Distinct retrograde microtubule motor sets drive early and late endosome transport
2020; Springer Nature; Volume: 39; Issue: 24 Linguagem: Inglês
10.15252/embj.2019103661
ISSN1460-2075
AutoresGiulia Villari, Chiara Enrico Bena, Marco Del Giudice, Noemi Gioelli, Chiara Sandri, Chiara Camillo, Alessandra Fiorio, Carla Bosia, Guido Serini,
Tópico(s)Autophagy in Disease and Therapy
ResumoArticle20 November 2020Open Access Source DataTransparent process Distinct retrograde microtubule motor sets drive early and late endosome transport Giulia Villari Giulia Villari orcid.org/0000-0002-4811-3990 Department of Oncology, University of Torino School of Medicine, Candiolo, Italy Candiolo Cancer Institute - Fondazione del Piemonte per l'Oncologia (FPO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Torino, Italy Search for more papers by this author Chiara Enrico Bena Chiara Enrico Bena orcid.org/0000-0002-3526-2858 Candiolo Cancer Institute - Fondazione del Piemonte per l'Oncologia (FPO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Torino, Italy IIGM - Italian Institute for Genomic Medicine, Candiolo, Italy Search for more papers by this author Marco Del Giudice Marco Del Giudice orcid.org/0000-0002-9231-6226 Candiolo Cancer Institute - Fondazione del Piemonte per l'Oncologia (FPO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Torino, Italy IIGM - Italian Institute for Genomic Medicine, Candiolo, Italy Search for more papers by this author Noemi Gioelli Noemi Gioelli orcid.org/0000-0002-4982-8410 Department of Oncology, University of Torino School of Medicine, Candiolo, Italy Candiolo Cancer Institute - Fondazione del Piemonte per l'Oncologia (FPO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Torino, Italy Search for more papers by this author Chiara Sandri Chiara Sandri orcid.org/0000-0003-2405-2297 Department of Oncology, University of Torino School of Medicine, Candiolo, Italy Candiolo Cancer Institute - Fondazione del Piemonte per l'Oncologia (FPO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Torino, Italy Search for more papers by this author Chiara Camillo Chiara Camillo orcid.org/0000-0001-8000-3188 Department of Oncology, University of Torino School of Medicine, Candiolo, Italy Candiolo Cancer Institute - Fondazione del Piemonte per l'Oncologia (FPO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Torino, Italy Search for more papers by this author Alessandra Fiorio Pla Alessandra Fiorio Pla Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy Search for more papers by this author Carla Bosia Carla Bosia orcid.org/0000-0002-8960-3443 IIGM - Italian Institute for Genomic Medicine, Candiolo, Italy Department of Applied Science and Technology, Polytechnic of Torino, Torino, Italy Search for more papers by this author Guido Serini Corresponding Author Guido Serini [email protected] orcid.org/0000-0002-3502-8367 Department of Oncology, University of Torino School of Medicine, Candiolo, Italy Candiolo Cancer Institute - Fondazione del Piemonte per l'Oncologia (FPO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Torino, Italy Search for more papers by this author Giulia Villari Giulia Villari orcid.org/0000-0002-4811-3990 Department of Oncology, University of Torino School of Medicine, Candiolo, Italy Candiolo Cancer Institute - Fondazione del Piemonte per l'Oncologia (FPO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Torino, Italy Search for more papers by this author Chiara Enrico Bena Chiara Enrico Bena orcid.org/0000-0002-3526-2858 Candiolo Cancer Institute - Fondazione del Piemonte per l'Oncologia (FPO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Torino, Italy IIGM - Italian Institute for Genomic Medicine, Candiolo, Italy Search for more papers by this author Marco Del Giudice Marco Del Giudice orcid.org/0000-0002-9231-6226 Candiolo Cancer Institute - Fondazione del Piemonte per l'Oncologia (FPO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Torino, Italy IIGM - Italian Institute for Genomic Medicine, Candiolo, Italy Search for more papers by this author Noemi Gioelli Noemi Gioelli orcid.org/0000-0002-4982-8410 Department of Oncology, University of Torino