EMBO/soda-nel huggingface dataset:
Mitochondria regulate autophagy by conserved signalling pathways (2011)
Figure 4

Role of TORC1 in autophagy regulation under amino‐acid starvation. (A) Wild‐type, rho0, Δnpr2, and Δnpr2 rho0 cells expressing prATG8‐GFP‐ATG8 (upper panel) or prATG8‐GFP (lower panel) were exposed to amino‐acid starvation medium supplemented with galactose in the absence or presence of rapamycin. Samples were analysed as described in Figure 1A. The means and s.d. of four (n=4) independent experiments are indicated. (B) Wild‐type, rho0, Δnpr2, and Δatg7 cells expressing prNPR1‐NPR1‐HA were exposed to amino‐acid starvation medium supplemented with galactose in the absence (upper panels) or presence (lower panels) of rapamycin. The hyperphosphorylated (Npr1‐P) and dephosphorylated (Npr1) forms of Npr1 are indicated. Cells were analysed at indicated time points by whole cell extraction and western blot analysis using α‐HA and α‐Cdc11 antibodies. (C) Wild‐type, rho0, Δnpr2, and Δatg7 cells were exposed to amino‐acid starvation medium supplemented with galactose. Additionally, wild‐type cells were treated with rapamycin during starvation (left panel). Phosphorylated (Atg13‐P) and dephosphorylated (Atg13) Atg13 was monitored at indicated time points by whole cell extraction and western blot analysis using α‐Atg13 and α‐Cdc11 antibodies.
Mitochondria regulate autophagy by conserved signalling pathways (2011)
Figure 5

Mitochondrial function controls autophagy by modulating PKA activity. (A) PKA‐dependent regulation of the autophagic response under amino‐acid starvation. Wild‐type, rho0, pka, and ras2G19V‐expressing cells harbouring prATG8‐GFP‐ATG8 (upper panels) or prATG8‐GFP (lower panels) were exposed to amino‐acid starvation medium supplemented with galactose. PKA activity in pka was inhibited by addition of 1NM‐PP1 (PP1; 1 μg/ml). Samples were analysed as described in Figure 1A; autophagic flux was determined after 3 and 6 h. (B) In vivo activity of PKA. Wild‐type, rho0, pka, and ras2G19V‐expressing cells harbouring 6xMYC‐cki12−200(S125/130A) (Cki1) were grown in galactose medium. When indicated, wild‐type cells were grown in galactose medium in the presence of antimycin A (AA) or oligomycin (O) for 6 h. PKA‐dependent phosphorylation of Cki1 was analysed by whole cell extraction and western blot analysis using a α‐Myc antibody (upper panels). Ratio of phosphorylated (Cki1‐P) and non‐phosphorylated (Cki1) forms of Cki1 relative to wild‐type cells (wt=1) (lower panels). (C) PKA inhibition restores autophagic flux, but not ATG8 induction in the presence of mitochondrial dysfunction. Wild‐type, rho0, pka, and pka rho0 cells expressing prATG8‐GFP‐ATG8 were treated as described in (A). Samples were analysed as described in Figure 1A. The means and s.d. of four (n=4) independent experiments are indicated in (A–C).
Mitochondria regulate autophagy by conserved signalling pathways (2011)
Figure 6

Mitochondrial respiratory deficiency impairs PAS recruitment of the Atg1–Atg13 complex under amino‐acid starvation. (A) ATG8 induction is independent of the Atg1–Atg13 complex and autophagic flux. Wild‐type, rho0, Δatg1, Δatg7, Δatg9, and Δatg11 cells harbouring prATG8‐GFP‐ATG8 were analysed as described in Figure 1A. The means and s.d. of four (n=4) independent experiments are indicated. (B) Atg1 and Atg13 recruitment to the PAS depends on mitochondrial function. Wild‐type and rho0 cells harbouring prATG8‐GFP‐ATG8 and prATG1‐ATG1‐mCherry (upper panels) or prATG13‐ATG13‐mCherry (lower panels) were exposed to amino‐acid starvation medium supplemented with galactose for 3 h. Wild‐type cells were treated with antimycin A (AA 30′) after 2.5 h of starvation for 30 min or with oligomycin (O) for 3 h of starvation. Arrowheads indicate the position of GFP‐Atg8 puncta. Transmission and fluorescence light microscopy images were superimposed to visualize cellular boundaries. Scale bar represents 1.5 μm. (C) Steady‐state levels of Atg1‐ and Atg13‐mCherry during amino‐acid starvation. Wild‐type, rho0, and Δatg7 cells expressing prATG1‐ATG1‐mCherry (upper panels) or prATG13‐ATG13‐mCherry (lower panels) were exposed and treated as described in (B) and analysed by whole cell extraction and western blot analysis using α‐dsRed and α‐Cdc11 antibodies.
Atg1‐mediated myosin II activation regulates autophagosome formation during starvation‐induced autophagy (2010)
Figure 1

