mcherry arp3 n  (Addgene inc)

Journal: bioRxiv

Article Title: ISG15-modification of the Arp2/3 complex restricts pathogen spread

doi: 10.1101/2022.12.27.522022

Figure Lengend Snippet: a-c Arp3 actin comet tail in Lm EGD infected MEFs at 6 h post infection (MOI 10). Bacteria are shown in green, and Arp3 in magenta. Fluorescent intensity profile (FIP) was taken of the dotted line along the midline of the cell in the inset image WT ( a ), USP18 C61A/C61A ( b ), and isg15 −/− ( c ), scale bar, 1 μm. d Percentage of bacteria with Arp3 surface localization at 6 HPI, representative data (WT n=5, CA n=9, KO n=10) analyzed with Kruskal-Wallis test followed by Dunn’s post hoc test. e Stimulated Emission Depletion (STED) images of bacteria in MEFs at 6 h post infection, bacteria are shown in green (a-ActA), and actin (Phalloidin) in magenta. scale bar, 1 μm. f Data from machine learning analysis of average length of actin comet tails formed by bacteria at 6 h post infection in wildtype, USP18 C61A/C61A , or isg15 −/− MEFs analyzed by Kruskal-Wallis test with Dunn’s post hoc. g Average thickness of actin comet tails formed by bacteria at 6 h post infection in wildtype, USP18 C61A/C61A , or isg15 −/− MEFs, compared by by Kruskal-Wallis test with Dunn’s post hoc. h Average signal intensity of actin structures along bacterial actin tails in wildtype, USP18 C61A/C61A , or isg15 −/− MEFs analyzed by Kruskal-Wallis test with Dunn’s post hoc. In f-g bar graphs represent the mean ± SEM and results analyzed from three independent experiments WT n=439, CA n=661, KO n=680. i Representative images of Lm actin comet tail formed at 24 HPI using STED imaging, bacteria in green (a-ActA), and actin (Phalloidin) in magenta; scale bar, 5 μm. j Wildtype, USP18 C61A/C61A , and isg15 −/− MEFs infected with Western Reserve strain of Vaccinia virus (VACV) for 8 h. Actin is shown in magenta, and VACV in green; scale bar,10 μm, insets, 1 μm. k Average length of VACV actin tail in MEFs; data represent the mean ± SEM, WT n=1183, CA n=3286, KO n=1752, significance determined by Kruskal-Wallis test with Dunn’s post hoc. l Representative confocal images of VACV actin comet tails at 12 HPI in WT and USP18 C61A/C61A using anti-anti-VACV 14K antibody (green) and phalloidin for actin (magenta); scale bar, 10 μm, insets, 1 μm. In d , f , g , h and k asterisks indicate p values with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Raw data are available in the source data file.

Article Snippet: Plasmid pBABEpuro-mCherry-ARP3-N-12 wildtype was constructed using mCherry-ARP3-N-12 plasmid as the template. mCherry-ARP3-N-12 was a gift from Michael Davidson (Addgene plasmid #54982; http://n2t.net/addgene:54982 ; RRID: Addgene_54982). mCherry-ARP3 fragment was amplified using primers mCh-ARP3 FWD and mCh-ARP3 REV, and then cloned into pBABE-puro vector, linearized with BamHI and SalI, via Gibson assembly (New England BioLabs Inc). pBABEpuro-mCherry-ARP3-N-12 K all R plasmid were generated in two steps. gblock fragment ARP3 K all R was digested with NheI and BamHI, column purified, and then co-ligated with the mCherry-ARP3-N-12 vector linearized with Nhel and BamHI. mCherry-ARP3 K all R fragment was amplified with primers mCh-ARP3 FWD and mCh-ARP3 REV and then cloned into pBABE-puro vector, linearized with BamHI and SalI, via Gibson assembly (New England BioLabs Inc).

