Journal: Nature methods

Article Title: Nuclear pores as versatile reference standards for quantitative superresolution microscopy

doi: 10.1038/s41592-019-0574-9

Figure Lengend Snippet: (a) Representative confocal x-z and (b) x-y image of the Nup96-GFP cell line. Green: Nup96-GFP, magenta: membranes (DiD). (c) EM density of the nuclear pore complex with C-termini of Nup96 indicated in red. (d) Side view and (e) top view schematic. (f) Widefield, (g) confocal and (h) airy scan images of Nup96-GFP. (i) Raw STED image of Nup96-GFP labeled with an AberriorStar635P-coupled anti-GFP nanobody. Resolution estimates based on Fourier power spectra for f-i can be found in . (j) Widefield expansion microscopy image of Nup96-GFP labeled with an Atto488-coupled anti-GFP nanobody. (k) As before, but imaged using structured illumination. Estimates of the expansion factor based on the analysis of the ring diameters can be found in . (l) As before, but imaged using SRRF. (m) SMLM image of Nup96-mMaple, (n, o) SMLM of Nup96-SNAP labeled with BG-AF647 in GLOX/MEA. (p, q) Dual-color SMLM image of Nup96-SNAP labeled with BG-AF647 (red) and WGA-CF680 (cyan) in GLOX/MEA. (r, s) Corners of the NPC can be used as a resolution target in x,y (r) and z (s). Resolution estimates based on Fourier Ring Correlation for m-q can be found in . Representative images of one (j-l) , two (a,b,i), three (p-s) , four (f-h,n,o) or six (m) independent experiments are shown. Scale bars 10 µm (b) , 1 µm (f-n,p) and 100 nm (o,q,r,s).

Article Snippet: Fixed cells were then further permeabilized using 0.2% Triton X-100 in PBS for 10 min, followed by two more washing steps in PBS for 5 min each before blocking in 2% (w/v) BSA in PBS for 1 h. Binding of primary rabbit anti-GFP antibodies (Cat#598; MBL International) to Nup96-GFP fusion proteins was achieved by placing the coverslips overnight upside-down onto a drop of primary antibody solution (diluted 1:250 in PBS containing 2% [w/v] BSA) at 4 °C.

Techniques: Labeling, Microscopy

Journal: Nature methods

Article Title: Nuclear pores as versatile reference standards for quantitative superresolution microscopy

doi: 10.1038/s41592-019-0574-9

Figure Lengend Snippet: (a-d) Workflow. (a) All NPCs in a cell are automatically segmented. (b) We fit a circle to the localizations and reject localizations outside a ring as background localizations. (c) We rotate the localizations to optimally fit an eightfold-symmetric template and count the number of slices that contain at least one localization. (d) We fit the histogram of the number of corners with a probabilistic model to directly obtain the absolute ELE. The statistical error is estimated by bootstrapping with 20 re-sampled data sets. (e-h) Gallery of NPCs. (e) Nup96-GFP labeled with an anti-GFP nanobody coupled to AF647. (f) Nup96-SNAP labeled with BG-AF647. (g) Nup96-Halo labeled with chloroalkane-AF647. (h) Nup96-mMaple . The numbers indicate the numbers of visible corners the algorithm detected. (i) Effective labeling efficiencies for various cell lines and ligands. Bars denote the mean, error bars the standard deviation and individual data points measurements of a single cell. These data are derived from N biologically independent experiments, n C imaged cells and n NPC analyzed NPCs: GFP-NB-Q-AF647: N = 2, n C = 6, n NPC = 2913; GFP-NB-Q-CF680: N = 2, n C = 5, n NPC = 1805; GFP-NB-X4-AF647: N = 2, n C = 9, n NPC = 4303; GFP-NB-X4-CF680: N = 2, n C = 6, n NPC = 2011; GFP-NB-S-AF647: N = 2, n C = 4, n NPC = 8768; GFP-NB-S-AF647 (2y): N = 2, n C = 3, n NPC = 1000; GFP-Antibody: N = 3, n C = 14, n NPC = 7380; SNAP-AF647: N = 4, n C = 11, n NPC = 5372; Halo-Cy5: N = 5, n C = 14, n NPC = 5967; Halo-O2-AF647: N = 2, n C = 5, n NPC = 1393; Halo-O4-AF647: N = 2, n C = 6, n NPC = 3395; Halo-PAJF549: N = 3, n C = 17, n NPC = 4066; mMaple: N = 6, n C = 16, n NPC = 8146; mMaple live: N = 3, n C = 6, n NPC = 1343; Example images for all labels can be found in , and imaging conditions are listed in and . Representative images of two (e,g) , four (a,f) or six (h) independent experiments are shown. Scale bars 1 µm (a) and 100 nm (e-h) . *labeled in live cells, imaged after fixation. **measured on Nup107-GFP.

