CEP90, may cause MCPH. The satellite proteins, with the exception of CCDC14, and MCPH proteins promote centriole duplication by recruiting CDK2 to the centrosome. Thus, centriolar satellites build a MCPH complex critical for human neurodevelopment that promotes CDK2 centrosomal localization and centriole duplication.Primary microcephaly (MCPH) associated proteins CDK5RAP2, CEP152, WDR62 and CEP63 colocalize at the centrosome. We found that they interact to promote centriole duplication and form a hierarchy in which each is required to localize another to the centrosome, with CDK5RAP2 at the apex, and CEP152, WDR62 and CEP63 at sequentially lower positions. MCPH proteins interact with distinct centriolar satellite proteins; CDK5RAP2 interacts with SPAG5 and CEP72, CEP152 with CEP131, WDR62 with MOONRAKER, and CEP63 with CEP90 and CCDC14. These satellite proteins localize their cognate MCPH interactors to centrosomes and also promote centriole duplication. Consistent with a role for satellites in microcephaly, homozygous mutations in one satellite gene, DOI:http://dx.doi.org/10.7554/eLife.07519.001 When a cell divides, the chromosomes that contain the genetic blueprint for the cell must be replicated and shared between the two new cells. A structure called the centrosome organizes the cellular machinery that separates the chromosome copies during cell division. At the center of each centrosome are two cylindrical microtubule-based structures called centrioles.Mutations in certain proteins that interact with the centrosome cause a neurodevelopmental disorder called primary microcephaly. People born with microcephaly have unusually small heads and brains. As a result, they may have difficulties with mental tasks. Scientists do not know exactly how these ‘microcephaly-associated’ proteins normally interact with the centrosomes or what they do at the centrosomes, so it is difficult to work out what goes wrong in people with microcephaly. One idea is that the proteins help to duplicate the centrioles before a cell divides. If this duplication does not occur, a cell cannot divide properly; so, people with mutations that interfere with centriole duplication cannot grow enough brain cells.Now, Kodani et al. have examined how these microcephaly-associated proteins work with ‘satellite’ proteins that congregate near the centrosome to duplicate centrioles. The satellite proteins help to recruit four microcephaly-associated proteins to the centrosome, where they are built into a ring. The microcephaly-associated proteins congregate at the centrosome in a particular order, with each protein recruiting the next one in the sequence. Once all four are in place near the centrosome, an enzyme that helps to duplicate the centrioles joins them.Further experiments suggest that mutations that affect one of the satellite proteins—known as CEP90—may cause microcephaly. Future analysis of how microcephaly-associated genes work may reveal the cell biological mechanisms by which centrioles participate in brain development.DOI:http://dx.doi.org/10.7554/eLife.07519.002 At the heart of the centrosome, the principle microtubule-organizing center of a mammalian cell, are two centrioles. During S phase, centrioles are duplicated through the coordinated assembly of centriolar complexes near the proximal ends of the preexisting centrioles. Centriole duplication is coupled to the cell cycle preventing under- or over-duplication, either of which can disrupt spindle formation and chromosomal segregation .Within centrosomes, a pair of centrioles are surrounded by pericentriolar material and centriolar satellites, 70–100 nm diameter electron dense structures that participate in microtubule-based transport . CentrioCaenorhabditis elegans  is required for centriole duplication, we repeated CDK5RAP2 knockdown using two previously published siRNAs  expressing a fusion between a truncated Cdk5rap2 and βGEO or decreased levels of Wdr62 exhibit centriole overaccumulation , suggestd siRNAs . Both ofntrioles . Insteaded cells . DepletiAs depletion of each of these four MCPH-associated proteins, CDK5RAP2, CEP152, WDR62 and CEP63, has similar effects on centriole duplication, we examined whether they co-localize. We discovered that, like CDK5RAP2, CEP152, and CEP63, WDR62 localizes to the interphase centrosome . To moreCDK5RAP2  efficiently depleted CEP63 and disrupted centriole duplication , a satellite from the Ian Fleming novel of the same name . In contAs CEP72, SPAG5, CEP131, MNR, CEP90 and CCDC14 localize to centriolar satellites throughout the cell cycle, similar to PCM1 , we usedPrevious studies have shown that centriolar satellites bring cargo proteins to centrosomes in a way that depends on intact microtubules . After bMass spectrometry of CDK5RAP2 coprecipitating proteins identified the centriolar satellite proteins, CEP72 and SPAG5 . We confCEP72 or SPAG5, there was a sharp decrease in the localization of CDK5RAP2 at the centrosome , recently described as a centriolar satellite component involved in centriole duplication . We confntrioles , a phenoMNR-depleted cells, indicating MNR is required for the centrosomal localization of WDR62  were born to parents who were first cousins and who had a total of 12 pregnancies , resulting in p.E89Q , a gene in which truncating mutations have been reported to cause a phenotype that overlaps that described in this family , a cell cycle-dependent kinase that is activated at the onset of S phase and participates in centriole duplication . Reciproed cells .CCDC14-depleted cells. We examined U2OS cells transfected with scrambled control (SC) or CCDC14 siRNA and treated with DMSO or the CDK2 inhibitor roscovitine. Following DMSO treatment, control cells in S phase had two pairs of centrioles while CCDC14-depleted cells had multiple Centrin foci  recruitment of ZYG-1 (PLK4) which, in turn, localizes SAS-5 (STIL) and SAS-6 to the centrosome, culminating in the recruitment of SAS-4  and CCDC14 (aa. 194–211) were developed by Pierce Technologies . Antibodies obtained for this study are the following: anti-Centrin1/2 20H5 , anti-CDK5RAP2 , anti-CEP63 , anti-MNR , anti-CCDC14 , anti-CEP131 , anti-CP110, anti-CEP72, anti-SPAG5, anti-CEP90, anti-Centrin1 , anti-PCM1 , anti-CDK2 , anti-WDR62 and anti-CEP152 , anti-Actin, anti-α-tubulin and anti-γ-tubulin , anti-SAS6 and anti-c-Myc . Alexa-conjugated secondary antibodies and Hoechst 33342 were obtained from Molecular Probes . DyLight and HRP-conjugated secondary antibodies were obtained from Jackson ImmunoResearch Laboratories  and Cell Signaling .CEP215 and CEP63 using previously published sequences, HeLa cells were grown in DMEM  supplemented with 10% FBS, and Glutamax-I.HeLa and U2OS cells (UCSF tissue culture facility) were cultured in Advanced DMEM  supplemented with 2% FBS  and Glutamax-I . Lyophilized roscovitine  was resuspended in DMSO. HeLa cells were treated with 17 μM of nocodazole for 45 min to depolymerize microtubules. To inhibit CDK2 function, U2OS cells were incubated with 2 μM of roscovitine for 24 hr. Cells were transfected with siRNA using Oligofectamine  according to the manufacturer's instructions and analyzed 48 hr later. Sequences for the siRNA oligonucleotides used in this study are described in ++ and Mg++ free Dulbecco's PBS , harvested using a cell scraper and lysed on ice for 10 min in lysis buffer  supplemented with protease and phosphatase inhibitors . Lysates were clarified  and the protein concentrations were determined using the Bradford assay . For each immunoprecipitation reaction analyzed by mass spectrometry, 5 mg of total lysate was incubated with 10 μg of antibody for 2 hr and then incubated with protein G-sepharose  for an additional 1.5 hr at 4°C. Immunocomplexes were washed three times in lysis buffer, once in detergent free lysis buffer and subsequently incubated with 0.2 M Glycine pH2.5 on ice for 10 min and quenched with 1 M Tris pH9. For smaller scaled immunoprecipitation reactions, 500 µg of total lysate was incubated with 2 µg of antibody for 2 hr and then incubated with protein G-Sepharose for an additional hour at 4°C. Complexes were washed three times in lysis buffer and subsequently boiled in 2× Laemmli reducing buffer. To detect WDR62, samples were incubated in 2× Laemmli reducing buffer, but were not boiled. Samples were separated on 4–15% gradient TGX precast gels , transferred onto nitrocellulose  and then subjected to immunoblot analysis using ECL Lightening Plus . For quantifications, samples were blotted with fluorescently labeled secondary antibodies and analyzed on a LiCOR Odyssey scanner. Specific protein signals were normalized to Actin.Asynchronous HeLa cells were incubated on ice for 5 min in chilled Ca2, and 0.1% β-mercaptoethanol) and clarified at 2500×g for 10 min. The resulting supernatant was layered atop a 60% sucrose cushion above a discontinuous sucrose gradient  and centrifuged at 40,000×g for 1 hr. Fractions were collected from the bottom and analyzed by Western blot.Centrosomes from asynchronously growing HeLa cells were isolated as previously described . HeLa ce3 in DPBS) overnight at 4°C. Primary and secondary antibodies were diluted in blocking buffer and incubated with cells at room temperature for 1 hr. To detect CDK2 using anti-CDK2 , cells were extracted with 0.1% Triton X-100 in a buffer consisting of 50 mM piperazine-1, 4-bis (2-ethanesulphonic acid) at pH 7.4, 5 mM MgCl2, and 5 mM EDTA prior to fixation in chilled methanol for 3 min. Alternatively, cells were fixed in ice cold methanol:acetone (1:1) for 7 min to detect CDK2 using anti-CDK2 . Coverslips were mounted using Gelvatol mounting media and imaged with an inverted Axio Observer D1 , image processing was completed with Adobe Photoshop. For fluorescence quantifications, specified sized regions of interest were selected and quantified using Fiji. The signal of the MCPH proteins were normalized to Centrin signal in HeLa or U2OS cells.To visualize centrosome and centriolar satellite proteins, cells were fixed in chilled methanol for 3 min. Following fixation; cells were incubated in blocking buffer  overnight at 37°C, denatured in 2 M urea, 10 mM NHSubjects were identified and evaluated in a clinical setting for medical history, cognitive impairment and physical abnormalities. Those with histories suggestive of hereditary disorders of brain development, epilepsy, and/or cognition were offered research enrollment. After obtaining written informed consent from participants or their legal guardian, phenotypic information and peripheral blood samples were collected for research purposes. The informed consent for this genetic study included permission for publication. Ethical review and approval was obtained from the Committee on Clinical