Atg1 induces myosin II activation and spaghetti‐squash activator (Sqa) identification. (A–F) Atg1‐induced myosin II activation depends on the kinase activity of Atg1. Third‐instar wing imaginal discs from ptc‐GAL4 UAS‐GFP controls or flies expressing indicated transgenes were stained with phospho‐MRLC (blue) and TRITC‐labelled phalloidin (red). Low level of phospho‐MRLC staining was observed in controls (A) and cells overexpressing kinase‐deficient Atg1‐KR (C), whereas a robust increase in phospho‐MRLC was found in cells overexpressing Atg1 (B). Co‐expression of Atg1 and spaghetti‐squash (SqhA20A21) exhibited low level of phospho‐MRLC staining (D). Atg1‐induced high level of phospho‐MRLC staining was not suppressed by expression of caspase inhibitor p35 (E) or by depletion of Atg12 (Atg12RNAi) (F). Bar, 20 μm. (G) Schematic diagram of Drosophila and mammalian MLCK family. The quantities within the kinase domains indicate the degree of amino acid identity to the kinase domain of Sqa (CG1776). Ank, ankyrin repeats; Death, death domain; Fn, fibronectin domain; Ig, immunoglobulin domain; NLS, nuclear localization signal. (H) Sqa, but not Atg1, directly phosphorylated Sqh in vitro. Flag–Atg1, Flag–Atg1‐KR, HA–Sqa and HA–Sqa‐KA were immunoprecipated from lysate of transfected cells and incubated in an in vitro kinase reaction mixture containing [γ‐32P]ATP and bacterially expressed recombinant wild‐type Sqh or SqhA20A21. As shown on the autoradiogram (top panel), wild‐type but not the kinase‐deficient Atg1 (Atg1‐KR) and Sqa (Sqa‐KA) was autophosphorylated. No phosphorylation was seen with SqhA20A21. The equal input of His‐fusion proteins is shown on the Coomassie staining. Anti‐Flag and anti‐HA immunoblottings (IBs) were used as controls to quantify the amount of proteins precipitated.
Atg1‐mediated myosin II activation regulates autophagosome formation during starvation‐induced autophagy (2010)
Figure 2

Genetic and biochemical interactions between Atg1 and spaghetti‐squash activator (Sqa). (A–K) Genetic interactions between Atg1, Sqa, and spaghetti‐squash (Sqh). Compared with the ptc‐GAL4 controls (A), expression of Atg1 or Sqa by ptc‐GAL4 resulted in missing anterior cross‐vein phenotypes (B, C). However, RNAi‐mediated downregulation of Atg1 (Atg1RNAi) or Sqa (SqaRNAi) did not cause wing vein defects (D, E). Depletion of Atg1 and Sqa suppressed Atg1 and Sqa‐induced wing vein defects, respectively (F, I). Atg1‐induced wing defects were modulated by depletion of Sqa (G) or by co‐expression of SqhA20A21 (H). Nevertheless, Sqa‐induced wing vein defects were suppressed by co‐expression of SqhA20A21 (J) but not Atg1RNAi (K). (L) Schematic presentation of the domain structures of Sqa and deletion mutants. (M–N) Atg1 physically interacted with Sqa. (M) 293T cells were transfected with HA‐tagged Sqa WT (wild‐type) or KA, together with Flag‐tagged Atg1 WT or KR. The cells were lysed 48 h after transfection and immunoprecipitated (IP) with anti‐Flag antibodies. The immunoprecipitated proteins and the total cell lysates (TCL) were analysed by immunoblotting (IB) with antibodies as indicated. (N) 293T cells transfected with Flag–Atg1‐KR and various HA‐tagged Sqa contracts were subjected to immunoprecipitations with anti‐HA antibody. The immunoprecipitated proteins and the total cell lysates were analysed by immunoblotting with antibodies as indicated. (O) Atg1 directly phosphorylated Sqa in vitro. Flag‐tagged Atg1 WT or KR immunoprecipated from lysate of transfected cells was used to phosphorylate bacterially expressed recombinant Sqa‐K1, Sqa‐K2, and Sqa‐C in an in vitro kinase assay. The lower panels represent equal input of His‐fusion proteins and Atg1 immunoprecipitates.
Atg1‐mediated myosin II activation regulates autophagosome formation during starvation‐induced autophagy (2010)
Figure 3