Article Snippet: Fixed cells were then further permeabilized using 0.2% Triton X-100 in PBS for 10 min, followed by two more washing steps in PBS for 5 min each before blocking in 2% (w/v) BSA in PBS for 1 h. Binding of primary rabbit anti-GFP antibodies (Cat#598; MBL International) to Nup96-GFP fusion proteins was achieved by placing the coverslips overnight upside-down onto a drop of primary antibody solution (diluted 1:250 in PBS containing 2% [w/v] BSA) at 4 °C.

Techniques: Labeling, Standard Deviation, Derivative Assay, Imaging

Journal: Nature methods

Article Title: Nuclear pores as versatile reference standards for quantitative superresolution microscopy

doi: 10.1038/s41592-019-0574-9

Figure Lengend Snippet: (a-d) Counting in diffraction limited microscopy. (a) Confocal image of the reference protein Nup96-GFP with the majority of nuclear pores resolved. (b) Confocal image of the target protein Nup107-GFP imaged with the same microscope settings. (c) Histograms of intensities of local maxima (see ) for the reference and target structures together with Gaussian fit to determine the mean intensity values. (d) Mean intensity values for several reference and target cells. These values show a small variation and are similar for reference (〈 I ref 〉 1552 = 55 ADU, N = 1, n C = 8, n R = 10104) and target complex (〈 I tar 〉= 1603 ± 77 ADU, N = 1, n C = 6, n T = 7178). (e-h) Counting with SMLM. (e) Reconstructed superresolution image for reference cell line Nup96-mMaple and (f) for target cell line Nup107-mMaple. NPC structures are automatically segmented to determine the numbers of localizations per NPC. (g) Histogram of number of localizations per NPC for reference and target. The number of Nup107-mMaple proteins per NPC is calculated from the average relative number of localizations. (h) The stoichiometry of Nup107 in the NPC ( n Nup107 = 32.1 ± 2.5, N = 5, n C = 13, n T = 1928) shows a high accuracy and low statistical errors of this counting approach. (i-m) Counting in yeast. (i) Mixture of Nup188-mMaple+Abp1-GFP reference cell lines with Nup82-mMaple+Nup188-mMaple target cell lines, which can be distinguished by the GFP signal. (j) Superresolution reconstruction and (k) individual nuclear pores. (l) Histograms of the number of localizations per nuclear pore, arrows indicate the mean (N = 2, n C = 508, n NPC = 1190 for Nup188 and n NPC = 1176 for Nup82+Nup188). (m) Copy number of several yeast nucleoporins per NPC, determined using Nup188 as a reference. These data are derived from: Nup82: N = 2, n C = 242, n T = 678, n R = 686; Nup82+Nup188: N = 2, n C = 508, n T = 1176, n R = 1190; Nup192: N = 2, n C = 558, n T = 992, n R = 916; Nic96C: N = 2, n C = 304, n T = 1102, n R = 1127; Nic96N: N = 2, n C = 532, n T = 1078, n R = 1079; Nic96N+Nup49GFP: N = 2, n C = 303, n T = 1137, n R = 1149; Nup188 (CHX treatment): N = 2, n C = 521, n T = 1157, n R = 1154. N denotes the number of biologically independent experiments, n C the number of analyzed cells, and n T /n R the number of analyzed NPCs for the counting target/reference. Bars denote the mean, error bars the standard deviation and data points individual acquisitions. Shown values depict weighted mean ± SD, based on n NPC . Representative images of one (b) , two (a,i-k) , five (f) or six (e) independent experiments are shown. Scale bars 10 µm (i) , 1 µm (a,b,e,f,j) , 100 nm (k).

Article Snippet: Fixed cells were then further permeabilized using 0.2% Triton X-100 in PBS for 10 min, followed by two more washing steps in PBS for 5 min each before blocking in 2% (w/v) BSA in PBS for 1 h. Binding of primary rabbit anti-GFP antibodies (Cat#598; MBL International) to Nup96-GFP fusion proteins was achieved by placing the coverslips overnight upside-down onto a drop of primary antibody solution (diluted 1:250 in PBS containing 2% [w/v] BSA) at 4 °C.

