Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1VEGF−). Nrp1VEGF− mutants survive to adulthood with normal vasculature revealing that NRP1 functions independent of VEGF-NRP1 binding during developmental angiogenesis. Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2. Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.During development, tissue repair, and tumor growth, most blood vessel networks are generated through angiogenesis. Vascular endothelial growth factor (VEGF) is a key regulator of this process and currently both VEGF and its receptors, VEGFR1, VEGFR2, and Neuropilin1 (NRP1), are targeted in therapeutic strategies for vascular disease and cancer. NRP1 is essential for vascular morphogenesis, but how NRP1 functions to guide vascular development has not been completely elucidated. In this study, we generated a mouse line harboring a point mutation in the endogenous DOI:http://dx.doi.org/10.7554/eLife.03720.001 Blood flows through blood vessels to carry oxygen and nutrients towards, and waste away from, the cells of the body. New blood vessels are formed not only during development but also throughout life as part of normal tissue growth and repair. However, blood vessels may also form as a consequence of diseases, such as cancer. For example, tumors often stimulate the growth of new blood vessels to ensure a good supply of blood carrying nutrients and oxygen. As such, some anti-cancer therapies try to stop blood vessels from developing in an attempt to slow down or prevent tumor growth.New blood vessels often form by branching off from existing vessels. One molecule that stimulates this branching process is called vascular endothelial growth factor (or VEGF for short). Three ‘receptor’ proteins found on the outside of cells can bind to the VEGF molecule and then trigger a response inside the cell that guides the development of new blood vessels. VEGF and its receptor proteins—including one called NRP1—are being investigated as a possible target for drugs that could treat cancer and other diseases affecting blood vessels. However, the exact mechanisms that control the formation of new blood vessels are not fully understood, which makes it difficult to develop these treatments.Now Gelfand et al. have created mice whose NRP1 receptors cannot bind VEGF. These mice unexpectedly survive to adulthood and develop normal blood vessels. This outcome is in contrast to mice that lack NRP1, which normally die as embryos and have severe defects with their nerves and blood vessels. Gelfand et al. instead found that mice that only lack NRP1 in the cells of their blood vessels had less of another receptor protein called VEGFR2 on the surface of these cells. This result suggests that NRP1 controls blood vessel development, not by binding to VEGF but by affecting how much of the VEGFR2 receptor is available to interact with VEGF.These findings challenge the long-held view of how NRP1 functions and lead Gelfand et al. to suggest a new mechanism: NRP1 interacts with VEGFR2, rather than with VEGF, to control the formation of new blood vessels. Future work will aim to uncover how these interactions regulate the normal development of blood vessels, and if other molecules that bind to NRP1 are involved in this process. Furthermore, these findings may help to guide the on-going efforts to develop drugs that target NRP1 into treatments that are effective against diseases that involve problems with blood vessels—including diabetes, immune disorders, and cancer.DOI:http://dx.doi.org/10.7554/eLife.03720.002 Nrp1−/− mice are embryonically lethal with both neural and vascular defects  recapitulate the devastating vascular defects observed in Nrp1−/− mice—the vascular network is poorly developed and large endothelial cell aggregates form within the brain . Surprisingly, although VEGF-NRP1 binding was successfully eliminated, the Nrp1VEGF− mutants survived into adulthood and did not display any of the severe vascular phenotypes seen in either the Nrp1−/− or the endothelial-specific NRP1 knock-out. Upon closer examination, NRP1-deficient blood vessels in the endothelial-specific NRP1 knock-out exhibited reduced VEGFR2 surface expression, a phenomenon not observed in the Nrp1VEGF− mutant. These results challenge the well-accepted view that NRP1 requires VEGF-NRP1 binding to facilitate developmental angiogenesis and points to a provocative new hypothesis that the angiogenic role of NRP1 lies in its capacity as a VEGFR2 co-receptor. Interestingly, retinal angiogenesis and blood flow recovery following hindlimb ischemia were mildly perturbed in the Nrp1VEGF− mutant suggesting that the postnatal vascular system is uniquely sensitive to the loss of VEGF-NRP1 binding. Together, this work not only significantly advances our basic scientific understanding of how NRP1 functions in VEGF-mediated angiogenesis, but also provides new insights that may facilitate the development of more effective NRP1-targeted anti-angiogenesis therapies.To delineate the role of VEGF-NRP1 interactions, we identified a single amino acid residue in the b1 domain of NRP1 that is necessary for VEGF-NRP1 binding and generated a mouse harboring this point mutation to abolish VEGF-NRP1 interactions in vivo  successfully abolished VEGF-NRP1 binding while conserving AP-SEMA3A binding as demonstrated through alkaline phosphatase histochemical staining on transfected COS-1 cells  and Vascular Endothelial Growth Factor B (VEGFB) (Nrp1D320K was co-expressed with PlexinA4 (Plex4A) to more accurately reflect the in vivo situation in which SEMA3A signals through a holoreceptor complex of both NRP1 and PlexinA. AP-SEMA3A binding levels to WT NRP1 and NRP1D320K were indistinguishable (D) of SEMA3A-NRP1D320K/PlexinA4 was unchanged from that of SEMA3A-NRP1/PlexinA4 further verifying that the SEMA3A-NRP1/PlexinA4 interaction was intact  . In a liuishable , and thes intact . Finallyrp1D320K . Taken tNrp1D320K mutation in the endogenous Nrp1 locus, delineated as Nrp1VEGF−. Specifically, two base pair mutations were introduced into exon 6 of the mouse Nrp1 gene to produce the D320K mutation in the endogenous Asp320 location  lung and adult heart, brain, lung and kidney  upon VEGF treatment  to visualize the retinal blood vessels and arteries, respectively. At P9, the termates . In the termates  indicatiNrp1VEGF− animals were also assessed for injury-induced arteriogenesis following femoral artery ligation. In this assay, the femoral artery was surgically severed in both Nrp1VEGF− and control mice, and blood flow recovery was monitored via deep penetrating laser Doppler imaging. Femoral artery ligation produced a comparable level of hindlimb ischemia in the Nrp1VEGF− mutants and controls  and fibroblast growth factor receptor-1 (FGFR1) . This prUltimately, our findings mark a pivotal step toward understanding the role of NRP1 in developmental angiogenesis and indicate that NRP1-VEGFR2 interactions rather than VEGF-NRP1 binding underlie NRP1's critical function in VEGF-mediated vascular development. Given the substantial resources invested in NRP1-targeted anti-angiogenesis therapies for vascular disease and cancer, the information gleaned from this study will be invaluable in identifying the cellular and molecular mechanisms underlying angiogenesis and ultimately using this information to instruct the development of new therapeutic approaches.Rat Neuropilin1 cDNA was re-cloned from pMT21 into pCS2+ using the original EcoRI and XhoI sites present in both vectors. Mutations were made using PCR, and the mutated fragment was subcloned back into pCS2-Nrp1 using endogenous restriction sites. The targeting vector (TV) was constructed using a combination of traditional cloning and recombineering along with point mutagenesis. Genomic DNA was obtained from the 129S7-AB2.2 BAC library, clone #bMQ-373E22. The short (3′) arm (1.3 kb) was cloned into the HpaI and EcoRI sites of 4600C-loxP. Two short homology arms  were created and cloned into the XhoI and NotI sites of 4600C-loxP, with the two arms joined by a SalI site. The homology arms were ligated in a triple ligation to 4600C-loxP as well as to each other. The vector was then linearized with SalI and electroporated into modified electrocompetent DH10B cells containing the previously mentioned BAC in order to facilitate homologous recombination to insert the remainder of the long arm. Recombineering was performed as described by the NCI-Frederick. After a full-length TV was made, the D320K mutation was introduced. The final TV was linerarized and electroporated into ES cells. All primer sequences used for the targeting vector construction are provided in HEK293T cells were transfected with AP-SEMA3A, AP-VEGF A, AP-VEGF B, or AP-PlGF expression constructs using a calcium phosphate transfection method. Media was changed after 6 hr. Cells were cultured for an additional 48 hr in DMEM + 10% FBS. After 48 hr the media were collected, filtered to remove the cell debris, and AP activity was measured. The ligands were frozen at −80°C until use.2, 224 mg L-homoarginine, 50 mg BSA, 445 mg p-nitrophenylphosphate) and measuring optical absorbance at 405 nm every 15 s for 1 min. Antibody staining of these cells was done as follows: non-specific binding was blocked with 5% Normal Goat Serum in DMEM for 30 min at 4°C. Cells were then incubated with primary antibody  for 2 hr at 4°C. They were then washed 6× with cold HBHA, then incubated with a secondary antibody (AP-tagged anti-rabbit) for 1.5 hr at 4°C. Cells were then washed 3× in cold HBHA, then lysed as described above. AP-activity was measured from lysed extracts. Binding of AP-tagged ligands was normalized to protein content of each well and to antibody staining with an anti-NRP1 antibody. Each AP-binding assay was independently repeated three times.COS-1 cells were grown in DMEM + 10% fetal bovine serum (FBS) + 1% Penicillin-Streptomycin. Cells were transfected with the indicated expression vectors using Lipofectamine-2000  in 6-well plates. 24 hr later, transfected cells were split into 24-well plates for parallel AP-binding and antibody staining. 24 hr after splitting, binding was performed using AP-tagged ligands . The binding protocol was as follows: cells were washed 1× with HBHA , then incubated for 75 min with 0.3 ml of 2 nM ligand. Cells were then washed 7× with HBHA on a rotating platform and 110 µl of cell lysis buffer (1% Triton X-100 and 10 mM Tris–HCl [pH 8]) was added to each well. Cells and buffer were scraped into Eppendorf tubes, then vortexed for 5 min to fully lyse them. The lysates were then spun down for 5 min, and the supernatant was heat inactivated at 65°C for 10 min to inactivate endogenous alkaline phosphatases. AP-activity was measured by adding 2× SEAP buffer . Sections were washed 3× in PBS and incubated in PBS at 65°C for 2 hr to heat inactive endogenous alkaline phosphatases and then incubated overnight in developing solution  with NBT (nitro-blue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3′-indolyphosphate p-toluidine). AP-ligand binding was analyzed in sections from at least three animals across two different litters per genotype.Embryos were dissected and frozen immediately in liquid nitrogen, then stored at −80°C until use. Sections were cut at 25 µm with a cryostat, then fixed for 8 min in ice-cold methanol. Sections were then washed 3× in PBS + 4 mM MgClFor immunoblotting, E14.5 lung samples were loaded on 8% polyacrylamide gels and run until the appropriate protein separation was achieved. Samples were electrophoretically transferred onto the PVDF membrane. Non-specific binding was blocked by a 1 hr incubation in 5% non-fat milk in TBST . The membranes were then incubated overnight with the following primary antibodies, as indicated below, at 4°C: anti-NRP1  were dissected, fixed, cryo-protected, and sectioned at 20 µm. Tissue sections were washed 3× for 5 min in 0.2% PBT (0.2% Triton X-100 in PBS), incubated with Isolectin GS-IB4  overnight at 4°C, washed 3× for 5 min in PBS, and coverslipped with using ProLong Gold/DAPI antifade reagent . Sections were imaged by fluorescence microscopy using a Nikon Eclipe 80i microscope equipped with a Nikon DS-2 digital camera. Quantification was performed using ImageJ. Vessel coverage delineates the percent of cortical pixel area covered by isolectin-positive pixels while vessel size quantifies the pixel area of each discrete vascular aggregate identified by isolectin staining.E14.5 mouse lungs were dissected in cold PBS and minced finely using a razor blade. The tissue was then incubated with plain EBM  or EBM containing 50 ng/ml VEGF for 15 min at 37°C. Lysis buffer  containing complete proteinase inhibitors , PhosSTOP , and sodium orthovanadate was added to the tissue, which was then pulverized with a pestle and incubated for 30 min while rotating at 4°C. Tissue was spun down and protein quantification was performed. The tissue was treated as described in the Western blotting section.Nrp1VEGF− animals treated with VEGF as described above.HEK293T cells were transfected with the indicated constructs using Lipofectamine-2000 . They were then grown in DMEM + 10% fetal bovine serum + 1% Penicillin-Streptomycin and 48 hr after transfection cells were washed and harvested in ice-cold PBS. Cells were lysed using lysis buffer  containing complete proteinase inhibitors . After 30 min of rotation in the cold room and subsequent centrifugation, protein was quantified and 20 µg of protein was frozen down as input controls. 0.5 µg of anti-VEGFR2 antibody  lysis buffer . Following a 5 min incubation at room temperature, 2 ml of ice-cold 5% FBS/PBS was added and cells were centrifuged at 1000×g for 5 min at 4°C. Cells were then blocked in Fc-blocking solution  for 20 min on ice, centrifuged, incubated with the labeled conjugated primary antibodies–PE-anti-CD31 (PECAM)  and APC-anti-Flk1-1 (VEGFR2) , for 30 min on ice with agitation every 10 min. After incubation, the cells were spun down, the supernatant was removed, and the cell pellet was resuspend in 1:10K Sytox in PBS/5%FBS. Cells were analyzed on a LSR II Flow Cytometer. Cells incubated with no antibody, APC-anti-Flk1, or PE-anti-CD31 only served as the control population.Analysis of E14.5 mouse embryos were performed on single cells from dissociated lungs. In brief, microdissection techniques were used to isolate the lung. Lungs were then rinsed in PBS and incubated in 2 mg/ml collagenase and 20 μg/ml DNase I 3× for 15 min at 37°C and gently pipetted. The collagenase was inactivated using 5 ml of ice-cold 10% FBS/PBS, centrifuged at 1000×Whole-mount retina immunohistochemistry was performed as previously described in Nrp1VEGF− and control littermates. After anesthesia was achieved, the bilateral hindlimbs and lower abdomen were cleared of hair and cleaned with 10% betadine and 70% alcohol. An incision of 3–4 mm was made in the right inguinal area to visualize the femoral artery. Two 6–0 silk sutures were tied in the proximal femoral artery and the deep femoral and epigastric artery branches were cauterized. The femoral artery was then ligated between the two sutures. The skin was sutured with one 4–0 prolene sutures. Immediately before and after surgery, each animal was scanned with a non-invasive laser doppler imaging system  under 1–3% isofluorane anesthesia. Blood flow recovery in the hindlimbs was further assessed on 3, 5, and 7 days post-surgery and quantified via Moor LDI Software.Ketamine (80–100 mg/kg) and xylazine (5–10 mg/kg) delivered by IP injection were used to anesthetize 12-week old male t-test was used to determine the statistical significance of differences