Fig 1.
Isoform-specific differences in recruitment of dynamin to CCPs.
(A) Diagram of Zinc-finger and CRISPR/Cas9n knock-in strategies for endogenous labeling of Dyn2 and Dyn1 in H1299 cells with C-terminal mRuby2 and eGFP tags, respectively. For Dyn2, a short linker and mRuby2 (red) were placed at the stop codon in exon 22. For canonical Dyn1 splice isoform “a,” the 19 C-terminal amino acids (blue) were inserted in exon 21, followed by a short linker, eGFP (green), with stop codon and a polyadenylation sequence (yellow). In both constructs, flanking homology arms (HAs) of roughly 800 bp were used to promote recombination (dashed lines). See S1 Fig for details. (B) Western blot analysis of tagged isoforms. The low levels of Dyn1 in H1299 cells could not be directly detected by western blotting but can be detected after pulldown with GST-Amphiphysin II SH3 domains. Representative TIRF images (see S1 and S2 Movies) showing membrane recruitment of endogenous Dyn2-mRuby2end (C) or Dyn1a-eGFPend (E) and corresponding lentiviral transduced SNAP(647)-CLCa images. (D,F) Clathrin labeled puncta were identified and thresholded to define bona fide CCPs [22]. Shown are the averaged kinetics of recruitment of SNAP-CLCa and Dyn2-mRuby2end (D) or Dyn1a-eGFPend (F) for all tracks with lifetimes between 40 and 60 s (831 CCPs from 5 movies containing a total of 15 cells for Dyn2-mRuby2end and 13,346 CCPs from 10 movies containing a total of 29 cells for Dyn1a-eGFPend). CLCa, clathrin light chain a; CRISPR/Cas9n, Clustered Regularly Interspaced Short Palindromic Repeats-associated nucleases 9 nickase; Dyn1, dynamin-1; GST, Glutathione S-transferase; HA, homology arm; SH3, SRC Homology 3.
Fig 2.
Activated Dyn1 regulates early stages of CME, even when recruited at low levels to CCPs.
(A) Schematic representation of Dyn1 regulation by phosphorylation/dephosphorylation and activation upon GSK3β kinase inhibition. (B) Dephosphorylation of Dyn1 S774 upon GSK3β inhibition by 20 μM CHIR99021 observed by immunoblotting using a Dyn1 phosphospecific antibody and the quantification of pDyn1/Dyn1 intensity ratios (mean ± SD, n = 3). Lysates were loaded at two different dilutions (1x and 0.3x). (C) Transferrin receptor (TfnR) internalization efficiency of parental H1299 cells and Dyn1KO cells and their sensitivity to GSK3β inhibition (mean ± SD, n = 3). (D) Initiation densities of bona fide CCPs and (E) their median lifetimes. Each dot represents the average value per movie, where each movie contained 1–5 cells (see Materials and methods). (F) The distribution of CCP lifetimes measured in the absence or presence of GSK3β inhibitor. Data are derived from 10 movies each; 13,346 CCPs of 40–60-s lifetimes were analyzed from 74,807 bona fide CCPs, and 13,494 CCPs of 40–60-s lifetimes were analyzed from 75,426 bona fide CCPs, respectively, for control and GSK3β inhibition. Similarly, the initiation densities (G), median lifetime (H), and the lifetime distribution of bona fide CCPs (I) were analyzed for H1299 Dyn1KO cells with or without GSK3β inhibition. Average recruitment of Dyn1a-eGFPend to CLCa-labeled CCPs with lifetimes of 40–60 s measured in the absence (J) or presence (K) of GSK3β inhibitor. (L) Maximum intensity of Dyn1a-eGFPend detected at any point throughout the lifetime of an individual CCPs measured in the absence or presence of GSK3β inhibitor. The underlying data of panels B–I and L can be found in S1 Data. (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001; see Materials and methods for description of statistical analysis used in this and other figures.) CCP, clathrin-coated pit; CLCa, clathrin light chain a; CME, clathrin-mediated endocytosis; Dyn1, dynamin-1; GSK3β, glycogen synthase kinase-3 beta.
Fig 3.
Dyn1 is inefficiently recruited to CCPs, even when overexpressed and activated in Dyn1KO H1299 cells.