School of Medicine, Candiolo, Italy Candiolo Cancer Institute - Fondazione del Piemonte per l'Oncologia (FPO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Torino, Italy Search for more papers by this author Chiara Sandri Chiara Sandri orcid.org/0000-0003-2405-2297 Department of Oncology, University of Torino School of Medicine, Candiolo, Italy Candiolo Cancer Institute - Fondazione del Piemonte per l'Oncologia (FPO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Torino, Italy Search for more papers by this author Chiara Camillo Chiara Camillo orcid.org/0000-0001-8000-3188 Department of Oncology, University of Torino School of Medicine, Candiolo, Italy Candiolo Cancer Institute - Fondazione del Piemonte per l'Oncologia (FPO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Torino, Italy Search for more papers by this author Alessandra Fiorio Pla Alessandra Fiorio Pla Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy Search for more papers by this author Carla Bosia Carla Bosia orcid.org/0000-0002-8960-3443 IIGM - Italian Institute for Genomic Medicine, Candiolo, Italy Department of Applied Science and Technology, Polytechnic of Torino, Torino, Italy Search for more papers by this author Guido Serini Corresponding Author Guido Serini [email protected] orcid.org/0000-0002-3502-8367 Department of Oncology, University of Torino School of Medicine, Candiolo, Italy Candiolo Cancer Institute - Fondazione del Piemonte per l'Oncologia (FPO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Torino, Italy Search for more papers by this author Author Information Giulia Villari1,2, Chiara Enrico Bena2,3,6, Marco Del Giudice2,3, Noemi Gioelli1,2, Chiara Sandri1,2, Chiara Camillo1,2, Alessandra Fiorio Pla4, Carla Bosia3,5 and Guido Serini *,1,2 1Department of Oncology, University of Torino School of Medicine, Candiolo, Italy 2Candiolo Cancer Institute - Fondazione del Piemonte per l'Oncologia (FPO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Torino, Italy 3IIGM - Italian Institute for Genomic Medicine, Candiolo, Italy 4Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy 5Department of Applied Science and Technology, Polytechnic of Torino, Torino, Italy 6Present address: Sorbonne Université, CNRS, Institut de Biologie Paris-Seine, Laboratoire Jean Perrin (LJP), Paris, France *Corresponding author. Tel: 39 0119933508; E-mail: [email protected] The EMBO Journal (2020)39:e103661https://doi.org/10.15252/embj.2019103661 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Although subcellular positioning of endosomes significantly impacts on their functions, the molecular mechanisms governing the different steady-state distribution of early endosomes (EEs) and late endosomes (LEs)/lysosomes (LYs) in peripheral and perinuclear eukaryotic cell areas, respectively, are still unsolved. We unveil that such differences arise because, while LE retrograde transport depends on the dynein microtubule (MT) motor only, the one of EEs requires the cooperative antagonism of dynein and kinesin-14 KIFC1, a MT minus end-directed motor involved in cancer progression. Mechanistically, the Ser-x-Ile-Pro (SxIP) motif-mediated interaction of the endoplasmic reticulum transmembrane protein stromal interaction molecule 1 (STIM1) with the MT plus end-binding protein 1 (EB1) promotes its association with the p150Glued subunit of the dynein activator complex dynactin and the distinct location of EEs and LEs/LYs. The peripheral distribution of EEs requires their p150Glued-mediated simultaneous engagement with dynein and SxIP motif-containing KIFC1, via HOOK1 and HOOK3 adaptors, respectively. In sum, we provide evidence that distinct minus end-directed MT motor systems drive the differential transport and subcellular distribution of EEs and LEs in mammalian cells. SYNOPSIS Distinct subcellular distribution of early and late endosomes is relevant for their specific functions. Here, motor proteins dynein and kinesin KIFC1 are found to be differentially required for retrograde transport of early endosomes vs late endosome/lysosomes towards microtubule (MT) minus ends. Endoplasmic reticulum transmembrane protein STIM1 interacts