between samples, and the genotype distribution was analyzed using a Chi-square test. Statistical analyses were performed with Prism 4 (GraphPad Software) and p values are indicated by * ≤ 0.05, ** ≤ 0.01, and *** ≤ 0.001.The standard error of the mean was calculated for each experiment and error bars in the graphs represent the standard error. A paired Student's  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 , Jeremy Nathans (Reviewing editor), and 3 reviewers.Thank you for sending your work entitled “Neuropilin-1 controls developmental angiogenesis by regulating VEGFR2 independent of VEGF-Neuropilin-1 binding” for consideration at The manuscript has been reviewed by three expert reviewers, and their assessments together with the Reviewing editor form the basis of this letter. I am also including the three reviews in their original form at the end of this letter, as there are many specific and useful suggestions in them that will not be repeated in the summary here.All of the reviewers were impressed with the incisive design and execution of your experiments. The mutant mouse that you constructed is the cleanest experimental approach to date to address the question of whether direct VEGF binding to Neuropilin1 (Nrp1) plays a role in vivo. Our consensus is that this is an important body of work but that a few key questions have been left open and that addressing them would greatly enhance the impact of the study.These are:Is the Nrp1 VEGF binding mutant mouse really normal? A close look at the retinal vasculature  and at health/fertility at later ages would be useful. You might consider stressing the system, for example with the oxygen induced retinopathy model  or with a test of tumor angiogenesis, both of which tie into the applied aspects of this work as related to the efficacy of anti-Nrp1 antibodies. Comparing the phenotypes to those reported by The decrease in VEGFR2 levels (and surface VEGFR2), which is presented as the mechanism by which the Nrp1 KO exerts its effects, could use more experimental backing. Checking the level of VEGFR2 phosphorylation in response to VEGF and an assessment of VEGFR2 trafficking with vs. without Nrp1 vs. with the Nrp1 VEGF binding mutant (in cell culture) would be very helpful.Is the VE cadherin level in Reviewer #1:eLife. The overall quality of the paper can be improved by addressing the following comments.Clear mechanistic understanding of the VEGF signaling pathway is of paramount importance since this pathway lies in the heart of vascular biology and is being targeted to treat a number of human diseases. The role of Neuropilin-1 is an important piece of the puzzle in VEGF signaling, and the central dogma about Neuropilin-1 is that binding to VEGFA is essential for its function despite lacking direct evidence to support it. Recent attempt to provide direct evidence by assessing a Neuropilin-1 allele that lacks VEGF-A binding capacity fell short to answer this question due to the severe reduction of expression . In this1) The degree of surface VEGFR2 reduction is much less than that of total VEGFR2 when Npn1 was deleted in ECs compare  with 5A,2) Since the reduction of surface VEGFR2 is modest ∼ 30%,  in the N3) Data from 4) Some mechanistic data related to how NPN1 regulates VEGFR2 level will significantly elevate this manuscript to a different level. The authors should consider including one or two in vitro analysis similar to the NCAM / FGFR1 study .Reviewer #2:Gelfand et al present evidence that a point mutation (D320K) in the extracellular domain of neuropilin 1 (Npn1) prevents its binding to VEGF while leaving relatively intact Nrp1 association with Sema 3A. By gene replacement strategy they also generated a mouse line harboring the D320K mutation in the Npn1 locus.The major finding of this paper is that, in absence of VEGF binding to Npn1, embryo vascular development is unaffected. Furthermore, it is reported that Npn1 co-immunoprecipitates with VEGFR2 and stabilizes VEGFR2 levels. The authors therefore hypothesize that Npn1 acts by controlling VEGFR2 levels through a still undefined mechanism.1) The authors are not giving a fair representation of the literature. In the Abstract they report: “Npn1 is essential for vascular morphogenesis, but how Npn1 functions to guide vascular development remains elusive”. This statement is unfair considering several previous papers: Lanahan et al Dev Cell 2013; Fantin et al Development 2014 or Raimondi et al J Exp Med 2014. Furthermore, similarly to what is reported here, 2) The reduction in VEGFR2 levels in Npn1 mutants is indeed striking but does not correspond to the findings of many other authors where the uncoupling or strong reduction on Npn1 did not modify VEGFR2 or VEGFR1 expression . In addition, while in Since Title, Abstract and Discussion are focused on this finding the authors should present more solid data to support their conclusions. Last but not least, if the decrease in VEGFR2 is in the range presented in 3) The authors do not investigate the possible effects of their Npn1 mutant on neurons. Taking into account that some types of neurons rely on VEGF165 and Npn1 signaling, these observations are needed for a more complete interpretation of the mutant phenotype.4) Introduction. Most of previously published data converge in saying that the domain of Npn1 responsible for the binding of Sema 3a is the b1 region and not the a1  only affects VEGF but not Sema3 binding. This approach is more sophisticated and better controlled than a previous study from another group who created a Nrp1Y297A mutation (PMID: 24401374), also aimed at ablating VEGF binding in Nrp1. The NrpY297A study suffered from a poor genetic targeting strategy that resulted in reduced Nrp1 expression (in vivo). Surprisingly, both studies found that VEGF binding to Nrp1 is not required for normal embryonic development. The current study shows this more convincingly, because the Nrp1Y297A study was more difficult to interpret because of the reduced Nrp1 levels caused by the Nrp1Y297A mutation. The novel Nrp1D320K strain described here will therefore be a very useful, well validated research tool.Furthermore, the authors found that the Nrp1D320K mutation caused a dramatic reduction of VEGFR expression. This is a very intriguing observation and it would be interesting to know the reason for this and what the functional consequences are. The study does not explore this but the authors conclude (in the Discussion) that “Nrp1 regulates angiogenesis by controlling the amount of VEGFR2 expression at the cell surface and consequently the level of VEGFR2-VEGF signaling.”There are several problems with this statement:1) It is logically wrong. Since the Npn1VEGF mice have reduced VEGFR2 levels but develop normally, Nrp1 obviously is not regulating angiogenesis via VEGFR2 levels, otherwise the mice would have a Nrp1 KO phenotype.2) The authors have not actually shown that VEGFR2 signalling is reduced. This would have to be shown more directly (e.g. VEGFR2 phosphorylation in Western blots).3) Although the flow cytometry data in 4) VEGFR2 levels were only studied in lung endothelial cells. Yet the main vascular phenotype in Nrp1 mutants is found in the brain and retinal vasculature.5) The Nrp1Y297A mice also developed normally but that study found abnormalities in postnatal animal, such as abnormal retinal vasculature development, reduced neovascularisation in the oxygen induced retinopathy model and reduced tumour growth. These postnatal phenotypes should be also checked in the Nrp1D320K mice.In summary, this study has been executed very nicely but the main drawback is that it ends prematurely. When I got to  Is the Nrp1 VEGF binding mutant mouse really normal? A close look at the retinal vasculature  and at health/fertility at later ages would be useful. You