Subdistal appendages (sDAPs) are centriolar elements that are observed proximal to the distal appendages (DAPs) in vertebrates. Despite the obvious presence of sDAPs, structural and functional understanding of them remains elusive. Here, by combining super-resolved localization analysis and CRISPR-Cas9 genetic perturbation, we find that although DAPs and sDAPs are primarily responsible for distinct functions in ciliogenesis and microtubule anchoring, respectively, the presence of one element actually affects the positioning of the other. Specifically, we find dual layers of both ODF2 and CEP89, where their localizations are differentially regulated by DAP and sDAP integrity. DAP depletion relaxes longitudinal occupancy of sDAP protein ninein to cover the DAP region, implying a role of DAPs in sDAP positioning. Removing sDAPs alter the distal border of centrosomal γ-tubulins, illustrating a new role of sDAPs. Together, our results provide an architectural framework for sDAPs that sheds light on functional understanding, surprisingly revealing coupling between DAPs and sDAPs. The mammalian centrosome is a microtubule organizing center (MTOC) that plays a crucial role in various biological processes, such as mitotic spindle organization and cell polarity regulation . A centrThe mother centriole is structurally differentiated from the daughter centriole by the presence of two appendage structures close to its distal end, namely distal appendages (DAPs) and subdistal appendages (sDAPs). DAPs are essential for centriole-membrane docking and primary cilia formation . We and Some EM images showed variations in the number of sDAPs, such as 2 to 12 sDAP stems in human endotheliocytes , illustrAlthough DAPs and sDAPs are mostly adjacent to each other in mammalian centrioles, their functions are distinct. DAPs are responsible for ciliogenesis and ciliary vesicle docking . sDAPs aStudies of the DAP–sDAP relationship are also very limited. As mentioned above, our previous super-resolution imaging result showed dual localizations of CEP89, potentially one localization in the DAP region and the other close to the sDAP region, but no functional studies have been performed to check whether CEP89 is associated with sDAPs or not. In RPE-1 cells, a genetic knockout of the most upstream sDAP protein ODF2 was shown to abolish the recruitment of other sDAP components , but neiIn this study, we systematically characterized the super-resolved molecular architecture of sDAPs using direct stochastic optical reconstruction microscopy (dSTORM). We mapped the spatial organization of multiple sDAP-related proteins and generated a three-dimensional model. Intriguingly, we found structural coupling between DAP and sDAP proteins. We have also directly observed the role of sDAPs on MT anchoring and further illustrated the selective involvement of γ‐tubulins on MT templating. Together, our studies systematically reveal the super-resolved architecture of sDAPs, suggest potential DAP–sDAP structural connections, and implicate a regulatory mechanism of MT anchoring around the centrosome.0 phase before proceeding to imaging.To characterize sDAP architecture systematically, we imaged multiple sDAP proteins reported previously  by dSTORWe first examined whether sDAP proteins form rings with a nine-fold symmetry like those formed by DAP proteins. We conducted two-color staining of the DAP protein SCLT1 and an sDAP protein of interest using dSTORM with Alexa Fluor 647 and Cy3B organic dyes. We used SCLT1 as a marker in widefield imaging to pick centrioles approximately viewed from the axial view by means of the well-defined ring structure of SCLT1 that is crudely observable in conventional widefield microscopy . We thenTo understand the architecture of sDAP proteins with nearly full occupancies at the sDAPs, we pre-selected centrioles based on whether we saw a ring or not for an sDAP protein of interest under widefield microscopy, and further analyzed these centrioles with super-resolution microscopy. As shown in When correlating the occupancies of sDAP proteins from ODF2 to CEP170 to the reported assembly hierarchy of sDAPs as illustrated in We defined the longitudinal position of sDAP proteins by means of dual color dSTORM super-resolution imaging. Each sDAP protein was immuno-stained together with the DAP protein SCLT1, which served as a position reference. As shown in Combining sDAP measurements from our axial and lateral dSTORM images , we gene–/– cells is larger than that of the wild-type cells, illustrating both narrowing and shifting of ODF2 occupancy upon sDAP CEP128 depletion. These observations imply that CEP128, as the binding partner of ODF2  and sDAP depletion (i.e. CEP128 CRISPR/Cas9 knockout) to check their effects on ODF2 localization with dSTORM. Successful knockout of CEP83 or CEP128 in RPE-1 cells were confirmed by immunoblotting . Lateral of ODF2 , regulats absent . This obs absent . Our ress absent . Note ths absent .We also examined the sDAP association of CEP89, a putative DAP protein that also exhibits a two-layered localization, by checking the effect of CEP89 localization upon sDAP depletion (CEP128 CRISPR/Cas9 knockout). Intriguingly, the proximal layer of CEP89 was absent when sDAP was depleted . While D–/CEP83–cells, ninein at the sDAPs covers a longitudinal region larger than that in wild-type cells (–/CEP83–cells (–/CEP83– Rescue), the sDAP distribution of ninein is restored  regrowth after nocodazole-induced MT depolymerization . Howevernetworks . Many MT–/–cells , but it /– cells . While p/– cells , this is/– cells . These D0 phase are mostly confined to a well-defined cylindrical position outside the mother centriole. This confined cylindrical distribution of γ-tubulins provides a better-defined localization of the previously known tightly packed PCM in the interphase, which is very different from the broad distribution of PCM-bound γ-tubulins during mitosis. When compared to the distribution of α-tubulin of wild-type centrioles shown in –/CEP128– cells , according to the manufacturer's protocol. This cell line is not in the list of commonly misidentified cell lines maintained by the International Cell Line Authentication Committee. Prior to immunofluorescence staining, cells were cultured on poly-L-Lysine coated coverslips and then serum starved for 24 hr before fixing with methanol at −20°C. For staining of CCDC68, CEP170 and ninein, cells were first extracted with PTEM buffer for 2 min before proceeding to ice-cold methanol fixation. The PTEM buffer contained 20 mM PIPES, 0.2% Triton X-100, 10 mM EGTA and 1 mM MgCl2 and was prepared at pH 6.8. After fixation, cells were permeabilized with 0.1% PBST (PBS with 0.1% Triton X-100) for 10 min before blocking with 3% BSA for 30 min. Primary antibodies at optimized dilution prepared with 0.1% BSA in PBST were then added to cells for 1 hr. To remove unbound antibodies, cells were washed three times with 0.1% PBST, and then incubated with optimally diluted secondary antibodies for 1 hr. Finally, cells were washed three times with 0.1% PBST and stored in PBS with sodium azide at 4°C until later use.Human retinal pigment epithelial cells (RPE-1) were purchased from ATCC  and were cultured in DMEM/F-12 medium  supplemented with 10% fetal bovine serum  at 37°C with 5% COCells were washed with ice-cold PBS twice and lysed with 1% NP-40 buffer . Total cell lysates were incubated on ice for 20 min and centrifuged . Following the protein quantification assay , 30 μg proteins from wild-type and knockout cells were analyzed by 8% SDS-PAGE. Proteins were transferred to PVDF membrane and incubated with the indicated antibodies, before signals were detected using the Western Lightning Plus reagent .A CRISPR/Cas9 gene targeting technique was used to inactivate CEP83 or CEP128 in RPE-1 cells as described previously . The tarGCTGCCAGATCAACGCACAGGG-3′), CEP128 gRNA4 (5′-GAGTCAGCTCTGAGATCTGAAGG-3′), CEP128 gRNA5 (5′ GCAGCTGAACTTCAGCGCAATGG-3′), CEP83 gRNA1 (5′-GGTGGAGACAGTGGATTGACAGG-3′), and CEP83 gRNA2 (5′-GATATTAACTCCACAAAAATTGG-3′). Multiple gRNAs targeting different exons for CEP128 were used to achieve complete protein depletion. Briefly, the targeting sequences were cloned into the gRNA cloning vector  via the Gibson assembly method . gRNA was co-expressed with Cas9 protein in RPE-1 cells using reagents from the Church group (http://www.addgene.org/crispr/church/). Knockout cell lines were obtained through clonal propagation and then confirmed by genotyping and immunoblotting.CEP128 gRNA2  cells together with V-SVG and pCMV-gag-pol plasmids for lentivirus generation. The virus was collected 48 hr post infection. 