Photoimmunotherapy is one of the most promising strategies in tumor immunotherapies, but targeted delivery of photosensitizers and adjuvants to tumors remains a major challenge. Here, as a proof of concept, we describe bone marrow mesenchymal stem cell-derived nanovesicles (NVs) displaying anti-PD-L1 antibodies (aPD-L1) that were genetically engineered for targeted drug delivery.The high affinity and specificity between aPD-L1 and tumor cells allow aPD-L1 NVs to selectively deliver photosensitizers to cancer tissues and exert potent directed photothermal ablation. The tumor immune microenvironment was programmed via ablation, and the model antigen ovalbumin (OVA) was designed to fuse with aPD-L1. The corresponding membrane vesicles were then extracted as an antigen–antibody integrator (AAI). AAI can work as a nanovaccine with the immune adjuvant R837 encapsulated. This in turn can directly stimulate dendritic cells (DCs) to boast the body's immune response to residual lesions.aPD-L1 NV-based photoimmunotherapy significantly improves the efficacy of photothermal ablation and synergistically enhances subsequent immune activation. This study describes a promising strategy for developing ligand-targeted and personalized cancer photoimmunotherapy.The online version contains supplementary material available at 10.1186/s12951-022-01266-3. Immunotherapy is a novel strategy for tumor therapy that works via an activated immune system –3. HowevPhotoimmunotherapy has shown value versus conventional immunotherapies. Recent studies have shown that photothermal-based immunotherapy can induce tumor cell death via high temperatures while simultaneously activating the tumor immune response –16. The Actively targeting nanoplatforms to the tumor microenvironment often requires conjugation of targeting ligands. Chemical covalent linkage of receptors or ligands to deliver photosensitizers or adjuvants may cause a loss of tumor targeting and lead to limited photoimmunotherapy. Antibodies can facilitate immunoregulation and binding to specific markers on tumor cells –21. NatuCell membrane-derived nanovesicles (NVs) are biogenic nanocapsule structures obtained by crushing or squeezing selected cell membranes –27. NVs To optimize the curative effect of malignant tumors, we describe here a novel method based on cell membrane-derived biomimetic nanovesicles displaying aPD-L1 for photoimmunotherapy , and regulation of the immune checkpoint. Thus, it is an immunotherapy for residual and distal lesions. Accordingly, the model antigen ovalbumin (OVA) served as a tumor-associated antigen and was designed to be co-expressed with aPD-L1 on the membrane of MSCs and the corresponding membrane vesicles. OVA was extracted as an antigen–antibody integrator (AAI) and is a promising multifunctional nanoplatform. With immune adjuvant (R837) encapsulated in AAI (AAI-R837), antigens and immune adjuvants are delivered to antigen-presenting cells (APCs) in a spatio-temporal way for follow-up immunotherapies. AAI can be a carrier for targeted delivery and can also carry antigen signal to APCs to promote the mature differentiation of DCs, thus boosting the immune response to tumors. Afterwards, AAI-R837 can work as a nanovaccine to facilitate immunotherapy. In this treatment strategy, the multi-functional nanoplatform was used in a combined way for photoimmunotherapy. The material achieved satisfactory inhibitory effects on malignant melanomas.A plasmid containing aPD-L1 fragments was first constructed to prepare aPD-L1 displaying MSCs. Then a lentiviral vector was used to infect MSCs extracted from the bone marrow cavity of C57BL/6 embryonic mice with the aPD-L1 displayed on the outside of the cell membrane Fig. b. SubseqTo test the binding ability of aPD-L1 NVs to the B16F10 melanoma cells, NVs and aPD-L1 NVs labeling fluorescent dye FITC were incubated with B16F10 cells for 3 h and then imaged with confocal laser scanning microscopy (CLSM). Significant fluorescence was observed in the periphery of B16F10 cells after being incubated with FITC-aPD-L1 NVs: They were five-fold brighter than FITC-NVs Fig. g. These 2, 5 min) was used to irradiate aPD-L1 NVs-ICG and free ICG at different concentrations; real-time detection used a NIR thermal imager  was implemented on the B16F10 melanoma cells with or without aPD-L1 NVs-ICG. The aPD-L1 NVs-ICG had good photothermal killing effects on B16F10 melanoma cells  test was used to verify the safety of different concentrations of aPD-L1 NVs-ICG on DC2.4 cells  and B16F10 melanoma cells (a representative cancer cell). The aPD-L1 NVs-ICG have no obvious toxic effects on DC2.4 cells and B16F10 melanoma cells Fig. d. In addlls Fig. e. The sulls Fig. f.Inflammation-induced PD-L1 propagates expression in the tumor microenvironment, thus inhibiting the antitumor cytotoxic T-cell response –41. TherThe drug distribution in vivo was monitored by an IVIS Lumina II imaging system. After 12 h and 24 h of administration, the aPD-L1 NVs-ICG had a significant fluorescence signal at the tumor site, and metabolites changed more slowly in vivo than free ICG and NVs-ICG  was used to irradiate the initial tumor on the left side for photothermal treatment, and the NIR thermal imager was used to monitor the temperature change of tumor site in real time. The results show that tumors treated with aPD-L1 NVs-ICG after laser irradiation, plus the warming capacity of the melanoma itself, led to a surface temperature of 60 ℃ that could ablate the tumor . The data indicated that the content of CD11c + CD86 + increased compared to PBS-treated groups Fig. e. HoweveFurthermore, after being co-incubated for 48 h, the cell culture medium was collected, and the content of immune-related cytokines  was detected by ELSA Fig. f–i. The Encouraged by the results in vitro, we further investigated the effect of AAI-R837 on the immune response in vivo. First, we verified the process of AAI uptake by DCs and migration to lymph nodes in vivo. AAI, free NVs, and OVA labeled with fluorescent dye Cy5.5 were injected subcutaneously into the right leg of mice, and the migration process of drugs in vivo was observed by an IVIS Lumina II imaging system. The injected free Cy5.5 and Cy5.5-OVA rapidly diffused and metabolized in the body although there was a certain concentration of inguinal lymph nodes in the early stage. The product was metabolized quickly over time Fig. a. Cy5.5-Next, photoimmunotherapy was done on the tumor. After that, various immunological materials were injected subcutaneously at the root of the tail on 13, 15, and 17 d as a vaccine for immunotherapy on remnant tumors Fig. d. After The immune effect after treatment was also studied. Three days after the final immunization, distal tumors, serum, and lymph nodes were collected, and immune cells and cytokines related to the immune response were detected. A cell suspension was prepared by homogenized the material, and the content of mature DCs in lymph nodes, as well as CTLs in tumor was analyzed by FCM Fig. g, h. CelIn summary, we constructed AAI that simultaneously express antibodies and tumor-related antigens using the cell membrane of bone marrow mesenchymal stem cells. Nano-vesicles expressing aPD-L1 can specifically bind to PD-L1 receptors on cancer cells and can be utilized to deliver photosensitizers to tumor sites for efficient photothermal therapy. Meanwhile, the aPD-L1 expressed on the modified nanocapsule can also specifically bind to the PD-L1 receptor on the DCs. In short, this design takes advantage of specific binding at the immune checkpoints. This provides a promising strategy for targeted drug delivery. Intriguingly, the immune environment in the tumor region was programmed via photothermal ablation. This led to improved immunotherapy to arrest the residual and distal tumor. Additionally, antibodies and antigens were co-expressed on AAI-R837 and delivered the immune adjuvant to the antigen presenting cells. Antigen signal was sent to the DCs correspondingly and the powerful immune response of the immune system was elicited. The experimental results indicated that the combined treatment of photoimmunotherapy on the melanoma model not only had a significant inhibiting effect on the initial tumor, but also inhibited distant tumor. The low immunogenicity of the materials extended the in vivo circulation time of the nanocarrier, and its biological safety suggests clinical applications.Imiquimod (R837) and Indooyanine gree (ICG) were purchased from Invivogen and BBI Life Science Corporation, respectively. Ovalbumin (OVA), calcein acetoxymethyl ester  and propidium iodide (PI) were purchased from Sigma-Aldrich Inc. FITC was purchased from MedChemExpress LLC and Cy5.5 was purchased from APExBIO Technology LLC. Anti-mouse CD274 , FITC-anti-mouse CD86, PE-anti-mouse CD11c, TITC-anti-mouse CD8 and PerCP-anti-mouse CD3ε for flow cytometry were purchased from Biolegend Inc. Basement Membrane Matrix was purchased from Becton, Dickinson and Company. TNF-α, IL-6, IL-12 and IFN-γ ELSA kit were purchased from Beyotime Biotech Inc.2. To make aPD-L1 be expressed on membrane of MSCs, the aPD-L1 gene with a signal peptide, flag tags, aPD-L1 ScFv and transmembrane sequence were synthesized. While to make aPD-L1 and OVA be co-expressed on membrane of MSCs, plasmids was constructed containing signal peptide, flag tags, aPD-L1 ScFv, transmembrane sequence as well as an intramembrane fragment containing an OVA sequence.DNA sequence of membrane-located signal peptide (from integrin beta 1):ATGAATTTGCAACTGGTTTCCTGGATTGGATTGATCAGTTTGATTTGTTCTGTATTTGGCCAAACAGATAAADNA sequence of Flag tag:GACTACAAGGACGACGACGACAAGDNA sequence of aPD-L1 ScFv:GCCCAGGCCGCCCTGACCCAGCCCAGCAGCGTGAGCGCCAACCTGGGCGGCACCGTGAAGATCACCTGCAGCGGCGGCAGCGGCAGCTACGGCTGGTACCAGCAGAAGGCCCCCGGCAGCGCCCCCGTGAGCCTGATCTACGACAACACCAACAGGCCCAGCGACATCCCCAGCAGGTTCAGCGGCGCCCTGAGCGGCAGCACCGCCACCCTGACCATCACCGGCGTGCAGGCCGAGGACGAGGCCGTGTACTACTGCGGCAGCAGGGACAGCAGCAACGCCGGCAGCGTGTTCGGCGCCGGCACCACCCTGACCGTGCTGGGCCAGAGCAGCAGGAGCAGCGGCGGCGGCGGCAGCAGCGGCGGCGGCGGCAGCGCCCTGACCCTGGACGAGAGCGGCGGCGGCCTGCAGACCCCCGGCGGCGCCCTGAGCCTGGTGTGCAAGGCCAGCGGCTTCACCTTCAGCGACAGGGGCATGCACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGGCGCCATCAGCAGGAGGGGCAGCACCACCACCTACGCCCCCGCCGTGAAGGGCAGGGCCACCATCACCAGGGACAACGGCCAGAGCACCGTGAGGCTGCAGCTGAACAACCTGACCGCCGAGGACACCGCCACCTACTTCTGCGCCAAGAACGACGACAGCGTGGGCATCGTGACCACCAGCACCATCGACGCCTGGGGCCACGGCACCGAGGTGATCGTGAGCAGCACCAGCGGCCAGGCCGGCCAGCACCACCACCACCACCACGGCGCCTACCCCTACGACGTGCCCGACTACGCCAGC.DNA sequence of transmembrane fragment:TTATGGGTCATCCTGCTGAGTGCTTTTGCCGGATTGTTGCTGTTAATGCTGCTCATTTTAGCACTGTGGDNA sequence of OVA:ATGGGCTCCATCGGCGCAGCAAGCATGGAATTTTGTTTTGATGTATTCAAGGAGCTCAAAGTCCACCATGCCAATGAGAACATCTTCTACTGCCCCATTGCCATCATGTCAGCTCTAGCCATGGTATACCTGGGTGCAAAAGACAGCACCAGGACACAGATAAATAAGGTTGTTCGCTTTGATAAACTTCCAGGATTCGGAGACAGTATTGAAGCTCAGTGTGGCACATCTGTAAACGTTCACTCTTCACTTAGAGACATCCTCAACCAAATCACCAAACCAAATGATGTTTATTCGTTCAGCCTTGCCAGTAGACTTTATGCTGAAGAGAGATACCCAATCCTGCCAGAATACTTGCAGTGTGTGAAGGAACTGTATAGAGGAGGCTTGGAACCTATCAACTTTCAAACAGCTGCAGATCAAGCCAGAGAGCTCATCAATTCCTGGGTAGAAAGTCAGACAAATGGAATTATCAGAAATGTCCTTCAGCCAAGCTCCGTGGATTCTCAAACTGCAATGGTTCTGGTTAATGCCATTGTCTTCAAAGGACTGTGGGAGAAAGCATTTAAGGATGAAGACACACAAGCAATGCCTTTCAGAGTGACTGAGCAAGAAAGCAAACCTGTGCAGATGATGTACCAGATTGGTTTATTTAG.AGTGGCATCAATGGCTTCTGAGAAAATGAAGATCCTGGAGCTTCCATTTGCCAGTGGGACAATGAGCATGTTGGTGCTGTTGCCTGATGAAGTCTCAGGCCTTGAGCAGCTTGAGAGTATAATCAACTTTGAAAAACTGACTGAATGGACCAGTTCTAATGTTATGGAAGAGAGGAAGATCAAAGTGTACTTACCTCGCATGAAGATGGAGGAAAAATACAACCTCACATCTGTCTTAATGGCTATGGGCATTACTGACGTGTTTAGCTCTTCAGCCAATCTGTCTGGCATCTCCTCAGCAGAGAGCCTGAAGATATCTCAAGCTGTCCATGCAGCACATGCAGAAATCAATGAAGCAGGCAGAGAGGTGGTAGGGTCAGCAGAGGCTGGAGTGGATGCTGCAAGCGTCTCTGAAGAATTTAGGGCTGACCATCCATTCCTCTTCTGTATCAAGCACATCGCAACCAACGCCGTTCTCTTCTTTGGCAGATGTGTTTCCCCTTAA.Bone