Techniques: Infection, Imaging, Western Blot

Journal: bioRxiv

Article Title: ISG15-modification of the Arp2/3 complex restricts pathogen spread

doi: 10.1101/2022.12.27.522022

Figure Lengend Snippet: a Time-lapse microscopy of Lm GFP-EGD (green) infected mTagRFP-T-lifeact-7 (magenta) expressing wildtype, USP18 C61A/C61A , or isg15 −/− MEFs at 4 h post infection imaged every 10s. Scale bar, 5 μm. SI movies 1-5. b Quantification of average speed per bacterium tracked from 3 independent experiments (WT n=61, CA n=35, KO n=75), analyzed by Kruskal-Wallis test with Dunn’s post hoc. c-e Model of ISG15 (in rose) C terminus attached to Lys18, Lys75, and Lys191 of Actin-related protein 3 (Arp3) in the inactive Arp2/3 complex PDB ID: 6YW7 ( c ); the active Arp2/3 complex PDB ID: 7AQK ( d ); the active Arp2/3 complex with branched actin filaments PDB: 7AQK ( e ). f Schematic of ActA domains with interacting proteins; inset amino acid sequence mutated to abrogate Arp2/3 recruitment and comparison of R148S with WT for motility and autophagy; created with Biorender.com. g Quantification of average speed of motility per bacterium of wildtype bacteria and Lm EGD DactA complemented with ActA R148S which unstably recruits the Arp2/3 complex tracked from three independent experiments (WT n=191, CA n=203, KO n=164) by Brown-Forsythe ANOVA test with Dunnett’s T3’s post hoc. h Representative time-lapse images of Lm EGD DactA ::ActA R148S in mTagRFP-T-lifeact-7 (magenta) expressing wildtype, USP18 C61A/C61A , or isg15 −/− MEF at 4 h post infection imaged every 10s. Scale bar, 5 μm. White arrows point to inconsistent actin tails. Yellow arrows highlight normal consistent bacterial actin tails. See also SI movies 6-8. i Representative Lm 10403s plaques in USP18 C61A/C61A mCherry-Arp3 and mCherry-Arp3 3KR at 3 DPI. j Plaque size (in square pixels) from 3 independent experiments (WT n=1073, KR n=768), analyzed with Kolmogorov-Smirnov’s t-test. In b, g, i and j asterisks indicate p values with **p < 0.01, ***p < 0.001, and ****p<0.0001. Raw data are available in the source data file.

Article Snippet: Plasmid pBABEpuro-mCherry-ARP3-N-12 wildtype was constructed using mCherry-ARP3-N-12 plasmid as the template. mCherry-ARP3-N-12 was a gift from Michael Davidson (Addgene plasmid #54982; http://n2t.net/addgene:54982 ; RRID: Addgene_54982). mCherry-ARP3 fragment was amplified using primers mCh-ARP3 FWD and mCh-ARP3 REV, and then cloned into pBABE-puro vector, linearized with BamHI and SalI, via Gibson assembly (New England BioLabs Inc). pBABEpuro-mCherry-ARP3-N-12 K all R plasmid were generated in two steps. gblock fragment ARP3 K all R was digested with NheI and BamHI, column purified, and then co-ligated with the mCherry-ARP3-N-12 vector linearized with Nhel and BamHI. mCherry-ARP3 K all R fragment was amplified with primers mCh-ARP3 FWD and mCh-ARP3 REV and then cloned into pBABE-puro vector, linearized with BamHI and SalI, via Gibson assembly (New England BioLabs Inc).

Techniques: Time-lapse Microscopy, Infection, Expressing, Sequencing

Journal: bioRxiv

Article Title: ISG15-modification of the Arp2/3 complex restricts pathogen spread

