-mediated endocytosis were identified by using sequences of individual human proteins to search for homologs in the C. elegans genome database. RNAi was performed as described in Methods. Germ cell corpses in one gonad arm of each animal were scored for at least 15 animals 60 h after the L4 stage. N/A indicates that hsp-1 RNAi caused defects in germline proliferation and no cell corpses could be scored.Cell corpse phenotype caused by RNAi of (DOC)Click here for additional data file.