Alnus glutinosa Gaertn.) belongs to dual mycorrhizal trees, forming ectomycorrhizal (EM) and arbuscular (AM) root structures, as well as represents actinorrhizal plants that associate with nitrogen-fixing actinomycete Frankia sp. We hypothesized that the unique ternary structure of symbionts can influence community structure of other plant-associated microorganisms , particularly under seasonally changing salinity in A. glutinosa roots. In our study we analyzed black alder root bacterial and fungal microbiome present at two forest test sites  in two different seasons . The dominant type of root microsymbionts of alder were ectomycorrhizal fungi, whose distribution depended on site : Tomentella, Lactarius, and Phialocephala were more abundant at the saline site. Mortierella and Naucoria (representatives of saprotrophs or endophytes) displayed the opposite tendency. Arbuscular mycorrhizal fungi belonged to Glomeromycota , however, they represented less than 1% of all identified fungi. Bacterial community structure depended on test site but not on season. Sequences affiliated with Rhodanobacter, Granulicella, and Sphingomonas dominated at the saline site, while Bradyrhizobium and Rhizobium were more abundant at the non-saline site. Moreover, genus Frankia was observed only at the saline site. In conclusion, bacterial and fungal community structure of alder root microsymbionts and endophytes depends on five soil chemical parameters: salinity, phosphorus, pH, saturation percentage (SP) as well as total organic carbon (TOC), and seasonality does not appear to be an important factor shaping microbial communities. Ectomycorrhizal fungi are the most abundant symbionts of mature alders growing in saline soils. However, specific distribution of nitrogen-fixing Frankia (forming root nodules) and association of arbuscular fungi at early stages of plant development should be taken into account in further studies.Black alder ( The global scarcity of water resources, environmental pollution and increased salinization of soil and water are the most pressing problems of the 21st century. Salinity affects more than 7% of global land surface and 70% of all irrigated agricultural soils worldwide . CurrentAlnus glutinosa Gaertn., commonly known as black alder, belongs to this plant family and is an actinorrhizal plant that forms symbiosis with nitrogen-fixing actinomycete Frankia sp.  and arbuscular (AM) fungi at the same time  and nitrogen (N), in moderately salt-tolerant plants, e.g., black alder , particularly under seasonally changing salinity. Specifically, we hypothesize that: (i) one type of symbiosis will dominate, (ii) salinity and seasonality will affect diversity and species richness of plant-associated bacteria and fungi, and (iii) salinity can preferentially promote occurrence of certain groups of symbionts or halotolerant bacterial and/or fungal taxa. Broadening of the knowledge on alder’s endophytes can open new horizons in plant-microbial interactions occurring in forests.Urtica dioica, Stachys sylvatica, and Ranunculus lanuginosus, but also Rubus occidentalis could be found. At the S site the groundcover was even less diverse consisting almost exclusively of U. dioica.The study was carried out in mid-northern Poland at two test sites: a non-saline one in Pszczółczyn  and a saline one in Słonawy  . SalinitRoots and soil samples  were collected from two test sites (NS and S) in two seasons of 2015 . In each test site three plots (10 m × 10 m) were selected, and within them three randomly selected trees were analyzed (9 trees per site). In total, 36 samples were analyzed   by potentiometric method, and phosphorus in 1% citric acid solution (Pca) by colorimetric method. The saturation paste extracts were prepared to evaluate the soil salinity level. The electrical conductivity (ECe) was measured conductometrically and saturation percentage (SP) – gravimetrically  and total nitrogen (TN) content using a CNS Vario MAX analyzer, pH  according to the manufacturer’s protocol with the number of washing steps increased to four. Three technical replicates (independent DNA isolations) were prepared for each sample. Tubes with sterile glass beads (Mo Bio Laboratories) were used for plant material homogenization. The amount of isolated DNA was quantified fluorometrically (Qubit 2.0) and the quality was assessed spectrophotometrically (NanoDrop 2000) and the preparations were diluted to 1 ng/μl.Table 1). Mock community sample with known composition of bacterial species  was also processed. Negative control (without DNA) and positive control (Escherichia coli K12 DH10B [NC_00930.1] and Paxillus involutus [GQ389624.1] purified DNA) were included in each PCR round.Bacterial 16S rRNA and fungal ITS amplicon libraries were generated in two-step PCR, first with the specific primers bearing M13/M13R overhangs   or 53°C (fungi) – 15 s, 72°C for 20 s; then 5 min at 72°C. The PCR products were checked on 1.5% agarose gels in TBE and then they were purified using DNA Clean-Up Purification Kit (EURx) according to the manufacturer’s protocol. Next, PCR products were quantified on Qubit 2.0 and diluted to 1 ng/μl.The first PCR reaction mix consisted of: 1 ng of DNA, 5 pmol of each primer, 4 nmol of each dNTP, 0.4 U of Phusion polymerase (Thermo Scientific), 100 μg of BSA and 1× concentrated buffer with 1.5 mM MgClThe second PCR round was performed using Taq PCR Master Mix Kit (Qiagen) according to the manufacturer’s protocol. The cycling conditions were as follows: 95°C – 5 min, 14 cycles of: 95°C – 30 s, 54°C –15 s, 72°C – 30 s; and finally 72°C for 1 min. The products were checked again on 1.5% agarose gel, quantified with Qubit 2.0 (Thermo Scientific) and pooled in equimolar amounts.Table 1).Libraries were purified twice with Agencourt AMPure XP (Beckman Coulter) according to the manufacturer’s protocol. The quality of the pooled libraries was assessed on a Bioanalyzer chip (Agilent) and they were quantified with KAPA Library Quantification Kit for Illumina Platform using LightCycler 480 (Roche) according to the manufacturers’ protocols. The final pool was diluted to 4 nM, denaturated, mixed with 5% of PhiX control library and sequenced with the use of 2 × 300 cycles kit v.3 on a MiSeq machine (Illumina). Sequencing was performed using HPLC-purified versions of forward and reverse primers as well as reverse-complement of the reverse primer  based on a BLAST analysis. Matches were considered significant when the alignment spanned over 99% of query length and identity was over 99% .Fungal sequences classified down to the species level were assigned to functional groups  based on information contained in NCBI’s databases including source of isolation, host and data contained in references pertaining to a given sequence .1. Then, the BIOM file was normalized with normalize_by_copy_number.py and metagenomes were predicted as well as NSTI scores calculated with predict_metagenomes.py, using KEGG Orthology2 as the base for predictions. Finally, an.spf file was prepared with biom_to_stamp.py. Predicted metagenomes were analyzed at the KEGG Orthologs (KO) level with STAMP v.2.1.3  in fall. At the NS site the values ranged between 0.55 to 1.13 dS⋅m-1. We supposed that the low ECe values at the saline site in fall were due to heavy rainfalls occurring right before sampling, which caused increase of soil water content and decreased salinity. It was confirmed by the higher level of soil moisture (M) at the S site, close to the values of SP. Similar phenomenon was observed at the NS site (Table 2). The soil samples from the S site were more acidic  than the NS ones . Soils at both sites were mineral, but the nutrients content  was consistently higher at the NS site (Table 2).Physico-chemical soil parameters of samples are presented in 3 158 772 read pairs were obtained, of which 2 317 906 met the quality criteria and were merged. Of these, 546 961 sequences were classified as bacterial, and the rest (1 770 945) was regarded to be ITS sequences. 319 355 putative chimeras and 43 184 doubletons and singletons were removed from the bacterial dataset, leaving 184 422 sequences in the analysis (1–29 056 per sample).Alnus glutinosa), demonstrating that the primers amplify not only fungal sequences. Finally, after removal of 11 264 singletons and doubletons 264 499 sequences were included in downstream analyses (2–18 341 per sample).ITSx correctly identified fungal ITS1 region in 863 931 putative ITS sequences. After classification with naive Bayesian classifier using the UNITE database, 588 198 non-fungal sequences were culled. They originated mainly from the host . The opposite tendency was observed for fungal community, however, in fall the values were comparable at both sites, and they decreased at the S site and increased at the NS site . Parameters measured for the bacterial community were always higher than for the fungal one: diversity ∼fourfold , species richness ∼100 times , and evenness ∼threefold  .Bacterial diversity, species richness as well as evenness for OTUs constructed at 0.03 dissimilarity threshold were lower at the S site both in spring and fall , however, the observed differences between the test sites were not significant. To the contrary, significant differences were observed at the genus level, where the most abundant genera differed between the NS and S sites: Bradyrhizobium and Rhizobium were more frequent at the NS site, while an unknown actinobacterial genus, Rhodanobacter, Granulicella, and Sphingomonas were more abundant at the S site . At the level of order, sites differed significantly in abundance of Frankiales, comprising bacteria of genus Frankia, and greater abundance was