Flat-to-curved transition during clathrin-mediated endocytosis correlates with a change in clathrin-adaptor ratio and is regulated by membrane tension

TL;DR: The results support the model that mammalian cells dynamically regulate the flat-to-curved transition in clathrin-mediated endocytosis by both biochemical and mechanical factors.

Abstract: Although essential for many cellular processes, the sequence of structural and molecular events during clathrin-mediated endocytosis remains elusive. While it was believed that clathrin-coated pits grow with a constant curvature, it was recently suggested that clathrin first assembles to form a flat structure and then bends while maintaining a constant surface area. Here, we combine correlative electron and light microscopy and mathematical modelling to quantify the sequence of ultrastructural rearrangements of the clathrin coat during endocytosis in mammalian cells. We confirm that clathrin-coated structures can initially grow flat and that lattice curvature does not show a direct correlation with clathrin coat assembly. We demonstrate that curvature begins when 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 model that mammalian cells dynamically regulate the flat-to-curved transition in clathrin-mediated endocytosis by both biochemical and mechanical factors.

Summary

Introduction

• Clathrin-mediated endocytosis (CME) is an essential uptake pathway that relocates membrane or extracellular cargo into the cell to regulate multiple cellular functions and cell homeostasis 1 .
• This process is coordinated by numerous adaptor and accessory proteins 1, 3 .
• For topological reasons this requires a major ultrastructural rearrangement of the clathrin lattice which appeared to be dynamically difficult and energetically costly [8] [9] [10] [11] [12] .
• Recently, correlative light and electron microscopy (CLEM) analyses provided experimental evidence that CCS first grow flat to their final size and then acquire curvature (constant area model, Fig. 1a ) 19 .
• The authors combine mathematical modelling of individual endocytic event dynamics and CLEM analysis to provide a comprehensive description of the dynamic ultrastructural rearrangement of the clathrin coat during CME.

EM and CLEM analysis of CCS do not support existing growth models

• To address whether CCP formation follows the constant curvature model or the constant area model (Fig. 1a ) 13 , the authors chose BSC-1 cells, a widely used cellular model to study CME 10, 14, 20 .
• In contrast, the constant area model implies that both projected and surface areas initially show similar growth but then the projected area should drop significantly as bending starts (Fig. 1b ).
• In contrast their EM data reveals that around 50% of the CCS in BSC-1 correspond to flat CCS (Fig. 1e ) and that a large fraction of the flat and dome structures have a projected area larger than the projected area of the pits (Fig. 1g ).
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• In conclusion, their TEM and CLEM analyses argue that neither of the proposed growth models fully explains the observed ultrastructural distribution and corresponding fluorescence intensities of CCS in BSC-1 cells.

Modelling of CCP assembly reveals bending of the coat before reaching full surface area

• It only provides snapshots of the dynamic process of CCP assembly 23 .
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• Mathematically there is only one type of growth equation that can explain why growth should stop at a finite patch radius, namely association over the edge and dissociation over the area of the patch (Supplementary Information).
• Given the high proportion of flat structures (Fig. 1e ), the authors reasoned that CCS start as flat structures and then acquire curvature before reaching the full clathrin content (Fig. 3d and Supplementary Information).
• As before, the ratio between the flat, dome, and pit structures was biased towards flat structures (Fig. 3g ) but now the calculated size and morphology distribution fit the EM data better than the distribution according to the constant area model.

Starting point of acquiring curvature is marked by a change in the AP2/clathrin ratio

• The flat-to-curved transition of a CCS requires major ultrastructural reorganisation of the coat 11 .
• To find the relationship between fluorescence intensity and the surface of CCS, the measured projected area needs to be corrected for the curvature to obtain the surface area of the CCS (Fig. 1b ).
• Assuming the geometry of a hemisphere for domes and an almost complete sphere for pits the authors expect a correction factor of ≤2 certified by peer review) is the author/funder.
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• These findings strongly support a model where flat-to-curved transition initiates at around 70% of its maximal clathrin content and correlates with the concomitant change in the AP2/clathrin ratio.

