EMBO/SourceData huggingface dataset:
Figure 1A. Confocal images show Iba1-immunoreactive microglia and macrophages in RPE/choroid whole mount preparations 48 hours after laser coagulation and intravitreal application of low (0.2 µg) and high (3 µg) dose polySia avDP20 or PBS-vehicle. Laser spots of vehicle-injected SIGLEC11 transgenic (tg) and wild type control animals revealed strong accumulation of activated microglia and macrophages, which was effectively decreased in polySia avDP20-injected SIGLEC11 tg animals and to a lower extent in wild type controls. Representative images out of at least three independent experiments are shown. Scale bar: 50µm.B. Quantification of the average pixel intensity of Iba1-positive area in laser spots reflects accumulation of reactive microglia/macrophages on RPE/choroid whole mounts. In comparison to the accumulation of reactive microglia/macrophages in control animals, there is a reduced pixel intensity found in polySia avDP20-treated SIGLEC11 tg animals. Notably, treatment with a high polySia avDP20 dose (3 µg) also reduced accumulation of microglia/macrophages in laser spots of wild type animals. Data show mean +/- SD. * p< 0.05, ** p< 0.01, *** p< 0.001, One-way ANOVA followed by Fisher's LSD. WT PBS (n = 24 spots), WT 0.2 µg Sia (n = 37 spots), WT 3 µg Sia (n = 20 spots), SIGLEC11 tg PBS (n = 25 spots), SIGLEC11 tg 0.2 µg Sia (n = 35 spots), SIGLEC11 tg 3 µg Sia (n = 31 spots), WT PBS vs WT 3 µg Sia p = 0.0147, WT 0.2 µg Sia vs SIGLEC11 tg 0.2 µg Sia p = 0.0046, SIGLEC11 tg PBS vs SIGLEC11 tg 0.2 µg Sia p = 0.0071, SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p < 0.0001.C. Confocal images of retinal whole mounts show Iba1-immunoreactive microglial cells. Retinal microglia of polySia avDP20-treated SIGLEC11 tg mice had a more ramified microglial morphology in the laser spot compared to PBS-vehicle treated mice. Interestingly, high polySia avDP20 dose (3 µg) also exerted a weak therapeutic effect in wild type animals. Representative images out of at least three independent experiments are shown. Scale bar: 20 µm.D. Percentage of retina showing activated microglial cells within the laser spots was quantified. PolySia avDP20 reduced the percentage of laser spots with activated microglia in SIGLEC11 tg animals and at high dose (3 µg) also in wild type controls. Data show mean +/- SEM. * p< 0.05, ** p< 0.01, ***p<0.001, One-way ANOVA followed by Fisher's LSD. WT PBS (n = 6 retinas), WT 0.2 µg Sia (n = 8 retinas), WT 3 µg Sia (n = 6 retinas), SIGLEC11 tg PBS (n = 8 retinas), SIGLEC11 tg 0.2 µg Sia (n = 7 retinas), SIGLEC11 tg 3 µg Sia (n = 6 retinas),WT PBS vs WT 3 µg Sia p = 0.0052, WT 0.2 µg Sia vs SIGLEC11 tg 0.2 µg Sia p = 0.0005, SIGLEC11 tg PBS vs SIGLEC11 tg 0.2 µg Sia p = 0.0159, SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p = 0.001.
