Journal: Cell reports

Article Title: Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin-mediated endocytosis

doi: 10.1016/j.celrep.2021.110008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Briefly, a guide RNA (5′-GCAGATGTAGTGTTTCCACA-3′) targeting the open reading frame in the immediate vicinity of the stop codon was cloned into the Cas9 expression vector pX330-U6-Chimeric_BB-CBh-hSpCas9 (gift from Feng Zhang; ), Addgene plasmid #42230).

Techniques: Derivative Assay, Recombinant, Electron Microscopy, Transfection, Expressing, Plasmid Preparation, Software, Cell Analysis, Gas Chromatography, Mass Spectrometry

Journal: The Journal of Cell Biology

Article Title: mTORC1 activity is supported by spatial association with focal adhesions

doi: 10.1083/jcb.202004010

Figure Lengend Snippet: RPTOR BioID2 reveals spatial association between mTORC1 and FAs. (A) Network representation of RPTOR BioID2 dataset. Proteins identified include well-known regulators of mTORC1 (protein complexes shown in green boxes), autophagy, and protein translation (black dashed boxes; see for a full list). ND, not detected in the proteomics dataset. Thick blue node border indicates BioID2-RPTOR. Edges (red lines) indicate reported physical protein–protein interactions. (B) Gene Ontology overrepresentation enrichment analyses of proteins identified by RPTOR BioID2. Molecular functions enriched with P < 10 −3 and cellular components enriched with P < 10 −5 are shown (hypergeometric tests with Benjamini–Hochberg correction; 5% FDR threshold). Bar color represents enrichment ratio of overrepresented terms. NTP, nucleoside triphosphatase. (C) Network representation of consensus adhesome proteins identified in the RPTOR BioID2 dataset. Edges (red lines) indicate reported physical protein–protein interactions. The largest interconnected subnetwork is shown. (D) Proportion of RPTOR-proximal proteins in the meta-adhesome database, including the consensus adhesome ( ; number of identified proteins indicated in parentheses). Segments are labeled with respective coverage of the meta-adhesome, consensus adhesome, and literature-curated adhesome by RPTOR-proximal proteins. See also . (E) Identification of 10 proteins significantly enriched in the RPTOR BioID2 dataset that were also identified in published BioID datasets of the FA proteins paxillin and kindlin-2 in U2OS cells .

Article Snippet: HeLa cells were cultured and cotransfected with the mTORC1 biosensor TORCAR (pcDNA3-TORCAR; a gift from Jin Zhang, The Johns Hopkins School of Medicine, Baltimore, MD; #64927, Addgene; ) and GFP-paxillin.

Techniques: Protein-Protein interactions, Labeling

Journal: The Journal of Cell Biology

Article Title: mTORC1 activity is supported by spatial association with focal adhesions

doi: 10.1083/jcb.202004010

Figure Lengend Snippet: RPTOR BioID2 reveals spatial association between mTORC1 and FAs. (A) Immunostaining of RPTOR-BioID2-expressing U2OS cells treated with 50 µM biotin overnight. Cells were stained for biotin and myc-tagged RPTR. Scale bars, 20 µm (insets, 10 µm). Nuclei were visualized with DAPI. (B) Representative immunoblots of streptavidin pull-downs following 50 µM biotin incubation overnight (left) and immunoprecipitation (IP) with anti-mTOR antibody (right). WCL, whole-cell lysate. (C) Unsupervised analysis of RPTOR BioID samples (BioID2-based proximity labeling) quantified by MS. Hierarchical cluster analysis of Spearman rank correlation coefficients of pairwise comparisons for all samples analyzed by MS (P < 2 × 10 −127 , Spearman’s test; left). Principal-component analysis of samples analyzed by MS (right). The first two principal components account for 88.6% of the total variance of the dataset. (D) Enrichment of noncontaminant proteins specifically enriched in RPTOR BioID samples ( n = 4 independent experiments; P < 0.01, two-sided Welch’s t test with permutation-based FDR correction, artificial within groups variance = 2). Proteins enriched by at least 256-fold or with P < 10 −6 (two-sided Welch’s t test) are labeled; enriched TOR signaling components (Gene Ontology accession 0031929) are labeled in bold. Putative contaminant proteins are indicated by gray crosses. (E) Interaction network analysis of TOR signaling components identified in U2OS cells by RPTOR BioID and MS. (F and G) KEGG pathway gene set enrichment analysis and Gene Ontology biological process overrepresentation enrichment analysis of noncontaminant proteins specifically enriched in RPTOR BioID samples. Biological processes enriched with P < 10 −9 (hypergeometric test with Benjamini–Hochberg correction; F) and pathways enriched with P < 0.0001 (Kolmogorov–Smirnov test with FDR correction; G) are shown. (H) Interaction network analysis of RPTOR-proximal adhesome components identified by RPTOR BioID. (I) Diagrammatic representation of mTORC1 and lysosomal proteins identified in meta-adhesome datasets. See and for more details. (J and K) Interaction network analysis of reported physical and predicted protein–protein interactions and pathway associations between mTOR signaling pathway components (J) and lysosome-associated proteins (K) detected in adhesion complex proteomes (meta-adhesome datasets). See and for additional details. (L) Identification of adhesion proteins significantly enriched in the RPTOR BioID2 dataset that were also identified in published BioID datasets of the FA proteins paxillin and kindlin-2 in mouse pancreatic fibroblasts .