High membrane tension inhibits the flat-to-curved transition of CCS

• By inducing curvature to the PM, the CCS needs to act against the plasma membrane tension (PMT).
• This effect was transient and cells quickly reverted to normal clathrin dynamics (Fig. 6b and d ).
• The authors found an accumulation of flat CCS under osmotic shock compared to normal conditions and the frequency was comparable to their predictions from the AP2 and CLC profiles certified by peer review) is the author/funder.
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• These flat structures, as well as the dome and pit structures, have the same size distribution as under normal conditions (Fig7d box/whiskers and 7e).

Discussion

• The complex coordination of CCS formation during CME has been investigated for decades 32, 33 and the field has been driven by the competition between the constant curvature versus the constant area models 13 .
• The authors model where a change in AP2/clathrin ratio drives the flat-to-curved transition is consistent with their recent observation that AP2 (and other adaptor/accessory proteins) partitions in different nanoscale area of the clathrin coat and that the concentration of AP2 varies within these zones at various stage of CCS assembly 22 .
• The authors growth behaviour of CCP assembly, determined in this work in BSC-1 cells, may also apply for other cell types.
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• Surprisingly, the increase in PMT only changes the ratio between the different morphologies of CCS in favour of flat structures but does not affect their size.

Methods

• Cell lines and cell culture BSC-1 cells were obtained from ATCC.
• BSC-1 cells stably expressing AP2-eGFP were created by transfecting BSC-1 cells with a plasmid expressing the sigma2 subunit of AP2 fused to eGFP 20 .
• After detaching, the cells were resuspended in complete medium.

Transfection

• Transfection of cells was done using Lipofectamine 2000 .
• The next day, cells were transfected at 70-80% confluence.
• 2µg DNA and 8µl Lipofectamine 2000 were separately mixed with 100µl OptiMEM .
• For generation of stable cell lines, the growth medium was exchange for fresh growth medium after 8 hours.
• For live-cell imaging of BSC-1 AP2-eGFP transiently expressing CLCa-tdtomato were seeded 8 hours after transfection.

Live-cell microscopy

• Glass coverslips (TH. Geyer, 25mm diameter, No. 1.5H) were coated with poly-Dlysine solution (Sigma-Aldrich, #P6407) at concentration 0.1mg ml -1 for 5 minutes at room temperature and washed three times with PBS.
• Cells were seeded on poly-D-lysine-coated coverslips and live-cell microscopy was performed 12-16 hours after seeding.
• Live-cell imaging of AP2-eGFP was performed with an inverted spinning-disk confocal microscope , with a 60x (1.42 numerical aperture, Apo TIRF, Nikon) or 100x (1.4 numerical aperture, Plan Apo VC, Nikon) oil immersion objective and a CMOS camera (Hamamatsu Ocra Flash 4).
• An environment control chamber was attached to the microscope to keep 37 °C and 5%CO2.
• 10 minutes-long movies of representative cells were taken with one frame every 3 seconds.

Osmotic shock experiments

• For live-cell microscopy of cells under osmotic shock, one cell was imaged for 5 minutes under normal conditions.
• Afterwards the medium was change to hypotonic certified by peer review) is the author/funder.
• 1 ratio of medium to water with 10% FBS) and the same cell was imaged for an additional 30 minutes, also known as medium (1.
• For TEM of cells under osmotic shock, cells were put into hypotonic medium and unroofed after 10 minutes of osmotic shock.

Immunofluorescence

• For immunofluorescence, intact cells growing on poly-D-lysine coated 12mm coverslips (#1.5, Thermo Scientific) or unroofed PMs were fixed with 2-4% paraformaldehyde for 20 minutes at room temperature.
• Intact cell were permeabilised with 0.5% Triton X in PBS for 15 minutes.
• After five washes with PBS, samples were incubated with the secondary antibody.
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Imaging of unroofed PMs for CLEM

• Widefield fluorescent images of unroofed PMs were taken with a Nikon N-STORM microscope with a 100x oil immersion objective and an EMCCD camera (Andor Ixon Ultra DU-897).
• The imaged area was marked with a circle (4mm in diameter) around the centre of the imaged area using an objective diamond scriber.
• The immersion oil was carefully removed from the bottom of the glass coverslip and the sample was prepared for EM.

TEM of metal replica

• Coverslips with unroofed membranes were fixed with 2% glutaraldehyde in PBS overnight.
• After two washes with water, samples were dehydrated with a series of ethanol solutions (15%-100%).
• For better orientation, the marked area of the coated samples was imaged with a phase contrast microscope.
• 5% hydrofluoric acid was used to remove glass from the metal replica.
• Certified by peer review) is the author/funder.

