Insights into the conformational organization and dynamics of proteins complexes at membranes is essential for our mechanistic understanding of numerous key biological processes. Here, we introduce graphene-induced energy transfer (GIET) to probe axial orientation of arrested macromolecules at lipid monolayers. Based on a calibrated distance-dependent efficiency within a dynamic range of 25 nm, we analyzed the conformational organization of proteins and complexes involved in tethering and fusion at the lysosome-like yeast vacuole. We observed that the membrane-anchored Rab7-like GTPase Ypt7 shows conformational reorganization upon interactions with effector proteins. Ensemble and time-resolved single-molecule GIET experiments revealed that the HOPS tethering complex, when recruited via Ypt7 to membranes, is dynamically alternating between a ‘closed’ and an ‘open’ conformation, with the latter possibly interacting with incoming vesicles. Our work highlights GIET as a unique spectroscopic ruler to reveal the axial orientation and dynamics of macromolecular complexes at biological membranes with sub-nanometer resolution. Proteins are part of the building blocks of life and are essential for structure, function and regulation of every cell, tissue and organ of the body. Proteins adopt different conformations to work efficiently within the various environments of a cell. They can also switch between shapes.One way to monitor how proteins change their shapes involves energy transfer. This approach can measure how close two proteins, or two parts of the same protein, are, by using dye labels that respond to each other when they are close together.For example, in a method called FRET, one dye label absorbs light and transfers the energy to the other label, which emits it as a different color of light. However, FRET only works over short distances , so it is not useful for larger proteins.Here, Füllbrunn, Li et al. developed a method called GIET that uses graphene to analyze the dynamic structures of proteins on membrane surfaces. Graphene is a type of carbon nanomaterial that can absorb energy from dye labels and could provide a way to study protein interactions over longer distances.Graphene was deposited on a glass surface where it was coated with single layer of membrane, which could then be used to capture specific proteins. The results showed that GIET worked over longer distances (up to 30 nm) than FRET and could be used to study proteins attached to the membrane around graphene. Füllbrunn, Li et al. used it to examine a specific complex of proteins called HOPS, which is linked to multiple diseases, including Ebola, measuring distances between the head or tail of HOPS and the membrane to understand protein shapes. This revealed that HOPS adopts an upright position on membranes and alternates between open and closed shapes.The study of Füllbrunn, Li et al. highlights the ability of GIET to address unanswered questions about the function of protein complexes on membrane surfaces and sheds new light on the structural dynamics of HOPS in living cells. As it allows protein interactions to be studied over much greater distances, GIET could be a powerful new tool for cell biology research. Moreover, graphene is also useful in electron microscopy and both approaches combined could achieve a detailed structural picture of proteins in action. Numerous fundamental cellular processes such as energy conversion, signal transduction, and transport occur in the context of lipid membranes. In animals, approximately one-third of the proteome is localized at the various membranes of the cell, while more than 50% of the currently available pharmaceuticals target membrane proteins . The mecThe structurally highly heterogeneous and dynamic systems are extremely difficult to tackle by traditional structural techniques that average ensembles. Single-molecule Förster resonance energy transfer (smFRET) has been used successfully to shed light on the structural heterogeneity of proteins lacking structural definition . Howeverd by 6 times the diffraction limit)  . Comparen limit) , with a n limit) . For expn limit) . We recon limit) , includin limit) . In animn limit) . Based oGI) to that on the silica substrate (I0) confirmed strong quenching by GIET with an efficiency of 83.6% for mEGFP bound to NB-H6. Direct immobilization of H6-tagged mEGFP on graphene resulted into even stronger GIET  was incorporated into the DOPC matrix. The probe strands have a common 20mer ssDNA sequence complementary to the anchor strand and were conjugated with 6-carboxyfluorescein (FAM) at either 3´- or 5´- end, respectively, for fluorescence read-out. By hybridization of the probe and anchor strands, eight distinct fluorophore distances from the monolayer surface were obtained, which are denoted as anchor strand-3´F or 5´F. For the 35mer and 50mer anchor strands, additional complementary 15mer and 30mer ssDNA were used as blockers, respectively, to obtain the fully length double strand DNA nanorulers  with 20mer, 25mer, 35mer, and 50mer nucleotides were designed as the scaffold of nanorulers . These snorulers . For thesn-glycero-3-phosphoethanolamine (OG488-DHPE), in which the dye molecule is directly linked to the lipid head group. In parallel, the same experiments were performed using silica-supported lipid bilayers. Simultaneous quantification of mass deposition and fluorescence intensity on both substrates ensured reliable quantification of GIET efficiencies (GI) were observed compared to those on silica (I0). The GI/I0 ratios increased with the end-to-end distances of the seven different FAM positions on DNA nanorulers. However, the increase was significantly lower than the theoretical prediction from electromagnetic simulations, which assumed fully perpendicular orientation on top of the lipid monolayer coating ° ° , confirmτG) to those on glass (τ0) closely matched the corresponding intensity ratios, which further corroborated the simulated distance-dependent GIET efficiency (GIET efficiencies were furthermore quantified by time-correlated single photon counting (TCSPC) using confocal laser-scanning microscopy cLSM, . Lifetimficiency . ResultsGI/I0. For the axial organization of mEGFP and mEGFP:NB complex tethered to the DOPC monolayer surface via trisNTA-DODA, distances 1.3 nm for H6-mEGFP directly tethered to the membrane (GI/I0: 7.6%) and 3.0 nm for mEGFP captured via NB-H6 (GI/I0: 16.4%) were estimated. The distance of 1.7 nm induced by the nanobody is in good agreement with the crystal structure of the GFP-NB complex , suggesting that the hybridized probe-anchor DNA strand collapses onto the surface of the lipid monolayer due to the flexible, unpaired 15mer gap. To test the hypothesis, a 15mer ssDNA blocker strand was added, resulting into a ~ sixfold increase of the fluorescence lifetime , the height corresponds to a tilting angle α = 45°, which is in good agreement with the tilting angle α obtained for the calibration. This observation not only supports the validation of the calibration, but also suggests loss in flexibility upon hybridization with the 15mer blocker strand forces the full duplex strand into an upstanding position . The purified protein was prenylated in vitro (mNeon-pYpt7) in the presence of the chaperone GDI (mNeon-pYpt7:GDI), and the complex was used for reconstitution into lipid layers . In paraτG/τ0 ratio in the absence of additional effector proteins and in the presence of Mon1-Ccz1 and HOPS (Efficient transfer of prenylated mNeon-pYpt7 to lipid monolayers (on graphene) and bilayers (on glass) and homogeneous distribution were confirmed by cLSM imaging . Bindingand HOPS . pYpt7 wand HOPS  and therand HOPS . The totThe HOPS complex binds Ypt7-GTP via its subunits Vps39 and Vps41 . Since tτG) and glass support (τ0). FLIM images of yEGFP-tagged HOPS bound to pYpt7-GTP showed homogeneous distribution on graphene, yielding a Gaussian-like distribution of fluorescence lifetimes  at very low concentration (50 pM) to sub-stochiometrically label yEGFP-tagged subunits in the HOPS complex at the head (Vps33) and the tail (Vps11), respectively. Under these conditions, signals from specifically labeled individual HOPS complexes were detected by TIRF microscopy, as confirmed by photobleaching analyses on glass and graphene  microscopy  have beeProof-of-concept experiments demonstrate the feasibility to reconstitute complex membrane-anchored multi-protein machineries onto graphene-supported lipid monolayers and to quantify their axial architecture and dynamics. We show that the HVD domain of Ypt7 adopts a strongly extended conformation, locating Ypt7 at a 4.5 nm distance above the membrane surface. This surprising observation could be explained by electrostatic repulsion of the negatively charged HVD. Significant changes in the axial positioning of Ypt7 were observed upon interaction with Mon1-Ccz1 and HOPS. Since Mon1-Ccz1 directly binds to the membrane , its Yptsn-glycero-3-phosphoethanolamine (OG488-DHPE) was purchased from Thermo Fisher Scientific. ATTO488-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (ATTO488-DPPE) was obtained from ATTO-TEC GmbH, Siegen, Germany. DY647P1 maleimide was purchased from Dyomics GmbH, Jena, Germany. TrisNTA-DODA was synthesized as previously reported . Glass microscopy coverslips with thickness #1.5 and diameter of 24 mm were purchased from Carl Roth (PK26.1). 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE), diacylglycerol (DAG) and DOGS-NTA were purchased from Avanti Polar Lipids, Alabama, USA. Phosphatidylinositol 3-phosphate (PI-3-P) was purchased from Echelon Biosciences Inc, Utah, USA. 2′,7′-difluoro-fluorescein conjugated with 1,2-dihexadecanoyl-reported . DNA oliEscherichia coli BL21 (DE 3) Rosetta cells, followed by purification of immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC)  Rosetta cells. After cell lyses by sonication, NB-H6 was purified by immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). For fluorescence labeling, a short linker including an additional cysteine residue followed by an ybbR-tag, a PAS repeat sequence and a terminal hexahistidine tag (a.a.: GSCGSGSKLDSLEFIASKLAPASPASPASPASPASLEHHHHHH) was C-terminally fused to the NB. Expression and purification was performed as described for NB-H6. The purified proteins were reacted with a twofold molar ratio of DY647P1 maleimide for 30 min and then purified by size exclusion chromatography. A degree of labeling close to 1.0 was obtained as quantified by UV-Vis spectroscopy.Protein expression, purification and labeling mEGFP with an N-terminal hexahistidine tag (H6-mEGFP) was cloned in the plasmid pET21a. The protein was expressed in hy (SEC) . mEGFP wnhancer’  with a Ceviously . For fluE. coli BL21 (DE 3) Rosetta cells. Rab GTPases and Gdi1 were purified as previously described with slight modifications (E. coli BL21 (DE 3) Rosetta cells. Cells were grown in Luria broth (LB) medium containing the specific antibiotics until an OD600 of around 0.8, before they were induced with 0.25 mM isopropyl-β-D-thiogalactoside (IPTG) for 18 hr at 16°C. For purification of the Rab GTPases, harvested cells were lysed by a Microfluidizer, Model M-110L  in 