Investigations at Beth Israel Deaconess Medical Center (current protocol 2001-P-000758). The study adheres to the state and federal regulations governing the conduct of human subject research (45 CFR Part 46 and 21 CFR Parts 50 and 56) and the ethical principles set forth in the Belmont Report.Patient DNA samples were subject to genome-wide SNP genotyping using Illumina 660W-Quad BeadChip (2.6M markers). Linkage analyses were performed using Allegro, MERLIN and homozygosity analysis was performed using custom scripts as previously described . Whole eCEP90 cDNA was obtained from Genecopoeia (EX-T9157-M12). The single–base pair mutation in CEP90 was introduced using the QuickChange site-directed mutagenesis kit (Agilent Technologies) with the primer pair: Forward: 5′-cttacaaagattgaagaattggacgagaaacttaatgatgcacttca-3′; Reverse: 5′-tgaagtgcatcattaagtttctcgtccaattcttcaatctttgtaag-3′.3X-FLAG tagged human  review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , a Reviewing editor, and three reviewers.Thank you for sending your work entitled “Centriolar satellites bring microcephaly-associated proteins to centrosomes to promote centriole duplication” for consideration at The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.This work by Kodani and colleagues from the Reiter lab explored interactions between different microcephaly-associated proteins and between microcephaly-associated proteins and centriolar satellite proteins. The authors seek to make the point that centriolar satellites are crucial for centrosome biogenesis and functionally link satellite proteins to MCPH. The paper is descriptive and functional studies are at the level of dependence of one protein for the localization of another. In spite of the title there is no data showing that satellites bring microcephaly proteins to centrosomes. The paper is difficult to read and will be a challenge for anyone not directly involved in this field. Perhaps a model or diagram would help to graphically illustrate the data.Major points:1) The hierarchical centrosome recruitment model has not been established thoroughly:A) The authors showed that depletion of centriolar satellite proteins SPAG5, CEP72, CEP131, MNR, and CEP90 disrupted centriole duplication. They claimed that these five centriolar satellite proteins participate in centriole duplication by interacting with different MCPH proteins and delivering them to centrosome. However, they failed to show any insightful mechanisms accounting for the recruitment and function. More specifically, the authors failed to provide direct evidence to support that SPAG5, CEP72, CEP131, MNR, and CEP90 participate in centriole duplication through interaction with MCPH proteins. The binding sites essential for these interactions could be mapped and the binding deficient mutants should be expressed in cells with knockdown background to test whether the interactions are the main reason for the phenotype.B) C) To support their sequential recruitment model, the authors need to demonstrate that CEP152, WDR62, and Cep63 are not co-depleted after Cep72 or SPAG5 RNAi. If they are, this may argue against the recruitment model and it would just be an issue of total protein level. Similar control experiments apply to the IPs and depletions of the other satellite proteins shown in subsequent figures.D) It is important to test interaction of the 4 MCPH proteins upon depletion of any of their respective centriolar satellite proteins. This would be required to test whether the recruitment hierarchy occurs at the centrosome, as proposed, or already in the cytoplasm, at the level of a pre-assembled large complex, for example. The authors could repeat the IPs shown in 2) None of the immunofluorescence data that investigate centrosome localization, and which extends over multiple figures, have been quantified. This is particularly problematic in cases in which the presented data clearly contradicts previously published work. Here it will be crucial to establish that the current data is correct. In addition, quantification of centrosome staining intensities in multiple cells and experiments should be shown after RNAi; this is standard in the field and required to determine how strongly one component depends on another and how variable the phenotype is.3) Centriole duplication was scored in cells that were in S-phase. That only S phase cells were scored may actually overestimate the phenotype, which could simply be a delay in duplication rather than a real duplication defect.4) The authors discuss acute vs. prolonged depletion to explain the fact that they see a role for CDK5RAP2 in centriole duplication, which was not observed in previous studies. However, depletion of CDK5RAP2  by RNAi has been used in previous studies, but centriole duplication defects were not been described. Rather CDK5RAP2 seems to have a role in regulating centrosome cohesion and centriole engagement. To support their finding, the authors should perform depletion experiments with siRNA and cells used in previous studies to see whether they can reproduce the duplication defect. This could also be used to confirm the dependency of Cep152 on CDK5RAP2. The same applies to Cep63 depletion. In 5) 6) 7) To confirm the general relevance of their study, and perhaps address inconsistencies with other studies (see #6), the authors should demonstrate that at least some key findings are not only valid in HeLa cells but also in other unrelated cell types.8) There is human patient data (sequence and phenotype), but it is not clear whether this is IRB approved or exempt.Other points:1) In 2) In 3) In 4) The single letter/number abbreviations for protein names used as labels for the IF panels are not very intuitive, since letters/numbers from random positions of the protein names are used, it is difficult to guess what “C”, “2” etc. means.5) 6) In 7) It is a bit confused why CEP135, STIL and SAS4 are not included in this study. Do these MCPH proteins also associate with centriolar satellite partners as proposed in this study?8) It is intriguing to propose the pairing hypothesis. However, the criteria for the paring proteins identified through MS are not revealed? For example, why CEP250 and CEP170 are not examined in this study? How to exclude the possibility of that these centriolar satellite proteins affect the hemostasis of centriolar satellite rather than the proposed protein-protein interaction?9) It will be more convincing to include another centrosome protein like CP110 in every panel of IP result as a proper negative control.eLife. Your revised article has been favorably evaluated by Vivek Malhotra (Senior editor), a Reviewing editor, and three reviewers. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below.Thank you for resubmitting your work entitled “Centriolar satellites assemble centrosomal microcephaly proteins to recruit CDK2 and promote centriole duplication” for further consideration at The reviewers of your revised manuscript are satisfied with the experimental aspects, however, one reviewer requests that you address the following item in your Discussion.This reviewer had raised this issue in the first round of review (reviewer #1 point 1D). The concern is with your proposal of the recruitment model by demonstrating that the sequential interactions between CDK5RAP2, CEP152, WDR62 and CEP63 occur at the centrosome rather than in the cytosol. In this reviewer's opinion the provided immunoprecipitations of MCPH proteins from soluble cell extract in the absence of specific satellite proteins support his/her speculation that the centrosomal recruitment of the MCPH proteins occurs in the form of large complexes that are pre-assembled in the cytosol, in a sequential manner and dependent on the respective satellite protein partner, rather than MCPH proteins being delivered by their satellite partner one by one to the centrosome. The reviewer is concerned with your conclusion that sequential assembly occurs at the centrosome?The reviewer assumes that the IPs were performed with soluble cell extracts that did not contain centrosomes (because these would end up in the pellet fraction when clearing the extract by high speed centrifugation). Or do you have data indicating the presence of centrosomes in your IPs? If centrosomes are not present during the IPs then the data clearly shows that all 4 MCPH proteins interact in the cytosol, and that the formation of this large complex depends on the sequential recruitment of each MCPH protein by its satellite partner. CDK5RAP2 could be considered the platform onto which all others sequentially and hierarchically assemble. And all of this can occur in the cytosol, in the absence of centrosomes. Obviously this will result in a hierarchical centrosome localization effect, as a result of changes in the pre-assembled cytosolic complexes. Unless you can exclude this possibility, the description and discussion of the recruitment data (and thus the model) needs to be changed. This work by Kodani and colleagues from the Reiter lab explored interactions between different microcephaly-associated proteins and between microcephaly-associated proteins and centriolar satellite proteins. The authors seek to make the point that centriolar satellites are crucial for centrosome biogenesis and functionally link satellite proteins to MCPH. The paper is descriptive and functional studies are at the level of dependence of one protein for the localization of another. In spite of the title there is no data showing that satellites bring microcephaly proteins to centrosomes.We have lengthened the title to more accurately reflect the data shown.The paper is difficult to read and will be a challenge for anyone not directly involved in this field. Perhaps a model or diagram would help to graphically illustrate the data.As suggested, we have extensively revised the text to make it easier for the casual reader to interpret, and have added a model to Major points:1) The hierarchical centrosome recruitment model has not been established thoroughly:A) The authors showed that depletion of centriolar satellite proteins SPAG5, CEP72, CEP131, MNR, and CEP90 disrupted centriole duplication. They claimed that these five centriolar satellite proteins participate in centriole duplication by interacting with different MCPH proteins and delivering them to centrosome. However, they failed to show any insightful mechanisms accounting for the recruitment and function. More specifically, the authors failed to provide direct evidence to support that SPAG5, CEP72, CEP131, MNR, and CEP90 participate in centriole duplication through interaction with MCPH proteins. The binding sites essential for these interactions could be mapped and the binding deficient mutants should