Transformation of images for CLEM

• The fluorescence microscopy image and the electron microscopy montage of the same membrane sheet were first manually and roughly overlaid using Photoshop.
• MATLAB was used to transform the fluorescence image according to the electron microscopy montage using three manually identified CCS.
• For the transformation the centre of the clathrin structure in the electron microscope montage and the centre of the fluorescence signal determined by a Gaussian fit were used as landmarks.

Tracking

• For tracking CME events, the authors used ilastik (http://ilastik.org).
• First the images were segmented using the pixel classification and object classification workflow.
• The maximal distance was put to 5 to avoid merging of close tracks.
• For particle detection, a Laplacianof-Gaussian filter and connected-component labelling was used.
• In addition, the lifetime of CCS was quantified and classified into different ranges.

STED

• STED nanoscopy was carried out using the Two-color-STED system (Abberior Instruments GmbH, Göttingen).
• Image acquisition was performed using a 100x Olympus UPlanSApo (NA 1.4) oil immersion objective and 70 % nominal STED laser power (l= 775 nm, max.
• Deconvolution of acquired images was done using certified by peer review) is the author/funder.

Constant area model

• As the fluorescence intensity of labelled clathrin triskelia is proportional to the number of incorporated clathrin triskelia or equivalently to the size of clathrin covered membrane area, the authors model the assembly of CCV as surface growth.
• In the observed fluorescence intensity tracks the intensity decreases after some time until the intensity vanishes completely.
• The constant area model assumes that a flat patch transforms into a spherical pit as soon as the patch reaches the area plateau.
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Data fits

• To test whether the constant area model correctly describes the shape and size of clathrin coated vesicles the authors fitted Eq. 1 to 4927 FM tracks of 4 different cells (Fig. 3b ) and calculated from the fitted area surface growth curves histograms which they could compare to EM histograms.
• Therefore, the authors related the intensity of an FM track to its corresponding area.
• Furthermore, the FM dataset was filtered before the fitting.
• The exact details of their procedure are described in the following.

Relate fluorescence intensity and surface area by means of CLEM

• To relate the fluorescence intensity of a clathrin FM track to the corresponding clathrin covered membrane area the authors use their clathrin CLEM data, relating the projected surface size of CCS to their fluorescence intensity.
• The authors analyse flat CCS for which the projected area directly corresponds to their surface.
• As the local intensity background is removed from the CLEM data the authors find 𝐼(𝐴) = 𝛽𝐴, (Eq. S2) where I is the intensity of the FM data, A is the area of the clathrin structure and 𝛽 is the proportionality constant.
• Fig. 4b shows the intensity of flat structures as a function of their projected area (blue).
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Relate different FM datasets

• To calculate a size histogram from fluorescence intensity tracks the authors analyse live cell FM data, that have a different intensity level than the fluorescence intensity of the CLEM data.
• Therefore, the authors need to relate these two different data sets.
• As the CLEM intensity I and the live cell FM intensity I' are both proportional to the number of labelled clathrin triskelia, both intensities, which are background corrected, can be related only by some factor 𝛼.
• In the live cell FM data set the authors register only detectable intensities, which exceed the local background signal.
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Data filtering

• To ensure that all structures correspond to real objects the authors assume for a productive object a minimal lifetime of 24 seconds.
• The authors filter their data set for FM tracks with multiple structures (defined as a FM track that shows at least two clear intensity maxima) to allow for direct fitting of single tracks.

Parameter choice and data fitting

• The parameters for the fit are restricted by assuming that growth curves should at least reach 90% and at most 120% of the maximal area value.
• Additionally, the authors assume that vesicle pinch off (corresponding to a decrease in the intensity to 10%) takes between 10 seconds to 20 seconds.
• Additionally, the authors require the fit to reach 99% of the steady state area before the area decrease happens and at least 10% of that time until the 99% area level are reached.
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Calculation of the size histogram

