Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 9 (1), 1109

Clathrin-adaptor Ratio and Membrane Tension Regulate the Flat-To-Curved Transition of the Clathrin Coat During Endocytosis

Affiliations

Clathrin-adaptor Ratio and Membrane Tension Regulate the Flat-To-Curved Transition of the Clathrin Coat During Endocytosis

Delia Bucher et al. Nat Commun.

Abstract

Although essential for many cellular processes, the sequence of structural and molecular events during clathrin-mediated endocytosis remains elusive. While it was long believed that clathrin-coated pits grow with a constant curvature, it was recently suggested that clathrin first assembles to form flat structures that then bend while maintaining a constant surface area. Here, we combine correlative electron and light microscopy and mathematical growth laws to study the ultrastructural rearrangements of the clathrin coat during endocytosis in BSC-1 mammalian cells. We confirm that clathrin coats initially grow flat and demonstrate that curvature begins when around 70% of the final clathrin content is acquired. We find that this transition is marked by a change in the clathrin to clathrin-adaptor protein AP2 ratio and that membrane tension suppresses this transition. Our results support the notion that BSC-1 mammalian cells dynamically regulate the flat-to-curved transition in clathrin-mediated endocytosis by both biochemical and mechanical factors.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Comprehensive ultrastructural characterisation of CCS in BSC-1 cells by TEM. a Schematic of the constant curvature and constant area models. The stages of different curvature (flat (blue), dome (red), pit (green)) and the variation of projected area, which can be assessed during TEM imaging, is depicted for both growth models. b Difference between projected area (black) and surface area (blue) during the course of CCP formation according to the two models. The schematic illustrates the relationship between projected area and surface area for flat, dome (approximately a hemisphere) and pit (approximately a complete sphere) CCSs. c TEM of metal replica from unroofed PM, overview of whole membrane, scale bar: 10 µm. d Examples of flat, dome and pit structures, scale bar: 100 nm. e Fraction of flat (blue), dome (red) and pit (green) CCSs in whole PM of BSC-1 cell. f Projected area distribution of all CCSs (black) measured by TEM. g Projected area distribution of the different clathrin morphologies (flat, dome, pit). A box/whisker plot of the projected area is shown in the inset. Mid-line represents median, cross represents the mean and the whiskers represent the 10 and 90 percentiles. Results are calculated from three different membranes (number of CCSs per membrane: 746, 869 and 739); means with SD are shown
Fig. 2
Fig. 2
CLEM of CCSs. a Schematic representation of the CLEM approach (upper part). Cells growing on poly-d-lysine (PDL)-coated coverslips were unroofed by sonication. Attached PMs were immunostained and imaged using FM. Samples were then critical-point dried and a metal replica was created and lifted from the sample onto a TEM grid for imaging in TEM. (Middle part) The FM and EM pictures were then correlated to combine their information (see Methods). Unroofed PMs were immunostained using an anti-clathrin heavy chains antibody. The white inset box represents the area observed by TEM. (Lower part) Examples of flat, dome, pit and multiple structures; top panel: CLEM, lower left: FM lower right: TEM; scale bar: 100 nm. b Fluorescence intensity distribution (clathrin heavy chain antibody, X22) of all CCSs (black line, left panel) and of flat (blue), dome (red), pit (green) CCSs and multiple structures which cannot be distinguished by fluorescence microscopy (orange) (right panel). A box/whisker plot of the fluorescence intensity is shown in the inset. Mid-line represents median, cross represents the mean and the whiskers represent the 10 and 90 percentiles. c Projected area distribution of all CCSs (black line, left panel) and of the different clathrin morphologies (right panel). d Correlation of size and fluorescence intensity of all CCSs sorted by their different morphologies. Graphs show one representative CLEM result with a total of 347 CCSs from one PM
Fig. 3
Fig. 3
Mathematical modelling of CCS growth from intensity profiles of individual CME events. a Mathematical representation of the constant area model, flat-to-curved transition happens at the time when clathrin reaches its final content. b Example of a clathrin intensity track fitted by the constant area model. Blue dots represent measured intensity of a single CME event; black line represents the fit with Eq. 1, dashed grey line marks the EM detection limit. The schematic on the top illustrates the calculated projected area and assigned curvature, flat (blue), dome (red) and pit (green). c Calculated projected area and curvature distributions of the CCSs according to the constant area model for 4927 FM tracks of 4 different cells. P-value of Welch’s t-test to compare the predicted to the measured distribution in i. A box/whisker plot of the projected area is shown in the inset. Mid-line represents median, cross represents the mean and the whiskers represent the 10 and 90 percentiles. d Mathematical representation of the updated growth model where a flat clathrin patch grows and the flat-to-curved transition happens before reaching the final clathrin content. e, f Same as b, c but using Eq. 2. g Comparison of the predicted ratio of flat, dome and pit structures from both growth model (Eq. 1 (a) and Eq. 2 (d)) and the distribution obtained from TEM imaging. Results are calculated for 4927 FM tracks of 4 different cells; means with SD are shown. h Direct comparison of the projected area distribution of flat and pit structures calculated by Eqs. 1 and 2 as well as measured in EM, box/whisker plot. i Measured projected area and curvature distributions of the CCSs from TEM data as shown in Fig. 1
Fig. 4
Fig. 4
