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VMP1 Establishes ER-Microdomains that Regulate Membrane Contact Sites and Autophagy

Tábara, Luis-Carlos, Escalante, Ricardo·2016·PLOS ONE
DOI 10.1371/journal.pone.0166499PMC PMC5115753
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Fig 1 VMP1 is associated to ER sliding events. (A) HeLa and Cos-7 cells transfected with a plasmid encoding VMP1 fused to GFP (VMP1-GFP) and visualized by confocal microscopy show a similar pattern. (B) Analysis of confocal microscopy of Cos-7 cells expressing VMP1-GFP and the ER marker Sec61. The punctated pattern is evident in the peripheral tubules and the perinuclear area. (C) In vivo time-lapse confocal microscopy of cells expressing VMP1-GFP and Sec61-mCherry. Different examples of sliding events are shown in frames. The corresponding movies are available as supplementary material (S1–S3 Movies). (D) ΔNt-VMP1-GFP were transfected in HeLa cells and colocalized with the ER marker Sec61 by confocal microscopy. The protein distributes throughout the ER loosing the punctated pattern in HeLa cells.
Fig 1 VMP1 is associated to ER sliding events. (A) HeLa and Cos-7 cells transfected with a plasmid encoding VMP1 fused to GFP (VMP1-GFP) and visualized by confocal microscopy show a similar pattern. (B) Analysis of confocal microscopy of Cos-7 cells expressing VMP1-GFP and the ER marker Sec61. The punctated pattern is evident in the peripheral tubules and the perinuclear area. (C) In vivo time-lapse confocal microscopy of cells expressing VMP1-GFP and Sec61-mCherry. Different examples of sliding events are shown in frames. The corresponding movies are available as supplementary material (S1–S3 Movies). (D) ΔNt-VMP1-GFP were transfected in HeLa cells and colocalized with the ER marker Sec61 by confocal microscopy. The protein distributes throughout the ER loosing the punctated pattern in HeLa cells.CC BY · PubMed Central
Fig 2 Analysis of colocalizations at the perinuclear area. (A) Confocal microscopy analysis of HeLa cells expressing VMP1-GFP and incubated with antibodies that recognize ERES (Sec31), ERGIC (Ergic53) and Golgi (GM130 and TGN46). No colocalizations were observed, although some regions showed close proximity. (B) Cells expressing VMP1-GFP were treated with nocodazole prior to fixation and detection of Golgi markers by confocal microscopy. Close localization and some degree of colocalization were observed with the trans-Golgi marker TGN46. The mean and the SD were obtained from two independent experiments (approximately 5–7 cells per experiment).
Fig 2 Analysis of colocalizations at the perinuclear area. (A) Confocal microscopy analysis of HeLa cells expressing VMP1-GFP and incubated with antibodies that recognize ERES (Sec31), ERGIC (Ergic53) and Golgi (GM130 and TGN46). No colocalizations were observed, although some regions showed close proximity. (B) Cells expressing VMP1-GFP were treated with nocodazole prior to fixation and detection of Golgi markers by confocal microscopy. Close localization and some degree of colocalization were observed with the trans-Golgi marker TGN46. The mean and the SD were obtained from two independent experiments (approximately 5–7 cells per experiment).CC BY · PubMed Central
Fig 3 VMP1 establishes close contacts with organelles. (A) Confocal analysis of colocalization of VMP1-GFP expressing HeLa cells with different organelle markers as specified in the images. Similar results were obtained in Cos-7 cells (not shown). (B) In vivo time-lapse microscopy showing examples of concerted movement between VMP1 and mitochondria, lipid droplets and endosomes (corresponding to S4–S7 Movies). (C) Quantification of the proportion of VMP1 puncta in contact with organelles (and the proportion of organelles in contact with VMP1). The mean and standard deviation (SD) were obtained from three independent transfections (approximately10 cells per transfection were analyzed). (D) Triple colocalization of VMP1 puncta with mitochondria and endosomes. VMP1 puncta colocalizing or in close proximity to mitochondria and endosomes are marked by M or EE respectively. Triple colocalizations are marked by arrows.