might consider stressing the system, for example with the oxygen induced retinopathy model  or with a test of tumor angiogenesis, both of which tie into the applied aspects of this work as related to the efficacy of anti-Nrp1 antibodies. Comparing the phenotypes to those reported by Nrp1VEGF- mutant.We thank the reviewers for this valuable suggestion. We have performed new experiments and now provide additional data describing the phenotype of the Nrp1VEGF- mutants in comparison to their littermates across several different developmental stages  We generated a viability table detailing the survival of Nrp1VEGF- mutants experience normal body growth.2) We grossly examined several organ systems  at P9 and adulthood and now include organ weights in Nrp1VEGF- heart at P9  We also generated whole mount images to illustrate the normal morphology of the rt at P9  and noteNrp1VEGF- mutants are fertile (pg 8) and in contrast to the Nrp1Y297A/Y297A hypomorph, Nrp1VEGF- mutants maintained appropriate NRP1 protein levels in several adult organ systems (4) We have also noted in the text that  systems .Nrp1VEGF- mutant at P9 and adulthood  We examined the retinal vasculature in the dulthood . ThroughNrp1VEGF- mutants and control littermates. In this challenge situation, the Nrp1VEGF- mutant initially exhibited reduced blood flow recovery to the injured hind limb post, but eventually caught up to control levels by seven days post-surgery (6) Finally, we performed femoral artery ligation surgeries to induce hind limb ischemia in -surgery .The decrease in VEGFR2 levels (and surface VEGFR2), which is presented as the mechanism by which the Nrp1 KO exerts its effects, could use more experimental backing. Checking the level of VEGFR2 phosphorylation in response to VEGF and an assessment of VEGFR2 trafficking with vs. without Nrp1 vs. with the Nrp1 VEGF binding mutant (in cell culture) would be very helpful.Tie2-Cre,Nrp1fl/- and Nrp1VEGF- mutants. We discovered that Nrp1VEGF- mutants have a modest reduction in VEGFR2 phosphorylation at the tyrosine residue 1175 (Y1175) upon VEGF treatment without a change in total VEGFR2 protein  to show that VEGFR2 protein level is decreased in the absence of NRP1 while Vegfr2 mRNA levels are unaffected by NRP1 siRNA. Moreover, the overexpression of Nrp1 led to an increase in VEGFR2 protein level as well as VEGFR2 cell surface expression.1) Vegfr2 mRNA levels.2) Similarly, Holmes et al., 2008 found that NRP1 siRNA consistently and significantly reduced VEGFR2 expression upon VEGF induced receptor-mediated endocytosis, but found no significant changes in 3) 4) in vitro studies are consistent with our in vivo results and provide clear mechanistic data related to how NRP1 regulates VEGFR2. Our current study provides the first in vivo evidence that NRP1 controls VEGFR2 levels at the cell membrane and offers the first in vivo phenotypic characterization linking NRP1 regulated VEGFR2 surface expression to vascular development.These Is the VE cadherin level inlower in the absence of Nrp1? If so, does this suggest other roles for Nrp1? Also, the decrease in VEGFR2 by Western blot seems more dramatic than the decrease shown by FACS. Is the lower level of VEGFR2 shown by FACS in the absence of Nrp1 really insufficient for VEGF signaling? Or is something more subtle at play (defective trafficking?)?Tie2-Cre,Nrp1fl/- mutants to determine if the reduction in VEGFR2 levels in the absence of NRP1 is sufficient to alter VEGF signaling. Indeed, we found a significant reduction in VEGFR2 phosphorylation  and included them in To further clarify the western blot data shown in Reviewer #1:1) The degree of surface VEGFR2 reduction is much less than that of total VEGFR2 when Npn1 was deleted in ECs (comparewith 5A), suggesting that the loss of Npn1 effect on surface VEGFR2 might be secondary to a reduction of total VEGFR2. Since the exiting data cannot distinguish between maintaining surface VEGFR2 levels versus regulating total VEGFR2 levels, the mechanism depicted inis not supported by actual data. The diagram should be modified to reflect a reduction in total VEGFR2, and the related text descriptions throughout the manuscript should be revised accordingly.To reconcile the level of total versus surface VEGFR2 protein, we have gone back and quantified the VEGFR2 levels by Western blot and found that the reduction in VEGFR2 is more in line with the results observed in the FACS analysis. We have updated 2) Since the reduction of surface VEGFR2 is modest  in the Npn1 null ECs, the functional significance of reducing VEGFR2 levels has not been clearly established. This question can be addressed by including a phospho-VEGFR2 blot or ELISA inTie2-Cre,Nrp1fl/- and Nrp1VEGF- mutants.This point is a great suggestion, and we have performed experiments to quantify phospho-VEGFR2 upon VEGF treatment. In particular, we have examined the level of VEGFR2 phosphorylation in lung lysates following VEGF treatment in the 3) Data fromindicated that either severely reduced NRP1 levels and/or abolishment of NRP1-VEGFA binding plays an important role in postnatal angiogenesis. This is an important issue in the angiogenesis field. The authors have the proper tool and data to address this question. They can contribute to the angiogenesis community by mentioning some aspects of postnatal development in their Npn1VEGFR- mice. For example, they can include a table that documents mouse numbers at different age after birth to demonstrate if there is reduced viability overtime, instead of only giving a statement about a single time point. They can also point out if they have seen overt phenotypes reported by, for example, have they ever seen evidence of cardiac failure in some of their knockin mice?in vivo tools to delineate whether the phenotypes described in Nrp1VEGF- mutant by including a table that documents survival at different developmental stages to demonstrate the sustained viability of the Nrp1VEGF- mutant. We have also examined organ weight, heart morphology, and retinal angiogenesis. As described above, we do not observe any of the impaired growth, cardiac failure, or postnatal fatality described in Nrp1Y297A/Y297A hypomorph rather than a lack of VEGF-NRP1 binding.We thank the reviewer for this suggestion. Indeed, we have the proper 4) Some mechanistic data related to how NPN1 regulates VEGFR2 level will significantly elevate this manuscript to a different level. The authors should consider including one or two in vitro analysis similar to the NCAM / FGFR1 study .in vitro work in multiple cell culture systems has used gain of function (overexpression) and loss of function (RNAi knock-down) to demonstrate that NRP1 is essential for the proper presentation, recycling and degradation of VEGFR2. These in vitro studies are consistent with our in vivo results and provide clear mechanistic data related to how NRP1 regulates VEGFR2. Our current study provides the first in vivo evidence that NRP1 controls VEGFR2 levels at the cell membrane and offers the first in vivo phenotypic characterization linking NRP1 regulated VEGFR2 surface expression to vascular development.As described in detail above, previous Reviewer #2:1) The authors are not giving a fair representation of the literature. In the Abstract they report: “Npn1 is essential for vascular morphogenesis, but how Npn1 functions to guide vascular development remains elusive”. This statement is unfair considering several previous papers: Lanahan et al Dev Cell 2013; Fantin et al Development 2014 or Raimondi et al J Exp Med 2014.Furthermore, similarly to what is reported here,already generated mice expressing Npn1 with a mutation that prevents VEGF binding. Homozygous mutated mice were viable and no severe and lethal cardiovascular phenotypes were observed implying that Npn1 could have