Journal: bioRxiv

Article Title: The Ebola virus matrix protein clusters phosphatidylserine, a critical step in viral budding

doi: 10.1101/2021.06.08.447555

Figure Lengend Snippet: a Representative confocal images of fluorescently labeled (TopFluor® TMR-PS) GUVs with varying lipid compositions (red) following incubation with eVP40-Alexa88 (green). Colocalization between eVP40 and PS within GUVs was indicated by plot profile analyses of fluorescence signals between TopFluor® TMR-PS (red dotted line) and eVP40-Alexa488 (green solid line) performed at indicated open yellow lines in a and shown in b-d . b Plot profile analysis of eVP40-Alexa488 and control GUVs (DPPC:Cholesterol:0.2mol% TopFluor® TMR-PS), c Plot profile analysis of eVP40-Alexa488 and PS GUVs (DPPC:Cholesterol:0.2mol% TopFluor® TMR-PS:DPPS). * indicates overlap in fluorescence signals. d Plot profile analysis of eVP40-Alexa488 and PS+PI(4,5)P 2 GUVs (DPPC:Cholesterol:0.2mol% TopFluor® TMR-PS:DPPS:PI(4,5)P 2 ). PS: phosphatidylserine; DPPC: dipalmitoyl-phosphatidylcholine; DPPS: dipalmitoyl-phosphatidylserine.

Article Snippet: GFP-PLCδPH was a kind gift from Tamas Balla (NIH). pCAG-GPI-GFP was a gift from Anna Katerina Hadjantonakis (Addgene #32601). pEGFP-N3-Annexin A2 was a gift from Volker Gerke & Ursula Rescher (Addgene #10796). pCAGGS-FLAG-eVP40 (NR49337) and pcDNA3.1-eGP (NR-19814) were obtained from BEI Services. pCAGGS-TIM-1 was from Heinz Feldmann[ ].

Techniques: Labeling, Incubation, Fluorescence

Journal: bioRxiv

Article Title: The Ebola virus matrix protein clusters phosphatidylserine, a critical step in viral budding

doi: 10.1101/2021.06.08.447555

Figure Lengend Snippet: a Representative confocal images from live cell imaging of HEK293 expressing various GFP-fused proteins (GFP; green) following supplementation with TopFluor® TMR-PS (red). Solid white lines indicate where plot profile analysis was performed; scale bar= 10 µm. b-c Validation of ability to detect exogenously added fluorescently labelled PS within the inner leaflet of the plasma membrane of cells b Plot profile analysis of HEK293 cells expressing cytosolic GFP. c Plot profile analysis of HEK293 cells expressing the PS sensor GFP-LactC2 d-e Investigation of functionally distinct eVP40 proteins ability to bind to fluorescently labelled PS within the inner leaflet of the plasma membrane in living cells. d Plot profile analysis of HEK293 cells expressing GFP-WT-eVP40. e Plot profile analysis of HEK293 cells expressing GFP-K224A-eVP40 (PS-binding residue mutant). f Plot profile analysis of HEK293 cells expressing GFP-WE/A-eVP40 (oligomerization deficient mutant). TopFluor TMR-PS fluorescence signal intensity (red dotted line) and GFP fluorescence signal intensity (green solid line).

Article Snippet: GFP-PLCδPH was a kind gift from Tamas Balla (NIH). pCAG-GPI-GFP was a gift from Anna Katerina Hadjantonakis (Addgene #32601). pEGFP-N3-Annexin A2 was a gift from Volker Gerke & Ursula Rescher (Addgene #10796). pCAGGS-FLAG-eVP40 (NR49337) and pcDNA3.1-eGP (NR-19814) were obtained from BEI Services. pCAGGS-TIM-1 was from Heinz Feldmann[ ].

Techniques: Live Cell Imaging, Expressing, Binding Assay, Mutagenesis, Fluorescence

Journal: bioRxiv

Article Title: The Ebola virus matrix protein clusters phosphatidylserine, a critical step in viral budding

doi: 10.1101/2021.06.08.447555

Figure Lengend Snippet: a Representative 3D reconstructed confocal images of immobilized GUVs (DPPC:Cholesterol:DPPS:PI(4,5)P 2 :TopFluor® TMR-PS(red)). Left panel: GUVs incubated without eVP40-Alexa488. Right three panels: GUVs incubated with 1.25 µM eVP40-Alexa488 (green). b Index of correlation (Mander’s coefficient) between TopFluor® TMR-PS and eVP40-Alexa488 of different GUVs compositions incubated with 1.25 µM eVP40-Alexa488. Values are reported as mean ± s.d. A one-way ANOVA with multiple comparisons was performed; ****p<0.0001. c % TopFluor® TMR-PS quenching by eVP40 using GUVs (DPPC:Cholesterol:TopFluor®TMR-PS + increasing mol% of PS), 2.5% PI(4,5)P 2 was added to GUVs with 60% PS. Fluorescence spectra were recorded (Ex: 547 nm; Em: 550-600 nm); n=2. d Representative confocal images of HEK293 cells expressing various GFP - fused proteins (green) and supplemented with TopFluor® TMR-PS (red); scale bar= 10 µm. Yellow arrows indicate high intensity PS fluorescence regions e %PM with PS clusters = area of high intensity fluorescent PS clusters over total plasma membrane area from images in panel (d). Black bars are control proteins and blue bars are eVP40 proteins. Values are reported as mean ± s.d.; N>18, n=3; A one-way ANOVA was performed with multiple comparisons compared to the control GFP %PS clustering (***p=0.0007, **p=0.004). DPPC: dipalmitoyl-phosphatidylcholine; DPPS: dipalmitoyl-phosphatidylserine; GUVs: giant unilamellar vesicles; PS: phosphatidylserine; PM: plasma membrane.