marrow mesenchymal stem cells were extracted from the bone marrow of suckling C57BL/6 mice (Seven days after birthed) referring to the established method . The micThen, Lentivirus vector encoding corresponding sequence was applied to infect MSCs. Infected MSCs displaying aPD-L1 with or without OVA were cultured in C57BL/6 mouse bone marrow mesenchymal stem cell complete culture medium maintained in 20% FBS and 1% Penicillin and streptomycin.6 in a petri dish, the medium in the culture dish with MSCs was removed, then the MSCs were scraped away by cell scraping, and were collected into the centrifuge tube with phosphate buffer solution (PBS). After 5% deoxysodium cholate, a surfactant, was added to the cell suspension (v:v = 1:100), MSCs were broken by a low power ultrasound  for 10 to 20 s using the ultrasonic crusher , and the protease inhibitor PMSF (10 mg/mL) was added (v:v = 1:200) to worked solution immediately. To remove the cytoplasm and nucleus, the suspending liquid was centrifuged at 3500 rpm (4 ℃) for 5 min. Supernatant was collected for further centrifugation at 15,000 rpm(4 ℃) for 20 min to acquire nanoscale membrane vesicles, then resulting vesicles were quantified by BCA kit (Beyotime Biotech Inc.) and dissolved in PBS and stored at 4 ℃ for standby application.In the preparation of nanovesicles, when the number of cells reaches 5╳106 MSCs, the ultrasonic crushing instrument (20 w) was used for ultrasonic concussion for three times at 4 ℃, each session lasts 5 to 10 s and interrupted at least 10 s. After resulting sample was centrifuged at 12,000 rpm (4 ℃) for 20 min, the content of ICG in the supernatant was detected by a microplate analyzer, and the loading efficiency of ICG was calculated. NVs loading drugs were dispersed in PBS and stored at 4℃ for further study. When loading the immune adjuvant, 100 μL R837 (0.5 mg/mL) was added to NVs suspension, and the loading was carried out according to the same method mentioned above. The loading efficiency of R837 was determined by HPLC (Waters 1525)-UV visible detector at 325 nm. Acetonitrile was used as the mobile phase. ICG encapsulation and the UV absorption spectra of NVs-ICG were measured by microplate reader.After 200 μL ICG aqueous solution (1 mg/mL) was mixed with NVs suspension come from 5×102) for 5 min. During the heating of each solution, an NIR thermal imaging camera was used to record the temperature changes.The expression of aPD-L1 on the MSCs membrane was verified by immunofluorescence. The morphology and structure of NVs were characterized by fetenay transmission electron microscopy (F20). The dynamic particle size and electric potential of NVs was measured with Zeta sizer Nano-ZS . The existence of aPD-L1 on the NVs was detected with coimmunoprecipitation (CO-IP) assay and Western blot (WB) analysis. In order to study photothermal performance of acquired materials, aPD-L1 NVs-ICG and ICG with different concentrations were dispersed in ultrapure water, then the samples were irradiated with 808 nm laser (1 W/cm4 cells/well) and cultured for 24 h. FITC-labeled aPD-L1 NVs or NVs were incubated with the cancer cells for 3 h, and cells in the other group were incubated with antiPD-L1 antibodies (10 μg/mL) for 2 h before the addition of aPD-L1 NVs to the culture medium. The NVs that were not bound to the cells were washed with PBS for three times, then the nuclei were stained with DAPI for 15 min. After the excess dye was washed and added new medium to the wells, confocal microscopy was performed under a confocal laser scanning microscope (LSM780) with 60 × oil-immersion lenses. The binding of AAI to DC2.4 cells was observed by the same method.To exam the ability of aPD-L1 NVs to binding to tumor cells, B16F10 cells were seeded in confocal culture dish (3 × 104 cells/well) for 24 h, respectively. After the cells were washed with PBS for three times, fresh medium containing different concentrations of aPD-L1 NVs-ICG and AAI-R837 was added for training another 24 h. The cell relative viability was detected by MTT assay and the cytotoxicity of the nanomedicine was determined.DC2.4 cells and B16F10 cells were selected to determine the cytotoxicity of the nanomedicine by standard MTT assay. DC2.4 cells and B16F10 cells were cultured in 96-well plates (1 × 104 cells/well) for 24 h. After washing the cells with PBS for three times, fresh medium containing 100 μg/mL of aPD-L1 NVs-ICG was added. Treated cells were irradiated with 808 nm laser (1.0 W/cm2) for 5 min. Viability of B16F10 cell was then assessed using standard MTT procedure. In order to intuitively evaluate the relative viability of B16F10 cells after different treatments, 2 μmol/L calcein acetoxymethyl ester  and 4 μ mol/L propidium iodide (PI) were used to stain the living and dead cells for 15 min, respectively. After washed with PBS, the cells were observed with inverted fluorescence microscope .To study the effect of photothermal therapy on B16F10 Cells with aPD-L1 NVs-ICG, B16F10 cells were cultured in 96-well plates(1 × 10Bone marrow derived dendritic cells (BMDCs)were extracted from the bone marrow of 6 ~ 8 weeks old C57BL/6 mice according to the established method . BMDCs w50 μg of ICG, 100 μg of NVs-ICG and aPD-L1 NVs-ICG (containing 50 μg of ICG) dispersed in 100 μL PBS were injected into subcutaneous melanoma burdened C57BL/6 mice via tail vein. Fluorescence imaging system  was used to obtain fluorescence images at 0, 6, 12, 24 and 48 h after injection, respectively. At 48 h, the main organs and tumors of the mice were collected, and fluorescence imaging and intensity records were collected.NVs, OVA and AAI were labeled with Cy5.5, subsequently, and 50 μL normal saline with equivalent 10 μg of OVA or approximately 50 μg of vesicular proteins injected into unilateral foot pad of C57BL/6 mice. Fluorescence imaging in vivo was obtained carrying out the system of fluorescence images at different time points . The left and right inguinal lymph nodes of all mice were collected at 96 h after injected, fluorescence images were obtained and the fluorescence signal intensity was calculated using the imaging system.6 OVA-expressing B16F10 cells were suspended in PBS and subcutaneously injected into the left posterior back of C57BL/6 mice to challenged primary tumor. On 6 d, 3 × 106 OVA-expressing B16F10 cells were subcutaneously injected into the right posterior back of C57BL/6 mice to challenged distant tumor. On 9 d after first inoculation, primary tumor was about 200 mm3 in size, various formulations were injected into the tail vein containing equivalent 4.5 mg/kg of ICG or approximately 100 μg of vesicular proteins (five mice per group). After 12 h, the primary tumor was irradiated by 808 nm laser (0.75 W/cm2) for 8 min, and the temperature of tumor surface was monitored by near-infrared thermal imaging instrument. On 13, 15 and 17 d after challenge of the first tumor, mice treated with photothermal therapy were subcutaneously injected with different immune drugs containing equivalent 10 μg of OVA or 10 μg of R837 or approximately 50 μg of vesicular proteins for immunotherapy of distant tumor (five mice per group). The body weight of the mice was recorded using electronic balance, and the tumor size and volume were measured using a vernier caliper every other day. The calculation formula (V) was V = d2*D/2, d and D was mm in the shortest and longest diameters of the tumors respectively. Animals were euthanized when they showed signs of impaired health or tumors larger than 2cm3.Female C57BL/6 mice (6 ~ 8 weeks) were purchased from Shanghai slake laboratory animal co., LTD and used according to the agreement approved by committee of Xiamen University Laboratory Animal Center. The mice were randomly divided into groups. 3 × 10To test the immune response in the treated mice, the mice were sacrificed and serum, tumor, and lymph nodes were collected on 3 d after photothermal treatment or final immunization. The levels of TNF-α, IL-6, IL-12 and IFN-γ in serum were detected by ELISA kit according to the instructions. The tissues were homogenized to prepare single-cell suspension, and tumor cells were labeled with anti-mouse CD3a and CD8 antibodies, and lymph node cells were labeled with anti-mouse CD11c and CD86 antibodies. Then, the content of CD8 + T lymphocytes and mature DC cells were detected by flow cytometry.The main organs  and tumor tissues of mice in each treatment group were collected and fixed in 4% paraformaldehyde. Hematoxylin and eosin staining were performed before electron microscopy.All the statistical figures are mean ± standard deviation, P values were calculated by Student's t test . All statistical analyses were performed using the IBM SPSS statistics 20.Additional file 1: Figure S1. (a) The standard curve of R837 was detected by HPLC; (b) The standard curve of ICG was measured by ultraviolet spectrophotometer; (c) Analysis of drug loading efficiency using nanometer delivery platform. Figure S2. The temperature change curve of the aPD-L1 NVs-ICG and ICG with corresponding concentration under the near infrared (NIR) laser irradiation at 808 nm wavelength . Figure S2. The temperature change curve of the aPD-L1 NVs-ICG and ICG with corresponding concentration under the near infrared (NIR) laser irradiation at 808 nm wavelength . Figure S4. (a) Temperature change curve of the tumor under different treatments under 808 nm laser irradiation . (b)Tumor volume growth curve for irradiated distant secondary tumors (in right flank) in mice after PTT with different drugs. (c) Survival curves of the mice after PTT with different drugs. Images of Hematoxylin and eosin (H&E) staining of irradiated primary tumors and distant secondary tumors sections after various treatments, respectively. Scale bar: 100 µm. (b)Weight curve after PTT with the different substances. Images of H&E staining of the main viscera after PTT, respectively. Scale bar: 100 µm. Figure S5. Productions of IL-6 (a), IL-12 (b), TNF-α (c) in serum from the mouse determined by ELISA after treated with PTT for 3 d. P values were calculated by Student's t test . Figure S6. Cell viability of the different concentrations of AAI-R837 on B16 cells and DCs after co-incubation for 24 h. Figure S7. The curve of fluorescence signal intensity in inguinal lymph nodes with time after subcutaneous injection of various drugs. Figure S8. (a) Images of Hematoxylin and eosin (H&E) staining of distant secondary tumors sections after photoimmunotherapy, respectively. Scale bar: 100 µm. (b) Weight curve after photoimmunotherapy with the different substances. (c) Images of H&E staining of the main viscera after photoimmunotherapy, respectively. Scale bar: 100 µm.