Article Snippet: HeLa cells were cultured and cotransfected with the mTORC1 biosensor TORCAR (pcDNA3-TORCAR; a gift from Jin Zhang, The Johns Hopkins School of Medicine, Baltimore, MD; #64927, Addgene; ) and GFP-paxillin.

Techniques: Immunostaining, Expressing, Staining, Western Blot, Incubation, Immunoprecipitation, Labeling, Protein-Protein interactions

Journal: The Journal of Cell Biology

Article Title: mTORC1 activity is supported by spatial association with focal adhesions

doi: 10.1083/jcb.202004010

Figure Lengend Snippet: mTORC1 is activated at the cell periphery. (A) HeLa cells grown in full-nutrient medium were FCS starved for 18 h (−FCS) or FCS starved and then recovered in FCS-containing medium for 10 min (10 min FCS). Immunostaining of mTOR and LAMP1 is shown. (B and C) Immunostaining for p-S6 and LAMP1 following starvation and refeeding as in A (B) or in response to amino acid starvation versus amino acid starvation and recovery (C). Arrows indicate mTORC1 activation at the cell periphery. The IntDens of peripheral or intracellular signal of p-S6 were quantified and the colocalization at the cell periphery between p-S6 and LAMP1 was quantified using Manders coefficient. (D) The proportion of cells with peripheral p-S6 staining was quantified in cells starved and recovered in FCS-containing medium (left) or amino acids (right) as indicated. (E) Quantification of the proportion of cells with peripheral LAMP1 in full-nutrient medium that exhibited peripheral p-S6 staining. (F) HeLa cells transfected with FLAG-ARL8B or empty FLAG plasmid were FCS starved for 18 h (−FCS) or FCS starved and then recovered in FCS-containing medium for 10 min (10 min FCS). Immunostaining of p-S6 and LAMP1 (left) or FLAG (right) is shown. The IntDens of peripheral or intracellular signal of p-S6 and the proportion of cells with peripheral p-S6 staining was quantified. (G) Cells were treated as in A, lysed, and subject to immunoblotting for mTORC1 activity (top). For quantification of relative p-S6 levels (bottom), error bars represent SEM; n = 3 independent experiments (for IntDens, n ≥ 10 cells were quantified per experiment). *, P < 0.05; **, P < 0.01; ***, P < 0.001; two-sided Student’s t test. Scale bars, 20 µm (insets, 10 µm). Nuclei were visualized with DAPI.

Article Snippet: HeLa cells were cultured and cotransfected with the mTORC1 biosensor TORCAR (pcDNA3-TORCAR; a gift from Jin Zhang, The Johns Hopkins School of Medicine, Baltimore, MD; #64927, Addgene; ) and GFP-paxillin.

Techniques: Immunostaining, Activation Assay, Staining, Transfection, Plasmid Preparation, Western Blot, Activity Assay

Journal: The Journal of Cell Biology

Article Title: mTORC1 activity is supported by spatial association with focal adhesions

doi: 10.1083/jcb.202004010

Figure Lengend Snippet: mTORC1 is localized at FAs. (A and B) HeLa cells grown in full-nutrient medium were FCS starved for 18 h (−FCS) or FCS starved and then recovered in FCS-containing medium for 10 min (10 min FCS) and immunostained for mTOR (A) or LAMP1 (B) and the FA protein paxillin. Colocalization (Manders coefficient) was analyzed. (C) PLAs for mTOR–paxillin interactions (and SDHA–paxillin as a control) were performed in HeLa cells under starvation–refeeding conditions as in A and B. Cells were counterstained with paxillin antibody to mark FAs, and the proportions of PLA-positive cells and PLA dots/cell were quantified. Error bars represent SEM; n = 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; two-sided Student’s t test. Scale bars, 20 µm (insets, 10 µm). Nuclei were visualized with DAPI.