observed at the S site  and Gammaproteobacteria , Actinobacteria  and Acidobacteria  . Similarly to bacteria, differences between sites and seasons at the class level were not significant, however, in general, greater abundance of Agaricomycetes was observed in fall, and Leotiomycetes were more frequent at the S site, and at the NS site in fall, while Mortierellomycotina Incertae Sedis and Sordariomycetes were most abundant at the NS site in fall, and at the S site in spring. At the genus level, Tomentella, Lactarius, Phialocephala, and Mortierella were the most frequent taxa, however, their share depended on site. Tomentella, Lactarius, and Phialocephala were more abundant at the S site, while Mortierella and Naucoria displayed the opposite tendency . Tomentella, Lactarius, Thelephora, and Naucoria were the most frequently identified ectomycorrhizal fungi in all variants of the experiment . Smaller number of sequences was noted for other EM fungal genera, such as Cortinarius or Amanita that were confounded within ‘other fungi’ group due to the low number of reads. Arbuscular mycorrhizal fungi were found in all samples and represented less than 1% of all identified fungi in each of them. They belonged to Glomeromycota .Fungal community was dominated by Agaricomycetes , Leotiomycetes , Mortierellomycotina Incertae Sedis , and Sordariomycetes  (p < 0.001) . Notably, grouping according to season was not significant (p > 0.05). CCA, a method of constrained ordination , showed that site together with Pca, pH, and TOC were significant environmental factors shaping the structure of bacterial communities in analyzed samples . Percent variance explained by the variables found in the CCA analysis was assessed with variance partitioning. Total variance explained by the variable in question is followed by variance explained solely by this variable: site – 25.00% (7.00%), Pca – 15.60% (0.10%), pH – 14.40% (3.20%), TOC – 9.30% (1.00%).Unconstrained ordination of bacterial community matrix (Bray–Curtis distance-based NMDS) demonstrated that the samples were divided according to site and this grouping appeared to be significant according to ANOSIM and PERMANOVA analyses (p < 0.001) . Similarly to bacterial communities, CCA analysis revealed significant effect of site, Pca and pH . Variance partitioning showed that season site explained 7.90% (3.70%), SP – 6.10% (3.90%), Pca – 5.80% (0.70%), pH – 5.10% (4.30%) of variance.Fungal communities, like bacterial ones, were grouped according to test site, and the grouping was significant . Sequences similar to those coming from denitrifiers were more frequent at the S site, while nitrifying and nitrogen-fixing ones were more abundant at the NS site. Counter intuitively, the potentially halotolerant taxa were more frequent at the NS site in spring (data not shown).Bacterial sequences were classified to functional groups potentially involved in nitrogen cycling based on their similarity to 16S rRNA genes of organisms of known function . The percentage of saprophytic and parasitic/pathogenic taxa appeared to be consistently low. Occurence of endophytes and mycorrhizal fungi turned out to be negatively correlated, although the correlation was not significant . Endophytes were more frequent in fall and at the NS site, while the opposite was true for mycorrhizal fungi.Fungal sequences were classified based on descriptions of species found in NCBI’s databases , five of them were represented more frequently in genomes of organisms coming from the S site. They were responsible for DNA replication and repair, biosynthesis and metabolism of pyrimidines as well as CoA biosynthesis. The remaining 61 categories were more frequent at the NS site. They were engaged in a plethora of processes, such as amino acids, peptides, carbohydrates, opines, and aromatic hydrocarbons metabolism, as well as type IV secretion, resistance to antibiotics and their biosynthesis  did not differ significantly between samples groups, nor between sites  that dynamically responds to the environment, e.g., soil quality . A recenPhoenix dactylifera. The lower average number of OTUs observed at the S site can be due to the fact that only a fraction of endophytes can thrive under conditions of increased salinity.In general, we have noted more bacterial than fungal OTUs in alder roots, which may be caused by the 10-fold greater number of bacterial than fungal species observed in most soils . ConsistPopulus deltoides. Moreover, our studies showed that Alphaproteobacteria, Actinobacteria, and Betaproteobacteria were found more frequently at the NS site, which is consistent with report of Black alder roots endophytic bacterial community was dominated by three phyla: Proteobacteria, Actinobacteria, and Acidobacteria that are the most common groups found in studies concerning soils e.g., , includiRhodanobacter, Granulicella, and Sphingomonas genera were significantly more abundant in libraries coming from black alder roots from the S site. Rhodanobacter is a Gram-negative, aerobic bacterium currently not regarded as halotolerant. However, this bacterial species was found as a relatively abundant organism in A. glutinosa nodules   was isolated from roots of silver birch and alder growing on heavy metals-contaminated sites ( legumes . However legumes . To daterasponia . Bradyrh1% NaCl)  or soil 1% NaCl) . A membeed sites . Howevered sites .Rhizobium and Bradyrhizobium genera). It might be connected to possible greater abundance of these taxa in non-saline soils, as it is known that environmental stress decreases level of Alphaproteobacteria in soils  salt-sensitive fungi may more readily enter plant roots where the environment is more stable, and (ii) plants may attract endophytes under stress conditions, as they might increase their tolerance to the stressors.These mechanisms are connected with plant reaction to abiotic stress. Differences in the outside environment put the plant metabolism out of homeostasis and impose the necessity to harbor specific genetic and metabolic traits within its cellular system . Plant mAlder’s endophytic fungal community was dominated by members of Agaricomycetes, Leotiomycetes, and Mortierellomycotina Incertae Sedis. In spite of large differences in taxa abundances between sites and seasons, statistical analysis indicated that the differences were not significant. This effect was caused by large variability among analyzed replicates, which might have been caused by specific spatial distribution of fungal mycelia, among them the mycorrhizal ones. We observed lower abundance of Agaricomycetes and Leotiomycetes at the NS site in spring. These groups comprise many ectomycorrhizal representatives that may be less abundant under conditions of high soil phosphorus content . Our anaTomentella, Lactarius, and Phialocephala members were more abundant at the S site. Tomentella was commonly found as an ectomycorrhizal partner of many trees, such as willow, birch, or alder, with tendency to dominate at unfavorable environmental conditions  and dominate the endophytic mycobiota in roots of conifers and members of the Ericaceae family in heathlands, forests, and alpine ecosystems  comprises many closely related endophytic species that colonize Alnus roots , and SP were found to be significant factors explaining variance in microbial communities. Seasonality is commonly regarded as an important parameter influencing plant-associated microbial communities . This isRhodanobacter, Sphingomonas, and Granulicella (bacteria); Tomentella, Lactarius, Phialocephala (fungi). We found a low number of sequences coming from obvious halotolerant/halophilic bacteria and fungi in our dataset. Ectomycorrhizal fungi  are an important component of endophytic community of alder roots at the saline site. The lasting challenge would be to evaluate microbiome influence on nutrients cycling and plant physiology of alder at a greater number of sites differing in salinity. This could be done by means of shotgun metatranscriptomic analysis coupled with marker amplicons sequencing both at the DNA and RNA levels.Salinity affects both bacterial and fungal diversity, albeit in different manners. Bacterial diversity decreases with salinity, while the response of fungi to this parameter is more complex. Bacterial and fungal community structure depends on salinity, and seasonality does not appear to be an important factor explaining variance in communities of root alder endophytes. There are taxa that are more abundant at the saline site: DT did all the laboratory analyses and wrote the first version of the manuscript. MG designed the sequencing system, managed the lab experiments, performed the bioinformatic and statistical analyses, and participated in the preparation of the manuscript. PH did the soil chemical analyses and interpreted the results. AP selected the areas for field experiments, analyzed the M level in soils, and interpreted the results of soil analysis. KH designed and managed the field and lab experiments as well as participated in the preparation of the manuscript. All authors revised the manuscript and approved the publication.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.