be expressed in cells with knockdown background to test whether the interactions are the main reason for the phenotype.To try to address the request to map the binding sites and perform rescue experiments to support insight into how satellite proteins participate in centriole duplication, we have misexpressed tagged centriolar satellite proteins. Unfortunately, we have found that low levels of expression of centriolar satellite proteins using minimal amounts of DNA leads to the formation of cytoplasmic aggregates, even in knockdown backgrounds. The aggregates started to form after less than 12 hours of expression , insuffiB): sinceshows that the four MCPH proteins interact with each other, the IPs of CEP72 and SPAG5 should be probed as well for CEP152, WDR62 and CEP63, to test if these two satellite proteins interact exclusively with CDK5RAP2, as suggested, or also with the other 3 MCPH proteins.To further investigate whether CEP72 and SPAG5 interact exclusively with CDK5RAP2, we examined whether they could co-precipitate CEP152, WDR62 and CEP63, as suggested. We determined that CEP72 and SPAG5 specifically interact with CDK5RAP2. The new data are included in C) To support their sequential recruitment model, the authors need to demonstrate that CEP152, WDR62, and Cep63 are not co-depleted after Cep72 or SPAG5 RNAi. If they are, this may argue against the recruitment model and it would just be an issue of total protein level. Similar control experiments apply to the IPs and depletions of the other satellite proteins shown in subsequent figures.We explored the possibility that CEP72 or SPAG5 might be required to stabilize CEP152, WDR62 and CEP63, which would certainly argue against our proposal that they function in CDK5RAP2 recruitment to the centrosome, and only indirectly in the centrosomal recruitment of CEP152, WDR62 and CEP63. As suggested, we checked whether CEP152, WDR62 and CEP63 were co-depleted upon knockdown of CEP72 or SPAG5 and found that they were not. The new data are included in CEP131, MNR, CEP90 or CCDC14 and found levels of the MCPH-associated proteins to be unaltered by depletion of any of these centriolar satellite protein, which is also consistent with the model that they participate in MCPH-associated protein recruitment to the centrosome. The new data are included as We also examined the stability of CDK5RAP2, CEP152, WDR62 and CEP63 in cells depleted of D) It is important to test interaction of the 4 MCPH proteins upon depletion of any of their respective centriolar satellite proteins. This would be required to test whether the recruitment hierarchy occurs at the centrosome, as proposed, or already in the cytoplasm, at the level of a pre-assembled large complex, for example. The authors could repeat the IPs shown inin the absence of SPAG5, CEP72, CEP131, MNR, or CEP90, to test whether the interactions between the 4 MCPH are maintained and, if not, which interactions become disrupted.As suggested, we repeated the immunoprecipitations in the absence of centriolar satellite proteins to examine whether the recruitment hierarchy occurs at the centrosome or as a pre-assembled complex in the cytoplasm. Following depletion of CEP131, MNR or CEP90, immoprecipitation of CEP152, WDR62 or CEP63, respectively, demonstrated that they were no longer able to co-precipitate the other MCPH-associated proteins. For example, CEP152 was unable to bind to CDK5RAP2 or WDR62 in the absence of CEP131. Similarly, WDR62 could not bind CEP152 or CEP63 in MNR-depleted cells and CEP63 failed to bind WDR62 in the absence of CEP90. Together, these data suggest that, in accordance with a possibility raised by the reviewers, the recruitment hierarchy of four MCPH-associated proteins occurs at the centrosome. These new data are included in 2) None of the immunofluorescence data that investigate centrosome localization, and which extends over multiple figures, have been quantified. This is particularly problematic in cases in which the presented data clearly contradicts previously published work. Here it will be crucial to establish that the current data is correct. In addition, quantification of centrosome staining intensities in multiple cells and experiments should be shown after RNAi; this is standard in the field and required to determine how strongly one component depends on another and how variable the phenotype is.As advised, we quantified the fluorescence intensities of the immunostaining of MCPH-associated proteins at centrosomes in cells depleted of MCPH-associated proteins or their cognate centriolar satellite protein. The data is included as 3) Centriole duplication was scored in cells that were in S-phase. That only S phase cells were scored may actually overestimate the phenotype, which could simply be a delay in duplication rather than a real duplication defect.To investigate the possibility that the duplication phenotype is due to a delay in duplication rather than a real centriolar duplication defect, we analyzed cells in mitosis treated with siRNA to SC, CDK5RAP2, CEP152, WDR62 or CEP63. We found that the duplication defect described in the text is also present during mitosis, suggesting that the phenotype is due to a duplication defect that is not attributable to a delay in duplication. The new data are included in 4) The authors discuss acute vs. prolonged depletion to explain the fact that they see a role for CDK5RAP2 in centriole duplication, which was not observed in previous studies. However, depletion of CDK5RAP2  by RNAi has been used in previous studies, but centriole duplication defects were not been described. Rather CDK5RAP2 seems to have a role in regulating centrosome cohesion and centriole engagement. To support their finding, the authors should perform depletion experiments with siRNA and cells used in previous studies to see whether they can reproduce the duplication defect. This could also be used to confirm the dependency of Cep152 on CDK5RAP2. The same applies to Cep63 depletion. In, and, CEP63 was shown to be required for CEP152 localization to the centrosomes in different cell types, in conditions of genomic deletion but also RNAi.CEP215 #282 or #283 did caused a failure of centriolar duplication, and a failure to recruit CEP152, WDR62 or CEP63 to the centrosome, in agreement with our findings using independent siRNAs (CDK5RAP2 #1 and 2). Interestingly, siRNA CEP215 #283 caused a centriole disengagement phenotype, but we did not observe an alteration in centriole engagement in cells treated with siRNA CEP215 #282 or either of our siRNAs (CDK5RAP2 #1 and 2) treated cells did not disrupt centriole engagement. We have included the new data as a We tested whether CEP215/CDK5RAP2 depletion using the siRNA previously described by Graser et al. altered centriole duplication. Cells treated with either the siRNA they call 5): what is the phenotypical consequence for the cells of expressing the mutant CEP90? Is centriole duplication indeed impaired? I propose that the authors deplete CEP90 and try to rescue the effects of the depletion with the WT CEP90 or with the mutant, analyzing the localization of CEP63 and centriole number.CEP90 mutation is not central to the main points of the paper, we are happy to remove these data and publish them in a subsequent work in which the phenotypical consequences can be more fully elucidated if that is the opinion of the reviewers. Please refer to the answer to point 1, in which we show cells misexpressing WT and E89Q mutant CEP90 that leads to the formation of cytoplasmic aggregates that misrecruit the centriolar satellite, PCM1.We agree with the reviewer that these are interesting questions. Unfortunately, even misexpression of wild-type or mutant CEP90 with a weak promoter  for less than a cell cycle leads to the formation of large cytoplasmic aggregates of CEP90. Given that these cytoplasmic aggregates may have cellular consequences independent of the normal functions of CEP90, we were unable to conclude whether centriole duplication is rescued specifically if the protein of interest is not localized properly. Given that the human 6), andshowed that CEP63 was required for CEP152 localization to the centrosomes in different cell types, upon RNAi knockdown and genomic deletion. In the current manuscript CEP63 depletion does not seem to affect CEP152. The argument in the discussion that acute vs. prolonged treatment may explain discrepancies with previous work is not valid, since acute depletion was also used in some of the previously published experiments. It is important that the authors rigorously address issues regarding previously published, conflicting data. The authors should test siRNA and cells that were previously tested.CEP63 NB disrupted centriole duplication, centrosomes failed to accumulate CDK5RAP2, CEP152, and WDR62, in accordance with what they reported and in contrast to our findings using other ways of depleting CEP63. Examining protein levels revealed that the CEP63 NB siRNA depleted endogenous CEP63 protein levels and also led to the co-depletion of CDK5RAP2, CEP152, and WDR62, again in contrast to our findings using other ways of depleting CEP63. The independent siRNAs CEP63 #1 and #2 deplete endogenous CEP63 to an equivalent degree but do not cause the co-depletion of these other proteins. Throughout this work, we have been careful to report only phenotypes that are caused by siRNAs that both efficiently deplete the target protein, and show phenotypes that are caused by multiple independent siRNAs. While our model would predict that destabilization of CDK5RAP2, as occurs upon knockdown with CEP63 NB, will cause a failure to recruit CEP152 to the centrosome, we are hesitant to make this conclusion as it is only observed with the use of this siRNA. These data are included as a Figure 1–figure supplement 8 and described the results in the subsection “MCPH-associated proteins interact and hierarchically localize to the centrosome”.We investigated whether our hierarchy model held true for CEP63 using siRNAs reported by Brown et al. While we would have liked to confirm the experiments from Sir et al., Dhamacon has discontinued the SMARTpool siRNA product referred to in their Nature Genetics paper, and was unable to tell us the siRNA sequences. We found that depletion of CEP63 using the siRNA that they call 7) To confirm the general relevance of their study, and perhaps address inconsistencies with other studies (see #6), the authors should demonstrate that at least some key findings are not only valid in HeLa cells but also in other unrelated cell types.As suggested, we tested key findings using an unrelated cell type. We confirmed that CDK5RAP2, CEP72 and SPAG5 are required for centriole duplication in U2OS cells. We also