Journal: Nature Communications

Article Title: ATP-dependent membrane remodeling links EHD1 functions to endocytic recycling

doi: 10.1038/s41467-018-07586-z

Figure Lengend Snippet: ATP- and curvature-sensitive membrane binding of EHD1. a Schematic of generation of membrane templates used in the study. b Distribution of tube radii from five independent preparations of templates. c Representative fluorescence micrographs showing distribution of EGFP-LactC2 on SLBs and tubes. Asteriks mark the bare glass surface. Scale bars = 5 μm. d Protein:membrane fluorescence ratios of EHD1-EGFP with various nucleotides (blue) and GFP-LactC2 (red) for the indicated numbers of tubes and SLBs sampled. Black lines denote means. Statistical significance was assessed using Mann–Whitney test and **** P < 0.0001. e Protein:membrane fluorescence ratios of AMP-PNP-bound EHD1-EGFP on tubes of varying starting sizes for the indicated numbers of tubes. Red lines denote means. Statistical significance was assessed using Mann–Whitney’s two-tailed test and * P = 0.010, ** P = 0.005, **** P < 0.0001

Article Snippet: LactC2 was a gift from Sergio Grinstein (Addgene plasmid # 22852).

Techniques: Membrane, Binding Assay, Fluorescence, MANN-WHITNEY, Two Tailed Test

Journal: Biomolecules

Article Title: Engineered Fluorescent Variants of Lactadherin C2 Domain for Phosphatidylserine Detection in Flow Cytometry.

doi: 10.3390/biom15050673

Figure Lengend Snippet: Figure 1. Recombinant protein PS sensors used in the current study. (A) Schematic representation of protein constructs. All proteins were N-terminally 6 × His-tagged. LactC2-based sensors contain the coding sequence of the bovine lactadherin C2 domain (residues 270–427). AxV-based sensors contain the coding sequence of human annexin V. (B) Coomassie Blue-stained SDS-PAGE gels of buffer- exchanged recombinant proteins. A total of 600 ng of protein was loaded onto a 12% SDS-PAGE gel. “M” indicates the protein molecular weight marker. Imaging was performed using a ChemiDoc MP Imaging system with Epi Far-Red illumination (Bio-Rad, Hercules, CA, USA). (C) A dot blot analysis of the lactC2-based sensors’ solubility. Three representative spots for the total fraction (T) and soluble fraction (S) for each protein are shown on the left. The dot blot was performed and analyzed as described in Section 2. The percentage of protein yield in the soluble fraction is shown on the right and was calculated as the ratio of the signal from the clarified lysate spots (soluble fraction) to the averaged signal from the crude lysate spots (total fraction). Data are represented as mean ± SEM. A statistical analysis was performed using Student’s t-test (** p < 0.01, *** p < 0.001).

Article Snippet: The coding sequence of the bovine lactadherin C2 domain (lactC2, residues 270–427) was obtained from the Lact-C2-GFP plasmid [22] (the Lact-C2-GFP was a gift from Sergio Grinstein, Addgene plasmid #22852; http://n2t.net/addgene:22852 (assessed on 27 March 2025); RRID:Addgene_22852).

Techniques: Recombinant, Construct, Sequencing, Staining, SDS Page, Molecular Weight, Marker, Imaging, Dot Blot, Solubility

Journal: bioRxiv

Article Title: INPP5E controls ciliary localization of phospholipids and odor response kinetics in a mouse model of Joubert syndrome