Clubionazilla-group is a relatively small species group, distributed exclusively in East Asia, with only three species clearly documented so far.The Clubionahooda Dong & Zhang, 2016, which was previously placed in the C.trivialis-group, is assigned to the C.zilla-group in the present paper. A new spider of the C.zilla-group from Jiugong Mountain in China is described under the name of C.jiugong sp. nov. Detailed descriptions and photographs of the new species are provided. Clubionazilla Dönitz & Strand, 1906 was reported by C.zilla-group, was established to accommodate C.zilla. The C.zilla-group was redeﬁned by Anaclubiona by Anaclubiona was rejected by zilla-group. The genus Anaclubiona Ono, 2010 is currently considered as a junior synonym of Clubiona by The female of zilla-group can be easily recognised by its tiny body (with body length 1.8–3.6 mm), in association with the characteristic genital organs. The male palp has a developed and occasionally branched embolic apophysis. The female epigyne has a pair of guide pockets (or hoods) or a transverse hood near the copulatory openings. The zilla-group is a relatively small taxon, with only three species having been clearly documented: C.zilla widespread in Japan, C.minima  endemic to Honshu in Japan, C.tanikawai Ono, 1989 from Ryukyu Is. in Japan and Hunan and Taiwan in China. C.hooda Dong & Zhang, 2016 was assigned to the trivialis-group in the original publication  and both sexes possess certain characters associated with the zilla-group. Therefore, it is very likely they are the opposite sexes of the same species. Based on that, as well as the DNA barcoding data, we matched the female and male together. This species is new to science and is described under the name of C.jiugong sp. nov. The aim of the current paper is to describe the new species, providing detailed morphological descriptions and illustrations.While examining spiders collected from Jiugong Mountain, Hubei Province, China Fig. A, we fouSpecimens in this study were collected by hand collecting from leaf-litter in Mt. Jiugong, Hubei. Spiders were fixed and preserved in 95% ethanol. Specimens were examined with an Olympus SZX7 stereomicroscope, details being studied with an Olympus CX41 compound microscope. Female epigynes and male palps were examined and illustrated after being dissected. Epigynes were removed and cleared in warm lactic acid before illustration. The vulva was also imaged after being embedded in Arabic gum. Photos were made with a Cannon EOS70D digital camera mounted on an Olympus CX41 compound microscope. The digital images were taken and assembled using Helifocus 3.10.3 software package . The disA DNA barcode was also obtained for the species matching. A partial fragment of the mitochondrial cytochrome oxidase subunit I (CO1) gene was amplified and sequenced for two specimens, using the primers LCOI1490 (5’-GGTCAACAAATCATAAAGATATTG-3’) and HCOI2198 (5’-TAAACTTCAGGGTGACCAAAAAAT-3’). For additional information on extraction, amplification and sequencing procedures, see All measurements were obtained using an Olympus SZX7 stereomicroscope and given in millimetres. Eye diameters are taken at the widest point. The total body length does not include chelicerae or spinnerets length. Leg lengths are given as total length . Most of the terminologies used in text and figure legends follow All specimens are deposited Museum of Guizhou Education University, Guiyang, Guizhou, China .Yu & Zhongsp. n.9B74C4BF-3DDC-5DB0-98D9-7D1EDCFDB51436f4d405-32d2-4119-97b6-9c3758c20f22Type status:Holotype. Occurrence: recordedBy: Qianle Lu; individualID: YHCLU0274; individualCount: 1; sex: male; lifeStage: adult; behavior: foraging; preparations: whole animal (EtOH); associatedSequences: MZ020606GenBank: ; Taxon: order: Araneae; family: Clubionidae; genus: Clubiona; specificEpithet: jiugong; scientificNameAuthorship: Yu & Zhong; Location: continent: Asian; country: China; countryCode: CHN; stateProvince: Hubei; county: Tongshan; locality: Jiugongshan Nature Reserve; decimalLatitude: 29.39; decimalLongitude: 114.65; Identification: identifiedBy: Hao Yu; dateIdentified: 2020-07; Event: samplingProtocol: by hand; samplingEffort: 10 km by foot; year: 2020; month: 7; day: 3; Record Level: institutionCode: MGEU; basisOfRecord: Preserved SpecimenType status:Holotype. Occurrence: recordedBy: Qianle Lu; individualID: YHCLU0275; individualCount: 1; sex: female; lifeStage: adult; behavior: foraging; preparations: whole animal (EtOH); associatedSequences: MZ020605GenBank: ; Taxon: order: Araneae; family: Clubionidae; genus: Clubiona; specificEpithet: jiugong; scientificNameAuthorship: Yu & Zhong; Location: continent: Asian; country: China; countryCode: CHN; stateProvince: Hubei; county: Tongshan; locality: Jiugongshan Nature Reserve; decimalLatitude: 29.39; decimalLongitude: 114.65; Identification: identifiedBy: Hao Yu; dateIdentified: 2020-07; Event: samplingProtocol: by hand; samplingEffort: 10 km by foot; year: 2020; month: 7; day: 4; Record Level: institutionCode: MGEU; basisOfRecord: Preserved SpecimenMale 5'CTTGATCTGCTATAGCAGGAACAGCTATAAGTGTTATAATTCGTATAGAATTAGGACAATCTGGAACATTTTTAGGAGATGATCATTTATATAATGTAGTAGTTACAGCTCATGCTTTTGTTATAATTTTTTTTATAGTAATACCTATTTTAATTGGAGGTTTTGGAAATTGAATAATTCCTATGATATTAGGAGCAGCTGATATAGCTTTTCCTCGTATAAATAATTTAAGTTTTTGATTATTACCTCCTTCGTTATTTATATTATTTATATCTTCTATAGCTGAAATAGGTGTGGGAGCAGGGTGAACTATTTATCCTCCTCTTGCATCTAGTATAGGTCATACAGGAAGAGCTATAGATTTTGCTATTTTTTCGTTACATCTAGCTGGAGCTTCTTCTATTATAGGGGCTGTAAATTTTATTACTACTATTATTAATATACGATATATTGGGATGAGAATAGAAAAAGTTCCATTATTTGTTTGGTCTGTTATAATTACTGCAGTACTCTTATTATTATCATTACCTGTATTAGCAGGTGCTATTACTATATTATTGACTGATCGAAATTTTAATACATCTTTTTTTGATCCAGCTGGAGGGGGAGATCCTATTTTATTTCAGCATTTATTTTGATTTTTTGG3' 5'TTTGATCTGCTATAGTAGGAACAGCTATAAGTGTTATAATTCGTATAGAATTGGGACAATCTGGAACATTTTTAGGAGATGATCATTTATATAATGTAGTAGTTACAGCTCATGCTTTTGTTATAATTTTTTTTATAGTAATACCAATTTTAATTGGAGGTTTTGGAAATTGAATAATTCCTATGATATTAGGAGCAGCTGATATAGCTTTTCCTCGTATAAATAATTTAAGTTTTTGATTATTACCCCCTTCGTTATTTATATTATTTATATCTTCTATAGCTGAAATAGGTGTGGGAGCAGGGTGAACTATTTATCCTCCTCTTGCATCTAGTATAGGTCATACAGGAAGAGCTATAGATTTTGCTATTTTTTCGTTACATCTAGCTGGAGCTTCTTCTATTATAGGGGCTGTAAATTTTATTACTACTATTATTAATATACGATATATTGGGATGAGAATAGAAAAAGTTCCATTATTTGTTTGGTCTATTATAATTACTGCAGTACTCTTATTATTATCATTACCTGTATTAGCAGGTGCTATTACTATATTATTGACTGATCGAAATTTTAATACATCTTTTTTTGACCCAGCTGGAGGAGGAGATCCTATTTTATTTCAGCATTTATTTTGATTTTTTGG3' , but is consistently separable by its genitalia. Male of the new species resembles that of C.hooda (C.hooda) and by the EPA originating from the prolateral portion of the tegulum, pointed to the retrolateral side  and differ from C.zilla by: (1) copulatory openings closely spaced and partly fused, situated at the medial portion of epigynal plate posterior margin ; (2) the proximal half of copulatory ducts close together  near the copulatory openings in female (C.zilla-group species with tiny bodies (less than 3.6 mm), C.trivialis-group is median sized clubionids . Although we have not examined the types of C.hooda, the species was well described with high-quality illustrations: developed embolar part apophysis in male and the paired epigynal hoods in female . The present study follows Anaclubiona as a synonym of Clubiona, rather than resurrecting the generic status of the zilla-group. Consequently, we temporarily place the new species in Clubiona sensu lato and assign them to C.zilla-group.The genus


Our results show that Rad53 protects replication forks in part by antagonising Mrc1 stimulation of CMG unwinding.The Rad53 DNA checkpoint protein kinase plays multiple roles in the budding yeast cell response to DNA replication stress. Key amongst these is its enigmatic role in safeguarding DNA replication forks. Using DNA replication reactions reconstituted with purified proteins, we show Rad53 phosphorylation of Sld3/7 or Dbf4-dependent kinase blocks replication initiation whilst phosphorylation of Mrc1 or Mcm10 slows elongation. Mrc1 phosphorylation is necessary and sufficient to slow replication forks in complete reactions; Mcm10 phosphorylation can also slow replication forks, but only in the absence of unphosphorylated Mrc1. Mrc1 stimulates the unwinding rate of the replicative helicase, CMG, and Rad53 phosphorylation of Mrc1 prevents this. We show that a phosphorylation-mimicking Mrc1 mutant cannot stimulate replication in vitro and partially rescues the sensitivity of a  Saccharomyces cerevisiae (ATR in humans)  . Mec1 th humans) . In addi humans) .rad53 or mec1 mutant cells , and finally firing factors, DNA polymerases, and accessory factors were added to initiate DNA replication. We followed replication progression by separating the products on alkaline agarose gels to visualise incorporation of radiolabelled dCTP. Rad53 inhibits late origin firing in vivo by phosphorylating two substrates: Dbf4 and Sld3 . To detely shown , whilst ly shown . As showly shown . Howeverly shown .To test whether Rad53 also inhibited Sld3, we took a similar approach by pre-incubating Sld3/7 with Rad53 prior to addition to the replication reaction. Similar to DDK, pre-incubation of Sld3/7 with Rad53 and ATP resulted in reduced mobility of Sld3 in SDS-PAGE  and inhiNext, we wanted to determine whether Rad53 could affect replication elongation. We pre-incubated the elongation factor mix  with Rad53 and ATP, then added this to reactions after MCM loading, DDK phosphorylation, and firing factor addition. We stopped the reactions at early time points so that any effects of elongation could be more easily seen by the size of the leading strand replication products. KD  are non-essential proteins known to directly stimulate replication fork rate . Their iKD , indicatecreased . This leecreased . These dKD was detected in Mrc1 immunoprecipitates  as a measure of CMG helicase activity, Devbhandari and Remus have recently shown that more U* product is generated in reactions containing M/C/T than in reactions lacking M/C/T . This co17AQ protein, which