Scaffold proteins modulate signalling pathway activity spatially and temporally. In budding yeast, the scaffold Bem1 contributes to polarity axis establishment by regulating the GTPase Cdc42. Although different models have been proposed for Bem1 function, there is little direct evidence for an underlying mechanism. Here, we find that Bem1 directly augments the guanine exchange factor (GEF) activity of Cdc24. Bem1 also increases GEF phosphorylation by the p21-activated kinase (PAK), Cla4. Phosphorylation abrogates the scaffold-dependent stimulation of GEF activity, rendering Cdc24 insensitive to additional Bem1. Thus, Bem1 stimulates GEF activity in a reversible fashion, contributing to signalling flux through Cdc42. The contribution of Bem1 to GTPase dynamics was borne-out by in vivo imaging: active Cdc42 was enriched at the cell pole in hypophosphorylated cdc24 mutants, while hyperphosphorylated cdc24 mutants that were resistant to scaffold stimulation displayed a deficit in active Cdc42 at the pole. These findings illustrate the self-regulatory properties that scaffold proteins confer on signalling pathways.DOI:http://dx.doi.org/10.7554/eLife.25257.001 Scaffold proteins are multivalent binding proteins that assemble and regulate the fidelity of signal transduction cascades, conferring properties ranging from signal amplification, specificity, localisation and switch-like behaviour . HoweverSaccharomyces cerevisiae provides a useful model for understanding the spatio-temporal control of scaffold signalling, since the scaffold protein Bem1 plays an important, yet incompletely defined role in the Cdc42-dependent establishment of polarised growth that the organism displays during the cell cycle. Early in the cell cycle, Cdc42 is activated at a specific site on the plasma membrane by the GEF Cdc24. Site-specific activation of Cdc42 involves landmark proteins and the scaffold Bem1, which binds Cdc24 and Cdc42 GTP  and a phospho-mimetic mutant in which 28 sites were mutated to aspartate (cdc24-28D)  . We alsoheromone . A portiheromone . Cell-cy at 37°C . The gro-folding . The temmaterial  . The morphological defects were also evident after F-actin visualisation  and bem1-PB1 (residues 469 to 550) were amplified by PCR with NdeI and XhoI restriction sites and cloned into a modified pGEX6P-2 vector, in which the BamHI site in the multiple cloning site was changed to NdeI. This generated pDM256, pDM516 and pDM502, respectively, in which the Bem1 constructs were tagged at the N-terminus with GST that could be subsequently removed by digestion with rhinovirus 3C protease.CDC24 was amplified by PCR with NdeI and XhoI sites and ligated into pET21a to generate pDM272, in which Cdc24 is C-terminally tagged with 6xHis. The nucleotide sequence encoding amino acids 75–854 were synthesised with HindIII and KpnI restriction sites . Four constructs were synthesised: one in which the 46 serines or threonines mapped by mass spectrometry were mutated to alanine (pDM615), one in which the 46 phosphosites were mutated to aspartic acid (pDM616), one in which only the 15 Bem1-specific phosphosites were mutated to alanine (pDM780) and one in which the 15 Bem1-specific sites were mutated to aspartic acid (pDM781). The HindIII-KpnI fragments were cloned into pDK51, a URA3 yeast vector enabling the in-frame fusion of the cdc24 mutant fragments with three copies of the HA epitope to generate pDM630, pDM632, pDM782 and pDM784 , a cdc24-28D-3xHA mutant (DMY2154), a cdc24-15A-3xHA mutant (DMY2333) and a cdc24-15D-3xHA mutant (DMY2134). Since the cdc24-46D mutant was not recovered, this mutant may not be viable. The cdc24-46A and cdc24-28D mutants were amplified by PCR from genomic DNA of DMY2151 and DMY2154 yeast strains, respectively, introducing NotI and XbaI sites, in addition to a ribosome-binding site. These PCR products were cloned into pET21a, generating pDM645 and pDM647, respectively, in which the cdc24 phospho-mutants were C-terminally tagged with 6xHis.d pDM784 . OligonuCDC24, cdc24-46A and cdc24-28D were amplified by PCR from genomic DNA prepared from DMY2147, DMY2151 and DMY2154 yeast, introducing HindIII and SpeI sites. The PCR products were cloned into a modified pRS416 plasmid, consisting of a CYC1 promoter between KpnI and SalI sites. Subsequently, mEOS, in addition to an amino-terminal GAGAGG linker and a carboxyl-terminal GAGAG linker-6xHis-tag, were added to the C-terminus of the constructs using SpeI and NotI sites. Finally, an ADH1 terminator sequence was introduced using a NotI site, generating pDM700, pDM701 and pDM704 for CDC24, cdc24-46A and cdc24-28D, respectively.To monitor the in vivo localisation of the Cdc24 phospho-mutants, and localisation of Cdc42 GTP in the control and cdc24 phospho-mutant strains, we generated a gic21-208-yEGFP fusion, containing a CRIB motif that was cloned into a modified pRS413 plasmid consisting of a CYC1 promoter (pDM842)  to generate pDM401 for expression of the fusion from the AOX1 promoter in Pichia pastoris. The pRS315 CDC42prom-mEOS-GAGA-CDC42 plasmid (pDM303) was constructed in three steps: the CDC42 promoter was cloned into pRS315 on a SalI-BamHI fragment. The CDC42 ORF and terminator were then ligated into this vector using an NdeI site that was engineered 5' of the BamHI site and a 3' SacII site. Finally, mEOS was ligated into the NdeI digested plasmid and checked for the correct orientation. All plasmids were verified by sequencing.To monitor the in vivo level (pDM842) . Full-leYeast strains were generated and grown using standard genetic techniques. Cells were grown in selective or rich media, depending upon the experiment. For cell viability assays, yeast were grown in rich medium at 25°C until mid-log phase. Ten-fold serial dilutions were spotted onto YPD plates, which were incubated at the indicated temperatures for 2 days.2+-immobilised metal affinity chromatography (IMAC), followed by Strep-Tactin affinity chromatography. Ground yeast powder was generated using a Retsch PM100 grinding mill  and a 125 ml steel bowl with 20 mm ball bearings chilled with liquid nitrogen. Greater than 85% lysis was typically observed after eight disruption cycles. For Cdc42 purification, room temperature lysis buffer (20 mM Tris-HCL (pH = 8.0), 1 M NaCl, 5% glycerol, 0.5% CHAPS supplemented with EDTA-free Protease inhibitor cocktail  and 1 mM PMSF) was added when the yeast powder showed signs of melting. The lysate was then stirred immediately for 10 min at 4°C and remained at 4°C for the subsequent purification. The cell lysate was centrifuged at 48,000 x g for 30 min in a JA25.5 rotor , and the supernatant was loaded into a Co2+-IMAC column. After extensive washing (20 mM Tris-HCL (pH = 8.0), 1 M NaCl, 5% glycerol and 0.05% Tween-20), 6xHis-Strep-tag-II-Cdc42 was eluted in Co2+-IMAC elution buffer (20 mM Tris-HCL (pH = 8.0), 150 mM NaCl, 5% glycerol and 250 mM imidazole and 0.05% Tween-20). In the second purification step, Strep-Tactin Superflow Plus  was added to the elution and agitated for 30 min at 4°C. The sample was loaded onto a column and then washed extensively with Strep-Tactin wash buffer (20 mM Tris-HCL (pH = 8.0), 150 mM NaCl, 5% glycerol and 0.05% Tween-20). Nucleotide-free Cdc42 was generated by washing the column with 50 column volumes of Strep-Tactin wash buffer supplemented with 25 mM EDTA, followed by an additional washing step using Strep-Tactin wash buffer. GDP-bound Cdc42 was generated by washing the column with Strep-Tactin wash buffer supplemented with 200 μM GDP and 10 mM MgCl2, then incubating for 1 hr at room temperature. The non-bound GDP and Mg2+ were removed in an additional washing step. GDP-loaded 6xHis-Strep-tag-II-Cdc42 was eluted in Strep-Tactin elution buffer (20 mM Tris-HCL (pH = 8.0), 150 mM NaCl, 5% glycerol supplemented with 5 mM d-Desthiobiotin and 0.05% Tween-20). Nucleotide-free Cdc42, which was produced for nucleotide binding experiments, was eluted in elution buffer before the GDP loading step. Protein concentration was estimated by absorption at 280 nm using ε = 21430 M−1cm−1 as the calculated molar excitation coefficient. Samples were aliquoted in 50 μl volume and flash frozen in liquid nitrogen for storage.Nucleotide-free and GDP-loaded 6xHis-Strep-tag-II-Cdc42 was purified in a two-step affinity purification method by Co3VO4, 100 mM β-glycerol phosphate, 200 mM potassium acetate and 1 mM PMSF). After centrifugation at 110,000 x g for 90 min, the supernatant was incubated with 300 µl IgG Sepharose for 2 hr. The beads were loaded onto a 1 ml column and washed three times in 10 ml lysis buffer then in 10 ml TEV buffer (10 mM Tris-HCL (pH = 8.0), 150 mM NaCl, 0.1% IGEPAL, 0.5 mM EDTA and 1 mM DTT). TEV protease was then added overnight at 4°C. The eluate was diluted three-fold in calmodulin binding buffer (10 mM Tris-HCL (pH = 8.0), 150 mM NaCl, 0.1% IGEPAL, 1 mM magnesium acetate, 1 mM imidazole, 2 mM CaCl2, and 10 mM β-mercaptoethanol), then incubated with 300 µl calmodulin Sepharose for 2 hr. The column was washed three times in 10 ml calmodulin binding buffer then eluted in kinase assay buffer (50 mM Hepes (pH = 7.6), 2 mM MgCl2, 0.05% Tween-20, 1 mM DTT, 10% glycerol and 2 mM EGTA). The eluted kinase was dialysed in kinase assay buffer lacking EGTA then flash frozen in liquid nitrogen for storage.Active Cla4-TAP was purified from 4 L of budding yeast after lysis in liquid nitrogen using a rotary mill, as described for the Cdc42 purification. Chilled, lysed yeast powder was resuspended in three volumes of lysis buffer (10 mM Tris-HCL (pH = 8.0), 1 M NaCl, 0.1% IGEPAL, 50 mM NaF, 2 mM Na600 ~2 , 1 M NaCl, 5 mM imidazole), Cdc24-6xHis was eluted (20 mM Tris-HCL (pH = 8.0), 150 mM NaCl, 500 mM imidazole). The protein was dialysed twice (20 mM Tris-HCL (pH = 8.0), 150 mM NaCl) and flash frozen in liquid nitrogen for storage.Cdc24-6xHis and GST-tagged Bem1 were expressed in Bl21-CodonPlus (DE3) cells. Briefly, cells were grown in terrific broth at 37°C until an OD600 ~2 . ExpressFor the Bem1 purification, cells were lysed by adding room temperature lysis buffer (50 mM Tris-HCL (pH = 7.5), 1 M NaCl, 0.1% Tween-20 and 5 mM DTT, an EDTA-free Protease inhibitor cocktail tablet and 1 mM freshly