Actin-generated force applied during endocytosis measured by Sla2-based FRET tension sensors

•FRET sensors in Sla2 report the actin-generated forces required for endocytosis•At least 8 pN force is transmitted by Sla2-Ent1 membrane-actin protein linker•Large amount of needed initial membrane bending and early invagination•Lowering cell turgor or tension reduces over linker Mechanical are integral to many cellular processes, including clathrin-mediated endocytosis, a principal trafficking route into cell. During provided endocytic proteins polymerizing actin cytoskeleton reshape plasma vesicle. Assessing requirements remodeling essential understanding endocytosis. Here, we determined applied during endocytosis using FRET-based inserted major force-transmitting yeast. We measured at molecule, hence possibly more than 300–880 vesicle formation. Importantly, decreasing pressure reduced Sla2. The measurements hypotonic conditions mutants lacking BAR-domain scaffolds then showed limits machinery. 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TSMs, mechanosensitive peptide connected two fluorophores undergoing Förster resonance transfer (FRET), reversibly extend low force, reporting thus about decrease FRET Cost 2019Cost A.L. Khalaji Grashoff Genetically encoded sensors.Curr. Protoc. 83: e85Crossref (16) Freikamp 2017Freikamp Mehlich Klingner Investigating piconewton microscopy.J. Struct. 2017; 197: 37-42Crossref (45) Further, sought analyze roles environmental transmission. estimate linkers its magnitude turgor, tension, proper function factors. yeast, first constructed strains expressing various (Sla2 FS), fusion containing different TSMs. comprising pair mTurquoise2 mNeonGreen (Mastop 2017Mastop Bindels D.S. Shaner N.C. Postma Gadella T.W.J. Goedhart Characterization spectrally diverse set fluorescent as acceptors mTurquoise2.Sci. Rep. 7: 11999Crossref (47) 2019Skruzny Pohl microscopy yeast.Biosensors. 122Crossref (12) sensitive distinct ranges, F40 (1–6 pN), HP35 (6–8 HP35st (9–11 pN) (Austen 2015Austen Ringer Chrostek-Grashoff Kluger Sabass Zent Rief Extracellular rigidity sensing talin isoform-specific linkages.Nat. 17: 1597-1606Crossref (218) 2010Grashoff Hoffman B.D. Brenner M.D. Zhou Parsons Yang M.T. McLean Sligar S.G. C.S. Ha al.Measuring across vinculin regulation focal adhesion dynamics.Nature. 2010; 466: 263-266Crossref (1057) were between coiled-coil dimerization motif actin-binding domain (Figure 1A). distinguish force-dependent from force-independent changes (Cost also TSMs integrated after domain, C terminus no controls, NF; Figure S1A). all actin-supplied linker, channel solely sensors. For that, deleted second, functionally redundant moiety Ent1, (ent1?ACB background; Resulting did not show any defect growth progression S1B; Video S1). Also, lifetimes fluorescence intensities sites similar wild-type protein, C-terminally-tagged (Sla2-mNG). Specifically, only slightly extended comparison Sla2-mNG (51.5 ± 3.7 s Sla2-HP35 versus 46.6 4.2 Sla2-mNG), reaching up 79%–87% intensity (Figures S1C S1D; eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIwMzBjMjFhMGRlMzMxYTYwYWUyZWE4MGU2NGRmOWMzZSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjg2ODQwOTIwfQ.H_pptjJ6lXGx_QeOocxGiUSvrh8uFhAX7UszJAQEOwmprVL0LqXeU59cMXkX28dq-Jf1Z3GP4iTEbjlgDjuuWb2xUeX7rvC1l_fS1MWIWTZ0MT7VlSJBZQze4aLhzj5C1n20p8Nl7irsIoNZz61Z2nPat6G8DiKpUD7bmChWM5SCVO-45o9wnn7s6OkE08Ef4Eu4H08pcg91xeqMyHwhV3Qbv3XG87_qjaOkFx_i0N1qje3m7xnvKXSzcTEbmTh94Kyvc_sKxB7ac3BWcjRnVcno_YXer5guRQ1Ig1TsKf21Css4XmB4ewak3wmdxOlWhFjl68RFcREtLMx7no2k7Q