Article Snippet: GFP-PLCδPH was a kind gift from Tamas Balla (NIH). pCAG-GPI-GFP was a gift from Anna Katerina Hadjantonakis (Addgene #32601). pEGFP-N3-Annexin A2 was a gift from Volker Gerke & Ursula Rescher (Addgene #10796). pCAGGS-FLAG-eVP40 (NR49337) and pcDNA3.1-eGP (NR-19814) were obtained from BEI Services. pCAGGS-TIM-1 was from Heinz Feldmann[ ].

Techniques: Incubation, Fluorescence, Expressing

Journal: bioRxiv

Article Title: The Ebola virus matrix protein clusters phosphatidylserine, a critical step in viral budding

doi: 10.1101/2021.06.08.447555

Figure Lengend Snippet: a GUVs of varying compositions were imaged prior to and following the addition of 1.25 µM eVP40-Alexa. eVP40-Alexa 488 enrichment ratios at the membrane of the GUVs was calculated by the ratio the Alexa488 fluorescence intensity at the GUV membrane / Alexa488 total fluorescence. Values are reported as mean ± s.d.; of n=3. A two-way ANOVA with multiple comparisons was performed. *p<0.0001. b Representative images of the step-wise image analysis workflow of quantifying PS clustering in living HEK293 cells expressing GFP-fused proteins using a custom ImageJ macro. scale bar= 10 µm. c Representative images from live cell imaging experiments of HEK293 cells expressing control GFP-fused proteins specific for the plasma membrane (GPI), and specific lipids, PS (LactC2) and PI(4,5)P 2 (PLCδ-PH). scale bar= 10µm.

Article Snippet: GFP-PLCδPH was a kind gift from Tamas Balla (NIH). pCAG-GPI-GFP was a gift from Anna Katerina Hadjantonakis (Addgene #32601). pEGFP-N3-Annexin A2 was a gift from Volker Gerke & Ursula Rescher (Addgene #10796). pCAGGS-FLAG-eVP40 (NR49337) and pcDNA3.1-eGP (NR-19814) were obtained from BEI Services. pCAGGS-TIM-1 was from Heinz Feldmann[ ].

Techniques: Fluorescence, Expressing, Live Cell Imaging

Journal: bioRxiv

Article Title: The Ebola virus matrix protein clusters phosphatidylserine, a critical step in viral budding

doi: 10.1101/2021.06.08.447555

Figure Lengend Snippet: a-c SPR demonstrates that eVP40 affinity to LUVs increases in relation to PS concentration. a Representative normalized sensorgram of His 6 -eVP40 binding to LUVs containing 1% PS indicating an apparent affinity of 2.5 µM. b Representative normalized sensorgram of His 6 -eVP40 binding to LUVs containing 11% PS indicating an apparent affinity of 0.65 µM. c Representative normalized sensorgram of His 6 -eVP40 binding to LUVs containing 22% PS indicating an apparent affinity of 0.18 µM. d-e PS concentration in LUVs enhances the ability of His 6 -eVP40 to oligomerize on membranes. d Representative western blot of chemical crosslinking performed on His 6 -WT-eVP40 following incubation with LUVs of varying PS content (detected by Mouse α-His antibody & HRP-Sheep α-Mouse). e Oligomerization capacity was determined from the western blot band density ratio of oligomers/(monomer + dimer) from chemical crosslinking experiments. A one-way ANOVA was performed with multiple comparisons compared to the control 0% PS LUVs control (30% PS *p= 0.021; 60% PS *p=0.017). n=3. Values are reported as mean ± s.d.; SPR: surface plasmon resonance; LUVs: large unilamellar vesicles; PS: phosphatidylserine; HRP: horseradish peroxidase.

Article Snippet: GFP-PLCδPH was a kind gift from Tamas Balla (NIH). pCAG-GPI-GFP was a gift from Anna Katerina Hadjantonakis (Addgene #32601). pEGFP-N3-Annexin A2 was a gift from Volker Gerke & Ursula Rescher (Addgene #10796). pCAGGS-FLAG-eVP40 (NR49337) and pcDNA3.1-eGP (NR-19814) were obtained from BEI Services. pCAGGS-TIM-1 was from Heinz Feldmann[ ].