Figure 2A. Fundus fluorescein angiography was performed 48 hours after laser coagulation and intravitreal application of low (0.2 µg) and high (3 µg) dose polySia avDP20 or PBS-vehicle. Late stage (10-11 minutes after fluorescein injection) fundus fluorescein angiography revealed that PBS-treated wild type controls and humanized SIGLEC11 mice showed normal levels of vessel leakage whereas polySia avDP20-treated SIGLEC11 transgenic (tg) mice had lower levels of vessel leakage compared to PBS-injected wild type or SIGLEC11 tg mice. High polySia avDP20 dose reduced vascular leakage also in wild type animals. Representative images out of at least eight independent experiments are shown.B. Fundus fluorescein angiography pictures were exported from Heidelberg Eye Explorer Software and fluorescein leakage was quantified with ImageJ software (NIH). Pixel intensities of 6 regions of interest per picture were quantified and background fluorescence was subtracted. PolySia avDP20-treated SIGLEC11 tg animals showed reduced vascular leakage compared to PBS-injected wild type control and SIGLEC11 tg mice. A reduction in vascular leakage was also observed in high dose polySia avDP20-injected wild type control mice. Data show mean +/- SD. * p< 0.05, *** p< 0.001, One-way ANOVA followed by Fisher's LSD. WT PBS (n = 20 eyes), WT 0.2 µg Sia (n = 17 eyes), WT 3 µg Sia (n = 9 eyes), SIGLEC11 tg PBS (n = 16 eyes), SIGLEC11 tg 0.2 µg Sia (n = 20 eyes), SIGLEC11 tg 3 µg Sia (n = 15 eyes), WT PBS vs WT 3 µg Sia p = 0.0193, WT 0.2 µg Sia vs SIGLEC11 tg 0.2 µg Sia p < 0.0001, SIGLEC11 tg PBS vs SIGLEC11 tg 0.2 µg Sia p < 0.0001, SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p = 0.0002.C. Anti-C5b-9 immunostaining of RPE/choroid whole mount preparations 48 hours after laser damage showed strong MAC deposition in the laser lesions of vehicle-injected controls. PolySia avDP20 treatment reduced MAC formation in a dose-dependent fashion independent of SIGLEC11 presence. Scale bar: 100 µm.D. Quantification of C5b-9 fluorescence signal intensity in the laser lesions. In comparison to the high amount of MAC deposition in vehicle-injected controls, the pixel intensity is reduced in polySia avDP20-treated eyes in a dose-dependent fashion and regardless of SIGLEC11 presence. Data show mean +/- SD. *** p< 0.001, One-way ANOVA followed by Fisher's LSD. WT PBS (n = 12 laser spots), WT 0.2 µg Sia (n = 12 laser spots), WT 3 µg Sia (n = 7 laser spots), SIGLEC11 tg PBS (n = 6 laser spots), SIGLEC11 tg 0.2 µg Sia (n = 10 laser spots), SIGLEC11 tg 3 µg Sia (n = 12 laser spots); all statistical comparisons p < 0.0001 except SIGLEC11 tg PBS versus SIGLEC11 tg 0.2 µg Sia p = 0.0004.
Figure 1Fluorescent images (left panels) and tracings (right panels) of PVD neurons of the indicated genotypes. PVD is visualized by the wdIs52 transgene. Primary (1º), secondary (2º), tertiary (3º), and quaternary (4º) dendrites are indicated, and the cell body is marked with an asterisk. Anterior is to the left and dorsal is up in all panels. Scale bars represent 20μm.Quantification of the number of quaternary branches (indicated in schematic) in different aman-2 alleles and in wild type control animals. All loss of function alleles of aman-2 display a significant decrease in PVD quaternary branch number. Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and is indicated (****p ≤ 0.0001). n = 15 for all genotypes and are biological replicates.Quantification of the number of full tertiaries (marked by secondary and tertiary branches as shown in schematic) in the genotypes indicated. Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and is indicated (****p ≤ 0.0001; ns = not significant). n = 15 for all genotypes and are biological replicates.Fluorescent images and tracings of PVD in mnr-1(dz213) hypomorphic animals alone and in combination with an aman-2(gk248486) null mutant. The dz213 introduces a L135F missense mutation in the DUF2181 domain of MNR-1/Menorin. PVD is visualized by the dzIs53 transgene. The cell body is marked with an asterisk. Scale bars represent 20μm. Quantification of the number of full tertiaries in genotypes indicated and traced in (F). The loss of aman-2 severely enhances the mnr-1(dz213) phenotype. Control indicates wild type control animals as shown in (A). Data are represented as mean ± SEM. Statistical significance was calculated using the Mann-Whitney test and is indicated (****p ≤ 0.0001; ns= not significant). n >23 for all genotypes and are biological replicates.Fluorescent images and tracings of PVD in kpc-1(dz254) hypomorph animals alone and in combination with an aman-2(gk248486) null mutant. PVD is visualized by the dzIs53 transgene. White arrows indicate self-avoidance defects in tertiary branches. Red arrows show gaps between tertiary branches (no self-avoidance defects). The cell body is marked with an asterisk. Scale bars represent 20μm. Quantification of the percent of self-avoidance defects in genotypes indicated and traced in (G). Control indicates wild type control animals as shown in (A). The loss of aman-2 suppresses defects in the kpc-1(dz254) phenotype. Data are represented as mean. Statistical significance was calculated using the Z-test and is indicated (****p ≤ 0.0001). n >15 for all genotypes and are biological replicates.