doi: 10.1101/451344

Figure Lengend Snippet: Loss of INPP5E causes redistribution of PIP2 and elongation of cilia in mouse OSNs. (A) PLCδ1-PH domain tagged with GFP (PLCPH-GFP), a probe for PIP2, was mostly restricted to the knob of the wild type (WT) OSNs. In a small percentage of OSNs proximal segment of varying length was also enriched in PIP2. Inert membrane bound lipid probe MyrPalm-mCherry was used as a counterstain to label the full length of axoneme opposing highly restricted localization of PLCPH-GFP resulting in overlapping colors (middle panel, white). PIP2 was evenly distributed in the plasma membrane of the knob as shown in z-stack view (right panel). Yellow lines denote z-stack projection shown at the bottom and right side of the image. (B) Opposite to the WT, in INPP5EOMP knockout PLCPH-GFP decorated the entire length of every cilium. This distribution resulted in a complete overlap of PLCPH-GFP and MP-mCherry labeling (middle panels, white color). The PIP2 redistribution is evident also in the z-stack view showing substantial enrichment of the proximal segment of cilia whereby PIP2 level in periciliary plasma membrane was not changed (right panel). (C) More than 50% of WT OSNs showed no PIP2 in their cilia. 18% of OSNs had only a single PIP2 positive cilium whereas three other groups of neurons equally represented remaining 30%. Conversely, PIP2 was detected in 100% of OSNs in INPP5E-OMP KO (green bar). (D) Length distribution of PLCPH-GFP positive aspects of cilia (PIP2 domain) in WT was substantially shifted to shorter values compared to the full cilia length measured with MyrPalmmCherry, yielding 29.5±0.5 µm (n=753). (E) Distribution of both PLCPH-GFP and MyrPalmmCherry length values showed complete overlap in INPP5E-OMP KO OSNs. Average ciliary length in the KO OSNs, 35.3±0.6 µm (n=495) was significantly longer than in the WT (unpaired t-test, t=7.363 df=1246, ****p<0.0001).

Article Snippet: Plasmids containing cDNA fragments were provided as follows (PLCδ1-PH-GFP, Addgene #51407, Btk-GFP Addgene #51463, mCherry-P4M-SidM, Addgene #51471, deposited by Tomas Balla; Tapp1-GFP, a gift from Takeshi Ijuin, Kobe University; D4H-mCherry, a gift from Gregory Fairn, University of Toronto; Lact-C2-GFP, Addgene 22852, deposited by Sergio Grinstein; Eqt2-SM-GFP, a gift from Christopher Burd, Yale University; TULP1 and TULP3, a gift from Saikat Mukhopadhyay, University of Texas Southwestern; Kir2.1, Addgene #32669, deposited by Matthew Nolan; PC2 (PKD2), Addgene #83451, deposited by Thomas Weimbs; Efhc1, a gift from Kazuhiro Yamakawa, RIKEN; IFT122, a gift from Jonathan Eggenschwiler, University of Georgia; GCaMP6f, Addgene 40755, deposited by Douglas Kim).

Techniques: Knock-Out, Labeling

Journal: bioRxiv

Article Title: INPP5E controls ciliary localization of phospholipids and odor response kinetics in a mouse model of Joubert syndrome

doi: 10.1101/451344

Figure Lengend Snippet: Virally induced ectopic expression of full-length wild type human INPP5E tagged with GFP (GFP-INPP5E-WT) completely reversed mislocalization of PIP2 in INPP5E-OMP KO mouse cilia. (A, B) INPP5E-OMP KO mice were infected at P8-P14 with a triple dose of Ad-GFP-INPP5EWT and tested 8-10 days later. GFP-INPP5E-WT is enriched in OSN knobs and also localized to cilia. The KO mice were co-infected with PLCPH-mCherry to measure rescue of the PIP2 localization. Several knobs of co-infected OSNs are indicated with arrowheads. (C) Zoomed-in view of the area marked with a square in (B) shows several knobs of OSNs co-infected with both viruses (arrowheads) resulting in a complete loss of ciliary PIP2 (green). (E) Rescue was quantified by measuring length of PIP2 positive ciliary aspect in the WT littermates and KO mice. The KO OSNs were identified within the same preparation by a strong ciliary distribution of PLCPH-mCherry, also lacking any detectable GFP-INPP5E-WT fluorescence. Rescue completely reversed INPP5E-OMP deficiency (cilia length 4.9±0.27 µm (n=110), WT; 28.5±1.37 µm (n=54), KO; 4.2±0.3µm (n=122), one-way ANOVA, F(DFn, DFd) 86.73 (2,283), ****p<0.0001).

