Clathrin

To construct mammalian expression plasmids, the respective genes of FPs were PCR-amplified as AgeI-NotI fragments and swapped with a gene encoding EGFP in the pEGFP-N1 plasmid (Clontech). IFP2.0-N1 and mIFP-N1 plasmids were acquired from Addgene (#54785 and #54620, respectively).
For protein tagging and labelling of intracellular structures study, miRFPs were amplified, digested with restriction enzymes and then swapped with mTagBFP2 either as C- (for α-tubulin and clathrin) or N-terminal fusions (for keratin, α-actinin, LifeAct, EB3, myosin, vimentin, clathrin, LAMP1, zyxin, H2B and mitochondrial signal) as previously described50 (link). C-terminal fusions (SGGGG)_n linker was increased to 30 amino acids. N-terminal fusions linker length was left unchanged.
To create an IκBα reporter plasmid (CMV-IκBα-miRFP703), we used a CMV-IκBα-FLuc plasmid kindly provided by S. Achilefu and D. Piwnica-Worms. A FLuc gene was replaced with one of the miRFP genes. Kozak sequence was deleted in the CMV-IκBα-miRFP703 and CMV-miRFP control plasmids.
miSplit670 and miSplit709 reporter plasmids, which are pC4-RHE-PAS, pC4EN-F1-mGAF670 and pC4EN-F1-mGAF709, were constructed from an iSplit plasmids25 (link) by swapping either PAS or GAF domains. A linker -ggggsggggs- was left unchanged. Where appropriate, an NLS sequence in the pC4EN-F1 plasmid was deleted by site-directed mutagenesis.
For mRNA labelling, a CMV-PAS-MCP plasmid was constructed as follows. PAS-ggggsggggs- without STOP codon was amplified as a single fragment and inserted into the C1 vector backbone using AgeI and KpnI sites, MCP was amplified from an ubc-nls-ha-MCP-VenusN-nls-ha-PCP-VenusC plasmid (Addgene, #52985) and inserted at KpnI and BamHI sites. The cmv-PCP-mGAF670 and cmv-PCP-mGAF709 plasmids were constructed as follows. A PCP without STOP codon was amplified from an ubc-nls-ha-MCP-VenusN-nls-ha-PCP-VenusC plasmid and then inserted into the C1 vector backbone using AgeI and EcoRI restriction sites. A -ggggsggggs-miGAF was amplified as a single fragment and inserted using EcoRI and KpnI sites. A phage-cmv-cfp-12xMBS-PBS was obtained by swapping a 12xMBS-PBS fragment from a Pcr4-12xMBS-PBS (Addgene, #52984) with 24xMS2 in a phage-cmv-cfp-24xms2 plasmid (Addgene, #40651). An ubc-nls-ha-MCP-VenusN-nls-ha-PCP-VenusC, a phage-cmv-cfp-24xMS2 and a Pcr4-12xMBS-PBS plasmids were gifts from B. Wu and R. Singer.
Plasmids encoding several green-red Fucci cell cycle reporters were provided by A. Miyawaki. The mKO2 and mAG genes fused with hCdt1(30–120), hCdt1(1/100), hGem(1/110) and hGem(1/60) sequences in the pCSII-EF-MCS plasmids were swapped with the miRFP709 or miRFP670v1 genes.

CLSs were detected and quantified using custom software developed in Matlab (MathWorks), as previously described (Aguet et al., 2013; Garay et al., 2015) (64 (link)). Briefly, diffraction-limited clathrin structures were detected using a Gaussian-based model method to approximate the point-spread function of eGFP-CLCa or antibody-labeled clathrin in TIRF-M images. The fluorescence intensity corresponding to the secondary channel such as that of a second label (e.g. CALM, EPSIN, AAK1) or of clathrin within epifluorescence images within CLSs was determined by the amplitude of the Gaussian model for the appropriate fluorescent channel for each structure. As such, the measurements of protein intensity within CLSs represent enrichment of the corresponding signal relative to the local background fluorescence in the vicinity of the detected CLS. Measurements (mean levels of various proteins within specified CLS subset for each cell) were subjected to either two-sided student’s t test or ANOVA followed by Tukey post-test, with a threshold of p < 0.05 for statistically significant differences between conditions.