with MT plus-end protein EB1 and the p150Glued subunit of dynactin/dynein via its SxIP motif and coiled coil domains. The STIM1/p150Glued/dynein complex drives accumulation of late endosomes in the perinuclear area of the cell. Correct cytosolic distribution of early endosomes depends on their simultaneous engagement with the STIM1/p150Glued/dynein and SxIP motif-containing KIFC1/p150Glued complexes. HOOK1 and HOOK3 adaptors mediate the interaction of p150Glued with early endosome-engaged dynein and KIFC1, respectively. Introduction In eukaryotic cells, the endolysosomal system is central in carrying out fundamental functions, such as plasma membrane (PM) remodeling, ligand-activated receptor signaling, and acquisition of nutrients (Wideman et al, 2014). Once internalized from the cell surface, cargos first localize into the early endosomal compartment (Naslavsky & Caplan, 2018). Early endosomes (EEs) are characterized by the presence of the small GTPase Rab5 that, via phosphatidylinositol 3 (PI3) kinase VPS34, elicits the synthesis of PI3-phosphate (PI3P), allowing the recruitment of PI3P-binding Rab5 effectors, such as early endosome antigen 1 (EEA1) on EEs (Galvez et al, 2012). Next, endocytosed cargos are either recycled back to the PM or kept in EEs that, moving along microtubules (MTs), from the cell periphery toward the juxtanuclear MT organizing center (MTOC), undergo maturation into Rab7, PI3, 5P2, and lysosomal-associated membrane protein 1 (LAMP-1) containing late endosomes (LEs) and then in degrading lysosomes (LYs). As a result, EEs, which are small (60–400 nm) and weakly acidic (pH 6.8–5.9), localize, at steady state, more peripherally than large (250–1,000 nm) and acidic (pH 6.0–4.9) perinuclear LEs/LYs (Huotari & Helenius, 2011). Several evidences indicate that the positioning of endosomes within the cytoplasm substantially affects their function (Bonifacino & Neefjes, 2017; Neefjes et al, 2017). Directed cell motility depends on the ability to recycle specific integrins and growth factor tyrosine kinase receptors with faster or slower kinetics, in response to their endocytic exit rate from more peripheral or perinuclear sorting compartment stations, respectively (Wilson et al, 2018). Cross-presentation to cytotoxic T cells of exogenous antigens, endocytosed by dendritic cells, relies on innate immunity signals that control EE movement along MTs and maturation into LEs/LYs (Weimershaus et al, 2018). Albeit so far described only in fungi, hitchhiking emerges as a novel strategy by which molecules (such as mRNA and proteins) and organelles (e.g., peroxisomes, endoplasmic reticulum, and lipid droplets) may connect to and exploit EEs to be evenly distributed throughout the cell (Higuchi et al, 2014; Salogiannis & Reck-Peterson, 2017). Recently, precursor miRNAs were also discovered to traffic along axons docked on LEs/LYs to reach growth cones and allow steering by guidance cues and the development of neural circuits (Corradi et al, 2020). Both in neuronal (Gowrishankar et al, 2015) and non-neuronal (Johnson et al, 2016) cells, more peripheral LYs are less proteolytic than the majority of LYs, more closely localized around the nucleus, because of either a reduced enzymatic amount (Gowrishankar et al, 2015) or activation (Johnson et al, 2016) of luminal proteases that have low pH optima (Mellman et al, 1986). Furthermore, lysosomal positioning controls the activation of mTOR complex 1 (mTORC1) signaling, which in turn influences autophagosome formation (Korolchuk et al, 2011; Poüs & Codogno, 2011). However, the molecular mechanisms responsible for the differential steady-state distribution of EEs and LEs/LYs in peripheral and perinuclear areas of eukaryotic cells, respectively, are unknown. The cytosolic logistics of endolysosomal cargos relies on motor proteins that move toward both peripheral fast-growing MT plus end and perinuclear slow-growing minus end (Bonifacino & Neefjes, 2017; Neefjes et al, 2017; Cross & Dodding, 2019). While endosomes are transported along MTs centrifugally (anterograde transport) by several kinesin motors, so far cytoplasmic multiprotein dynein 1 complex (hereafter referred as dynein) has been identified as the only main MT motor in charge of the opposite centripetal movement (retrograde transport) (Bonifacino & Neefjes, 2017; Neefjes et al, 2017; Cross & Dodding, 2019). Indeed, specific kinesins drive the anterograde motion of either EEs and LEs/LY, whereas their retrograde transport is thought to depend on the coupling of the same dynein motor to different adaptor proteins (Reck-Peterson et al, 2018). To function as a highly processive motor, dynein must undergo conformational transition from an auto-inhibited to an active conformational state (Zhang et al, 2017; McKenney, 2018). The multiprotein asymmetric complex dynactin is the main cofactor that releases dynein from its auto-inhibition at MT plus end and activates its minus end-directed retrograde motility (Ketcham & Schroer, 2018; McKenney, 2018). Distinct adaptor proteins containing a long coiled coil (CC) domain, such as bicaudal D cargo adaptor 2 (BICD2) (Hoogenraad & Akhmanova, 2016) as well as the Rab11 family interacting protein 3 (FIP3), HOOK3, and the spindle apparatus coiled coil protein 1 (SPDL1) (McKenney et al, 2014), are required to stabilize the formation at the MT plus end of the dynein–dynactin complex, bound to specific cargos, and prompted to drive their retrograde transport (McKenney, 2018; Reck-Peterson et al, 2018). Proteins localized at the MT plus end, acting as scaffolds where dynactin is recruited and thereby contributing to dynein conformational activation, play a key role in the initiation of movement toward the MT minus end (Akhmanova & Steinmetz, 2015; McKenney, 2018). Dynactin is formed by a short both-side capped actin-related protein 1 (ARP1) polymer and a projecting p150Glued side arm, kept together by a shoulder complex (Ketcham & Schroer, 2018; Reck-Peterson et al, 2018). Three CC stretches allow p150Glued to extend from the shoulder complex and interact with the MT plus end via its basic and cytoskeleton-associated protein glycine-rich (CAP-Gly) domain (Ketcham & Schroer, 2018; Reck-Peterson et al, 2018). The CAP-Gly domain located at the N-terminus of p150Glued interacts with the C-terminal Glu-Glu-Tyr (EEY) motif of tyrosinated α-tubulin and end-binding (EB) proteins, both enriched at the MT plus end (Akhmanova & Steinmetz, 2015; McKenney et al, 2016; McKenney, 2018; Rupam & Surrey, 2018). Yet, how interactions among MT plus end proteins, dynactin, dynein, and different endosomal cargos are coordinated and regulated in cells is still under investigation (Olenick et al, 2019; Saito et al, 2020). Moreover, differently from fungi (Steinberg, 2014), the molecular machinery that moves EEs toward the nucleus in animal cells is only in part understood (Neefjes et al, 2017). Here, we reveal a new role for the endoplasmic reticulum (ER) transmembrane protein stromal interaction molecule 1 (STIM1), previously shown to bind via its Ser-x-Ile-Pro (SxIP) motif the protein EB1 (Grigoriev et al, 2008), in promoting the association of the p150Glued subunit of dynactin to EB1 and its ensuing recruitment to the plus end of MTs in mammalian cells. As a result, STIM1 plays a key role in the regulation of endosomal cargo loading and dynein-dependent transport. We also unveil that, while LEs are transported toward the nucleus by the STIM1-dependent recruitment of the dynactin/dynein complex, the retrograde transport of EEs depends on the antagonistic cooperation between dynein and the minus end-directed kinesin-14 KIFC1, which also binds EB1 through a SxIP motif (Braun et al, 2013). The cooperative antagonism between dynein and KIFC1 may thus represent a molecular strategy to differentially regulate MT-based transport and subcellular distribution of specific vesicular components of the endolysosomal system, a general feature that is central for fundamental functions of eukaryotic cells. Results STIM1 forms a triple protein complex with p150Glued and EB1 to promote dynactin loading at MT plus ends The dynactin subunit p150Glued plays a key role in the release of the MT plus end protein EB1 auto-inhibition and subsequent engagement of the dynein/dynactin complex at MT plus ends (Hayashi