Article Snippet: Plasmids containing cDNA fragments were provided as follows (PLCδ1-PH-GFP, Addgene #51407, Btk-GFP Addgene #51463, mCherry-P4M-SidM, Addgene #51471, deposited by Tomas Balla; Tapp1-GFP, a gift from Takeshi Ijuin, Kobe University; D4H-mCherry, a gift from Gregory Fairn, University of Toronto; Lact-C2-GFP, Addgene 22852, deposited by Sergio Grinstein; Eqt2-SM-GFP, a gift from Christopher Burd, Yale University; TULP1 and TULP3, a gift from Saikat Mukhopadhyay, University of Texas Southwestern; Kir2.1, Addgene #32669, deposited by Matthew Nolan; PC2 (PKD2), Addgene #83451, deposited by Thomas Weimbs; Efhc1, a gift from Kazuhiro Yamakawa, RIKEN; IFT122, a gift from Jonathan Eggenschwiler, University of Georgia; GCaMP6f, Addgene 40755, deposited by Douglas Kim).

Techniques: Expressing, Infection, Fluorescence

Journal: bioRxiv

Article Title: INPP5E controls ciliary localization of phospholipids and odor response kinetics in a mouse model of Joubert syndrome

doi: 10.1101/451344

Figure Lengend Snippet: Other INPP5E-related phosphoinositides in mouse OSNs were almost exclusively restricted to the OSN knobs and change level in the INPP5E dependent manner. (A, D) PI(4)P probe, mCherry-P4M-SidM was not significantly affected by loss of INPP5E showing only insignificant trending decrease in the knobs (179±26 relative units, WT, n=94; 143±17 relative units, KO, n=54; unpaired t-test, t=0.9777 df=146, p=0.3298). (B, E) Tandem PH domain, Tapp1 tagged with GFP was used to specifically label membrane PI(3,4)P2 enriched mostly in the knobs and in cilia only in a small fraction of OSNs. Importantly, overall pattern of PI(3,4)P2 distribution did not change in INPP5E-OMP KO. Fluorescence intensity, however, measured in OSN knobs showed significant decrease in the KO compared to WT mice (280±11 relative units, n=830, WT; 174±7 relative units, n=858; unpaired t-test, t=8.453 df=1686, p<0.0001). (C, F) PIP3 detected with Btk-PH domain tagged with GFP, was restricted mostly to the knobs with relatively low presence in cilia of the WT and KO. Quantitative analysis of fluorescence showed increase of the intensity in the knobs of the KO (668±64 relative units, n=60, WT; 1,495±185 relative units, n=91, KO; unpaired t-test, t=3.536 df=149, ***p=0.0005). (G) Fluorescence intensity of MP-mCherry used as a negative control, was not significantly different in the OSN knobs of WT and KO mice (340±31 relative units, n=46, WT; 378±23 relative units, n=70, KO; unpaired t-test, t=1.001 df=114, p=0.3188).

Article Snippet: Plasmids containing cDNA fragments were provided as follows (PLCδ1-PH-GFP, Addgene #51407, Btk-GFP Addgene #51463, mCherry-P4M-SidM, Addgene #51471, deposited by Tomas Balla; Tapp1-GFP, a gift from Takeshi Ijuin, Kobe University; D4H-mCherry, a gift from Gregory Fairn, University of Toronto; Lact-C2-GFP, Addgene 22852, deposited by Sergio Grinstein; Eqt2-SM-GFP, a gift from Christopher Burd, Yale University; TULP1 and TULP3, a gift from Saikat Mukhopadhyay, University of Texas Southwestern; Kir2.1, Addgene #32669, deposited by Matthew Nolan; PC2 (PKD2), Addgene #83451, deposited by Thomas Weimbs; Efhc1, a gift from Kazuhiro Yamakawa, RIKEN; IFT122, a gift from Jonathan Eggenschwiler, University of Georgia; GCaMP6f, Addgene 40755, deposited by Douglas Kim).