Fig 3 VMP1 establishes close contacts with organelles. (A) Confocal analysis of colocalization of VMP1-GFP expressing HeLa cells with different organelle markers as specified in the images. Similar results were obtained in Cos-7 cells (not shown). (B) In vivo time-lapse microscopy showing examples of concerted movement between VMP1 and mitochondria, lipid droplets and endosomes (corresponding to S4–S7 Movies). (C) Quantification of the proportion of VMP1 puncta in contact with organelles (and the proportion of organelles in contact with VMP1). The mean and standard deviation (SD) were obtained from three independent transfections (approximately10 cells per transfection were analyzed). (D) Triple colocalization of VMP1 puncta with mitochondria and endosomes. VMP1 puncta colocalizing or in close proximity to mitochondria and endosomes are marked by M or EE respectively. Triple colocalizations are marked by arrows.CC BY · PubMed Central
Fig 4 VMP1 is an ER protein in close proximity to vesicles and mitochondria. Cells expressing VMP1-GFP were subjected to inmuno-gold labeling with an antibody against GFP and visualized by TEM. Labeling is found in nuclear membrane (NM) (A) and ER tubules (B) as well as in close proximity to vesicles (C) and mitochondria (M)
Fig 4 VMP1 is an ER protein in close proximity to vesicles and mitochondria. Cells expressing VMP1-GFP were subjected to inmuno-gold labeling with an antibody against GFP and visualized by TEM. Labeling is found in nuclear membrane (NM) (A) and ER tubules (B) as well as in close proximity to vesicles (C) and mitochondria (M)CC BY · PubMed Central
Fig 5 VMP1 associates simultaneously with organelles and autophagosomes. (A) Confocal microscopy analyses showing the level of colocalization of VMP1 puncta with the omegasome marker DFCP1(detected by expression of DFCP1-HA and subsequent inmunolocalization using anti-HA antibody) and LC3 (detected with LC3 Ab). Cells were under starvation conditions to induce autophagy (EBSS during 2 hours) (B) Quantification of VMP1 colocalizations with DFCP1 and LC3. The mean and standard deviation (SD) were obtained from three independent transfections (approximately 7 cells per transfecction). (C) Confocal analysis of colocalization between VMP1-GFP, autophagosome markers (LC3 or DFCP1) and organelles (Mitochondria labeled by MitoBlue, ERES detected by Sec31 Ab and early endosomes labeled by Rab5-mCherry). (D, E) Quantification of colocalization between VMP1 and mitochondria or endosomes as indicated in the figure. The mean and SD were obtained from 2 independent transfections (approximately 10 cells per transfection).
Fig 5 VMP1 associates simultaneously with organelles and autophagosomes. (A) Confocal microscopy analyses showing the level of colocalization of VMP1 puncta with the omegasome marker DFCP1(detected by expression of DFCP1-HA and subsequent inmunolocalization using anti-HA antibody) and LC3 (detected with LC3 Ab). Cells were under starvation conditions to induce autophagy (EBSS during 2 hours) (B) Quantification of VMP1 colocalizations with DFCP1 and LC3. The mean and standard deviation (SD) were obtained from three independent transfections (approximately 7 cells per transfecction). (C) Confocal analysis of colocalization between VMP1-GFP, autophagosome markers (LC3 or DFCP1) and organelles (Mitochondria labeled by MitoBlue, ERES detected by Sec31 Ab and early endosomes labeled by Rab5-mCherry). (D, E) Quantification of colocalization between VMP1 and mitochondria or endosomes as indicated in the figure. The mean and SD were obtained from 2 independent transfections (approximately 10 cells per transfection).CC BY · PubMed Central
Fig 6 VMP1 depletion block starvation-induced autophagy in HeLa and Cos7 cells. (A) Confocal microscopy analysis of control and VMP1-depleted HeLa cells expressing the omegasome marker DFCP1 and the ER marker Sec61. Aberrant morphology of the omegasome marker DFCP1 is shown. Detection of LC3 and the ER-marker Calnexin in control and VMP1-depleted HeLa cells to show the accumulation of LC3 structures at the ER. (B) Detection by confocal microscopy of LC3 and the ER-marker Calnexin of control and VMP1-depleted Cos-7 cells to show the accumulation of LC3 structures at the ER. (C) Triple colocalization of Mitochondria, ER and LC3 in control and VMP1-depleted HeLa cells. Below the quantification of colocalization events between LC3 and mitochondria is shown. All experiments were under starvation conditions to induce autophagy (EBSS during 2 hours). The mean and the SD were obtained from two independent transfections (approximately 10 cells per transfection) and analyzed using the Student t-test.