confirmed that the protein stability of CDK5RAP2 depends CEP72 and SPAG5 in U2OS cells. We were also able to confirm that the centrosomal localization of CDK5RAP2 depends on CEP72 and SPAG5 where it serves as a scaffold to recruit CEP152, WDR62, and CEP63. We have included these new data as 8) There is human patient data (sequence and phenotype), but it is not clear whether this is IRB approved or exempt.The human patient data is IRB approved and this approval is described in the Materials and methods section.Other points:1) In, the localization/pattern of CDK5RAP2 is entirely different from the one shown in. The authors need to provide better images or explain the differences.The appearance of CDK5RAP2 localization does differ using diffraction limited microscopy  versus s2) In, in the DMSO panel, CDK5RAP2 appears more focused than on. This needs to be clarified.We thank the reviewer for bringing this to our attention. We have replaced the panel with a more representative image.In the nocodazole panel, the signal intensity for each of the 4 MCPH proteins appears brighter than in the DMSO panel. Is there indeed more protein co-localizing with PCM1 or is this just due to increased exposure/brightness settings? Here the signal of each protein at the centrioles (centrin marker) versus satellite (PCM1 marker) should be quantified for each condition in a number of cells, to show that there is a de-localization upon nocodazole treatment.We concur that the signal intensity of each of the four MCPH-associated proteins appears brighter upon nocodazole treatment. We were careful to image the DMSO and nocodazole-treated samples at the same exposure settings and process all images in parallel and in the same way. As suggested by the reviewers, we quantified the fluorescence signal of each MCPH-associated protein at the centrioles and satellites and found that nocodazole increased the proportion of the MCPH-associated proteins at the satellites. This quantitation is now included as a graph in 3) In, there is a reference to a CCDC14 siRNA #3, which doesn't appear before (only #1 and #2). This needs to be clarified. Another issue is that when CCDC14 is depleted, the CEP63 signal is restricted to the centrioles, contrary to what is shown in).CCDC14-depleted cells, we quantified the fluorescence intensity of CEP63 and the other three MCPH-associated proteins and found that only CEP63 co-localized with the majority of the Centrin positive foci. This quantitation is included in We thank the reviewers for bringing this mistake to our attention. We have changed the reference to siRNA #3 to siRNA #2. We apologize for the mistake. Although the domain of CEP63 localization expands with the increase in Centrin foci upon CCDC14 depletion, we do not perceive the CEP63 to be strictly localized to centrioles. To further assess the accumulation of CEP63 to Centrin positive foci in 4) The single letter/number abbreviations for protein names used as labels for the IF panels are not very intuitive, since letters/numbers from random positions of the protein names are used, it is difficult to guess what “C”, “2” etc. means.We apologize for not using single letter/number abbreviations that were not intuitive. To rectify this, we replaced all labels with their protein names. Additionally, the color of the font used for label of each protein either on the top or left of each panel reflects the channel used to image that protein, making it clear that Centrin is always depicted in green and the other centrosomal protein is depicted in red.5): the channel color legend is missing.We thank the reviewer for bringing this to our attention. We have added the channel color legend to the figure.6) In, the authors use U2OS cells instead of HeLa (which were used in the previous experiments), without any reference to why the change in cell line.While both U2OS and HeLa cells displayed supernumerary Centrin foci upon CCDC14 depletion, supernumerary Centrin foci of HeLa cells are tightly packed, making them difficult resolve and assess how they co-localize with other centrosomal proteins. Therefore, we displayed U2OS cells, in which the supernumerary Centrin foci are more dispersed. We made note of this rationale in subsection “Centriolar satellite component CCDC14 restrains the centrosomal accumulation of CEP63 and CDK2”.7) It is a bit confused why CEP135, STIL and SAS4 are not included in this study. Do these MCPH proteins also associate with centriolar satellite partners as proposed in this study?We agree with the reviewer that CEP135, STIL and SAS4 would have been nice to include in this study. However, because we do not have reagents of sufficient quality to fully assess whether CEP135, STIL and SAS4 associate with still other centriolar satellite proteins, we will need to work on developing better tools. It is possible that CEP135, STIL and SAS4 bind cognate centriolar satellite proteins similar to CDK5RAP2, CEP152, WDR62 and CEP63, but for this work, we focused on the four MCPH-associated proteins that we were confident biochemically interacted with each other, and the six satellite proteins that we were confident interacted with them.8) It is intriguing to propose the pairing hypothesis. However, the criteria for the paring proteins identified through MS are not revealed? For example, why CEP250 and CEP170 are not examined in this study? How to exclude the possibility of that these centriolar satellite proteins affect the hemostasis of centriolar satellite rather than the proposed protein-protein interaction?The criteria for our focusing on the proteins we identified were twofold: we focused on proteins that both localized to the centrosome and which were previously implicated in centriole duplication. While both CEP250 and CEP170 localize to the centrosome, neither was strongly required for centriole duplication in our hands. Whereas CEP250 and CEP170 have roles in centriole cohesion and subdistal appendage organization, respectively, we were not able to discern any roles for them in MCPH-associated protein function, and thus were not included in this study. Inhibition of the function of centriolar satellite proteins may well be affecting aspects of centrosomal biology beyond the interaction with MCPH-associated proteins, including homeostasis of the centriolar satellites. However, we have focused on the aspects of centrosomal function for which we have positive evidence, and will continue to assess whether these centriolar satellite proteins participate in other aspects of centrosomal function in the future. A more complete description of these possibilities is now included in the Discusssion.9) It will be more convincing to include another centrosome protein like CP110 in every panel of IP result as a proper negative control.We thank the reviewers for suggesting these sets of experiments. As an extra measure of specificity, we now include CP110 as a negative control in The reviewers of your revised manuscript are satisfied with the experimental aspects, however, one reviewer requests that you address the following item in your Discussion.This reviewer had raised this issue in the first round of review (reviewer #1 point 1D). The concern is with your proposal of the recruitment model by demonstrating that the sequential interactions between CDK5RAP2, CEP152, WDR62 and CEP63 occur at the centrosome rather than in the cytosol. In this reviewer's opinion the provided immunoprecipitations of MCPH proteins from soluble cell extract in the absence of specific satellite proteins support his/her speculation that the centrosomal recruitment of the MCPH proteins occurs in the form of large complexes that are pre-assembled in the cytosol, in a sequential manner and dependent on the respective satellite protein partner, rather than MCPH proteins being delivered by their satellite partner one by one to the centrosome. The reviewer is concerned with your conclusion that sequential assembly occurs at the centrosome?The reviewer assumes that the IPs were performed with soluble cell extracts that did not contain centrosomes (because these would end up in the pellet fraction when clearing the extract by high speed centrifugation). Or do you have data indicating the presence of centrosomes in your IPs? If centrosomes are not present during the IPs then the data clearly shows that all 4 MCPH proteins interact in the cytosol, and that the formation of this large complex depends on the sequential recruitment of each MCPH protein by its satellite partner. CDK5RAP2 could be considered the platform onto which all others sequentially and hierarchically assemble. And all of this can occur in the cytosol, in the absence of centrosomes. Obviously this will result in a hierarchical centrosome localization effect, as a result of changes in the pre-assembled cytosolic complexes. Unless you can exclude this possibility, the description and discussion of the recruitment data (and thus the model) needs to be changed.Our data show that each MCPH protein cannot interact with other MCPH proteins in the absence of its cognate centriolar satellite interactor, and that there is a sequential MCPH protein recruitment to the centrosome.Our immunoprecipitation experiments were performed with soluble cell extracts that likely contain the majority of centrosomal proteins aside from the salt-insoluble centriole core . As most non-centriolar components are extracted off of the centrosome by the combination of salt and detergent that we use, we surmise that the MCPH components are part of the soluble cell extract used in our IPs. Thus, while centrosomes are not present in our lysates, many centrosomal proteins are.We concur with the reviewer that, based on the available information, it is not possible to discriminate between formation of the MCPH protein complex at the centrosome and pre-assembly into a complex in the cytoplasm prior to being delivered to the centrosome. We agree that it is possible that the failure to assemble the MCPH complex in the cytoplasm could result in the failed hierarchical assembly at the centrosome. Because we cannot definitively distinguish between assembly of the MCPH complex in the cytoplasm and at the centrosome, we have refined the Discussion and the legend for the final model figure to specifically discuss both possibilities.


