Journal: eLife

Article Title: Evolutionary conservation of centriole rotational asymmetry in the human centrosome

doi: 10.7554/elife.72382

Figure Lengend Snippet: Figure 1. LRRCC1 is localized in a rotationally asymmetric manner at the distal end of centrioles in the human centrosome. (a) LRRCC1 localization in non-treated RPE1 cells (left) or in cells treated with nocodazole to disperse the pericentriolar satellites (right). LRRCC1 (Ab2, yellow), γ-tubulin (PCM, magenta), and DNA (cyan). Bar, 5 µm (insets, 2 µm). (b) Longitudinal view of centrioles and procentrioles in the duplicating centrosome of an RPE1 cell analyzed by ultrastructure expansion microscopy (U-ExM). LRRCC1 (Ab2, yellow), acetylated tubulin (magenta). Bar, 0.5 µm. (c) Centrioles from WT RPE1 cells as seen from the distal end. LRRCC1 (Ab2, yellow), acetylated tubulin (magenta). Images are maximum intensity projections of individual z-sections encompassing the LRRCC1 signal. Note that an apparent shift between channels occurs when centrioles are slightly angled with respect to the imaging axis. Bar, 0.2 µm. (d) Lateral distance between LRRCC1 (left, yellow) or hPOC5 (middle, cyan) signal intensity peaks and the centriole center (given by the position of acetylated tubulin intensity peak, magenta) in ciliated RPE1 cells. Individual intensity profiles were measured along the green lines. The approximate position of the centriole is shown (white cylinders). Note that LRRCC1 and hPOC5 were also detected at the periphery of the centriole, towards the proximal end for LRRCC1 and in the appendage region for hPOC5. Bar, 0.2 µm. Right: interpeak distance (d). Bars, mean ± SD, 31 cells from two different experiments (Kolmogorov–Smirnov test). (e) Workflow for calculating the average staining from 3D-reconstructed individual centrioles generated from confocal z-stacks. The brightest part of LRRCC1 signal was used as a reference point to align the centrioles. (f) Average LRRCC1 staining obtained from 34 individual centrioles viewed from the distal end, in transverse and longitudinal views. A diagram representing the average pattern in transverse view is also shown.

Article Snippet: Antibodies were affinity- purified over the corresponding GST- LRRCC1 fusion bound to Affi- Gel 10 resin (Bio- Rad).

Techniques: Microscopy, Imaging, Staining, Generated

Journal: eLife

Article Title: Evolutionary conservation of centriole rotational asymmetry in the human centrosome

doi: 10.7554/elife.72382

Figure Lengend Snippet: Figure 2. The LRRCC1 rotationally asymmetric pattern is conserved in mouse multiciliated cells (MCCs). (a) Centrioles in the cytoplasm of mouse ependymal cells differentiating in vitro analyzed by ultrastructure expansion microscopy (U-ExM), in longitudinal and transverse view. Lrrcc1 (Ab2, yellow), γ-tubulin (basal foot cap, cyan), and acetylated tubulin (magenta). Of note, γ-tubulin was also detected in the proximal lumen of centrioles. Bar, 0.2 µm. (b) Procentrioles assembling via the centriolar (right) or the deuterosome pathway (left and center) in ependymal cells. Lrrcc1 (Ab2, yellow), acetylated tubulin (magenta). Bar, 0.2 µm. (c) Transverse view of centrioles docked at the apical membrane in fully differentiated mouse tracheal cells, viewed from the distal end. Lrrcc1 (Ab2, yellow), γ-tubulin (cyan), and acetylated tubulin (magenta). Bar, 0.2 µm. (d) Average image generated from 35 individual centrioles from mouse trachea, viewed from the distal end, shown in transverse and longitudinal views. The position of the basal foot (cyan dotted line) stained with γ-tubulin was used as a reference point to align the centrioles. A diagram of the average pattern in transverse view is shown, in which the direction of ciliary beat (Schneiter et al., 2021) is represented by a dotted arrow and the basal foot axis by a green line. Triplets are numbered counterclockwise from the LRRCC1 signal.

Article Snippet: Antibodies were affinity- purified over the corresponding GST- LRRCC1 fusion bound to Affi- Gel 10 resin (Bio- Rad).