50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.05-fold protease inhibitor cocktail. For purification of Gdi1, lysis was performed in PBS supplemented with 5 mM β-mercaptoethanol and 1 mM PMSF. The lysate was centrifuged at 40,000 g for 30 min at 4°C, and the supernatant was incubated for 2 hr at 4°C with pre-equilibrated Ni-NTA agarose  for His-fused proteins or Glutathione Sepharose (GSH) 4B beads  for GST-fused proteins. Ni-NTA beads were washed with 50 ml lysis buffer lacking PMSF and protease inhibitor but supplemented with 10 mM imidazole. For elution, the imidazole concentration was increased to 300 mM. After elution, the buffer was exchanged via a PD10 column  containing no imidazole. GSH beads were extensively washed with 120 ml lysis buffer lacking PMSF and protease inhibitor cocktail, and proteins were eluted by cleaving the affinity-tag with TEV- or Precision-protease, respectively, for 2 hr at 16°C.Endocytic proteins and protein complexes: (1) Rab GTPases and Gdi1. pET24b-Ypt7 , pET24d-ications . The pETE. coli BL21 (DE 3) Rosetta cells. The Rab GGTase and the Rab escort protein were expressed and induced as Rab GTPases and Gdi1. The Rab GGTase (pCDF-Duet-1-Bet2-Bet4) and the Rab escort protein (pET-Mrs6) were expressed and induced as described in 1.2. Harvested cells were lysed in 50 mM Tris, pH 8.0, 300 mM NaCl, 2 mM β-mercaptoethanol and 1 mM PMSF and centrifuged as described above. The supernatant was loaded on a pre-equilibrated Hi-Trap Ni-Sepharose column . After extensive washing of the column with lysis buffer containing 30 mM imidazole but lacking PMSF, bound protein was eluted in a linear 30–300 mM imidazole gradient over 30 column volumes. Protein-containing fractions were pooled and dialyzed against buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, and 1 mM MgCl2. The buffer was exchanged twice.(2)Rab GGTase and Rab Escort Protein. pET30-Mrs6  and pCDFSaccharomyces cerevisiae and purified essentially as described before , and 10% glycerol. For purification of Mon1-Ccz1, the salt concentration was decreased to 150 mM NaCl. Cell lysis in the presence of glass beads was conducted in a FastPrep-24 5G . After removal of the glass beads, the supernatant was centrifuged at 120,000 g for 1 hr at 4°C. Centrifuged lysate was incubated with pre-equilibrated immunoglobulin G (IgG) Sepharose  for 2 hr at 4°C. IgG beads were washed with 15 ml lysis buffer lacking PMSF and FY, and bound proteins were eluted by cleavage with TEV protease for 1 hr at 16°C.(3) HOPS, yEGFP-fused HOPS and Mon1-Ccz1. HOPS, yEGFP-fused HOPS and Mon1-Ccz1 were expressed in d before . For yEGStrains and plasmids used in this study are listed in 2, 1 mM DTT) for 1 hr at 37°C. Mrs6 and Bet2-Bet4 fused to a His6 tag were removed by subsequent incubation of the reaction with Ni-NTA Agarose  for 1 hr at 4°C. The stoichiometric complex containing the prenylated Rab and GDI was isolated from the supernatant by size exclusion chromatography. The functionality of prenylated Rabs was tested in membrane association and tethering assays as described before with modifications  and resuspended in HEPES-KOAc (HK) buffer . Liposomes as unilamellar vesicles were obtained by five freeze and thaw cycles in liquid nitrogen. For tethering assays, the 2 mM liposome suspension was extruded through polycarbonate filters  using a hand extruder . To analyze the membrane association of mNeon-Ypt7, 50 pmole mNeon-Ypt7 complexed with GDI was incubated with 50 nmole liposomes  in the presence or absence of GTP for 30 min at 27°C. Liposomes were sedimented by centrifugation for 20 min at 20,000 g. The fraction of membrane-bound mNeon-Ypt7 was determined by the fluorescent signal of the supernatant before and after sedimentation, which was quantified in a SpectraMax M3 fluorescence plate reader .For analysis of HOPS-mediated tethering, liposomes containing 69 mol % POPC, 18 mol % POPE, 8 mol % ergosterol, 1 mol % DAG, 1 mol % ATTO488-DPPE, and 3 mol % DOGS-NTA or compensatory amounts of POPC were used. Ypt7 C-terminally fused to a His6 tag was loaded with GDP or GTP and afterwards targeted to liposomes via the lipid analogue DOGS-NTA . Liposom2 silica-coated transducers for TIRFS-RIF detection and glass coverslips for microscopy imaging. Before coating, the substrates were cleaned by plasma cleaner . Coating of graphene monolayer on substrate was based on the manufacturer’s instruction. Briefly, the Easy Transfer Monolayer containing graphene monolayer with a thin protecting polymer film was cut into 0.5 × 0.5 cm2 pieces. The obtained piece was slowly emerged into MilliQ water to float on top of water. The polymer-protected graphene monolayer was fished out by a clean glass coverslip or TIRFS-RIF transducer from below, resulting in face-to-face contact of graphene monolayer with the substrate. The obtained substrate was left drying at room temperature for 30 min, followed by heating in a 150°C oven for 2 hr. The hot substrate was taken out and immediately stored under vacuum for 24 hr for cooling down. With the protecting polymer film, the obtained graphene-coated substrate could be stored at ambient conditions for weeks. Immediately before the experiments, the graphene-coated substrate was incubated in acetone for 1 hr, sequentially in isopropanol for another 1 hr, to remove the protecting polymer film on graphene. By blow-drying with N2 stream, the graphene monolayer-coated substrate was ready for use.Silica-type substrates were used for coating with graphene monolayer. These include 1 × 1 cm2+ ions. The coverslip was rinsed with 250 mM EDTA and 200 mM imidazole, sequentially. After incubation with 10 mM NiCl2 for 10 min, the coverslip was washed by HBS buffer and 200 mM imidazole to remove possible non-specifically bound Ni2+ ions.For preparation of DOPC liposomes, 2.5 µmol DOPC was dissolved in chloroform in a 50 ml round bottom flask. For preparation of liposomes containing DOPC:DOPS (95:5 molar ratio), 2.4 µmol DOPC and 0.12 µmol DOPS dissolved in chloroform were mixed in a 50 ml round bottom flask. Similarly, a lipid mixture of 2.4 µmol DOPC and 0.125 µmol trisNTA-DODA was used for preparing liposomes of DOPC:trisNTA-DODA (95:5 molar ratio). Liposomes were prepared as small unilamellar vesicles (SUVs) by probe sonication as described before . To form2+ ion loading follows the same protocol as in vitro by using the flow-through system in the TIRFS-RIF setup. On the Ni2+-loaded trisNTA-DODA/DOPC lipid mono-/bilayers, 1 µM NB-H6 was injected, followed by injections of 100 nM tagless mEGFP, HBS buffer rinsing and imidazole washing for surface regeneration. For direct immobilization of H6-mEGFP, 1 µM H6-mEGFP was injected followed by imidazole washing. Mass signals of protein binding and fluorescence signals were recorded simultaneously in RIF channel and TIRFS channel, respectively. The ratio of fluorescence intensity G/II0 was normalized to the amount of immobilized mEGFP according to A home-built set-up for simultaneous total internal reflection fluorescence spectroscopy (TIRFS)  and refl2+ for DNA hybridization . Fluorescence ratio of G/II0 was normalized to the amount of immobilized DNA according to G/I0I was obtained by normalizing to the mass signals according to For DNA hybridization, liposomes with 250 µM DOPC/DOPS (95:5 molar ratio) was injected to the surface of TIRFS-RIF transducer to form lipid layers on the solid support. Anchor strands with cholesterol modification were injected for immobilization on the surface. Sequentially, probe strand labeled with FAM was injected for hybridization with the anchor strand. All DNA concentrations were 1 µM. The running buffer was HBS buffer containing 5 mM MgBinding of Mon1-Ccz1 to lipid layers was probed as described before . 250 µM S, of the electric dipole emitter as a function of dipole distance d and orientation  to the substrate. Considering the flexible linker and rapid rotation of the fluorophore in biomolecules, S was averaged over random orientations as a simplified S(d). The relative fluorescence lifetime (Gτ/τ0) was calculated as:τ0 is the free-space lifetime in the absence of GIET, S0 is the free-space emission power of an ideal electric dipole emitter, c being the vacuum speed of light, k0is the wave vector in vacuum, n is the refractive index of water (n = 1.33), and p is the amplitude of the dipole moment vector.For simulating the electrodynamic coupling of the excited fluorophore to graphene, the excited fluorophore was treated as an ideal electric dipole emitter and the graphene as a layer of matter with specific thickness and  bulk refractive index as described before . SolvingGτ/τ0 as a function of distance d was calculated for the following geometry: glass substrate (n = 1.52) covered with single sheet graphene  coated with lipid monolayer , topped with water (n = 1.33). The emission maximum, QY, and τ0 of the fluorophores were: FAM , EGFP , mNeonGreen , and Dy647P1 . The emission maximum and QY were taken from the literature d=lDN5 counts per sample for robust lifetime analyses. If not mentioned elsewhere, the TCSPC histograms were tail-fit with mono-exponential decay function using SymPhoTime64 integrated in the PicoQuant system. Only for mNeon-Ypt7 on graphene, the TCSPC histograms were fit with bi-exponential decay functions by fixing one component to the obtained average lifetime of mNeon-Ypt7 on glass (<15%).Lifetime measurements were carried out on a confocal laser-scanning microscope  equipped with a FLIM/FCS upgrade kit from PicoQuant using a 60× (NA 1.2) water-immersion objective . EGFP/mNeonGreen was excited either by the 488 nm line of an argon laser (Olympus) for cLSM/FRAP or by a picosecond pulsed 485 nm laser diode at 40 MHz repetition rate . Time-correlated single photon counting (TCSPC) was performed using the TCSPC module PicoHarp 300 (PicoQuant together with a picosecond laser driver Sepia II (PicoQuant) and a single photon avalanche detector (PicoQuant)). Emission photons were filtered by a 500–550 nm bandpass filter . TCSPC was acquired using point measurement or image mode (i.e. FLIM mode). Acquisition time was more than 90 s to obtain >10800 µl 250 µM DOPC/DOPS (95:5 molar ratio) was added on top of graphene-coated glass coverslips. After incubation at room temperature for 5 min, the coverslips were washed for 5 times with 1 ml HBS-Mg buffer. The cholesterol-modified ssDNA anchor strands were added in the solution with a final concentration of 200 nM. After 5 min incubation, excess anchor strands were removed by washing for 5 times with HBS-Mg buffer. Probe strands of FAM-labeled ssDNA were added with a final concentration of 200 nM. After 5 min incubation, excess probe strands were removed by washing for 10 times with HBS buffer. For fluorescence lifetime measurements, a final concentration of 10 µM EDTA was added to the solution for preventing possible metal ion-mediated fluorescence quenching. TCSPC was acquired by point measurement mode. Lifetimes were obtained by monoexponential fitting of TCSPC curves. For FRAP experiments of DNA strands, a region of interest (ROI) was placed in areas with and without graphene, respectively.In FRAP experiments of DNA strands, a circular region of interest (ROI) with a radius of 10 μm was selected. Five