3 ml of the viral stock was used to infect 5 × 105–/CEP83–RPE-1 cells seeded onto a 60 mm plate. Cells were selected 2 days after infection and maintained in culture medium containing 2 µg/ml puromycin .CEP83 construct synthesis and generation of CEP834 in PBS buffer for 30 min on ice. After dehydrating in a graded series of ethanol, the cells were then infiltrated and embedded in EPON812 resin  to generate a resin sample. A microtome  was used to cut the sample into serial sections (~90 nm thickness), which were then stained with 1% uranyl acetate and 1% lead citrate. Samples were imaged using FEI Tecnai Spirit G2 and a JOEL JEM-1400plus transmission electron microscope.RPE-1 cells were grown on Aclar film -based coverslips and fixed in 4% paraformaldehyde  and 2.5% glutaraldehyde  with 0.1% tannic acid in PBS buffer at 37°C for 30 min. Cells were then further postfixed in 1% OsO2 to quench most of the fluorescence from Alexa 647 and Cy3B, respectively. A weak 405 nm beam was used to activate a portion of the dyes, converting them from a dark state to an excitable state. For single-color imaging of Alexa 647, signals were filtered with a bandpass filter ; for two-color imaging, the Alexa 647 channel was first recorded, and then the Cy3B channel was acquired with the corresponding filter .For dSTORM imaging, Alexa Fluor 647 antibody (1:200) and Cy3B-conjugated antibody (1:100) were used as secondary antibodies. In general, the protein of interest was stained with Alexa Fluor 647 antibody whereas the reference protein, for example SCLT1, was stained with Cy3B-conjugated antibody. The dSTORM imaging system included a modified inverted microscope  and a laser merge module  with individual controllers for three light sources. To illuminate samples in the wide field, photon beams from a 637 nm laser , a 561 nm laser  and a 405 nm laser  were homogenized  and focused with a 100 × 1.49 NA oil immersion objective . To minimize z-axis drift, a perfect focusing system  was used. During dSTORM imaging, the 647 nm and 561 nm laser lines were operated at a high intensity of ~1–5 kW/cmThe collected fluorescent signal was filtered through a single-band emission filter and acquired on an EMCCD  with a pixel size of 93 nm. For each dSTORM image, 10,000–20,000 frames were acquired every 20 ms (exposure time). Individual single-molecule peaks were localized using MetaMorph Superresolution Module  based on a wavelet segmentation algorithm. Super-resolution images were cleaned with a Gaussian filter with a radius of 1 pixel. For imaging sample preparation, cells grown on coverslips were placed in an imaging chamber  and immersed in dSTORM imaging buffer. The buffer included TN buffer at pH 8.0, and an oxygen-scavenging system consisting of 60–100 mM mercaptoethylamine  at pH 8.0, 0.5 mg/mL glucose oxidase , 40 mg/mL catalase , and 10% (w/v) glucose .To correct lateral position drift, fiducial markers  were added to the sample at a dilution of 1/200 before imaging. The drift was measured and corrected with ImageJ via frame-by-frame correlation of the fiducial markers. Chromatic aberration between the long and short wavelength channels was compensated with a customized algorithm relocating each pixel of a 561 nm image to its targeted position in the 647 nm channel with a predefined correction function obtained by a parabolic mapping of multiple calibration beads. For axial imaging of the DAPs, a ring pattern of SCLT1 was used to identify the top view orientation. For lateral imaging, the rod-like pattern of SCLT1 was used as an indicator of the lateral view of the centriole-cilium. To determine the diameter of the sDAP proteins, a radial position of an individual super-resolved punctum was measured and the distance between each punctum and the center was defined as the radius. To correlate dSTORM with EM images, signal of CEP170 was used to align with the tip of the sDAP contour on the electron micrograph.The 3D mother centriole structure model was drawn with the 3D illustration software Blender . The dimensions of the model were based on the mean localization positions measured from the dSTORM images  in this For expansion microscopy, cells were fixed with 4% PFA  for 10 min before blocking. ATTO647N  and Alexa Fluor 488 secondary antibodies were used in immunostaining. Stained cells were then incubated in 0.1 mg/ml acryloyl-X  solution in DMSO overnight at 4°C, followed by washing twice for 15 min each with PBS. For in-situ polymer synthesis, monomer solution  was prepared as described previously . Samples In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Centrioles are cylindrical microtubule-based structures that form the core of the major microtubule-organizing center, the centrosome, and also serve as basal bodies of cilia and flagella. Centriole functions partly depend of protrusions emerging from centriole surface, which are termed distal and subdistal appendages. This paper nicely combines super-resolution microscopy and CRISPR-Cas9 knockouts in human cells to reveal the molecular organization of centriole appendages. These data confirm previous observations and provide novel insight into the architecture of centriole appendages and also contribute to the ongoing discussion about the functional role and interdependence of these structures.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Anna Akhmanova as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Laurence Pelletier (Reviewer #3).Thank you for submitting your article "Super-resolution microscopy reveals coupling between mammalian centriole subdistal appendages and distal appendages" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:In this manuscript, Wong and colleagues present imaging of sub-distal appendage (SDAP) architecture using quantitative dSTORM. The authors show that different SDAP proteins display different radial distributions around the mother centrioles. The authors then use similar analyses to map the lateral position occupied by each of the SDAP analysed and generate an axial and lateral composite view of SDAP organization, which suggests that ODF2 and CEP89 distribution are localized to both DAPs and SDAP region consistent with a potential positional relationship between these structures. The authors then go on to show that ODF2, CEP89 and Ninein distribution is differentially regulated upon the perturbation of DAP and SDAP integrity using CRISPR KO cell lines. The authors then demonstrate a small but reproducible reduction in the number of microtubules anchored in the vicinity of SDAP in CEP128 KO cells. Finally, the authors present data suggesting that the majority of γ-tubulin adjacent to the mother centriole does not appear to correlate with anchored microtubules at SDAPs in cells that are in G0 phase. Overall, this is a very nice paper, with high-quality quantitative sub-diffraction imaging of centriolar appendages. Although some of the data is somewhat preliminary, the results presented here expose both a positional and functional relationship between DAPs and SDAPs and begin to reveal a more direct role for SDAPs in microtubule anchoring at centrosomes, which will stimulate further work in these areas.Essential revisions:1) The most important problem with this work is that conclusions derived from STORM-based analyses are not corroborated by ultrastructural analysis.Specifically, Figure 3 shows a 3D model of SDAPs. The model shows nine subdistal appendages. Since SDAPs occupy more than one microtubule triplet, the model presented in Figure 3 might not reflect the actual organization of SDAPs in RPE-1 cells. In addition, it is not obvious how this model was deduced from presented STORM data, which itself did not reveal a nine-fold distribution of most SDAP proteins. The authors will either need prove their model by performing EM analysis, or clarify that this is a speculative model. As it is now, it may suggest the wrong concept of SDAP architecture in mammalian cells. Please see PMID: 30045886, a recent review on this subject.Further, in Figure 4I, which summarizes the localization changes of SDAP proteins in Cep128-/- and Cep83-/- cells, some type of appendage is drawn at the distal centriole end. Do authors mean to say that in the absence of distal appendages, subdistal appendages form at the centriole's very distal ends? If so, it would be critical to perform an EM analysis of wild type and knockout centrioles and to show whether there are any structural changes associated with centriole distal ends. Otherwise, it needs to be clear that the model is speculative. The cartoon in Figure 6G is not corroborated by STORM or ultrastructural analysis either.In summary, it should be possible to perform some classical EM to, at minimum, to investigate whether in knockout cells lacking DAPs sub distal-like structures form at the distal ends of centrioles. If the models and cartoons cannot be proven by EM, then it would be essential to state very clearly that these models and cartoons are speculative.2) Figure 4A-C. The authors show that after Cep128 and Cep83 knockout ODF2 STORM signal changes, losing its proximal portion after Cep128 removal. The same seems to occur after Cep83 depletion but it is not clear from Figure 4D which portion of ODF2 is lost after Cep83 depletion. However, based on 4B and C, there is a ~50 nm gap between Sclt-1 and ODF2 protein signals in