Article Snippet: HeLa cells were cultured and cotransfected with the mTORC1 biosensor TORCAR (pcDNA3-TORCAR; a gift from Jin Zhang, The Johns Hopkins School of Medicine, Baltimore, MD; #64927, Addgene; ) and GFP-paxillin.

Techniques: Control

Journal: The Journal of Cell Biology

Article Title: mTORC1 activity is supported by spatial association with focal adhesions

doi: 10.1083/jcb.202004010

Figure Lengend Snippet: mTORC1 is activated in FAs. (A) HeLa cells grown in full-nutrient medium were FCS starved for 18 h (−FCS) or FCS starved and then recovered in FCS-containing medium for 10 min (10 min FCS) in the absence or presence of the mTORC1 inhibitor rapamycin (100 nM) and immunostained for p-S6 and the FA protein paxillin. (B) Colocalization (Manders coefficient) between p-S6 and paxillin was analyzed. (C) Fluorescence intensity line profile plots corresponding to lines exemplified by an arrow in A. (D and E) Analysis of p-S6 in ROIs corresponding to FAs and adjacent (control) areas. Representative confocal image of paxillin (top) and p-S6 (bottom) in refed HeLa cells (D). The ROIs corresponding to FAs (top inset) and adjacent (control) areas (bottom inset) are indicated by white borders in zoom insets (D; right), and p-S6 IntDens in ROIs was quantified (E). (F) Diagram demonstrating the principle of the TORCAR biosensor . (G) TORCAR biosensor analysis. Representative confocal image of GFP-paxillin (top) and GFP-paxillin intensity-scaled ratio image of TORCAR FRET-based biosensor (bottom) in serum-starved and refed HeLa cells. Note that to visualize the differences, the images were gamma adjusted, and white borders in GFP-paxillin zoom insets highlight the ROIs corresponding to FAs (top inset) and control areas (bottom inset) used for quantification. (H and I) mTORC1 activity was quantified in serum-starved versus refed conditions (H) and before and after stimulation with 25 mM membrane-permeable leucine methylester (I), presented as the normalized ratio of TORCAR biosensor in FAs and control areas. Each data point represents a coverslip including several cells. A/D, acceptor-to-donor ratio. (J) HeLa cells grown in full-nutrient medium were starved of amino acids and FCS (−aa −FCS) and then recovered in amino acid– and insulin-containing medium for 30 min (30 min aa +ins) in the presence of L-HPG with and without rapamycin. HPG incorporation was visualized via Click-IT reaction with Alexa Fluor 488 azide before cells were immunostained for p-S6 and paxillin. (K) Colocalization (Manders coefficient) between p-S6 and HPG was analyzed. (L) Fluorescence intensity line profile plots corresponding to lines as exemplified by an arrow in J. (M) Quantification of HPG IntDens in ROIs corresponding to FAs and adjacent (control) areas. Scale bars, 20 µm (insets 10 µm). Nuclei were visualized with DAPI. Error bars represent SEM; n = 3 independent experiments. For B, E, K and M, *, P < 0.05; **, P < 0.01; ***, P < 0.001; two-sided Student’s t test. For H and I, ****, P < 0.0001; two-way ANOVA with Sidak's multiple comparisons test.

Article Snippet: HeLa cells were cultured and cotransfected with the mTORC1 biosensor TORCAR (pcDNA3-TORCAR; a gift from Jin Zhang, The Johns Hopkins School of Medicine, Baltimore, MD; #64927, Addgene; ) and GFP-paxillin.

Techniques: Fluorescence, Control, Activity Assay, Membrane

Journal: The Journal of Cell Biology

Article Title: mTORC1 activity is supported by spatial association with focal adhesions

doi: 10.1083/jcb.202004010

Figure Lengend Snippet: mTORC1 signaling is activated in the vicinity of FAs. (A) Fluorescence intensity line profile plots corresponding to lines exemplified by an arrow in . (B) U2OS, HEK293, and Cos7 cells grown in full-nutrient medium were FCS starved for 18 h (−FCS) or FCS starved and then recovered in FCS-containing medium for 10 min (10 min FCS) and immunostained for p-S6 and the FA protein paxillin. Colocalization (Manders coefficient) between p-S6 and paxillin was analyzed. (C) Representative confocal image of GFP-paxillin intensity-scaled ratio image of TORCAR FRET-based biosensor in serum-starved HeLa cells before and after stimulation with 25 mM membrane-permeable leucine methylester. Note that, to visualize the differences, the images were gamma adjusted, and black borders in zoom insets highlight the ROIs corresponding to FAs. (D) Quantification of ratio change (percentage) of TORCAR biosensor signal before stimulation (−FCS), after stimulation with FCS (+FCS), in the presence of rapamycin (+FCS +Rapamycin), and in response to leucine methylester (+Leucine). Each data point represents a coverslip including several cells; box-and-whisker plot whiskers represent minimum and maximum values. Error bars represent SEM; n = 3 independent experiments. ****, P < 0.0001; two-way ANOVA with Sidak correction. For B, **, P < 0.01, ***, P < 0.001 two-sided Student’s t test. Scale bars, 20 µm (insets, 10 µm). Nuclei were visualized with DAPI.