(A) Western blot showing overexpression of Dyn1WT-eGFP or Dyn1S774/8A-eGFP in Dyn1KO H1299 cells. Note that endogenous Dyn1 is not detected in parental cells at this loading level (see Fig 1B). (B) Effect of siRNA knockdown of Dyn2 on TfnR internalization in Dyn1KO cells reconstituted with Dyn1aWT-eGFP and treated or not with GSK3β inhibitor. Results are normalized to rates of endocytosis in parental H1299 cells. The data represents mean ± SEM of n = 3 experiments containing four replicates each (*p ≤ 0.05, **p ≤ 0.01 and ****p ≤ 0.0001). Initiation densities (C), median lifetimes (D), and the lifetime distribution (E) of bona fide CCPs analyzed in H1299 Dyn1KO cells reconstituted with Dyn1WT-eGFP with or without GSK3β inhibition, determined as in Fig 2. (F) Representative TIRFM images of overexpressed Dyn1WT-eGFP and mRuby2-CLCa and (G) quantification of the average recruitment of Dyn1WT-eGFP to CCPs, identified by mRuby2-CLCa, with lifetimes between 40 and 60 s (14,495 CCPs from a pool of 100,050 bona fide Dyn1-positive CCPs from 18 movies and 9,651 CCPs from a pool of 68,909 bona fide CCPs from 12 movies were analyzed from control and GSK3β, respectively). (H) Maximum Dyn1aWT-eGFP intensity averaged among individual bona fide CCP tracks in the absence or presence of GSK3β inhibitor. The underlying data of panels B–E and H can be found in S1 Data. CCP, clathrin-coated pit; CLCa, clathrin light chain a; Dyn1, dynamin-1; GSK3β, glycogen synthase kinase-3 beta; siRNA, small interfering RNA; TfnR, transferrin receptor; TIRFM, total internal reflection fluorescence microscopy.
Fig 4.
Nonphosphorylatable Dyn1 mutant mimics GSK3β effects and can partially substitute for Dyn2.
CCP initiation densities (A), median lifetimes (B), and the lifetime distribution (C) of bona fide CCPs analyzed in H1299 Dyn1KO cells reconstituted with Dyn1WT- or Dyn1S774/8A-eGFP, determined as described in Fig 2. (D) Representative TIRFM images of overexpressed Dyn1WT-eGFP or Dyn1S774/8A-eGFP and mRuby2-CLCa and (E) quantification of their average recruitment to CCPs with lifetimes between 40 and 60 s. (F) Maximum intensities of Dyn1WT-eGFP or Dyn1S774/8A-eGFP averaged among individual bona fide CCP tracks. (G) Effect of siRNA knockdown of Dyn2 on TfnR endocytosis in parental and Dyn1KO H1299 cells and Dyn1KO cells reconstituted with either Dyn1aWT-eGFP or Dyn1aS774/8A-eGFP. (H) Representative TIRFM images of Dyn2 siRNA-treated Dyn1KO cells overexpressing Dyn1aWT-eGFP and mRuby2-CLCa treated or not with GSK3β inhibitor and (I) quantification of the average recruitment of Dyn1WT-eGFP to CCPs with lifetimes between 40 and 60 s in Dyn2 knockdown cells treated or not with GSK3β inhibitor. The underlying data of panels A–C, F, and G can be found in S1 Data. CCP, clathrin-coated pit; CLCa, clathrin light chain a; Dyn1, dynamin-1; GSK3β, glycogen synthase kinase-3 beta; siRNA, small interfering RNA; TfnR, transferrin receptor; TIRFM, total internal reflection fluorescence microscopy.
Fig 5.
Dyn1 and Dyn2 are differentially recruited to CCPs and differentially required for GSK3β-regulated CME.
(A) Immunoblot validation of Dyn1 and Dyn2 KO A549 cells and their corresponding reconstitution at near endogenous levels with eGFP-labelled Dyn1 or Dyn2. GFP blot shows that in A549 cells Dyn1 is expressed at approximately 5-fold higher levels than Dyn2. CCP initiation densities (B), and median lifetimes (C) in Dyn1 or Dyn2 knockout cells with or without GSK3β inhibition, determined as described in Fig 1. (D) Representative TIRFM and epi images of co-cultured Dyn1KO:Dyn1a-eGFP:SNAP-CLCa and Dyn2KO:Dyn2-eGFP:mRuby2-CLCa cells allowing direct comparison of Dyn1a-eGFP versus Dyn2-eGFP recruitment to CCPs in A549 cells. (E) Quantification of the average recruitment of Dyn1a-eGFP or Dyn2-eGFP to CCPs with lifetimes between 40 and 60 s (4,420 CCPs from a pool of 12,555 Dyn1a-eGFP-positive CCPs and 3,961 CCPs from a pool of 12,766 Dyn2-eGFP-positive CCPs from a total of 11 movies were identified to have a lifetime between 40 and 60 s). Data are obtained from cells co-imaged either for SNAP(647)-CLCa (and Dyn1a-eGFP) or mRuby2-CLCa (and Dyn2-eGFP). (F) Maximum intensities of Dyn1a-eGFP or Dyn2-eGFP averaged among individual bona fide CCP tracks. (G) Subcellular fractionation of parental A549 cells into membrane (P) versus cytosolic (S) fractions and western blotted for the indicated proteins. Cytosolic MEK1/2 and membrane-associated TfnR serve as controls for fractionation. Quantification is shown in red above each band as the fraction of total protein in the P versus S fraction. Results are representative of 3 independent experiments. The underlying data of panels B, C, and F can be found in S1 Data. CCP, clathrin-coated pit; CLCa, clathrin light chain a; CME, clathrin-mediated endocytosis; Dyn1, dynamin-1; epi, epifluorescent; GSK3β, glycogen synthase kinase-3 beta; KO, knockout; TfnR, transferrin receptor; TIRFM, total internal reflection fluorescence microscopy.
Fig 6.
Generation and characterization of dual genome-edited Dyn1a-eGFP and Dyn2-mRuby2 A549 cells.