• From the growth curves, the authors calculated a histogram (Fig. 3c ) to compare the constant area model to the measured projected size EM histogram (Fig. 3h ).
• The authors classified the chosen times and corresponding areas into three categories.
• The authors computed the projected area by assuming that the transformation within the dome and pit phase is a linear function of time.
• From the growth curves the authors calculated a histogram (Fig. 3f ) to compare "the curvature acquisition during growth model" to the measured projected size EM histogram (Fig. 3h ).
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Curvature acquisition during growth: updated model

• In the updated growth model the authors assume that CCS first grow flat, start to invaginate as they reach 70 % of their final size (which they determine by taking the inverse of the intensity ratio of pit and flat structures in clathrin CLEM, which is 1.44) and finally grow as a spherical cap until a full pit has formed.
• As before the authors model the assembly of clathrin coated vesicles as surface growth.
• The authors mathematical description of the "curvature acquisition during growth model " considers a spherical cap with radius 𝑅 that grows at its edge with rate 𝑘 "# which can be expresses by sizes.
• After reaching 70% of the maximal area the authors classify CCS in the first 40% of the remaining time to be domes and pits otherwise.
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Data fits, parameter choice and data fitting

• To test whether the "curvature acquisition during growth" model correctly describes the shape and size of clathrin coated vesicles the authors fitted Eq. S4 to 4927 FM tracks of 4 different cells (Fig. 3e ) and calculated from the fitted area surface growth curves histograms which they could compare to EM histograms.
• Therefore, the authors related the intensity of an FM track to its corresponding area.
• The exact details of their procedure are the same as before.
• The parameters for the fit are restricted by assuming that growth curve should at least reach 90% and maximal 120% of the maximal area value and that the vesicle pinching off (corresponding to a decrease in the intensity to 10%) takes between 10 seconds to 20 seconds.
• The authors implement the Python module 'lmfit' for fitting the area tracks where they use the method 'nelder' of the minimiser function.

Live cell FM analysis resampling

• To determine the ratio of clathrin and AP2 during the process of CME, the authors performed TIRF microscopy of BSC-1 AP2-eGFP cells transiently expressing CLCatdtomato.
• The authors analysed the excess of clathrin in comparison with AP2 during the formation of CCVs.
• Therefore, the authors calculated the ratio of the maximum clathrin intensity divided by the intensity of clathrin at the time when AP2 shows an intensity plateau (95% intensity level) and subtract this ratio from the ratio, which they get for AP2.
• The authors calculate the plateau time, defined as the time when 95% of the AP2 plateau intensity is reached by fitting the constant certified by peer review) is the author/funder.
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Calculation of the ratio histogram during the osmotic shock

• To determine the ratio histogram of flat, dome and pit CCS during the osmotic shock (Fig. 7a and 7c ) the authors first defined a transition time 𝑡 D@A#0$"@ADƒ"# , when flat CCS start to invaginate, where the normalised clathrin intensity exceeds the normalised AP2 intensity by 5% (blue region in Fig. 6c ).
• The found ratios of flat, dome and pit CCS were then plotted as a function of the time after the osmotic shock (Fig. 7a ).
• Averaging over all tracks and considering only tracks with lifetimes shorter than 90s and with AP2/clathrin discrepancy the authors obtained 47.8% flat CCS, 18.0% dome CCS and 34.2% pit CCS which is close to the determined ratios in (Fig. 5g ). certified by peer review) is the author/funder.
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Figures

Figure 5: Change in the AP2/clathrin ratio is associated with flat-to-curved 962 transition. (a) Example of an AP2 (blue) and clathrin (red) intensity profile from an 963 individual CME event. The AP2 profile was fitted to equation 1 to find the time when 964 AP2 signal plateaus. For more information see Supplementary Information. The 965 fluorescence intensity of AP2 and clathrin were normalised to the fluorescence 966 intensity of the time when the fitted AP2 profile reaches its plateau. Time offset 967 (difference between the time AP2 plateaus and clathrin reaches its maximum 968 intensity) and intensity offset (excess of maximal clathrin signal over AP2 maximum 969 intensity) are indicated in the profiles. Quantification of the time offset (b) and the 970 intensity offset (c) for 754 tracks of one single cell. (d) Quantification of the clathrin 971 content at the time when AP2 reaches its plateau from 4927 FM tracks. The clathrin 972 signal was normalised to the maximal clathrin signal in each track. (e) Example of an 973 AP2 (blue) and clathrin (red) profile fitted to equation 1 and 2, respectively. These 974 fits where used to calculate the size and curvature distributions of the CCS in (f and 975