Figure 2Fluorescent images and tracings of wild type control (top) and lect-2(gk846764) hypomorphic animals (bottom) fed on plates with 300µm swainsonine vs a DMSO control. PVD is visualized by the wyIs581 transgene. The cell body is denoted with an asterisk. Anterior is to the left and dorsal is up in all panels. Vesicular gut autofluorescence is visible as white circular staining. Scale bars represent 20μm. Quantification of the number of full tertiaries in denoted genetic backgrounds (aman-2 null is gk248486). Black data points indicate DMSO and orange data points show swainsonine treated animals. Data are represented as mean ± SEM. Statistical significance was calculated using the Mann-Whitney test and is indicated (****p ≤ 0.0001; ns= not significant). n = 12 for all genotypes and are biological replicates. Quantification of the number of quaternary dendrites in wild type control animals fed on plates with and without 300µm swainsonine. Animals treated with swainsonine show a significant decrease in quaternary branch number, Data are represented as mean ± SEM. Statistical significance was calculated using the Mann-Whitney test and is indicated (****p ≤ 0.0001). n = 12 for each experiment and are biological replicates.
Figure 3(B) Fluorescent composite images (top) and tracings (bottom) of mnr-1(dz213) in an aman-2(gk248486) and an aman-2(gk248486); MGAT-1(null) background. An MGAT null mutant lacks the three C. elegans paralogs: gly-12, gly-13, and gly-14. PVD is visualized by the wyIs581 transgene. The cell body is denoted with an asterisk. Anterior is to the left and dorsal is up in all panels. Scale bars represent 20μm. (C) Quantification of full tertiaries of denoted genotypes. Data are represented as mean ± SEM. Statistical comparisons were performed using the Kruskal-Wallis test. Statistical significance is indicated (***p ≤ 0.001, ****p ≤ 0.0001, ns=not significant). n=15 for all genotypes and are biological replicates. (D) Quantification of the number of quaternary branches in wild type control, aman-2(gk248486) null ,and MGAT1(null) animals. Loss of MGAT1 suppresses the decrease in quaternary branch number in the aman-2(null) background. Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and is indicated (**p ≤ 0.01, ****p ≤ 0.0001). n = 15 for all genotypes and are biological replicates.Western blot against GFP in C. elegans lysate expressing no transgenes (N2) and expressing DMA-1::GFP (qyIs369), after precipitating with anti-GFP antibody. The red boxed plus sign indicates that the lysate is treated with the PNGase F glycosidase. The downwards size shift reveals that N-glycan structures are present on DMA-1. Ladder is marked in kilodaltons (kDa). The GFP tag contains no N-glycosylation sites. The experiment was repeated four times with biological replicates.Western blot against GFP in C. elegans lysate DMA-1::GFP (qyIs369), after precipitating with anti-GFP antibody. Control indicates an otherwise wild type background as opposed to an aman-2(gk248486) null background. The upward size shift in the mutant reveals that the loss of aman-2 alters the identity of N-glycan structures on DMA-1. The experiment was repeated four times with biological replicates.Western blot against GFP in C. elegans lysate DMA-1::GFP (qyIs369), after precipitating with anti-GFP antibody. Control indicates an otherwise wild type background as opposed to an aman-2(gk248486) null background. The red boxed +F indicates that the lysate is treated with the PNGase F glycosidase, while the green boxed +H corresponds to the Endo H glycosidase, which cleaves high-mannose and hybrid type N-glycans. For complementary experiments using the Endo D glycosidase, which cleaves paucimannose type N-glycans, Size shifts indicate that some hybrid/high-mannose structures are present on DMA-1 (left), and that the aman-2 mutant results in only hybrid/high-mannose structures on DMA-1 (right). The experiment was repeated four times with biological replicates.