Techniques: In Vitro, Microscopy, Membrane, Generated, Staining

Journal: eLife

Article Title: Evolutionary conservation of centriole rotational asymmetry in the human centrosome

doi: 10.7554/elife.72382

Figure Lengend Snippet: Figure 3. Procentriole assembly site is partly correlated with centriole rotational polarity. (a) Diagram showing the localization of Vfl1p (cyan) in the centrioles/basal bodies (gray) and procentrioles/probasal bodies (pink) of C. reinhardtii. The microtubule roots are also shown. (b) Early stage of procentriole assembly stained for LRRCC1 (Ab2, cyan), SAS-6 (yellow), and acetylated tubulin (magenta) in a HEK 293 cell. The brightness of the acetylated tubulin labeling was increased in the insets. Bar, 0.1 µm. (c) Successive stages of centriole elongation in HEK 293 cells stained for LRRCC1 (Ab2, cyan) and acetylated tubulin (magenta). Bar, 0.1 µm. (d) Location of LRRCC1 in the procentrioles (top panels) and position of the procentriole relative to its parent centriole polarity (bottom panels), in RPE1 and HEK 293 centrioles analyzed by ultrastructure expansion microscopy (U-ExM). For each diplosome, the angle between LRRCC1 in the procentriole and the centriole long axis (top panels), or between the procentriole and LRRCC1 in the centriole (bottom panels) was measured. The number of diplosomes analyzed is indicated. p-Values are indicated when statistically different from a random distribution (χ2 test).

Article Snippet: Antibodies were affinity- purified over the corresponding GST- LRRCC1 fusion bound to Affi- Gel 10 resin (Bio- Rad).

Techniques: Staining, Labeling, Microscopy

Journal: eLife

Article Title: Evolutionary conservation of centriole rotational asymmetry in the human centrosome

doi: 10.7554/elife.72382

Figure Lengend Snippet: Figure 4. LRRCC1 is required for ciliary assembly and signaling. (a) Left: LRRCC1 staining (Ab2) of WT or LRRCC1-defficient RPE1 cells obtained by CRISPR/Cas9 editing (clones 1.1, 1.2, and 1.9). Bar, 2 µm. Right: quantification of fluorescence intensity in WT or CRISPR clones treated with control or LRRCC1 siRNAs. Bars, mean ± SD, three independent experiments. p-Values are provided when statistically significant from the corresponding control (one-way ANOVA). (b) Quantification of LRRCC1 distal pool at the mother centriole of ciliated WT or CRISPR cells. Left: Airyscan images showing the region of interest (circled). LRRCC1 (yellow), acetylated tubulin (magenta). Bar: 0.5 µm. Right: quantification of the corresponding signal. Bars, mean ± SD, ≥47 cells from two independent experiments. p-Values are provided when statistically significant from the corresponding control (one-way ANOVA). (c) Percentage of ciliated cells in WT or CRISPR cells treated with control or LRRCC1 siRNAs and serum-deprived during 24 hr. Bars, mean ± SD, ≥204 cells from three independent experiments for each condition. p-Values are provided when statistically significant from the corresponding control (one-way ANOVA). (d) Left: SMO (yellow) accumulation at primary cilia (ARL13B, magenta) following SMO-agonist (SAG)-induction of the Hedgehog pathway, in WT or CRISPR cells. Bar, 2 µm. Right: quantification of ciliary SMO expressed as a percentage of the SAG-treated WT mean. Bars, mean ± SD, three independent experiments. p-Values are provided when statistically significant from the corresponding control (one-way ANOVA). (e) Ciliary SMO expressed as a percentage of the SAG-induced control mean in RPE1 cells treated with control or LRRCC1 siRNAs. Bars, mean ± SD, three independent experiments. p-Values are provided when statistically significant from the corresponding control (one-way ANOVA).

Article Snippet: Antibodies were affinity- purified over the corresponding GST- LRRCC1 fusion bound to Affi- Gel 10 resin (Bio- Rad).