Techniques: Fluorescence, Negative Control

Journal: bioRxiv

Article Title: INPP5E controls ciliary localization of phospholipids and odor response kinetics in a mouse model of Joubert syndrome

doi: 10.1101/451344

Figure Lengend Snippet: Distribution of integral membrane lipids was not changed in OSNs and cilia in INPP5E-OMP KO mice. (A) D4H-mCherry, a cholesterol binding probe was enriched in the proximal segment of olfactory cilia equally in the WT and KO OSNs (arrowheads). Cholesterol was detected albeit at a lower level also in the full-length ciliary axoneme. (B) Phosphatydilserine, probed with C2 motif of lactadherin, was uniformly distributed along the cilia and was also enriched in the dendritic knobs of OSNs. (C) Sphingomyelin specific probe, Eqt2-SMP-GFP was mostly enriched in the OSN knobs and detected at a low level in cilia. (D) Glycosylated phosphatydilinositol was probed in OSNs with a human folate 1 receptor, GPI-GFP which failed to detect any presence in cilia whereby mostly restricted to the knobs in both the WT and INPP5E-OMP KO mouse.

Article Snippet: Plasmids containing cDNA fragments were provided as follows (PLCδ1-PH-GFP, Addgene #51407, Btk-GFP Addgene #51463, mCherry-P4M-SidM, Addgene #51471, deposited by Tomas Balla; Tapp1-GFP, a gift from Takeshi Ijuin, Kobe University; D4H-mCherry, a gift from Gregory Fairn, University of Toronto; Lact-C2-GFP, Addgene 22852, deposited by Sergio Grinstein; Eqt2-SM-GFP, a gift from Christopher Burd, Yale University; TULP1 and TULP3, a gift from Saikat Mukhopadhyay, University of Texas Southwestern; Kir2.1, Addgene #32669, deposited by Matthew Nolan; PC2 (PKD2), Addgene #83451, deposited by Thomas Weimbs; Efhc1, a gift from Kazuhiro Yamakawa, RIKEN; IFT122, a gift from Jonathan Eggenschwiler, University of Georgia; GCaMP6f, Addgene 40755, deposited by Douglas Kim).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: INPP5E controls ciliary localization of phospholipids and odor response kinetics in a mouse model of Joubert syndrome

doi: 10.1101/451344

Figure Lengend Snippet: Soluble and polytopic proteins with affinity to PIP2 mislocalize in olfactory cilia of INPP5E-OMP KO. (A) Tubby-like proteins tagged with GFP, TULP1-GFP and TULP3-GFP were preferentially restricted to the knobs in the WT (upper left panel). Build-up of PIP2 in cilia of the KO resulted in complete redistribution of TULP1 (bottom panels). Note that loss of INPP5E activity led to a depletion of TULP1 within knobs revealing proximal segment of cilia decorated with TULP1-GFP (right bottom panel). Quantification of a percentage of the OSN knobs having TULP1-positive cilia per analyzed image showed significant increase in the KO (25.49±0.06%, n=4, WT; 100%, n=6, KO; Mann-Whitney t-test, p=0.0048). (B) TULP3-GFP, like TULP1, also showed dramatic redistribution between the knob and cilia resulting in a significant increase of percentage of knobs with TULP3-positive cilia (30.87±0.12%, n=6, WT; 100%, n=4, KO; Mann-Whitney t-test, p=0.0095). (C) Potassium inward rectifier ion channel, Kir2.1-mCherry, a polytopic protein with two membrane-spanning loops and a known affinity to PIP2 also changed its ciliary distribution in INPP5E-OMP OSNs. Kir2.1-mCherry moved into the ciliary membrane in a significantly larger fraction of OSNs in the KO (right upper panel, 3.02±0.02%, n=12, WT; 24.34±5.89%, n=10, KO; Mann-Whitney t-test, p=0.0023). (D, E) As a negative control we used a different ion channel, PC2 (PKD2 or TRPP1) tagged with mCherry (mCherry-PC2) and a microtubule binding protein Efhc1 (GFP-Efhc1) both of which, however, did not change their distribution in the knobs of INPP5E-OMP KO (upper panels, WT; bottom panels, KO).