VEGF-independent role during vascular development. Although the experimental approaches used in the two papers are different, the final conclusion remains the same.Nrp1VEGF- mutant maintains appropriate levels of NRP1 protein . A detailed point-by-point description of these results is provided above.We have updated this sentence in the Abstract to provide a fairer representation of the previously published work. As Reviewer 1 points out, the phenotypes described in 2) The reduction in VEGFR2 levels in Npn1 mutants is indeed striking but does not correspond to the findings of many other authors where the uncoupling or strong reduction on Npn1 did not modify VEGFR2 or VEGFR1 expression . In addition, while inthe drop in receptor expression is almost complete; init is around 35% only.Since Title, Abstract and Discussion are focused on this finding the authors should present more solid data to support their conclusions. Last but not least, if the decrease in VEGFR2 is in the range presented inthe phenotype of Npn1 KO should be comparable to that of VEGFR2 KO and this is not the case.We agree with this sentiment and have gone back to quantify the VEGFR2 levels in our Western blots. Indeed, the decrease in VEGFR2 levels is more on par with the FACS analysis and we have replaced the Western blot image with a more representative data. Furthermore, we have included our quantification in 3) The authors do not investigate the possible effects of their Npn1 mutant on neurons. Taking into account that some types of neurons rely on VEGF165 and Npn1 signaling, these observations are needed for a more complete interpretation of the mutant phenotype.Nrp1-/- mutants have a defect in which the migration of the facial motor neurons is disrupted. The phenotype was thought to be a result of VEGF-NRP1 binding in neurons because Nrp1Sema- and Tie2-Cre;Nrp1fl/- mutants do not have this phenotype while VEGF-120 mice phenocopy the Nrp1-/-. With in situ hybridization for Isl1, we analyzed the migratory path of motor neurons at E12.5. To our surprise, we did not see a migration defect in the Nrp1VEGF- mutant. However, we also failed to see a migration defect in the neuronal-specific NRP1 knock-out  demonstrating that NRP1 is not required in neurons for this phenotype. In this regard, our data bring the claims of previous work into question and resolving this discrepancy is beyond the scope of our current manuscript. In addition, we also examined gonadotropin-releasing hormone (GnRH) neurons because Carboni et al., 2011 showed that VEGF164 promotes the survival of migrating GnRH neurons by co-activating the ERK and AKT signaling pathways through NRP1. However, as with the facial motor neurons, we did not detect any GnRH survival phenotype in our Nrp1VEGF- mutants.First, we looked at the most robust neural phenotype that has been linked to VEGF-NRP1 function. Specifically, facial motor neurons express NRP1, and their migration during development is dependent on the presence of VEGF164 . According to the literature, 4) Introduction. Most of previously published data converge in saying that the domain of Npn1 responsible for the binding of Sema 3a is the b1 region and not the a1 (see for instanceNat. Rev. Immunol. 2103 by Kikutani). This should be introduced and discussed in a better way in the text.Nrp1 variants containing smaller deletions in the b1 domain were engineered with site-directed mutagenesis to identify a region specific for VEGF-NRP1 binding  that “Nrp1 regulates angiogenesis by controlling the amount of VEGFR2 expression at the cell surface and consequently the level of VEGFR2-VEGF signaling.”There are several problems with this statement:1) It is logically wrong. Since the Npn1VEGF mice have reduced VEGFR2 levels but develop normally, Nrp1 obviously is not regulating angiogenesis via VEGFR2 levels, otherwise the mice would have a Nrp1 KO phenotype.Nrp1VEGF- mice have normal VEGFR2 levels, as seen in Tie2-Cre,Nrp1fl/- mutants have reduced VEGFR2 levels which is consistent with the Nrp1-/- phenotype shown in Based on the critique, it is apparent that Reviewer 3 misunderstood the results from our study. We clearly show that 2) The authors have not actually shown that VEGFR2 signalling is reduced. This would have to be shown more directly (e.g. VEGFR2 phosphorylation in Western blots).Tie2-Cre,Nrp1fl/- and Nrp1VEGF- mutants. Please see our detailed description above.We have performed additional experiments to determine the level of VEGFR2 phosphorylation in lung lysates following VEGF treatment in the 3) Although the flow cytometry data inindicates reduced surface expression of VEGFR2 this seems only to be the case for around 50% of the cells. On the other hand, the Western blot data  indicates a much stronger reduction of around 90% (contradicting the flow cytometry data somewhat). This demonstrates that also intracellular VEGFR2 must be dramatically reduced and not only surface VEGFR2Tie2-Cre,Nrp1fl/- mutant is comparable to the reduction seen in our FACS analysisAs previously described above, we have reassessed VEGFR2 levels in our Western blots and now have included quantification in 4) VEGFR2 levels were only studied in lung endothelial cells. Yet the main vascular phenotype in Nrp1 mutants is found in the brain and retinal vasculature.Tie2-Cre;Nrp1fl/- mutants are embryonic lethal and finding a tissue with an adequate source of cells was of paramount importance. We attempted to analyze brain endothelial cells via FACS, but the tissue was difficult to dissociate and formed clumps during the immunolabeling process. These clumps were unable to be analyzed via the FACS equipment and resulted in a significant loss of endothelial cells from our samples.We examined VEGFR2 levels in lung endothelial cells for practical purposes, in that we were able to harvest the greatest number of endothelial cells from this organ to perform our analysis. The 5) The Nrp1Y297A mice also developed normally but that study found abnormalities in postnatal animal, such as abnormal retinal vasculature development, reduced neovascularisation in the oxygen induced retinopathy model and reduced tumour growth. These postnatal phenotypes should be also checked in the Nrp1D320K mice.Nrp1VEGF- mutant. In particular, we have examined the retinal vasculature at two time points (P9 and adult), challenged the mutant with a hindlimb ischemia assay, and included additional information about fertility, organ size, and viability.We appreciate this constructive criticism and have now included a broader phenotypic analysis of the


Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin , and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid partitioning and suggest underlying biological roles shared by such elements.The ability of plasmids to propagate in  Saccharomyces cerevisiae, episomal plasmids are powerful tools for defining DNA sequences that govern chromosomal functions. Notably, plasmid-based assays have helped identify and characterize S. cerevisiae DNA replication origins and centromeres, the DNA regions essential for chromosome duplication, and segregation during cell division, respectively , capable of linking chromosomal regions to the inner nuclear membrane (INM) via its interactions with the silencing protein Sir4  to identify dozens of putative plasmid partitioning elements in the yeast genome  origin and revealed four distinct modular elements, a conserved A-element and poorly conserved B-elements, B1, B2, and B3, and helped establish ARS1 as the paradigm ARS used in many subsequent biochemical studies , the protein complex that selects origins in all eukaryotes . Thus, y studies . Biochem studies . The B2 es (ARS1 . The confunction . HoweverARS1 (“mut-ARSseq”) . Briefly, sheared DNA from a comprehensive library of functional ARSs was subcloned and