Journal: bioRxiv

Article Title: Endocytic vacuole formation by WASH-mediated endocytosis

doi: 10.1101/2021.06.18.448076

Figure Lengend Snippet: (A) HaCaT cells were seeded on HPV16-AF568 bound to ECM, treated with 10 µg/ml cytoD or DMSO and unroofed. Fluorescent virus particles imaged by spinning disk microscopy and depicted in the small insets were correlated with structures identified in TEM images of platinum/carbon replicas of unroofed membranes. Arrowheads indicate endocytic pits. (B) Depicted is the percentage of particles associated with the indicated structure ± variation between the membranes (DMSO: 134 viruses/ 7 membranes; cytoD: 101 viruses/5 membranes). Of note, about 20-25% of particles were not associated with any prominent structure. (C) HeLa ATCC cells were infected with HPV16-GFP in presence of cytoD. Infection was scored by flow cytometry and normalized to DMSO treated controls. The mean of three experiments ± SD is shown. (D) HeLa ATCC cells treated with 10 µg/ml CytoD or left untreated (ctrl.) were infected with HPV16-GFP and processed for ultra-thin section TEM at 6 h p.i.. All scale bars are 100 nm.

Article Snippet: HeLa ATCC cells (2-3 x 10 5 cells) were seeded in 6 cm dishes.

Techniques: Virus, Microscopy, Infection, Flow Cytometry

Journal: bioRxiv

Article Title: Endocytic vacuole formation by WASH-mediated endocytosis

doi: 10.1101/2021.06.18.448076

Figure Lengend Snippet: (A) HeLa ATCC cells were infected with HPV16-GFP, subjected to immunogold labeling of actin on ultra-thin cryosections, and analyzed by TEM. Asterisks indicate HPV16 in endocytic pits. Scale bars are 100 nm. (B) HeLa ATCC cells were transfected with lifeact-EGFP, infected with HPV16-AF594 and imaged by live cell TIRF-M at 1 h p.i.. Movies were acquired with 0.5 Hz frame rate for 5 min. HPV16 entry events were identified manually after background subtraction and filtering. The yellow box indicates the virus entry event shown as a kymograph (right) and intensity profile (C). Scale bar is 2 µm. (C) Plotted are the intensity profiles of HPV16 and lifeact (light red/green) as well as moving averages (intense red/green). Values are depicted relative to the half time of virus loss (t = 0). The time points of the onset of actin polymerization (1) and of virus signal loss (2) from the cell surface, as well as of actin peak intensity (3) and of completion of virus uptake (4) were determined manually. (D) Time points were determined for 21 virus entry events as indicated in (C), averaged and are depicted as box plots.

Article Snippet: HeLa ATCC cells (2-3 x 10 5 cells) were seeded in 6 cm dishes.

Techniques: Infection, Labeling, Transfection, Virus

Journal: bioRxiv

Article Title: Endocytic vacuole formation by WASH-mediated endocytosis

doi: 10.1101/2021.06.18.448076

Figure Lengend Snippet: Branched actin polymerization mediated HPV16 endocytosis (A) HeLa ATCC cells were infected with HPV16-GFP or VSV-GFP in presence of the formin inhibitor SMIFH2. Infection was scored by flow cytometry, normalized to solvent treated controls and is depicted as mean ± SD (HPV16: n = 3, VSV: n = 4). (B) HeLa ATCC cells transfected with Scar-W-GFP or Scar-WA-GFP were infected with HPV16-RFP. Infection of transfected cells was analyzed by microscopy, normalized to Scar-W-GFP control cells and is depicted as the mean of three experiments ± SD. (C) HeLa Kyoto cells were depleted of Arp3 and infected with HPV16-GFP, VV-GFP or SFV. Infection was scored by automated microscopy or flow cytometry, normalized to cells transfected with a non-targeting control siRNA (ctrl.) and is depicted as mean ± SD (HPV16/SFV: n = 3, VV: n = 4). (D) Arp3 depletion was followed by infection with HPV16-pHrodo and live cell spinning disk microscopy at 6 h p.i.. Shown are average intensity projections of the HPV16-pHrodo signal with cell outlines (yellow), scale bar is 25 µm. Virus signal intensities per cell were measured using a CellProfiler pipeline and normalized to ctrl. (n = 4, mean ± SD). Knock down levels were analyzed by Western blotting against Arp3.

Article Snippet: HeLa ATCC cells (2-3 x 10 5 cells) were seeded in 6 cm dishes.