et al, 2005; Akhmanova & Steinmetz, 2015; McKenney et al, 2016; McKenney, 2018). However, how those molecular dynamics, so far investigated in in vitro studies with recombinant proteins or in crystals (Duellberg et al, 2014), are regulated in intact living cells is unknown. Considering the ability of the ER protein STIM1 to bind, similarly to p150Glued, EB1 (Grigoriev et al, 2008), we verified whether STIM1 silencing by short hairpin (sh) RNA (shSTIM1; Fig EV1A) may affect p150Glued association with the MT plus end protein in human primary endothelial cells (ECs). The lack of STIM1 significantly reduced the amount of p150Glued that interacts with EB1 at steady-state in living ECs (Fig 1A), thus suggesting a potential new role for STIM1 in the ordinated recruitment of proteins at MT plus ends and, potentially, the loading and retrograde transport of cargos (Ayloo et al, 2014; Reck-Peterson et al, 2018). Yet, STIM1 silencing did not affect the assembly of the dynein/dynactin complex (Fig EV1B). Click here to expand this figure. Figure EV1. STIM1 drives late endosome retrograde transport without affecting the assembly of the dynein/dynactin complex, showing an additional function other than ER calcium sensor Representative Western blot analysis of the endogenous STIM1 in ECs silenced with a control shRNA (shCTL) or three different tested shRNAs targeting STIM1 (shSTIM1) (left) and its quantification by normalized densitometry (right). Results are the average ± SD of three independent assays and were analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA P ≤ 0.001; Bonferroni for shCTL and sh#490 P ≤ 0.001***, for shCTL and sh#718 P ≤ 0.01** and for shCTL and sh#780 P ≤ 0.01**. In this manuscript, only the #780 was used. Representative Western blot analysis of endogenous light-intermediate chain (LIC) of cytoplasmic dynein 1 co-immunoprecipitated with p150Glued in shCTL or shSTIM1 ECs (left) and its quantification by normalized densitometry (right). Results are the average ± SD of three independent assays. ShCTL value of each biological replicate was normalized on itself and so shSTIM1 experimental value. Results were analyzed by a two-tailed heteroscedastic Student's t-test, P > 0.05 not significant (ns). Representative Western blot analysis of GFP-STIM1 WT, ΔCC1, ΔCC2, or ΔCC3 co-immunoprecipitated with mCherry-p150Glued WT in cotransfected HEK 293T cells. Negative control (CTL) was performed incubating cell lysate from HEK 293T cotransfected with an empty GFP vector together with mCherry-P150Glued WT with pre-cleared protein A or G-Sepharose and the rabbit GFP antibody. Right, its quantification by normalized densitometry. Results are the average ± SD of three independent assays. The value of p150Glued co-immunoprecipitated with GFP-STIM1 WT from each biological replicate was normalized on itself and so those immunoprecipitated with GFP-STIM1 ΔCC1, ΔCC2, or ΔCC3. Results were analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA P ≤ 0.01**; Bonferroni for STIM1 WT and ΔCC1 P > 0.05 not significant (ns), for STIM1 WT and ΔCC2 P > 0.05 not significant (ns) and for STIM1 WT and ΔCC3 P > 0.05 not significant. Representative Western blot analysis of the endogenous STIM1 in ECs silenced with a control siRNA (siCTL) or one targeting STIM1 (siSTIM1) (left) and its quantification by normalized densitometry (right). Results are the average ± SD of three independent assays. Results were analyzed by a two-tailed heteroscedastic Student's t-test, P ≤ 0.001***. Confocal microscopy images of untreated (UT) or treated with Thapsigargin (TG) ECs and stained for endogenous LAMP-1 (in green) to visualize LEs and DAPI (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm. Distribution of distance to nucleus, normalized on cell size, quantified by image (as in D) segmentation (see Materials and Methods, Confocal microscopy and early/late quantification) of LAMP-1+ endosomes. Results are from three independent experiments for a total of 441 late endosomes in 13 UT cells (34 ± 4 endosomes per cell) and 519 late endosomes in 18 TG cells (29 ± 3 endosomes per cell) and analyzed by a two-tailed heteroscedastic