The results of our study suggest that genes involved in general resistance mechanisms of plants contribute to variation of BYDV resistance in maize.Barley yellow dwarf virus (BYDV) is expected to become a prominent problem in maize cultivation. Breeding for resistance is the best strategy to control the disease and break the transmission cycle of the virus. The objectives of our study were (1) to determine genetic variation with respect to BYDV resistance in a broad germplasm set and (2) to identify single nucleotide polymorphism (SNP) markers linked to genes that are involved in BYDV resistance. An association mapping population with 267 genotypes representing the world’s maize gene pool was grown in the greenhouse. Plants were inoculated with BYDV-PAV using viruliferous Rhopalosiphum padi. In the association mapping population, we observed considerable genotypic variance for the trait virus extinction as measured by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and the infection rate. In a genome-wide association study, we observed three SNPs significantly [false discovery rate (FDR) = 0.05] associated with the virus extinction on chromosome 10 explaining together 25 % of the phenotypic variance and five SNPs for the infection rate on chromosomes 4 and 10 explaining together 33 % of the phenotypic variance. The SNPs significantly associated with BYDV resistance can be used in marker assisted selection and will accelerate the breeding process for the development of BYDV resistant maize genotypes. Furthermore, these SNPs were located within genes which were in other organisms described to play a role in general resistance mechanisms. This suggests that these genes contribute to variation of BYDV resistance in maize.With increasing winter temperatures in Europe, The online version of this article (doi:10.1007/s00122-014-2400-1) contains supplementary material, which is available to authorized users. Barley yellow dwarf virus (BYDV) is a group of plant viruses which are of high commercial relevance in small grain cereals  (Clark and Adams R genes that are involved in the resistance to viruses belong to the largest class of R genes with a nucleotide-binding site plus leucine-rich repeat (NB-LRR) , two major resistance genes have been mapped to chromosomes 6 (Scm1) and 3 (Scm2)  resistance. Jones et al. (mcd2 to contribute to resistance against Maize chlorotic dwarf virus (MCDV). BYDV resistance is described to be quantitatively inherited in barley  determine genetic variation with respect to BYDV resistance in a broad germplasm set of maize and (2) to identify single nucleotide polymorphism (SNP) markers linked to genes that are involved in BYDV resistance.® Classic) with a day–night temperature regime of 16 h/24 In our study, we used 267 inbred lines from a set of 302 genotypes which represent the world wide diversity of maize breeding programs . One week after inoculation, the plant protection fleece was removed and the plants were sprayed with the insecticide “Lizetan Plus” (Bayer) to stop inoculation.Viruliferous To analyze the BYDV content, six weeks after the inoculation start, plant material from the sixth leaf was collected separately for each plant and each leaf was analyzed by DAS-ELISA according to Clark and Adams  using thThe two F3 sib-pollinated families, Mu06347 and Mu06527, from the Maize Genetics Cooperation Stock Center with a 9 bp insertion in the 5mentclass2pt{minimTwo replicates were inoculated with BYDV and two served as a non-inoculated control. The inoculation and the analyses of EX were performed as described above. To test whether the insertions are present, we designed the following polymerase chain reaction (PCR) primers: forward primer (5′-GCGAAACCGAGTAGGTGGA-3′) and reverse primer (5′-GAGGGAATGGAGGAAGGAAG-3′) for the 5′UTR of the gene using the software Primer3 , which extends 2,000 bp in maize  associated with the trait EX Table ; Fig. 3aThese three SNPs were also significantly associated with the trait IR and explained between 11 and 18 % of the phenotypic variance each Table ; Fig. 3bIn the mutant genotype experiment, the mean EX values differed between the mutant genotypes and the wild type genotype. The EX value of the mutant genotype Mu06347 was with 0.47 two times higher compared to the wild type W22 (0.23) and Mu06527 even three times higher (0.70). The same was true for IR with values from 37.5 % for the mutant genotype Mu06347 and 50.5 % for Mu06527 compared to W22 with 15.7 %. Nevertheless, the observed differences were not significantly different from 0  associated with the two traits EX and IR were all located in the sequences of previously described genes. The genes which are physically close to significantly associated SNPs are not necessarily the causal genes for the observed trait variation. Nevertheless, we found evidence that suggests such a functional relationship in physical proximity to the associated SNPs. The three SNPs on chromosome 10 which are significantly associated with EX and IR are located in the sequence of the gene GRMZM2G018027. The best hit for this gene in Besides the callose deposition, He et al.  reportede et al.  or the ce et al. .Scmv1 on chromosome 6 in maize. This candidate gene Zmtrx-h encodes a putative 119-residue protein that is highly similar to h-Type Thioredoxin. Thioredoxin proteins regulate redox pathways and induce systemic acquired resistance (SAR). Therefore, its overexpression leads to resistance to Tobacco mosaic virus and Cucumber mosaic virus and also improved tobacco resistance to oxidative stress , UniformMu genotypes are available containing an insertion in the gene GRMZM2G018027. To test whether this mutation changes the resistance to BYDV, we compared the mutant genotypes to their wild type inbred W22 with regard to their virus extinction. In the mutant genotypes we found considerably higher EX values compared to the wild type, which suggests that a mutation in the gene could reduce the BYDV resistance. In our study, this difference was, however, not statistically significant. One reason for the non-significance could be that the gene is not the causal gene and therefore, it has no influence on BYDV resistance. Another reason could be, however, that the insertion is located in the 5In the Maize Genetics Cooperation Stock Center . This makes it difficult to observe statistically significant differences. UniformMu genotypes can carry multiple insertions in multiple genes. This can also lead to a high error in the analyses of these genotypes. Therefore, we suggest to study further the effect of Arabidopsis thaliana and Oryza sativa. It was reported by Uzarowska et al.  (86424631-137500030bp) from the study of Zambrano et al.  associations with the BYDV resistance traits. Therefore, our results suggest that the eukaryotic translation initiation factors are no candidates for BYDV resistance in the examined maize population.Kang et al. , ZambranThe evaluation of maize genotypes for their BYDV resistance by aphid inoculation and DAS-ELISA analyses is very labor- and cost intensive and therefore, its integration in commercial breeding programs is not promising. Therefore, the development of molecular markers linked to the traits EX and IR is the basic requisite for an effective improvement of BYDV resistance levels. BYDV resistant maize inbreds would contribute to secure a sustainable agriculture. The SNPs which were significantly (FDR = 0.05) associated with the traits EX and IR explained with 24.86 and 33.63 % a considerable proportion of the phenotypic variance. That means their use for marker-assisted selection in breeding maize to improve BYDV resistance is promising.From the DNA sequence flanking the SNPs Table , marker If the allele combination which was found to increase the BYDV resistance in our study is not present in the proprietary genetic material, our results (given in Online Resource Table S1) can be used to identify resistance sources for the different heterotic pools of interest.The broad and continuous variation with regard to BYDV resistance in the association mapping population shows that an improvement of BYDV resistance by breeding is possible and provides the condition for the identification of molecular markers linked to BYDV resistance by association mapping. The identified markers are strongly associated with the traits virus extinction and infection rate and explain a considerable part of the phenotypic variation of the studied association mapping population. The results of our study suggest that genes involved in general resistance mechanisms are also involved in BYDV resistance in maize.FH participated in the design of the experiment, carried out most of the experimental work, performed the statistical analyses, and drafted the manuscript. AH participated in the design of the experiment, experimental work, and drafted the manuscript. BS conceived the study, participated in its design and statistical analyses, and drafted the manuscript. All authors read and approved the final manuscript.Supplementary material 1 (pdf 39 KB)Below is the link to the electronic supplementary material.


Ruminococcaceae and Lachnospiraceae lower up to 6 folds, Prevotellaceae lower by ~40%, and Clostridiaceae and Veillonellaceae higher up to 10 folds compared to microbiota without protozoa. An orthogonal partial least squares-discriminant analysis of urinary metabolome matched differences in microbiota structure. Discriminant metabolites were mainly involved in amino acids and protein metabolic pathways while a negative interaction was observed between methylotrophic methanogens Methanomassiliicoccales and trimethylamine N-oxide. These results stress the influence of gut microbes on animal phenotype and show the potential of metabolomics for monitoring rumen microbial functions.The rumen microbiota is an essential part of ruminants shaping their nutrition and health. Despite its importance, it is not fully understood how various groups of rumen microbes affect host-microbe relationships and functions. The aim of the study was to simultaneously explore the rumen microbiota and the metabolic phenotype of lambs for identifying host-microbe associations and potential biomarkers of digestive functions. Twin lambs, separated in two groups after birth were exposed to practices (isolation and gavage with rumen fluid with protozoa or protozoa-depleted) that differentially restricted the acquisition of microbes. Rumen microbiota, fermentation parameters, digestibility and growth were monitored for up to 31 weeks of age. Microbiota assembled in isolation from other ruminants lacked protozoa and had low bacterial and archaeal diversity whereas digestibility was not affected. Exposure to adult sheep microbiota increased bacterial and archaeal diversity independently of protozoa presence. For archaea, Methanomassiliicoccales displaced Methanosphaera. Notwithstanding, protozoa induced differences in functional traits such as digestibility and significantly shaped bacterial community structure, notably  The gut microbiota of higher animals provides a myriad of ecosystem services to the host-microbe holobiont. In the case of livestock animals, these ecosystem