Figure 4Quantification of the number of quaternary branches in DMA-1::2xFLAG control animals and animals with combinations of DMA-1::2xFLAG N-glycan attachment sites mutated. Note that DMA-1::2xFLAG control animals display a slightly reduced number of quaternary dendrites compared to wild type animals Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and Mann-Whitney between individual comparisons, and is indicated (**p ≤ 0.01, ****p ≤ 0.0001, ns=not significant). n = 15 for all genotypes and are biological replicates.Quantification of the number of quaternary branches in DMA-1::2xFLAG control animals alone and in combination with an aman-2(gk248486) null mutant (shaded in red). Control data is identical as in (B) and shown for comparison only. Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and Mann-Whitney between individual comparisons, and is indicated (**p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns=not significant). n = 15 for all genotypes and are biological replicates.Fluorescent images (left) and tracings (center) of PVD in animals of denoted backgrounds (aman-2(null) is gk248486). S4 corresponds to the endogenous alteration of N-glycan attachment site N386 of DMA-1. PVD is visualized by the dzIs117 transgene. The cell body is marked with an asterisk. Scale bars represent 20μm.
Figure 1A. Histograms of spindle angle of GFP-H2B U2OS cells transfected with either GAPDH or Spindly siRNA and filmed on rectangular micropatterns. Cells were filmed 48 hours post-transfection.B. Mitotic timing of control-depleted U2OS cells or CLIP-170-depleted cells. CLIP-170-depeleted cells are split in two categories; 'aligned' and 'misaligned', relating to the observed chromosome misalignment phenotypeC. The histograms of spindle angles for the two different categories of CLIP-170-depleted U2OS cells (Left graph with alignment defect, right graph without alignment defect).D and E. Mitotic timing and spindle angle histograms of control (GAPDH)-depleted U2OS cells and CENP-E-depleted cells.
Figure 2A. HeLa cell stably expressing GFP-LGN and transiently expressing RFP-H2B was treated with noscapine to induce chromosome misalignments. Cells were imaged every 5 minutes. The time is relative to NEB. The red arrows indicate cortical regions where the chromosomes are in close proximity to the cell boundaries, accompanied by the displacement of LGN from the cortex.B. Similar assay as described in supplementary figure 2D was carried out in the presence of a small molecule inhibitor against Aurora-B (ZM447439) in combination with the proteasome inhibitor MG-132 to keep cells arrested in mitosis and in B. with an inhibitor against Plk1 (BI2536). Drugs were added at the same timing as Hoechst, 30 minutes prior to imaging.
Figure 3A. Immunofluorescence images and quantifications of KT-localized Plk1 in U2OS cells after PBIP1 depletion. Cells were either Mock transfected or with PBIP1 siRNA for 72 hours. Cells were treated with nocodazole for 1 hour and fixed. Cells were stained with the centromere marker ACA, Plk1 and DAPI.B. Representative images and histograms showing the effect of chromosome position on cortical LGN enrichment after 72 hours of Mock and PBIP1 depletion.C. Live cellimages and histograms of GFP-H2B U2OS cells Mock transfected or with siPBIP1 filmed on rectangular micropatterns in the presence of a CENP-E inhibitor. The spindle angles for both conditions were scored at 32 minutes after NEB.. D. The total spindle angle displacement within the first hour of mitosis was determined by measuring the spindle angle at each time point and calculating the sum of the Δ spindle angle between each time points.