Fig 6 VMP1 depletion block starvation-induced autophagy in HeLa and Cos7 cells. (A) Confocal microscopy analysis of control and VMP1-depleted HeLa cells expressing the omegasome marker DFCP1 and the ER marker Sec61. Aberrant morphology of the omegasome marker DFCP1 is shown. Detection of LC3 and the ER-marker Calnexin in control and VMP1-depleted HeLa cells to show the accumulation of LC3 structures at the ER. (B) Detection by confocal microscopy of LC3 and the ER-marker Calnexin of control and VMP1-depleted Cos-7 cells to show the accumulation of LC3 structures at the ER. (C) Triple colocalization of Mitochondria, ER and LC3 in control and VMP1-depleted HeLa cells. Below the quantification of colocalization events between LC3 and mitochondria is shown. All experiments were under starvation conditions to induce autophagy (EBSS during 2 hours). The mean and the SD were obtained from two independent transfections (approximately 10 cells per transfection) and analyzed using the Student t-test.CC BY · PubMed Central
Fig 7 VMP1 dependent alterations in ER structure and lipid droplets. (A) TEM image of whorl structures from VMP1-depleted Cos-7 cells. (B) TEM image of altered ER structures in VMP1-depleted cells. (C) To analyze the abnormal ER features the mean and the SD were obtained fromof two independent inhibitions using 50 sections of 25 μm2 for each condition (25 sections per inhibition). (D) Control and VMP1-depleted HeLa and Cos-7 cells were treated with oleic acid and stained for LDs with Bodipy. Representative images of at least three independent experiments are shown. (E) Quantification analysis of LD number and clusters. The mean and the SD were obtained from three independent experiments (approximately 10 cells per experiment). The level of significance were analyzed using the Student t-test.
Fig 7 VMP1 dependent alterations in ER structure and lipid droplets. (A) TEM image of whorl structures from VMP1-depleted Cos-7 cells. (B) TEM image of altered ER structures in VMP1-depleted cells. (C) To analyze the abnormal ER features the mean and the SD were obtained fromof two independent inhibitions using 50 sections of 25 μm2 for each condition (25 sections per inhibition). (D) Control and VMP1-depleted HeLa and Cos-7 cells were treated with oleic acid and stained for LDs with Bodipy. Representative images of at least three independent experiments are shown. (E) Quantification analysis of LD number and clusters. The mean and the SD were obtained from three independent experiments (approximately 10 cells per experiment). The level of significance were analyzed using the Student t-test.CC BY · PubMed Central
Fig 8 VMP1 regulates ER-mitochondria MCS. (A) Hela and Cos-7 cells were transfected with siRNAs of control, Atg5 and VMP1 (two different hairpins), stained with mitotracker and analyzed by confocal microscopy. Alterations in size and morphology of mitochondria were observed in VMP1-depleted cells. Representative images of at least three independent experiments. To the right, a representative western-blot analysis of the mitochondrial protein Tim23 form HeLa and Cos-7 cells treated with the different siRNAs is shown. Representative western blots of three independent experiments are shown. (B) TEM analysis of control and VMP1-depleted Cos-7 cells show altered mitochondria with dilated cristae and abundant MCS (marked by arrows). (C) Several parameters of MCS and mitochondria morphology between control and VMP1-depleted cells are compared. The mean and the SD were obtained from two independent inhibitions (150 and 100 mitochondrial profiles respectively were analyzed). (D) Model of VMP1 function regulating MCS of multiple organelles and the impact in autophagy. Accumulation of PtdIns3P and autophagic structures at the ER as a consequence of enlarged VMP1 depletion.
Fig 8 VMP1 regulates ER-mitochondria MCS. (A) Hela and Cos-7 cells were transfected with siRNAs of control, Atg5 and VMP1 (two different hairpins), stained with mitotracker and analyzed by confocal microscopy. Alterations in size and morphology of mitochondria were observed in VMP1-depleted cells. Representative images of at least three independent experiments. To the right, a representative western-blot analysis of the mitochondrial protein Tim23 form HeLa and Cos-7 cells treated with the different siRNAs is shown. Representative western blots of three independent experiments are shown. (B) TEM analysis of control and VMP1-depleted Cos-7 cells show altered mitochondria with dilated cristae and abundant MCS (marked by arrows). (C) Several parameters of MCS and mitochondria morphology between control and VMP1-depleted cells are compared. The mean and the SD were obtained from two independent inhibitions (150 and 100 mitochondrial profiles respectively were analyzed). (D) Model of VMP1 function regulating MCS of multiple organelles and the impact in autophagy. Accumulation of PtdIns3P and autophagic structures at the ER as a consequence of enlarged VMP1 depletion.CC BY · PubMed Central

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Metadata

Authors
Tábara, L.-C., & Escalante, R.
ORCID
Escalante, Ricardo
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2016-11-18
Volume
11
Issue
11
Pages
e0166499
ISSN
1932-6203
DOI
10.1371/journal.pone.0166499
PMC
PMC5115753
Cited by
89
References
2
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
MINECO (BFU2015-64440-P)

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