Techniques: Concentration Assay, Binding Assay, Western Blot, Incubation, SPR Assay

Journal: bioRxiv

Article Title: The Ebola virus matrix protein clusters phosphatidylserine, a critical step in viral budding

doi: 10.1101/2021.06.08.447555

Figure Lengend Snippet: a CellTiter-Glo® viability results of HEK293 cells. a HEK293 cells were treated with fendiline for 24 hours (black line) and 48 hours (blue line) and viability was assessed as a % viability of control. b PS saturation analysis from lipidomic analysis (LC/MS/MS) of total lipids extracted from HEK293 cells treated with the indicated concentration of fendiline (48 hours). Values are normalized to DMSO control and are reported as mean ± s.d.; n=3; A one-way ANOVA was performed with multiple comparisons compared to the control DMSO. c PA level analysis from lipidomic analysis (LC/MS/MS) of total lipids extracted from HEK293 cells treated with 5 µM fendiline (48 hours). Values are normalized to DMSO control and are reported as mean ± s.d.; n=3; A two-tailed t-test was performed. d-e Analysis of PS plasma membrane localization in response to 24 hour fendiline treatment. d Representative confocal images from live cell imaging of HEK293 cells expressing GFP-LactC2 and treated with fendiline for 24 hours; scale bars= 10 µm. d Effect of fendiline on PS plasma membrane localization was calculated by the ratio of GFP fluorescence at the (plasma membrane intensity/intracellular intensity). Values are normalized to DMSO control and are reported as mean ± s.d.; N>15, n=3; A one-way ANOVA was performed with multiple comparisons compared to the DMSO control. **p=0.0045; ***p=0.0003. f N&B analysis of HEK293 cells expressing the control GFP. Analysis was performed at 48 hours post treatment (DMSO) to align with N&B analysis performed on experiments with HEK293 cells expressing GFP-LactC2 or GFP-eVP40 and treated with the control or fendiline. Left panel: Representative images from time-lapse (30 frames) of HEK293 expressing EGFP and treated with fendiline for 48 hours. scale bar = 5 µm. Middle panel: Brightness and Intensity plots for representative image. Right panel: Selection map correlating each pixel in the representative image to an oligomerization state (b value) (red: monomer). PS: phosphatidylserine; PA: phosphatidic acid; N&B: number & brightness analysis.

Article Snippet: GFP-PLCδPH was a kind gift from Tamas Balla (NIH). pCAG-GPI-GFP was a gift from Anna Katerina Hadjantonakis (Addgene #32601). pEGFP-N3-Annexin A2 was a gift from Volker Gerke & Ursula Rescher (Addgene #10796). pCAGGS-FLAG-eVP40 (NR49337) and pcDNA3.1-eGP (NR-19814) were obtained from BEI Services. pCAGGS-TIM-1 was from Heinz Feldmann[ ].

Techniques: Liquid Chromatography with Mass Spectroscopy, Concentration Assay, Two Tailed Test, Live Cell Imaging, Expressing, Fluorescence, Selection

Journal: bioRxiv

Article Title: The Ebola virus matrix protein clusters phosphatidylserine, a critical step in viral budding

doi: 10.1101/2021.06.08.447555

Figure Lengend Snippet: a CellTiter-Glo® viability results of Vero cells. Cells were treated with control or fendiline for 48 hours according to the BSL-4 infection model; d-1/0 (black line), e.d. (blue line) and e.o.d (gray line) and viability was assessed as a % viability of control. b-c Effect of fendiline on eVP40 PM localization in HEK293 cells after 24 hours of treatment. b Representative confocal images from live cell imaging experiments of HEK293 cells expressing EGFP-WT-eVP40 after 48 hours of fendiline treatment. scale bars= 10 µm. c Effect of fendiline on eVP40 PM localization was quantified by the ratio of EGFP fluorescence intensity at the PM / total GFP fluorescence intensity (and normalized to DMSO control). N>15, n=3. Values are reported as mean ± s.d. A one-way ANOVA with multiple comparisons was performed compared to the DMSO control. PM: plasma membrane.

Article Snippet: GFP-PLCδPH was a kind gift from Tamas Balla (NIH). pCAG-GPI-GFP was a gift from Anna Katerina Hadjantonakis (Addgene #32601). pEGFP-N3-Annexin A2 was a gift from Volker Gerke & Ursula Rescher (Addgene #10796). pCAGGS-FLAG-eVP40 (NR49337) and pcDNA3.1-eGP (NR-19814) were obtained from BEI Services. pCAGGS-TIM-1 was from Heinz Feldmann[ ].