Techniques: Staining, CRISPR, Clone Assay, Fluorescence, Control

Journal: eLife

Article Title: Evolutionary conservation of centriole rotational asymmetry in the human centrosome

doi: 10.7554/elife.72382

Figure Lengend Snippet: Figure 5. Depleting LRRCC1 induces defects in centriole structure. (a) Centriole length in mother (MC) and daughter (DC) centrioles analyzed by ultrastructure expansion microscopy (U-ExM) in WT or LRRCC1-deficient clones (1.1 and 1.9). Left: centrioles were stained for acetylated tubulin (magenta) and CEP164 (yellow) to measure centriole length (arrows). Bar, 0.5 µm. Right: quantification. Bars, mean ± SD, ≥38 centrioles from three independent experiments. p-Values are provided when statistically significant from the corresponding control (one-way ANOVA). (b) Centriole length in control cells or CRISPR cells treated with LRRCC1 siRNA-1 and stained for acetylated tubulin and CEP83. Bars, mean ± SD, ≥43 centrioles from three independent experiments. p-Values are provided when statistically significant from the corresponding control (one-way ANOVA). (c) Transmission electron microscopy view of centrioles in WT and CRISPR (clone 1.9) RPE1 cells. Note that the 1.9 centrioles are from the same cell. N = 9 centrioles from eight different cells for clone 1.9, 3 centrioles from two different cells for WT. Bar, 0.5 µm. (d) Examples of normal distal appendages (DAs), DAs with abnormal morphology (white arrowhead: abnormal spacing between consecutive DAs; cyan arrowhead: abnormal DA shape) or missing DAs (gray arrowhead) in RPE1 cells stained with CEP164 (yellow) and analyzed by U-ExM. Images are maximum intensity projections of individual z-sections encompassing the CEP164 signal. Note that an apparent shift between channels occurs when centrioles are slightly angled with respect to the imaging axis. Bar, 1 µm. (e) Percentages of centrioles presenting anomalies in CEP164 staining in WT or CRISPR RPE1 cells. ≥87 centrioles from eight independent experiments for each condition. p-Values are provided when statistically significant from the corresponding control (two-way ANOVA).

Article Snippet: Antibodies were affinity- purified over the corresponding GST- LRRCC1 fusion bound to Affi- Gel 10 resin (Bio- Rad).

Techniques: Microscopy, Clone Assay, Staining, Control, CRISPR, Transmission Assay, Electron Microscopy, Imaging

Journal: eLife

Article Title: Evolutionary conservation of centriole rotational asymmetry in the human centrosome

doi: 10.7554/elife.72382

Figure Lengend Snippet: Figure 6. C2CD3 localizes asymmetrically at the distal end of centrioles and is affected by LRRCC1 depletion. (a) C2CD3 levels at the centrosome of WT or CRISPR RPE1 cells. Bars, mean ± SD, three independent experiments. p-Values are provided when statistically significant from the corresponding control (one-way ANOVA). (b) C2CD3 levels at the centrosome in RPE1 cells treated with control or LRRCC1 siRNAs. Bars, mean ± SD, three independent experiments. p-Values are provided when statistically significant from the corresponding control (one-way

Article Snippet: Antibodies were affinity- purified over the corresponding GST- LRRCC1 fusion bound to Affi- Gel 10 resin (Bio- Rad).

Techniques: CRISPR, Control

Journal: eLife

Article Title: Evolutionary conservation of centriole rotational asymmetry in the human centrosome

doi: 10.7554/elife.72382

Figure Lengend Snippet: Figure 7. C2CD3 and LRRCC1 partially colocalize at the distal end of centrioles. (a) RPE1 centrioles processed for ultrastructure expansion microscopy (U-ExM) and stained for LRRCC1 (Ab2, yellow), C2CD3 (cyan), and acetylated tubulin (magenta). Bar, 0.1 µm. (b) Model showing the possible location of LRRCC1 and C2CD3 relative to each other within human centrioles. Right panel: diagram showing the respective positions of the acorn (Geimer and Melkonian, 2004) and Vfl1p (Silflow et al., 2001) in C. reinhardtii. The direction of the flagellar beat is indicated by a dotted arrow, and the distal striated fiber is in gray. (c) Evolution of the roles played by Vfl1p/LRRCC1 proteins and associated rotationally asymmetric centriolar substructures. In C. reinhardtii, Vfl1p is required for proper ciliary assembly (1), as well as for the formation of fibers and microtubular roots (2) that control the position of centrioles and procentrioles (3), and overall cellular organization (Adams et al., 1985; Silflow et al., 2001). In human cells, LRRCC1 and C2CD3 are required for primary cilium assembly (1) – this study and Thauvin-Robinet et al., 2014; Ye et al., 2014 – and a role in asymmetric anchoring of cytoskeletal elements to the centriole may also be conserved (2), which could indirectly affect the determination of procentriole assembly site.