prepared PMSF) to ground bacterial powder. The lysate was processed by sonication and centrifugation in the same manner as the Cdc24 −6xHis lysate. The supernatant was loaded onto a glutathione column. The column was extensively washed (50 mM Tris-HCL (pH = 7.5), 250 mM KCl, 0.1% Tween-20 and 5 mM DTT), and then GST-Bem1 was eluted (50 mM Tris-HCL (pH = 7.5), 250 mM KCl and 5 mM reduced glutathione). For GST pull-down experiments, GST-Bem1 was dialysed twice against 20 mM Tris-HCL (pH = 7.5), supplemented with 150 mM NaCl and flash frozen in liquid nitrogen for storage. For enzymatic experiments, GST-Bem1 was dialysed extensively in protease buffer (20 mM Tris-HCL (pH = 7.5), 150 mM NaCl, 1 mM DTT and 1 mM EDTA), the GST was digested using rhinovirus 3C protease then removed by re-loading the protein onto a glutathione agarose column. The flow-through, which contained Bem1, was dialysed extensively in 20 mM Tris-HCL (pH = 7.5), 150 mM NaCl then flash frozen in liquid nitrogen for storage.2, 100 nM mant-GTP, supplemented with 100 μM GMP-PNP nucleotide to maintain a low concentration of the mant-GTP fluorophore. Cdc24 was used at a final concentration of 60 nM after 30 min room temperature pre-incubation with Bem1 at 5 µM. The reaction was started by adding GDP-bound Cdc42 to 9 µM final concentration and exchange was monitored for at least 2000 s with 15 s intervals. For each sample, a mock reaction was used in the absence of GDP-loaded Cdc42 to normalise for bleaching and to subtract possible sources of background noise such as Cdc24-mant-GTP interaction. The intrinsic GDP to mant-GTP exchange rate of Cdc42 was determined in the absence of Cdc24.Förster resonance energy transfer (FRET) between Cdc42 and N-methylanthraniloyl-GTP (mant-GTP) was measured to monitor the Cdc24-mediated GDP to mant-GTP exchange reaction on Cdc42 in real time. Trp97 of Cdc42, which is in close proximity to the GTP binding site, was excited using 280 nm wavelength light using a 5 nm bandwidth. The FRET signal was detected at the emission peak of mant-GTP, at 440 nm using an 8 nm bandwidth. All fluorescence measurements were performed at 27°C on a Tecan Infinite M1000PRO plate reader  in 384-well, non-binding microplates , in a 10 μl reaction volume. The final buffer conditions were 20 mM Tris-HCl (pH = 8.0), 150 mM NaCl, 1 mM DTT, 5 mM MgClA single exponential equation  was fittI is the fluorescence intensity change, MaxI is the maximal fluorescence intensity, MinI is the minimal fluorescence intensity, obsk is the observed kinetic rate constant and t is the time in seconds.where All reactions were performed at least three times with proteins from two different purifications.A 100 nM final concentration of mant-GDP or mant-GTP were titrated with increasing concentration of nucleotide-free Cdc42 in buffer containing 20 mM Tris-HCl (pH = 7.5),150 mM NaCl and 1 mM DTT at 27°C. The interaction was monitored for 30 min by FRET at 440 nm using 280 nm wavelength light for excitation. Maximal fluorescence changes (-) were obtained by fitting a single exponential onto each trace:I is the fluorescence intensity change, MaxI is the maximal fluorescence intensity, MinI is the minimal fluorescence intensity, obsk is the observed kinetic rate constant and t is the time in seconds.where Maximal fluorescence changes of each trace were plotted against the nucleotide-free Cdc42 concentration and the dissociation constants were determined by fitting a quadratic binding equation:I is the maximal fluorescence change observed in each trace, dK is the dissociation constant, [Cdc42] is the total Cdc42 concentration and [mant] is the total mant-GDP or total mant-GTP concentration.where All reactions were performed at least three times with proteins from two different purifications.2, 10% glycerol and 1 mM ATP) at 30°C. SDS loading buffer was added to samples at the indicated times and analysed by SDS-PAGE and Western blotting using mouse anti-His antibody  and AlexaFluor532 goat anti-mouse IgG(H+L)  as primary and secondary antibodies, respectively. Fluorescence was detected using a Typhoon TRIO+ scanner . The result was analysed by ImageQuant TL 1D version 8.1 software . The time-resolved kinase reactions were repeated at least three times.For the time-resolved kinase reactions shown in 2, 10% glycerol and 1 mM ATP) at 30°C for 2 hr. Phosphorylation of Cdc24 was validated by Western blot using anti-His antibody. A 60 nM final concentration of phosphorylated samples were used directly in the GEF assay, as described above.A 600 nM final concentration of Cdc24 was treated with 3 nM Cla4 in the presence or absence of 2 μM Bem1 - under these conditions more than 80% of Cdc24 is estimated to exist in a complex with Bem1 - in kinase buffer (20 mM Tris (pH = 8.0), 2 mM MgClFor cell synchronisation, 0.5 μg/ml α-factor was added to mid-log phase yeast cultures at 25°C for 3 hr, as described previously . Cells wTo characterise the mobility of Cdc24 by SDS-PAGE during the cell cycle, 1.5 ml cells were removed at the indicated time points. After centrifugation, small glass beads were added to the pellet and the samples were flash frozen in liquid nitrogen. Samples were vigorously agitated with glass beads in 100 μl SDS loading buffer supplemented with 1 mM PMSF, 50 mM NaF and 75 mM β-glycerol phosphate. Samples were immediately boiled and analysed by SDS-PAGE and Western blotting. For actin staining, yeast were grown to early log phase, fixed and then stained with Alexa 546-phalloidin, as previously described .Equal amounts of Cdc24, cdc24-46A and cdc24-28D were added to excess amounts of GST or GST-Bem1 immobilised on glutathione agarose beads . After incubation on ice for 1 hr, beads were washed extensively  and proteins were eluted . Proteins were then TCA precipitated, and analysed by SDS-PAGE and Western blotting. All pull-down experiments were repeated three times.2, 10% glycerol and 1 mM ATP) in a 25 μl reaction volume at 30°C for 45 min. Under these conditions in the presence of Bem1, maximal Cdc24 phosphorylation was observed. After the kinase reaction, disulphide bonds in the proteins were reduced by adding DTT to a final concentration of 5 mM at 56°C for 25 min. Samples were then cooled to room temperature before alkylating cysteines at room temperature for 30 min in the dark in 14 mM of freshly prepared iodoacetamide. Excess iodoacetamide was quenched by adding DTT to 5 mM and incubating at room temperature for 15 min. In order to isolate phosphorylated Cdc24, 0.5 μg protein was analysed by 12% SDS-PAGE and Coomassie-stained Cdc24 bands were excised for trypsin digestion.In order to identify phospho-sites on Cdc24, phospho-peptides of Cdc24 after trypsin digestion were enriched by strong cation exchange chromatography, followed by IMAC, and then analysed by mass spectroscopy. For kinase reactions, a 600 nM final concentration of Cdc24 was treated with 3 nM Cla4 in the presence or absence of 2 μM Bem1 in kinase assay buffer (20 mM Tris (pH = 8.0), 2 mM MgClExcised gel bands were sliced into approximately 1 mm cubes, and destained by incubation in 50% acetonitrile/50 mM ammonium bicarbonate at 37°C. Destained gel slabs were dehydrated by incubation in neat acetonitrile, and re-hydrated in digestion buffer . Digestions were incubated overnight at 37°C. The digestion supernatant was decanted to a fresh tube, and the gel pieces were washed two times with 50% acetonitrile/50 mM ammonium bicarbonate. The washes were combined with the digestion supernatant, and evaporated via SpeedVac . Digests were resuspended in 5% formic acid/5% acetonitrile, and cleaned via c18 microextraction . SamplesCDC42. To detect Cdc42 GTP in the cdc24 phospho-mutant strains, DMY2147, DMY2151, DMY2154, DMY2333 and DMY2334 were transformed with pDM842, encoding 1-208gic2-yEGFP. Transformants were plated on selective minimal media containing dextrose and were grown at 25°C for imaging in mid-logarithmic phase.In order to visualise the Cdc24 phospho-mutant proteins in vivo, the DMY570 strain, containing Cdc24 under the control of a galactose-inducible promoter as the sole source of Cdc24 was transformed with pDM700, pDM701 and pDM704 plasmids, encoding Cdc24-mEOS, cdc24-46A-mEOS and cdc24-28D-mEOS, respectively. To visualise Cdc42 in the cdc24 phospho-mutant strains, DMY2147, DMY2151 and DMY2154 were transformed with pDM303, encoding mEOS-1-208 at the pole, the average fluorescence intensity of the pole (AFIP) and the cytosol (AFIC) were determined for each cell using an empirically determined threshold value that enabled the cell pole to be identified. Next, the AFIP was normalised as follows: normalised fluorescence intensity of the pole= (AFIP-AFIC)/AFIC). The values were plotted using Prism software.All Images were analysed and processed using ImageJ software. To calculate the enrichment of Cdc24, Cdc42 and gic2 In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Mohan Balasubramanian as Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your work entitled "Scaffold-mediated gating of Cdc42 signaling flux contributes to GTPase nanoclustering" for consideration by eLife.Our decision has been reached after extensive consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in The referees agree that the reconstitution of Cdc42-Cdc24-Bem1, using Pichia expressed Cdc42, is well done and linking the phosphorylation status to self-limitation of Cdc42 activation (i.e. GEF activity of phosphorylated Cdc24 is not activated further by Bem1) is an exciting conclusion (with the proviso that there are a number of controls that need to be done). The genetic experiments and associated imaging with the phosphomimetic mutants are interesting, but