Cep128-/- cells. Also, the brightest portion of the ODF2 signal is shifted for additional ~50 nm toward centriole proximal end. This is not consistent with the authors' interpretation of the data. Since it has been previously shown that ODF2 interacts with Cep128 via its N terminus which extends further away from centrioles, one interpretation why ODF2 signal changes in Cep128-/- cells could be that without its natural binding partner, it changes its configuration from more to less extended, changing the shape of the STORM signal.3) The quality of some STORM images raises concerns. For instance, even a well characterized distal appendage protein SCLT-1, which is known to reproducibly localizes to nine discrete foci here on longitudinally analyzed centrioles shows a highly variable and irreproducible pattern. This raises a question about the reproducibility and reliability of other STORM signals, specifically in Figure 4G-H and 6C where Ninein and Γ-tubulin signals are very variable from one centriole to another. CP110 signal in Figure 4G is also of a poor quality, showing nonspecific signals. It is not clear how reliably it detects centriole distal ends. RPE-1 cells used in this study are supposed to be serum starved and, based on published data, majority of cells should have a cilium and should not have CP110 at centrioles.Related to this: The authors use 24hours of FBS to synchronize cells in G0 and allow for, or not, cilia formation. Do the authors know how robust the G0 arrest and what % ciliation they achieve? It is unclear from looking at the data if they are imaging ciliated cells or not. More robust cell cycle arrest protocols could have been used or an additional cilia marker incorporated in their analysis to ensure differences observed in the +/- FBS conditions represents cell cycle modulation or the presence or not of a primary cilium.4) Introduction: The authors claim that no clear EM image of SDAPs has been reported for human RPE-1 cells. This statement seems inaccurate, considering that there is a plethora of electron micrographs showing both sets of appendages in this cell line . It thus appears that SDAPs of RPE-1 cells are documented across literature appear to be present in variable number, that they can adopt various morphology (based on electron densities) and that they are associated with microtubules at their ends.eLife. Your revised article has been evaluated by Anna Akhmanova (Senior Editor) and two reviewers.Thank you for resubmitting your work entitled "Super-resolution microscopy reveals coupling between mammalian centriole subdistal appendages and distal appendages" for further consideration by The manuscript has been improved but there are some remaining issues raised by reviewer 1 that need to be addressed before acceptance, as outlined below. Addressing these comments should not require any new experiments, but please take the various concerns and suggestions seriously and modify the paper accordingly. Please provide a point-by-point rebuttal explaining the changes that you have made.Reviewer #1:In the revised manuscript, the biggest issue remains the interpretation of the data and the model.The authors have performed TEM analysis to understand the organization of subdistal appendages in starved RPE-1 cells . They concluded that the TEM analysis supports the nine-ness in the organization of sDAS. However, there are several issues with this interpretation and, consequently, with the model.Subsection 'ODF2 is close to the centriole microtubule wall whereas ninein and CEP170 are close to the sDAP tips' states that "The TEM images reveal that sDAP backbone can be mostly observed in the same section with a nine-fold arrangement". It appears that this conclusion was derived from only one cross-sectioned centriole (which is not adequate). The number of centrioles analyzed in cross section is not indicated But I saw only one analyzed centriole with eight instead of nine distinguishable sDA densities . So, in my view, presented electron micrograph clashes with author's interpretation. It demonstrates the lack of heterogeneity in the appearance of SDA in EM and questions the nine-fold arrangement of sDAs,The authors explained  that they used two studies to model SDAs. These are: Anderson et al., (PMID: 5064817), a study performed in cells from Rhesus monkey oviduct, and Paintrand et al., (PMID: 1486002), a study conducted on isolated centrioles from lymphoblastoid cell line.Anderson study explores basal bodies which sport only one basal foot positioned in the middle of the basal body longitudinal axes. Moreover, the authors say that the "structure at the longitudinal position proximal to the DAPs is the basal foot, a 'badminton-shaped' structure largely different from the sDAPs in the mother centriole". It is, therefore, confusing how can Anderson model was used to model SDAs in RPE-1.Paintrand study, another work used to model SDAs, describes centrioles isolated from lymphoblastoid cells. Since during centriole isolation the heads of SDAs are frequently lost, their conical appearance may as well be perturbed So, again, it is not clear how SDAs from this study can be taken as a benchmark for modeling od sDAs in intact cells. It is not obvious whether authors realize that most centriole electrographs presented in Paintrand study were additionally digitally modified  with the purpose to highlight specific discussion points. Such averaging will inevitably result in the appearance of nine subdistal appendages. Centrioles from the same study that were not rotationally averaged clearly show a variable number of sDAs and their more conical shape of sDA's densities Therefore, the following interpretation of sDA morphology in the introduction paragraph needs to be changed. After all, author's own EM data is not in agreement with their own description.Introduction: "When sDAPs do form a complete ring of a nine-fold symmetry, its symmetric pattern is different from that of DAPs , with one pairs of parallel electron-dense "spokes" in each arm, where one of the spokes is associated with the A-tubule of a MT triplet of the mother centriole and the other is associated with the C-tubule of an adjacent MT triplet...". It is also unclear why was Uzbekov and Alieva cited here, the centriole in question is taken over from Paintrand study.EM analysis of SDAs of longitudinally positioned centrioles is now included in the manuscript . It reveals variability in the shape of sDAs. All six appendages in wild type RPE cells are different in their morphology  and they don't point in the same direction. It would be useful to accordingly adjust the interpretation in subsection 'Ninein covers a broadened longitudinal region toward the centriole distal end upon DAP removal'.EM analysis of sDAs in Cep83-/- cells has revealed centrioles with somewhat uneven length and with sDAs positioned at various distances from both centriole's ends. Fewer sDA EM densities are also present in Cep83-\- cells. How this asymmetry is not reflected in the radial distribution of STORM Ninein signal, which remained more-less symmetrical organized around mother centriole? It is surprising that, given this revelation, the analysis of additional sDAPs has not been added to the analysis.Introduction: "Loncarek group further used correlative super-resolution microscopy and EM to show precise localization of DAP proteins relative to the electron dense blade structure , improving the architectural mapping of the DAPs". Please note that Bowler at al. used tomography to elucidate the organization of DA's electron densities, which argued against the notion that DAs are organized as blades.The text says: "To understand the architecture of protein complexes at their mature stage in terms of structural occupancy, we further analyzed super-resolution images of those with nearly full ring occupancies for each sDAP protein.What does it mean their mature stage? Were centrioles somehow pre-selected for imaging based on whether they saw a ring or not by wide field microscopy? Structural occupancy is unclear. I think that I understand the meaning, but the sentence could be re-phrased.Arbitrary terminology seems to be used to describe the morphology of sDAs . It is confusing. sDA's parts are usually described as a head and a stem. At least these terms need to be defined.Further proofreading is necessary. Some sentences are illogical; it is hard to understand what they meant. For instance: The title of the Figure 6 says: "γ tubulins around mother centriole do not nucleate MT anchoring at the sDAPs in the G0 phase". What does it mean that anchoring is not nucleated? The abbreviation "sDAP" appears erroneously used on some places. "sDAP protein" and "DAP protein" is also used, although "P" already stands for "protein"... Essential revisions:1) The most important problem with this work is that conclusions derived from STORM-based analyses are not corroborated by ultrastructural analysis.Specifically, Figure 3 shows a 3D model of SDAPs. The model shows