The relative amount of AP2 and clathrin molecules per surface unit of a CCS is curvature dependent. CLEM analysis of CCSs labelled with AP2-eGFP (a) or clathrin heavy chain antibody (b). Flat (blue), dome (red) and pit (green). Lines in the corresponding colour show linear regression of the projected area and of the fluorescence intensity. CLEM analysis of CCS corrected according to the regression of flat structures labelled with AP2-eGFP (c) or clathrin heavy chain antibody (d). Projected areas of dome and pit structures of the CLEM analysis were multiplied by a correction factor to fit the linear regression of flat CCS. Lines in the corresponding colour show linear regression of the calculated surface and the fluorescence intensity. e Table shows correction factors for dome and pit structures for AP2-eGFP or clathrin heavy chain labelling used in (c) and (d)
Fig. 5
Fig. 5
Change in the AP2/clathrin ratio is associated with flat-to-curved transition. a Example of an AP2 (blue) and clathrin (red) intensity profile from an individual CME event. The AP2 profile was fitted to Eq. 1 to find the time when AP2 signal plateaus. For more information see Supplementary Information. The fluorescence intensity of AP2 and clathrin was normalised to the fluorescence intensity of the time when the fitted AP2 profile reaches its plateau. Time offset (difference between the time AP2 plateaus and clathrin reaches its maximum intensity) and intensity offset (excess of maximal clathrin signal over AP2 maximum intensity) are indicated in the profiles. Quantification of the time offset (b) and the intensity offset (c) for 754 tracks of one single cell. d Quantification of the clathrin content at the time when AP2 reaches its plateau from 4927 FM tracks. The clathrin signal was normalised to the maximal clathrin signal in each track. e Example of an AP2 (blue) and clathrin (red) profile fitted to Eqs. 1 and 2, respectively. These fits were used to calculate the size and curvature distributions of the CCS in (f, g). For more information see Supplementary Information. f Comparison of the calculated ratio of flat, dome and pit structures to the measured ratio in TEM. Results calculated from 4927 FM tracks from 4 different cells; means with SD are shown. g Calculated projected area of the CCSs using a growth model where the flat-to-curved transition corroborates with the change of clathrin/AP2 ratio (when AP2 signal reaches its plateau phase) for 4927 FM tracks of 4 different cells. P-value of Welch’s t-test to compare the predicted to the measured distribution in (h). A box/whisker plot of the projected area is shown in the inset. h Measured projected area and curvature distributions of the CCSs from TEM data as shown in Fig. 1. A box/whisker plot of the projected area is shown in the inset. i Direct comparison of the projected area distribution of flat and pit structures calculated according to the AP2/clathrin ratio as well as measured in EM, box/whisker plot
Fig. 6
Fig. 6
Osmotic shock induces stalling of CCSs. a Illustration of the effect of osmotic shock on BSC-1 cells. Hypotonic medium was applied to BSC-1 cells, inducing osmotic swelling that results in an increase in PMT. The same BSC-1-expressing fluorescently tagged clathrin light chain and AP2 proteins were followed from 5 min prior (internal control) until 30 min post hypotonic medium application using spinning disc confocal microscopy. b Kymograph of AP2-eGFP (green) and clathrin light chain a-tdtomato (red)-expressing BSC-1 cells. The dynamics of CCSs was recorded during 5 min prior to osmotic shock until 30 min post osmotic shock. The time after applying the hypotonic medium can be divided into latency, stalling and osmotic shock reversion time depending on the effect on CME dynamics. Scale bar: 5 min. c Representative AP2 (blue) and clathrin (red) intensity profile from an individual CME event during the time of stalling fitted to Eq. 1 to quantify the plateau time. d Quantification of the lifetime of CME events during osmotic shock experiments for 1607 tracks of one single cell. CME events were binned in 3 min intervals in respect to the onset of osmotic shock. Red line indicates lifetime of CME prior to osmotic shock. e Quantification of the plateau time of AP2 of individual CME events during osmotic shock experiments (as defined in Fig. 5a) for 1607 tracks of one single cell. CME events were binned in 3 min intervals in respect to the onset of osmotic shock. Red line indicates plateau time of CME prior to osmotic shock. f Quantification of the time offset between AP2 plateau and clathrin maximum of individual CME events during osmotic shock experiments (as defined in Fig. 5a) for 1607 tracks of one single cell. CME events were binned in 3 min intervals in respect to the onset of osmotic shock. Red line indicates time offset of CME prior to osmotic shock
Fig. 7
Fig. 7
Osmotic shock blocks flat-to-curved transition of CCSs. a Predicted ratio of flat (blue), dome (red) and pit (green) structures calculated from the binned AP2 and clathrin profiles of CME events (Fig. 6c) during osmotic shock for 1357 tracks. b Examples of CCSs under normal and osmotic shock conditions. Blue arrows point to flat structures. c Comparison of measured and predicted frequency of flat, dome and pit structures under normal and osmotic shock conditions. d Projected area distribution of the different clathrin morphologies under normal and osmotic shock conditions. A box /whisker plot of the projected area is shown in the inset. Mid-line represents median, cross represents the mean and the whiskers represent the 10 and 90 percentiles. e Comparison of projected area distributions of flat CCSs under normal and osmotic shock conditions. Results are calculated from four different membranes (number of CCSs per membrane: normal conditions 267, 308, 229 and 323; osmotic shock: 395, 99, 351 and 201); means with SD are shown
Fig. 8
Fig. 8
Model of CCP assembly. Schematic representation of the growth model of CCSs. CCSs initiate as flat clathrin array. They first grow in size in a flat morphology with a constant AP2/clathrin ratio. When they reach around 70% of their full clathrin content, the AP2/clathrin ratio starts to decrease and the CCSs start acquiring their curvature. CCPs keep growing by adding additional clathrin molecules until formation and release of CCVs into the cytoplasm. The flat-to-curved transition of CCSs can be inhibited by increasing PMT, resulting in an accumulation of flat structures. We propose that flat-to-curved transition is concomitant with bypassing the energy barrier necessary to curve the PM and that this critical step in CME is coordinated by the uncoupling of clathrin and AP2 characterised by their abrupt ratio decrease