need to be strengthened.The problem is in Figures 6 and 7 (super-resolution microscopy) and this seems like a separate paper that has been appended to the rest of the work, which has technical and interpretational issues, all listed below.eLife. However, the referees believe that the in vitro reconstitution and the associated genetics could be submitted as a short-article (please take a look at eLife contribution types for more information). If you choose this approach, please address the substantive points raised by the referees pertaining to Figures 1-5 (most of which are control experiments /new experiments that should strengthen the findings). The referees’ comments are appended verbatim.In light of this, we will be unable to consider this paper further for publication in Reviewer #1:In this manuscript, Rapali et al. study the regulation of an important complex that regulates cell polarization through Cdc42 GTPase. Cdc42 is activated by its GEF Cdc24, and also binds the scaffold Bem1. The authors show that Bem1 acts in vitro in two opposing ways: it promotes Cdc24 GEF activity towards Cdc42 and also increases the rate of phosphorylation of Cdc24 by the PAK kinase Cla4. Phosphorylated Cdc24 still activates Cdc42, but cannot be promoted by Bem1. This thus likely limits the positive effect that Bem1 has on Cdc42 activation, which the authors interpret as Bem1 contributing to signaling flux through Cdc42. They also identify all phosphorylation sites on Cdc24 and analyze the effects of phospho-mutants and phospho-mimetic mutants in vivo. In a second part, the authors use super-resolution imaging approaches to probe the diffusion and cluster-forming ability of Cdc42, probing both the function of Bem1 and of Cdc24 phosphorylation sites in these processes.In my view, the first half of the manuscript is very interesting, clean and refreshing. The biochemistry is performed to high standards and largely convincing (a few missing controls are mentioned below). The interpretation that the contrasting roles of Bem1 increase flux is possible, but only one of several interpretations. For instance, another interpretation could be that Bem1 contributes to reaching rapidly a high, but not excessive Cdc42 activity. By contrast, the second half of the manuscript, including the analysis of Cdc24 phosphomutants and especially the super-resolution imaging, is less convincing and does not fit very well with the first half. It reads more like two stories lumped together. The consequence is that I do not feel the statement made by the title of the paper is well supported. Specific comments are below.Regarding the effect of Cdc24 phosphorylation status on Bem1-dependent activity enhancement, one missing control in 46A mutant are interpreted relative to Bem1-Cla4 function. However, this mutant also includes the CDK sites, and thus some effects, such as delay in budding may be due to CDK1 action. It is also unclear in the text how the 28D sites were selected. The text could also sometimes be clearer. For instance, the double-negation in paragraph five of the section is convoluted. What the authors may mean is that the phospho-blocking and phospho-mimetic mutations have distinct phenotypes and thus are likely to affect Cdc24 each in a specific manner. Clarification on all these points would help the reader.The interpretation of the Cdc24 phospho-mutants is complicated and difficult to link with the biochemistry shown in the first part of the paper. They appear to conclude from their in vivo analysis "in keeping with their in vitro biochemical observations" that the 46A is active and the 28D less active (subsection “Phospho-regulation of Cdc24 impacts polarized growth in vivo” and further). If this is "in keeping with the biochemical observations", the difference in activity in the two alleles should also be Bem1-dependent in vivo, not intrinsic to Cdc24 GEF activity , but this is not tested. More generally, all effects of the The mobility shift of Cdc24 from synchronized cultures is also difficult to interpret. The authors claim that "wild type Cdc24 undergoes a dramatic reduction in electrophoretic mobility due to phosphorylation at around 60 minutes after synchronous release from a G1-arrest". However, the The effect of Cdc24 phosphorylation on Cdc42 distribution and activity in vivo are not clear. The authors interpret the enrichment of Cdc24 and Cdc42 at the polar cap as sign of Cdc42 activity. This could be more directly evaluated by using probes for Cdc42-GTP, such as CRIB, which would be predicted to be in increased amounts in the 46A mutant, and decreased in the 28D mutant. Pull-down of Cdc42-GTP could also be performed to probe this point.Regarding the super-resolution analysis of Cdc42, one major question I have is which version of mEos is being used? Some versions have the tendency to dimerize, which would make fusion proteins prone to oligomerization. For instance, mEos2 has been shown to still dimerize, which raises questions about mEos, from which mEos2 is derived. mEos3.1 and 3.2 appear to be truly monomeric. Because the authors do not have a Cdc42 mutant that blocks cluster formation, it is difficult to be very conclusive about the validity of the observed nanoclusters. I also feel that the link between nanocluster size and Cdc42 activity status is unclear and not directly supported by evidence. The 46A and 28D mutants show changes in Cdc42 cluster sizes at cell poles, which the authors interpret as a link between Cdc42 activity and cluster size, but (as mentioned above) they have not directly tested Cdc42 activity in these mutants in vivo. To probe for a link between activity and cluster size, the authors should look at the distribution of constitutively active and inactive Cdc42 mutants.I have similar difficulties with the analysis of diffusion coefficients. The differences between wt and bem1∆ are also very slight, which does not help to affirm the position that the observed nanoclusters are physiologically relevant. I find for instance surprising that, in Figure 6A-B, the diffusion coefficients seem to be altered in bem1∆ both at the pole and in the rest of the cell compared to wildtype . I would expect a faster diffusion at the pole of bem1∆ cells, as shown , but not a slower diffusion elsewhere. More generally, the analysis of Cdc42 trajectories is not particularly convincing. To convince that there are actually four distinct classes (as opposed to a continuum), the authors should show a plot of frequencies versus α value, which should reveal four distinct peaks/modes. From the examples shown, I am for instance not convinced that there is any directed movement: the examples show only two data points in a line before a more confined-looking behavior.Reviewer #2:In this manuscript the authors study the role of the scaffold protein Bem1 and its interactions with the Cdc42 GEF Cdc24 and the PAK kinase Cla4, in the context of budding yeast polarization. The first part of the manuscript focuses on biochemical experiments with purified proteins and shows that Bem1 increases GEF activity of Cdc24 on Cdc42 and at the same time supports phosphorylation of Cdc24 by the PAK kinase Cla4. Interestingly, the authors then find that phosphorylated Cdc24 can no longer be stimulated by Bem1, providing a possible self-limiting loop to Cdc42 activation – a critical feature for efficient cell polarization that has remained elusive on the molecular level in all existing models of spontaneous symmetry breaking. The authors then confirm the relevance of the identified reactions in vivo, using a Cdc24 mutant lacking 46 MS-mapped phosphorylation sites. In particular, using single particle tracking of Cdc42 in cells they propose that Bem1 and Cdc24-phosphorylation contribute to dynamics of Cdc42 by affecting nanocluster formation at the polarization site.eLife. However, there are a couple of issues that definitely need to be clarified or improved before publication (see major points below). There are also a couple of technical concerns that can be easily fixed.The topic of this study connects central aspect of signal transduction and cell polarization research. Bem1 has been proposed to be a key factor for a positive feedback loop driving symmetry breaking and the earliest steps in yeast polarization. However, the specific mechanisms and consequences of Bem1-Cdc24-Cla4 interactions have been subject to a long and controversial debate in the field. The current study clarifies a few important molecular aspects of this debate in a convincing manner, and provides elegant in vivo support for their main biochemical findings. I therefore think that this work is of great relevance and provides the kind of combination between conceptual advance and technical innovation aimed for at 46A mutant + Dcla4 .1) The demonstrations of Bem1 effects on Cdc24 activity and phosphorylation are convincing but they mostly confirm or incrementally improve on previous studies. The idea of a self-limitation and negative effect of phosphorylation on GEF-stimulation is however a really new and therefore very important part of this study. As I interpret the current results the negative effect on Bem1-stimulation could either be due to the phosphorylation itself (as the authors suggest) or due to the presence of Cla4  The second key point of this study is the proposed effect of Bem1 on Cdc42 nanocluster formation. While I have no doubts that such clustering occurs for Cdc42 – as it probably does for every protein of the PM  – the proposed link to Bem1 and phosphorylation is less convincing to me. My main concern stems from the single molecule localization analysis. The signal density on the cell surface (for both Cdc42 