cannot be phosphorylated by Mec1  that indeed exhibited faster replication than wild-type Mrc1 after Rad53 phosphorylation . Unfortunately, these mutants — especially Mrc119A — did not stimulate replication to the rate of unphosphorylated wild-type Mrc1 . This is likely due, in part, to the fact that these mutants exhibited defects in promoting faster replication in the absence of Rad53 indicating that they are not completely functional . Moreover, even Mrc119A was still inhibited slightly by incubation with Rad53 (compare lanes 5 and 6), suggesting that additional sites not mutated in this construct can still be phosphorylated by Rad53 and inhibit Mrc1 function. Cells expressing 19AMRC1 as the sole copy of MRC1 were not sensitive to the replication stress agent hydroxyurea (HU)  . Another8D) was unable to stimulate replication even in the absence of Rad53  were identified in a phosphoproteomics screen of MMS-treated cells , whilst the mrc1∆, rad9∆ double mutant was completely defective in Rad53 phosphorylation (lanes 12–15). mrc1Δ rad9Δ mutants are inviable  or MMS (0.006%), although they did not promote additional survival to higher concentrations (8 mM HU and 0.01% MMS)  relative to MRC1 wild type  . Similar01% MMS)  arguing 1Δ cells . MRC18D ild type . These dOur results show that, in addition to its role in checkpoint activation upstream of Rad53, Mrc1 also has a role downstream of the checkpoint, as a substrate of Rad53. Phosphorylation of Mrc1 by Rad53 prevents Mrc1-dependent stimulation of CMG unwinding, leading to a reduced replication fork rate. We suggest that linking these two roles could allow Rad53 to slow replication speed specifically at a stressed or damaged fork, and, therefore, act efficiently at stochastic fork stalling events without initiating a global checkpoint response. But under more severe replication stress, where more Rad53 is active, Rad53 could phosphorylate Mrc1 at all forks to slow replication globally.Using a novel assay to measure DNA unwinding activity, we found that unwinding by CMG is not very synchronous, as evidenced by shallow unwinding curves. Moreover, sites as close as 1 kb from the origin were not completely unwound after 50 min. In the presence of Mrc1, unwinding was faster and the curves were steeper, suggesting more synchronous unwinding leading to even the site 2 kb from the origin approaching 100% unwinding by 30 min. In single-molecule experiments, CMG frequently paused and backtracked while unwinding DNA . The abi8D mutant showed normal checkpoint activation supporting the idea that it remains bound to replication forks. However, we note that Rad53 can target more than these eight sites and full phosphorylation may affect more functions and protein-protein interactions than those disrupted in the Mrc18D mutant.A recent structure of CMG bound to M/C/T suggests the C-terminus of Mrc1, containing the majority of its Rad53 phosphorylation sites, may contact Cdc45 and Mcm2 . Other wmrc1∆ cells treated with HU, suggesting that Mrc1 has some role in restraining CMG at stalled forks . The peptides were synthesised using FMOC for temporary α-amino group protection. Protecting groups used were Pbf for arginine, OtBu for glutamic acid and aspartic acid, Trt for asparagine, glutamine, histidine, and cysteine, tBu for serine, threonine and tyrosine, and Boc for lysine and tryptophan. Each amino acid was coupled by activating its carboxylic acid group with DIC in the presence of HOBT. Individual aliquots of amino acids were spotted on to a cellulose membrane which has been derivatised to have 8 to 10 ethylene glycol spacers between the cellulose and an amino group. Synthesis was accomplished by cycles of coupling of amino acids, washing then removal of the temporary α-amino protecting group by piperidine followed by more washing. Once the required number of cycles of coupling and deprotection and washing had been completed, the membranes were treated with a solution of 20 ml containing 95% TFA, 3% TIS, and 2% water for 4 hr. Following this treatment, membranes were washed four times with DCM, four times with ethanol, and twice with water to remove side chain protecting groups and TFA salts and once again with ethanol for easier drying. Just prior to kinase assay, membranes were washed extensively in reaction buffer, then incubated with 80 nM Rad53, 10 µM ATP, and 0.02 µCi/µl γexo- , 20 nM GINS, 15 nM CDK, 20 nM Csm3/Tof1, 20 nM Mrc1, 20–30 nM Sld3/7, 20 nM Mcm10, 20–50 nM Sld2, and 350 nM RPA. At each time point, 2.5 µl of reaction was added to a tube containing 5 µl replication buffer and 1 µl MseI (NEB) for 3 min, and then the reaction was quenched with the addition of EDTA. Samples were then deproteinated with SDS and proteinase K followed by column clean-up (QIAquick PCR purification kit) according to manufacturer’s instructions. The final elution was done with 300 µl 10 mM Tris pH 8. Then qPCR was performed in triplicate using 4 µl sample in 8–9 µl reaction with FastStart Universal SYBR Green Master Mix (Roche) and primers flanking each MseI cassette  was then fit with a spline and normalised to the unwound DNA in the last timepoint of the closest site (200 bp from origin). The unwinding rate was then calculated by integrating the spline and averaging over the last three sites .AGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGGACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGGGGAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTGGCCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCCTCGATTTTTTTATGTTTAGTTTCGCGGACGACGGTTTCGAGGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGAGGCGGTGTTCGCCGAGATCGGCTCGCGCAAAGCCGAGTTGAGCGAACTAAACATAAAAATACAGCATCAGATGGTAGGCCTCCTGGCGCCGCACCGGCCTCAGCATCCGGTACCTCAGCTGGCCACATCACTGTCTTTCTTATGACGGTACTACCGGTGTTCACTGCACCAAGGTAACACTCATTAAATTAAGGTTAAATTAATCTACACAATTCTCTTTTGCTATTGGTACCGGATTCTCCAGCTCTGACTTCAGCGTCTCTGAAGGAATCTTTGCAGGTGCTTACGCTTACTACCTAAACTACAATGGTGTTGTCGCTACTAGTGCCGCTTCTTCAACCACTGGATCTGGTCCTAGGGCTTCGGTCCGCCCCTACTTCAGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAATTCGGTTAAGTTAAGTTAAGGTTAAAGAAGCTAACGCCAGGGTTTTCCCAGTCACGATTCGGTGCTTCCGTTACCGGTTCAACTGCTTCCACTTCATGGGCTACTTTTTGGACCGGAACGGCTGGTACTATCGGCCTGGTATCATCCTTTACCGAAGCAACATCTGTTTACACTACAACACTAGACCAAGCACAGTCGTAGTTTCTTGTTCAGAGATGACTCCAATGGTAACGTCTATACCATTACCACAATCATTAAGTTAAATTAAGTTAATCAAACCGTTCCATGCTCATCCACTACCGCCACTATTACTTCTTGTGATGAAACTGGATGTCACGTTAGTACATCAACCGGTGCTGTTGTAACTGAAACCGTTTCTTCCAAGGCATACACAACTGCCAAAGTAACTCGTTGTGACGCCAATCAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCATGGTTAAAGTTAACTTAAATTAAGGTACGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAGGCTCAATGTAACTTAGCCACTGTCAATTGGGAATGTTCCAGGGATTCATGGACAACAACTGCAACTGGAGTATCATACACCACTGTCACCGTAACCCACTGTGACGACAATGGCTGTAACACCAAGACTAAGCTCCTGAAGCTACCACCACAACTATCGCCCACCAGGACCACCGTCACCTTTAGTGATGACAATGAAGGTAAGACCTTGGGTGAGTCTGGTCCAGCGGAGGGCCACTACTGTTTCTCCAAAGACATACACCACCGCTACTGTTACTCAGGGGGATAAAAATGCCTGCCTCACCAAGACTGTCACTTCTGAATGTCCTGAAGAAACTTCAGCAACTACTACTGTCACTTCTGAGGGTTCTAAAGCAACCTCATTGAGTCGACGCGGGGGCGACGATTAACCTTAACGTTAAGTTAAGCTAGCACGACTACGCATCCCTCTGACTACTTCTCGGGGTGGGACTATACTGGTACCGATACGGGCTGTGATGACAACGATGTGTAGAACTGGGACAATCAGATCTGAGGCCCCTGAAGCCACAACGGGTACTGTTTCTAACAACAGATACAACATGGAGGGCCAACATTGTCACAATAGAAGCTCCGCCAGAAACAGTAGAAACTTCAGAAACCAGTGCTGCCCCTAAGGACATACACTACTGCCACTGGTTACTCAATGGTTTAGAGGGTGGTTGCCACGTCAAGATAATCACCTCTAAAATACCTGAAGCTACTTCAACCGTCACGGGTGCTTCTCCAAAACGGCCTTACATAGCCGGATACAGTGACTTTGACAGGTTTGCGGGGCACAGCAATGACTTGCATAGCTGCGTGCGGGGGAAGGAACTCTTGCGTCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCATTATGGTTAACTTAAGTTAATTTAAGCTATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCAGTGAGTATCTCTGCTTGACGACCCCTTGGCGCAGAGGTGCTGGCCGCGTGCTAAGTTGAAGCGGCTGCACTGCTGCAAGGTCCGTCACGGAGGCGTCGGACCGGCAGGAGCACTAGCCCATCGACCCGTACGGGAACACTCTATATCGCTCTCGGACGGACATTCTGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTGGAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTG.600 0.5 and arrested with 20 µg/ml of alpha-factor for 2–3 hr at 25°C. Cells were washed two times with YPD and then resuspended in YPD + 200 mM HU. Cells were then harvested at the indicated times, and protein was extracted with 10% trichloroacetic acid. Extracts were then processed by 3–8% Tris-acetate SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-Flag  and anti-Rad53  antibodies.Log-phase yeast cultures in YPD were diluted to OD600 0.3 and arrested with 20 µg/ml of ɑ-factor for 3 hr at 25°C. Cells were washed two times with YPG and then resuspended in YPG + 200 mM HU + 200 μg/ml BrdU. After 1 hr, cells were washed two times with YPG and then resuspended in YPG + 2 mM thymidine. Cells were harvested at the indicated times, and DNA was extracted with phenol-chloroform. DNA was run on a 0.8% alkaline agarose gel, then alkaline transferred to a positively charged nylon membrane with the VacuGene XL Vacuum Blotting System (Amersham), and immunoblotted with anti-BrdU antibody .Log-phase yeast cultures in YP-raffinose were centrifuged and resuspended in YP-galactose to ODK227A,K339A was constructed by PCR with mutated oligos on pET21b-RAD53  and the 3xFLAG tag between the BamHI and NotI sites of pRS40N and then was cut with Blp1 to modify the already integrated codon-optimised MRC1 at the his3 locus. pVP14 (MRC117AQ) contains all S/T residues followed by Q sites mutated to A as in 14A) contains the following mutations: S911A, S918A, S920A, T952A, S957A, T996A, T997A, S1006A, S1033A, T1036A, S1039A, S1040A, S1043A, T1045A. pAWM25 (MRC119A) contains the following mutations: T882A, S911A, S918A, S920A, S924A, T932A, T952A, S957A, S958A, T996A, S997A, S1006A, S1010A, S1033A, T1036A, S1039A, S1040A, S1043A, T1045A. pAWM18 (Mrc141A) contains the following mutations: T882A, S911A, S918A, S920A, S924A, T932A, S937A, T952A, S957A, S958A, S961A, T963A, S965A, T967A, S969A, T970A, T971A, S972A, T974A, T977A, T996A, S997A, S1006A, S1010A, S1013A, T1015A, T1027A, S1033A, T1036A, S1039A, S1040A, S1043A, T1045A, T1050A, T1060A, T1063A, T1079A, T1081A, S1083A, S1089A, S1093A. pAWM17 (Mrc18D) contains the following mutations: S911D, S918D, S920D, T952D, S957D, T996D, S997D, S1006D. pAWM47 and pAWM48 contained base pairs 1424–3288 of MRC1 between XhoI and BamHI and 127 bp of the 3’ UTR of MRC1 between BamHI and NotI. The 3xFLAG tag was added with flanking BamHI sites, and the plasmids were integrated into yeast after cutting with XbaI. The template used in the CMG helicase assay was made by modifying a plasmid with a pBlueScript vector and contained a synthetic origin with either two ORC binding sites 70 bp apart (pAWM36) or no ORC binding sites (pAWM37) (Yeast strains are listed in 1b-RAD53 . Mrc1 fr53 (MRC1  and modi(pAWM37)  and a sy public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Acceptance summary:S. cerevisiae  in blocking both initiation of DNA replication and replication fork progression via inhibition of Mrc1 activation of the replicative DNA helicase. The paper elegantly utilizes the power of biochemical reconstitution of complete DNA replication with purified proteins and its regulation by checkpoint kinases.This paper addresses an important role for the DNA replication checkpoint kinase Rad53 in the yeast Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Bruce Stillman as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Kevin Struhl as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Oscar M Aparicio (Reviewer #3).Thank you for submitting your article "Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.We all felt that this is a very strong paper that confirms the effect of Rad53 kinase on Sld3 and Dbf4 activities in initiation of DNA replication, but goes further and identifies Mrc1 and Mcm10 as substrates for the kinase in regulating replication fork progression and CMG activity. The results are clear and support the conclusions.Essential revisions:The reviewers made a number of suggestions, but only a subset of these are listed below and we ask that you consider adding the requested data.(1) Page 10 and Figure 5E. The observation that Mrc119A can still be inhibited by Rad53 kinase, albeit less than the wild type Mrc1 protein, might suggest (1) that there are additional phosphorylation sites (as suggested by the authors) or (2) that Rad53 binds directly to Mrc1 and this interaction may partially block its stimulation of CMG. Can the authors test if the Rad53 binds to purified Mrc1? The reason for asking this question is that it is possible that both Mrc1 and Rad53, which are both known to be located at active DNA replication forks, create a solid-state system, feedback control at the replication fork for Mec1 activating Rad53 kinase and Rad53 phosphorylating Mrc1.(2) How selective is the effect of Rad53 on the Mrc1-dependent replisome slowdown in the biochemical assays? It would be useful to exclude the possibility that any active kinase could phosphorylate Mrc1 and cause the same phenotype.(3) In addition to the viability assays shown in Figure 6, it would be useful if the authors could provide an assay that monitors replication fork progression in vivo to ensure that the Mrc1-8D protein restrains fork progression even in the absence of Rad53.Reviewer #1 (Recommendations for the authors):1. Page 10 and Figure 5E. The observation that Mrc119A can still be inhibited by Rad53 kinase, albeit less than the wild type Mrc1 protein, might suggest (1) that there are additional phosphorylation sites (as suggested by the authors) or (2) that Rad53 binds directly to Mrc1 and this interaction may partially block its stimulation of CMG. Can the authors test if the Rad53 binds to purified Mrc1? The reason for asking this question is that it is possible that both Mrc1 and Rad53, which are both known to be located at active DNA replication forks, create a solid-state system, feedback control at the replication fork for Mec1 activating Rad53 kinase and Rad53 phosphorylating Mrc1.Reviewer #2 (Recommendations for the authors):The key point that needs to be addressed, in my opinion, is whether the phenomena identified in the biochemical studies are reflective of the in vivo situation. I offer below a few comments that should help addressing this issue.(1) How selective is the effect of Rad53 on the Mrc1-dependent replisome slowdown in the biochemical assays? I ask this since recombinant Rad53 is highly active and it would be useful to exclude the possibility that any active kinase could phosphorylate Mrc1. Alternatively, does the effect of Rad53 on in vitro DNA replication dependent on its two FHA domains?(2) While I do have sympathy with the authors for their struggle in identifying the key Rad53 target sites on Mrc1, I feel they should nevertheless provide evidence that one or more of the sites mutated in the Mrc1-8D variant are indeed phosphorylated in cells, in a Rad53-dependent manner, in response to DNA replication stress.(3) In addition to the viability assays shown in Figure 6, it would be useful if the authors could provide an assay that monitors replication fork progression to ensure that the Mrc1-8D protein restrains fork progression even in the absence of Rad53.Reviewer #3 (Recommendations for the authors):I would really appreciate a figure showing the locations of the 8/14/19 mutations. I feel these should be added to the diagram in S3B.I suggest the authors construct and test an 8A version. If this allele exhibits more limited effects than 14A, it will strengthen the inference that the 8D changes are not just breaking the protein.I think a bit more in vivo analysis of these mutants would be useful and appreciated, especially by experts such as bulk analysis of DNA content during S phase progression. One would predict that the 8D allele would cause slow replication even without drugs. However, it might have little effect given the apparently intact checkpoint signaling and presumably intact origin firing control.One set of easy experiments that seems to be missing is more analysis of the 14A and 19A alleles in vivo. These alleles might be predicted to exhibit sensitivity to HU and MMS. Should effects be noted, it will probably be important to examine Rad53 activation in these mutants similar to analyses in 6B and C to rule out possible effects on transducing the signal to activate Rad53 as opposed to defects in receiving effector signals from activated Rad53. It is possible that the 14A and 19A mutations will have no sensitivities due to incomplete penetrance or redundant mechanisms, but worth a try. Essential revisions:The reviewers made a number of suggestions, but only a subset of these are listed below and we ask that you consider adding the requested data.(1) Page 10 and Figure 5E. The observation that Mrc119A can still be inhibited by Rad53 kinase, albeit less than the wild type Mrc1 protein, might suggest (1) that there are additional phosphorylation sites (as suggested by the authors) or (2) that Rad53 binds directly to Mrc1 and this interaction may partially block its stimulation of CMG. Can the authors test if the Rad53 binds to purified Mrc1? The reason for asking this question is that it is possible that both Mrc1 and Rad53, which are both known to be located at active DNA replication forks, create a solid-state system, feedback control at the replication fork for Mec1 activating Rad53 kinase and Rad53 phosphorylating Mrc1.KD  does not slow replication. However, we have now included new data  showing that neither wild type Rad53 nor Rad53KD co-immunoprecipitate appreciably with Mrc1. Under the same buffer conditions — identical to the buffer conditions in the replication reactions — Csm3/Tof1 co-immunoprecipitate nearly stoichiometrically. We think that, together, these results indicate that Rad53 inhibition of Mrc1 is likely via phosphorylation and not binding. We have added this point in the section “Rad53 inhibition of replication elongation via Mrc1 and Mcm10” on page 6.The reviewer correctly points out that Rad53 might inhibit Mrc1 by binding and not just by phosphorylating Mrc1, as has been suggested for another Rad53 target, DDK . We think this is unlikely because pre-incubation of Mrc1 with Rad53(2) How selective is the effect of Rad53 on the Mrc1-dependent replisome slowdown in the biochemical assays? It would be useful to exclude the possibility that any active kinase could phosphorylate Mrc1 and cause the same phenotype.It is, of course, very difficult to exclude the possibility that some other kinase(s) may also inhibit Rad53. Nonetheless, we think this is an interesting point, especially given the fact that DDK has been implicated in Mrc1 regulation in fission yeast. We, therefore, tested whether the two main replicative kinases, DDK and CDK, can phosphorylate and inhibit Mrc1. In a new Figure 2 —figure supplement 1B we show that neither DDK nor CDK were able to inhibit Mrc1 in replication elongation. We have added this point to section “Rad53 inhibition of replication elongation via Mrc1 and Mcm10” on page 6.