Atg1-mediated myosin II activation regulates autophagosome formation during starvation-induced autophagy

Abstract

Autophagy is a membrane-mediated degradation process of macromolecule recycling. Although the formation of double-membrane degradation vesicles (autophagosomes) is known to have a central role in autophagy, the mechanism underlying this process remains elusive. The serine/threonine kinase Atg1 has a key role in the induction of autophagy. In this study, we show that overexpression of Drosophila Atg1 promotes the phosphorylation-dependent activation of the actin-associated motor protein myosin II. A novel myosin light chain kinase (MLCK)-like protein, Spaghetti-squash activator (Sqa), was identified as a link between Atg1 and actomyosin activation. Sqa interacts with Atg1 through its kinase domain and is a substrate of Atg1. Significantly, myosin II inhibition or depletion of Sqa compromised the formation of autophagosomes under starvation conditions. In mammalian cells, we found that the Sqa mammalian homologue zipper-interacting protein kinase (ZIPK) and myosin II had a critical role in the regulation of starvation-induced autophagy and mammalian Atg9 (mAtg9) trafficking when cells were deprived of nutrients. Our findings provide evidence of a link between Atg1 and the control of Atg9-mediated autophagosome formation through the myosin II motor protein.

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 (Sqh^A20A21) 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 (Atg12^RNAi) (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 Sqh^A20A21. 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 Sqh^A20A21. 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.

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 (Atg1^RNAi) or Sqa (Sqa^RNAi) 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 Sqh^A20A21 (H). Nevertheless, Sqa-induced wing vein defects were suppressed by co-expression of Sqh^A20A21 (J) but not Atg1^RNAi (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.

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.

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, Sqa^RNAi, Sqa-T279A, spaghetti-squash (Sqh^A20A21), and Atg7^RNAi 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 Atg12^RNAi (E), Sqa-T279A (F), Sqa^RNAi (G), or Sqh^A20A21 (H) markedly suppressed GFP–Atg8a puncta formation during starvation. The autophagic defects caused by Sqa-T279A and Sqa^RNAi were rescued by co-expression of the constitutively active Sqh^E20E21 (I) and Sqh^D20D21 (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; ^***P<0.001). (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). ^*P<0.05, ^**P<0.01, ^***P<0.001. See Supplementary data for genotypes.

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.

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, ^***P<0.001). (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.

Figure 7

Redistribution of myosin II during nutrient starvation. (A) The fat body of spaghetti-squash (sqh^AX3); 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. ^*P<0.05, ^**P<0.01.

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. ^*P<0.05, ^**P<0.01. Bar, 10 μm.