Article Snippet: HeLa cells were cultured and cotransfected with the mTORC1 biosensor TORCAR (pcDNA3-TORCAR; a gift from Jin Zhang, The Johns Hopkins School of Medicine, Baltimore, MD; #64927, Addgene; ) and GFP-paxillin.

Techniques: Fluorescence, Membrane, Whisker Assay

Journal: The Journal of Cell Biology

Article Title: mTORC1 activity is supported by spatial association with focal adhesions

doi: 10.1083/jcb.202004010

Figure Lengend Snippet: Functional FAs are required for mTORC1 activation. (A–C) Control and TLN1/2 DKO mouse kidney fibroblasts grown in full-nutrient medium were amino acid and FCS starved (−aa −FCS) and then recovered in amino acid–containing medium (10 min aa) or amino acid– and FCS-containing medium (10 min aa +FCS) for 10 min. Cells were analyzed by immunostaining for LAMP1 and p-S6 (A), colocalization (Manders coefficient) was quantified (B), and cell lysates were analyzed by immunoblotting to monitor changes in the mTORC1 pathway (C). (D) Control and TLN1/2 DKO cells subjected to the starvation and refeeding as in A–C were lysed and analyzed by liquid chromatography MS to measure intracellular levels of amino acids. Amino acid concentrations were normalized to protein levels. (E) HeLa cells were FCS starved for 18 h (−FCS), treated with 50 µM ROCKi or 5 µM integrin antagonist (cilengitide) for 1 h in −FCS medium, starved of amino acids (−aa −FCS) for 1 h in the presence of inhibitors, and then recovered in full-nutrient medium for 10 min. Cells were subjected to immunostaining for paxillin and p-S6, and the IntDens of peripheral or intracellular p-S6 staining was quantified. (F–H) Control and senescent (30 d after 20-Gy x-ray irradiation [IR]) primary human fibroblasts were subjected to FCS starvation for 18 h (−FCS) or FCS starved and then recovered in FCS-containing medium for 10 min (10 min FCS), fixed and immunostained for p-IGFR1 and paxillin (F). Number of FAs per pixel (G) and CTCF of p-IGFR1 staining (H) were quantified. Note that CTCF was used because it takes into account cell size as senescent cells are significantly larger than proliferating fibroblasts. (I and J) Senescent (30 d after 20-Gy x-ray irradiation) primary human fibroblasts were subjected to FCS starvation for 18 h (−FCS); during last 2 h of starvation, cells were treated with DMSO (control) or 50 µM ROCKi. Cells were subjected to immunostaining for paxillin and p-IGFR1 or p-S6 (I), and the IntDens of p-IGFR1 and p-S6 staining was quantified (J). Error bars represent SEM; n = 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; two-sided Student’s t test. Scale bars, 20 µm (insets, 10 µm). Nuclei were visualized with DAPI.

Article Snippet: HeLa cells were cultured and cotransfected with the mTORC1 biosensor TORCAR (pcDNA3-TORCAR; a gift from Jin Zhang, The Johns Hopkins School of Medicine, Baltimore, MD; #64927, Addgene; ) and GFP-paxillin.

Techniques: Functional Assay, Activation Assay, Control, Immunostaining, Western Blot, Liquid Chromatography, Staining, Irradiation

Journal: The Journal of Cell Biology

Article Title: mTORC1 activity is supported by spatial association with focal adhesions