Article Snippet: Antibodies were affinity- purified over the corresponding GST- LRRCC1 fusion bound to Affi- Gel 10 resin (Bio- Rad).

Techniques: Microscopy, Staining, Control

Journal: The Journal of Biological Chemistry

Article Title: The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression

doi: 10.1016/j.jbc.2022.101699

Figure Lengend Snippet: rs4903064 resides in a renal cancer–associated regulatory element. A , ATAC-seq tracks from primary renal cancer cells at the DPF3 locus. The top track indicates SNPs in high LD with rs4903064 (r 2 > 0.8). ATAC-seq was performed in cells from three individuals with different rs4903064 genotypes (TT, CT, CC). KIRC RE indicates r egulatory e lements defined by ATAC-seq in ccRCC TCGA samples. B , zoomed in view of ( A ). SNP rs4903064 resides in open chromatin and in KIRC_67261. Please note that open chromatin is only detected in cells carrying the risk allele C. C , sequences of the two alleles at rs4903064. The risk allele C creates a putative hypoxia-responsive element (HRE). D , allele-specific qPCR assay for rs4903064 indicates a shift toward the risk allele C in DNA fragments captured by ATAC experiments in tumor cells ( red triangle ) or tubular cells exposed to 1 mM DMOG ( orange triangle ) when compared to input DNA or DNA from ATAC experiments in untreated tubular cells. Cells were isolated from an individual heterozygous for rs4903064. E , allelic ratio of rs4903064 in ATAC samples from tubular cells isolated from three individuals heterozygous for the SNP performed in triplicates. Cells were exposed to 1 mM DMOG for 16 h or left untreated. Bars indicate mean values ± SD. t test, ∗∗∗∗ p < 0.0001. F , allelic ratio of rs4903064 in FAIRE samples from cells of four heterozygous individuals. Cells were exposed to 1 mM DMOG for 16 h, and values were normalized to corresponding input DNA. The mean value for each individual is shown. Bars indicate mean values ± SD. ∗one sample t test, p < 0.05. G , reporter assay in HeLa cells using the pGL3 promoter vector with or without a 415 bp sequence covering the protective or the risk allele of rs4903064. Cells were treated with 1 mM DMOG for 16 h as indicated. Bars indicate mean values ± SD of one experiment performed in triplicates. H , HeLa cells were depleted for the indicated HIF-α isoforms using siRNA and transfected with the reporter plasmid including the risk allele. Cells were exposed to 1 mM DMOG for 16 h. Reporter activity was normalized to the activity of co-transfected β-galactosidase. Bars indicate mean values ± SD of one experiment performed in triplicates. I and J , DPF3 protein levels are high in HIF-positive ccRCC tumors. A total of 330 ccRCC tumors were stratified according to the immunoreactive score for HIF-1α, HIF-2α, and DPF3 staining in no, low, medium, or strong staining. Fractions of DPF3 signals are shown for each subgroup of HIF staining. Pearson χ 2 -test; p = 1.8 × 10 −8 for HIF-1α ( I ) and p = 0.003 for HIF-2α ( J ). ATAC, Assay for Transposase-Accessible Chromatin; ccRCC, clear cell renal cell carcinoma; DMOG, dimethyloxalylglycine; DPF3, double PHD fingers 3; FAIRE, formaldehyde-assisted isolation of regulatory elements; HIF, hypoxia-inducible transcription factor; KIRC, kidney renal clear cell carcinoma; SNP, single nucleotide polymorphism.

Article Snippet: ChIP experiments were performed as previously described using antibodies directed against HIF-1α (Cay10006421, rabbit polyclonal, Cayman Chemicals), HIF-1β (NB100–110, rabbit polyclonal, Novus Biologicals), RNApol 2 (SC-899, rabbit polyclonal, Santa Cruz), or H3K27ac (ab4729, rabbit polyclonal, Abcam) ( , ).