Techniques: Microscopy, Derivative Assay, Standard Deviation

Journal: Autophagy

Article Title: Podocytes maintain high basal levels of autophagy independent of mtor signaling

doi: 10.1080/15548627.2019.1705007

Figure Lengend Snippet: Podocytes exhibit high levels of basal autophagy and autophagic flux. (A) Representative images obtained from cryosections of 4-month old Gfp-Lc3 mice stained for the basement membrane marker NID1/nidogen-1/entactin (red) and GFP-LC3 (green). (B) Two-photon images of glomeruli from Gfp-Lc3 mice perfused with dextran as a marker for glomerular capillaries (in red) displaying autophagosomes in vivo (in green, marked with arrow). (C) Representative images obtained from cryosections of 4-month-old Rfp-Gfp-Lc3 mice stained for NID1 (purple). RFP-LC3 and GFP-LC3 fluorescence is endogenous. (D) Quantification of (C) out of 30 glomeruli from 3 mice each with surrounding tubular cells (** ≤ 0.01, * ≤ 0.05). (E) Cryosections displaying glomeruli (upper panel) and tubular system (lower panel) stained for NID1 (red) and GFP-LC3 (anti-GFP antibody, green) in 4-month-old WT mice with and without chloroquine (4 h after chloroquine [Cq] administration i.p. 100 mg Cq/kg BW). (F) Western blot from immortalized human podocyte cell line and proximal tubular cell line (HK2) for MTORC1 downstream targets and LC3 abundance. (G) Densitometry obtained from (F) (** ≤ 0.01)

Article Snippet: Sections were incubated for 1 h with primary antibodies (rat anti-NID1/nidogen-1/entactin [Novus, NBP1-977001], rabbit anti-GFP [Biozol/MBL, 598]).

Techniques: Staining, Marker, In Vivo, Fluorescence, Western Blot

Journal: Autophagy

Article Title: Podocytes maintain high basal levels of autophagy independent of mtor signaling

doi: 10.1080/15548627.2019.1705007

Figure Lengend Snippet: Basal autophagy is independent of MTOR activity in podocytes in vivo . (A) Schematic of generating podocyte-specific deletion of Rptor or Tsc1 using Nphs2-Cre mice and Cre-Lox technique. (B) Cryosections from 2-week-old mice bearing podocyte-specific knockout for Rptor and transgenic for Gfp-Lc3 compared to Gfp-Lc3 WT mice (NID1 in red, GFP-LC3 in green). (C) Quantification of GFP-LC3 autophagosomes per glomerular area out of 30 glomeruli each from 3 mice (ns, not significant). (D) Cryosections from 2-week-old mice bearing podocyte-specific knockout for Tsc1 and transgenic for Gfp-Lc3 compared to Gfp-Lc3 WT mice (NID1 in red, GFP-LC3 in green). (E) Quantification of GFP-LC3 autophagosomes per glomerular area out of 30 glomeruli each from 3 mice (ns, not significant). (F) Western blot out of glomerular lysates obtained from 2-week-old mice for MTORC1 downstream targets and LC3 and SQSTM1 abundance. (G) Densitometry for LC3-II, SQSTM1 and p-RPS6 obtained from 3 WT glomerular lysates and 3 glomerular lysates obtained from 2-week-old mice bearing a podocyte-specific deletion of Rptor or Tsc1 , respectively (** ≤ 0.01, * ≤ 0.05, ns, not significant)

Article Snippet: Sections were incubated for 1 h with primary antibodies (rat anti-NID1/nidogen-1/entactin [Novus, NBP1-977001], rabbit anti-GFP [Biozol/MBL, 598]).

Techniques: Activity Assay, In Vivo, Knock-Out, Transgenic Assay, Western Blot

Journal: Autophagy

Article Title: Podocytes maintain high basal levels of autophagy independent of mtor signaling

