bioimagesUnknownFubito NakatsuThe Journal of Cell Biologyhttps://www.ebi.ac.uk/biostudies/studies/S-JCBD-201206095JCB

Imaging of Golgi complex PtdIns4P in control MEFs using GFP-GOLPH3.

PtdIns(4,5)P2-positive intracellular vesicles, visualized with PH(PLC[delta]) (green), colocalize with the endocytic clathrin machinery component Dynamin 2 (red) in PI4KIII[alpha] KO MEFs.

TIRF microscopic analysis of PI4KIII[alpha] KO MEFs transfected with STIM1-mRFP before the addition of thapsigargin.

F-actin in control MEFs as revealed by staining with fluorescently-labeled phalloidin.

Confocal imaging of live HeLa cells transfected with C6S/C7S/C8S/C9S EFR3A-tdTomato.

PtdIns(4,5)P2-positive intracellular vesicles, visualized with PH(PLC[delta]) (green), does not colocalize with the endosomal marker EEA1 (red) in PI4KIII[alpha] KO MEFs.

TIRF microscopic analysis of control MEFs transfected with STIM1-mRFP 8 min after the addition of 1 µm thapsigargin.

Mislocalization of plasma membrane proteins in cells lacking PI4KIII[alpha]. PI4KIII[alpha] KO MEFs were transfected with EFR3B-GFP and imaged by confocal microscopy.

TIRF microscopic analysis of GFP-M1-PI4KIII[alpha] shows transient visits to the plasma membrane (1 s interval between frames).

TIRF microscopic analysis of GFP fluorescence in HeLa cells treated with siRNA against TTC7A/B and transfected with GFP-M1-PI4KIII[alpha].

Chronic depletion of plasma membrane PtdIns(4,5)P2 from HeLa cells by transfection with a membrane-anchored inositol 5-phosphatase (mRFP-IPP1-CAAX, red, see cell on right) results in diffuse intracellular fluorescence of the PtdIns(4,5)P2 probe (iRFP-PH(PLC[delta], blue), but no PtdIns(4,5)P2-positive intracellular vesicles are apparent 16 h post-transfection. Note that the myristoylated/palmitoylated plasma membrane marker L10-GFP (green) is retained at the plasma membrane.

Mislocalization of plasma membrane proteins in cells lacking PI4KIII[alpha]. PI4KIII[alpha] KO MEFs were transfected with M1R-YFP and imaged by confocal microscopy.

Immunofluorescence labeling of endogenous clathrin in control MEFs, as visualized by confocal microscopy.

Imaging analysis of HeLa cells double-transfected with GFP-M1-PI4KIII[alpha] (green) and EFR3B-HA (red) following anti-HA immunfluorescence labeling.

PtdIns(4,5)P2-positive intracellular vesicles, visualized with PH(PLC[delta]) (green), does not colocalize with the endosomal marker Rab5 (red) in PI4KIII[alpha] KO MEFs.

PtdIns(4,5)P2-positive intracellular vesicles, visualized with PH(PLC[delta]) (green), does not colocalize with the PtdIns3P marker 2xFYVE(Hrs) (red) in PI4KIII[alpha] KO MEFs.

Confocal imaging of live HeLa cells transfected with C5S/C7S/C8S EFR3B-tdTomato.

TIRF microscopic analysis of GFP fluorescence in HeLa cells treated with negative control siRNA and transfected with GFP-M1-PI4KIII[alpha].

PtdIns(4,5)P2-positive intracellular vesicles, visualized with PH(PLC[delta]) (green), does not colocalize with the endosomal marker Rab9 (red) in PI4KIII[alpha] KO MEFs.

TIRF microscopic analysis of GFP fluorescence in HeLa cells treated with siRNA against EFR3A/B and TTC7A/B and transfected with GFP-M1-PI4KIII[alpha].

Imaging of Golgi complex PtdIns4P in PI4KIII[alpha] KO MEFs using GFP-GOLPH3.

Palmitoylation of EFR3B is required for recruitment of TTC7B and PI4KIII[alpha] to the plasma membrane. Spinning-disc confocal imaging analysis of HeLa cells transfected with GFP-M1-PI4KIII[alpha] (green), TTC7B-mCherry (red), and the C5S/C7S/C8S triple mutant of EFR3B-FLAG (blue).

TIRF microscopic analysis of PI4KIIIa KO MEFs transfected with STIM1-mRFP 8 min after the addition of 1 µm thapsigargin.

Confocal imaging of live HeLa cells transfected with TTC7B-GFP.

TIRF microscopic analysis of control MEFs transfected with STIM1-mRFP before the addition of thapsigargin.

Immunofluorescence labeling of AP-2 in control MEFs.

Imaging of PtdIns(4,5)P2 distribution in control MEFs using RFP-PH(PLC[delta]).

PtdIns(4,5)P2-positive intracellular vesicles, visualized with PH(PLC[delta]) (green), colocalize with the endocytic clathrin machinery component epsin 1 (red) in PI4KIII[alpha] KO MEFs.