nine subdistal appendages. Since SDAPs occupy more than one microtubule triplet, the model presented in Figure 3 might not reflect the actual organization of SDAPs in RPE-1 cells. In addition, it is not obvious how this model was deduced from presented STORM data, which itself did not reveal a nine-fold distribution of most SDAP proteins. The authors will either need prove their model by performing EM analysis, or clarify that this is a speculative model. As it is now, it may suggest the wrong concept of SDAP architecture in mammalian cells. Please see PMID: 30045886, a recent review on this subject.Originally we used TEM images of centrioles from previous studies (PMID: 5064817 by Anderson and PMID: 1486002 by Bornens' team) to deduce the architecture of the centriole and sDAP backbone as well as our dSTORM data to deduce the localization of sDAP proteins. Here to confirm the organization of sDAP backbone in RPE-1 cells, we have followsfollows the reviewer's suggestion and performed serial TEM sectioning of RPE-1 centrioles in the axial view, shown in Figure 1I. The TEM data reveals that sDAP backbone can be mostly observed in the same section with a nine-fold arrangement. It is also interesting to note that the sDAP arms are mostly wider than those of DAPs, similar to the observation recorded in Bornens' paper. Therefore, in our model, we constructed a nine-fold distribution of sDAPs. We have added this figure to the main text to support our model . From the EM image, it seems that the arms of sDAPs are more flexible in shape and distribution than those of DAPs, potentially reflecting why the nine-fold symmetry is less obvious for the localization of sDAP proteins. We have stated the structural characteristics of the sDAP EM image as discussed above in the main text.Our 3D model was constructed by combining measurements from previous studies, our current TEM results, and our dSTORM data, aiming to provide a view of the potential sDAP structure. Nonetheless, we also cannot rule out that sDAPs in RPE-1 cells may also be a dynamic structure as reported in PMID: 30045886, and therefore this sDAP structure may possibly reflect the organization of sDAPs at a subset of centrioles. We have thus edited the manuscript from subsection 'Axial and lateral images compose a 3D molecular map of sDA' to clarify this.Further, in Figure 4I, which summarizes the localization changes of SDAP proteins in Cep128-/- and Cep83-/- cells, some type of appendage is drawn at the distal centriole end. Do authors mean to say that in the absence of distal appendages, subdistal appendages form at the centriole's very distal ends? If so, it would be critical to perform an EM analysis of wild type and knockout centrioles and to show whether there are any structural changes associated with centriole distal ends. Otherwise, it needs to be clear that the model is speculative. The cartoon in Figure 6G is not corroborated by STORM or ultrastructural analysis either.In summary, it should be possible to perform some classical EM to, at minimum, to investigate whether in knockout cells lacking DAPs sub distal-like structures form at the distal ends of centrioles. If the models and cartoons cannot be proven by EM, then it would be essential to state very clearly that these models and cartoons are speculative.-/- RPE-1 cells, as shown in Figure 4J. In wild type cells, sDAPs  mostly maintain their longitudinal position, with the triangular arms pointing toward the direction close to perpendicular to the centriole orientation . Surprisingly, we found that the positions and shapes of the arms of sDAPs are largely varying in CEP83 depleted cells, showing with more examples to illustrate the variations . That is, the sDAP structure is much less stable when DAPs are missing. Specifically, a few examples show that sDAP tips may point toward the distal end with a thicker arm, consistent with what we observed in ninein super-resolution images of CEP83-/- cells. This is in line with the previous TEM study of CEP164 knockdown RPE-1 centrioles (PMID: 23253480), where EM images show that sDAPs are titled toward the distal end. In other cases, sDAPs can localize at the middle region of the centriole, occupy the same side of the centriole, miss one side of occupancy, or localize at different longitudinal positions in the same centriole. These large variations imply that the loss of DAPs via CEP83 depletion affects the structural stability of sDAPs, reassuring that DAPs and sDAPs are structurally related to each other.Thanks for the reviewer's comment. We have followsfollows the suggestion and performed TEM to examine the differences of sDAP structures between wild type and CEP83-/- centrioles to demonstrate possible structural variations .In accordance with these TEM results, we have revised our cartoon model for CEP832) Figure 4A-C. The authors show that after Cep128 and Cep83 knockout ODF2 STORM signal changes, losing its proximal portion after Cep128 removal. The same seems to occur after Cep83 depletion but it is not clear from Figure 4D which portion of ODF2 is lost after Cep83 depletion. However, based on 4B and C, there is a ~50 nm gap between Sclt-1 and ODF2 protein signals in Cep128-/- cells. Also, the brightest portion of the ODF2 signal is shifted for additional ~50 nm toward centriole proximal end. This is not consistent with the authors' interpretation of the data. Since it has been previously shown that ODF2 interacts with Cep128 via its N terminus which extends further away from centrioles, one interpretation why ODF2 signal changes in Cep128-/- cells could be that without its natural binding partner, it changes its configuration from more to less extended, changing the shape of the STORM signal.-/- cells is larger than that of the wild type cells, illustrating both narrowing and shifting of ODF2 occupancy upon sDAP CEP128 depletion. These observations imply that CEP128, as the binding partner of ODF2, regulates the organization of ODF2. We have added these results to Figure 4B-4D in the main text . It remains to be examined what exactly the thinner layer of ODF2 in CEP128-/- cells belongs to. One speculation is that the sDAP depletion results in the removal of ODF2 in the sDAP layer. We have stated clearly that this is only a speculation in the main text.We thank the reviewer for providing us insight into the potential change of ODF2 configuration upon CEP128 depletion. To further examine the idea, we used two different antibodies targeting both the N terminus and C terminus of ODF2 to examine their distribution differences in wild type and CEP128 depleted cells. The ODF2 antibody  we used in the manuscript targets the N terminus of ODF2, i.e. aa #39-200, out of its 829 amino acids, referred to as ODF2-N. We used another commercial antibody  that targets the C terminus of ODF2 (aa #800 to the C terminus), referred to as ODF2-C. dSTORM imaging of ODF2-C reveals that in wild type cells, ODF2-C distribution is wider than ODF2-N distribution in the radial direction . In addition, ODF2-N extends more toward the proximal end than ODF2-C in the longitudinal direction, consistent with the CEP128 interaction result in PMID30623524. The distal edge of ODF2-C is closer to SCLT1 than that of ODF2-N, more reaching the centriole distal end. Interestingly, when CEP128 is depleted, distributions of both ODF2-N and ODF2-C become thinner. The gap between ODF2-N/C and SCLT1 of CEP1283) The quality of some STORM images raises concerns. For instance, even a well-characterized distal appendage protein SCLT-1, which is known to reproducibly localizes to nine discrete foci here on longitudinally analyzed centrioles shows a highly variable and irreproducible pattern. This raises a question about the reproducibility and reliability of other STORM signals, specifically in Figure 4G-H and 6C where Ninein and Γ-tubulin signals are very variable from one centriole to another. CP110 signal in Figure 4G is also of a poor quality, showing nonspecific signals. It is not clear how reliably it detects centriole distal ends. RPE-1 cells used in this study are supposed to be serum starved and, based on published data, majority of cells should have a cilium and should not have CP110 at centrioles.In dual color dSTORM imaging, we used Alexa Fluor 647 (AF647) and Cy3b dyes for immunostaining. Out of the two dyes, AF647 performs better than Cy3b in terms of its blinking property, which provides images with a more homogeneous intensity. Therefore, we always mark our protein of interest with AF647 and use Cy3b as the reference marker. The aim of Figure 6B was to measure the diameter of γ-tubulin. Therefore, in Figure 6B, γ-tubulin was marked by AF647 dye while SCLT1 was used as a reference for the axial orientation. To illustrate the difference, here we have also imaged the SCLT1/γ-tubulin pair in a reverse manner, i.e. marking SCLT1 with AF647 and γ-tubulin with Cy3b as shown in With regard to the imaging of CP110, we understand that serum starvation triggers ciliogenesis