Techniques: Infection, Live Cell Imaging, Expressing, Fluorescence

Journal: bioRxiv

Article Title: The Ebola virus matrix protein clusters phosphatidylserine, a critical step in viral budding

doi: 10.1101/2021.06.08.447555

Figure Lengend Snippet: a-c Effect of fendiline on eVP40 and mVP40 PM localization in HEK293 cells after 48 hours of treatment. a Representative confocal images from live cell imaging experiments of HEK293 cells expressing EGFP-WT-eVP40 (top panel) and EGFP-WT-mVP40 (bottom panel) after 48 hours of fendiline treatment. scale bars= 10 µm. Effect of fendiline on eVP40 (b) and mVP40 (c) PM localization was quantified by the ratio of EGFP fluorescence intensity at the PM / total EGFP fluorescence intensity (and normalized to DMSO control). N>15, n=3. Values are reported as mean ± s.d. A one-way ANOVA with multiple comparisons was performed compared to the DMSO control. d-e Analysis of eVP40 oligomerization in HEK293 cells in response to 48 hour fendiline treatment using N&B analysis. d Left panel: Representative images from time-lapse (30 frames) of HEK293 expressing EGFP-WT-eVP40 and treated with fendiline for 48 hours. scale bar = 5 µm. Middle panel: Brightness and Intensity plots for each representative image. Right panel: Selection map correlating each pixel in the representative image to an oligomerization state (b value) (red: monomer-hexamer, green: hexamer-12mer, blue: 12mer-24mer, pink: >24mer). e Average % pixel quantification from panel (d)= % of GFP-WT-eVP40 with brightness values corresponding to monomer-hexamer (∼1.-1.6), hexamer-12mer (∼1.6-2.0), 12mer-24mer (2.0-3.2) and >24mer (>3.2) over the total pixels within each image. Values are reported as mean ± s.d.; N≥9, n=3; A two-way ANOVA was performed with Dunnett’s multiple comparisons compared to the control DMSO % average pixels (**p=0.0035). PM: plasma membrane; N&B: Number & Brightness analysis.

Article Snippet: GFP-PLCδPH was a kind gift from Tamas Balla (NIH). pCAG-GPI-GFP was a gift from Anna Katerina Hadjantonakis (Addgene #32601). pEGFP-N3-Annexin A2 was a gift from Volker Gerke & Ursula Rescher (Addgene #10796). pCAGGS-FLAG-eVP40 (NR49337) and pcDNA3.1-eGP (NR-19814) were obtained from BEI Services. pCAGGS-TIM-1 was from Heinz Feldmann[ ].

Techniques: Live Cell Imaging, Expressing, Fluorescence, Selection

Journal: bioRxiv

Article Title: The Ebola virus matrix protein clusters phosphatidylserine, a critical step in viral budding

doi: 10.1101/2021.06.08.447555

Figure Lengend Snippet: a-d Functional budding assays assessed at 24 hours (a-b) and 48 hours (c-d) post treatment. a Representative western blot of budding assays performed at 24 hours. VLP samples (top panel) and cell lysate samples (bottom panel) collected from HEK293 cells and immunoblotted for eVP40 expression; GAPDH served as a loading control. eVP40 detected by (Rabbit α-eVP40 and HRP-Goat α-Rabbit); GAPDH detected by mouse α-GAPDH and HRP-Sheep α-Mouse) b Quantification of relative budding index at 24 hours post fendiline treatment. Relative budding index was determined by the western blot band density of eVP40 in the VLP fraction/(total eVP40 cell lysate + eVP40 VLP band density) and was normalized to the DMSO control. Cell lysate eVP40 band density was normalized to GAPDH band density prior to use in budding index quantification. n=3. Values are reported as mean ± s.d. A one-way ANOVA was performed with multiple comparisons compared to the DMSO control. c Representative western blot of budding assays performed at 48 hours. VLP samples (top panel) and cell lysate samples (bottom panel) collected from HEK293 cells and immunoblotted for eVP40 expression; GAPDH served as a loading control. eVP40 detected by (Rabbit α-eVP40 and HRP-Goat α-Rabbit); GAPDH detected by (Mouse α-GAPDH and HRP-Sheep α-Mouse) b Quantification of relative budding index at 48 hours post fendiline treatment. Relative budding index was determined by the western blot band density of eVP40 in the VLP fraction/(total eVP40 cell lysate + eVP40 VLP band density) and was normalized to the DMSO control. Cell lysate eVP40 band density was normalized to GAPDH band density prior to use in budding index quantification. n=3. Values are reported as mean ± s.d. A one-way ANOVA was performed with multiple comparisons compared to the DMSO control. (*p=0.0260) e-f SEM micrographs of HEK93 cells. e Representative micrographs of mock transfected HEK293 cells harvested after 48 hours of no treatment or DMSO treatment. f Representative micrographs of HEK293 cells expressing FLAG-eVP40 and harvested after 48 hours of no treatment, DMSO treatment, or the indicated concentration of fendiline. VLPs: virus like particles; SEM: scanning electron microscopy; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; HRP: horseradish peroxidase.

Article Snippet: GFP-PLCδPH was a kind gift from Tamas Balla (NIH). pCAG-GPI-GFP was a gift from Anna Katerina Hadjantonakis (Addgene #32601). pEGFP-N3-Annexin A2 was a gift from Volker Gerke & Ursula Rescher (Addgene #10796). pCAGGS-FLAG-eVP40 (NR49337) and pcDNA3.1-eGP (NR-19814) were obtained from BEI Services. pCAGGS-TIM-1 was from Heinz Feldmann[ ].