Techniques: Infection, Flow Cytometry, Solvent, Transfection, Microscopy, Control, Virus, Knockdown, Western Blot

Journal: bioRxiv

Article Title: Endocytic vacuole formation by WASH-mediated endocytosis

doi: 10.1101/2021.06.18.448076

Figure Lengend Snippet: (A) HeLa Kyoto cells were depleted of WASH and infected with HPV16-GFP or VV-GFP. Infection was determined by automated microscopy and flow cytometry and normalized to cells transfected with a control siRNA (ctrl.) (n = 4, mean ± SD.) (B) After depletion of WASH or Arp3, cells were infected with HPV16-pHrodo and imaged live by spinning disk microscopy at 6 h p.i.. Shown are average intensity projections of the HPV16-pHrodo signal with cell outlines (yellow), scale bar is 25 µm. The intensity of virus signal per cell was normalized to ctrl. and is depicted as the mean of three experiments ± SD. (C) EGFP (ctrl.) or EGFP-WASH were expressed in NIH-3T3 wild type and WASH KO cells. Infection with HPV16-RFP was scored by flow cytometry and normalized to wild type cells expressing EGFP (ctrl.). Values are depicted as the mean ± SD (n = 3). (D) NIH-3T3 wild type and WASH KO infected with HPV16-GFP were subjected to ultra-thin section TEM at 6 h p.i.. The number of virus filled plasma membrane invaginations was determined for 43 cells per cell line in two independent experiments. Total pit numbers were normalized to wild type cells and are depicted as mean ± SD. Scale bars are 100 nm. (E) HeLa ATCC cells transfected with EGFP-WASH were infected with HPV16-AF647. Cells were imaged by live cell TIRF-M at 1 h p.i.. Movies were acquired with 0.5 Hz frame rate for 5 min. HPV16 entry events were identified manually after background subtraction and filtering. Shown is a kymograph of the virus entry event highlighted by the yellow box, and the corresponding EGFP-WASH signal. Scale bar is 2 µm. (F) Intensity profiles of HPV16 and WASH (light red/green) as well as moving averages (intense red/green) of the virus entry event shown in (E) depicted relative to the half time of virus loss (t = 0). (G) HeLa ATCC cells were transfected with EGFP-WASH and infected with HPV16. At 6 h p.i., cells were subjected to immunogold labeling of GFP (WASH, 15 nm gold) and actin (10 nm gold) on ultra-thin cryosections analyzed by TEM. Asterisks indicate HPV16 particles, black and white arrowheads indicate WASH staining and the membrane, respectively. Scale bars are 100 nm.

Article Snippet: HeLa ATCC cells (2-3 x 10 5 cells) were seeded in 6 cm dishes.

Techniques: Infection, Microscopy, Flow Cytometry, Transfection, Control, Virus, Expressing, Clinical Proteomics, Membrane, Labeling, Staining

Journal: bioRxiv

Article Title: Endocytic vacuole formation by WASH-mediated endocytosis

doi: 10.1101/2021.06.18.448076

Figure Lengend Snippet: (A) HeLa Kyoto cells were depleted of Vps26a, Vps29, SNX2 or CtBP1 and infected with HPV16-GFP. Infection was scored by automated microscopy and normalized to ctrl. (n = 3, mean ± SD). (B) Cells were depleted of Arp3 or SNX2 and infected with HPV16-pHrodo followed by live cell imaging at 6 h p.i.. Shown are average intensity projections of the HPV16-pHrodo signal acquired by spinning disk microscopy. Cell outlines in yellow were manually created with brightfield images, scale bar is 25 µm. The intensity of virus signal per cell was normalized to ctrl. and is shown as mean ± SD (SNX2: n = 3, Arp3: n = 2). (C) HeLa ATCC cells were transfected with EGFP-SNX2, infected with HPV16-AF647 and imaged live by TIRF-M at 1 h p.i.. Movies were acquired with 0.5 Hz frame rate for 5 min. HPV16 entry events were identified manually after background subtraction and filtering. Shown is a kymograph of the virus entry event highlighted with the yellow box and the corresponding EGFP-SNX2 signal. Scale bars is 2 µm. (D) Intensity profiles of HPV16 and SNX2 (light red/green) as well as moving averages (intense red/green) of the virus entry event shown in (C) depicted relative to the half time of virus loss (t = 0). (E) HeLa ATCC cells were transfected with EGFP-SNX2, infected with HPV16 and subjected to immunogold labeling of EGFP-SNX2 on ultra-thin cryosections analyzed by TEM at 6 h p.i.. Black and white arrowheads indicate SNX2 staining and the membrane, respectively, asterisks mark virus particles. Scale bars are 100 nm.

Article Snippet: HeLa ATCC cells (2-3 x 10 5 cells) were seeded in 6 cm dishes.

Techniques: Infection, Microscopy, Live Cell Imaging, Virus, Transfection, Labeling, Staining, Membrane