Article Snippet: Plasmids containing cDNA fragments were provided as follows (PLCδ1-PH-GFP, Addgene #51407, Btk-GFP Addgene #51463, mCherry-P4M-SidM, Addgene #51471, deposited by Tomas Balla; Tapp1-GFP, a gift from Takeshi Ijuin, Kobe University; D4H-mCherry, a gift from Gregory Fairn, University of Toronto; Lact-C2-GFP, Addgene 22852, deposited by Sergio Grinstein; Eqt2-SM-GFP, a gift from Christopher Burd, Yale University; TULP1 and TULP3, a gift from Saikat Mukhopadhyay, University of Texas Southwestern; Kir2.1, Addgene #32669, deposited by Matthew Nolan; PC2 (PKD2), Addgene #83451, deposited by Thomas Weimbs; Efhc1, a gift from Kazuhiro Yamakawa, RIKEN; IFT122, a gift from Jonathan Eggenschwiler, University of Georgia; GCaMP6f, Addgene 40755, deposited by Douglas Kim).

Techniques: Activity Assay, MANN-WHITNEY, Negative Control, Binding Assay

Journal: bioRxiv

Article Title: INPP5E controls ciliary localization of phospholipids and odor response kinetics in a mouse model of Joubert syndrome

doi: 10.1101/451344

Figure Lengend Snippet: INPP5E is responsible for shaping the odor-evoked intracellular calcium transient in the knob of OSNs. (A, B) Ectopically expressed GCaMP6F was visualized in the en face preparation of mouse OE by wide-field fluorescence microscopy. Bright spots represent numerous OSN knobs. (B) Stimulation micropipette filled with a mixture of 132 different odorants diluted to 1:10,000 in ACSF was positioned as indicated. A single 100-ms pulse at 10psi pressure generated a plume of fluorescein covering an area over the epithelial surface demarcated by a dotted line. (C) Repetitive application of a single odor pulse (arrowheads) evoked nearly identical responses. GCaMP6F fluorescence corrected for background was calculated as (F-Fo)/Fo. (D) Individual traces measured in more than 100 OSNs across several areas and animals were averaged to create the graph. Traces were normalized to the peak value before averaging. (E, F) Odor-evoked GCaMP6F response had faster decay in INPP5E-OMP KO OSNs than the response in the WT control group (WT: 6.49±0.37 s, n=167; KO: 3.59±0.18 s, n=110, unpaired t-test, t=6.077, df=275, ****p<0.0001). The response in the KO also had faster rising phase (WT: 1.12±0.07 s, n=46; KO: 0.80±0.08 s, n=30, unpaired t-test, t=2.936 df=74, p=0.0044). To calculate termination phase time constant (decay tau) each individual trace was fit to an exponential function. Rise time 10-90% was defined as time to reach from 10% to 90% of the response peak level.

Article Snippet: Plasmids containing cDNA fragments were provided as follows (PLCδ1-PH-GFP, Addgene #51407, Btk-GFP Addgene #51463, mCherry-P4M-SidM, Addgene #51471, deposited by Tomas Balla; Tapp1-GFP, a gift from Takeshi Ijuin, Kobe University; D4H-mCherry, a gift from Gregory Fairn, University of Toronto; Lact-C2-GFP, Addgene 22852, deposited by Sergio Grinstein; Eqt2-SM-GFP, a gift from Christopher Burd, Yale University; TULP1 and TULP3, a gift from Saikat Mukhopadhyay, University of Texas Southwestern; Kir2.1, Addgene #32669, deposited by Matthew Nolan; PC2 (PKD2), Addgene #83451, deposited by Thomas Weimbs; Efhc1, a gift from Kazuhiro Yamakawa, RIKEN; IFT122, a gift from Jonathan Eggenschwiler, University of Georgia; GCaMP6f, Addgene 40755, deposited by Douglas Kim).

Techniques: Fluorescence, Microscopy, Generated