Techniques: Functional Assay, Western Blot, Expressing, Transfection, Concentration Assay, Electron Microscopy

Journal: bioRxiv

Article Title: The Ebola virus matrix protein clusters phosphatidylserine, a critical step in viral budding

doi: 10.1101/2021.06.08.447555

Figure Lengend Snippet: a-c TEM analysis of eVLP morphology. a Representative transmission electron micrographs of eVLPs purified from HEK293 cells expressing FLAG-eVP40 and eGP following 48 hours of DMSO (left panel) or 5 µM fendiline treatment (right panel). b Quantification of eVLP length (µm) of DMSO-derived eVLPs (black) and fendiline-derived eVLPs (blue). N>50, n=3. Values are reported as mean ± s.d. A two-tailed t-test was performed (**p=0.0139). c Quantification of eVLP diameter (nm) of DMSO-derived eVLPs (black) and fendiline-derived eVLPs (blue). N>50, n=3. Values are reported as mean ± s.d. A two-tailed t-test was performed (*p=0.0430). d-e Fluorescence based DiI TIM-1 dependent entry assay. d Representative confocal images from the DiI-entry assay comparing entry of eVLPs produced from DMSO (top panel) and fendiline-treated HEK293 cells (bottom panel) into target cells (HEK293 cells transiently expressing increasing amounts of TIM-1; 0.0 µg, 0.5 µg, 1.0 µg). A stack of 10 frames was acquired for each image. DiI (initially red) was recolored to yellow for easier observation in print; blue (Hoechst 3342 stain); scale bar = 10 µm. e Quantification of eVLP entry was performed by calculating the total number of DiI punctate / the total number of DiI-positive cells. Three images from each z-stack was quantified. N=9, n=3. Values are reported as mean ± s.d. A one-way ANOVA was performed with multiple comparisons against the 0.0 µg TIM-1 condition for both DMSO- and fendiline derived eVLPs.(****p<0.0001; **p=0.0093). eVLP: entry-competent viral like particles; TEM: transmission electron microscopy; TIM-1: t-cell immunoglobulin receptor-1; eVLPs: entry-competent VLPs; eGP: Ebola glycoprotein; DiI: 1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindocarbocyanine Perchlorate.

Article Snippet: GFP-PLCδPH was a kind gift from Tamas Balla (NIH). pCAG-GPI-GFP was a gift from Anna Katerina Hadjantonakis (Addgene #32601). pEGFP-N3-Annexin A2 was a gift from Volker Gerke & Ursula Rescher (Addgene #10796). pCAGGS-FLAG-eVP40 (NR49337) and pcDNA3.1-eGP (NR-19814) were obtained from BEI Services. pCAGGS-TIM-1 was from Heinz Feldmann[ ].

Techniques: Transmission Assay, Purification, Expressing, Derivative Assay, Two Tailed Test, Fluorescence, Produced, Staining, Electron Microscopy

Journal: bioRxiv

Article Title: The Ebola virus matrix protein clusters phosphatidylserine, a critical step in viral budding

doi: 10.1101/2021.06.08.447555

Figure Lengend Snippet: a Ebola virus-like particles (eVLPs) were produced in DMSO treated HEK293 cells expressing FLAG-eVP40 and eGP constructs which were then collected, purified, and melted while measuring the loss in polarized light absorption at 220 nm. b Prior to melting, the circular dichroism spectra of DMSO VLPs was measured alongside the absorbance and detector (hv) signals. c Ebola VLPs were produced in 5 µM fendiline treated HEK293 cells using the same FLAG-eVP40 and eGP constructs which were then collected, purified and melted while measuring the loss in polarized light absorption at 220 nm. d Prior to melting, the circular dichroism spectra of Fendiline VLPs was measured alongside the absorbance and detector (hv) signals.

Article Snippet: GFP-PLCδPH was a kind gift from Tamas Balla (NIH). pCAG-GPI-GFP was a gift from Anna Katerina Hadjantonakis (Addgene #32601). pEGFP-N3-Annexin A2 was a gift from Volker Gerke & Ursula Rescher (Addgene #10796). pCAGGS-FLAG-eVP40 (NR49337) and pcDNA3.1-eGP (NR-19814) were obtained from BEI Services. pCAGGS-TIM-1 was from Heinz Feldmann[ ].

Techniques: Produced, Expressing, Construct, Purification

Journal: Nature Communications

Article Title: Intracellular hydrogelation preserves fluid and functional cell membrane interfaces for biological interactions

doi: 10.1038/s41467-019-09049-5

Figure Lengend Snippet: Examination of membrane protein lateral mobility on GCs. a Representative trajectories from CD80-GFP fluorescence tracking on different GCs and control cells examined by TIRF microscopy. b Diffusion coefficients of CD80-GFP were calculated from the TIRF fluorescence tracking data. Error bars represent geometric mean with 95% confidence interval. c Representative images showing recovery of CD80-GFP fluorescence in 4 wt% GC following photobleaching. Red rectangles indicate the photobleached area of interest. Scale bars = 2 μm. d A schematic illustration of membrane proteins with different sizes of intracellular domains, including CD80-GFP, EGFP-GPI, TfR, Lyn-GFP, and EGFR-GFP, which were assessed for their lateral mobility on GCs. e Recovery kinetics of CD80-GFP, EGFP-GPI, TfR, Lyn-GFP and EGFR-GFP on GCs and control cells assessed by FRAP. For GCs with 4 to 20 wt% hydrogel densities, all examined membrane proteins had similar lateral mobility as on live cells. Error bars represent geometric mean with 95% confidence interval. f Membrane protein mobility on 40 wt% GCs relative to their corresponding mean mobility on live cells. Error bars represent geometric mean with 95% confidence interval ( n = 7–12). Statistical analysis was performed using a two-tail Student t test, **** p <0.001