Student's t-test, P ≤ 0.001***. Source data are available online for this figure. Download figure Download PowerPoint Figure 1. STIM1 forms a triple protein complex with p150Glued and EB1 to promote dynactin loading at MT plus ends Representative Western blot analysis of the endogenous p150Glued co-immunoprecipitated with EB1 in shCTL or shSTIM1 ECs (left) and its quantification by normalized densitometry (right). Negative control (CTL) was performed incubating cell lysate with protein A- or G-Sepharose and empty mouse IgG. Results are the average ± SD of three independent assays. shCTL value of each biological replicate was normalized on itself and so shSTIM1 experimental value. Results were analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA P ≤ 0.001; Bonferroni for shCTL and shSTIM1 P ≤ 0.05*. Representative of three Western blot analysis of endogenous p150Glued, light-intermediate chain (LIC) of cytoplasmic dynein 1, and EB1 immunoprecipitated with STIM1 in wild-type ECs. Negative control (CTL) was performed incubating cell lysate with protein A- or G-Sepharose and empty rabbit IgG. Schematic model of the molecular interactions among STIM1, EB1, and p150Glued at the MT plus ends. Black arrows point at already known binding motifs, whereas red parts show interaction domains studied in this manuscript. TM, transmembrane domain. Differentially colored labels with bars correspond to the purified protein fragment with its tag and molecular weight in kDa. Representative Western blot analysis of increasing amount of GST-STIM1 (cytoplasmic domain) pulled down by Cap-Gly-CC1-p150Glued-V5-coated beads, in the absence or presence of FLAG-EB1 C-terminal. Colors label the protein as in (C). Negative control was performed using equal amount of empty GST protein, together with Cap-Gly-CC1-p150Glued. Representative Western blot analysis of mCherry-p150Glued WT co-immunoprecipitated with GFP-STIM1 WT, NN, ΔCC1–3/WT (ΔCC/WT), or ΔCC1–3/NN (ΔCC/NN) in cotransfected HEK 293T cells. Negative control (CTL) was performed incubating cell lysate from HEK 293T cotransfected with an empty GFP vector together with mCherry-p150Glued WT with pre-cleared protein A or G-Sepharose and the rabbit GFP antibody. Below, its quantification by normalized densitometry. Results are the average ± SD of three independent assays. The value of p150Glued co-immunoprecipitated with GFP-STIM1 WT from each biological replicate was normalized on itself and so those immunoprecipitated with GFP-STIM1 NN, ΔCC/WT or ΔCC/NN. Results were analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA P ≤ 0.001; Bonferroni for STIM1 WT and NN P > 0.05 not significant (ns), for STIM1 WT and ΔCC/WT P > 0.05 not significant (ns) and for STIM1 WT and ΔCC/NN P ≤ 0.05*. Representative Western blot analysis of GST-STIM1 (cytoplasmic domain) pulled down by Cap-Gly-CC1-p150Glued-V5-coated beads, in the presence of FLAG-EB1 C-terminal WT or ΔY. Negative control was performed using equal amount of empty GST protein, together with Cap-Gly-CC1-p150Glued. Confocal microscopy analysis of wild-type ECs, transiently transfected with GFP-STIM1 WT or, NN or ΔCC1–3/WT (ΔCC/WT) together with mCherry-p150Glued. Scale bar = 10 μm. Right insets are shown to highlight MT-bound p150Glued+ punctae (arrows), colocalized with STIM1. Scale bar = 5 μm. Average number of the mCherry-p150Glued+ punctae in the same cells as in (G). Counts are the average ± SEM of three independent experiments for a total of 150 punctae (30 cells). Results were analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA P ≤ 0.001; Bonferroni for STIM1 WT and NN P ≤ 0.001*** and for STIM1 WT and ΔCC/WT P > 0.05 not significant (ns). Colocalization analysis of mCherry-p150Glued with GFP-STIM1 WT, NN, or ΔCC1–3/WT (ΔCC/WT), transiently cotransfected in ECs, as in (G). Results are the average ± SEM of three independent experiments for a total of 150 punctae (30 cells) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA P ≤ 0.001; Bonferroni for STIM1 WT and NN P ≤ 0.001*** and for STIM1 WT and ΔCC/WT P > 0.05 not significant (ns). Source data are available online for this figure. Source Data for Figure 1 [embj2019103661-sup-0008-SDataFig1.zip] Download figure Download PowerPoint Next, we wondered whether, other than with EB1 (Grigoriev et al, 2008), STIM1 may also associate with p150Glued in ECs. We found that indeed STIM1 co-immunoprecipitates with p150Glued (Fig 1B), but not with dynein light-intermediate chain (LIC), thus suggesting a triple complex formed by the ER protein, EB1, and p150Glued. To directly assess the possible formation of this ternary complex, we performed in vitro interaction assays with the corresponding purified proteins. In particular, we generated and purified the wild-type FLAG-tagged C-terminal EB1 portion containing the STIM1-binding EBH domain (Grigoriev et al, 2008) and the p150Glued-interacting EEY motif (Akhmanova & Steinmetz, 2015) (FLAG-EB1 WT C-term, orange bar in Fig 1C), the GST-tagged cytoplasmic domain of STIM1 (GST-STIM1 cyto, green bar in Fig 1C), and the Cap-Gly domain of p150Glued fused with its first CC (CC1a), V5-tagged (Cap-Gly-CC1a-p150Glued-V5, blue bar in Fig 1C). In the last construct, we included the CC1a domain alone, as we posited it would have been the only one, among the three CC regions of p150Glued (Tripathy et al, 2014), available for the binding to STIM1, being the second (CC1b) and third (CC2) domain known to interact with dynein IC and the Arp1 filament of dynactin, respectively (McKenney, 2018; Reck-Peterson et al, 2018). Moreover, since the CC1a is known to exist in an inhibited form folded with the second CC1b motif, using the first part only of the whole CC1 of p150Glued would have allowed us to avoid any inactivation of the protein (Wang et al, 2014; Saito et al, 2020). We confirmed in vitro the binding between purified Cap-Gly-CC1a-p150Glued-V5 and GST-STIM1 cyto, with increasing amount of the latter detected to associate with immunoprecipitated Cap-Gly-CC1a-p150Glued-V5 (Fig 1D). Of note, the in vitro interaction between p150Glued and STIM1 was clearly stabilized by the addition of purified FLAG-EB1 WT C-term, which is known to bind both the Cap-Gly domain of p150Glued (Akhmanova & Steinmetz, 2015; McKenney et al, 2016; McKenney, 2018; Rupam & Surrey, 2018) and the SxIP motif of STIM1 (Grigoriev et al, 2008). Hence, the ER protein STIM1 forms a ternary complex together with the MT plus end protein EB1 and the dynactin subunit p150Glued both in living ECs and in vitro. Then, we verified whether, similarly to what observed in vitro with purified recombinant proteins (Fig 1D), also in living ECs the interaction between STIM1 and p150 Glued relies on the simultaneous binding of STIM1 to EB1. The SxIP motif mediates the association of STIM1 to the EB homology (EBH) domain of EB1 and the ensuing STIM1 tracking of MT plus ends (Yao et al, 2012; Chowdhury et al, 2015). To verify the possible involvement of STIM1 SxIP motif in the interaction with p150Glued, we employed a mutant version of STIM1 unable to bind EB1 (Grigoriev et al, 2008; Honnappa et al, 2009) and dubbed GFP-STIM1 NN (Fig 1C) as the Ile and Pro residues of the SxIP motif were replaced by two Asn (N). Since STIM1 also owns three CC domains (Novello et al, 2018), which are known ubiquitous protein–protein interaction domains (Woolfson et al, 2012) and key in releasing the auto-inhibition of MT plus end proteins (Hayashi et al, 2005), we asked whether STIM1 CC motifs may also mediate its association with p150Glued. Hence, we generated GFP-STIM1 mutants lacking each of the CC domain (ΔCC1, ΔCC2, and ΔCC3; Fig EV1C) or all of them (ΔCC1–3; Fig 1E) in both wild-type (WT) or NN-STIM1 backbones (ΔCC1–3/WT and ΔCC1–3/NN), cotransfected them with mCherry-p150Glued WT in HEK239T cells, and verified their interactions. Neither the deletion of single (Fig EV1C) or all three CC domains (ΔCC1–3/WT; Fig 1E) nor the mutation of the SxIP motif (NN; Fig 1E) affected STIM1 binding to p150Glued, compared to WT STIM1 in living cells. However, the GFP-STIM1 ΔCC1–3/NN mutant, which simultaneously lacks all CC domains and the ability to bind EB1 via the SxIP motif, did not co-immunoprecipitate with p150Glued (Fig 1E). Thus, in agreement with what we
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