(3) In addition to the viability assays shown in Figure 6, it would be useful if the authors could provide an assay that monitors replication fork progression in vivo to ensure that the Mrc1-8D protein restrains fork progression even in the absence of Rad53.Drosophila melanogaster deoxyribonucleoside kinase (dmdNK) and the human equilibrative nucleoside transporter (hENT1) which together allow efficient BrdU incorporation and rapid chasing of BrdU from nucleotide pools . In these experiments, cells are released from α factor into HU in medium containing BrdU. This results in a smear of short, labelled replication intermediates from early firing origins on alkaline agarose gels detected by ‘western’ blotting with anti-BrdU antibody. We can then estimate fork rates from the extension of this labelled smear to larger sizes after HU is washed out and BrdU is replaced with thymidine. We have done this in +RAD53 cells carrying wild-type MRC1 or 8D.MRC1 The replication intermediates of 8DMRC1 cells were shorter and were extended at a slower rate consistent with the idea that 8DMRC1 promotes slower replication forks in vivo . We have added this to the “Mrc18D slows fork rate in vitro and in vivo and partially rescues rad53 mutant sensitivity” section on page 11. Unfortunately, this approach requires synchronisation with HU, and therefore cannot be used with rad53 mutants because of the extensive fork collapse and continued origin firing in HU . Rad53 becomes rapidly dephosphorylated after release from HU, so the latter part of the curve in the new Figure 6E represents forks moving in the absence of active Rad53. Although we recognise this does not exactly address the reviewer’s question, it is probably the best we can do at the moment, and it shows that the 8D mutant does indeed have slower forks in vivo.This is a good point, but not a trivial request. We include new data that we believe at least partially addresses this issue. We previously showed that we could do ‘pulse-chase’ experiments in vivo using yeast strains that express the Reviewer #1 (Recommendations for the authors):1. Page 10 and Figure 5E. The observation that Mrc119A can still be inhibited by Rad53 kinase, albeit less than the wild type Mrc1 protein, might suggest (1) that there are additional phosphorylation sites (as suggested by the authors) or (2) that Rad53 binds directly to Mrc1 and this interaction may partially block its stimulation of CMG. Can the authors test if the Rad53 binds to purified Mrc1? The reason for asking this question is that it is possible that both Mrc1 and Rad53, which are both known to be located at active DNA replication forks, create a solid-state system, feedback control at the replication fork for Mec1 activating Rad53 kinase and Rad53 phosphorylating Mrc1.Addressed in Essential revisions.Reviewer #2 (Recommendations for the authors):The key point that needs to be addressed, in my opinion, is whether the phenomena identified in the biochemical studies are reflective of the in vivo situation. I offer below a few comments that should help addressing this issue.(1) How selective is the effect of Rad53 on the Mrc1-dependent replisome slowdown in the biochemical assays? I ask this since recombinant Rad53 is highly active and it would be useful to exclude the possibility that any active kinase could phosphorylate Mrc1. Alternatively, does the effect of Rad53 on in vitro DNA replication dependent on its two FHA domains?Addressed in Essential revisions.(2) While I do have sympathy with the authors for their struggle in identifying the key Rad53 target sites on Mrc1, I feel they should nevertheless provide evidence that one or more of the sites mutated in the Mrc1-8D variant are indeed phosphorylated in cells, in a Rad53-dependent manner, in response to DNA replication stress.8D  have been detected by proteomics from cells treated with MMS  showing they are indeed phosphorylated in response to replication stress. Another phosphoproteomics screen identified nearby residues  that were also dependent on the presence of Rad53 . We have added this to the “Mrc18D slows fork rate in vitro and in vivo and partially rescues rad53 mutant sensitivity” section on page 11. It would be very difficult/impossible to get the kind of mass spec coverage we would need to validate all of the sites, even in vitro.Apologies – we realise that we neglected to include some important references related to previous phosphoproteomic studies. Three of the eight sites in Mrc1(3) In addition to the viability assays shown in Figure 6, it would be useful if the authors could provide an assay that monitors replication fork progression to ensure that the Mrc1-8D protein restrains fork progression even in the absence of Rad53.Addressed in Essential revisions.Reviewer #3 (Recommendations for the authors):I would really appreciate a figure showing the locations of the 8/14/19 mutations. I feel these should be added to the diagram in S3B.We have now added figures with the location of the mutations to new Figure 5 —figure supplement 1B.I suggest the authors construct and test an 8A version. If this allele exhibits more limited effects than 14A, it will strengthen the inference that the 8D changes are not just breaking the protein.14A mutant is nearly wild-type in its ability to promote replication speed in vitro, it would be very surprising if the Mrc18A mutant, which is effectively a subset of the 14A mutant wasn’t also like wild type. Because we thought it would be highly unlikely the 8A mutant would work less well than the 14A mutant and because of time constraints we decided not to test this mutant.Because the Mrc1I think a bit more in vivo analysis of these mutants would be useful and appreciated, especially by experts such as bulk analysis of DNA content during S phase progression. One would predict that the 8D allele would cause slow replication even without drugs. However, it might have little effect given the apparently intact checkpoint signaling and presumably intact origin firing control.8D strains. Because S phase progression by FACS analysis reflects both the state of origin firing and replication fork speed, we instead used a system using BrdU incorporation and replication intermediate size measurements to determine replication fork rates in these cells. This experiment is described in Essential revisions.The reviewer makes a great suggestion to look at replication fork rate in the Mrc1One set of easy experiments that seems to be missing is more analysis of the 14A and 19A alleles in vivo. These alleles might be predicted to exhibit sensitivity to HU and MMS. Should effects be noted, it will probably be important to examine Rad53 activation in these mutants similar to analyses in 6B and C to rule out possible effects on transducing the signal to activate Rad53 as opposed to defects in receiving effector signals from activated Rad53. It is possible that the 14A and 19A mutations will have no sensitivities due to incomplete penetrance or redundant mechanisms, but worth a try.19A mutant in response to hydroxyurea and didn’t detect any phenotype . As the reviewer points out, this could be due to incomplete penetrance  or redundant mechanisms. We have added this to the section “Identification of Rad53 phosphorylation sites in Mrc1” on page 10.We tested the sensitivity of the Mrc1


Correction to: BMC Genomics 22, 771 (2021)https://doi.org/10.1186/s12864-021-08057-4Hsp23_E2 and Prx2540-1_E, were not entered correctly into this table.Following the publication of the original article , the corThe correct sequence of YB320 is 5’-TAGTTGGGGATGTCTTCGAATGTACATATGTTCCAAATCG -3’ and of YB362 is 5’- TAGTTGGGGATGTCTTCCATTTAGCTCATCTCCACGCTAG -3’.The correct Additional file Additional file 23: Table S10. Excel file with description of synthetic DNA fragments (oligos and gene blocks).


Exosomes are extracellular vesicles of endosomal origin that are released by practically all cell types across metazoans. Exosomes are active vehicles of intercellular communication and can transfer lipids, RNAs, and proteins between different cells, tissues, or organs. Here, we describe a mechanism whereby proteins containing a KFERQ motif pentapeptide are loaded into a subpopulation of exosomes in a process that is dependent on the membrane protein LAMP2A. Moreover, we demonstrate that this mechanism is independent of the ESCRT machinery but dependent on HSC70, CD63, Alix, Syntenin-1, Rab31, and ceramides. We show that the master regulator of hypoxia HIF1A is loaded into exosomes by this mechanism to transport hypoxia signaling to normoxic cells. In addition, by tagging fluorescent proteins with KFERQ-like sequences, we were able to follow the interorgan transfer of exosomes. Our findings open new avenues for exosome engineering by allowing the loading of bioactive proteins by tagging them with KFERQ-like motifs. LAMP2A targets cytosolic proteins to extracellular vesicles to modulate interorgan communication. Exosomes are nanosized vesicles of 40 to 160 nm in diameter that are secreted by most cell types to the extracellular space , referred to as multivesicular bodies (MVBs)  machinery  the LAMP2A gene in a human cell line with normal chromosome number, ARPE-19. LAMP2A originates from the alternative splicing of the LAMP2 gene, which contains nine exons, including three different splice variants of the exon 9  . All splExtracellular vesicles (EVs) from the media supernatants of WT and LAMP2A KO ARPE-19 cells were isolated for mass spectrometry (MS)–based proteome analysis. Note that there is still a lack of consensus on specific markers for exosomes, and the most common method used for exosome isolation, ultracentrifugation, is unable to separate exosomes from other populations of small EVs such as microvesicles. Following EV guidelines . TEM of 6 cells confirmed the presence of membrane proteins of endosomal origin, CD63 and LAMP2A, and the cytosolic proteins associated to EVs Flotillin-1 (FLOT1) and HSC70, as well as the absence of the endoplasmic reticulum marker calnexin and the plasma membrane marker Na+- and K+-dependent adenosine triphosphatase  of 2.5 μg of whole-cell lysates and 100% of isolated sEVs secreted by 40.0 × 10P < 0.05) in LAMP2A KO sEVs and that 203 of the down-regulated proteins (67%) contained a putative KFERQ motif . Accordingly, the identified proteins were enriched in EV markers to the same level as in previously published EV studies (fig. S1D) . InitialRQ motif . These fTo address the mechanisms involved in the targeting of proteins containing KFERQ motifs to EVs, we used chimeric proteins consisting of a fluorescent protein, such as mCherry, and the KFERQ motifs of α-synuclein (VKKDQ) and ribonuclease A (KFERQ) separated by a peptide spacer , based oWe hypothesized that HSC70 is involved in the targeting of proteins into nascent EVs by binding and delivering proteins containing KFERQ-like motifs to endosomes. To