Figure 1Expression levels of the autophagosome marker LC3B-II were evaluated in wild-type (WT) and Ctns−/− fibroblasts by Western blot (WB). Data are representative of five different experiments with similar results.WT and Ctns−/− fibroblasts were stained using LC3B antibodies and analyzed by fluorescence microscopy. The number of LC3B-positive puncta was quantified by the ImagePro software and normalized per area arbitrary units. Results are mean ± SEM (n = 27 WT and 24 Ctns−/− cells). ***P < 0.001 (unpaired t-test).Immunofluorescence analysis of liver (C) and kidney (D) tissues from WT and Ctns−/−GFP-LC3 transgenic mice using anti-GFP antibodies. Quantification of LC3 puncta was obtained by counting the number of GFP-positive structures relative to the number of nuclei in the same field, using the ImagePro software. GFP-LC3 puncta were counted by analyzing 6 to 10 fields per tissue section (200-400 nuclei per field), in a total of 3 WT and 3 Ctns−/−mice expressing GFP-LC3. Results are mean ± SEM. In (C), **P = 0.0029; in (D), **P = 0.0025 (unpaired t-test). Scale bar: 5 μm.
Figure 2Representative images of wild-type (WT) and Ctns−/− mouse fibroblasts transfected with the ptfLC3 vector under resting conditions (fed), or after serum starvation (Serum Starv.) in the presence or absence of the alkalinizing drug chloroquine (CQ). GFP and RFP staining was analyzed by confocal microscopy. Examples of red-only puncta (mature autophagosomes) are indicated with arrows. Scale bar: 5 μm.The percentage of mature autophagosomes (red-only vesicles) was calculated based on the ratio between the number of red-only puncta and the total number of autophagosomes (number of green and red + red-only puncta). The graph is representative of three different experiments with similar results. Results are mean ± SEM (n = 7 cells per condition). In addition to the experimental conditions shown in (A), the Aa/serum starvation condition is included.WT and Ctns−/− fibroblasts under resting conditions were treated with 100 nM bafilomycin A (BafA) for 2 h and LC3B-II levels were analyzed by Western blot (WB).Phosphorylation levels of the mTOR complex kinase 1 substrate S6K and LC3B-II levels in WT and Ctns−/− fibroblasts were measured by WB under resting conditions (−), withdrawal of both amino acids and serum (Aa/Ser. Starv.) and subsequent recovery by replacement of starvation medium with normal cell growth medium (Rec), in the presence or absence of 100 nM BafA for the indicated time.WB analysis showing phosphorylation levels of the mTOR substrate S6K and expression of LC3B-II in resting or serum-starved WT,Ctns−/− and Ctns−/− fibroblasts expressing GFP-CTNS.
Figure 3Analysis of the expression of endogenous SQSTM1 in wild-type or Ctns−/− mouse fibroblasts under fed (serum) or starved (15 h serum starvation) conditions. In these assays, 20 μg of total protein lysates were resolved by SDS-PAGE and analyzed by Western blot (WB; A). Equal loading was established by silver staining (B). Quantification (mean ± SEM) of five different areas of each silver-stained lane are shown in (C).Degradation of long-lived proteins using metabolic labeling was performed as described in Supplementary Materials and Methods. The data represent the mean ± SEM of nine independent samples analyzed in three independent experiments. *P = 0.009 for WT and *P = 0.0013 for Ctns−/−, respectively. NS, not significant (unpaired t-test).
Figure 4The expression levels of the endogenous LAMP2A and LAMP1 proteins were analyzed in wild-type (WT) and Ctns−/−mousefibroblasts (A), kidney (B) and liverlysosomes (C) by Western blot. Quantitative densitometry analysis of immunoblots obtained from different experiments was performed by calculating the ratio between LAMP2A and actin signals in each lane. Results are mean ± SEM of four independent experiments with mousefibroblasts, 5 WT and 6 Ctns−/−mousekidneys, 3 WT and 3 Ctns−/−mouselivers (lysosomal extracts).Comparative analysis of the amount of LAMP2A in wild-type or Ctns−/−liverlysosomes was performed using two different anti-LAMP2A antibodies. 15 μg of lysosomal proteins were loaded in each lane. Ab1, anti-LAMP2A antibody raised against the twelve amino acids of the cytosolic region of ratLAMP2A largely validated to recognize mouseLAMP2A but not other LAMP2 isoforms (Cuervo Dice, 1996). Ab2, anti-LAMP2A Abcam Ab18528. Decreased lysosomalLAMP2A was evident using either antibody. LAMP1 was used as a loading control. Representative of two independent experiments with similar results.