doi: 10.1101/2022.12.27.522022

Figure Lengend Snippet: a Validation of ActA expression in Lm wild-type EGD, EGD ΔactA , and EGD ΔactA strain complemented with wildtype ActA, ActA R148S, or ActA R148S GFP constructs. b Representative image of plaque formation of EGD ΔactA::actA R148S infection (MOI 0.001) in WT, USP18C64A and isg15 −/− A549 at 4 DPI. c Plaque size (in square pixels) from four independent experiments (WT n=742, CA n=945, KO n=653), analyzed with one-way ANOVA followed by Tukey’s post hoc (*p < 0.05, and **p < 0.01, raw data are available in the source data file). d Validation of deletion of endogenous Arp3 in usp18 C61A/C61A MEFs. usp18 C61A/C61A actr3 −/− MEF clone was complemented with mCherry-Arp3 wildtype or mCherry-Arp3 that harbors lysine mutations to arginine at Lys18, Lys75, Lys191. e Representative image of plaque formation of Lm 10403s in USP18 C61A mCherry-Arp3 and mCherry 3KR Arp3 (K18/75/191R) at 3 DPI. Infected cell monolayers were stained with a-InlC (black) and crystal violet (pink). f Immunoprecipitation of mCherry tag in mCherry-Arp3 complemented usp18 C61A/C61A actr3 −/− MEFs treated with Type I IFN (1000 units/mL) for 24 h; SDS-PAGE for both Arp3 and ISG15. The experiment was repeated three times, and one representative blot is shown. g Immunoprecipitation of mCherry in mCherry-Arp3 complemented usp18 C61A/C61A actr3 −/− MEFs infected with Lm EGD (MOI 10) for 24 h; SDS-PAGE for both Arp3 and ISG15. Blot from one representative experiment is shown. In f and g , asterisks indicate background bands from strip and reprobe.

Article Snippet: Plasmid pBABEpuro-mCherry-ARP3-N-12 wildtype was constructed using mCherry-ARP3-N-12 plasmid as the template. mCherry-ARP3-N-12 was a gift from Michael Davidson (Addgene plasmid #54982; http://n2t.net/addgene:54982 ; RRID: Addgene_54982). mCherry-ARP3 fragment was amplified using primers mCh-ARP3 FWD and mCh-ARP3 REV, and then cloned into pBABE-puro vector, linearized with BamHI and SalI, via Gibson assembly (New England BioLabs Inc). pBABEpuro-mCherry-ARP3-N-12 K all R plasmid were generated in two steps. gblock fragment ARP3 K all R was digested with NheI and BamHI, column purified, and then co-ligated with the mCherry-ARP3-N-12 vector linearized with Nhel and BamHI. mCherry-ARP3 K all R fragment was amplified with primers mCh-ARP3 FWD and mCh-ARP3 REV and then cloned into pBABE-puro vector, linearized with BamHI and SalI, via Gibson assembly (New England BioLabs Inc).

Techniques: Expressing, Construct, Infection, Staining, Immunoprecipitation, SDS Page, Stripping Membranes

Journal: bioRxiv

Article Title: ISG15-modification of the Arp2/3 complex restricts pathogen spread

doi: 10.1101/2022.12.27.522022

Figure Lengend Snippet: a Representative image of Lm GFP-EGD infected wildtype mCherry-Arp3 expressing usp18 C61A/C61A actr3 −/− MEFs at 12 HPI. Insets demonstrate bacteria with actin tails from two connected daughter cells. Actin (phalloidin) in blue, bacteria (green), mCherry-Arp3 (magenta) and nuclei (yellow). b Representative image of Lm GFP-EGD infected usp18 C61A/C61A actr3 −/− MEFs expressing mCherry-Arp3 3KR at 12 HPI. Insets demonstrate bacteria no longer associate with actin tails cells on both poles of connected daughter cells. Actin (phalloidin) in blue, bacteria (green), mCherry-Arp3 (magenta) and nuclei (yellow). Scale bars in image and insets are respectively 10 μm and 1 μm.