and CP110 should be absent on ciliated centrioles. We chose to use 24-hour starvation on purpose because it will give us ~60% ciliated cells  with ninein at the sDAP region perpendicular to the centriole, whereas CP110 on a mother centriole localizes at the edge of the centriole 'rod' close to ninein at sDAP region  Introduction: The authors claim that no clear EM image of SDAPs has been reported for human RPE-1 cells. This statement seems inaccurate, considering that there is a plethora of electron micrographs showing both sets of appendages in this cell line . It thus appears that SDAPs of RPE-1 cells are documented across literature appear to be present in variable number, that they can adopt various morphology (based on electron densities) and that they are associated with microtubules at their ends.We thank the reviewer for the advice. We have removed the sentence 'no clear EM image of SDAPs has been reported for human RPE-1 cells' in the Introduction to avoid confusion. The list of articles suggested by the reviewer includes TEM images of PRE-1 centriole in the lateral view upon CEP120 knockout (PMID: 30988386), CEP164 knockdown (PMID: 23253480), WDR8 knockdown (PMID: 26675238), EHD1 knockdown (PMID: 25686250) and NEDD1 overexpression (PMID: 26880200). CEP164 is a DAP protein among these proteins. Interestingly, from the siCEP164 EM images (PMID: 23253480), we also observed tilted sDAP structure similar to that of our CEP83 depleted RPE-1 centrioles . This data echoes with our observation that loss of DAP affects sDAP stability, suggesting a structural relationship between them.Reviewer #1:In the revised manuscript, the biggest issue remains the interpretation of the data and the model.The authors have performed TEM analysis to understand the organization of subdistal appendages in starved RPE-1 cells . They concluded that the TEM analysis supports the nine-ness in the organization of sDAS. However, there are several issues with this interpretation and, consequently, with the model.Subsection 'ODF2 is close to the centriole microtubule wall whereas ninein and CEP170 are close to the sDAP tips' states that "The TEM images reveal that sDAP backbone can be mostly observed in the same section with a nine-fold arrangement". It appears that this conclusion was derived from only one cross-sectioned centriole (which is not adequate). The number of centrioles analyzed in cross section is not indicated but I saw only one analyzed centriole with eight instead of nine distinguishable sDA densities . So, in my view, presented electron micrograph clashes with author's interpretation. It demonstrates the lack of heterogeneity in the appearance of SDA in EM and questions the nine-fold arrangement of sDAs,We thank the reviewer for the comments. The serial cross-sectioned TEM images in Figure 1I are from one centriole  that they used two studies to model SDAs. These are: Anderson et al., (PMID: 5064817), a study performed in cells from Rhesus monkey oviduct, and Paintrand et al., (PMID: 1486002), a study conducted on isolated centrioles from lymphoblastoid cell line.Anderson study explores basal bodies which sport only one basal foot positioned in the middle of the basal body longitudinal axes. Moreover, the authors say that the "structure at the longitudinal position proximal to the DAPs is the basal foot, a 'badminton-shaped' structure largely different from the sDAPs in the mother centriole". It is, therefore, confusing how can Anderson model was used to model SDAs in RPE-1.Paintrand study, another work used to model SDAs, describes centrioles isolated from lymphoblastoid cells. Since during centriole isolation the heads of SDAs are frequently lost, their conical appearance may as well be perturbed So, again, it is not clear how SDAs from this study can be taken as a benchmark for modeling od sDAs in intact cells. It is not obvious whether authors realize that most centriole electrographs presented in Paintrand study were additionally digitally modified  with the purpose to highlight specific discussion points. Such averaging will inevitably result in the appearance of nine subdistal appendages. Centrioles from the same study that were not rotationally averaged clearly show a variable number of sDAs and their more conical shape of sDA's densities Therefore, the following interpretation of sDA morphology in the introduction paragraph needs to be changed. After all, author's own EM data is not in agreement with their own description.We thank the reviewer for the comments. The two models from Anderson's TEM study and Paintrand's study, which we used as references for our 3D centriole model, were actually used to construct (1) the centriole wall with the distal appendages and (2) the subdistal appendages, respectively. The same as we mentioned above, we agree with the reviewer that the number of sDAP varies as shown in Figure 9 of Paintrand's paper Figure , in which a range of around 3-9 sDAPs can be observed. We also realize that some of the images in Paintrand's study were rotationally averaged. However, the presence of nine sDAP stems was also observed in a few of the non-averaged images as shown in Figure 3 Figure and Figure 9 from Paintrand's work. Again, we do not intend to emphasize a consistent nine-fold arrangement of sDAP. Our model represents one of the potential arrangements existing in a subset of sDAPs, as we clarified in subsection 'Axial and lateral images compose a 3D molecular map of sDAPs'. The main goal of constructing a 3D model is to provide a schematic view on the potential positioning of sDAP proteins on the mother centriole.To avoid confusion, we have edited the sentences to further clarify the construction of our 3D model and emphasize the dynamic nature of sDAP structure starting from subsection 'Axial and lateral images compose a 3D molecular map of sDAPs' as follows: "The model of the centriole and the DAPs are based on previous TEM serial sections of the monkey oviduct basal body ; the sDAP model is constructed based on the TEM study of centrioles from a human lymphoblastoma cell line  and our sDAP TEM results . Note that because the structures of sDAPs are dynamic , this model only represents one possible organization of sDAPs in a subset of centrioles, such that other settings may also exist."Introduction: " When sDAPs do form a complete ring of a nine-fold symmetry, its symmetric pattern is different from that of DAPs , with one pairs of parallel electron-dense "spokes" in each arm, where one of the spokes is associated with the A-tubule of a MT triplet of the mother centriole and the other is associated with the C-tubule of an adjacent MT triplet...". It is also unclear why was Uzbekov and Alieva cited here, the centriole in question is taken over from Paintrand study.With regard to the reference, the reason we cite the article  is because it is the first comprehensive review paper comparing and summarizing the difference between sDAPs and DAPs, supporting our statement that the symmetric pattern of sDAPs is different from that of DAPs. We have also included Paintrand work as a reference for nine-fold symmetric morphology of the sDAPs.To clarify, we have edited the Introduction as follows:"Some EM images showed variations in the number of sDAPs, such as 2 to 12 sDAP stems in human endotheliocytes , illustrating the dynamic nature of sDAPs. That is, in contrast to the exact number of nine DAPs per centriole, the number of sDAPs may be different in different centrioles . Even when sDAPs do form a complete ring of a nine-fold symmetry, its morphology is different from that of DAPs . Each sDAP stem is composed of one pair of electron-dense signals on the sides, where one of them is associated with the A-tubule of a MT triplet of the mother centriole and the other is associated with the C-tubule of an adjacent MT triplet ."EM analysis of SDAs of longitudinally positioned centrioles is now included in the manuscript . It reveals variability in the shape of sDAs. All six appendages in wild type RPE cells are different in their morphology  and they don't point in the same direction. It would be useful to accordingly adjust the interpretation in subsection 'Ninein covers a broadened longitudinal region toward the centriole distal end upon DAP removal'.We thank the reviewer's suggestion and have changed the sentence in subsection 'Ninein covers a broadened longitudinal region toward the centriole distal end upon DAP removal' as follows:"In wild type cells, sDAPs mostly maintained their longitudinal position close to and proximal to the DAPs, but their shapes varied in different centrioles or even in the same centriole."EM analysis of sDAs in Cep83-/- cells has revealed centrioles with somewhat uneven length and with sDAs positioned at various distances from both centriole's ends. Fewer sDA EM densities are also present in Cep83-\- cells. How this asymmetry is not reflected in the radial distribution of STORM Ninein signal, which remained more-less symmetrical organized around mother centriole? It is surprising that, given this revelation, the analysis of additional sDAPs has not been added to the analysis.