Figure 5Immunofluorescence analyses of endogenous LAMP1, LAMP2A (A, B), LAMP2B (C) and total LAMP2 (D) localization in wild-type (WT) and Ctns−/− mouse fibroblasts. (A) Confocal microscopy analysis. White arrows represent LAMP1-positive circular structures (lysosomes) which were visible in both WT and Ctns−/− cells. Colocalization of LAMP1 and LAMP2A was observed in WT fibroblasts (white arrows, merged panel) but rarely in Ctns−/− cells (red arrow). Most LAMP2A showed a punctate pattern and lack of colocalization with LAMP1 in Ctns−/− cells (arrowheads, lower panels). Scale bars: 5 μm. Inset scale bars: 2 μm.Immunofluorescence analyses of endogenous LAMP1, LAMP2A (A, B), LAMP2B (C) and total LAMP2 (D) localization in wild-type (WT) and Ctns−/− mouse fibroblasts.(B) High-resolution stochastic optical reconstruction microscopy (STORM) analysis of the localization of LAMP1 and LAMP2A in wild-type and Ctns−/− cells. In Ctns−/− cells, LAMP2A was detected near (arrowheads, estimated distance > 50 nm) but not always adjacent (10-50 nm) to LAMP1 or at LAMP1-negative structures (arrows). Scale bar: 0.5 μm.Immunofluorescence analyses of endogenous LAMP1, LAMP2A (A, B), LAMP2B (C) and total LAMP2 (D) localization in wild-type (WT) and Ctns−/− mouse fibroblasts. (C) Immunofluorescence showing colocalization of endogenous LAMP1 and LAMP2B in both wild-type and CTNS-deficient fibroblasts (arrows). Scale bar: 20 μm.Immunofluorescence analyses of endogenous LAMP1, LAMP2A (A, B), LAMP2B (C) and total LAMP2 (D) localization in wild-type (WT) and Ctns−/− mouse fibroblasts. (D) Immunofluorescence analysis of LAMP1 and total LAMP2 (antibody ABL93 DSHB). Arrows show colocalization. Arrowheads show LAMP2-positive LAMP1-negative vesicles.
Figure 6Immunofluorescence analyses of endogenous LAMP2A, ER (Grp78/94) (A), which is expanded in cystinotic cells (white arrow), VAMP7-positive vesicles (B) and Rab11a-positive structures (C) was performed using wild-type or Ctns−/− fibroblasts as described in Materials and Methods. At least 2 independent experiments were performed for each vesicular trafficking or ER marker. (B, C) Results are expressed as the percentage of LAMP2A that colocalizes with VAMP7 or Rab11. For Rab11/LAMP2A colocalization, 29 wild-type and 15 Ctns−/− cells were quantified. For VAMP7/LAMP2A colocalization (white arrows), 49 WT and 57 Ctns−/− cells were analyzed. Data are expressed as mean ± SEM. *P < 0.001 (unpaired t-test). Scale bars: 20 μm.
Figure 7WT,Ctns−/− or Ctns−/− mouse fibroblasts treated for 20 h with either a combination of both leupeptin and chloroquine (CQ/Leu) or bafilomycin A (BafA) alone were fixed and immunostained with antibodies recognizing endogenous LAMP1 and LAMP2A proteins and samples were analyzed by confocal microscopy as described in Material and Methods. Lysosomal colocalization of LAMP1 and LAMP2A was evident in treated (middle lower and bottom panels) but not in untreated (middle upper panels) Ctns−/− cells. Scale bars: 5 μm. Inset scale bars: 2 μm.