assess whether HSC70 has a role in the loading of proteins into sEVs, we used Pifithrin-μ (Pifi), a compound that acts by inhibiting the interaction of HSC70 with its substrates  and a deWhile some EVs such as microvesicles originate from shedding of the plasma membrane, exosomes are formed by the inward invagination of the endosomal limiting membrane followed by fusion of the endosome with the plasma membrane. To investigate whether ExoSignal-tagged proteins are present in endosomes, we used the constitutively active mutant of Rab5 (Q79L) that blocks the conversion of early endosomes (EEs) into LEs, resulting in the formation of very large hybrid endosomes that accumulate large ILVs . LAMP2A Subsequently, we isolated endosomal compartments using a discontinuous sucrose gradient, according to protocol . The mat2 showed that HIF1A is present in sEVs only when cells express LAMP2A (fig. S4A). Isolation of sEVs from 769-P cells, KO for HIF1A and the ubiquitin ligase Von Hippel–Lindau (AA) showed that only WT HIF1A is present in sEVs (fig. S4B). Moreover, subcellular fractioning using an OptiPrep linear gradient showed that endogenous HIF1A localizes to endosomal fractions and that LAMP2A KO decreases HIF1A presence in those fractions (fig. S4C). We next incubated endosomal fractions with glutathione S-transferase (GST) (no KFERQ motif) and HIF1A-GST, in the presence or absence of recombinant HSC70 and adenosine triphosphate (ATP), followed by trypsin treatment to degrade recombinant HIF1A protein unprotected by the endosomal membrane  independently of LAMP2A , which expresses mCherry in endothelial cells, showed that GFP-ExoSignal was present in the lumen of the blood vessels and inside endothelial cells (Tg(mpeg1:mCherry), which expresses mCherry in macrophages, we observed that macrophages moving within the caudal plexus incorporated GFP-ExoSignal , expressing GFP in endothelial cells, with sEVs from WT cells, we were able to increase the number of vascular branches, including longer blood vessels with greater volume, in the retinal vascular network . The targeted proteins contain amino acid sequences, biochemically related to the KFERQ motif, a pentapeptide sequence previously involved in two selective forms of autophagy: chaperone-mediated autophagy (CMA) , 7.5 μM Pifi (Merck), and 50 nM bafilomycin A1 (Apollo Scientific).The human cell lines ARPE-19  and 769-P  were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with glutamine (Biowest) supplemented with 10% fetal bovine serum  and penicillin-streptomycin . Cells were cultured at 37°C under 5% CO+,K+-ATPase, dilution of 1:1000 ; goat anti-GAPDH, dilution of 1:2000 ; mouse anti-ExoC2, dilution of 1:500 ; rabbit anti–histone 3, dilution of 1:1000 ; anti-Vps4b, dilution of 1:500 , goat anti-GFP, dilution of 1:1000 , goat anti-Rab31, dilution of 1:500 ; mouse anti–syntenin-1 ; and horseradish peroxidase (HRP)–conjugated secondary goat anti-mouse , goat anti-rabbit , rabbit anti-goat , and goat anti-rat , dilution of 1:5000. The following were also used: Alexa Fluor 568–conjugated donkey anti-goat , Alexa Fluor 488–conjugated donkey anti-goat , Alexa Fluor 488–conjugated donkey anti-rabbit , Alexa Fluor 546–conjugated donkey anti-mouse , donkey anti-rabbit Cy3 , and donkey anti-mouse Cy5, dilution of 1:250. The following were also used: dextran, Alexa Fluor 488, 10,000 molecular weight (MW), anionic, fixable ; dextran, Alexa Fluor 647, 10,000 MW, anionic, fixable ; wheat germ agglutinin, Alexa Fluor 488 conjugate ; cathepsin B assay kit ; DAPI ; Protein G–Sepharose ; Dynabeads Protein G ; nitrocellulose membranes ; ECL ; protease inhibitor cocktail ; OptiPrep iodixanol density media ; ONE-Glo Luciferase Assay System ; and Pierce BCA Protein Assay Kit .The following antibodies were used: mouse anti-LAMP2 clone H4B4, dilution of 1:1000 (WB) and 1:100 ; rabbit anti-LAMP2A, dilution of 1:500 (WB) and 1:100 ; rabbit anti-LAMP2B, dilution of 1:500 ; mouse anti-actin, dilution of 1:2000 ; goat anti-HIF1A, dilution of 1:1000 ; goat anti-CD63, dilution of 1:1000 ; mouse anti-CD63, dilution of 1:100 ; rabbit anti–Flotillin-1, dilution of 1:500 ; goat anti-CTSB/cathepsin B clone S-12, dilution of 1:500 ; mouse anti-Alix, dilution of 1:500 ; mouse anti-TSG101, dilution of 1:500 ; goat anti-GST, dilution of 1:500 ; goat anti-EEA1, dilution of 1:500 ; goat anti-mCherry, dilution of 1:500 (WB) and 1:100 ; rabbit anti-DsRed, dilution of 1:100 ; mouse anti-m6pR, dilution of 1:100 ; rat anti-HSC70 clone 1B5, dilution of 1:1000 ; goat anti-Rab27, dilution of 1:500 ; goat anti-tubulin, dilution of 1:2000 ; mouse lamin B1, dilution of 1:500 ; goat anti-calnexin, dilution of 1:1000 ; goat anti–NaThe sEV protein solution containing SDS and dithiothreitol (DTT) was loaded onto filtering columns and washed exhaustively with 8 M urea in Hepes buffer (m/z) was followed by higher-energy collisional dissociation (HCD) fragmentation and Orbitrap detection of the 15 most intense ions observed in the MS scan. Target value in the Orbitrap for MS scan was 1,000,000 ions at a resolution of 70,000 at m/z 200. Fragmentation in the HCD cell was performed at normalized collision energy of 31 eV. Ion selection threshold was set to 25,000 counts, and maximum injection time was 100 ms for MS scans and 300 and 500 ms for MS/MS scans. Selected sequenced ions were dynamically excluded for 45 s.Peptide samples were analyzed by nano–LC-MS/MS (Dionex RSLCnano 3000) coupled to a Q-Exactive Orbitrap mass spectrometer (Thermo Scientific). Briefly, the samples (5 μl) were loaded onto a custom-made fused capillary precolumn  with a flow of 5μl/min for 7 min. Trapped peptides were separated on a custom-made fused capillary column  packed with ReproSil-PurC18 3-μm resin  with a flow of 300 nl/min using a linear gradient from 92% A (0.1% formic acid) to 28% B (0.1% formic acid in 100% acetonitrile) over 93 min followed by a linear gradient from 28 to 35% B over 20 min at a flow rate of 300 nl/min. Mass spectra were acquired in positive ion mode, applying automatic data-dependent switch between one Orbitrap survey. MS scan in the mass range of 400 to 1200 mass/charge ratio  transformation; (ii) removing common MS contaminants followed by log2 (x + 1) transformation and quantile normalization; and (iii) removing common MS contaminants followed by log2 (x + 1) transformation, quantile normalization, and abundance filtering to optimize overall Gaussian distribution of the quantitative values. For simplicity, only the quantile-normalized quantitative data are presented here. Statistical differences were calculated by using the R package limma (https://rshine.einsteinmed.org/).Statistical and bioinformatics analyses of MS data were performed in the statistical programming language R. Quantitative data from MaxQuant and VEMS were analyzed in R statistical programming language. IBAQ and protein spectral counts from the two programs were preprocessed by three approaches: (i) removing common MS contaminants followed by logFor this work, we used the plasmids pT81 HRE (x3)-Luciferase  (https://zlab.bio/guide-design-resources).ARPE-19 cells were transduced with the lentiviral vector pCW-Cas9  protein] and psPAX2 (Rev and Pol proteins) into a producer cell line, 293STAR RDPro [American Type Culture Collection (ATCC)]. Recombinant viral particles were harvested 48 days later, cleared for cell debris by centrifugation at 3200The sequences were synthesized and cloned into pUC57 (GeneCust). PA-mCherry and PA-mCherry ExoSignal were subcloned into pLenti6 (Thermo Scientific) using a Gateway adapted vector using BP/LR Clonase II (Thermo Scientific) according to the manufacturer’s instructions.Human LAMP2A was synthesized into pUC57 plasmid with the sequence 5′-CAAGTTTGTACAAAAAAGCAGGCTCTCGAGCAatgGTGTGCTTCCGCCTCTTCCCGGTTCCGGGCTCAGGGCTCGTTCTGGTCTGCCTAGTCCTGGGAGCTGTGCGGTCTTATGCATTGGAACTTAATTTGACAGATTCAGAAAATGCCACTTGCCTTTATGCAAAATGGCAGATGAATTTCACAGTACGCTATGAAACTACAAATAAAACTTATAAAACTGTAACCATTTCAGACCATGGCACTGTGACATATAATGGAAGCATTTGTGGGGATGATCAGAATGGTCCCAAAATAGCAGTGCAGTTCGGACCTGGCTTTTCCTGGATTGCGAATTTTACCAAGGCAGCATCTACTTATTCAATTGACAGCGTCTCATTTTCCTACAACACTGGTGATAACACAACATTTCCTGATGCTGAAGATAAAGGAATTCTTACTGTTGATGAACTTTTGGCCATCAGAATTCCATTGAATGACCTTTTTAGATGCAATAGTTTATCAACTTTGGAAAAGAATGATGTTGTCCAACACTACTGGGATGTTCTTGTACAAGCTTTTGTCCAAAATGGCACAGTGAGCACAAATGAGTTCCTGTGTGATAAAGACAAAACTTCAACAGTGGCACCCACCATACACACCACTGTGCCATCTCCTACTACAACACCTACTCCAAAGGAAAAACCAGAAGCTGGAACCTATTCAGTTAATAATGGCAATGATACTTGTCTGCTGGCTACCATGGGGCTGCAGCTGAACATCACTCAGGATAAGGTTGCTTCAGTTATTAACATCAACCCCAATACAACTCACTCCACAGGCAGCTGCCGTTCTCACACTGCTCTACTTAGACTCAATAGCAGCACCATTAAGTATCTAGACTTTGTCTTTGCTGTGAAAAATGAAAACCGATTTTATCTGAAGGAAGTGAACATCAGCATGTATTTGGTTAATGGCTCCGTTTTCAGCATTGCAAATAACAATCTCAGCTACTGGGATGCCCCCCTGGGAAGTTCTTATATGTGCAACAAAGAGCAGACTGTTTCAGTGTCTGGAGCATTTCAGATAAATACCTTTGATCTAAGGGTTCAGCCTTTCAATGTGACACAAGGAAAGTATTCTACAGCTCAAGACTGCAGTGCAGATGACGACAACTTCCTTGTGCCCATAGCGGTGGGAGCTGCCTTGGCAGGAGTACTTATTCTAGTGTTGCTGGCTTATTTTATTGGTCTCAAGCACCATCATGCTGGATATGAGCAATTTTAGGGTACCACCCAGCTTTCTTGTACAAAGTGGACGCGT-3′. Human LAMP2A was subcloned into pLenti6 (Thermo Scientific), a Gateway adapted vector using BP/LR Clonase II (Thermo Scientific), according to the manufacturer’s instructions.P2A-GFP-ExoSignal was synthesized and subcloned into the p3E plasmid with the sequence 5′-TACAGGTCACTAATACCATCTAAGTAGTTGATTCATAGTGACTGCATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAACTTTCTTGTACAAAGTGGAGATCTatgGGAAGCGGACGTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTGCTCGAGCAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGAAAGCTTTGTTAAAAAAGACCAAGCAGAACCACTACACCGAAAATTCGAACGACAATGAGGTACCGTCGACCAACTTTATTATACAAAGTTGGCATTATAAAAAAGCATTGCTTATCAATTTGTTGCAACGAACAGGTCACTATCAGTCAAAATAAAATCATTA-3′. Subsequently, the sequence was subcloned into a pDestTol2pA5 plasmid (gift from C.-H. Yuhm’s laboratory) according to the manufacturer’s instructions (Thermo Scientific), along with p5E-Ubi  and pME-mCherry (gift from C.