(A) Immunoblot validation of Dyn1a-eGFP and Dyn2-mRuby2 single- and dual-genome-edited A549 cells. (B) TfnR endocytosis in dual genome edited A549 cells compared to parental A549 cells and their sensitivity to siRNA-mediated Dyn2 knockdown. (C) Representative TIRF images of Dyn1 and Dyn2 distribution relative to CLCa in dual genome-edited A549 cells. CCP initiation densities (D), median lifetimes (E), and the lifetime distribution (F) of bona fide CCPs in dual-genome-edited A549 cells with or without GSK3β inhibition, determined as in Fig 2. The underlying data of panels B and D–F can be found in S1 Data. Dyn1, dynamin-1; GSK3β, glycogen synthase kinase-3 beta; TfnR, transferrin receptor.
Fig 7.
Tracking clathrin and dynamins in dual genome-edited Dyn1a-eGFP and Dyn2-mRuby2 A549 cells.
(A) Representative TIRF images and corresponding kymographs of dynamic behavior of overexpressed SNAP-CLCa, Dyn2-mRuby2end, and Dyn1a-eGFPend in dual genome-edited A549 cells. See S3 Movie. (B) Examples of Dyn1 and Dyn2 dynamics at individual CCPs (i–iv) and (C) their corresponding quantitative traces. CLCa, clathrin light chain a; Dyn1, dynamin-1.
Fig 8.
Dyn1 and Dyn2 are recruited to the same CCPs and Dyn1 activation alters the dynamics of all CCP subpopulations.
(A) Triple-color master/slave analyses of average dynamics of recruitment of Dyn2-mRuby2endo and/or Dyn1a-eGFPend to lifetime cohorts of SNAP-CLCa labeled CCPs identifies Dyn1/Dyn2 positive, Dyn1 only, Dyn2 only, and Dyn1/2-negative subpopulations of CCPs. The percentage of detected CCPs in each class is indicated. (B) Effect of GSK3β inhibition on the median lifetimes of compositionally distinct CCP subpopulations. The underlying data of panel B can be found in S1 Data. CCP, clathrin-coated pit; CLCa, clathrin light chain a; Dyn1, dynamin-1; GSK3β, glycogen synthase kinase-3 beta.
Fig 9.
SNX9 preferentially binds activated Dyn1 and is required for Dyn1-dependent changes in the lifetime distribution of CCPs.
(A) Effect of siRNA knockdown of the indicated dynamin SH3 domain-containing endocytic accessory proteins on TfnR endocytosis in parental, Dyn1KO, and Dyn2KO A549 cells. siEndo refers to siRNA knockdown of endophilin A1, 2, and 3; siITSN refers to siRNA knockdown of intersectins 1 and 2; all others were single siRNAs. Knockdown efficiencies were determined to be >85% by western blotting. Data are normalized to the extent of TfnR uptake in control siRNA-treated parental, Dyn1KO, and Dyn2KO cells, which is set to 100, to allow direct comparison of the relative effects of siRNA knockdowns. (B) eGFP pulldown of Dyn1aWT-eGFP, Dyn1aS774/8A-eGFP, or Dyn2WT-eGFP expressed in Dyn1KO or Dyn2KO A549 cells, respectively, using anti-eGFP nAbs. Parental cells that do not express an eGFP-tagged protein (Dyn1end) are used as control. The pulldown fractions were analyzed by immunoblot. Effect of SNX9 siRNA-mediated knockdown on (C) CCP initiation densities, (D) median lifetimes, and (E) lifetime distribution of bona fide CCPs in Dyn1KO H1299 cells overexpressing either Dyn1aWT-eGFP or Dyn1aS774/8A-eGFP (data are derived from 7 movies for each condition, with each movie consisting of 1–3 cells). Each data point is the average value from a single movie. The underlying data of panels A, C–E, and L can be found in S1 Data. (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001) CCP, clathrin-coated pit; Dyn1, dynamin-1; SH3, SRC Homology 3; siRNA, small interfering RNA; SNX9, sorting nexin 9; TfnR, transferrin receptor.
Fig 10.
EGF stimulation alters CCP dynamics in a Dyn1-dependent manner.
(A) Immunoblot analysis of changes in phosphorylation state of Dyn1 S774 and GSK3β upon EGF stimulation of parental A549 cells. (B,C) Quantification of the EGF-triggered changes in phosphorylation state (i.e., ratio of phosphorylated/ total protein) of GSK3β and Dyn1 (mean ± SD of n = 3 experiments, data are normalized to 0 min time point). CCP initiation densities (D), median lifetimes (E), and the lifetime distribution (F) of bona fide CCPs in serum starved A549 cells before (Control, Ctrl) or after incubation with EGF (20 ng/ml) for 10 min prior to imaging. CCP initiation densities (G) and median lifetimes (H) in serum-starved Dyn1KO A549 cells before (Ctrl) or 10 min after incubation with EGF (EGF). The underlying data of panels B–H can be found in S1 Data. CCP, clathrin-coated pit; Dyn1, dynamin-1; GSK3β, glycogen synthase kinase-3 beta.