screened for ARS function by a similar library sequencing method as described above. This experiment delineated the A element and flanking sequences important for propagation in yeast for > 100 ARSs, providing the raw material for further dissection of these elements.Recently, a suite of high throughput methods was developed to map and delineate ARSs on a genome-wide scale (“ARSseq”) and to perform deep-scanning mutagenesis of ARSs, starting with HMR and HML, contain ARS elements closely associated with a plasmid partitioning function mediated by the silencer RAP1 site. These and other studies provide strong evidence that an association between plasmids and the INM is a mechanism for enhancing centromere-independent plasmid distribution during cell division, but additional mechanisms may exist that reflect distinct features of chromosome biology, including mechanisms that promote intra- and interchromosome interactions  or by “hitching a ride” on a chromosome  , representing over 100 yeast origins. While the initial study assayed the ability to transform yeast , here we generated quantitative information about plasmid propagation efficiency by subjecting the pool of transformed yeast to a competitive growth experiment coupled to deep sequencing. The data generated by this experiment defined the chromosomal regions required for maximal plasmid propagation for 112 individual ARSs in a single experiment. They also allowed us to compare the efficiency between different ARSs. Strikingly, the HMR-E (miniARS317) and HML-E (miniARS301) transcriptional silencers were among the most effective ARSs in the library. We performed deep mutational scanning of miniARS317 and miniARS301, direct ARS assays and numerical simulations that all supported the conclusion that the RAP1 site-mediated partitioning intrinsic to these ARSs was important for their high efficiency in the miniARS experiment. Significantly, additional ARS assays of several nonsilencer origins revealed previously undefined putative plasmid-partitioning elements. To define such elements more precisely and address their relative frequency among yeast origins, we transferred the miniARS library to a centromere-containing plasmid and performed a new competition experiment. Computational approaches to compare the data generated from the two different miniARS experiments allowed the parsing of the replication and putative partitioning functions on multiple ARS loci. A substantial fraction of the origins represented in these miniARS libraries, ∼25%, contained regions important for plasmid propagation in the absence of a centromere. However, in the presence of a centromere, these regions are dispensable for efficient plasmid propagation. We propose that these regions enhance plasmid partitioning to the daughter cell and therefore called them “putative partitioning regions.” Motif analyses identified 74 new putative partitioning elements within these regions in 26 nonsilencer origins out of a total of 112 origins examined. Many of these motifs were predicted to bind proteins involved in negative regulation of a number of cellular processes, including filamentous growth and gene expression, suggesting underlying linkages among chromosomal elements capable of substituting for centromere function on plasmids.In this study, we adapted the ARS assay to examine both replication and potential partitioning elements in budding yeast at a genomic scale  and individual yeast transformants were pooled to generate the initial miniARS library for a competitive growth experiment. All competitions were performed in shaking liquid cultures at 30° in growth medium lacking uracil, such that the uracil selectable marker on the plasmid was demanded throughout the experiment. Approximately 10 OD of cells were used to extract DNA at the start of the competition and at set time intervals throughout the competition. The DNA was extracted and purified and used as a source of total plasmid DNA for deep sequencing as described (The appropriate library DNA purified from ARS317-mut and miniARS301-mut libraries were constructed using synthetic oligos from TriLink BioTechnologies. Randomly mutagenized oligos spanning miniARS317-full (153 bp) or miniARS301-full (135 bp) were synthesized with a mutation frequency of 1.2% and 1.8% at each position for miniARS317-mut and miniARS301-mut, respectively. The mutagenized oligos contained invariant 5′ and 3′ elements for subsequent primer annealing and PCR amplification. PCR products were cloned using Gibson assembly into the Acen-vector pIL22, and E. coli clones were pooled and used to prepare the miniARSmut libraries. The plasmid libraries were transformed into W303 yeast, and yeast clones were pooled to generate the starting miniARSmut library in yeast. Each miniARS-mut library was grown competitively in liquid batch culture as described for the Acen- and Cen-miniARS libraries above. For these ARSmut experiments, competitive fitness was defined as the ratio of a given fragments’ frequency at the end of the competition to its frequency at the start of the competition.The minihttp://cerevisiae.oridb.org). For all analyses in this study, only fragments from confirmed yeast origins that contained a high confidence ORC binding site  annotated in a previous study were included . The code is provided as a text file in RAP1 site was derived in Weblogo from the RAP1 Position Specific Frequency Matrix (PFSM). A consensus sequence for the ORC site was created in Weblogo using the sequences of the 232 ORC sites annotated previously (http://meme-suite.org) with a P-value cut-off of 0.05. MiniARS fragments were mapped, aligned and analyzed using standard functions in LibreOffice Calc spreadsheets. All data except for numerical simulations were graphed in R. Supplemental Material Table S1 contains the complete list of motifs identified within the 26 partitioning ARSs  http://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-7-113. Genotype and Phenotype Ontology: A Gene Ontology term finder algorithm was performed in the Saccharomyces Genome Database  using GO process terms, demanding a P-value cutoff of 0.01. Table S2 includes the complete GO analyses. 84 Phenotype Ontologies (PO), lists of genes associated with a particular phenotype, were downloaded from SGD. The fraction of genes identified by various motif collections that belonged to the PO terms was determined and normalized to the fraction of sequence specific DNA binders (GO: 0043565). Table S3 includes the complete PO analyses.A consensus Table S4 contains the names and sequences of all plasmids. Sequencing data for this project are available at the NCBI Sequence Read Archive BioProject Accession #SRP065331.Plasmids are available upon request. URA3 gene on the plasmid (Yeast transformed with the previously described miniARS library were grown competitively under conditions that selected for the  plasmid  (Liachkoy-axis) was plotted against the fragment’s length in base pairs (x-axis). Fragments overlapping selected origins were then examined for their behavior in the population . From these analyses, it was clear that the transcriptional silencer HMR-E (miniARS317) generated a large number of highly competitive miniARS fragments . HML-E associated fragments (miniARS301) were also enriched among the most competitive miniARSs, though to a lesser extent than miniARS317 . HMR-I (miniARS318), a weaker silencer than either of the E-silencers, was also enriched among competitive miniARSs, although to a lesser extent than either of the E-silencers . Thus, silencer-associated fragments were among the most competitive miniARSs and their overall competitiveness correlated with their relative strength as silencers .In ARS317 associated with the most competitive miniARSs, the ARS317-associated fragments were binned into three