and Cdc24) seems very low, especially in the non-polarized regions. This could be due to the limitations of mEOS2  compared to chemical dyes used in STORM applications. How can the authors exclude that the cluster results are affected by labeling density (larger cluster in the denser polarized cap area)? This would perfectly correlate with the observed effects (increase at pole for 42A). An additional concern is the current resolution limit (40 nm), which is very close to the identified cluster size (76 nm). Considering additional factors such as mobility of the fluorophore (GAGA linker) and variable polarization of fluorescence  clusters might not be readily resolvable any more. In summary, at the moment the shown PALM data definitely needs some sort of additional validation. Possible experimental controls with/without predicted clustering would be Cdc42 in LatA treated cells, constitutive active Cdc42 or markers of known clusters such a eisosomes  or endocytic patches .3) A majority of the track in Figure 6 seem to occur at the edge of cells. If using HiLo (beter to use TRIRF) the focus should be on the actual cell surface so that all tracks can be followed in 2D. At the moment I see a bias to shorter tracks as the movement in z can no longer be tracked. Also the observed differences (pole vs. rest) are not very strong and the effect of Bem1 deletion seems to be rather increasing motility in non-polarized areas than decreasing the polar motility. To support the single particle tracking it would therefore be important to provide an additional approach. The authors could perform careful FRAP experiments in cap vs. non-polarized regions of the cell. This should allow detection of the observed effects. After all, the average MSD that was obtained is quite similar to the effective FRAP recovery rate.4) The current data support a reduction in stimulation of Cdc24 by Bem1 after Cla4-mediated phosphorylation. To really support the sequence of events proposed in the model , Cla4 would need to act after Bem1 recruitment (fits the idea of Bem1 as recruitment platform for Cla4). Can the authors perform such an experiment with their reconstituted system? 1. Measure basal GEF activity, 2. Add Bem1 for 30 min, 3. Measure stimulated GEF activity, 4. Add Cla4 (high levels) for 1 h, 5. Measure GEF activity again to see basal levels again. This would provide a perfect final experiment and nicely relate to the proposed model.Reviewer #3:This manuscript investigates how the Cdc42 scaffold Bem1 regulates Cdc42 activation via positive and negative regulation in the budding yeast model system. Reports have shown that Cdc42 activation undergoes oscillations/fluctuations during polarization in the fission yeast  and budding yeast  model systems. These findings indicated that Cdc42 undergoes both positive feedback as well as a time delayed negative feedback. Further Das et al., 2012 suggested that the negative feedback was mediated by the Cdc42 target Pak1 kinase and in Pak1 kinase mutants Cdc42 activation and GEF-scaffold complex (Scd1-Scd2) is increased at the cell tips leading to increased Cdc42 activation and dampening of Cdc42 pulsing at these sites. Later Kuo et al., 2014 demonstrated that Cdc24 is phosphorylated by the kinase Cla4 and this leads to a decrease in Cdc24 GEF activity. This manuscript provides further evidence that Cla4 phosphorylates Cdc24 and thus leading to dampening of GEF activity. The authors claim that Bem1 promotes Cla4 mediated phosphorylation of Cdc24 and describe this as a "Bem1 dependent mechanism". They also claim that this leads to desensitization of Cdc24 to Bem1 interaction. The evidence in support of these claims is not strong. Further, in support of previous reports, the authors use single particle tracking to show that the Cdc42 complex at the cell tips is less dynamic in the presence of Bem1. The authors suggest that Cdc24 phosphorylation decreases clustering of Cdc42 complexes at sites of polarization. While the findings presented here do not provide a novel insight into how Cdc42 is negatively regulated, it does provide additional evidence for previous findings.1) The authors state that phosphorylation of Cdc24 reduces its activity, but do not directly test whether this is due to decreased affinity between Cdc24 and Cdc42. While the authors test the GEF activity of Cdc24 in various mutants, they fail to use the most direct  approach of measuring Cdc42 activity in vivo. Later they present phenotypes of these mutants, but only indirectly link them to Cdc42 by observing cell morphology and cytoskeleton organization .2) Previous reports  have shown that the Pak kinase forms a complex with Cdc42-Cdc24-Bem1 to positively regulate Cdc42 activation and constitutes a positive feedback loop that leads to symmetry breaking. The authors have not discussed their findings in the context of this report. Moreover, Cdc24-Cla4 fusion was sufficient to normally activate Cdc42 suggesting that Bem1 acts as a scaffold and may stabilize the Cdc42 ternary complex. This could explain increased Cdc24 phosphorylation in the presence of Bem1. Thus it is possible that Bem1 does not specifically influence Cla4 phosphorylation but rather any scaffold capable to bringing together Cdc42-Cdc24-Cla4 in a complex would work.3) The authors include Cdk1 phosphorylation sites in their D and A cdc24 mutants, but do not describe whether these residues are phosphorylated during bud emergence, which would indicate their relevance. Inclusion of these residues in the S and A cdc24 mutants is problematic, because it prevents the changes in protein activity and localization to be solely attributed to the proposed Bem1-Cdc24-Cla4-dependent mechanism. Additionally, the authors do not provide rationale stating why only 28 residues were mutated in the D mutants, while 46 were used in the A mutants.Cdc24-46A mutants.4) The authors do not mention time-delayed negative feedback regulation of Cdc42 as reported by Das et al. 2012 and Howell et al., 2012. Could their findings provide a molecular explanation for this feedback regulation? Kuo et al. 2014 have shown that Cdc24 non-phosphorylatable mutants show stabilized Cdc42 activation during bud emergence. Do the authors expect a similar phenotype with their cdc24-28D and 46A mutants? This would further explain the outcome of the Cdc24 phosphorylation on Cdc42 dynamics and polarization.5) How does Cdc42 dynamics change with 6) If Pak mediated Cdc24 phosphorylation does not alter GEF activity or Cdc24-Bem1 interaction what exactly does Cdc24 phosphorylation lead to? The authors claim that Cdc24 phosphorylation renders it insensitive to Bem1. However, they also show that Cdc24 non-phosphorylatable Cdc24 requires Bem1 for enhanced GEF activity. While these findings are not necessarily contradictory, they do lead to questions about how Cdc24 responds to Bem1 interaction. The authors claim that Cdc42 phosphorylation does not disrupt Cdc24-Bem1 interaction. Could it be that Cdc24 phosphorylation destabilizes that Cdc24-Cdc24-Bem1-Cla4 ternary complex instead?[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your revised article has been favorably evaluated by Tony Hunter as the Senior editor, Mohan Balasubramanian as Reviewing editor, and three reviewers.Thank you for resubmitting your work entitled "Scaffold-mediated gating of Cdc42 signaling flux" for further consideration at The referees have found your revised short format article significantly improved and are satisfied that you have provided a compelling biochemical reconstitution of Cdc42 GTPase signaling pathway, especially around the negative feedback regulation of the pathway and the role of Cla4 phosphorylation of Cdc24-GEF in a Bem1 dependent manner.However, a single major and several minor points have been raised. I would like you to address the major point highlighted below, without fail. I will leave it to your judgement about the rest of the minor points, although several of these are very easily fixed with writing, doing which will improve the readability.Essential revisions:The authors have also added a more direct measure of Cdc42-GTP local levels by use of the Gic2 CRIB localization. They report that CRIB levels are higher in the Cdc24-A mutant and undetectable in the Cdc24-D mutants . This is [Editors’ note: the author responses to the first round of peer review follow.]Reviewer #1:In this manuscript, Rapali et al. study the regulation of an important complex that regulates cell polarization through Cdc42 GTPase. Cdc42 is activated by its GEF Cdc24, and also binds the scaffold Bem1. The authors show that Bem1 acts in vitro in two opposing ways: it promotes Cdc24 GEF activity towards Cdc42 and also increases the rate of phosphorylation of Cdc24 by the PAK kinase Cla4. Phosphorylated Cdc24 still activates Cdc42, but cannot be promoted by Bem1. This thus likely limits the positive effect that Bem1 has on Cdc42 activation, which the authors interpret as Bem1 contributing to signaling flux through Cdc42. They also identify all phosphorylation sites on Cdc24 and analyze the effects of phospho-mutants and phospho-mimetic mutants in vivo. In a second part, the authors use super-resolution imaging approaches to probe the diffusion and cluster-forming ability of Cdc42, probing both the function of Bem1 and of Cdc24 phosphorylation sites in these processes.In my view, the first half of the manuscript is very interesting, clean and refreshing. The biochemistry is performed to high standards and largely convincing (a few missing controls are mentioned below). The interpretation that the contrasting roles of Bem1 increase flux is possible, but only one of several interpretations. For