Article Snippet: HeLa cells expressing EGFP-GPI (Addgene, pCAG: GPI-GFP, #32601), CD80-GFP (Sino Biological Inc., pCMV3-mCD80-C-GFPSpark, MG50446-ACG), Transferrin Receptor (TfR) (Sino Biological Inc., pCMV3-hTfR-C-DDK (flag) tag, HG11020-CF), Lyn-GFP (Sino Biological Inc., pCMV3-hLyn-C-GFPSpark, HG10829-ACG), EGFR-GFP (Sino Biological Inc., pCMV3-mEGFR-C-GFPSpark, MG51091-ACG), and GFP-beta-actin (Sino Biological Inc., pCMV3-hbeta-actin-N-GFPSpark, HG10962-ANG) were prepared via transfection.

Techniques: Fluorescence, Microscopy, Diffusion-based Assay

Journal: Nature

Article Title: Subcellular transcriptomes and proteomes of developing axon projections in cerebral cortex

doi: 10.1038/s41586-018-0847-y

Figure Lengend Snippet: The sub-proteome |P|GC of proteins detected by mass-spec in purified cortical callosal GCs from P3 mouse brains electroporated with membrane-GFP at E15, visualized in a protein-interaction network according to the STRING database. Links indicate known interactions. Colors highlight identifiable protein complexes, as indicated in insets.

Article Snippet: DNA constructs The following plasmid DNA expression constructs were used for in utero electroporations: membrane-GFP (used in , , and ; as well as , , and ) is a GFP-GPI fusion construct kindly provided by Anna-Katerina Hadjantonakis 40 (Memorial Sloan Kettering); membrane-RFP and nuclear-GFP (used in ) corresponds to a 2A bi-cistronic gene encoding myristoylated-tdTomato and Histone2B-GFP from plasmid pCAG-TAG (Addgene plasmid # 26771), generously provided by Shankar Srinivas 4 (Oxford).

Techniques: Mass Spectrometry, Purification

Journal: Nature

Article Title: Subcellular transcriptomes and proteomes of developing axon projections in cerebral cortex

doi: 10.1038/s41586-018-0847-y

Figure Lengend Snippet: a, Selective labeling of upper layer callosal projection neurons with nuclear-GFP (green) and membrane-RFP (red) by in utero electroporation. Nascent callosal projection at postnatal day 3 displays ipsilateral somata with green nuclei, and trans-hemispheric axons with red GCs; scale bar is 600 µm for main panel, 100 μm for insets. b, Schematic of subcellular RNA-proteome mapping workflow: neuron labeling by in utero electroporation at E15; postnatal day 3 harvesting; cell dissociation of ipsilateral hemispheres for sorting of GFP+ cell bodies; contralateral hemispheres undergo subcellular fractionation to isolate GC fraction for RFP+ GC sorting. c, FACS plots with gates for GFP+ somata and RFP+ GC used to collect trans-hemispheric GCs and their parent cell bodies; RNA and protein extracted from sorted GC and soma samples were analyzed by RNA-seq and mass-spec to yield paired measurements of sub-transcriptomes |R|GC and |R|soma, and sub-proteomes |P|GC and |P|soma, respectively. GC-to-soma ratios of mRNA (ΛR) and protein (ΛP) from corresponding paired sub-transcriptome and sub-proteome measurements were calculated for each gene. ΛR and ΛP correlate and map sub-proteomes and sub-transcriptomes within single neuronal projections directly from brain.

Article Snippet: DNA constructs The following plasmid DNA expression constructs were used for in utero electroporations: membrane-GFP (used in , , and ; as well as , , and ) is a GFP-GPI fusion construct kindly provided by Anna-Katerina Hadjantonakis 40 (Memorial Sloan Kettering); membrane-RFP and nuclear-GFP (used in ) corresponds to a 2A bi-cistronic gene encoding myristoylated-tdTomato and Histone2B-GFP from plasmid pCAG-TAG (Addgene plasmid # 26771), generously provided by Shankar Srinivas 4 (Oxford).