-/- cells with our dSTORM images for longitudinal distribution of ninein , we agree with the reviewer that the ninein dSTORM signal does not show a high level of asymmetry. This is related to our centriole pre-selection process prior to dSTORM imaging. Since the DAP proteins are absent in CEP83-/- cells, SCLT1 or any other large diameter DAP protein cannot be used as a marker for the centriole orientation. Therefore, when we imaged ninein in CEP83-/- cells, ninein itself was also used as an orientation marker. In our experiment, we co-stained CP110, ninein and centrin in CEP83-/- cells as shown in Figure -/- cells that lack one of the electron density signals at sDAPs) would not be selected because it was challenging to assure that these centrioles were parallel to the imaging plane and thus to image them from a lateral view point. The ninein selected for dSTORM imaging would be the one with both left and right signals. Indeed, in the dSTORM imaging, the ninein signal is less asymmetric , but their distribution pattern is comparable to one of the EM images in CEP83-/- cells  in which sDAPs are present on both sides and occupy a wider longitudinal space than those of the wild type cells. Thus, our ninein images reflect its distribution in a subset of centrioles, and this centriole pre-selection process should be the reason why the left-right asymmetry is less obvious in dSTORM images of ninein. To clarify this, we have added a sentence in subsection 'Ninein covers a broadened longitudinal region toward the centriole distal end upon DAP removal' as follows: "Note that laterally oriented centrioles were preselected by the pattern of centrin and ninein under widefield imaging prior to dSTORM imaging; therefore, unlike the asymmetry of sDAP stems observed in the EM images of CEP83-/- cells , asymmetry of ninein is less obvious in the ninein super-resolution images of CEP83-/- cells ."Comparing our EM analysis of Cep83Introduction: "Loncarek group further used correlative super-resolution microscopy and EM to show precise localization of DAP proteins relative to the electron dense blade structure , improving the architectural mapping of the DAPs". Please note that Bowler at al. used tomography to elucidate the organization of DA's electron densities, which argued against the notion that DAs are organized as blades.We thank the reviewer's comment. Bowler's paper suggested that each DAP is anchored to the centriole by a triangle shaped base with their centriole EM images, and indeed only stated that their finding of the triangle shaped base "gives a perception of a 'blade'". To avoid confusion, we changed the sentence in the Introduction as follows: "Loncarek group further used correlative super-resolution microscopy and EM to show precise localization of DAP proteins relative to the electron dense structure of DAPs , improving the architectural mapping of the DAPs."The text says: "To understand the architecture of protein complexes at their mature stage in terms of structural occupancy, we further analyzed super-resolution images of those with nearly full ring occupancies for each sDAP protein.What does it mean their mature stage? Were centrioles somehow pre-selected for imaging based on whether they saw a ring or not by wide field microscopy? Structural occupancy is unclear. I think that I understand the meaning, but the sentence could be re-phrased.When we mapped the radial distribution of sDAP proteins, we purposely searched for the ring-like structure of sDAP proteins by widefield microscopy prior to dSTORM imaging. That is, in the reviewer's word, a subset of centrioles was pre-selected based on whether we saw a ring or not by widefield microscopy. Our goal was to understand the radial arrangement of sDAP proteins when they formed a ring-like structure. The term 'mature stage' was used to define such centrioles. To clarify, we have rephrased the sentences in subsection 'ODF2 is close to the centriole microtubule wall whereas ninein and CEP170 are close to the sDAP tips' as follows."To understand the architecture of sDAP proteins with nearly full occupancies at the sDAPs, we pre-selected centrioles based on whether we saw a ring or not for an sDAP protein of interest by widefield microscopy and further analyzed them with super-resolution microscopy."Arbitrary terminology seems to be used to describe the morphology of sDAs . It is confusing. sDA's parts are usually described as a head and a stem. At least these terms need to be defined.We used 'DAP arm' as in the Boren's paper  to represent a single sDAP stem, 'DAP backbone' to describe the entire sDAP structure on the mother centriole and 'spokes' to refer to the electron dense signal on each side of a sDAP stem. To avoid confusion, we have adapted the term 'sDAP stem(s)' for sDAP arm or backbone and removed the term 'spokes'.The term 'root' means the portion of a sDAP stem which connects the centriole wall. We have further clarified its definition when it first appeared in subsection 'Lateral super-resolution images reveal sDAP as a triangular structure'.Further proofreading is necessary. Some sentences are illogical; it is hard to understand what they meant. For instance: The title of the Figure 6 says: "γ tubulins around mother centriole do not nucleate MT anchoring at the sDAPs in the G0 phase". What does it mean that anchoring is not nucleated?We thank the reviewer for the reminder. To avoid confusion, we have corrected the title of Figure 6 to "A majority of γ-tubulins around the mother centriole are not associated with MT anchoring at sDAPs in the G0 phase". We have also proofread the entire manuscript carefully and made several changes in the manuscript.The abbreviation "sDAP" appears erroneously used on some places. "sDAP protein" and "DAP protein" is also used, although "P" already stands for "protein"...We also noticed that various short forms exist in the literature for distal appendages  and subdistal appendages . In our work, we used sDAP and DAP as the abbreviation for subdistal appendage and distal appendage, respectively, following papers such as PMID: 24882706, PMID: 24231678 and PMID: 29514088. These terms are defined when they first appear in the Introduction.


Our aim was to develop and evaluate a German adaptation of myfood24, a fully automated, web-based 24-h dietary recall (24HDR). To complete a self-administered 24HDR with myfood24, users have to search and enter consumed foods within the underlying database by a free text search. The adaptation process thus mainly consisted of the development of an appropriate food database. myfood24-Germany was evaluated in 92 adults aged 17–78 years (study 1). Participants completed four non-consecutive 24HDRs and answered an evaluation questionnaire after the final recall. The System Usability Scale Score  was calculated. Users’ search behavior was examined with screen recordings in 15 adults aged 20–60 years (study 2). Participants had to enter three sample meals presented as food packaging or pictures. The final database included 11,501 food items (7203 generic and 4298 branded items) with up to 131 nutrients. In study 1, the median completion time for a 24HDR was 15 min. The median SUS score of 78 indicated good usability. The majority of participants considered the overall user-friendliness as good (46%) or very good (21%), and 75% were willing to use myfood24-Germany regularly. Both studies showed that finding and choosing an appropriate item within the database was a major challenge. A German version of myfood24 was successfully developed. The user evaluation indicated a short completion time, good usability and acceptability of the tool, and confirmed its feasibility for repeated short-term application. The repeated application of short-term dietary assessment instruments such as 24-h dietary recalls (24HDRs) has been recommended to accurately estimate the usual dietary intake in large-scale studies ,2,3. TheMyfood24 is a fully automated online dietary assessment tool, which was first developed for use in the UK population . It can To our knowledge, so far there is no web-based 24HDR available for use in the German population. Therefore, we aimed to develop a German adaptation of myfood24 and evaluate its feasibility and usability in a German study sample.The adaptation process of myfood24 for use in German populations included three major steps: (1) the compilation of a German food composition database suitable for self-administered use; (2) its formatting according to the predefined myfood24 database structure; and (3) the translation of all implemented text modules to the German language. The compilation process was conducted by a team of experienced researchers in nutritional epidemiology with knowledge of German consumption habits. All translations were double-checked.The myfood24-Germany database was built from two German food composition