Figure 2: CLEM of CCS. (a) Schematic representation of the CLEM approach (Upper 906 part). Cells growing on poly-D-lysine (PDL)-coated coverslips were unroofed by 907 sonication. Attached PMs were immunostained and imaged using FM. Samples were 908 then critical-point dried and the metal replica was created and lifted from the 909 sample onto a TEM grid for imaging in TEM. (Lower part) The FM and EM pictures 910 were then correlated to combine their information (See methods). Unroofed PMs 911 were immunostained using an anti-clathrin heavy chains antibody. The white inset 912 box represents the area observed by TEM. (b) Fluorescence intensity distribution 913 (clathrin heavy chain antibody, X22) of all CCS (black line, left panel) and of flat 914 (blue), dome (red), pit (green) CCS, and multiple structures which cannot be 915

Figure 7: Osmotic shock blocks flat-to-curved transition of CCS. (a) Predicted 1018 ratio of flat (blue), dome (red), and pit (green) structures calculated from the 1019 binned AP2 and clathrin profiles of CME events (Fig.6c) during osmotic shock for 1020 1357 tracks. (b) Examples of CCS under normal and osmotic shock conditions. 1021 Blue arrows point to flat structures. (c) Comparison of measured and predicted 1022 frequency of flat, dome, and pit structures under normal and osmotic shock 1023 conditions. (d) Projected area distribution of the different clathrin morphologies 1024 under normal and osmotic shock conditions. A box /whiskers plot of the 1025 projected area is shown in the inset. Mid-line represents median, cross represents 1026 the mean and the whiskers represent the 10 and 90 percentiles. (e) Comparison 1027 of projected area distributions of flat CCS under normal and osmotic shock 1028 conditions. Results are calculated from four different membranes (number of CCS 1029 per membrane: normal conditions 267, 308, 229, and 323; osmotic shock: 395, 1030 99, 351, and 201); means with SD are shown. 1031

Figure 4: The relative amount of AP2 and clathrin molecules per surface unit 948 of a CCS is curvature dependent. CLEM analysis of CCS labelled with AP2-eGFP (a) 949 or clathrin heavy chain antibody (b). Flat (blue), dome (red), and pit (green). Lines 950 in the corresponding colour show linear regression of the projected area and of the 951 fluorescence intensity. CLEM analysis of CCS corrected according to the regression 952 of flat structures labelled with AP2-eGFP (c) or clathrin heavy chain antibody (d). 953 Projected areas of dome and pit structures of the CLEM analysis were multiplied by 954 a correction factor to fit the linear regression of flat CCS. Lines in the corresponding 955 colour show linear regression of the calculated surface and the fluorescence 956 intensity. (e) Table shows correction factors for dome and pit structures for AP2-957 eGFP or clathrin heavy chain labelling used in (c) and (d). 958

Figure 6: Osmotic shock induces stalling of CCS. (a) Illustration of the effect of 991 osmotic shock on BSC-1 cells. Hypotonic medium was applied to BSC-1 cells, 992 inducing osmotic swelling that results in an increase in PMT. The same BSC-1 993 expressing fluorescently tagged clathrin light chain and AP2 proteins was followed 994 from 5 minutes prior (internal control) until 30 minutes post hypotonic medium 995 application using spinning disc confocal microscopy. (b) Kymograph of AP2-eGFP 996 (green) and clathrin light chain a-tdtomato (red) expressing BSC-1 cells. The 997 dynamics of CCP was recorded during 5 min prior to osmotic shock until 30 minutes 998 post-osmotic shock. The time after applying the hypotonic medium can be divided 999 into latency, stalling, and osmotic shock reversion time depending on the effect on 1000 CME dynamics. Scale bar: 5 minutes. (c) Representative AP2 (blue) and clathrin 1001 (red) intensity profile from an individual CME event during the time of stalling fitted 1002 to equation 1 to quantify the plateau time. (d) Quantification of the lifetime of CME 1003 events during osmotic shock experiments for 1607 tracks of one single cell. CME 1004 events were binned in 3 min intervals in respect to the onset of osmotic shock. Red 1005 line indicates lifetime of CME prior to osmotic shock. (e) Quantification of the 1006