services provide food and income to hundreds of millions of people worldwide  that was constant throughout the trial and hay was fed at libitum. In period 1, sampling and individual digestibility measures were performed in week 14. At the age of 15 weeks, lambs in group 1 (G1) were inoculated with 100 ml rumen fluid obtained from two rumen-cannulated wethers containing a conventional microbiota. Donor wethers were fed a hay diet once a day. Rumen contents were taken before feeding, strained through a polyester monofilament fabric (250 μm mesh aperture) and pooled to produce the inoculum. The concentration of protozoa was 5.3 logThe second group (G2) was inoculated 1 week later with the same inoculum (100 ml/lamb) that was kept frozen as a way to induce changes in the microbial community. Protozoa in particular are labile to freeze-thawing . Feeding and any manipulation on lambs was done in the morning before handlers contacted other animals. It started always with G2 and included a change of protective clothing before tending to G1. Bed cleaning was done with minimal contact by grouping the lambs in one corner of the pen.Liquid chromatography (LC) separation was performed by reversed phase chromatography on a 10 cm Luna C18(2) columns using an acidic water-acetonitrile system as described in detail in the Supplementary Material. The LC was interfaced to a Micromass LCT Time of flight MS instrument using an ESI interface and running in positive mode scanning m/z 100–900  were clustered at 97% of similarity and filtered using USEARCH PCR using primers targeting the 16S rRNA gene . The model included the fixed effect of inoculum, lambs age, group, and period × group interaction. The animal was considered as a random effect. Best fitting covariance structure was compound symmetry. Differences were tested with the LSMEANS statement for period, group and their interaction and with the LSMESTIMATE statement for testing the effect of inoculum at 20 and 26 weeks of age. Significance was declared at The Kruskal-Wallis test as implemented in SAS was used to estimate the difference between samples in the number of sequenced reads. Differences in the abundance of reads attributed to phyla, families and genera were done using Metastats  and S-plots. The significance of individual variables between groups (G1 vs. G2) was further assessed using ANOVA test . Consensus OPLS-DA was used to combine metabolomics and microbial data . The Good's coverage estimator returned the highest coverage values for 14 weeks lambs at 99.3%. As lambs aged, the coverage value decreased in subsequent periods. However, the coverage at the end of the 31-week experiment was still good at =97.8% meaning that over 45 reads were needed to detect a new phylotype. Accordingly, rarefactions curves for younger animals started to flatten earlier than for older animals . In contrast, changes in Veillonellaceae could not be attributed to S. ruminantium, which remained constant throughout the experiment . The qPCR data also confirmed that decreases in Ruminococcaceae numbers could not be attributed to the common fibrolytic genera, R. albus and R. flavefaciens that showed no changes or even a tendency to increase for the latter. Total bacteria, 16S rRNA gene copies per unit of DNA were logically higher in the absence/low density of protozoa  to 69 and 73% for 20 vs. 26 and 26 vs. 31 weeks, respectively. For G2 the lowest similarity, although not statistically different from the other periods, was also observed when lambs were allowed to be colonized by protozoa (26 vs. 31 weeks = 61%). The similarity between 14 and 20 and between 20 and 26 weeks of age were 63 and 72%, respectively. Protozoa seemed to produce the biggest shift in bacterial β-diversity substantiating the Unifrac results.Beta-diversity analysis using the UniFrac metric . There was a general trend for increased counts with aging, pairwise comparison between groups revealed that the difference was due to variance between 14 and 31 weeks for G1. Both the average Good's coverage estimator of 99.6 ± 0.4% and leveled off rarefactions curves  and G2 was less diverse than G1 for the first three sampling ages (Table P < 0.05). A total of 77 OTUs were identified at 97% similarity but many of these OTUs were present in low numbers and only in some lambs and were not further considered. Based on their abundance, 29 OTUs were retained as they represented ≥1% of the total number of archaeal sequences and were present in ≥50% of the rumen samples from a group at a given time (Supplementary Table S2). These were grouped using the main clades reported in the rumen  discriminated on the first axis samples from lambs at 14 weeks from those taken at other periods indicating a less marked effect of protozoa on methanogens than on bacteria. The combined effect of age and inoculation were more important determinants of the archaeal community than protozoa alone  that were included throughout the analysis, were first examined by PCA to obtain a global overview of the different sources of variability. A clear separation between lambs mainly explained by the presence or absence of protozoa was observed along the first component highlighting the massive effect of colonization events on urinary metabolites  of 32.8% and a cross-validated predictive ability Q2Y(cum) of 78.1%. The explained variance was split into predictive variation (R2p(X) = 13.7%) and the uncorrelated variations  = 19.4%). A separate comparison of samples at each time point was then made using PCA and OPLS-DA models. They showed negligible differences between the two groups of lambs at 14 weeks of age despite the fact that they were physically separated but, otherwise, had the same initial contact with their dams and were similarly treated. At 20 and 26 weeks of age both groups were well separated, similarly to the global PCA and OPLS-DA models (figures not shown). However, at 31 weeks, when lambs were all in the same lot, previous differences were effaced. The most discriminant metabolites were selected using a VIP threshold of 2. Figure Supervised multivariate analysis was then carried out using OPLS-DA to focus on discriminative metabolic patterns between faunated and non-faunated animals. The assessment of a global OPLS-DA model accounting for all time points resulted in one predictive and two orthogonal components Figure , with a n = 65). A stringent trimming was performed on the bacterial OTUs for obtaining a core set of phylotypes. Only OTUs that were in ≥50% of the rumen samples from a group at a given time and that represented ≥0.1% of the total community counts were selected. From an initial 3946 OTUs, 517 fitted these criteria. For archaea, the dataset contained the 29 OTUs described in the text. A model with two latent variables  was obtained using leave-one-animal-out cross validation with satisfactory goodness of fit (R2Y = 0.95) and goodness of prediction indices (Q2 = 0.78). The faunation effect was clearly visible on the score plot of the two predictive latent variables (tp1 vs. tp2), separating the two groups  and for the rest the closest relatives had between 95 and 82% identity. These observations are further described in the Discussion Section.To better comprehend the changes induced by treatments we also applied a novel data fusion approach for the joint analysis of metabolomic and microbial data, namely consensus OPLS-DA analysis , particularly in week 26 in conjunction with OTA challenge with a 6% difference observed between G1 and G2, with and without a standard protozoal population, respectively. Despite the differences described above, growth was not different between groups.Table The composition of the rumen microbiota of the isolated lamb groups at the first measurement was markedly different of the expected microbiota of lambs reared in contact with adult ruminants. The low diversity of bacterial phyla and the absence of protozoa indicate that the experimental conditions succeeded in maintaining a microbiota that resembled that of 2 to 4-week-old ruminants  than at later time points. It is possible that differences observed in bacterial communities between G1 and G2 at weeks 20 and 26 could be influenced by the freeze-thaw treatment imposed to the inoculum used in G2. Freeze-thaw might have affected some populations but only marginally as suggested by the absence of α-diversity differences between G1 and G2. β-diversity discrimination between groups was mainly driven by the relative abundance of taxa rather than their presence/absence indicating that freeze-thaw was not deleterious to major bacteria. It is interesting to note the contrasting abundance at different measuring periods between Fibrobacteres and Ruminococcaceae, two bacterial groups associated to plant fiber digestion. Fibrobacteres, absent in 14 week-old lambs, were not acquired during the first few hours after birth, when in contact with the mother, or through the environment. Their absence at 14 weeks and presence in older lambs was confirmed by qPCR targeting Methanomicrobium spp, all main methanogens clades described in the rumen . Total tract digestibility was also adequate for this type of diet, although it is noted that the measure does not differentiate between rumen and the intestine and less rumen activity may be compensated by greater digestion in the hindgut. Rumen fermentation was stimulated by the microbial inoculum, particularly for G1 containing protozoa. This is in accord with the microbial ecology tenet that higher diversity would improve ecosystems functions and stability . Some clearly increased following inoculation with the complex rumen microbiota such as sebacic acid or indole-3-carboxylic acid, a metabolite associated to tryptophan metabolism, and while others decreased, such as trimethylamine N-oxide (TMAO). Trimethylamine N-oxide is synthesized in the liver from trimethylamine produced by gastrointestinal microorganisms from dietary sources. In ruminants, trimethylamine is a common rumen metabolite that is mainly originated from dietary choline and glycine-betaine (Neill et al., The presence of protozoa also seems to modify the host metabolome, either because of their own metabolic activity or by modulating the bacterial community. Purine derivatives, allantoic acid and hypoxanthine, are markers of microbial protein synthesis (Chen and Gomes, Many animal species including humans exhibit individual differences in their microbiota. These differences have been ascribed to diet, intrinsic host genetic and immunological factors and to ecological processes of microbial community composition (Mulder et al., We observed that growth in lambs was not affected by a reduced rumen microbial diversity, by the presence or not of protozoa or by a mild stress treatment. In spite of that, rumen microbial diversity had a major influence on rumen fermentation parameters, digestibility, and the general metabolism of the host as monitored through the urinary metabolome profile. These results suggest that under controlled and unchanged sanitary, environmental, and dietary conditions an adequate rumen function can be provided by a microbiota with reduced diversity. The evolutionary advantages for ruminants to maintain an extremely diverse microbial community in the rumen could be regarded as an insurance policy against dietary changes and occasional aggressions such as xenobiotics. The interaction found between TMAO and methanogens from the order Methanomassiliicoccales shows potential for the metabolomics approach for the discovery and monitoring of biomarkers of rumen microbial functions of interest.