Immunofluorescence labeling of AP-2 in PI4KIII[alpha] KO MEFs.

Acute depletion of plasma membrane PtdIns(4,5)P2 using an optogenetic approach results in diffuse intracellular fluorescence of the PtdIns(4,5)P2 probe iRFP-PH(PLC[delta]) but no detectable PtdIns(4,5)P2-positive intracellular vesicles. This image is of iRFP-PH(PLC[delta]) fluorescence before light-mediated PtdIns(4,5)P2 depletion.

TIRF microscopic analysis of PI4KIII[alpha] KO MEFs transfected with STIM1-mRFP and GFP-M1*-PI4KIII[alpha] to rescue before the addition of thapsigargin.

Imaging of PtdIns(4,5)P2 distribution in PI4KIII[alpha] KO MEFs using RFP-PH(PLC[delta]).

Acute depletion of plasma membrane PtdIns(4,5)P2 using an optogenetic approach results in diffuse intracellular fluorescence of the PtdIns(4,5)P2 probe iRFP-PH(PLC[delta]) but no detectable PtdIns(4,5)P2-positive intracellular vesicles. This image is of iRFP-PH(PLC[delta]) fluorescence 1 min after light-mediated PtdIns(4,5)P2 depletion.

Localization of GFP-PH(Osh2) in PI4KIII[alpha] KO MEFs.

PtdIns(4,5)P2-positive intracellular vesicles, visualized with PH(PLC[delta]) (green), colocalize with the endocytic clathrin machinery component AP-2 (red) in PI4KIII[alpha] KO MEFs.

TIRF microscopic analysis of GFP fluorescence in HeLa cells treated with siRNA against EFR3A/B and transfected with GFP-M1-PI4KIII[alpha].

Confocal imaging of live HeLa cells transfected with wild-type EFR3A-tdTomato.

Immunofluorescence labeling of endogenous clathrin in PI4KIII[alpha] KO MEFs, as visualized by confocal microscopy.

Imaging of Golgi complex morphology in PI4KIII[alpha] KO MEFs using anti-GM130 immunofluorescence.

Immunofluorescence labeling of epsin 1 in control MEFs.

Imaging analysis of HeLa cells triple-transfected with GFP-M1-PI4KIII[alpha] (green), TTC7B-mCherry (red), and EFR3B-HA (blue). EFR3B-HA immunofluorescence was performed with an anti-HA antibody followed by an Alexa Fluor 647-conjugated anti-rat secondary antibody.

TIRF microscopic analysis of PI4KIII[alpha] KO MEFs transfected with STIM1-mRFP and GFP-M1-PI4KIII[alpha] to rescue before the addition of thapsigargin.

Acute depletion of plasma membrane PtdIns(4,5)P2 using an optogenetic approach results in diffuse intracellular fluorescence of the PtdIns(4,5)P2 probe iRFP-PH(PLC[delta]) but no detectable PtdIns(4,5)P2-positive intracellular vesicles. This image is of iRFP-PH(PLC[delta]) fluorescence 30 min after light-mediated PtdIns(4,5)P2 depletion.

Confocal imaging of HeLa cells transfected with GFP-M1-PI4KIII[alpha].

TIRF microscopic analysis of GFP-M1*-PI4KIII[alpha] shows no transient visits to the plasma membrane (1 s interval between frames).

Colocalization of GFP-PIPK1[gamma] (green) and RFP-PH(PLC[delta]) (red) on intracellular sites in PI4KIII[alpha] KO MEFs as assessed by confocal microscopy.

Acute depletion of plasma membrane PtdIns(4,5)P2 using an optogenetic approach results in diffuse intracellular fluorescence of the PtdIns(4,5)P2 probe iRFP-PH(PLC[delta]) but no detectable PtdIns(4,5)P2-positive intracellular vesicles. This image is of iRFP-PH(PLC[delta]) fluorescence 60 min after light-mediated PtdIns(4,5)P2 depletion.

Confocal imaging of live HeLa cells transfected with EFR3B-tdTomato.

TIRF microscopic analysis of PI4KIII[alpha] KO MEFs transfected with STIM1-mRFP and GFP-M1*-PI4KIII[alpha] to rescue 8 min after the addition of 1 µm thapsigargin.

Imaging analysis of HeLa cells double-transfected with TTC7B-mCherry (red) and EFR3B-HA (green) following anti-HA immunfluorescence labeling.

Imaging of Golgi complex morphology in control MEFs using anti-GM130 immunofluorescence.

TIRF microscopic analysis of GFP fluorescence in HeLa cells treated with negative control siRNA and transfected with GFP-M1*-PI4KIII[alpha].

Localization of GFP-PH(Osh2) in control MEFs.

Immunofluorescence labeling of epsin 1 in PI4KIII[alpha] KO MEFs.