pre-bleach images were recorded before the ROI was illuminated by 405 nm in 10 s. A background signal 2 and incubation for 10 min at room temperature. The coverslip was washed extensively with HBS (>15 times) to remove excess EDTA and Mg2+ ions. Where indicated, 100 nM Mon1-Ccz1 or HOPS were added to mNeon-pYpt7-GTP-loaded membranes for 5 min at room temperature. Before fluorescence lifetime determination, the unbound complexes in solution were removed by washing for 8 times with HBS. For loading of HOPS complexes containing yEGFP-fused subunits on membranes, 50 nM HOPS carrying one subunit  fused to yEGFP was added to pYpt7-GTP-loaded membranes for 5 min at room temperature. Before fluorescence lifetime determination, the coverslip was washed 5 times with HBS to remove unbound HOPS in solution. Fluorescent lifetimes of mNeon and yEGFP, respectively, were determined by TCSPC acquired in image mode (FLIM). Fluorescence lifetime ratios were determined based on lifetimes obtained on graphene and glass, respectively, and the distance from the graphene surface d were calculated based on the calibration curve .Graphene-coated glass coverslips were incubated with 800 µl DOPC and washed as described above. Membrane association of 200 nM prenylated mNeon-Ypt7 or 150 nM prenylated Ypt7 complexed with GDI was conducted in the presence of 20 mM EDTA, pH 8.0 and 1 mM GTP for 30 min at 30°C. The loading reaction was stopped by addition of 25 mM MgClon curve . The disFor FRAP experiments of mNeon-pYpt7-GTP and mNeon-pYpt7-GTP in complex with Mon1-Ccz1 and HOPS, respectively, a ROI with a radius of 7.5 µm was illuminated with a 405 nm laser for 8.2 s. The fluorescence recovery was monitored for 72 s at a time resolution of 1.8 s (excitation: 488 nm). The 2.Single-molecule imaging experiments were conducted by total internal reflection fluorescence (TIRF) microscopy with an inverted microscope (Olympus IX71) equipped with a triple-line total internal reflection (TIR) illumination condenser (Olympus) and a back-illuminated electron multiplying (EM) CCD camera  as described before . A 150 ×Photobleaching steps were counted manually from time-lapse single-molecule intensity traces as before . The sinSingle emitters in time-lapse TIRFM images were localized via a 2D Gaussian mask . IntensiG/I0I, which has a propagated relative error of The axial localization precision of smGIET was determined by comparing the root mean square deviation (RMSD) with the mean intensity of individual molecules. To avoid intensity fluctuations on graphene due to GIET, RMSD and mean intensities were quantified based on single-molecule detections on glass coverslips . For intAll data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The authors use graphene-induced energy transfer as a unique spectroscopic ruler to reveal the axial orientation and dynamics of macromolecular complexes in biological membranes with sub-nanometer resolution. This development is a very significant contribution that can help in future mechanistic studies of numerous key biological processes. To illustrate this approach, the authors determine the orientation of the Rab7-like GTPase Ypt7 and of the multi-protein HOPS tethering complex at a lipid monolayer surface.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Felix Campelo as Reviewing Editor and José Faraldo-Gómez as Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Volker Kiessling (Reviewer #1).Thank you for submitting your article "Nanoscopic anatomy of dynamic multi-protein complexes at membranes resolved by graphene induced energy transfer" for consideration by eLife. The Editors has drafted this decision letter to help you prepare a revised submission.The reviewers have discussed the reviews with one another and with the Reviewing Editor. Based on these discussions, we have concluded that a revised version of your manuscript might be suitable for publication in eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either (a) include the new data, if this is possible; otherwise, (b) limit claims to those supported by data in hand; or (c) explicitly state that the relevant conclusions require additional supporting data. In the latter case, our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in eLife, either of which would be linked to the original paper.We would like to draw your attention to changes in our revision policy made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in SummaryThe authors use GIET as a unique spectroscopic ruler to reveal the axial orientation and dynamics of macromolecular complexes at biological membranes with sub nanometer resolution, which is a very significant contribution that can help in the future mechanistic understanding of numerous key biological processes. GIET is based on the fluorescence quenching in close proximity to a graphene surface. The system is calibrated by utilizing "DNA nanorulers" of different length and with fluorescent labels at different specific positions. Measuring the fluorescence intensities and fluorescence lifetimes relative to unquenched samples. The calibrated system was then used to determine the orientation of the Rab7-like GTPase Ypt7 and of the multiprotein HOPS tethering complex at a lipid monolayer surface. The normal distances of specific residues to the monolayer surface are determined in ensemble measurements and the transition between three distinguishable conformations are measured in single molecule imaging experiments.All three reviewers highlighted the importance and the quality of the research presented in this manuscript. However, the reviewers also raised important questions and concerns, detailed below, which must be satisfactorily addressed before the manuscript can be accepted for publication. Given that this manuscript is being considered for a "Tools and Resources" article, it will be crucial for the authors to carefully address the reviewers' comments to further establish the validity and scope of the methodology and the clarity of the manuscript.In particular, it will be important to fully clarify the results shown in Figure 2 , in the context of reviewer #2 comments. It will also be important that the authors discuss in more detail the limitations of their technique and the expected errors in the measurements. The authors are also very strongly encouraged to perform the additional control experiments suggested by the reviewers, which appear relatively straightforward and not very time-consuming, as they would greatly strengthen the significance and impact of the study.Essential revisionsReviewer #1:Nadia Fuellbrunn and co-workers present their work about a new fluorescence method to determine the conformation and its dynamic of protein complexes by measuring the normal distance of site-specific fluorophores relative to a lipid monolayer. The graphene induced energy transfer (GIET) assay is based on the fluorescence quenching in close proximity to a graphene surface. The system is calibrated by utilizing "DNA nanorulers" of different length and with fluorescent labels at different specific positions. Measuring the fluorescence intensities and fluorescence lifetimes relative to unquenched samples. The calibrated system was then used to determine the orientation of the Rab7-like GTPase Ypt7 and of the multiprotein HOPS tethering complex at a lipid monolayer surface. Both proteins are part of the yeast vacuolar fusion and tethering machinery. The normal distances of specific residues to the monolayer surface are determined in ensemble measurements and the transition between three distinguishable conformations are measured in single molecule imaging experiments.The authors present a very nice and useful new method with a lot of potential in particular if it could be used to follow conformational changes of membrane proteins. The approach of measuring the normal distance of site-specific labeled residues is very similar to the method of site-specific fluorescence interference microscopy , 911), where the conformation of SNARE proteins reconstituted in supported membranes had been investigated. Although the overall strategy seems very similar, sdFLIC hasn't been mentioned in the Introduction where the authors discuss several methods by which protein conformations in membranes can be investigated.The authors clearly show the usefulness of this new method especially in the dynamic measurements on single molecules. However, there are a couple of open questions and issues that should be addressed and clarified:A lipid monolayer is not a membrane and the authors should be clear and consistent in their writing in the whole manuscript . Proteins are not reconstituted into a membrane on graphene. The fact that the HOPS complex clusters in a diffusible membrane shows that this situation  is very different from the one at the monolayer. This raises another question. Is it justifiable to use the membrane system to measure the unquenched intensities/lifetimes to normalize the monolayer data? Shouldn't this be done on a monolayer too?A thorough and extensive error discussion is missing. Since this work is presented as a first application of this method, a detailed discussion and quantification of all error sources  is necessary. One would expect these errors to be different for distance changes of one fluorophore compared to the errors of absolute distances.The data points in the calibration Figure 2B are already deviating from the fit curve especially in the range between 3 and 8 nm. This should be quantified and reported. Wouldn't this be a minimum systematic error? The deviation looks systematic, so there might be something missing in the theory? Could a fluorescein labeled lipid be used to determine the 0 nm (2.5 nm from graphene) point in the calibration curve?Related to the previous point. Without error discussion can the absolute distances really be reported with Å accuracy as in Figures 3 and 4.Could some of the observed distance changes be due to orientation changes of the dipole? The theoretical curve assumes isotropic orientation. Is this justified under all conditions ?In ensemble experiments where a distance change upon binding of a ligand is observed like in Figure 2D or in Figure 3G, the authors should show the specificity and concentration dependence of the result. Titrating the ligand should confirm that the reaction and with that the observed distance changes are in saturation. A comparison with a non-specific membrane binding protein or protein domain should confirm the specificity of the observed distance change on pYpt7.Related to this issue: How reproducible are the pYpt7 densities in the monolayer or membrane? If ligand binding is in saturation small density differences might not matter, but the authors should show that.Figure 3—figure supplement 4 shows that the measured lifetimes of mNeon-pYpt7 in membranes on glass are split into two populations. This is not discussed, and it looks like the mean value of the two components was used to normalize the graphene data. The graphene data either doesn't show or resolve different populations on a monolayer.Similar to the glass data above, the graphene data of Vps33-yEGFP in Figure 4—figure supplement 2 shows two populations (possibly