Journal: bioRxiv

Article Title: Clathrin-associated carriers enable recycling through a kiss-and-run mechanism

doi: 10.1101/2024.07.09.601372

Figure Lengend Snippet: Clathrin-associated recycling carriers are derived from early endosomes. a , AP2-TagRFP +/+ CLTA-670nano +/+ cells stably expressing EGFP-sensor were treated with ethanol (control) or latrunculin A, and then imaged at 1.5-s intervals by TIRF microscopy. Shown are tracks of AP2-negative sensor-positive fusion events from representative cells, and the frequency of fusion events from the cells (n = 25 and 30 cells). b , AP2-TagRFP +/+ CLTA-670nano +/+ cells stably expressing EGFP-sensor were treated with DMSO (control) or the myosin II inhibitor blebbistatin, and then imaged by TIRF microscopy. The frequency of fusion events from the cells is shown (n = 24 and 24 cells). c , AP2-TagRFP +/+ CLTA-670nano +/+ cells stably expressing EGFP-sensor were treated with DMSO (control) or nocodazole, and then imaged at 1.5-s intervals by TIRF microscopy. Left: Spatial frequency distribution of fusion events from DMSO- or nocodazole-treated cells (n = 22 and 23 cells). Middle: Frequency of fusion events. Right: Relative frequency of fusion events occurring from the cell edge (set as 0) to the center. d , AP2-TagRFP +/+ CLTA-670nano +/+ cells stably expressing EGFP-sensor were treated with either control siRNA or siRNA targeting Rab5a, Rab5b, Rab5c, or Rab5a/5b/5c, and then imaged at 1-s intervals by TIRF microscopy. Tracks of all fusion events from representative cells and the frequency of fusion events from the siRNA-treated cells are shown (from left to right, n = 43, 40, 41, 41, and 41 cells). e , AP2-670nano +/+ cells stably expressing EGFP-sensor were transiently transfected with mScarlet-I-Rab5a or mScarlet-I-Rab5a(S34N), and then imaged at 1-s intervals by TIRF microscopy. The plots show the frequency of fusion events from the cells (n = 24 and 24 cells). f , AP2-mScarlet +/+ cells were subjected to knock out of the expression of the indicated proteins and then transiently transfected with EGFP-sensor, and then imaged at 1-s intervals by TIRF microscopy. The plots show the frequency of fusion events from the cells (from left to right, n = 22, 23, 23, 24, 22, 22, 36, 38, 36, 37, 36, 36, and 36 cells). g , Cells stably expressing EGFP-sensor and genome-edited to express Halo-EEA1 (pool) were pretreated with DMSO (control) or nocodazole for 1 h and then imaged at 1.5-s intervals by spinning-disk confocal microscopy near the bottom surface. The EEA1-positive endosomes in the time series were detected and tracked. The overlay of all tracks (color-coded based on the velocity of endosome movement) from representative cells treated with DMSO or nocodazole is shown. h-l , Cells stably expressing EGFP-sensor and genome-edited to express Halo-EEA1 (pool) were transiently transfected with AP1σ1-mScarlet-I ( h ), CLTA-mScarlet-I ( i ), mScarlet-I-Rab1a ( j ), mScarlet-I-Rab11a ( k ), or Arf1-mScarlet-I ( l ), stained with the JFX 650 -HaloTag ligand, treated with nocodazole for 1 h, and then imaged at 1.5-s intervals by spinning-disk confocal microscopy near the bottom surface. Montages show the recruitment of EGFP-sensor to the AP1/CLTA/Rab1a/Rab11a/Arf1-positive structures budded from the EEA1-positive endosomes close to the plasma membrane (arrows). m-p , Genome-edited cells expressing Arf1-mEGFP and Halo-EEA1 (pool) were transiently transfected with mScarlet-I-Rab1a ( m ), mScarlet-I-Rab11a ( n ), CLTA-mScarlet-I ( o ), or VPS35-mScarlet-I ( p ), and then imaged near the middle plane by SIM. Left: Sub-organelle distribution of Arf1 with the indicated protein on an EEA1-positive early endosome. Middle: Intensity profiles of the proteins (between the arrows) on the early endosome. Right: Polar plots of the relative spatial distribution of Rab1a, Rab11a, clathrin, or VPS35 with respect to Arf1 on individual early endosomes. Statistical analysis was performed using the unpaired, two-tailed Student’s t -test in a - c and e , and the ordinary one-way ANOVA with Tukey’s multiple comparisons test in d and f ; n.s., no significance; ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are shown as mean ± 95% CI in a - f . Scale bars, 10 μm in g and 0.5 μm in m - p .

Article Snippet: Actin and microtubule networks were disrupted by treating cells with latrunculin A (Cayman; 0.5 μM for 30-60 min), blebbistatin (Targetmol; 25 μM for 2-3 h), or nocodazole (Sigma-Aldrich; 10 μM for 1-2 h) in the SUM159 culture medium.

Techniques: Derivative Assay, Stable Transfection, Expressing, Control, Microscopy, Transfection, Knock-Out, Confocal Microscopy, Staining, Membrane, Two Tailed Test