Techniques: Isolation, Reporter Assay, Plasmid Preparation, Sequencing, Transfection, Activity Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression

doi: 10.1016/j.jbc.2022.101699

Figure Lengend Snippet: HIF binding to the RCC risk allele. A , HIF ChIP qPCR results for the DPF3 locus. Experiments were performed from cells isolated from 11 TT, 11 CT, and 1 CC individuals. Bars indicate mean values ± SD. Student’s t test, ∗ p < 0.05, ∗∗ p < 0.01. B , binding of HIF to the control locus at EGLN3 is not affected by the rs4903064 genotype. Bars indicate mean values ± SD. C , HIF ChIP-seq tracks in two different ccRCC cell lines at the rs4903064-associated enhancer (TT: RCC4 and CC: individual 40,911,432 from Yao et al. ). HIF binding is only detectable in cells from a patient carrying the C allele ( red , HIF-1α: 12 × C from 12 reads in total, HIF-2α 99 × C from 99 reads in total). D , same tracks as in ( C ) covering the EGLN3 control locus. E and F , H3K27ac ChIP-seq tracks from corresponding normal ( blue ) and ccRCC ( red ) tissue indicates increased activity of the disease-associated regulatory element at DPF3 ( E , highlighted in yellow ) and of the EGLN3 locus ( F ) in tumors. G , H3K27ac ChIP-seq tracks for two different ccRCC cell lines at the rs4903064-associated enhancer (TT: 786-O and CC: individual 40,911,432 from Yao et al. ). Levels of the active enhancer mark H3K27ac are high in the individual carrying the C allele and reduced upon VHL re-expression. H , same tracks as in ( G ) covering the EGLN3 control locus. Note that H3K27ac levels are reduced upon VHL re-expression in both cell lines. ccRCC, clear cell renal cell carcinoma; ChIP, chromatin immunoprecipitation; DPF3, double PHD fingers 3; EGLN3, Egl-9 family hypoxia inducible factor 3; HIF, hypoxia-inducible transcription factor; KIRC, kidney renal clear cell carcinoma; SNP, single nucleotide polymorphism; VHL, von Hippel-Lindau.

Article Snippet: ChIP experiments were performed as previously described using antibodies directed against HIF-1α (Cay10006421, rabbit polyclonal, Cayman Chemicals), HIF-1β (NB100–110, rabbit polyclonal, Novus Biologicals), RNApol 2 (SC-899, rabbit polyclonal, Santa Cruz), or H3K27ac (ab4729, rabbit polyclonal, Abcam) ( , ).

Techniques: Binding Assay, ChIP-qPCR, Isolation, Control, ChIP-sequencing, Activity Assay, Expressing, Chromatin Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression

doi: 10.1016/j.jbc.2022.101699

Figure Lengend Snippet: Allele specific interactions at rs4903064. A – C , allele-specific qPCR on ChIP samples generated from primary tubular cells exposed to 1 mM DMOG for 16 h. A , HIF ChIP indicates enrichment for the C allele in DNA fragments captured with HIF-1α or HIF-1β antibodies. n = 7 individuals. B , H3k27ac as a marker for active chromatin is enriched at the C allele. n = 4 individuals. C , RNApol2 is enriched at the C allele of rs4903064 indicating increased interaction with the transcriptional machinery from this allele. n = 4 individuals. D , quantification of the results in ( A – C ). The allelic ratio is shifted to the risk allele C, which indicated increased interaction with HIF and the transcriptional machinery as well as increased activity of this allele. Values are mean ± SD. One sample t test, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. ChIP, chromatin immunoprecipitation; DMOG, dimethyloxalylglycine; HIF, hypoxia-inducible transcription factor.

Article Snippet: ChIP experiments were performed as previously described using antibodies directed against HIF-1α (Cay10006421, rabbit polyclonal, Cayman Chemicals), HIF-1β (NB100–110, rabbit polyclonal, Novus Biologicals), RNApol 2 (SC-899, rabbit polyclonal, Santa Cruz), or H3K27ac (ab4729, rabbit polyclonal, Abcam) ( , ).

Techniques: Generated, Marker, Activity Assay, Chromatin Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression

doi: 10.1016/j.jbc.2022.101699

Figure Lengend Snippet: Regulation of DPF3 expression and effect of DPF3 on tubular cell growth. A , relative expression levels of DPF3 in primary renal tubular cells exposed to 1 mM of DMOG for 16 h. Samples were stratified according to the rs4903064 genotype (TT = 42, CT/CC = 44). Values were normalized to expression levels of the housekeeping gene HPRT and to values from corresponding untreated cells. Each dot represents the mean value from cells of one individual. qPCR was performed in duplicates per individual. Bars indicate mean values ± SD. Student’s t test, ∗∗∗∗ p < 0.0001. B , DPF3 expression levels in isolated tumor cells compared to corresponding untreated tubular cells from 8 (TT) and 14 (CT/CC) individuals, respectively. Bars indicate mean values ± SD. One sample t test, ∗ p < 0.05. C , top , allelic ratios of rs4903064 in cells from heterozygous individuals exposed to DMOG 1 mM or left untreated. One sample t test, ∗∗∗ p < 0.001. Bottom , allelic ratios of rs4903064 in ccRCC cells with high (CA9+) or low (CA9−) CA9 expression as a marker for HIF-1α activity. Values are mean from technical triplicates, and each dot represents values derived from one individual. Bars indicate mean values ± SD. t test, ∗ p < 0.05. D , Western blot for DPF3 and beta actin from lysates of different clones of HKC-8 cells with or without defective DPF3. E , Western blot for DPF3 and beta actin from lysates of two different huPTC lines transfected either with nontargeting (nt) guides or guides targeting DPF3. F , proliferation assays of the clones of cells indicated in ( D ). Dark lines and dots indicate the mean value from the three different clones of cells, which were tested in biological triplicates. Light gray and light blue lines indicate individual values for the three different clones of cells. G , proliferation assay from cells indicated in ( E ). H , Western blot for DPF3 and beta actin from lysates of primary renal tubular cells (PTC) transfected either with nontargeting (nt) guides or guides targeting DPF3. Cells were harvested at day 3 after transfection. I , cyst volume of PTC 7 days after plating in a Matrigel matrix (n = 3 wells per condition, 4 quadrants per well). J , representative images of the cysts in the Matrigel matrix. Values are mean ± SD. t test, ∗ p < 0.05, ∗∗ p < 0.01 for F , G , and I . CA9, carbonic anhydrase 9; ccRCC, clear cell renal cell carcinoma; DMOG, dimethyloxalylglycine; DPF3, double PHD fingers 3; huPTC, human urinary primary tubular cells.

Article Snippet: ChIP experiments were performed as previously described using antibodies directed against HIF-1α (Cay10006421, rabbit polyclonal, Cayman Chemicals), HIF-1β (NB100–110, rabbit polyclonal, Novus Biologicals), RNApol 2 (SC-899, rabbit polyclonal, Santa Cruz), or H3K27ac (ab4729, rabbit polyclonal, Abcam) ( , ).

Techniques: Expressing, Isolation, Marker, Activity Assay, Derivative Assay, Western Blot, Clone Assay, Transfection, Proliferation Assay

Journal: iScience

Article Title: Roles of class III phosphatidylinositol 3-kinase, Vps34, in phagocytosis, autophagy, and endocytosis in retinal pigmented epithelium

doi: 10.1016/j.isci.2025.112371

Figure Lengend Snippet: Identification and phosphoinositide contents of autophagy-associated membrane aggregates in chloroquine-treated cultured RPE cells (A–L) In cultured RPE cells treated with 25 μg/mL chloroquine overnight, autophagy marker LC3 accumulated (A–D and H) and co-localized with WIPI2 (C), WIPI1 (D), and Atg9 (H). In these cells p62 and Atg16L co-localized with WIPI2 (E–F), and Atg9 co-localized with p62 (G). PI(3)P was observed in membranes surrounding, but not truly co-localized with, LC3 puncta (B). Probes for PI(4)P (EGFP-FAPP1, (I)), PI(5)P (DsRed-3xING2, (J)), and PI(3,4)P2 (DsRed-TAPP1, (K)) did not co-localize with LC3. A probe for PI(4,5)P2 (EGFP-PLCδ, (L)) was partially found in membranes surrounding LC3 puncta. Line profiles of intensity for the indicated puncta ( insets ) are plotted on the far right . Arrows indicate puncta visible in more than one channel for alignment and comparison in adjacent panels.

Article Snippet: Two tandem copies of Hrs, FAPP1, or TAPP1, three tandem copies of ING2, or one copy of PLCδ phosphoinositide-binding domains, or WIPI1 or WIPI2 cDNA were cloned with N-terminal EGFP or DsRed fusions in pCAG vector (a gift from Connie Cepko (Addgene plasmid #11150)) Matsuda & Cepko, using EcoRI and NotI sites. All constructs were verified by sequencing. , Addgene , #11150.

Techniques: Membrane, Cell Culture, Marker, Comparison