groups based on their competitive fitness values, and the terciles were ranked from lowest (+) to highest (+++) based on their average competitive fitness value   , left. T fitness , right. Rap1 binding site (RAP1 site) 5′ of the silencer ORC site. In addition, both silencer miniARSs contained sequences 3′ of this site that are not part of the defined transcriptional silencers and have no known role in silencing  and previously uncharacterized nonsilencer sequences  were associated with miniARS317-full and miniARS301-full. Statistical analyses of the motifs  tied to the function of miniARS317-full are shown in Figure S2.This mapping revealed obvious similarities between the sequence organizations of the silencer miniARSs that generated the highest competitive fitness. In particular, both silencer miniARS-max fragments contained the silencer Figure S3). In terms of fragment distance 3′ of the ORC site, the silencer ARSs were, in fact, quite similar to this average ARS structure, and to the structure of the paradigm ARS, ARS1. However, the most competitive silencer miniARSs were distinct because they contained a RAP1 site positioned 5′ of their ORC site. Given the known roles of Rap1 on silencer-containing plasmids, these data suggested that Rap1-mediated partitioning was relevant to the high competitive fitness of the silencer miniARSs in the miniARS competition experiment.We performed a similar analysis with all of the fragments present in the miniARS data set combined to define an “average” ARS  and miniARS301-mut libraries were cloned into the same plasmid backbone used for the miniARS library and used to transform yeast. The yeast population was then subjected to competitive growth, and deep sequencing of the plasmid population was performed at the start and finish of the competition. The single nucleotide changes and their effects on miniARS competitive fitness for the two silencer miniARSs are summarized in In a previous study, we used deep mutational scanning of a minie.g., ORC and RAP1) and putative  sites present in the maximally competitive silencer miniARSs contained nucleotides that contributed to competitive fitness. A previous study provided evidence that the region containing the putative FKH site contains a second weaker ORC binding site . In Figure S3, the mutational profile of the two different ORC binding sites is presented in an expanded form to allow more detailed examination. For miniARS317-full, several nucleotide positions within the conserved core A-element were unexpectedly tolerant of nucleotide substitutions that deviated from the consensus. In addition, miniARS317-full was tolerant of mutations within the WTW motif, the conserved nucleotides within the B1 element of yeast origins  or contained (Cen) a centromere  (average PLR = 16% ± 3%), whereas the three distinct fragments with high competitive fitness values generated lower PLRs (average PLR = 6% ± 2%) , reducing their PLRs by ∼sixfold and converting them into efficient ARSs. Analyses of variants of ARS317-full containing single nucleotide substitutions in the RAP1 site confirmed these results, and suggested that the RAP1 site was the only site on this fragment that was essential for Cen-independent partitioning . These data provided evidence that the silencer RAP1 site could at least partially substitute for the function of a centromere in an ARS assay. These data provided evidence that competitive fitness in the miniARS experiment could be modulated substantially by a fragment’s intrinsic partitioning ability.The ARS assays performed in the Acen plasmid used for the miniARS library revealed that the five distinct mini6% ± 2%) . These rABF1 and RAP1 sites. Hence, we suspect that this fragment associated more strongly with the Sir proteins and thus promoted a level of Sir4-Esc1-mediated partitioning that is capable of competing with (antagonizing) centromere function . If a fragment with replicator function exhibits Cen-antagonism, then it should reduce propagation of a Cen plasmid but not an Acen plasmid. While we did not rigorously test for Cen-antagonism, we note that fragment 6 in Rap1-mediated mechanism that links silenced regions to the INM is considered robust. Consistent with this observation, silencer Rap1-mediated plasmid partitioning is so robust that under some circumstances it can compete against a centromere (referred to as centromere antagonism) (Kimmerly and Rine 1987). Although the data described above supported the idea that silencer Rap1-mediated partitioning could have a profound consequence on plasmid abundance in the competitive miniARS experiment, it remained unclear how plasmid partitioning might affect any particular miniARS fragment’s competitive fitness in a miniARS experiment. To address this issue, numerical simulations were performed , then the probability would be high that each of the mother and daughter cells will receive a fragment after cell division. In contrast, a fragment with low partitioning fitness  would have a high probability of being retained by the mother cell after cell division. Crowding penalty was described as the probability that the replication fitness of a miniARS fragment, regardless of the intrinsic origin efficiency of the element, will decrease as the numbers of the miniARS fragments increase in a given cell. In this simulation, a crowding penalty of one means that, once a given cell has acquired 100 miniARS fragments, the replication fitness of each of those fragments is reduced by 50%. The crowding penalty was treated as a constant within each individual simulation.Replication fitness was defined as the probability that all copies of a particular miniARS fragment present in a cell would be duplicated in a given S-phase. In the example shown, the miniARS is present in four copies in the starting cell. If the replication fitness of this fragment were 1.0, then the probability would be high that the four fragments would produce eight fragments after S-phase. A lower replication fitness of 0.5 would mean that the probability would be low that all four fragments would be copied. Partitioning fitness was defined as the probability that miniARS fragments in a cell would distribute equitably to mother and daughter cells after cell division. In the example shown, if the two fragments have a high partitioning fitness (x-axis) and partitioning fitness (y-axis) were allowed to divide for twelve generations. To account for natural variations among the cells in a population, Gaussian noise was introduced for these two parameters in the model. Importantly, the model was robust, producing similar results with different levels of Guassian noise. The predicted frequency  of each miniARS fragment at the end of simulation was depicted in a heat map. In the absence of a crowding penalty , only replication fitness had a large impact on the competitive fitness of a miniARS fragment  , we examined fragments of low and high competitive fitness for six different nonsilencer miniARSs by standard ARS assays, as described for the miniARS317 experiments in ARS317.Based on the data discussed thus far, the miniARS experiment was sensitive to both replication and partitioning abilities. Natural, chromosomal-based plasmid partitioning elements other than the ARS428, ARS410, and ARS503—the PLRs were similar for their fragments regardless of whether the plasmid contained a centromere. Thus providing a robust plasmid distribution mechanism via a centromere had no effect on ARS stability for these miniARS fragments, suggesting that their competitive fitness differences were due primarily to replication fitness differences. However, for the three remaining ARSs in this group— ARS523, ARS1007, and ARS1018—the fragments that produced low competitive fitness in the miniARS experiment (and high PLRs on an Acen