Mislocalization of plasma membrane proteins in cells lacking PI4KIII[alpha]. PI4KIII[alpha] KO MEFs were transfected with L10-GFP and imaged by confocal microscopy.

TIRF microscopic analysis of PI4KIIIa KO MEFs transfected with STIM1-mRFP and GFP-M1-PI4KIIIa to rescue 8 min after the addition of 1 µm thapsigargin.

F-actin in PI4KIII[alpha] KO MEFs as revealed by staining with fluorescently-labeled phalloidin.

Palmitoylation of EFR3B is required for recruitment of TTC7B and PI4KIII[alpha] to the plasma membrane. Spinning-disc confocal imaging analysis of HeLa cells transfected with GFP-M1-PI4KIII[alpha] (green), TTC7B-mCherry (red), and wild-type EFR3B-FLAG (blue).

PtdIns(4,5)P2-positive intracellular vesicles, visualized with PH(PLC[delta]) (green), do not colocalize with the Golgi PtdIns4P marker PH(OSBP) (red) in PI4KIII[alpha] KO MEFs.

Confocal imaging of HeLa cells transfected with GFP-M1*-PI4KIII[alpha].

bioimagesImage 139031 (Figure 7 - G)Figure 8 - AFigure 8 - BFigure 3 - NoneImage 139021 (Figure 6 - I)Image 139005 (Figure 4 - C)Image 139013 (Figure 6 - A and B)Image 139280 (Figure 7 - C)Supplemental Figure 2 - ASupplemental Figure 2 - BImage 139398 (Figure 1 - E)Image 139007 (Figure 5 - G)Image 138986 (Figure 2 - C)Image 139056 (Supplemental Figure 5 - A)Image 139291 (Supplemental Figure 2 - B)Figure 1 - AImage 139284 (Figure 8 - A)Figure 1 - EImage 139011 (Figure 5 - D)Image 139285 (Figure 8 - A)Figure 6 - A and BImage 139023 (Figure 6 - J)Image 139027 (Figure 7 - A)Image 139001 (Figure 3 - None)Image 138999 (Figure 3 - None)Image 139032 (Figure 7 - G)Image 139052 (Figure 8 - B)Image 139010 (Figure 5 - D)Image 139003 (Figure 4 - C)Image 138964 (Figure 1 - A)Figure 6 - G and HFigure 5 - FImage 139008 (Figure 5 - F)Figure 5 - GImage 139054 (Figure 8 - B)Image 139399 (Figure 1 - E)Figure 5 - DImage 139288 (Figure 8 - A)Figure 5Figure 4Figure 7Image 139293 (Figure 2 - E)Figure 6Figure 8Figure 1Supplemental Figure 5 - AFigure 3Figure 2Supplemental Figure 5Supplemental Figure 2Figure 7 - AFigure 7 - CImage 139286 (Figure 8 - A)Figure 7 - GFigure 7 - HFigure 6 - C and DImage 139025 (Figure 6 - K)Image 139009 (Figure 5 - F)Image 139283 (Figure 8 - A)Image 138987 (Figure 2 - C)Image 138998 (Figure 3 - None)Image 139290 (Supplemental Figure 2 - A)Figure 2 - EFigure 2 - FImage 139006 (Figure 5 - G)Image 139030 (Figure 7 - G)Figure 2 - CFigure 2 - DImage 139014 (Figure 6 - C and D)Image 139055 (Supplemental Figure 5 - A)Image 139282 (Figure 8 - A)Image 139017 (Figure 6 - E and F)Figure 6 - E and FImage 139019 (Figure 6 - G and H)Image 139002 (Figure 3 - None)Figure 4 - CImage 139287 (Figure 8 - A)Figure 6 - KImage 139018 (Figure 6 - G and H)Image 139033 (Figure 7 - G)Image 139015 (Figure 6 - C and D)Figure 6 - IImage 139294 (Figure 2 - F)Figure 6 - JImage 139012 (Figure 6 - A and B)Image 139053 (Figure 8 - B)Image 139024 (Figure 6 - K)Image 138965 (Figure 1 - A)Image 139020 (Figure 6 - I)Image 139016 (Figure 6 - E and F)Image 139000 (Figure 3 - None)Image 139022 (Figure 6 - J)Image 139026 (Figure 7 - A)Image 139292 (Figure 2 - D)Image 139004 (Figure 4 - C)Image 139289 (Figure 8 - A)Image 139281 (Figure 7 - H)Lukas B. TannerMingming HaoMarkus R. WenkFubito NakatsuGuanghou ShuiMichelle PirruccelloSang Yoon LeeNicholas T. IngoliaJeeyun ChungPietro De CamilliJeremy M. BaskinfalsePtdIns4P synthesis by PI4KIIIα at the plasma membrane and its impact on plasma membrane identity2012-12-10T11:23:39Z2018-11-29T11:23:39Z2018-11-29T11:23:39ZS-JCBD-20120609510.1083/jcb.201206095