Download .mp4 (1.34 MB) Help files S1. Comparison Sla2-mNeonGreen behavior sites, related 1The signal was acquired mixed cultures indicated Sla2-mNeonGreen- sensors-expressing cells. identified subsequent acquisition (last frame). Images every 1 180 s. Scale bar, 2 ?m. follow sensors, analyzed individual events. simultaneously recorded signals until point scission associated rapid move cytoplasm concomitant disappearance 1B). Decrease ratio (from here ratio) events three 1C). observed respective controls Similarly, change detected (Sla2?THATCH NF controls) channeling Ent1 S1E S1F). Sla2?THATCH strongly suggests that molecules conformational intermolecular occurring invagination. approximately 13 before timing coincided very well appearance marker Abp1-mScarlet-I (12.7 2.6 scission; Figures 1B 1D), indicating outset correlates beginning (compare 1C 1D). starting drop continuous decrease, furthermore fraction engaged transmission increases they recruited growing When compared found while Sla2-F40 behaved similarly, significantly smaller Sla2-HP35st 1E). As module detect higher structurally pN; Austen indicates fully some them might experience above 11 pN. Altogether, these comparisons suggest minimally value, connection recently number (45–133 molecules; 2015Picco Mund Ries Visualizing functional architecture machinery.eLife. 4: e04535Crossref (82) 2019Sun X. Jiang Pollard T.D. Direct budding conserved evolvable features.eLife. 8: e50749Crossref (13) (approximately 84% level; S1D), results single event (assuming under time see discussion). 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Remarkably, reducing 3A , S2B, S3). corroborate means, employed described approach reduce modifying gradient (Riggi 2018Riggi Niewola-Staszkowska Chiaruttini Colom Kusmider Mercier V. Soleimanpour Stahl Matile Loewith triggers PtdIns(4,5)P2 separation inactivate TORC2.Nat. 20: 1043-1051Crossref (78) this, incubated soluble lipid palmitoylcarnitine (PalmC), incorporated tension. Again, ratio, 3B S2C). summary, Finally, capacity conditions, intensify opposing use fps1? lack aquaglyceroporin cannot adapt hypoosmotic glycerol efflux (Tamás 1999Tamás M.J. Luyten Sutherland F.C.W. Hernandez Albertyn Valadi Li Prior B.A. Kilian Ramos al.Fps1p accumulation release compatible solute osmoregulation.Mol. Microbiol. 1999; 31: 1087-1104Crossref (296) cultivated fps1?, sorbitol exposed shifts exchange media osmolarity 0.5 M, 0.4 0.25 sorbitol. medium, normal levels lifetime FS (84% completed min; 63.3 3.5 51.5 sorbitol; compare 4A S1C). Contrary shifted clear increase stalled extending min (32% S2). (likely conditions) observe significant profile non-shifted S4). Osmotic shift further exacerbated (22% completed; condition bigger, yet decreased 4B S2D). almost longer precluding (less 1% completed, increasing environment led events, could there remarkable adaptation force-generating and/or -transmitting turgor. either can provide limited sustain conditions. latter possibility uncoupling 2019Riggi Bourgoint Macchione TORC2 tension.J. 218: 2265-2276Crossref (33) eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJmNWI4Njk2OTVjYWVhZjg4ZjYwY2NhZTU0YzlkZjNiYyIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjg2ODQwOTIwfQ.YHhsQunEcqTDUJXxsCAUBFq77RpKAy40nB3wqnA1K4b-Ag5yO0xZVcyVuhPNNEdC7AxYjdMURH_d-NULT2kJD2eTJS-QsBkEarZgXcHjHhixOzzva-x8OL0e1mtakIFeocQlwitjgxjvZN0iOyczF8Cn6VpIU2RyGyaaFmbvpicXIZSD58EjNoGP0_3uhI8fCsW0unx_