even Vps39). The mean of the two doesn't seem to represent any data points but it was used for the result. Please comment and discuss.The statistics of the results are not clear. How many measurements were performed per sample, how many independently prepared samples were used?Reviewer #2:This is a well-written and comprehensive manuscript describing a novel methodological approach to probe axial organization of proteins labeled with fluorescence dyes in the nanometer range and in the context of membranes. For this, the authors developed a lipid monolayer assembly on graphene for site-specific protein capturing. Graphene-induced energy transfer (GIET) was adapted by replacing metal with graphene, an optical transparent material. GIET has advantages analogous to FRET as well as TIRF, such as a strong distance dependence and the ability to measure nanometer-range distances. The specific properties of graphene lead to a ~30 nm axial resolution, according to the author's calculations. The authors first validated GIET's ability to measure distance within a dynamic range of 25 nm on graphene-supported lipid monolayers using DNA oligonucleotides as a nanoscale ruler. The authors applied this approach to dissecting the axial organization and dynamics of HOPS-mediated large multi-protein complex regulated by Rab7-like Ypt7, involved in vesicular transport and fusion.Strengths of this manuscript are the comprehensive nature of the methodological approach described. The authors provide validation of GMIET distance-based measurements. The authors indicate that HOPS complex adopts an upright orientation on membranes with interesting axial dynamics. Their results introduce GIET to significant biological problems as a powerful technique to characterize nanoscale spatiotemporal organization of multi protein complexes at membranes. Another strength of this paper is the ability to use GIET distance measurements to monitor conformational transitions dynamics, e.g. "breathing" of the HOPS complex.Weaknesses are inherent to the probing of higher-order complexes at membranes using a complex methodology like GMIET, which requires taking those complexes out of their biological membrane background for the analysis. In fact, that contradiction reveals itself in the Introduction, where the authors first mention that "mechanistic understanding of large membrane protein complexes has been addressed by techniques […] that […] take them out of their biological membrane context". Later the authors similarly propose to use distance dependent fluorescence quenching by graphene as a novel technique to quantify the axial organization and dynamics of large membrane protein complexes upon taking them out of their native membrane context. It would be good for the authors to accept that their methodology does indeed provide an improvement in the ability to provide spatiotemporal solution in the 20-30nm but still works within a reconstructed system framework that as some of the other experimental approaches that the authors criticize, attempts to reconstruct large membrane complexes in an artificial lipid monolayer and thus out of their native environment and thus out of context.Nevertheless, this is a tour de force by the authors to completely reconstruct the HOPS-complex regulated by Rab7-like Ypt7, to highlight the value of the GIET technique to measure distances between fluorophore and graphene layer.A few comments should be addressed:– Figure 2 is the core of the paper and the ability to validate the distances of the ssDNA designed as nanorulers sustains all the remaining results. If the results in Figure 2 are questionable, then all other results would be problematic. So, it is important to question why the authors need to include undefined parameters to match their results to the ground truth distance measurements provided by the DNA nanoruler system. The consideration of the tilting parameter is unfortunate and would be important to find a system in which ground truth can be measured without data processing and calculation to match model to experimental data.– Also is not clear why the authors rely on the ratio between the measurements generated from graphene vs silica layers?There should be a theoretical explanation for these statements:– "Strikingly, the obtained correlation of IG/I0 vs d matches the predicted GIET curve well with a globally fitted tilting angle of (43 ±1)o , confirming the distance-dependent GIET on graphene-supported lipid monolayer."– "Ratios of fluorescence lifetimes on graphene (τG) to those on glass (τ0) closely matched the corresponding intensity ratios, which also confirmed the simulated distance dependent GIET efficiency ".– Similarly, for longer distances >10nm it would be important to have a DNA ruler than could directly be measured as ground truth using GIET on graphene-supported lipid monolayer without needing comparison with silica substrate measurements.– Also it is interesting that a similar angle of 45o is found for calibration curve as well as for the measurements of 35mer DNA nanoruler: "Comparing to the length of the 35mer DNA nanoruler (11.9 nm), the height corresponds to a tilting angle α = 45{degree sign}, which is in good agreement with the tilting angle α obtained for the calibration." Is there an explanation for this finding/coincidence?Reviewer #3:In brief, this work highlights GIET as a unique spectroscopic ruler to reveal the axial orientation and dynamics of macromolecular complexes at biological membranes with sub nanometer resolution, which is a very significant contribution to the mechanistic understanding of numerous key biological processes.Surface plasmon enhancement of electromagnetic field on metals, semiconductors and graphene has been implemented in various biosensing applications, covering a spectral region from UV-visible to IR. Comparing to the broad applications of surface enhancement, the nearfield radiationless electromagnetic coupling between the surface plasmon and excited fluorephores is less harnessed for biosensing. The situation is changing. Metal induced energy transfer (MIET) and graphene-based MIET  have emerged recently. In this manuscript, Fullbrunn et al. have set up the first step of using GIET in the field of protein structural biology. In contrast of detecting the structures of DNA origami or likely robust antibody-based biomolecular analysis, the current work draws a high standard by using lipidated membrane association protein and a tricky multiprotein complex. The authors' strategy is straightforward which achieved their goals efficiently: biofunctionalizing graphene on glass, validating the distance dependent GIET, and determining the structure of protein complexes. With the interdisciplinary expertise, the authors successfully used GIET as a spectroscopic ruler mapping the axial distance of the ~20 nm long HOPS complex on graphene. Very impressively, the structural dynamics of HOPS complexes was quantified by single molecule GIET on graphene-coated microscopy coverslips. I found this research was very well designed and carried out carefully. The manuscript was written in a good logical flow. The conclusions were supported by sufficient figures, tables and their supplementary.The longer sensitive range of GIET than FRET is indeed very suitable to obtain the structural overview of many extended proteins. As demonstrated in this work, GIET could be a powerful tool for determining the structure of membrane proteins. However, being a surface plasmon-based method, GIET is limited by immobilizing target proteins on the surface. From this aspect, one must point out GIET is not as feasible as FRET for applications in solution. Another disadvantage of the current method is that the coating on graphene is a non-fluidic lipid monolayer . The authors has noticed in the conclusions “GIET will allow to dissect.… large protein complexes on membranes upon interaction with membrane-bound ligands, and thus test molecular hypotheses in real time”. Actually, instead of considering “interaction of membrane-bound ligands”, surface architectures of fluidic lipid bilayer will be urgently needed for the applications of a large number of biologically important transmembrane proteins. Essential revisionsReviewer #1:Nadia Fuellbrunn and co-workers present their work about a new fluorescence method to determine the conformation and its dynamic of protein complexes by measuring the normal distance of site-specific fluorophores relative to a lipid monolayer. The graphene induced energy transfer (GIET) assay is based on the fluorescence quenching in close proximity to a graphene surface. The system is calibrated by utilizing "DNA nanorulers" of different length and with fluorescent labels at different specific positions. Measuring the fluorescence intensities and fluorescence lifetimes relative to unquenched samples. The calibrated system was then used to determine the orientation of the Rab7-like GTPase Ypt7 and of the multiprotein HOPS tethering complex at a lipid monolayer surface. Both proteins are part of the yeast vacuolar fusion and tethering machinery. The normal distances of specific residues to the monolayer surface are determined in ensemble measurements and the transition between three distinguishable conformations are measured in single molecule imaging experiments.The authors present a very nice and useful new method with a lot of potential in particular if it could be used to follow conformational changes of membrane proteins. The approach of measuring the normal distance of site-specific labeled residues is very similar to the method of site-specific fluorescence interference microscopy , 911), where the conformation of SNARE proteins reconstituted in supported membranes had been investigated. Although the overall strategy seems very similar, sdFLIC hasn't been mentioned in the Introduction where the authors discuss several methods by which protein conformations in membranes can be investigated.The sdFLIC is indeed a very useful method to detect distance changes of proteins on membranes that we, while being aware of the pioneering work, unfortunately neglected to include into our considerations. In the revised discussion, we now included a thorough discussion of sdFLIC including key references.The authors clearly show the usefulness of this new method especially in the dynamic measurements on single molecules. However, there are a couple of open questions and issues that should be addressed and clarified:A lipid monolayer is not a membrane and the authors should be clear and consistent in their writing in the whole manuscript . Proteins are not reconstituted into a membrane on graphene. The fact that the HOPS complex clusters in a diffusible membrane shows that this situation  is very different from the one at the monolayer. This raises another question. Is it justifiable to use the membrane system to measure the unquenched intensities/lifetimes to normalize the monolayer data? Shouldn't this be done on a monolayer too?We fully agree with the reviewer that the behavior of proteins in a diffusive membrane is likely very different from their behavior on a lipid monolayer. We particularly emphasized this for the HOPS complex “Given the