instance, another interpretation could be that Bem1 contributes to reaching rapidly a high, but not excessive Cdc42 activity. By contrast, the second half of the manuscript, including the analysis of Cdc24 phosphomutants and especially the super-resolution imaging, is less convincing and does not fit very well with the first half. It reads more like two stories lumped together. The consequence is that I do not feel the statement made by the title of the paper is well supported. Specific comments are below.cdc24 phospho-mutants in light of the comments raised during review. These are explained in detail below.We thank the reviewer for their encouraging comments. The title of the manuscript has now been modified to reflect the focus on the biochemistry. We have also performed numerous additional experiments and controls to analyze the Regarding the effect of Cdc24 phosphorylation status on Bem1-dependent activity enhancement, one missing control in As requested, we performed GEF reactions with 0.2 µM Bem1 and have included the data in new The interpretation of the Cdc24 phospho-mutants is complicated and difficult to link with the biochemistry shown in the first part of the paper. They appear to conclude from their in vivo analysis "in keeping with their in vitro biochemical observations" that the 46A is active and the 28D less active (subsection “Phospho-regulation of Cdc24 impacts polarized growth in vivo” and further). If this is "in keeping with the biochemical observations", the difference in activity in the two alleles should also be Bem1-dependent in vivo, not intrinsic to Cdc24 GEF activity , but this is not tested. More generally, all effects of the 46A mutant are interpreted relative to Bem1-Cla4 function. However, this mutant also includes the CDK sites, and thus some effects, such as delay in budding may be due to CDK1 action. It is also unclear in the text how the 28D sites were selected. The text could also sometimes be clearer. For instance, the double-negation in paragraph five of the section is convoluted. What the authors may mean is that the phospho-blocking and phospho-mimetic mutations have distinct phenotypes and thus are likely to affect Cdc24 each in a specific manner. Clarification on all these points would help the reader.cdc24 alleles in a bem1 mutant. In new BEM1 in cdc24-46A or cdc24-28D strains resulted in an indistinguishable temperature sensitive growth phenotype at 37ºC  The demonstrations of Bem1 effects on Cdc24 activity and phosphorylation are convincing but they mostly confirm or incrementally improve on previous studies. The idea of a self-limitation and negative effect of phosphorylation on GEF-stimulation is however a really new and therefore very important part of this study. As I interpret the current results the negative effect on Bem1-stimulation could either be due to the phosphorylation itself (as the authors suggest) or due to the presence of Cla4  The current data support a reduction in stimulation of Cdc24 by Bem1 after Cla4-mediated phosphorylation. To really support the sequence of events proposed in the model , Cla4 would need to act after Bem1 recruitment (fits the idea of Bem1 as recruitment platform for Cla4). Can the authors perform such an experiment with their reconstituted system? 1. Measure basal GEF activity, 2. Add Bem1 for 30 min, 3. Measure stimulated GEF activity, 4. Add Cla4 (high levels) for 1 h, 5. Measure GEF activity again to see basal levels again. This would provide a perfect final experiment and nicely relate to the proposed model.We agree that this is a great test of the model, but it has been extremely technically challenging. We perform the assays in 10 µl volumes, so the extended times required to extract the Kobs from the reaction curves meant that the reactions become prone to evaporation. The problems are not remedied by increasing the reaction volumes, since the next problem encountered is judging the effect of phosphorylation. The effect of adding Bem1 to the GEF activity is clear, but the effect of adding kinase is to subtly dampen the rate of GEF activity. There are additional problems here: most of the substrate, Cdc42, has been consumed early in the reaction, resulting in increased signal-to-noise as the reaction proceeds. Moreover, the reaction rate can no longer be fit to a single exponential, but must instead be judged manually. In conclusion, we cannot currently do this experiment, and in fact, it was for this reason that we initially broke this complex reaction into more approachable constituents.Reviewer #3:[…] 1) The authors state that phosphorylation of Cdc24 reduces its activity, but do not directly test whether this is due to decreased affinity between Cdc24 and Cdc42. While the authors test the GEF activity of Cdc24 in various mutants, they fail to use the most direct  approach of measuring Cdc42 activity in vivo. Later they present phenotypes of these mutants, but only indirectly link them to Cdc42 by observing cell morphology and cytoskeleton organization .cdc24- 46A mutant compared to wild type CDC24, while the levels of active Cdc42 in the cdc24-28D mutant were below the detection limit of our imaging system. Thus, the in vitroGEF assays, actin cytoskeleton staining, Cdc24 localization, Cdc42 localization and active Cdc42 localization are consistent.We have now analyzed a marker for active Cdc42 in vivo, as requested by the reviewer. The results are presented in new 2) Previous reports  have shown that the Pak kinase forms a complex with Cdc42-Cdc24-Bem1 to positively regulate Cdc42 activation and constitutes a positive feedback loop that leads to symmetry breaking. The authors have not discussed their findings in the context of this report. Moreover, Cdc24-Cla4 fusion was sufficient to normally activate Cdc42 suggesting that Bem1 acts as a scaffold and may stabilize the Cdc42 ternary complex. This could explain increased Cdc24 phosphorylation in the presence of Bem1. Thus it is possible that Bem1 does not specifically influence Cla4 phosphorylation but rather any scaffold capable to bringing together Cdc42-Cdc24-Cla4 in a complex would work.The reviewer raises the complexity of the budding yeast polarity system, which has generated considerable debate in the field . As the reviewer rightly points out, in the Kozubowski manuscript, the role of Bem1 in symmetry breaking was attributed to its tethering of Cdc24 and Cla4, since a Cdc24-Cla4 fusion bypassed the requirement for Bem1 and Bud1 in polarity establishment. This argued that Cla4 plays a positive role in polarity establishment. However, the role of Cla4 has since been shown to be more complex, as the Lew lab have published two manuscripts in which Cla4 was found to contribute to negative feedback during polarity establishment (PMID 24631237 and 26523396). The two findings are not incompatible; it is possible that Cla4 phosphorylates a subset of Cdc24 sites that boost Bem1-dependent GEF activity, but that the ensuing phosphorylation of all sites attenuates this stimulation. A similar mechanism has been reported for the Cdk1 inhibitor Wee1 in budding yeast (PMID 16096060). The initial phosphorylation of Wee1 by Cdk1 activates Wee1, while subsequent full phosphorylation of Wee1 by Cdk1 is inhibitory. It is not possible for us to perform the exhaustive mutational analyses required to test this hypothesis in the short article format of our resubmission, but we have cited the Kozubowski paper, as requested. In the Discussion section, we have now added that, "While a previous study found that the PAK Cla4 played a positive role in polarity establishment in S. cerevisiae , subsequent work found that phosphorylation of Cdc24 by Cla4 resulted in reduced GEF activity, while increased Cdc42 recruitment to the cell pole was observed in Cdc24 phosphorylation mutants ."The reviewer asks if any scaffold that tethers Cdc42-Cdc24-Cla4 might suffice to stimulate Cdc24 phosphorylation. We have not tested whether other scaffolds might induce the same pattern of phosphorylation in Cdc24 as Bem1 and we have stated this in the Discussion section of the resubmitted manuscript: "It will also be important to test whether any scaffold that increases the local Cdc24-Cla4 concentration would stimulate Cdc24 phosphorylation, or whether Bem1 exerts specific effects." While Bem1 presumably does increase the local Cdc24 concentration available for phosphorylation by Cla4, our data suggest that it does so in a specific fashion. Even when Cla4 and Cdc24 are incubated together for extensive periods (6 hours), the extent of Cdc24 phosphorylation is markedly reduced compared with the inclusion of Bem1 in the reaction. Since no other proteins are present in these reactions, it seems likely that such extensive incubations in the absence of Bem1 would eventually result in full phosphorylation of Cdc24, but it does not. This point was also borne out by our mass spectrometry analysis in which we identified Bem1-specific phosphorylation sites in Ccd24.3) The authors include Cdk1 phosphorylation sites in their D and A cdc24 mutants, but do not describe whether these residues are phosphorylated during bud emergence, which would indicate their relevance. Inclusion of these residues in the S and A cdc24 mutants is problematic, because it prevents the changes in protein activity and localization to be solely attributed to the proposed Bem1-Cdc24-Cla4-dependent mechanism. Additionally, the authors do not provide rationale stating why only 28 residues were mutated in the D mutants, while 46 were used in the A mutants.cdc24-15A and cdc24-15D. These mutants resulted in enrichment of Cdc42-GTP at the pole in the case of the cdc24-15A mutant  The authors do not mention