Techniques: Labeling, In Utero, Electroporation, Fractionation, RNA Sequencing Assay, Mass Spectrometry

Journal: Nature

Article Title: Subcellular transcriptomes and proteomes of developing axon projections in cerebral cortex

doi: 10.1038/s41586-018-0847-y

Figure Lengend Snippet: a, Schematic of two-color sorting to separate GFP GCs from RFP GCs. 1. Brains from a GFP mouse and an RFP mouse were homogenized together. 2. Subcellular fractionation of the homogenate yields a GC fraction, containing the red and green GCs in a suspension of diluted cytosol. 3. This suspension of mixed GCs was sorted on a customized small-particle fluorescence flow cytometer to collect pure green GCs from the mix. b, Analysis of GC fraction (GC fr.) RNA and protein marker enrichment versus the starting homogenate (input). Top: native gel electrophoresis shows de-enriched presence of large (28S) and small (18S) ribosomal subunit rRNA in GC fraction. Middle: RT-PCR detects β-Actin mRNA (ubiquitous) but not GFAP mRNA (progenitor and glial marker) from GC fraction. Bottom: Western blot detects enrichment of GAP43 (GC protein marker) and depletion of MAP2 (somato-dendritic protein marker) in GC fraction. c-d, GC protection assays with non membrane permeable degrading enzymes (RNAse in c and protease in d) to test GC integrity and GC-specific membrane encapsulation of RNA and proteins in isolated GCs (see schematic Extended Data Figure 1a). Treatment with enzyme plus detergent, but neither alone, completely abolishes RNA and protein signal from GC fractions. Signals persisting in treatments with enzyme alone (lanes 3) correspond to RNA and protein encapsulated (protected) by GC membrane, and correspond to the specific molecular content of isolated GCs. Treatment with protease alone has no effect on signal from GAP43, a known GC marker, confirming GC-specificity. Conversely, signal from GM130, a Golgi matrix protein known to be excluded from GCs, is abolished with protease treatment alone, indicating no non-specific encapsulation in GCs. Reduced presence of both actin and rRNA in samples treated with enzyme alone is consistent with their ubiquitous presence in both GCs and elsewhere in the homogenate. e, Small particle sorter plot of forward- and side-scatter of GC sample (blue) overlaid on plot of size-standard beads (grey) for size comparison. Isolated GCs are submicron particles with a size range centered on 0.5 µm. f, FACS plot of the mixed GFP and RFP GC suspension (as schematized in a), showing separation of GCs into two monochrome red and green populations. Collection gate used to isolate pure green GCs is indicated by the shaded green square. g, Collected green GCs from f were re-sorted and reveal specific purification of only GFP GCs, demonstrating that exceptionally pure labeled GCs can be isolated as individual “singlet” GCs from a heterogenous sample using GC sorting directly from brain.

Article Snippet: DNA constructs The following plasmid DNA expression constructs were used for in utero electroporations: membrane-GFP (used in , , and ; as well as , , and ) is a GFP-GPI fusion construct kindly provided by Anna-Katerina Hadjantonakis 40 (Memorial Sloan Kettering); membrane-RFP and nuclear-GFP (used in ) corresponds to a 2A bi-cistronic gene encoding myristoylated-tdTomato and Histone2B-GFP from plasmid pCAG-TAG (Addgene plasmid # 26771), generously provided by Shankar Srinivas 4 (Oxford).

Techniques: Fractionation, Fluorescence, Flow Cytometry, Marker, Nucleic Acid Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Western Blot, Isolation, Purification, Labeling

Journal: Nature

Article Title: Subcellular transcriptomes and proteomes of developing axon projections in cerebral cortex

doi: 10.1038/s41586-018-0847-y

Figure Lengend Snippet: a, Volcano plot of GC-soma RNA mapping, ΛR (log2) values are plotted for each transcript versus statistical significance (-log P-value). Significance thresholds indicate Benjamini-Hochberg False Discovery Rate (FDR) of 0.05. Transcripts are colored by mRNA class as indicated in b. Example transcripts from each class are labeled and further verified in c and d. Full transcript values are listed in Supplementary Table 4. b, legend and schematics of mRNA classes based on known mTOR dependence: mRNAs containing a TOP motif are mTOR-dependent (green), schema indicates direct binding to LARP1 and mTOR; mRNAs containing Internal Ribosome Entry Sites (IRES) or lacking poly-A tails are mTOR-independent (blue); canonical mRNAs that undergo cap-dependent translation (grey) display moderate responses to mTOR. c, Verification of RNA-seq mapping values (x-axis) with qPCR measurements (y-axis). Error bars show SEM, n=3. The two data sets cross-validate with correlation coefficient R2=0.736. d, Single-molecule RNA chromogenic in situ hybridization (ISH, red) of two TOP transcripts: Rack1 (non-ribosomal TOP) and Rplp0 (ribosomal TOP), compared to a control transcript Ppib (soma-mapped canonical) in callosal projection neurons. Neurons were labeled with mem-GFP (green) via in utero electroporation at E15, cultured at P0, fixed and hybridized at DIV3. DNA in nuclei stained with DAPI (blue). Soma and GC closeups shown in insets as overlays of transcript (red) with mem-GFP (green) in upper rows, or with traced GC outlines in lower rows. Five example GCs are shown per sample to capture the representative range. Scale bars indicate 10 µm.