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Additionally, there is a robust ~10 Hz rhythmic burst of retinal ganglion cell (RGC) spikes on the trough of the oscillatory LFP. In rd1 mice, the rhythmic burst of RGC spikes is always phase-locked with the oscillatory LFP and this phase-locking property is preserved regardless of postnatal ages. However, in rd10 mice, the frequency of the oscillatory rhythm changes according to postnatal age, suggesting that this rhythm might be a marker of the stage of degeneration. Furthermore when a biphasic current stimulus is applied to rd10 mice degenerate retina, distinct RGC response patterns that correlate with the stage of degeneration emerge. This review also considers the significance of these response properties.Characterization of the electrical activity of the retina in the animal models of retinal degeneration has been carried out in part to understand the progression of retinal degenerative diseases like age-related macular degeneration (AMD) and retinitis pigmentosa (RP), but also to determine optimum stimulus paradigms for use with retinal prosthetic devices. The models most studied in this regard have been the two lines of mice deficient in the β-subunit of phosphodiesterase ( One theory posits that substantial remodeling and rewiring processes upon photoreceptor degeneration transform the retina into a self-signaling neuronal network . The frequency of oscillation becomes stable around 4–5 Hz from PNW 8 onwards, when a strong correlation is also established between the first peak of bursting RGC spikes and oscillatory LFP  and PNW8 (4.5 ± 0.6 Hz)).In a previous study with rd10, that could explain some of the differences we found in the rd10 with regard to when the oscillatory rhythm is first being consistently observed (PNW 8 or PNW 6.5). Although the same strain of rd10 (B6.CXB1-Pde6brd10/J), is used in the data of Goo et al.  were found to respond to epiretinal stimulation . With the application of glutamate antagonists CNQX and AP-7, electrically evoked activity in RGCs in wt and rd1 mice was abolished or greatly diminished, suggesting that the RGC responses were indeed network-mediated indirect activation not the direct activation of RGC. Correlation between functional type of RGCs and their electrical responses was however equivocal; in wt, only type II cells, all of which were ON-center RGCs, could be linked to a physiological cell type, while in rd1 mouse retinas, the only 2 out of 50 RGCs that were responsive to light were of different classes . Recently, Im and Fried  in wt mice typically has a single peak with latency of less than 100 ms. In rd1 mice at PNW 8 however, the PSTH has multiple peaks (~4 peaks), with ~10 Hz rhythm (in-phase with the LFPs) within 400 ms  stimulation, the temporal pattern of RGC responses is very different in wt and rd10 retina. Recent work .To address whether oscillation is noise, in this review, we would like to focus on the role of oscillatory rhythm affecting RGC spikes response to electrical stimulus in p < 0.001 in Figure The spontaneous firing rate varies according to postnatal age. It peaks during PNW 4.5–6.5 (p < 0.01). Similarly, PNW 20 showed higher evoked spikes than all other age groups , than first peak in PNW 8 mice, there is a high possibility of conveying visual information through these remnant peaks at this age. This finding suggests that not only the first peak but also the remnant peaks should be considered as meaningful response. Although no pharmacological dissection of the network-mediated activation of RGCs had been tried in Park et al. , from thin vitro MEA recording, in vivo recording could shed more light on the role of multiple peaks resulting from oscillating LFP. Recording in vivo from the superior colliculus (SC), Ivanova and coworkers have reported significantly more spontaneous activity in the light-stimulated rd10 mouse retina than in age-matched wt mice  in the rd10 visual cortex have not been reported, it is not clear whether a corollary of the PSTH multiple peaks seen in the retina will be observed in the visual cortex following electrical stimulation of the retina. This might warrant future studies.Since all the data in Figure rd1 and rd10 degenerate retinas exhibit spontaneous activity consisting of bursting RGC spikes, superimposed and phase-locked with concurrent LFPs. In rd1, oscillation occurs at a frequency of 10 Hz, independent of postnatal age while in rd10 it occurs at 5 Hz, but varies with postnatal age with less robust phase locking. The rd10 oscillation has the highest frequency at PNW 4, it is stable at 4–5 Hz from PNW 8 onwards.Both The response to electrical stimulation shows the best correlation between stimulus amplitude and evoked RGC spikes occurring at PNW 8, especially for the remnant peaks (at 100–1000 ms post-stimulus). This is interpreted to mean that the first post- stimulus response peak (100 ms), may not be the only information carrying response of the degenerate retina and that stimulus paradigms for retinal prosthetic devices may need to take into account the stage of retinal degeneration.YSG, DJP, JRA, SSS conceived and designed the experiments; YSG, DJP performed the experiments; DJP, JRA analyzed the data; YSG, DJP, SSS wrote the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lpcat3 in the intestine fail to thrive during weaning and exhibit enterocyte lipid accumulation and reduced plasma TGs. Mice lacking Lpcat3 in the liver show reduced plasma TGs, hepatosteatosis, and secrete lipid-poor very low-density lipoprotein (VLDL) lacking arachidonoyl PLs. Mechanistic studies indicate that Lpcat3 activity impacts membrane lipid mobility in living cells, suggesting a biophysical basis for the requirement of arachidonoyl PLs in lipidating lipoprotein particles. These data identify Lpcat3 as a key factor in lipoprotein production and illustrate how manipulation of membrane composition can be used as a regulatory mechanism to control metabolic pathways.The role of specific phospholipids (PLs) in lipid transport has been difficult to assess due to an inability to selectively manipulate membrane composition in vivo. Here we show that the phospholipid remodeling enzyme lysophosphatidylcholine acyltransferase 3 (Lpcat3) is a critical determinant of triglyceride (TG) secretion due to its unique ability to catalyze the incorporation of arachidonate into membranes. Mice lacking DOI:http://dx.doi.org/10.7554/eLife.06557.001 Living cells are surrounded by a membrane that forms a barrier between the cell and its external environment. This membrane is largely made up of a variety of molecules known as lipids. The particular lipid molecules found in a cell membrane strongly influence its mobility, flexibility and other physical properties.The liver and intestine can package lipids gained from the diet into molecules called lipoproteins, which are released into the bloodstream for use by the body. An enzyme known as Lpcat3 is found in high levels in the liver and intestine and it appears to be involved in the production of lipoproteins. Altering the amount of Lpcat3 in cells can change the types of lipids found in the cell membranes, connected to the production of lipoproteins.Rong et al. studied newborn mice that were missing the Lpcat3 protein in either the liver or intestine. Mice lacking Lpcat3 in the intestine had higher levels of lipids inside their intestine cells and grew more slowly than normal mice. Mice lacking Lpcat3 in the liver also accumulated lipids in their cells and their bloodstream had lower levels of lipids that contain a molecule called arachidonic acid than normal mice. Further experiments showed that the loss of Lpcat3 reduces the ability of lipids to move within the cell membrane.The experiments show that Lpcat3 plays a key role in attaching arachidonic acid to membrane lipids to promote the release of lipoproteins into the bloodstream. Rong et al.'s findings reveal that changing the type of lipids in the cell membrane plays an important role in regulating the levels of lipids in the blood.DOI:http://dx.doi.org/10.7554/eLife.06557.002 The Lpcat-dependent remodeling process is essential for the diversity and asymmetric distribution of acyl chains because the de novo PL synthesis pathway has little substrate specificity (Phospholipids (PLs) are important components of biological membranes and also serve as precursors for the generation of diverse signaling molecules . In mammcificity .We previously identified the sterol-activated nuclear receptor LXR as an integrator of cellular lipid levels and membrane PL composition. LXR controls the expression of Lpcat3, which is the most abundant Lpcat family member in liver and intestine. Cell-based assays suggest that Lpcat3 preferentially catalyzes the synthesis of phosphatidylcholine (PC) species containing an unsaturated fatty acyl chain at the sn-2 position , but theMoreover, the larger topic of the regulatory potential of dynamic phospholipid remodeling has been largely unexplored. It has been reported that modifications of PL composition influence a range of cellular processes . HoweverThe rate of lipoprotein production has been linked with the availability of PC, but the mechanisms underlying this link are not clear . PC is tWe demonstrate here that Lpcat3 is uniquely required for the incorporation of arachidonic acid into membranes in vivo, and that an absence of arachidonoyl PLs profoundly affects lipid transport and lipoprotein production. Biophysical, electron microscopy (EM) and biochemical studies indicate that Lpcat3-dependent production of arachidonoyl PLs is important for lipid movement within membranes and for the efficient lipidation of apoB–containing lipoproteins. We also show that induction of Lpcat3 activity is required for the ability of LXRs to promote hepatic VLDL production. These data identify Lpcat3-dependent