doi: 10.1080/15548627.2019.1705007

Figure Lengend Snippet: Effects of acute and long-term pharmacological inhibition of MTORC1 activity on autophagy. (A) Schematic of the short-term treatment regimen (rapamycin vs. vehicle, n = 5 each, rapamycin dose: 10 mg/kg BW i.p. for 3 d). (B) Quantification of GFP-LC3 autophagosomes per glomerular area out of 30 glomeruli each from 5 mice per group (vehicle vs. rapamycin). (C) Representative cryosections from 16-week-old mice transgenic for GFP-LC3 with and without rapamycin treatment (NID1 in red, GFP-LC3 in green, rapamycin dose: 10 mg/kg BW i.p. for 3 d). (D) Representative western blot from glomerular lysates from rapamycin and vehicle treated mice (WT mice, n = 3 each, rapamycin dose: 10 mg/kg BW i.p. for 3 d). (E) Densitometric quantification of (D) (** ≤ 0.01, * ≤ 0.05). (F) Schematic of the long-term treatment regimen (rapamycin dose: 4 mg/kg BW i.p. for 3 weeks). (G) Quantification of serum levels of rapamycin (n = 6 each) (*** ≤ 0.001). (H) Representative western blot from glomerular lysates from rapamycin long-term, short-term and vehicle treated mice. (I) Densitometry from 3 glomerular lysates of each treatment and vehicle controls for LC3-II, SQSTM1 and p-RPS6 Ser235/236 (** ≤ 0.01, ns, not significant). LT, long-term; ST, short-term

Article Snippet: Sections were incubated for 1 h with primary antibodies (rat anti-NID1/nidogen-1/entactin [Novus, NBP1-977001], rabbit anti-GFP [Biozol/MBL, 598]).

Techniques: Inhibition, Activity Assay, Transgenic Assay, Western Blot

Journal: The Journal of Cell Biology

Article Title: Trafficking of plasmepsin II to the food vacuole of the malaria parasite Plasmodium falciparum

doi: 10.1083/jcb200307147

Figure Lengend Snippet: BFA induces a reversible accumulation of proPM II–GFP in the ER. (A and B) GFP fluorescence in live B7 parasites treated for 2 h with BFA: (A) a trophozoite and (B) a schizont undergoing nuclear division. The arrowhead in B indicates food vacuole fluorescence. (C) Redistribution of GFP 10 min after release of the BFA block. Fluorescent spots reappear at the periphery of the parasite (arrowheads). 100 μg/ml cycloheximide was present to inhibit protein synthesis after BFA washout. Similar results were obtained in the absence of cycloheximide. In A–C, fluorescence from the nuclear stain Hoechst 33342 is pseudocolored red. Bar, 2 μm. (D) Cryosection of a BFA treated B7 trophozoite double-labeled with an antibody against GFP (18-nm colloidal gold) and an antibody recognizing the ER marker BiP (12-nm colloidal gold). Most of the 18-nm gold label is associated with the nuclear envelope (arrowhead), whereas the 12-nm gold label is associated with the peripheral ER (asterisk) extending away from the nucleus. A low magnification image of this parasite is provided in Fig. S2. n, nucleus. Bar, 200 nm. (E) B7 trophozoites were 35 S-labeled for 2 h in the presence of 5 μg/ml BFA (“BFA” lane). Both BFA and unincorporated 35 S were washed out either in the absence (no inhib) or presence (ALLN) of an inhibitor of PM II maturation. proPM II–GFP and GFP were immunoprecipitated with an anti-GFP antibody. The low intensity of the GFP band in “no inhib” lane relative to proPM II–GFP in the “BFA” lane is likely due to two factors: GFP contains one third of the label present in proPM II–GFP, and may be slowly degraded in the food vacuole. Sizes of molecular mass markers are indicated in kD.

Article Snippet: GFP was immunoprecipitated in the absence of SDS using living colors full-length A.v. polyclonal anti-GFP antibody (1:500; BD Biosciences).

Techniques: Fluorescence, Blocking Assay, Staining, Labeling, Marker, Inhibition, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Trafficking of plasmepsin II to the food vacuole of the malaria parasite Plasmodium falciparum

doi: 10.1083/jcb200307147

Figure Lengend Snippet: ALLN treatment results in accumulation of proPM II–GFP in the food vacuole membrane. (A) GFP fluorescence in a live B7 trophozoite treated with BFA for 2 h followed by replacement of BFA with 100 μM ALLN for a further 2 h. A bright rim of fluorescence circumscribes the food vacuole (arrow). A local concentration of fluorescence on the food vacuole membrane is indicated with an arrowhead. Two cytostomal vacuoles above the food vacuole are also visible. Bar, 2 μm. (B) A trophozoite treated as in A in which the Hoechst 33342–stained nucleus is pseudocolored red. (C and D) Trophozoites treated as in A and labeled with either (C) anti-GFP or (D) anti–PM II antibody. Low magnification images of these parasites are provided in Fig. S2. Abbreviations are given in the legend to . Bars, 200 nm.

Article Snippet: GFP was immunoprecipitated in the absence of SDS using living colors full-length A.v. polyclonal anti-GFP antibody (1:500; BD Biosciences).

Techniques: Fluorescence, Concentration Assay, Staining, Labeling