Phosphorylation of spaghetti‐squash activator (Sqa) at Thr‐279 is required for Atg1‐mediated myosin II activation. (A) Characterization of Atg1‐dependent phosphorylation sites on Sqa. 293T cells transfected with Flag‐tagged Atg1 or Atg1‐KR were subjected to immunoprecipitation with anti‐Flag antibodies, followed by in vitro kinase assays with bacterially expressed Sqa‐K2, Sqa‐K2‐T194A, Sqa‐K2‐T239A, and Sqa‐K2‐T279A as substrates. Atg1 but not Atg1‐KR was autophosphorylated (top panel). Relative phosphorylation levels of substrates were quantified. Data are represented as mean±s.e. of triplicates. (B–D) Phosphorylation of Sqa at Thr‐279 is critical for the kinase activity of Sqa. (B) HA‐tagged Sqa or Sqa‐T279A immunoprecipated from lysate of transfected cells was used to phosphorylate bacterially expressed recombinant spaghetti‐squash (Sqh) WT and A20A21, in an in vitro kinase assay. (C) Clonal expression of Sqa but not Sqa‐T279A (GFP‐positive cells) in the larval wing imaginal discs resulted in a marked increase in phospho‐MRLC staining (blue) and actin reorganization (red). Bar, 20 μm. (D) The larval fat body of denoted genotypes were dissected, lysed, and subjected to western blot analysis using anti‐phospho‐MRLC and anti‐Sqh antibodies. For quantification, the levels of MRLC phosphorylation in each genotype were measured with ImageJ and normalized to the Sqh levels. Data are expressed as a fold change compared with the wild‐type controls. Each value represents mean±s.e. from triplicate experiments. See Supplementary data for genotypes.
Atg1‐mediated myosin II activation regulates autophagosome formation during starvation‐induced autophagy (2010)
Figure 4

Atg1–spaghetti‐squash activator (Sqa)‐mediated myosin II activation is required for starvation‐induced autophagy. (A, B) Activation of myosin II on nutrient deprivation. The larval fat body of denoted genotypes under fed or starved conditions were dissected, lysed, and subjected to western blot analysis using antibodies specific for phospho‐myosin regulatory light chain (MRLC) and total MRLC. The Rheb, SqaRNAi, Sqa‐T279A, spaghetti‐squash (SqhA20A21), and Atg7RNAi transgenes were expressed under the control of hs–GAL4 driver (B). For quantification, the relative phosphorylation levels of MRLC were quantified as in Figure 3D. Data are represented as mean±s.e. of triplicates. (C–J) Starvation‐induced autophagosome formation was compromised by inhibition of myosin II activation. Compared with the fed condition (C), starvation induced a robust formation of GFP–Atg8a puncta in larval fat‐body cells (D). Clonal expression of Atg12RNAi (E), Sqa‐T279A (F), SqaRNAi (G), or SqhA20A21 (H) markedly suppressed GFP–Atg8a puncta formation during starvation. The autophagic defects caused by Sqa‐T279A and SqaRNAi were rescued by co‐expression of the constitutively active SqhE20E21 (I) and SqhD20D21 (J), respectively. Bar, 20 μm. (K) The average number of GFP–Atg8a marked autophagosomes per cell is shown (data are represented as mean±s.e. of 20 fat‐body samples imaged per genotype; ***P0.001). (L) Inhibition of myosin II activity increased sensitivity to starvation. Flies carrying transgenes under the control of fat body‐specific Cg–GAL4 driver were used for further analysis. Histogram illustrating the survival curve of adult female flies of denoted genotypes when placed under starved conditions. Data are mean±s.e. from triplicate experiments (n=100 flies/genotype/treatment). *P0.05, **P0.01, ***P0.001. See Supplementary data for genotypes.
Atg1‐mediated myosin II activation regulates autophagosome formation during starvation‐induced autophagy (2010)
Figure 5