Figure 3: Mathematical modelling of CCS growth from intensity profiles of 924 individual CME events. (a) Mathematical representation of the constant area 925 model, flat-to-curved transition happens at the time when clathrin reaches its final 926 content. (b) Example of a clathrin intensity track fitted by the constant area model. 927 Blue dots represent measured intensity of a single CME event; black line represents 928 the fit with equation 1. The schematic illustrates the calculated size and curvature, 929 flat (blue), dome (red), and pit (green). (c) Calculated projected area and curvature 930 distributions of the CCS according to the constant area model for 4927 FM tracks of 931 4 different cells. P-value of Welch’s t-test to compare the predicted to the measured 932 distribution in panel h. A box/whiskers plot of the projected area is shown in the 933 inset. Mid-line represents median, cross represents the mean and the whiskers 934 represent the 10 and 90 percentiles. (d) Mathematical representation of the updated 935 growth model where a flat clathrin patch grows and the flat-to-curved transition 936

Figure 1: Comprehensive ultrastructural characterisation of CCS in BSC-1 cells 886 by TEM. (a) Schematic of the constant curvature and constant area models. The 887 stages of different curvature (flat (blue), dome (red), pit (green)) and the variation 888 of projected area, which can be assessed during TEM imaging, is depicted for both 889 growth models. (b) Difference between projected area (black) and surface area 890 (blue) during the course of CCP formation according to the two models. The 891 schematic illustrates the relationship between projected area and surface area for 892

Figure 8: Model of CCP assembly. Schematic representation of the growth model 1034 of CCS. CCS initiate as flat clathrin array. They first grow in size in a flat morphology 1035 with a constant AP2/clathrin ratio. When they reach around 70% of their full 1036 clathrin content, the AP2/clathrin ratio starts to decrease and the CCS start 1037 acquiring their curvature. CCPs keep growing by adding additional clathrin 1038 molecules until formation and release of CCV into the cytoplasm. The flat-to-curved 1039 transition of CCS can be inhibited by increasing PMT resulting in an accumulation of 1040 flat structures. We propose that flat-to-curved transition is concomitant with 1041 bypassing the energy barrier necessary to curve the PM and that this critical step in 1042 CME is coordinated by the uncoupling of clathrin and AP2 characterised by their 1043 abrupt ratio decrease. 1044

Full text

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!4
Delia! Bucher
1,2*
,! Felix! Frey
3,4*
,! Kem! A.! Sochacki
5
,! Susann! Kummer
1
,! Jan-Philip!5
Bergeest
2,3,6
,!William!J.!Godinez
2,3,6
,
!
Hans-Georg!Kräusslich
1
,!Karl!Rohr
2,3,6
,!Justin!W.!6
Taraska
5!
,!Ulrich!S.!Schwarz
3,4
,!Steeve!Boulant
1,2
!7
!8
1!
Department! of! Infectious! Diseases,! Virology,! University! Hospital! Heidelberg,! Im!9
Neuenheimer!Feld!324,!69120!Heidelberg,!Germany!10
2!
German! Cancer! Research! Center! (DKFZ),! Im! Neuenheimer! Feld! 581,! 69120!11
Heidelberg,!Germany!12
3!
BioQuant-Center,!Im!Neuenheimer!Feld!267,!69120!Heidelberg,!Germany!13
4!
Institute!for!Theoretical!Physics,!Heidelberg!University,!Philosophenweg!19,!69120!14
Heidelberg,!Germany!
!
15
5!
National! Heart! Lung! and! Blood! Institute,! National! Institutes! of! Health,! Bethesda,!16
U.S.A.!17
6!
Department!of!Bioinformatics!and!Functional!Genomics,!Heidelberg!University,!Im!18
Neuenheimer!Feld!267,!69120!Heidelberg,!Germany!19
!20
*!equal!contributions!21
!22
!23
!24
Correspondence!should!be!addressed!to!Steeve!Boulan t:!25
s.boulant@dkfz.de!26
!27
!28
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was notthis version posted July 11, 2017. ; https://doi.org/10.1101/162024doi: bioRxiv preprint