doi: 10.1083/jcb.202004010

Figure Lengend Snippet: Disruption of FAs or impairment of peripheral lysosomal distribution inhibits mTORC1 activation by nutrients. (A and B) HeLa cells transfected with scrambled (Scr) or TLN1/2 siRNAs grown in full-nutrient medium were amino acid and FCS starved for 1 h (−aa −FCS) and then recovered in amino acid–containing medium (10 min aa) or amino acid– and FCS-containing medium (10 min aa +FCS). Cells were analyzed by immunostaining for LAMP1 and p-S6 (A) or immunoblotting to detect mTORC1 activity (B). (C) HeLa cells subjected to amino acid and FCS starvation for 1 h were scraped (suspension) or left adherent and then refed as described in A and subjected to immunoblot analysis to detect mTORC1 activity. (D and E) HeLa cells were FCS starved for 18 h (−FCS); treated with DMSO (control), ROCKi, or integrin antagonist (cilengitide) for 1 h in −FCS medium; starved of amino acids (−aa −FCS) for 1 h in the presence of inhibitors; and then recovered in full-nutrient medium for 10 min. Cells were subjected to immunoblot analysis to detect mTORC1 activity (D). The IntDens of peripheral paxillin staining was quantified (E). (F) Scrambled (Scr) or ARL8B siRNA–transfected HeLa cells were FCS starved for 18 h (−FCS) and then recovered in FCS-containing medium for 10 min (10 min FCS). Immunostaining of p-S6 and LAMP1 is shown. (G) HeLa cells expressing FLAG-RPTOR and FLAG-RPTOR FAT grown in full-nutrient media were lysed, immunoprecipitated with FLAG antibody, and immunoblotted for FLAG and mTOR (left) and FLAG and paxillin (right). (H) Fluorescence intensity line profile plots corresponding to lines exemplified by arrows in are shown. (I) Scrambled or ARL8B siRNA–transfected HeLa cells were FCS starved for 18 h (−FCS) and then recovered in FCS-containing medium for 10 min (10 min FCS). Immunostaining for paxillin (left) and fluorescence intensity line profile plots corresponding to lines exemplified by arrows (right) are shown. (J) Scrambled or ARL8B siRNA–transfected HeLa cells expressing FLAG-RPTOR or FLAG-RPTOR FAT grown in full-nutrient medium were FCS starved for 18 h (−FCS) or FCS starved and then recovered in FCS-containing medium for 10 or 60 min (10, 60 min FCS), lysed, and subjected to immunoblot analysis using antibodies as shown. Error bars represent SEM; n = 3 independent experiments. For J, n = 2 independent experiments and error bars represent SD. *, P < 0.05; **, P < 0.01; two-sided Student’s t test (performed between groups). Scale bars, 20 µm (insets, 10 µm). Nuclei were visualized with DAPI.

Article Snippet: HeLa cells were cultured and cotransfected with the mTORC1 biosensor TORCAR (pcDNA3-TORCAR; a gift from Jin Zhang, The Johns Hopkins School of Medicine, Baltimore, MD; #64927, Addgene; ) and GFP-paxillin.

Techniques: Disruption, Activation Assay, Transfection, Immunostaining, Western Blot, Activity Assay, Suspension, Control, Staining, Expressing, Immunoprecipitation, Fluorescence

Journal: The Journal of Cell Biology

Article Title: mTORC1 activity is supported by spatial association with focal adhesions

doi: 10.1083/jcb.202004010

Figure Lengend Snippet: Constitutive targeting of mTORC1 to FAs uncouples it from regulation by lysosomal positioning. (A) HeLa cells expressing FLAG-RPTOR or FLAG-RPTOR FAT grown in full-nutrient medium were immunostained for FLAG and the FA protein paxillin. Representative images and fluorescence intensity line profile plots corresponding to lines exemplified by arrows are shown. (B) Scrambled (Scr) or ARL8B siRNA–transfected HeLa cells expressing FLAG-RPTOR or FLAG-RPTOR FAT grown in full-nutrient medium were FCS starved for 18 h (−FCS) or FCS starved and then recovered in FCS-containing medium for 10 min (10 min FCS) and immunostained for p-S6 and FLAG. (C) The IntDens of peripheral or intracellular signal of p-S6 was quantified. (D) Cells treated as in B were subjected to immunoblot analysis using antibodies as shown. Error bars represent SEM; n = 3 independent experiments (for IntDens, n ≥ 10 cells were quantified per experiment). **, P < 0.01; two-sided Student’s t test. Scale bars, 20 µm (insets, 10 µm). Nuclei were visualized with DAPI. (E) Diagram illustrating the proposed role of FAs in the activation of mTORC1 in response to growth factor–promoting stimuli. aa trans., amino acid transporter; GFR, growth factor receptor. See Discussion for further details.

Article Snippet: HeLa cells were cultured and cotransfected with the mTORC1 biosensor TORCAR (pcDNA3-TORCAR; a gift from Jin Zhang, The Johns Hopkins School of Medicine, Baltimore, MD; #64927, Addgene; ) and GFP-paxillin.

Techniques: Expressing, Fluorescence, Transfection, Western Blot, Activation Assay