impaired diffusion in the graphene-supported lipid monolayer, however, the conformationally highly dynamic HOPS complex observed in our studies may represent an intermediate state prior to lateral HOPS clustering, which in turn could increase the efficiency of HOPS to tether incoming vesicles”. This description is based on our observations of: (1) identical binding behavior of Ypt7 and Mon1-Ccz1 on diffusive membranes on glass and on a lipid monolayer on graphene , and (2) pYpt7-specific binding of HOPS on lipid mono- and bilayers . These results suggest that HOPS maintains its functionality and the conformation on both the diffusive membrane and on a lipid monolayer.We have carefully revised the manuscript to only use the term “membrane” in case of lipid bilayers and otherwise stick to the term “lipid monolayer”. Particularly, the description has been changed to “quantifying the axial position of proteins onto lipid monolayers by GIET” in the revised manuscript.Regarding the use of the unquenched intensities/lifetimes on a silica-supported membrane for normalization: the lipid composition  is the same for lipid mono- and bilayers ensuring very similar dielectric environment that may alter photophysical properties of fluorophores in addition to the effects caused by the molecular context within labeled DNA or proteins. However, these lipids do not absorb visible light and therefore are not expected to affect the fluorescence by inelastic interaction. Therefore, we can safely assume that fluorescence intensity and lifetime are similarly affected on lipid mono- and bilayers. We prefer lipid bilayers for practical reasons, because lipid monolayer formation on silanized, hydrophobic glass surfaces in our hands is much more prone for defect formation, probably due to lack of a perfect hydrophobic silane monolayer.A thorough and extensive error discussion is missing. Since this work is presented as a first application of this method, a detailed discussion and quantification of all error sources  is necessary. One would expect these errors to be different for distance changes of one fluorophore compared to the errors of absolute distances.The data points in the calibration Figure 2B are already deviating from the fit curve especially in the range between 3 and 8 nm. This should be quantified and reported. Wouldn't this be a minimum systematic error? The deviation looks systematic, so there might be something missing in the theory? Could a fluorescein labeled lipid be used to determine the 0 nm (2.5 nm from graphene) point in the calibration curve?d within ±5% τG. By plotting the derivative of FAM’s lifetime to distance, a distance dependent sensitivity was obtained, which is presented in panel D. Even though the systematic error increases with distance, accurate distance measurements can still be obtained by increasing the precision of measurement . Panel C summarizes the GIET curves of four different fluorophores: FAM, mNeonGreen, EGFP and DY647P1. These curves share a similar distance dependency. Moreover, statistical error of single-molecule GIET was evaluated using the root mean square deviation (RMSD). A relative 10% axial localization precision was obtained for single molecule detection at video rate (Supplementary file 5). Information of these error analyses was mentioned in the manuscript: Similarity of GIET curves for different dyes was described in Discussion paragraph two. Relative axial localization precision for single molecule detection was mentioned in Discussion paragraph one and in Materials and methods . In the revised manuscript, we now emphasize the results in Figure 2—figure supplement 2 regarding the error analysis in the context of its first citation in subsection “DNA nanoruler calibration confirms distance-dependent GIET efficiency”. We furthermore now cite Figure 2—figure supplement 2 in paragraph two of the Discussion to guide the readers to GIET curves of different fluorophores.We thank the reviewer for his suggestions. A detailed theoretical error analysis was presented in Figure 2—figure supplement 2: The systematic error is shown in panel B using FAM dye as an example, where the yellow regions mark the variation of sn-glycero-3-phosphoethanolamine) integrated into the lipid monolayer. OG488 and fluorescein share largely identical excitation/emission spectra, yielding very similar calculated GI/I0 for the “zero position” measurement (2.5% for FAM and 2.4% for OG488). However, we obtained GI/I0 = 3.8 ± 0.2% for OG488-DHPE  which is in line with the tendency of systematically decreased GIET-efficiency close to the lipid monolayer surface. Reliable determination of the fluorescence lifetime was not possible for OG488-DHPE integrated into graphene-supported lipid monolayers  The dramatically decreased fluorescence signals at short distance induce errors in measurements. For distances below 5 nm above graphene, less than 10% of the fluorescence signal is left due to strong GIET and therefore potential background signals have stronger bias to both intensity and lifetime measurements. (2) Several flexible linkers are involved in our nanoruler system, which allow increased distances of the fluorophores. Since the fluorophores and the lipid monolayer surface are negatively charged, strong electrostatic repulsion can be expected when fluorophores are dragged into close proximity of the lipid headgroups. Therefore, we took up the reviewer’s excellent suggestion and performed an additional GIET calibration with OG488-DHPE . Under this assumption, we consider it being justified to provide distance data based on the experimental errors of the measurements. For the results of mNeon-Ypt7 , the standard deviation (s.d.) is less than 2%. For HOPS-Vps-yEGFP , the s.d. is less than 4.4%. All the distances of proteins were calculated by considering the s.d. of lifetime measurements. There results were summarized as mean ± s.d. in Supplementary file 2. As shown in Supplementary file 2, the distances can be reported with sub-nanometer precision except for HOPS Vps33-yEGFP.In the revised manuscript, we have stressed that all experimental distances are based on the assumption that the theoretical curve applies.Could some of the observed distance changes be due to orientation changes of the dipole? The theoretical curve assumes isotropic orientation. Is this justified under all conditions ?Since both yEGFP and mNeonGreen are fused to proteins via long flexible amino acid linkers , we can confidently assume largely isotropic orientations. While the linker sequence of mNeon-fused Ypt7 was already specified  we have now also included the linker sequence in yEGFP-fused HOPS subunits. In case of the systems used for calibration – specifically the variants with surface-proximal fluorophore localization – bias by limited flexibility cannot be ruled out entirely. This may also contribute to the systematic deviations observed for these data points.In ensemble experiments where a distance change upon binding of a ligand is observed like in Figure 2D or in Figure 3G, the authors should show the specificity and concentration dependence of the result. Titrating the ligand should confirm that the reaction and with that the observed distance changes are in saturation. A comparison with a non-specific membrane binding protein or protein domain should confirm the specificity of the observed distance change on pYpt7.Related to this issue: How reproducible are the pYpt7 densities in the monolayer or membrane? If ligand binding is in saturation small density differences might not matter, but the authors should show that.We agree with the reviewer that it is important to point out that the observed distance changes upon ligand binding are in saturation and specific. We should emphasize that the transfer of pYpt7 to the lipid mono- and bilayers is rather less efficient and thus densities are far below a monolayer and therefore cannot be reliably quantified by our label-free detection. By contrast, binding of Mon1-Ccz1 is very efficient with a density >1 ng/mm² achieved under our experimental conditions , i.e. more than 1/5 of a protein monolayer. This means that Mon1-Ccz1 is available in at least 10-fold excess over pYpt7, thus ensuring pseudo-first order reaction conditions. We actually refrained from further increasing the Mon1-Ccz1 density to prevent bias by crowding the surface.Since we do not have available another protein that efficiently binds to lipid mono- or bilayer surfaces and we could not use Ni-trisNTA lipids due to His-tagged proteins involved in the reconstitution of pYpt7, we could not perform meaningful negative control experiments. However, we would like to emphasize that Figure 3G itself displays the specificity of observed distance changes for mNeon-Ypt7. Whereas interaction with the guanine nucleotide exchange factor Mon1-Ccz1 leads to an increase in the axial distance by 0.7 nm, the addition of the tethering complex HOPS leads to a decrease by 0.7 nm. Thus, observed distance changes for mNeon-Ypt7-GTP are likely to be related to the function of the ligand and are highly specific.Figure 3—figure supplement 4 shows that the measured lifetimes of mNeon-pYpt7 in membranes on glass are split into two populations. This is not discussed, and it looks like the mean value of the two components was used to normalize the graphene data. The graphene data either doesn't show or resolve different populations on a monolayer.This is indeed a somewhat surprising result that may be related to two fluorescent forms of mNeonGreen observed at physiological pH : a neutral form  and a basic form . The population of basic form increases with pH value. The observed two lifetimes in Figure 3—figure supplement 4 have a ratio of 0.9 that matches the previous report. Detection of two lifetimes could be due to changes of the dominant form by heterogeneous surface properties of lipid bilayers on glass. However, the two-population behavior was not repeatedly observed on other glass substrates. We thus treated the two populations as variations in the experiment and used the mean value for normalization. We did not observe two populations of lifetime on graphene, indicating that the possible difference of lifetimes cannot be resolved on graphene. In the revised Figure caption of Figure 3—figure supplement 4, we have noted that mean values of fluorescence lifetimes were used for calculating the ratios.Similar to the glass data above, the graphene data of Vps33-yEGFP in Figure 4—figure supplement 2 shows two populations (possibly even Vps39). The mean of the two doesn't seem to represent any data points but it was used for the result. Please comment and discuss.The observation of two populations of the lifetime for Vps33-yEGFP on graphene is indeed intriguing. The situation is different to the abovementioned situation that yEGFP has shown a consistent fluorescence lifetime on glass as shown in Figure 4—figure supplement 2. Most prominently, repeating the experiments of Vps33-yEGFP on graphene resulted in the same two-lifetime profile. Thus, accidental heterogeneity of surface properties can be excluded. Together with the broad lifetime distribution of Vps16-yEGFP shown in Figure 4D, these results may be related to