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

TLDR: 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.

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

Full text

!"#$%$&%'()*+,- $)#./0$0&.- ,()0.1- '"#$2)0.%3+,0#$+,-1
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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
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

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.
The copyright holder for this preprint (which was notthis version posted July 11, 2017. ; https://doi.org/10.1101/162024doi: bioRxiv preprint

:+/("$/--84
;<-#.,-=>;<-#.#"4/0/-&?-==@-,&-.&$-/(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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Frequently asked questions

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.