Automated detection, tracking, and analysis of CCPs (as in Figs. 1 and 2) was as previously described (4 (link), 5 (link), 6 (link), 64 (link)) following time-lapse TIRFM of RPE cells stably expressing eGFP-CLCa. Diffraction-limited clathrin structures were detected using a Gaussian-based model method to approximate the point-spread function (6 (link)), and trajectories were determined from clathrin structure detections using u-track (76 ). sCLSs were distinguished from bona fide CCPs based on unbiased analysis of clathrin intensities in the early CCP stages (5 (link), 6 (link)). Both sCLSs and CCPs represent nucleation events, but only bona fide CCPs represent structures that undergo stabilization, maturation, and in some cases scission to produce vesicles (5 (link), 6 (link)). We report the sCLS nucleation, CCP initiation, CCP lifetime distribution, and the density of persistent CCPs, as well as the intensity of eGFP-CLC within structures as the ‘plateau intensity’ of eGFP-clathrin within these structures. CCPs exhibit several phases including initiation, growth/assembly, plateau, and disassembly/scission (77 (link)). Here, we define the ‘plateau intensity’ of eGFP-CLC in TIRF or epifluorescence microscopy images as the mean fluorescence of that protein within each detected clathrin structure, measured within timepoints corresponding to 30% and 70% of the total lifetime of that structure, during which time CCPs exhibit minimal growth or disassembly (64 (link)). Because CCPs are diffraction-limited objects, the amplitude of the Gaussian model of the fluorescence intensity of eGFP-CLC informs about CCP size (Figs. 1, E and F and 2, E and F). All measurements were subjected to ANOVA followed by Tukey post-test with a threshold of p < 0.05 for statistically significant differences between conditions.

WJ460 (DMSO) was purchased from Glixx Laboratories Inc. or MedChem Express; A-485 (DMSO), vacuolin-1 (DMSO) and apilimod (DMSO) were purchased from MedChem Express; EGA (DMSO), leupeptin (H2O) and pepstatin A (DMSO) were purchased from MilliporeSigma; E64D (DMSO) was purchased from Cayman Chemical. Compounds were purchased in solution or reconstituted and were aliquoted and stored at -20°C or -80°C as recommended by the manufacturers. Viability following treatment with WJ460 was assessed using alamarBlue (Bio-Rad) according to the manufacturer’s instructions. Cells were transfected using Turbofect (Thermo Fisher Scientific) according to the manufacturer’s instructions and harvested 24 h later. Whole cell extracts were prepared, and western blotting was done as described [98 (link)]. Affinity enrichment of HBZ using GST-KIX was done as described [34 (link)]. Antibodies used were as follows: anti-HTLV-1 Env (ARP-1578) and anti-Tax (hybridoma 168B17-46-92) were obtained from NIH AIDS Research and Reagent Program; anti-HBZ has been described [99 (link)]; anti-6x His (ab9108) was purchased from Abcam; anti-MyoF (HPA014245) and anti-Myc (06–340) were purchased from MilliporeSigma; anti-HTLV-1 Env gp46 (1C11, sc-53890; 65/6C2.2.34, sc-57865), anti-Gag p19 (TP7, sc-57870) and anti-β-actin (C4, sc-47778) were purchased from Santa-Cruz; anti-clathrin (D3C6, #4796), anti-caveolin-1 (D46G3, #3267), anti-EEA1 (C45B10, #3288), anti-Rab5a (E6N8S, #46449), anti-Rab7 (D95F2, #9367), anti-Rab11 (D4F5, #5589), anti-LAMP-1 (D2D11, #9091) were purchased from Cell Signaling; anti-EHD2 (PA5-80576) was purchased from ThermoFisher Scientific. All blots were developed using Pierce ECL 2 (Thermo Fisher Scientific) and scanned with a Typhoon RGB imager (Cytiva). Immunoblot quantification was performed using ImageQuant TL v8.1 (Cytiva). To enrich Env SU/gp46 when transiently expressed in HEK293T cells, whole cell extracts were immunoprecipitated with Lens Culinary Agglutinin lectin bound to agarose beads (Vector Laboratories) [26 (link)] and beads resuspended in SDS loading dye.