the different HOPS conformations that we identified by single molecule GIET analysis.In the revised manuscript, we have now specified the reason by choosing HOPS Vps33-yEGFP for single molecule GIET analysis in the revised manuscript  to emphasize this observation.The statistics of the results are not clear. How many measurements were performed per sample, how many independently prepared samples were used?eLife’s transparent reporting form during submission of the manuscript. With the information now provided in the manuscript and summarized in the transparent reporting form, this information should be provided in sufficient detail.Thank you for pointing this out. Within our manuscript, we describe the sample volumes for all experiments. They are shown in figure captions and notes for tables : see Figures 2, 3, 4 and 5, and their figure supplements; Supplementary files 1, 2, and 4; Table 1, etc. To highlight the robustness of our work, we summarized all information on statistics in Reviewer #2:This is a well-written and comprehensive manuscript describing a novel methodological approach to probe axial organization of proteins labeled with fluorescence dyes in the nanometer range and in the context of membranes. For this, the authors developed a lipid monolayer assembly on graphene for site-specific protein capturing. Graphene-induced energy transfer (GIET) was adapted by replacing metal with graphene, an optical transparent material. GIET has advantages analogous to FRET as well as TIRF, such as a strong distance dependence and the ability to measure nanometer-range distances. The specific properties of graphene lead to a ~30 nm axial resolution, according to the author's calculations. The authors first validated GIET's ability to measure distance within a dynamic range of 25 nm on graphene-supported lipid monolayers using DNA oligonucleotides as a nanoscale ruler. The authors applied this approach to dissecting the axial organization and dynamics of HOPS-mediated large multi-protein complex regulated by Rab7-like Ypt7, involved in vesicular transport and fusion.Strengths of this manuscript are the comprehensive nature of the methodological approach described. The authors provide validation of GMIET distance-based measurements. The authors indicate that HOPS complex adopts an upright orientation on membranes with interesting axial dynamics. Their results introduce GIET to significant biological problems as a powerful technique to characterize nanoscale spatiotemporal organization of multi protein complexes at membranes. Another strength of this paper is the ability to use GIET distance measurements to monitor conformational transitions dynamics, e.g. "breathing" of the HOPS complex.Weaknesses are inherent to the probing of higher-order complexes at membranes using a complex methodology like GMIET, which requires taking those complexes out of their biological membrane background for the analysis. In fact, that contradiction reveals itself in the Introduction, where the authors first mention that "mechanistic understanding of large membrane protein complexes has been addressed by techniques… that.… take them out of their biological membrane context". Later the authors similarly propose to use distance dependent fluorescence quenching by graphene as a novel technique to quantify the axial organization and dynamics of large membrane protein complexes upon taking them out of their native membrane context. It would be good for the authors to accept that their methodology does indeed provide an improvement in the ability to provide spatiotemporal solution in the 20-30nm but still works within a reconstructed system framework that as some of the other experimental approaches that the authors criticize, attempts to reconstruct large membrane complexes in an artificial lipid monolayer and thus out of their native environment and thus out of context.Nevertheless, this is a tour de force by the authors to completely reconstruct the HOPS-complex regulated by Rab7-like Ypt7, to highlight the value of the GIET technique to measure distances between fluorophore and graphene layer.We agree with the reviewer’s insightful comments that our method as currently presented is limited to reconstituting protein complexes after taking them out of their biological membrane context. This approach is similar to traditional high-resolution structural analysis and consequently we aim in the future to combine GIET with single particle transmission electron microscopy using the same graphene supported lipid monolayer system for reconstitution of these complexes on an TEM grid. This will enable powerful correlation of structure and dynamics. Reconstitution furthermore ensures full control on the system with respect to protein and lipid composition and enables sequentially building up the system in situ. In addition, site-specific labeling of purified compounds will in the future enable to interrogate conformational changes much more specifically.However, our goal is also to extend GIET to analyzing axial organization of membrane proteins in their physiological context, e.g. signaling complexes in the plasma membrane, which are extremely challenging to isolate and reconstitute in a functional manner. We are confident that we can in the future interface cells with glass-supported graphene in a manner that will enable such measurements.In light of the reviewer’s comment for avoiding possible misleading, we have strengthened the inherent advantage of exploring heterogeneity by single molecule methods in the revised Introduction. It is thus more specific to the present work of characterizing structural dynamics of HOPS by using single molecule GIET with extended sensitive range.A few comments should be addressed:– Figure 2 is the core of the paper and the ability to validate the distances of the ssDNA designed as nanorulers sustains all the remaining results. If the results in Figure 2 are questionable, then all other results would be problematic. So, it is important to question why the authors need to include undefined parameters to match their results to the ground truth distance measurements provided by the DNA nanoruler system. The consideration of the tilting parameter is unfortunate and would be important to find a system in which ground truth can be measured without data processing and calculation to match model to experimental data.We agree with the reviewer that a calibration without a variable parameter would be preferable. However, we have previously confirmed the validity of our electromagnetic simulations of GIET efficiencies using coating with silica layers with different thicknesses . Based on this “ground truth”, we here needed to confirm the possibility of applying this method for distance measurements over a large dynamic range under biologically relevant conditions on a graphene-supported lipid monolayer. This was achieved by using the DNA nanoruler system, for which a robust tilting angle of 43±1 degree was obtained for both intensity and lifetime measurements. In addition, the structural dynamics observed for a 35mer DNA after hybridizing with the 15mer blocker strand confirmed a very similar tilting angle of 45 degree. These self-consistent results furthermore support our calibration by introducing the tilting angle. While additional approaches using cholesterol-modified DNA origami could be envisaged for a more robust distance measurement, establishing such a method is beyond the scope of this study. Rather, we took up the suggestion from reviewer 1 and added one more calibration data using dye-labeled lipid OG488-DHPE . When use the additional data point for global fitting, we have obtained the same tilting angle as before, i.e. average 43±1 degree. The new results furthermore confirm the robustness of calibration. In the revised manuscript, we have updated the calibration curve shown in Figure 2B, Figure 2—figure supplement 1, and Supplementary file 1.– Also is not clear why the authors rely on the ratio between the measurements generated from graphene vs silica layers?0τ of FAM when attached to 3´and 5´ ends of the oligonucleotides (cf. Supplementary file 1C). Such effects were reliably referenced out by measuring intensities (0I) and lifetimes (0τ) under exactly the same conditions (i.e. for the same molecules at a lipid-water interface) but in the absence of GIET. Therefore, we determined 0I and 0τ on silica-supported membranes, thus ensuring that the ratios G/I0I and G/τ0τ only comprise GIET-based quenching effects. Such reference measurements were particularly important for intensity measurements, which strongly depend on molecule densities and acquisition properties that may vary between different measurements. To robustly consider these effects, we applied simultaneous label-free and fluorescence detection side-by-side on silica and graphene surfaces.Our measurements are based on the quantum yield (intensity) and the excited state decay kinetics (fluorescence lifetime) as key photophysical properties of the fluorophore that are altered in vicinity of graphene. These properties, however, are also affected by the molecular environment of the fluorophore – as nicely demonstrated by the different In the revised manuscript, we have added an explanation in Materials and methods: Electromagnetic simulation of GIET to clarify this point.There should be a theoretical explanation for these statements:– "Strikingly, the obtained correlation of IG/I0 vs d matches the predicted GIET curve well with a globally fitted tilting angle of (43 ±1)o , confirming the distance-dependent GIET on graphene-supported lipid monolayer."– "Ratios of fluorescence lifetimes on graphene (τG) to those on glass (τ0) closely matched the corresponding intensity ratios, which also confirmed the simulated distance dependent GIET efficiency ".These two comments are related to whether the ratios of fluorescence intensity and lifetime can be used for GIET experiments. As mentioned above, the ratio of intensities measured on graphene and glass normalizes the influence of experimental conditions, and the fluorescence intensity is proportional to the lifetime since there is no static quenching in our experiments. Thus the relative fluorescence intensity and relative lifetime both allow reliable quantification of the distance.We have now added the necessary explanation in the revised Materials and methods: Electromagnetic simulation of GIET.– Similarly, for longer distances >10nm it would be important to have a DNA ruler than could directly be measured as ground truth using GIET on graphene-supported lipid monolayer without needing comparison with silica substrate measurements.0I and 0τ, which were applied for all rulers, independent on the fluorophore distance from the surface. Such reference measurements are integral part of determining the GIET efficiencies and thus the distances from the graphene surface.As we have answered to the previous comments, measurements on silica were performed to robustly determine In the revised manuscript, we have updated the results of calibration shown in Figure 2B, Figure 2—figure supplement 1 and Supplementary file 1. We have also added the necessary explanations in Materials and methods: Electromagnetic simulation of GIET for using the ratios.