time-delayed negative feedback regulation of Cdc42 as reported by Das et al. 2012 and Howell et al., 2012. Could their findings provide a molecular explanation for this feedback regulation? Kuo et al. 2014 have shown that Cdc24 non-phosphorylatable mutants show stabilized Cdc42 activation during bud emergence. Do the authors expect a similar phenotype with their Cdc24-46A mutants.cdc24-46A mutant.This is a very good point and is something that we are currently investigating using standard widefield imaging and SPT-PALM. The reviewer is correct that the current models from the Verde and Lew labs would predict that Cdc42 might be stabilized in the 5) How does Cdc42 dynamics change with cdc24-28D and 46A mutants? This would further explain the outcome of the Cdc24 phosphorylation on Cdc42 dynamics and polarization.cdc24-46A mutant, both at the pole and non-pole, but this data has been removed from the resubmitted manuscript at the request of the editor.We have only looked at Cdc42 dynamics in the mutants by single particle tracking. Cdc42 diffusion was reduced in the 6) If Pak mediated Cdc24 phosphorylation does not alter GEF activity or Cdc24-Bem1 interaction what exactly does Cdc24 phosphorylation lead to? The authors claim that Cdc24 phosphorylation renders it insensitive to Bem1. However, they also show that Cdc24 non-phosphorylatable Cdc24 requires Bem1 for enhanced GEF activity. While these findings are not necessarily contradictory, they do lead to questions about how Cdc24 responds to Bem1 interaction. The authors claim that Cdc42 phosphorylation does not disrupt Cdc24-Bem1 interaction. Could it be that Cdc24 phosphorylation destabilizes that Cdc24-Cdc24-Bem1-Cla4 ternary complex instead?We apologize for giving the wrong impression on this point. The pulldown experiment that we present only allows us to conclude that phosphorylation of Cdc24 does not markedly influence its affinity for Bem1. Since the sensitivity of a pulldown assay is limited, further biophysical investigation will be required to fully address this point. It is possible that phosphorylation may increase the off-rate of Cdc24 for Bem1 or decrease the on-rate, but our results do not currently enable definitive conclusions on this point. Similarly, we are looking at how phosphorylation may affect the ternary Cdc42-Cdc24-Bem1-Cla4 complex. However, the labile nature of the complex, involving low affinity interactions between most components, makes this a very challenging project.[Editors' note: the author responses to the re-review follow.]Essential revisions:The authors have also added a more direct measure of Cdc42-GTP local levels by use of the Gic2 CRIB localization. They report that CRIB levels are higher in the Cdc24-A mutant and undetectable in the Cdc24-D mutants . This iscdc24-Ala and cdc24-Asp phosphorylation site mutants. Importantly, the optimization of this Cdc42-GTP reporter enabled us to ensure that quantitative imaging was only performed on polarizing cells, as requested by the reviewers. The conclusion of these experiments is unchanged from our previous submission, but we are now able to present corroborating quantitative analyses for the cdc24-28D and cdc24- 15D mutants.We redesigned the marker used to assay levels of Cdc42-GTP in vivoand we repeated the experiments shown in


P > 0.05). Our present study found that, with the help of CAD software, it is feasible to design a TPTD screw custom drill guide that could guide the accurate TPTD screw trajectory on 3D-printed lumbar models.Transpedicular transdiscal screw fixation is an alternative technique used in lumbar spine fixation; however, it requires an accurate screw trajectory. The aim of this study is to design a novel 3D-printed custom drill guide and investigate its accuracy to guide the trajectory of transpedicular transdiscal (TPTD) lumbar screw fixation. Dicom images of thirty lumbar functional segment units  of L1–L4 were acquired from the PACS system in our hospital  and imported into reverse design software for three-dimensional reconstructions. Images were used to print the 3D lumbar models and were imported into CAD software to design an optimal TPTD screw trajectory and a matched custom drill guide. After both the 3D printed FSU models and 3D-printed custom drill guide were prepared, the TPTD screws will be guided with a 3D-printed custom drill guide and introduced into the 3D printed FSU models. No significant statistical difference in screw trajectory angles was observed between the digital model and the 3D-printed model ( Bilateral pedicle screw (BPS) fixation was recognized as the “gold standard” surgical technique for spinal arthrodesis ; howeverThe three-dimensional (3D) printing technique was developed in recent decades . It faciIt was reported that with CT DICOM data, the 3D digital model can be reconstructed and used for the printing of accurate 3D models . Lu et aThirty CT DICOM data sets of double lumbar segments  were exported from the PACS system. The CT data were confirmed to be without any spinal abnormality such as fracture, tumor or spinal deformity. First, the CT DICOM data were imported into the reverse design software for three-dimensional reconstruction, the threshold value was set at “Bone (CT)”, “226-Max”, which is optimal for bone reconstruction; the “Max” value is auto recognized by reverse design software dependent on the bone mineral density of participants. After the 3D digital images were reconstructed, the data were saved in an STL (stereolithography) format. Second, the 3D digital images in STL format were imported into CAD software. As reported in a previous study , we simuAfter the optimal TPTD screw trajectory was simulated, the feature of the bony surface around the TPTD screw entry point was extracted, and a new surface with the best fit to the bony surface was created . The cusThe TPTD screw custom drill guide in STL format was imported into the 3D-printing code software, the position was adjusted, saved in Gcode format, and imported into the 3D printer ; curable resin was used to print the plastic TPTD screw custom drill guide . At the The 3D-printed lumbar model was fixed at the base of support on the table, and the TPTD screw custom drill guide was placed on the TPTD screw entry point. To introduce the 1.2 mm diameter K-wire (Kirschner wire), a guide tube with a 5.5 mm or 6.0 mm outer diameter and a 1.3 mm inner diameter was inserted inside the hollow custom drill guide, then a 1.2 mm diameter K-wire with a sharp tip was placed through the guide tube and forward to the intervertebral space and upper vertebral body using a power bit drill . Once thThe radiographic lateral and anteroposterior (AP) films of the TPTD screw fixed 3D printed lumbar models were obtained at this time . Four anThis research was performed in accordance with the principles described in the Declaration of Helsinki and was approved by the Institutional Ethics Review Board of The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University (No. 2015-30). Written informed consent was obtained from all participants.t-tests, with the level of significance set at P < 0.05.The data were analyzed with statistical analysis software. The “α” and “β” angles of the simulated trajectory from the CAD software and the “α1” and “β1” angles of the trajectory from radiographic films of the 3D-printed model were compared by paired P = 0.138 (Left α vs. α1), 0.516 (Right α vs. α1), 0.133 (Left β vs. β1), 0.674 (Right β vs. β1)). All TPTD screws were successfully introduced into the 3D-printed model guided by our novel TPTD screw custom drill guide. The angle values of the simulated trajectory in 3D digital images and the screw trajectory in 3D-printed models are shown in Internal screw fixation has been widely used in spinal surgery. Many methods can be used to place an internal screw; some surgeons prefer to place the pedicle screw by free hand, referring to the anatomic landmarks . To imprFor TPTD screw fixation, the trajectory was different from the transpedicular screw; the entry point was lower, and the screw should be introduced obliquely upward to meet the superior margin of the pedicle where it joins the vertebral body. Therefore, it is more challenging to achieve an optimal TPTD screw . BirkenmHowever, the intra-operative radiation exposure associated with C-arm or O-arm use could increase the risk of cancer in surgeons . SeveralIn this study, the novel TPTD screw custom drill guide was designed based on the CT scan data. Compared to the custom drill guide in studies by The creation of an accurate fitted-surface to bony surface was important to achieve an accurate screw trajectory. The CAD software was used to design the TPTD screw custom drill guide in this study; the “Shape Sculptor” and “Quick Surface Reconstruction” functions could help us extract the aimed bony surface conveniently and accurately. Additionally, the custom drill guide designed in CAD software could be exported in STL format, which is the exact format used for further 3D printing.in vitro study; the lumbar model used in this study was a 3D-printed model, not a cadaveric specimen. 3D-printing technology could provide an accurate morphometric lumbar spinal model (There were some limitations of this study. First, this was an al model , and theal model . Second,The construction of a TPTD screw custom drill guide was convenient with CAD software. Additionally, the 3D-printed TPTD screw custom drill guide could help us achieve an accurate TPTD screw trajectory at a lower cost and could reduce the radiation exposure for clinical investigations.10.7717/peerj.3564/supp-1Supplemental Information 1Raw dataClick here for additional data file.