-H. Yuhm’s laboratory).g for 10 min, and used. For TSG101, VPS4b, CD63, and Alix, pLKO.1 plasmids with shRNA sequences were obtained from the RNAi Consortium . Control used was a nontargeting sequence . The target sequences used were as follows: TSG101 , VPS4b , CD63 , and Alix . For Syntenin-1 target, pLKO.1 plasmids with shRNA sequences were obtained from Sigma Mission library .All lentiviral particles were produced by cotransfection of pLenti6 and pMD 2.G (VSV-G protein) and psPAX2 (Rev and Pol proteins) into a producer cell line, 293STAR RDPro (ATCC). Recombinant viral particles were harvested 48 days later, cleared for cell debris by centrifugation at 3200For the knockdowns, Rab27a and Rab27b, as well as Rab31 microRNA (miRNA)–expressing vectors, were constructed by inserting specific nucleotide sequences into pcDNA6.2-GW/EmGFP-miR plasmid harboring a Pol II promoter obtained from Thermo Scientific. These sequences are fused with GFP coding sequence. The synthesized oligonucleotides were annealed and ligated into pcDNA6.2-GW/EmGFP-miR according to the manufacturers’ instructions. For Rab27a and Rab27b, tandem miRNA sequences were transferred into pAd adenoviral vector from Thermo Scientific using Gateway technology. Sequences used were as follows: Rab27a miRNA1, 5′-AAACTTTGCTCATTTGTCAGG-3′; Rab27a miRNA2, 5′-TTAACTGATCCGTAGAGGCAT-3′; Rab27b miRNA1, 5′-ATTGACTTCCCTCTGATCTGG-3′; and Rab27b miRNA2, 5′-TTTCCCTGAAGATCCATTCGG-3′. For Rab31, miRNA sequences were transferred into pAd adenoviral vector from Thermo Scientific using Gateway technology. Sequences used were as follows: miRNA1, 5′-TTTCTTTGCAGGAAACGTCCC-3′ and miRNA2, 5′-TAAACTGAAGGCCATGTTGCG-3′. Viral particles were produced, and cells were infected as described before  in 0.1 M phosphate buffer (pH 7.4). Cells were processed for ultracryomicrotomy and contrasted as described . Samples were washed with phosphate-buffered saline (PBS) and fixed with 1% GA for 5 min. After washing with distilled water, grids were contrasted with uranyl oxalate (pH 7) for 5 min and transferred to methylcellulose-uranyl acetate for 10 min on ice. Observations were carried out using a Tecnai G2 Spirit BioTwin electron microscope (FEI) at 80 kV.g for 10 min, followed by centrifugation at 16,500g for 20 min. From this pellet, the microvesicle fraction was collected. To remove larger particles, the supernatant was filtered with a 0.22-μm filter unit, after which it was ultracentrifuged at 120,000g for 70 min, using an SW 32 Ti rotor. The resulting pellet was washed with PBS, and after ultracentrifugation, exosomes were resuspendedin PBS.Exosomes derived from 40 million cultured cells were isolated from 40 ml of conditioned medium. Cells were cultured in exosome-depleted medium for 48 hours, prepared in accordance with the work of Lasser and colleagues  containing the exosomes was collected using a syringe with a 18-gauge needle. Exosomes were then transferred to a new ultracentrifuge tube, diluted in 35 ml of PBS, and centrifuged overnight at 100,000g and 4°C. The resulting pellet was resuspended in 100 μl of PBS, and protein quantification was performed.For MS, an extra step of purification was introduced using a 30% sucrose cushion . Cells were lysed by sonication (three rounds of 3 s at 4°C) and centrifuged at 1000g for 5 min (2×). Continuous 5 to 30% OptiPrep gradients were prepared in working solution using a gradient mixer. Postnuclear supernatant (PNS) was added to the top of 9-ml gradients and centrifuged at 100,000g for 16 hours using a 70.1 Ti rotor. Sequential fractions were collected . Fractions were recovered by ultracentrifugation at 100,000g for 30 min in an SW 32 Ti rotor. For WB, fractions were collected in PBS and further denaturated in Laemmli buffer.Continuous density gradients were performed as described previously with adaptations. Briefly, 8 × 10et al. was used (6), cultured in DMEM with 10% FBS, were washed with ice-cold PBS and collected with a cell scraper. Cells were collected to a 2-ml microfuge tube and centrifuged at 300g for 5 min at 4°C. Cell pellet was loosened using cold finger in a homogenization buffer . Samples were centrifuged at 1300g for 10 min at 4°C. Supernatant was discarded, and the pellet was gently resuspended with a wide-cut tip in three times the pellet volume of HB. The suspension was passed through a 25-gauge needle, attached to a 1-ml syringe, 10 times.For the separation of EEs and LEs, a protocol described by de Araujo g for 10 min at 4°C. The supernatant was collected and centrifuged again. The PNS that originated from the second centrifugation is used for organelle isolation. PNS (5%) is centrifuged at 4°C for 10 min at 16,000g. The recovered supernatant was collected for cytoplasmatic fraction, free of vesicles. For the remaining PNS, the sucrose concentration was adjusted to 40.6%. The PNS was then loaded at the bottom of an ultracentrifuge tube. The 40.6% solution was overlaid with 1.5 volumes of 35% sucrose solution and 1 volume of 25% sucrose solution, and the tube was filled to the top with HB. The gradient was centrifuged at 210,000g for 16 hours at 4°C, using a 70.1 Ti rotor. Each interface was collected. LEs are found in the 25%/HB interface. EEs are present in the 35%/25% interface. The endosomal fractions were diluted in 35 ml of HB solution and centrifuged at 100,000g for 1 hour in an SW 32 Ti rotor. The organelle pellets were resuspended in an appropriate buffer depending on the downstream procedures.The homogenate was then diluted in HB (1 part homogenate to 0.7 parts HB) and centrifuged at 16002PO4, pH 7.25 was adjusted with KOH) buffer according to protocol ] for 45 min at 37°C. After the incubation period, samples were cooled on ice for 1 min followed by a 30-min incubation with trypsin at 37°C. Samples were put back to ice and denaturated in Laemmli buffer, heated at 95°C for 5 min, and resolved on a 10% SDS–polyacrylamide gel electrophoresis (PAGE) gel. The gel was transferred to a nitrocellulose membrane for 75 min at 100 V. The membranes were blocked with TBS-T containing 5% skim milk, followed by incubation with antibodies against the proteins of interest.Recombinant proteins GST (SICGEN) and GST-HIF1A  were incubated with 5 μg of freshly isolated vesicles and recombinant protein HSC70  in Mops buffer  and ATP regeneration buffer  and sonicated three times, 1 s each at 4°C. Afterward, samples were centrifuged at 15,000g for 10 min, and pellets were discarded. All samples were incubated with 2 μg of the antibody against the protein of interest overnight at 4°C. Subsequently, 30 μl of Protein G–Sepharose was added to the sample, and incubations proceeded at 4°C for 2 hours. Beads were washed three times with lysis buffer containing 0.15% NP-40, denatured with Laemmli buffer, and boiled at 95°C for 5 min. Samples were then analyzed by SDS-PAGE. The membranes were blocked with 5% nonfat milk in TBS-T and probed for the proteins of interest.Cells were collected from dishes with ice-cold PBS using a cell scraper and centrifuged at 15,0002PO4; pH 7.25 was adjusted with KOH). Magnetic beads were incubated with anti-LAMP2A or anti-LAMP2B antibodies for 1 hour. Subsequently, the endosomes were incubated with the magnetic beads for an additional hour at 4°C. Immunoprecipitates were then gently washed three times with KPBS. The samples were denatured with Laemmli buffer and boiled at 95°C for 5 min. Samples were then analyzed by SDS-PAGE. The membranes were blocked with 5% nonfat milk in TBS-T and probed for the proteins of interest.Afterward, endosomal immunoprecipitation was adapted from protocol , 150 mM NaCl, 10 mM iodoacetamide, 2 mM PMSF, and 1× cocktail inhibitor from Sigma-Aldrich]. Samples were then centrifuged at 4°C for 10 min at 1000g. Supernatants were removed to a new 1.5-ml microfuge tube, and pellet (nuclear pellet) was lysed with Laemmli buffer. Supernatant was centrifuged at 4°C for 10 min at 16,000g. The resultant pellet (vesicular pellet) and the second supernatant (cytoplasm) were also lysed with Laemmli buffer. Samples were then analyzed by SDS-PAGE. The membranes were blocked with 5% nonfat milk in TBS-T and probed for the proteins of interest.To evaluate the presence of exosomal HIF1A in the nuclei of receiving cells, exosomes were isolated from ARPE-19 cells that were under hypoxia with 300 μM CoClHIF1A activity was measured by transducing cells with the reporter gene Luciferase under the control of the HRE, using ONE-Glo Luciferase Assay System (Promega), according to the manufacturer’s specifications.casper, Tg(kdrl:mCherry), or Tg(mpeg1:mCherry) embryos, at 100-cell stage, were injected in the YSL with pUbi-mCherry-p2A-GFP-ExoSignal in the presence or absence of MOs at 150 nM concentration, with a microinjector under a stereomicroscope. We used a Syntenin-a MO oligonucleotide directed against the translation start site [5′-ACAACGACATCCTTTCTGCTTTCA-3′ (Tg(fli1a:EGFP) zebrafish with a microinjector under a stereomicroscope. At 5 dpf, embryos were fixed with 4% PFA overnight at 4°C. Embryos were washed in PBS and mounted in glycerol mounting media. Images were acquired in a Zeiss LSM 710 confocal microscope using a 40× 1.2 C-Apochromat water-immersion objective. When required, optical slices were acquired, and 3D reconstruction of the images and quantifications were performed using Imaris software.Zebrafish lines were maintained in a circulating system with 14-hour day and 10-hour night cycle periods at 28°C. The mating and spawning of zebrafish were incited by the change of dark to light. Embryos were collected before they start independent feeding, at 5 days postfertilization (dpf). Therefore, no ethical approval was necessary according to the Council Directive 2010/63/EU on the protection of animals used for scientific purposes  with Tukey’s multiple comparisons tests, using GraphPad Prism 8.0 software (GraphPad Software). For comparison between two groups, the paired