– Also it is interesting that a similar angle of 45o is found for calibration curve as well as for the measurements of 35mer DNA nanoruler: "Comparing to the length of the 35mer DNA nanoruler (11.9 nm), the height corresponds to a tilting angle α = 45{degree sign}, which is in good agreement with the tilting angle α obtained for the calibration." Is there an explanation for this finding/coincidence?The “coincidence” as reviewer pointed out is indeed a very important evidence supporting the validation by using the DNA nanorulers. We have shown global fitting of 7 intensity measurements  and 7 lifetime measurements to a highly conserved 43±1 degree tilting angle. Based on the calibration, an independent experiment for exploring the structural dynamics of 35mer DNA obtains a tilting angle of 45 degree. The self-consistent result of the tilting angles, i.e. 45 degree vs the globally fitted 43±1 degree, thus confirm the justification of introducing tilting of DNA nanorulers in the calibration.In the revised manuscript, we have now emphasized this point.Reviewer #3:In brief, this work highlights GIET as a unique spectroscopic ruler to reveal the axial orientation and dynamics of macromolecular complexes at biological membranes with sub nanometer resolution, which is a very significant contribution to the mechanistic understanding of numerous key biological processes.Surface plasmon enhancement of electromagnetic field on metals, semiconductors and graphene has been implemented in various biosensing applications, covering a spectral region from UV-visible to IR. Comparing to the broad applications of surface enhancement, the nearfield radiationless electromagnetic coupling between the surface plasmon and excited fluorephores is less harnessed for biosensing. The situation is changing. Metal induced energy transfer (MIET) and graphene-based MIET  have emerged recently. In this manuscript, Fullbrunn et al. have set up the first step of using GIET in the field of protein structural biology. In contrast of detecting the structures of DNA origami or likely robust antibody-based biomolecular analysis, the current work draws a high standard by using lipidated membrane association protein and a tricky multiprotein complex. The authors' strategy is straightforward which achieved their goals efficiently: biofunctionalizing graphene on glass, validating the distance dependent GIET, and determining the structure of protein complexes. With the interdisciplinary expertise, the authors successfully used GIET as a spectroscopic ruler mapping the axial distance of the ~20 nm long HOPS complex on graphene. Very impressively, the structural dynamics of HOPS complexes was quantified by single molecule GIET on graphene-coated microscopy coverslips. I found this research was very well designed and carried out carefully. The manuscript was written in a good logical flow. The conclusions were supported by sufficient figures, tables and their supplementary.The longer sensitive range of GIET than FRET is indeed very suitable to obtain the structural overview of many extended proteins. As demonstrated in this work, GIET could be a powerful tool for determining the structure of membrane proteins. However, being a surface plasmon-based method, GIET is limited by immobilizing target proteins on the surface. From this aspect, one must point out GIET is not as feasible as FRET for applications in solution. Another disadvantage of the current method is that the coating on graphene is a non-fluidic lipid monolayer . The authors has noticed in the conclusions “GIET will allow to dissect.… large protein complexes on membranes upon interaction with membrane-bound ligands, and thus test molecular hypotheses in real time”. Actually, instead of considering “interaction of membrane-bound ligands”, surface architectures of fluidic lipid bilayer will be urgently needed for the applications of a large number of biologically important transmembrane proteins.It is true that GIET is a surface-based structural analysis tool and therefore we clearly identified its potential for structural analysis of protein complexes in the context of membranes. We also agree with the importance of extending this methodology towards fluid lipid bilayer architectures stressed by the reviewer and we are currently pushing our efforts into this direction. For our proof-of-concept experiments, however, the well-defined monolayer architecture used in this study clearly provided important advantages for quantifying distances with minimum bias.


Three-dimensional (3D) in vitro skin models have been widely used for cosmeceutical and pharmaceutical applications aiming to reduce animal use in experiment. This study investigate capability of ovine tendon collagen type I (OTC-I) sponge suitable platform for a 3D in vitro skin model using co-cultured skin cells (CC) containing human epidermal keratinocytes (HEK) and human dermal fibroblasts (HDF) under submerged (SM) and air-liquid interface (ALI) conditions. Briefly, the extracted OTC-I was freeze-dried and crosslinked with genipin (OTC-I_GNP) and carbodiimide (OTC-I_EDC). The gross appearance, physico-chemical characteristics, biocompatibility and growth profile of seeded skin cells were assessed. The light brown and white appearance for the OTC-I_GNP scaffold and other groups were observed, respectively. The OTC-I_GNP scaffold demonstrated the highest swelling ratio (~1885%) and water uptake (94.96 ± 0.14%). The Fourier transformation infrared demonstrated amide A, B and I, II and III which represent collagen type I. The microstructure of all fabricated sponges presented a similar surface roughness with the presence of visible collagen fibers and a heterogenous porous structure. The OTC-I_EDC scaffold was more toxic and showed the lowest cell attachment and proliferation as compared to other groups. The micrographic evaluation revealed that CC potentially formed the epidermal- and dermal-like layers in both SM and ALI that prominently observed with OTC-I_GNP compared to others. In conclusion, these results suggest that OTC_GNP could be used as a 3D in vitro skin model under ALI microenvironment. Recently, the use of non-animal models to evaluate the safety of materials in industry, agriculture and biomedical applications has gradually increased . The acuBriefly, the current RHS has been used for various applications in the cosmeceutical, theranostic, pharmaceutical, biomedical and clinical settings. Moreover, RHS has been useful as an in vitro model for toxicity testing drugs, chemical and advanced materials. The Organisation for Economic Cooperation and Development (OECD) guideline 428 clearly mentions that the preparation of human skin, including full-thickness skin, split-thickness skin and heat-separated human epidermis, can be imitated by an in vitro human skin equivalent. However, the quality of reconstructed 3D skin should be comparable with human skin, so any further testing would be closely related to native skin.Traditionally, safety testing is performed in animal models, such as rodents (rats and mice) or non-rodents (dogs and mini pigs) because they represented systemic organisms resembling physiologically and functionally as of human being . HoweverThe innovative medical and material research has led to novel technologies in the quest for improved cell culture methods. Pioneering research in three-dimensional (3D) culture models from the Bissell laboratory has opened the way for more sophisticated and relevant culture models than the traditional 2D methods . 3D cellCurrently, 3D skin models can be generated by culturing keratinocytes onto a cellular matrix (fibroblast-collagen matrix), acellular matrix  or inert plastic filler. As a replacement to chemically synthesized biopolymers, such as chitosan  and sodiPrevious studies have described the successful extraction of collagen type I from ovine tendon. Ovine collagen type I is able to form various 3D scaffold designs, and its application as a rapid treatment for full thickness skin loss has been explored ,22,23. TThis study was approved by the Universiti Kebangsaan Malaysia (UKM) Research Ethics Committee (Code No. FF-2015-087).2HPO4; 0.2 M) and phosphate buffered saline  as the dialysis buffer every 12 h. Dialysed collagen was frozen at −80 °C for 6 h followed by freeze-drying for 24–48 h [Ovine tendon collagen type I (OTC-I) was extracted and purified as previously described by Fauzi and co-workers (2016) . Crude t 24–48 h . OTC-I tThe OTC-I solution was poured into the desired mould and frozen at −80 °C for 6 h, followed by a freeze-drying process  for 24–48 h. Commercialised atelocollagen (ATC) was used as a control.w/v) genipin , which is a natural crosslinking agent and 1 M (w/v) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide , which is a synthetic crosslinking agent. The sponge scaffolds were dissolved in the GNP and EDC working solution at room temperature for 6 h, followed by several washing steps using PBS. Furthermore, both the crosslinked scaffolds were then frozen at −80 °C for 6 h prior to freeze-drying for 24–48 h before use for further analysis. The OTC-I sponge crosslinked with genipin (GNP) and carbodiimide (EDC) were denoted as OTC-I_GNP and OTC-I_EDC sponge, respectively. A non-crosslinked OTC-I sponge and atelocollagen (ATC) were used as the controls.OTC-I sponge scaffolds were crosslinked with 0.1%  for at least 1 h and until it reached near-equilibrium staining. The non-crosslinked and crosslinked OTC-I sponges were soaked in solution A (phosphomolybdic acid hydrate) for 2 min, in solution B  for 60 min and subsequently in solution C (0.1% hydrochloric acid) for 2 min as mentioned in the manufacturer’s protocol. Most of the excessive water from the slides was physically removed by vigorous shaking.d). Then, the scaffolds were immersed in PBS (pH 7.2) and incubated at 37 °C for 1 h. The excessive liquid from the sponge scaffolds surface were gently removed using filter paper, followed by weighing to obtain the weight after swelling (Ss). The water uptake ability was evaluated after incubation for 48 h at 37 °C to permit water adsorption therefore as mentioned in previous publication [d) followed by weighing to obtain their weight after water uptake (Ws). The swelling ratio and water uptake were calculated using the equations below:The swelling ratio was performed as mentioned in a previous publication by Yang and co-workers (2018) with minor modifications . Freeze-lication  the sponv/v) for 10 min each. Finally, the OTC-I sponges were dried overnight using a critical point dryer  followed by nanogold coating. The scaffolds were observed by SEM and the pore size was measured randomly using Phenom Pro X integrated software .The surface topography and cross-section microstructure of non-crosslinked and crosslinked OTC-I sponges were evaluated via scanning electron microscopy  as mentioned in a previous study . The fab3 pieces and the spectral data were recorded by a PE Spectrum 100 FT-IR spectrometer  in a wavelength range of 700 to 4000 cm−1. The absorbance peaks were analysed to identify any chemical structural changes after the crosslinking process.The