Keywords:! clathrin-mediated! endocytosis,! clathrin-coated! pit s,! flat! clathrin! lattice,!29
CLEM,!membrane!curvature!30
87/$)#'$-31
Although! essential! for! many! cellular! processes,! the! sequence! of! structural! and!32
molecular! events! during! clathrin-mediated! endocytosis! remains! elusive.! While! it!33
was! believed! that ! clathrin-coated! pits! grow! with! a! constant! curvature,! it! was!34
recently! suggested! that! clathrin! first! assembles! to! form! a! flat! structure! and! then!35
bends! while! maintaining! a! constant! su rfa ce! area.! Here,! we! combine! correlative!36
electron!and!light!microscopy!and!mathematical!modelling!to!quantify!the!sequence!37
of! ultrastructural! rearrangements! of! the! clathrin! coat! during! endocytosis! in!38
mammalian!cells.!We!confirm!that!clathrin-coated!structures!can!initially!grow!flat!39
and! that! lattice! curvature! does! not! show! a! direct! correlation! with! clathrin! coat!40
assembly.! We! demonstrate! that! curvature! begins! when! 70%! of! the! final! clathrin!41
content!is!acquired.!We!find!that!this!transition!is!marked!by!a!change!in!the!clathrin!42
to! clathrin-adaptor! protein! AP2! ratio! and! that! membrane! tension! suppresses! this!43
transition.! Our! results! support! the! model! that! mammalian! cells! dynamically!44
regulate! the! flat-to-curved! transition! in! clathrin-mediated! endocytosis! by! both!45
biochemical!and!mechanical!factors.!!46
! !47
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was notthis version posted July 11, 2017. ; https://doi.org/10.1101/162024doi: bioRxiv preprint