UNC‐51‐like kinase (Ulk1) and zipper‐interacting protein kinase (ZIPK) are essential for starvation‐induced myosin II activation. (A) Ulk1 interacted with ZIPK. 293T cells transfected with HA‐tagged ZIPK together with Flag‐tagged Ulk1 or Ulk1‐KI were subjected to immunoprecipitations with anti‐Flag antibody. The immunoprecipitated proteins and the total cell lysates were analysed by immunoblotting with antibodies as indicated. (B) HA‐tagged kinase‐inactive ZIPK‐KA (K42A) was expressed in 293T cells with Flag‐tagged Ulk1 or Ulk1‐KI. Cell lysates were incubated with or without alkaline phosphatase (CIP) and analysed by immunoblotting with antibodies as indicated. (C) Myosin II was activated upon nutrient deprivation in MCF7 cells. MCF7 cells were cultured in serum containing DMEM medium (nutrient‐rich condition) or in Earle's balanced salt solution (EBSS; starved condition) for 2 h. The myosin II activity was significantly downregulated after EBSS‐starved cells were replenished with fresh DMEM medium containing 10% FBS for 2 h. The myosin II activity was quantified and expressed as a fold changes compared with the DMEM controls. Each value represents mean±s.e. of triplicates. (D) MCF7 cells stably infected with lentivirus expressing control (shLuc), Ulk1 or ZIPK shRNA were cultured in nutrient‐rich DMEM medium (F) or EBSS (S) for 2 h. Effects of Ulk1 and ZIPK knockdown on starvation‐induced myosin II activation were analysed by immunoblotting with antibodies as indicated. Data are mean±s.e. of triplicates.
Atg1‐mediated myosin II activation regulates autophagosome formation during starvation‐induced autophagy (2010)
Figure 6

Involvement of zipper‐interacting protein kinase (ZIPK) and myosin II in starvation‐induced autophagy. (A) MCF7/GFP–LC3 cells stably infected with lentivirus expressing control (shLuc), ZIPK, or non‐muscle myosin heavy chain‐IIA (NMHC‐IIA) shRNA were cultured in nutrient‐rich medium (DMEM) or starvation medium (Earle's balanced salt solution; EBSS) in the presence or absence of lysosomal inhibitor bafilomycin A1 (BafA1) for 2 h. Depletion of ZIPK and NMHC‐IIA markedly inhibited starvation‐induced GFP–LC3 puncta formation. Quantification of the number of GFP–LC3 dots per cell (lower panel) was shown (data are represented as mean±s.e. of 100 cells, ***P0.001). (B) Cells as in (A) were cultured in nutrient‐rich medium (F) or EBSS (S) with or without BafA1 for 2 h. Effects of ZIPK and NMHC‐IIA knockdown on starvation‐induced GFP–LC3 conversion were assessed by immunoblotting with anti‐LC3, anti‐ZIPK, anti‐NMHC‐IIA, and anti‐tubulin antibodies. The relative ratio of LC3II/LC3I is shown at the right panel. Data are mean±s.e. of triplicates.
Atg1‐mediated myosin II activation regulates autophagosome formation during starvation‐induced autophagy (2010)
Figure 7