phospholipid remodeling as a critical, LXR-regulated step in TG secretion, and suggest that this step might be further explored as a strategy to treat hyperlipidemias.Lpcat3 transcripts in liver and intestine  by breeding the global heterozygous knockout mice with mice expressing FLPe recombinase  to be indistinguishable from control  mice . Consistent with that idea, Lpcat3 expression was reduced by >90% in primary hepatocytes from L-Lpcat3 KO mice  for 9 weeks. Mice lacking hepatic Lpcat3 again showed lower total plasma TG levels  and a stLpcat3 expression is regulated by the sterol-activated nuclear receptor LXR . Furthermore, we observed compensatory increases in the abundance of other PC species in L-Lpcat3 KO livers, notably those containing monounsaturated chains . Analysis of PC species in the pooled VLDL fractions by ESI-MS/MS revealed highly selective reductions in 16:0, 20:4 PC and 18:0, 20:4 PC . Thus, tInterestingly, the severe loss of arachidonate in livers of Lpcat3 KO mice on chow diet was observed in PLs, but not in TG  nor in cPltp and the lipid transport protein Cd36. A number of additional genes involved in lipid metabolism were induced to a more modest degree, including Txnip and Fabp2. Interestingly, many of the genes that were induced are known targets for the lipid-activated nuclear receptor PPARα, suggesting a compensatory gene expression response to increased cellular lipid levels in the absence of Lpcat3  impairs VLDL secretion and induces hepatosteatosis . Data analysis was performed with GenespringGX . The GEO accession numbers for the microarray data are GSE65352 and GSE65353.Total RNA was isolated from cells and tissues with Trizol . cDNA was synthesized, and gene expression was quantified by real-time PCR with SYBR Green  and an ABI 7900. Gene expression levels were normalized to 36B4 or GAPDH. Primer sequences are listed in Cells and tissue lysates were prepared by homogenization in RIPA buffer  supplemented with protease and phosphatase inhibitors . Lysates were cleared by centrifugation. Plasma samples were diluted with RIPA buffer. Protein lysate were then mixed with NuPAGE LDS Sample Buffer, size-fractionated on 4–12% Bis-Tris Gels (Invitrogen), transferred to hybond ECL membrane , and incubated with an apoB antibody . Primary antibody binding was detected with a goat anti-rabbit secondary antibody and visualized with chemiluminescence .Lpcat3 was generated with a sequence replacement ‘knock-out first/conditional-ready’ gene-targeting vector. The vector was electroporated into JM8A1.N3 ES cell line from C57BL/6N mice. Positive clones were identified by genotyping and long-range PCR at both the 5′ and 3′ ends. Two targeted ES cell clones were injected into C57BL/6 blastocysts to generate chimeric mice. High-percentage male chimeras were obtained, and resulting chimeras bred with female C57BL/6 mice to obtain heterozygous knockout mice (Lpcat3+/−). Lpcat3+/− mice were intercrossed to produce homozygous knockout mice (Lpcat3−/−). To create a conditional knockout allele, Lpcat3+/− mice were mated with mice expressing a Flpe recombinase deleter transgene . That transgene excises the gene-trapping cassette in intron 2 of Lpcat3, producing a conditional knockout allele containing loxP sites in intron 2 and intron 3. Lpcat3Fl/Fl mice were crossed with albumin-Cre or villin-Cre transgenic mice from The Jackson Laboratory .A conditional knockout allele for Mice were fasted for 4 hr and then injected intravenously with 500 mg/kg body weight of tyloxapol . Blood was subsequently collected, and the plasma separated by centrifugation for the measurement of TGs.Lpcat3−/−, Lpcat3fl/fl, Lpcat3fl/fl; Albumin-Cre, and Lpcat3fl/fl; Villin-Cre mice were generated as described above. All the mice were housed under pathogen-free conditions in a temperature-controlled room with a 12-hr light/dark cycle. Mice were fed a chow diet, a western diet (Research Diets #D12079B), or a high-sucrose diet (Research Diets #D07042201). Liver tissues were collected and frozen in liquid nitrogen and stored at −80°C or fixed in 10% formalin. Blood was collected by retro-orbital bleeding, and the plasma was separated by centrifugation. Small intestines were excised and cut into three segments with length ratios of 1:3:2 . For adenoviral infections, VQAd-CMV Cre/eGFP and VQAd-Empty eGFP were purchased from Viraquest. 8- to 10-week-old male Lpcat3fl/fl mice were injected (via the tail vein) with 2 × 109 plaque-forming units (pfu). Mice were sacrificed or used for TG secretion studies 3–4 weeks after the adenovirus injection. Blood was collected from mice by cardiac puncture. Plasma lipids were measured with the Wako L-Type TG M kit, the Wako Cholesterol E kit; and the Wako HR series NEFA-HR(2) kit. Tissue lipids were extracted with Bligh-Dyer lipid extraction . After an initial centrifugation at 5000×g for 15 min, most of the supernatant was removed and the yellow-brown portion (approximately the upper one-third) of the pellet was suspended in homogenization buffer, layered over 2.7 ml of 1.2 M sucrose and spin for 30 min at 100,000×g in a swinging bucket rotor (SW50.1). The Golgi fraction was collected at the buffer/1.2 M sucrose interface. The Golgi was suspended in distilled water and pelleted by centrifugation at 5500×g for 20 min. Aliquots of the Golgi fraction were used to measure TG and protein concentration. Golgi apoB was analyzed as described . Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and three reviewers, one of whom, Tobias Walther, has served as a guest Reviewing editor.Thank you for sending your work entitled “Lpcat3-dependent production of arachidonyl phospholipids is a key determinant of triglyceride secretion” for consideration at The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.All reviewers noted the high quality of data, as well as the interesting findings presented. For a number of experiments, the reviewers feel there is a need for clarification.First, the interpretation of Lpcat3 deficiency leading to membrane fluidity changes which in turn leads to the observed lipoprotein secretion defects is interesting, but not convincingly and fully shown here. Laurdan staining could reflect other changes in the membrane besides fluidity  and it is unclear really how this would lead to the phenotype described. The authors should consider revising this point and elaborate the Discussion. For example, in the Discussion the authors state that lipid changes “increase membrane fluidity and curvature”(does this mean the ER structure changes? Where does the curvature change? How do they know that happens?).Second, the experiment fractionating Golgi and microsomes should be quantified and clarified. Was tauro-cholate or another detergent used here? Earlier data from the Olofsson laboratory indicate that in the absence of the detergent significant amounts of apoB lipoproteins associated with inner leaflets of microsomes. This might indicate that the authors in their experiments may select for a subpopulation of particles. It would also be of interest to see whether the ER and Golgi apoB-lipoproteins both were skewed in buoyant density to denser particles in the absence of Lpcat3. While the reviewers agree that most lipid is added to VLDL in the Golgi, it is also possible that there are some larger particles also formed in the ER (as implied by classic EM studies by Bob Hamilton) and they may be similarly affected by Lpcat3 deficiency.Third, since the group has previously suggested that ablation of hepatic Lpcat3 expression promotes inflammatory response and ER stress it is important to address whether these conditions might cause of the phenotype observed in liver-specific Lpcat3 knockout mice. First, the interpretation of Lpcat3 deficiency leading to membrane fluidity changes which in turn leads to the observed lipoprotein secretion defects is interesting, but not convincingly and fully shown here. Laurdan staining could reflect other changes in the membrane besides fluidity  and it is unclear really how this would lead to the phenotype described. The authors should consider revising this point and elaborate the Discussion. For example, in the Discussion the authors state that lipid changes “increase membrane fluidity and curvature”(does this mean the ER structure changes? Where does the curvature change? How do they know that happens?).We acknowledge the limitations of the Laurdan studies and we have revised our Discussion to be more circumspect in the conclusions drawn from this data.Second, the experiment fractionating Golgi and microsomes should be quantified and clarified. Was tauro-cholate or another detergent used here? Earlier data from the Olofsson laboratory indicate that in the absence of the detergent significant amounts of apoB lipoproteins associated with inner leaflets of microsomes. This might indicate that the authors in their experiments may select for a subpopulation of particles. It would also be of interest to see whether the ER and Golgi apoB-lipoproteins both were skewed in buoyant density to denser particles in the absence of Lpcat3. While the reviewers agree that most lipid is added to VLDL in the Golgi, it is also possible that there are some larger particles also formed in the ER (as implied by classic EM studies by Bob Hamilton) and they may be similarly affected by Lpcat3 deficiency.We thank the reviewers for this insightful comment. We have responded in two ways. First, we used detergent (0.025% deoxycholic acid) when we isolated the Golgi luminal contents. We have now included these missing details in the Methods. Second, we have added new EM data that directly assess the size of nascent lipoprotein particle in the ER and Golgi. These studies provide definitive support for our conclusion that the particles are less lapidated in the absence of Lpcat3.Third, since the group has previously suggested that ablation of hepatic Lpcat3 expression promotes inflammatory response and ER stress it is important to address whether these conditions might cause of the phenotype observed in liver-specific Lpcat3 knockout mice.This was also an excellent point. We do not see prominent induction of ER stress responses in the Lpcat3 KO mice, and we believe that this is related to the difference between the experimental approaches (acute Lpcat3 knockdown vs genetic deletion). We have now added this data to the revised manuscript. We point out that we do in fact observe increased inflammation in the Lpcat3 KO mice in the setting of western diet feeding, and these data are included in the paper. The fact that the lipoprotein production defect is observed in the absence of inflammation (e.g. on chow diet), supports the conclusion that this defect is not secondary to inflammatory changes.