Article Snippet: DNA constructs The following plasmid DNA expression constructs were used for in utero electroporations: membrane-GFP (used in , , and ; as well as , , and ) is a GFP-GPI fusion construct kindly provided by Anna-Katerina Hadjantonakis 40 (Memorial Sloan Kettering); membrane-RFP and nuclear-GFP (used in ) corresponds to a 2A bi-cistronic gene encoding myristoylated-tdTomato and Histone2B-GFP from plasmid pCAG-TAG (Addgene plasmid # 26771), generously provided by Shankar Srinivas 4 (Oxford).

Techniques: Labeling, Binding Assay, RNA Sequencing Assay, Chromogenic In Situ Hybridization, In Utero, Electroporation, Cell Culture, Staining

Journal: Nature

Article Title: Subcellular transcriptomes and proteomes of developing axon projections in cerebral cortex

doi: 10.1038/s41586-018-0847-y

Figure Lengend Snippet: a, Biochemical analysis of GC enrichment of mTOR pathway proteins and controls, shown in triplicate western blots of homogenate (input) and GC fraction (GC fr.) pairs, derived from six independent preps. GC marker GAP43 is positive control for enrichment, Golgi marker Gm130 is negative control. b, Quantification of GC enrichment blots in a expressed as ratios of GC fr. signal over input signal, normalized to the corresponding GAP43 ratio (marked by horizontal line). Error bars indicate SEM, n≥3 litters. TSC1, Rictor, and Lamp1 are present in GCs comparable to actin and tubulin, while mTOR, LARP1, and Raptor display high GC enrichment comparable to GC marker GAP43. c, Closeups of GCs from callosal projection neurons immunostained for endogenous mTOR pathway proteins (red in overlays, heat mapped in underlying panels). Five example GCs are shown per sample to capture the representative range. Neurons were labeled via in utero electroporation at E15 with membrane-GFP (green in overlays, outlined in underlying panels), cultured at P0, fixed and stained at DIV 3. mTOR, LARP1, TSC1, and Raptor (mTORC1 marker) appear in dense local foci within GCs. Rictor (mTORC2 marker) and Lamp1 (lysosome marker) appear in fine granules distinct from GC foci. Bar (lower right) indicates heat-map color range, as well as 10 µm scale.

Article Snippet: DNA constructs The following plasmid DNA expression constructs were used for in utero electroporations: membrane-GFP (used in , , and ; as well as , , and ) is a GFP-GPI fusion construct kindly provided by Anna-Katerina Hadjantonakis 40 (Memorial Sloan Kettering); membrane-RFP and nuclear-GFP (used in ) corresponds to a 2A bi-cistronic gene encoding myristoylated-tdTomato and Histone2B-GFP from plasmid pCAG-TAG (Addgene plasmid # 26771), generously provided by Shankar Srinivas 4 (Oxford).

Techniques: Western Blot, Derivative Assay, Marker, Positive Control, Negative Control, Labeling, In Utero, Electroporation, Cell Culture, Staining

Journal: Nature

Article Title: Subcellular transcriptomes and proteomes of developing axon projections in cerebral cortex

doi: 10.1038/s41586-018-0847-y

Figure Lengend Snippet: a, Electroporation of callosal projection neurons with GFP and genetic payloads at E15, fixation and analysis at P3. Control electroporations (left column, grey in quantifications) show soma migration into upper layers (middle row insets, examples from four brains), and callosal projections well into the contralateral cortex (bottom insets, examples from four brains). Electroporation with dominant negative PI3 kinase expression construct (PI3K-DN, middle column, green in quantifications) results in hindered migration of somata, and failure of callosal axon growth. Electroporation of Cre expression construct in mice with homozygous floxed-mTOR alleles for conditional mTOR gene deletion (right column, Cre + mTORfl/fl, blue in quantifications) resultes specifically in failure of callosal axon growth. Scale bars indicate 100 µm. b, Quantification of the location of the electroporation field shows comparable mediolateral electroporation positions across all samples. Plotted are histograms of binned GFP intensities along the tangential axis of the ipsilateral cortex ending at the midline. c, Quantification of extent of migration, with percentage of somata in layers II/III (dark colors) vs. somata still en route in deeper layers (light colors). Inhibiting PI3K signaling (green) interferes with migration, while acute mTOR deletion (blue) does not significantly affect migration. d, Quantification of callosal axon extension showing that PI3K inhibition (green) as well as knockout of mTOR (blue) disrupt the formation of axon projection across the corpus callosum. Plotted are binned GFP intensity histograms within the corpus callosum from ipsilateral, through the midline (indicated dotted line) to contralateral side. All error bars show SEM, n=4 mice.

Article Snippet: DNA constructs The following plasmid DNA expression constructs were used for in utero electroporations: membrane-GFP (used in , , and ; as well as , , and ) is a GFP-GPI fusion construct kindly provided by Anna-Katerina Hadjantonakis 40 (Memorial Sloan Kettering); membrane-RFP and nuclear-GFP (used in ) corresponds to a 2A bi-cistronic gene encoding myristoylated-tdTomato and Histone2B-GFP from plasmid pCAG-TAG (Addgene plasmid # 26771), generously provided by Shankar Srinivas 4 (Oxford).

Techniques: Electroporation, Migration, Dominant Negative Mutation, Expressing, Construct, Inhibition, Knock-Out