9.$)&,('$ 0& .-48
! Clathrin-mediated! endocytosis! (CME)! is! an! essential! uptake! pathway! that!49
relocates!membrane!or!extracellular!cargo!into!the!cell!to!regulate!multiple!cellular!50
functions!and!cell!homeostasis
1
.!During!CME,!the!clathrin!coat!is!assembled!to!form!51
a! clathrin-coated! pit! (CCP)! that! after! dynamin-mediated! scission! from! the! plasma!52
membrane! (PM)! leads! to! the! formation! of! a! clathrin-coated! vesicle! (CCV)
2
.! This!53
process! is! coordinated! by! numerous! adaptor! and! accessory! proteins
1,3
.! Electron!54
microscopy! (EM)! of! clathrin! coated! structures! (CCS)! has! shown! the! architectural!55
complexity! of! the! clathrin! meshwork! organized! into! hexagons! and! pentagons
4,5
.!56
From!this!EM!analysis,!it!was!proposed!that!a !CCV!initiates!as!a!flat!clathrin!lattice!57
that!is!then!rearranged!to!form!a!curved!CCP
4,6,7
.!However,!for!topological!reasons!58
this! requires! a! major! ultrastructural! rearrangement! of! the! clathrin! lattice! which!59
appeared!to!be!dynamically!difficult!and!energetically!costly
8–12
.!For!these!reasons,!60
this!notion!was!replaced!by!a!general!belief! that!CCS!grow!with!a!constant!curvature!61
(constant!curvature!model,!Fig.1a)
8,9,13
!and!that!flat!CCS!are!distinct!from!CCPs!and!62
serve!different!purposes
14–16
.!This!model!was!supported!by!the!finding!that!purified!63
clathrin! triskelia! self-assemble! into! curved! clathrin! baskets! in# vitro
17,18
.! Recently,!64
correlative! light! and! electron! microscopy! (CLEM)! analyses! provided! experimental!65
evidence! that! CCS! first! grow! flat! to! their! final! size! and! then ! acquire! curvature!66
(constant!area!model,!Fig.1a)
19
.!However,!this!study!did!not!measure!the!dynamics!67
of! CCP! formation! directly,! and! it! did! not! identify! the! cellular! factors! that! might!68
determine! when! the! flat-to-curved! transition! occurs.! Thus! a! comprehensive!69
understanding! of! t he! dynamic! process! of! coat! rearrangement,! of! the! temporal!70
aspects! of! flat-to-curved! transition! and! of! what! governs! this! ultrastructural!71
rearrangement!during!CME!is!still!missing.!!72
In! this! work,! we! combine! mathematical! modelling! of! individual! endocytic!73
event!dynamics!a nd! CLEM! analysis! to!provide!a! comprehensive! description! of!the!74
dynamic! ultrastructural! rearrangement! of! the! clathrin! coat! during! CME.! We!75
demonstrate! that ! CCPs! indeed! initially! grow! as! flat! arrays,! but! that! their!76
reorganisation! into! curved! structures! occurs! before! reaching! their! full! clathrin!77
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content.! We! correlate! this! flat-to-curved! transition! with! a! change! in! the!78
AP2/clathrin!ratio!and!show!that!it!is!governed!by!biophysical!properties!of!the!PM.!79
Our!findings!provide!a!unifying!view!of!the!dynamic!process!of!coat!rearrangement!80
during! CME! and! our! approach! constitutes! a! methodological! framework! to! further!81
study!the!fine-tuned!spatio-temporal!mechanism!regulating!coat!assembly.!82
! -83
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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:+/("$/--84
;<-#.,-=>;<-#.#"4/0/-&?-==@-,&-.&$-/(66&)$-+A0/$0.1-1)&5$2-3&,+"/-85
!86
To! address! whether! CCP! formation! follows! the! constant! curvature! model! or! the!87
constant!area!model!(Fig.1a)
13
,!we!chose!BSC-1!cells,!a!widely!used!cellular!model!to!88
study! CME
10,14,20
.! BSC-1! cells! present! homogenous! CME! events! in! regard! to! both!89
lifetime! as! well! as! intensity! profiles! and! lack! the! long-live! flat! clathrin-coated!90
plaques
10,15
! (Supplementary! Fig.1! and! Fig.1c-g).! Both! models! predict! different!91
growth! profiles! for! the! surface! and! projected! area! during! CCP! formation.! The!92
constant! curvature! model! implies! that! the! projected! area! will! quickly! be! smaller!93
than! the! surface! area.! In! contrast,! the! constant! area! model! implies! that! both!94
projected! and! surface! areas! initially! show! similar! growth! but! then! the! projected!95
area!should!drop!significantly!as!bending!starts!(Fig.1b).!96
To! comprehensively! characterise! the! ultrastructural! organisation! of! CCS! in! BSC-1!97
cells,! we! performed! TEM! of! metal! replicas! from! unroofed! PMs! (Fig.1c).! We!98
confirmed! that! CCS! are! not! altered! by! the! unroofing! procedure! using! stimulated!99
emission!depletion!(STED)!super-resolution!microscopy!of!intact!and!unroofed!cells.!100
The! number! and! size! distribution! of! CCS! were! indeed! similar! between! intact! and!101
unroofed!cells!(Supplementary!Fig.2).!CCS!in!TEM!images!of!whole!PM!sheet s!were!102
counted,! categorised! as! flat,! dome! or! pit! structures! (Fig.1d-e)! and! t heir! size! was!103
measured!as!projected!area!(Fig.1a,!f,!and!g).!For!the!constant!curvature!model,!we!104
would! expect! no! flat! structures! at! all! and! no! dome! structures! that! exceed! the!105
projected!area!of!pits!(Fig.1a-b).!In!contrast!our!EM!data!reveal s!that!around!50%!of!106
the!CCS!in!BSC-1!correspond!to!flat!CCS!(Fig.1e)!and!that!a!large!fraction!of!the!flat!107
and!dome!structures!have!a!projected!area!larger!than!the!projected!area!of!the!pits!108
(Fig.1g).! Since! BSC-1! cells! do! not! have! clathrin-coated! plaques
10,15
,! these! results!109
demonstrate! that! the! constant! curvature! model! cannot! explain! the! CCS! size!110
distribution,!in!agreement!with!the!recent!results!by!Avinoam!et!al
19
.!Although!the!111
existence!of!flat!CCS! seems!to!argue!in!favour!for!the!constant!area!model,!we!would!112
expect!that!some!flat!structures!have!the!same!projected!area!as!the!surface!area!of!113
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Q1. What are the contributions in "Flat-to-curved transition during clathrin-mediated endocytosis correlates with a change in clathrin-adaptor ratio and is regulated by membrane tension" ?

The authors confirm that clathrin-coated structures can initially grow flat 39 and that lattice curvature does not show a direct correlation with clathrin coat 40 assembly. The authors demonstrate that curvature begins when 70 % of the final clathrin 41 content is acquired. 46 47 certified by peer review ) is the author/funder.