Redistribution of myosin II during nutrient starvation. (A) The fat body of spaghetti‐squash (sqhAX3); sqh–GFP larva under fed or starved conditions were dissected and subjected to immunofluorescence analysis. Sqh–GFP and phospho‐myosin regulatory light chain (MRLC) were enriched in cell–cell junction under nutrient‐rich (fed) condition. Under starvation conditions, Sqh–GFP and phospho‐MRLC were localized to both cell–cell junction and perinuclear region. Actin was stained with TRITC‐labelled phalloidin (red) and nucleus was stained with DAPI (blue). Bar, 20 μm. (B) Starvation induced redistribution of myosin II in MCF7/GFP–LC3 cells. Both phospho‐MRLC and MRLC were localized in peripheral region of cells in nutrient‐rich medium (DMEM), whereas they redistributed to the perinuclear region and co‐localized with GFP–LC3 under starvation conditions (Earle's balanced salt solution; EBSS). Bar, 10 μm. (C) MCF7/GFP–LC3 cells cultured in nutrient‐rich (DMEM) or starved (EBSS) conditions were homogenized and subjected to centrifugation, and the resulting post‐nuclear supernatant (PNS) was fractionated by high‐speed centrifugation into membrane pellet and cytosol. Proteins were resolved by SDS–PAGE and immunoblotted with anti‐TGN46 antibody as a control for membrane‐association proteins, anti‐caspase‐3 as a control for cytosolic proteins. The levels of phospho‐MRLC and MRLC in each fraction were quantified using ImageJ and plotted relative to their amounts in PNS (n=3). Each value represents mean±s.e. of three experiments. *P0.05, **P0.01.
Atg1‐mediated myosin II activation regulates autophagosome formation during starvation‐induced autophagy (2010)
Figure 8

The starvation‐dependent cycling of mammalian Atg9 (mAtg9) is blocked by inhibition of myosin II activity. (A) Immunofluorescence analysis of GFP–mAtg9 and phospho‐myosin regulatory light chain (MRLC) in MCF7/GFP–mAtg9 cells. GFP–mAtg9 was enriched in the trans‐Golgi network (TGN) labelled with anti‐TGN46 antibody and phospho‐MRLC localized to cell peripheral in nutrient‐rich condition (DMEM). GFP–mAtg9 redistributed and co‐localized with phospho‐MRLC in starvation condition (Earle's balanced salt solution; EBSS). (B) mAtg9 interacted with non‐muscle myosin heavy chain‐IIA (NMHC‐IIA) under starvation conditions. 293T cells (right panel) or 293T cells stably infected with lentivirus expressing control (shLuc), UNC‐51‐like kinases (Ulk1) or zipper‐interacting protein kinase (ZIPK) shRNA (left panel) were transfected with V5‐tagged mAtg9 and GFP–tagged NMHC‐IIA. At 48 h after transfection, cells were incubated in either serum‐containing medium (DMEM) or EBSS for 2 h with or without the treatment of 50 μM ML‐7 and blebbistatin (Blebb). Cell lysates were immunoprecipitated with anti‐V5 antibody and immunoblotted with indicated antibodies. (C) MCF7/GFP–mAtg9 cells were stably infected with lentivirus expressing control (shLuc), ZIPK, or NMHC‐IIA shRNA. Under starved conditions, mAtg9 was redistributed from the TGN to a dispersed peripheral pool in control cells (shLuc). Starvation‐induced GFP–mAtg9 redistribution was blocked in ZIPK and NMHC‐IIA knockdown cells. The images were analysed by quantifying mAtg9 localization in cells for either a TGN‐enriched or dispersed pattern. Data are represented as mean±s.e. of 70 cells. *P0.05, **P0.01. Bar, 10 μm.
LC3 and GATE‐16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis (2010)
Figure 1

The LC3 protein subfamily is essential for autophagy. (A) HeLa cells stably expressing GFP‐LC3B (left panel) or GFP‐GATE‐16 (right panel) were transfected with either non‐targeting siRNA (control siRNA) or a pool of LC3 siRNAs (A, B, C), using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in EBSS medium in the absence or presence of 0.1 μM Baf A and subjected to western blot analysis after lysis with RIPA extraction buffer. (B) Quantification of relative p62 and GFP‐GATE‐16 level (upper panel) and degradation (lower panel) was performed as described in ‘Materials and methods’. Mean±s.d. of three independent experiments is presented. (C) HeLa cells stably expressing GFP‐GATE‐16 were transfected with non‐targeting siRNA or a pool of LC3 siRNAs and treated as in (A). The cells were subjected to immunostaining with anti‐p62 after fixation, and analysed by confocal microscopy. The GFP intensity of three independent experiments was measured as described in ‘Materials and methods’ (right panel). Scale bar: 20 μm.
LC3 and GATE‐16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis (2010)
Figure 2

