Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Nov 1;130(21):3631-3636.
doi: 10.1242/jcs.205930. Epub 2017 Sep 18.

Mechanoregulation of Clathrin-Mediated Endocytosis

Affiliations
Free PMC article

Mechanoregulation of Clathrin-Mediated Endocytosis

Joshua P Ferguson et al. J Cell Sci. .
Free PMC article

Abstract

We characterized the tension response of clathrin-mediated endocytosis by using various cell manipulation methodologies. Elevated tension in a cell hinders clathrin-mediated endocytosis through inhibition of de novo coat initiation, elongation of clathrin coat lifetimes and reduction of high-magnitude growth rates. Actin machinery supplies an inward pulling force necessary for internalization of clathrin coats under high tension. These findings suggest that the physical cues cells receive from their microenvironment are major determinants of clathrin-mediated endocytic activity.

Keywords: Actin dynamics; Clathrin-mediated endocytosis; Hypotonic shock; Membrane tension; Microaspiration.

Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Aspiration of the plasma membrane slows down clathrin coat dynamics. (A) Kymograph showing the clathrin activity at the ventral surface of a BSC1 cell expressing AP2–eGFP. Clathrin coat traces elongate gradually upon microaspiration (dashed line; Movie 1). Blue and red arrowheads mark the initiation and conclusion of a clathrin-coated structure, respectively. Δt is its lifetime. (B) Clathrin coat lifetime distributions are shown for nine BSC1 cells imaged before and during microaspiration (ntraces=40,943). (C) For the same nine cells, the standard deviation of the clathrin growth rate distributions are shown in boxplots. Lines connect the standard deviation values obtained from the same cell before and during aspiration. The narrower growth rate distributions indicate slower clathrin coat dynamics. (D) Box plots are the initiation and conclusion densities of clathrin-coated structures before and during aspiration. In the boxplots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers show the 10–90th percentiles. P-values were obtained with a two-tailed t-test.
Fig. 2.
Fig. 2.
Cell squeezing induces fast and reversible alterations in clathrin coat dynamics. (A) Cartoon and representative frames of a BSC1 cell are shown at different stages of squeezing (Movie 2). (B) Kymograph showing the temporal evolution of the clathrin traces detected at the ventral surface of the cell shown in A. Dashed lines mark the squeezing steps. (C) For the cell in A, normalized distributions of clathrin growth rates are plotted for different squeezing levels (Fig. S1). The standard deviation of the distribution reduces as the tension increases. (D) For the same cell, the time variation of the ventral surface area (upper) and the standard deviation of the clathrin growth rates (lower). The stepwise changes in these parameters are due to discrete levels of squeezing. (E) The response of the same cell to squeezing is shown as the mean clathrin lifetime (upper) and initiation and conclusion densities (lower). Dashed lines indicate change in squeezing (ntraces=8217). (F) Standard deviation of clathrin growth rates (upper), mean lifetime (middle), and initiation and conclusion densities (lower) from a cell that undergoes increased stepwise squeezing (orange dashed lines) and relaxation (blue dashed lines) (Movie 3) (ntraces=8255).
Fig. 3.
Fig. 3.
Hypotonic swelling inhibits clathrin coat dynamics temporarily. (A) Change in the volume (normalized to the initial value) is plotted for three BSC1 cells during hypotonic swelling (i.e. osmoshock). (B) Mean clathrin coat lifetime (upper), and initiation and conclusion densities (lower) are plotted against time for a BSC1 cell treated with hypotonic shock (dashed line). (C) Clathrin lifetime distributions are assembled pre- and post-osmoshock for 12 gene-edited SUM159 cells expressing AP2–eGFP (ntraces=34,113). (D,E) For the same cells, the standard deviation of clathrin growth rates (D) and initiation and conclusion densities of clathrin-coated structures pre- and post-osmotic shock (E) are shown in boxplots. Lines connect the standard deviation values obtained from the same cell pre- and post-osmoshock. In the boxplots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers show the 10–90th percentiles. P-values were obtained with a two-tailed t-test.
Fig. 4.
Fig. 4.
Actin dynamics mediate the inward movement of clathrin coats prior to disassembly. (A) Mean±s.e.m. values for of normalized AP2 intensity traces (determined for a SUM159 cell before and after hypotonic swelling; ntraces=3728). The traces are aligned at the end point before averaging. (B) Mean±s.e.m. z displacements are shown for the two trace groups in A. (C) Top, growth rate distributions are assembled for eight SUM159 cells before and after hypotonic swelling (ntraces=30,409). Different growth phases (ff, fast formation; sf, slow formation; p, plateau; sd, slow dissolution; fd, fast dissolution) were determined by quantifying the change in the clathrin coat signal over 12-s-long time windows (Ferguson et al., 2016). Bottom, for the same cells, bar plots show the mean+s.e.m. of the z velocities of the trace fragments (12 s long) that are used to generate the growth rate distributions above. Trace fragments that have the highest z velocity are found in the fast dissolution (fd) phase. (D) Top, representative intensity traces of AP2 (green) and LifeAct (red) fluorescence during the formation of a clathrin-coated vesicle at the ventral surface of a BSC1 cell. Bottom, the relative LifeAct intensity (mean±s.e.m.) colocalizing with clathrin coats is shown for different growth phases (ncells=4, ntraces=28,795). Note that the growth phases are determined by using the master (AP2–eGFP) signal. (E) Bar plots show the z velocities (mean±s.e.m.) corresponding to different growth phases for AP2 traces obtained from BSC1 cells in the absence and presence of jasplakinolide (Jasp) (Control, ncells=7, ntraces=20,204; Jasp, ncells=6, ntraces=35,972). *P<0.0001; **P<0.001 (two-tailed t-test).

Similar articles

See all similar articles

Cited by 7 articles

See all "Cited by" articles

MeSH terms

LinkOut - more resources

Feedback