Article Snippet: Plasmid pBABEpuro-mCherry-ARP3-N-12 wildtype was constructed using mCherry-ARP3-N-12 plasmid as the template. mCherry-ARP3-N-12 was a gift from Michael Davidson (Addgene plasmid #54982; http://n2t.net/addgene:54982 ; RRID: Addgene_54982). mCherry-ARP3 fragment was amplified using primers mCh-ARP3 FWD and mCh-ARP3 REV, and then cloned into pBABE-puro vector, linearized with BamHI and SalI, via Gibson assembly (New England BioLabs Inc). pBABEpuro-mCherry-ARP3-N-12 K all R plasmid were generated in two steps. gblock fragment ARP3 K all R was digested with NheI and BamHI, column purified, and then co-ligated with the mCherry-ARP3-N-12 vector linearized with Nhel and BamHI. mCherry-ARP3 K all R fragment was amplified with primers mCh-ARP3 FWD and mCh-ARP3 REV and then cloned into pBABE-puro vector, linearized with BamHI and SalI, via Gibson assembly (New England BioLabs Inc).

Techniques: Infection, Expressing

Journal: bioRxiv

Article Title: ISG15-modification of the Arp2/3 complex restricts pathogen spread

doi: 10.1101/2022.12.27.522022

Figure Lengend Snippet: a Quantification of average sensitivity to 0.05% trypsin in wildtype, Usp18 C61A/C61A , or isg15 −/− MEFs under standard tissue culture conditions; results represent mean +/-SEM from 3 individual experiments (WT n=3, CA=3, KO=3), significance determined by Tukey’s multiple comparisons. b Area under the curve analysis of trypsin sensitivity in MEFs, compared with Brown-Forsythe ANOVA test with Dunnett’s T3 post hoc. c Representative images of actin organization and Arp3 localization in mCherry-Arp3 wildtype or Arp3 3KR expressing usp18 C61A/C61A actr3 −/− MEFs. Actin (phalloidin) in green, mCherry-Arp3 in magenta, endogenous Arp3 (a-Arp3) in blue, and DAPI in yellow. Scale bars, 10 μm. d Representative image of migration pattern of Type I IFN treated (1000 U/mL for 24H) mCherry-Arp3 wildtype or Arp3 3KR expressing usp18 C61A/C61A actr3 −/− MEFs on fibronectin; scale bars, 10 μm, see SI movies 17-19. e Quantification of average population of cells present with rapidly turning over filopodia-like structure (“jazz hands”). Mean -/+ SEM from 2 individual experiments, 49-95 cells were counted and tracked per genotype per repeat. f Area under the curve analysis of MEFs with “jazz hands”, average -/+ SEM compared with twotailed unpaired t test. g Representative histological images of neonatal skin (Postnatal Day 1), H and E stain, from wildtype, usp18 C61A/C61A , and isg15 −/− animals. Scale bar = 88 μm. h Quantification of keratin layer average thickness from six neonates from each genotype; oneway ANOVA with Tukey’s post hoc test. i Quantification of average total thickness from six neonates from each genotype; one-way ANOVA with Tukey’s post-hoc test. In a , b , and f asterisks indicate p values with *p < 0.05, and ****p < 0.0001. Raw data are available in the source data file.

Article Snippet: Plasmid pBABEpuro-mCherry-ARP3-N-12 wildtype was constructed using mCherry-ARP3-N-12 plasmid as the template. mCherry-ARP3-N-12 was a gift from Michael Davidson (Addgene plasmid #54982; http://n2t.net/addgene:54982 ; RRID: Addgene_54982). mCherry-ARP3 fragment was amplified using primers mCh-ARP3 FWD and mCh-ARP3 REV, and then cloned into pBABE-puro vector, linearized with BamHI and SalI, via Gibson assembly (New England BioLabs Inc). pBABEpuro-mCherry-ARP3-N-12 K all R plasmid were generated in two steps. gblock fragment ARP3 K all R was digested with NheI and BamHI, column purified, and then co-ligated with the mCherry-ARP3-N-12 vector linearized with Nhel and BamHI. mCherry-ARP3 K all R fragment was amplified with primers mCh-ARP3 FWD and mCh-ARP3 REV and then cloned into pBABE-puro vector, linearized with BamHI and SalI, via Gibson assembly (New England BioLabs Inc).

Techniques: Expressing, Migration, Staining