The GABARAP/GATE‐16 protein subfamily is essential for autophagy. (A) HeLa cells stably expressing GFP‐GATE‐16 (left panel) or GFP‐LC3B (right panel) were transfected with either non‐targeting siRNA (control siRNA) or a pool of GABARAP siRNAs (GABARAP, GABARAPL1, and GATE‐16) using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in EBSS medium in the absence or presence of 0.1 μM Baf A and subjected to western blot analysis after lysis with RIPA extraction buffer. (B) Quantification of relative p62 and GFP‐LC3B level (upper panel) and degradation (lower panel) were performed as described in ‘Materials and methods’. Mean±s.d. of three independent experiments is presented. (C) HeLa cells stably expressing GFP‐LC3B were transfected with non‐targeting siRNA or a pool of GABARAP siRNAs and treated as in (A). The cells were subjected to immunostaining with anti‐p62 after fixation, and analysed by confocal microscopy. The GFP intensity of three independent experiments was measured as described in ‘Materials and methods’ (right panel). Scale bar: 20 μm.
LC3 and GATE‐16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis (2010)
Figure 3

Both GABARAP/GATE‐16 and LC3 protein subfamilies are essential for autophagic flux. (A) Bulk protein degradation was tested in HeLa cells that were transfected with non‐targeting siRNA (control siRNA), a pool of GABARAP/GATE‐16 siRNAs, or a pool of LC3 siRNAs. The rate of long‐lived protein degradation was measured in cells incubated in either αMEM (control) or EBSS (starvation) medium, in the presence or absence of 10 mM of 3‐MA, 72 h after siRNA transfection. Values expressing the protein degradation percentage are represented as the mean±s.d. of three determinations. (B) HeLa cells stably expressing GFP‐LC3B (left panel) or GFP‐GATE‐16 (right panel) were transfected with a pool of GABARAP/GATE‐16 siRNAs or a pool of LC3, respectively, and with non‐targeting siRNA. Seventy‐two hours after transfection, the cells were collected after 5 h of starvation and the relative level of GFP‐LC3B or GFP‐GATE‐16 was measured using flow cytometry. Values represent the mean±s.d. of three experiments.
LC3 and GATE‐16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis (2010)
Figure 4

Both Atg8 subfamilies are required for autophagosome maturation. (A) HeLa cells stably expressing YFP‐Atg5 were transfected with non‐targeting siRNA (control siRNA), a pool of LC3 siRNAs, or a pool of GABARAP/GATE‐16 siRNAs using DharmaFect reagent. Seventy‐two hours after transfection, the cells were incubated for 2 h in EBSS medium, fixed, and immunostained with anti‐Atg16 antibodies. Quantification of Atg5 and Atg16‐labelled puncta structures from three independent experiments is presented on the right panel. Scale bar: 20 μm. (B) HeLa cells stably expressing YFP‐Atg5 were treated as in (A) and monitored by live microscopy. Quantification of the lifespan of Atg5‐labelled puncta structures is presented. Mean±s.d. of three independent experiments is presented. *P0.05, **P0.001. (C) YFP‐Atg5 cells were transfected with siRNA pool and starved as in (A). Cryo sections of fixed cells were immunolabelled using anti‐GFP antibodies and analysed by TEM as described in ‘Materials and methods’. Quantification of the Atg5‐labelled structures size is presented at the right panel. *P0.05, **P0.001.
LC3 and GATE‐16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis (2010)
Figure 5

LC3B and GATE‐16 overexpression differently affects phagophore appearance. (A) Control HeLa cells and HeLa cells stably expressing GFP‐LC3B or GFP‐GATE‐16 were starved for 2 h in EBSS medium fixed and immunostained with anti‐Atg16 antibodies. Quantification of Atg16‐labelled puncta structures from three independent experiments is presented at the right panel. Arrowheads represent Atg‐16‐labelled puncta. Scale bar: 20 μm. (B) HeLa cells stably expressing GFP‐LC3B (left panel) or GFP‐GATE‐16 (middle panel) were starved for 2 h in EBSS medium. The cells were fixed and their cryo sections were immunolabelled as in (Figure 4B). Quantification of the size of GFP‐labelled structures (right panel) from three independent experiments is presented. *P0.05, **P0.001.