databases—the German Food Code and Nutrient Data Base (Bundeslebensmittelschlüssel (BLS) version 3.02) and the in-house database of the Dortmund Nutritional and Anthropometric Longitudinally Designed (DONALD) study LEBTAB ,13,14. TAs the BLS includes generic food items only, selected branded food items from LEBTAB  were addTo be able to use the myfood24 features, further formatting steps were necessary. myfood24 includes prompts for often forgotten food items. Therefore, common accompaniments to foods were added to certain items . Second, a number of synonyms  and potential misspellings  were added. Third, filters were formatted by assigning information on type (branded vs. generic) and on food category  to each food item. Finally, information on average portion sizes and household measures for myfood24-Germany were obtained from two scientific German publications. A nutrition table published by Wahrburg and Egert  and the The evaluation study of myfood24-Germany was embedded in the validation study of the tool . Participants were recruited at the campus of the University of Bonn by oral advertisement and flyers as well as in the general German population in and around the city of Bonn by social media and press releases. Eligible subjects had to be ≥18 years old, fluent in German, and had to have regular high-speed Internet access and a valid email address. Further, they had to be stable in bodyweight , free from metabolic diseases, and willing to maintain their current dietary and activity behavior during the time of the study. On a first study visit, individuals were screened for eligibility and informed about the study procedures. During the study, participants were asked to keep a three-day weighed FR and completed four 24HDRs with myfood24-Germany. The first 24HDR was completed in the study center where participants received brief instructions in using myfood24-Germany by study staff. The introduction included a short explanation of the main features  for all participants before they started the recall. A researcher was present while participants completed the recall and participants were allowed to ask questions on functionalities and to ask for help when any problems occurred. The following three 24HDRs were completed at home. After the final recall, participants were asked to answer an online evaluation questionnaire.Data on usability and acceptability of myfood24-Germany was collected by an online evaluation questionnaire including 68 questions on six different categories . Usability was assessed by the System Usability Scale (SUS), which is a common method for evaluating digital technologies, conceived by Brooke et al. ,23,24. IIn order to assess the acceptance of myfood24-Germany, study participants were asked whether they would consider using myfood24 regularly (yes/no) and, if yes, in what time intervals they would use it .Further, participants were asked about technical details , technical problems , their subjective opinion on the user-interface and about the application of the search function, and other features in myfood24 Germany. Finally, participants were able to give free text comments and to make overall suggestions at the end of the questionnaire.Demographic characteristics and information on lifestyle factors were assessed by an online questionnaire that was filled in by participants in the study center. Bodyweight was measured to the nearest 100 g using a calibrated electronic scale (Kern MPC 250K100M) with participants dressed in underwear only. Standing height was measured to the nearest of 0, 1 cm using a stadiometer (seca 217). Body Mass Index (BMI) was calculated as the bodyweight (kg) divided by the square of the body height (m).®) was used to identify characteristics of participants that were potential predictors for the SUS score or the average completion time for a 24HDR with myfood24-Germany. The following variables were tested: sex , age (years), BMI (kg/m2), education level , nutritional education/profession ?” yes/no), experience in completing 24HDRs , experience in using computer (“Do you work with a computer/laptop regularly (>5×/week)?” yes/no), type of diet (“Are you a vegetarian or vegan?” no/yes vegetarian/yes vegan), smoker (yes/no)). The significance level was set at p < 0.05. The completion time for a 24HDR was determined by calculating the minutes between the addition of the first and the last item in myfood24-Germany. Of note, participants were free to postpone the completion of their 24HDR and finalize it at a later time point. Therefore, for all 24HDRs completed at home, outliers (>1.5× IQR) in completion time were manually checked by reviewing the automatic myfood24-Germany nutrient data output (n = 27). As a result, recalls with time intervals of >60 min between the entry of one item to the next were excluded for analysis of the completion time (n = 5).Since only a few variables were normally distributed, descriptive data are presented as medians with their lower- (Q1) and upper-quartile (Q3) for continuous variables or as frequencies and percentages for categorical variables. A backward selection procedure  were not specifically available in the myfood24-Germany database so that participants had to find a suitable equivalent . Participants received written instructions and had to enter the items without any further help. The entering process was documented with the screen recorder software Apowersoft Free [To examine users’ search behavior in myfood24-Germany, a convenience sample of students and employees was recruited by oral advertisement and flyers at the University of Bonn (independent of study 1). Subjects had to be ≥18 years old and fluent in German. Nutrition professionals and students of Nutritional Sciences were excluded. Written informed consent from all participants was obtained before enrolment. At the study visit, participants were requested to enter three sample meals  in myfood24-Germany . The meaoft Free . DemograTo investigate users’ search behavior, screen videos were analyzed . First, the number of entered search terms before an item was selected was counted. Second, it was examined overall and stratified by meal, whether participants searched a food item by product name and/or brand name. Finally, it was analyzed how often participants omitted an item  instead of choosing an equivalent product, and how often a food item from the table was forgotten . To investigate the impact of individual search behaviors on hypothetical energy and nutrient intakes, mean energy and nutrient intake according to the automatic myfood24-Germany output were compared to the nutrient reference values from the packaging for branded food items or to nutrient information from the BLS for generic products . The mean of absolute individual differences was calculated.From 18 recruited participants, 15 entered the sample meals following the study protocol . For anaA German version of myfood24 including features and functionalities of the original UK version was successfully developed. The current version of the underlying food composition database comprises data from the BLS and LEBTAB . ResearcFrom 97 study participants, 92 completed the evaluation questionnaire . All of p = 0.04). Median SUS score was lower in women, representing the majority of study participants (median SUS score (Q1–Q3) women vs. men: 75 (65–83) vs. 83 (70–90)). The overall SUS score did not change when nutritionists (n = 19) and participants with experience in completing a 24HDR (n = 10) were excluded from the calculation  = 78 (68–83)). In accordance with the good SUS score, the majority of participants considered the overall user-friendliness of myfood24-Germany as good (46%) or very good (21%) (od (21%) . Seventyp < 0.0001) was a potential predictor for the individual mean completion time in the examined sample. The completion time did not change after the exclusion of nutritionists and participants with experience in conducting 24HDRs  = 15 (8–23)). In concordance with the actual time requirement, the self-reported median completion time was 15 min (Q1–Q3: 10–20).The calculated median time was between the addition of the first and the last food item, and thus the median time it took participants to complete a 24HDR with myfood24-Germany was 15 min (Q1–Q3: 9–23). However, in some cases, >60 min passed from the entry of one food item to the next, suggesting that participants took a break during the entering process. Time requirement increased with age . Backwarn = 1), that the correct portion size could not be entered (n = 1) and that no confirmation was received after the completion of the recall (n = 2).Ninety percent of participants used a computer or laptop to conduct the 24HDRs and three percent used tablets. Although participants were informed that myfood24 is an online tool, which at the time of study had not been optimized for smartphone application, six participants (7%) reported completing the 24HDRs on a smartphone. All 92 participants agreed that they were