Similar articles

See all similar articles

Cited by 14 articles

See all "Cited by" articles

References

    1. McMahon HT, Boucrot E. Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nat. Rev. Mol. Cell Biol. 2011;12:517–533. doi: 10.1038/nrm3151. - DOI - PubMed
    1. Ferguson SM, De Camilli P. Dynamin, a membrane-remodelling GTPase. Nat. Rev. Mol. Cell Biol. 2012;13:75–88. doi: 10.1038/nrm3266. - DOI - PMC - PubMed
    1. Taylor MJ, Perrais D, Merrifield CJ. A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis. PLoS Biol. 2011;9:e1000604. doi: 10.1371/journal.pbio.1000604. - DOI - PMC - PubMed
    1. Heuser J. Three-dimensional visualization of coated vesicle formation in fibroblasts. J. Cell Biol. 1980;84:560–583. doi: 10.1083/jcb.84.3.560. - DOI - PMC - PubMed
    1. Maupin P, Pollard TD. Improved preservation and staining of HeLa cell actin filaments, clathrin-coated membranes, and other cytoplasmic structures by tannic acid-glutaraldehyde-saponin fixation. J. Cell Biol. 1983;96:51–62. doi: 10.1083/jcb.96.1.51. - DOI - PMC - PubMed

Publication types

MeSH terms

Feedback