chemical characterisation of the OTC-I sponges was performed using Fourier transform infrared spectrometry (FT-IR), as described in Busra et al. (2019) . The sca2 in size was minced into small pieces and digested by dual enzymatic approaches with 0.6% collagenase type I  for 5–6 h at 37 °C in a shaker incubator, followed by cell dissociation using 0.05% trypsin-EDTA  for 10 min. The cell suspension was then centrifuged. Co-cultured skin cells were seeded in three wells of a six-well polystyrene plate  and maintained at 37 °C in a 5% CO2 incubator, with medium changes every 2–3 days. Both co-cultured epidermal keratinocytes (HEK) and dermal fibroblasts (HDF) were cultured in an equal amount of serum-free Epilife medium  and F12:DMEM (Ham’s F-12 nutrient: Dulbecco’s Modified Eagle Medium)  medium which was supplemented with 10% foetal bovine serum (FBS) , 1% antibacterial-antimycotic (AA) , 1% Glutamax  and 2% HEPES . Once skin cells reached 70–80% confluence, differential trypsinisation was performed to dissociate HDF from culture plate using 0.05% Trypsin-EDTA. The HDF were seeded in 75 cm2 culture flasks with F12:DMEM medium containing 10% FBS while HEK were cultured in a six-well plate with Epilife  medium.Redundant skin tissue samples were processed as previously described by Busra and co-workers (2019) . A skin 4 cells/cm3 were seeded on the surface of all of the fabricated bioscaffolds and incubated at 37 °C, with 5% CO2 for 24 h. The fabricated bioscaffolds seeded with co-culture HEK and HDF then were incubated with calcein AM (2 mM) and EthD-1 (4 mM) in PBS for 30 min as recommended by the manufacturer’s protocol. Subsequently, the stained cells were observed using a Nikon A1R-A1 confocal laser scanning microscope . The live and dead cells were stained green  and red (EthD-1), respectively.The live/dead cell viability assay  was used to analyse the cytotoxic effect of the non-crosslinked and crosslinked OTC-I sponges according to the manufacturer’s protocol. On the culture plate, co-culture HEK and HDF at a density of 1 × 104 cells/cm3. Unattached cells in the culture suspension were quantified at 2 h after seeding using the trypan blue exclusion test. Subsequently, the percentage of cell attachment on the non-crosslinked and crosslinked OTC-I sponges was evaluated using the following equation:For cell attachment analysis, co-culture HEK and HDF at passage 2 were seeded on the non-crosslinked and crosslinked OTC-I sponges at a density of 1 × 104 co-culture of HEK and HDF were seeded on the non-crosslinked and crosslinked OTC-I sponges in a 48-well culture plate for different time points . The quantification of cell proliferation was performed by using the Vybrant® MTT cell proliferation assay kit  and, hence, the manufacturer’s instructions were followed. At day 1, 3 and 5 the seeded sponges were transferred to a new 48-well culture plate to avoid the possibility of false positive results from the seeded cells that might have attached on the surface of the well. The spent medium was replaced with 100 µL of fresh co-culture medium containing Epilife-F12:DMEM at a 1:1 ratio. Then, 10 µL of MTT reagent was added into each well and incubated at 37 °C for 4 h. Next, 100 µL of 10% (w/v) SDS in 0.01 M HCl solution was added into each well, followed by 4 h of incubation. Finally, 100 µL of the solution was transferred into a 96-well plate and the absorbance was read at 570 nm using a spectrophotometer .A 1 × 102 and a density of 5 × 105 cells/cm2. For the submerged microenvironment, the co-cultured HEK and HDF were maintained as follows: co-culture medium containing Epilife  and F12:DMEM in a 1:1 ratio was added in each well to a volume such that the medium level reached slightly higher than the sponge’s height. The scaffolds seeded with co-cultured HEK and HDF were maintained in an incubator with 5% CO2, at 37 °C and the medium was changed on a daily basis for 30 days of the culture period.The non-crosslinked and crosslinked OTC-I sponge scaffolds were inserted into a 24-well culture plate. The co-culture HEK and HDF were then seeded onto the fabricated sponges with a surface area of 0.33 cmThe seeding of co-cultured HEK and HDF was done similar to the submerged conditions and the seeded scaffolds were maintained in a submerged microenvironment for two days. Afterward, to initiate the air-liquid interface (ALI) microenvironment, the media was completely aspirated from the 24-well culture plate and from inside the culture insert. Then, fresh co-culture medium containing Epilife  and F12:DMEM in a 1:1 ratio was added to the lower compartment (culture plate), allowing the co-culture HEK and HDF to be exposed to the air from the surface and for the media to be perfused only from the lower side. To allow the co-cultured cells on the surface to remain exposed to the air, subsequent media replacement for the next 30 days was performed in the lower compartment only.The non-crosslinked and crosslinked OTC-I sponges were seeded with co-cultured HEK and HDF and remained in culture for 30 days. Next, the cells were fixed with 3% glutaraldehyde and a serial chemical process and observed via SEM for the microscopic evaluation of cell morphology. The morphological evaluation was done separately for submerged conditions and the ALI microenvironment.p < 0.05.The statistical evaluation was performed using GraphPad Prism 5.0 . The data are expressed as the mean ± standard deviation (SD). The comparison between multiple groups was analysed by ANOVA and any statistically significant difference was deemed when The gross appearance of atelocollagen (ATE), non-crosslinked and crosslinked (OTC-I) sponges is illustrated in The swelling ratio and water uptake analysis were determined as shown in The photomicrographs of surface topography and cross-sections of the fabricated sponges were examined via scanning electron microscopy (SEM), as shown in −1), amide I due to C–O stretching (1634 cm−1), amide II due to N–H deformation (1547 cm−1) and amide III due to N–H deformation (1235 cm−1), which correspond to the characteristics of collagen type I. Obviously, there was no major shift in the FT-IR spectra of OTC-I_GNP and OTC-I_EDC regarding the absorbance peaks of amide A, I, II and III, as presented in Chemical characterisation of the non-crosslinked and crosslinked OTC-I sponge scaffolds was performed using FT-IR spectrometry. The FT-IR spectra of fabricated sponges are illustrated in Co-cultured human epidermal keratinocytes (HEK) and human dermal fibroblasts (HDF) were seeded on the non-crosslinked and crosslinked OTC-I sponge scaffolds to evaluate their cytotoxicity a; ATC waThe attachment of co-cultured HEK and HDF 24 h post-seeding on the non-crosslinked OTC-I, OTC-I_GNP, OTC-I_EDC and ATC sponges are shown in The proliferation of co-cultured HEK and HDF on the non-crosslinked OTC-I, OTC-I_GNP and OTC-I_EDC sponges was evaluated at different time points: day 1, 3 and 5 c. The ATThe evaluation of co-cultured HEK and HDF on the fabricated OTC-I sponges is illustrated in The goal of this study was to fabricate a three-dimensional (3D) in vitro skin model or reconstructed human skin (RHS) comprising of ovine tendon collagen type I (OTC-I) as a bioscaffold seeded with co-culture HEK and HDF. Development of an RHS should take into consideration several factors, including a suitable provisional bio-template, cell type, microenvironment and related culture medium. Direct contact of HEK with HDF (co-culture) is critical for the proliferation, migration and stratification of the epidermis ; therefoPrevious studies have shown that treatment of collagen with genipin induces colour changes from yellowish to the dark bluish range dependent on treatment duration. The colour changes of collagen indicated successful cross-linking of genipin with primary amines , and canThe swelling ratio is an important parameter because it indicates how the sponge scaffold will respond when it comes into contact with blood or other body fluids; this can also affect the cell differentiation process . The swe−1 indicated that OTC-I had a β-sheet and triple helix structure. Moreover, the peak intensity at 1546 cm−1 to 1235 cm−1 indicates the triple helical content in collagen. The results were consistent with previous studies conducted by Riaz et al. (2018) [As described in our previous study, the FT-IR peak at 1635 cm. (2018) , and Fau. (2018) , that peCell toxicity in evaluating the suitability of construct in supporting the attachment and growth of the seeded cells demonstrated that the non-crosslinked OTC-I and OCT-I_GNP were equally favourable for cell attachment and they did not interfere with cell growth. Cell toxicity associated with genipin had been found to be dose-dependent , so thesThe cytotoxicity and unfavourable properties of OTC-I_EDC in supporting cell attachment and proliferation might be primarily due to the interaction of EDC with collagen binding sites. Previous studies have shown that EDC-crosslinked collagen scaffolds consume carboxylate-containing acid side chains, which are essential for recognition by the cell surface. Thus, it adversely affects the attachment of cells to the scaffold through collagen-binding integrins ,38. A loTo date, there are no data available on the direct measurement of changes in cell polarisation in response to their surrounding 3D-environment and the mechanical forces generated by neighbouring cells. However, Giavazzi and co-workers in 2018 reported that a change in the surrounding environment induced direct effects on the collective migration of epithelial cells. This cell flocking caused the rearrangement of cells and induced the alignment of cells perpendicularly to the migration direction . A similThe observation of an effect of the submerged and ALI microenvironments with the micromorphology of seeded HEK and HDF were consistent with the cytotoxicity results. In both microenvironment conditions, the OTC-I_EDC sponge did not favour the seeded cells and imposed toxicity on skin cells, followed by apoptosis. We found a synergistic enhancement of cell growth in HDF and HEK co-culture; however, this observation was in contrast with previous findings by one of the co-authors in which the absence of cell growth in co-cultured constructs was reported . This wo+ ions and lower incubation temperatures can be investigated.In conclusion, these preliminary results provide valuable information on a three-dimensional (3D) in vitro skin model using ATE, non-crosslinked and crosslinked OTC-I sponge scaffolds seeded with HEK and HDF in submerged (SM) and air-liquid interface (ALI) microenvironments. Even though OTC-1_GNP was found to be the optimal construct for supporting co-cultured HEK and HDF in an ALI microenvironment and could be a potential candidate for future 3D skin reconstruction modelling. Moreover, to improve the current findings, the authors suggest further histological and immunohistochemical analysis, to confirm multi-layer formation and stratification of keratinocytes and the detection of keratinocytes terminally-differentiated markers, respectively. In future models of 3D skin, to further stimulate multi-layer keratinocyte formation, culturing HEK in higher concentrations of Ca