able to load the website and 95% agreed that no technical problems occurred during the application. Technical problems described included that food items could not be entered because they have not been displayed properly (The majority of participants agreed that the website is easy to navigate (97%) and the layout is appealing (88%). Only three participants stated that they had difficulties with the application of myfood24. The difficulties described by these participants mostly concerned the search for food items. The search function in myfood24 was rated as easy to use by 79% of participants and 84% stated that they were able to find food items quickly. Although 71% of participants agreed that they could not find all the foods they consumed, the majority of these participants (74%) agreed that they found appropriate alternatives for the respective food item. Nevertheless, slightly more than half of all participants (53%) stated that they had difficulties in finding an appropriate food item. Mostly, participants described that the database was incomplete, that there should be more basic food items to choose from, better possibilities to enter out-of-home consumption, and the opportunity to add food items that are apparently missing in the database. Further, the alphabetically sorted display and the high number of search results was criticized and a lack of synonyms and (miss)spellings was mentioned. Some participants stated that they would prefer using myfood24-Germany on a smartphone.The available portion images and portion sizes were found to be helpful by the vast majority of participants . Some participants mentioned that creating recipes in the recipe builder was very laborious. Although myfood24-Germany features were briefly explained to participants during the first recall, some participants mentioned that they did not recognize many of the features at all or only after repeated usage. Features in myfood24 used and considered helpful by the users can be seen in Characteristics of participants in study 2 are shown in n = 44 of n = 238 possible searches given by n = 17 products × n = 14 participants), participants tried to find the product by further entering the brand name or using the filter option for brands. A sensitivity analysis stratified by meals showed that the entered search terms at the beginning of the entering process included the brand name more often than at the end (breakfast: 31% (30 of 98); lunch: 19% (13 of 70); dinner: 9% (6 of 70); data not shown). When searching for a branded product not identically available in the database, participants either chose a related generic item (69%), another item of the same brand (8%), an equal or similar item from another brand (19%), or they did not choose any item at all (3%). In the remaining cases (1%), the participants forgot to enter the presented food items. When searching for an available branded item (n = 2), participants chose the appropriate product in 50% of the cases. For the remaining cases, a related generic item (47%) or a similar product from another brand (3%) was selected.On average, participants entered two search terms before they selected an item  (data not shown). Most participants searched for branded food items by entering the generic descriptions only. Overall, in 21% of the searches for branded items  [n = 20) [n = 4 ≥ 65 years old) compared to younger age groups [n = 299 aged 60–85 years) [The SUS score had been developed as a means to measure the overall perceived usability of a system . Althougn = 20)  and othe[n = 20) . Moreovee groups  and the e groups . The aute groups ,30,31. O5 years) . Howevern = 5), large time gaps between the entry of one food item to the next were observed within study 1. The self-administered 24HDR variant of myfood24-Germany evaluated here enables participants to decide at what time of the day to start and complete the 24HDR and thus offers great flexibility and time saving compared to a traditional interviewer-based 24HDR. The individual completion procedure , however, does not always comply with the typical approach of a traditional 24HDR and might affect participants’ memory. The quality of the assessed nutritional data will be determined separately and will give insights on the validity of the self-administered variant of myfood24-Germany. The additional use of the interviewer-administered variant might be beneficial for the inclusion of study participants without Internet access or low computer literacy. However, we cannot draw a conclusion on time requirements or the usability and feasibility of this variant of myfood24-Germany from the present studies.The median completion time of 15 min for myfood24-Germany in the evaluation study was short and comparable to other web-based 24HDRs within every age group ,33. For n = 6) used a smartphone to complete the recall. So far, smartphone-based tools mainly reflected digital counterparts of FR that enable real-time recording. In contrast, in 24HDRs, the diet is recalled from the previous day. To take into account the ongoing digitalization, some web-based 24HDRs have already been optimized for mobile applications [Some participants  was developed in an elaborate process [n = 2319 items). Database maintenance seems more feasible for a concise variant with generic items only, since the inclusion of branded products in the database involves the need for regular updates. However, data on user-experience to assess the actual level of burden associated with a lower versus a higher range of food choices in self-administered 24HDRs are missing. The myfood24-Germany database comprises a large number of generic items and branded products. Although most participants agreed that the search function in myfood24-Germany was easy to use, many reported having problems with finding a correct item and criticized the incompleteness of the database. This has also been observed for other web-based 24HDRs. For example, during the testing of myfood24-UK, 89% of the users stated that the search function was easy to use but only 29% agreed that they could find food items easily [The most challenging part in the adaptation process of myfood24-Germany was the development of an appropriate food database. The underlying database determines the users’ success of searching for food items and is essential for the functionality and accuracy of automated web-based dietary assessment tools ,36. Diff process . For Foo process . The auts easily . Rowlands easily  than myfs easily . In the s easily .Our examination of users’ search strategy in myfood24-Germany (study 2) showed that branded products were mostly searched for by entering a generic description, suggesting that the products’ brand plays a minor role when searching for food items. Further, participants preferred to choose a related generic item as an equivalent for an absent branded product. Even when an exact match for a branded product was available in the database, a related generic item was chosen in nearly half of the cases. However, this behavior might be explained by the fact that generic items are displayed first in the search results. Users of online 24HDRs tend to choose items from the top of the list when several search results are displayed  and our The comparison of hypothetical energy and nutrient intake showed that the difference between mean energy and macronutrient intake for the entered sample meals and the reference was low. However, the interindividual variation was high. The variation in hypothetical energy intakes primarily resulted from the different alternatives that were chosen for absent manufactured products. For example, for a branded smoothie product, two alternatives were chosen that differed around 150% in energy content. These findings support the presumption that searching and choosing an appropriate equivalent for products is a major challenge for participants. This is supported by results from ASA24, showing that participants substitute items they cannot find by potentially inappropriate other items or omit them .Our study had some strengths and limitations. The study 1 design allowed a detailed evaluation of myfood24-Germany after repeated use by adult participants across a large age range in a study setting. The number of participants that answered an evaluation questionnaire exceeded the minimum number of five users necessary to identify usability problems within every age group . Due to Myfood24 was successfully adapted for use in the German adult population. To our knowledge, it is now the first fully automated online system for self-administered 24HDRs available in Germany. The user evaluation in the examined sample of adults indicated a short completion time, good usability and acceptability of the tool across age groups, and confirmed its feasibility for repeated short-term application. Search mechanisms should be optimized to facilitate the successful search for specific food items in the underlying database and decrease the potential risk of misreporting.