plasmid) generated low PLRs when present on a Cen plasmid. These results suggested that the ARS523, ARS1007, and ARS1018 fragments with low competitive fitness actually possessed intrinsically high core replication abilities, but lacked efficient partitioning abilities, behaving similarly to miniARS317 fragments that lacked a RAP1 site.As expected, the six distinct miniARS fragments with high competitive fitness generated relatively low plasmid loss rates (PLRs) compared to the corresponding fragments with low competitive fitness . For thrThe data described above revealed that as yet undefined partitioning elements could influence the competitive fitness of miniARSs. To address this issue using the high throughput power of the miniARS experiment described, we transferred the miniARS library to a plasmid containing a centromere. The resulting Cen-miniARS library was used to transform yeast and a competitive growth experiment was performed as for the Acen-miniARS library. The Acen-miniARS and Cen-miniARS data sets from these competitive growth experiments were then compared .RAP1 site, then it should exhibit a comparatively lower competitive fitness in the Cen-miniARS experiment because partitioning ability would no longer be an advantage. Thus, in theory, simply comparing the most competitive fragments between the two different experiments should reveal chromosomal regions that contain plasmid-partitioning element(s). For example, for miniARS317, the prediction was that the RAP1 site would not be required for high competitive fitness of miniARS317 fragments in the Cen-miniARS experiment. To test this prediction, all of the miniARS317 fragments with low competitive fitness  in a given miniARS experiment were grouped and compared to all of the miniARS317 fragments with high competitive fitness  in the same experiment  that performed a partitioning function, such as a periment . For thex-axis) and Cen-miniARS (y-axis) experiments. A perfect correlation between competitive fitness in the two different miniARS competition experiments would be expected to produce fragments only within the central diagonal portion of the graph. Such an outcome would occur if differences in partitioning bias between fragments played no role in competitive fitness, which we know from the experiments described above is not the case. Of the fragments present in the experiments, 863 (38%) fell near this diagonal line, but many other fragments fell in regions suggesting that partitioning might affect their competitive fitness. For example, 103 fragments fell within the bottom right corner of the graph  . These fragments showed high competitive fitness in the Acen-miniARS competition but low competitive fitness in the Cen-miniARS competition and represented 32 distinct ARSs, 28% of the ARSs analyzed in this experiment. Fragments in this region of the heat map likely contained (a) DNA sequence element(s) that contributed to partitioning fitness, giving them a high competitive fitness value in the Acen-miniARS experiment even though their intrinsic replication fitness was relatively low. Conversely, the 172 miniARS-fragments, representing 24 ARSs, within the top left corner  likely contained intrinsically strong replication ability that was masked in the Acen-miniARS experiment by being out-competed by miniARS fragments with robust partitioning ability.The comparative approach was used in a combined analysis of all miniARS fragments to determine how many miniARSs were affected substantially by an associated partitioning function in the Acen-miniARS experiment. In vs. replication, we initially focused on regions that we interpreted to enhance partitioning. For this purpose, fragments in the bottom left corner of the graph  were grouped together and compared to fragments in the bottom right corner . These fragments should differ from each other at the level of partitioning ability. As seen in the top pair of heat maps in ARS1. Thus, at this level of combined analyses, selecting for either enhanced partitioning or replication abilities led to similar, though not identical, average fragments. A similar outcome was obtained from reverse analyses, that is examining loss of replication abilities or partitioning abilities rather than acquisition of these abilities   . Each ofARS523, ARS1007, ARS1018). Each of these miniARSs generated several fragments that fell on the far right side of the graph, including some in the bottom right corner, indicative of fragments containing relatively strong partitioning capacity relative to replication. In contrast, the example “nonpartitioner” miniARS422 did not generate any fragments that fell within the far right bottom corner of the graph. In fact, most miniARS422 fragments fell on the left side of the diagonal line, suggesting that many ARS422 fragments had high intrinsic replication ability compared to other fragments in the data set, but relatively weak partitioning ability.Analogous heat maps for four nonsilencer miniARSs are also shown in ARS318 generated many fragments distributed throughout the heat map in i.e., high competitive fitness in the Cen-miniARS experiment) to one maximally fit for partitioning . To map nucleotides important for the partitioning ability of miniARS318, fragments from the lower left corner of the ARS318 graph in ARS318 acquired partitioning ability was on the 5′ side of the ORC binding site and included the silencer ABF1 site, which is essential for its silencer function. These data indicate a role for another silencer binding protein in partitioning, and are consistent with the current model that links intrinsic aspects of silencer function to plasmid partitioning  which had been shown by direct ARS assays to contain a region(s) required for partitioning . In the total set of 26 nonsilencer ARSs, including the three shown in We extended this mapping approach to the 23 additional nonsilencer partitioning origins that were defined as such because they contained at least one fragment within the bottom right corner of the heat map in Figure S4 revealed 74 discrete motifs within partitioning regions, and 26 discrete motifs within nonpartitioning regions of these ARSs (Table S1). Of the 74 putative partitioning motifs, 10 were identified in ≥ 10 of the 26 partitioning ARSs and were therefore called “high frequency” partitioning motifs (Table S1). Interestingly, while the ABF1 site was a high frequency partitioning motif, a RAP1 site was not identified in these analyses, consistent with the observation that RAP1 and ORC site juxtaposition is a defining feature of the E-silencer ARSs. Regardless, these analyses suggested that multiple different sequence-specific DNA binding proteins might influence plasmid partitioning, perhaps some as strongly as the silencer Rap1-based mechanism . Notably, the 26 nonpartitioning motifs were not enriched in any GO category relative to the 256 sequence-specific DNA binding proteins and thus are not shown in Table S2). Thus, for example, DNA binding proteins defined by the high frequency partitioning motifs were enriched 12-fold over all sequence-specific DNA binding proteins in the GO category “negative regulation of filamentous growth,” while the larger group of all partitioning motifs were enriched ∼threefold in this category. All together, these analyses indicated that the putative partitioning motifs were not a random group of sequence-specific DNA binding motifs, but potentially linked to negative regulation of RNA metabolism and particular growth strategies.Because a large number of putative partitioning motifs were identified (n = 74) within the partitioning regions queried, we looked for shared features among the DNA binding proteins predicted to bind these motifs by using annotated data sets available on SGD. Gene Ontology (GO) analyses were performed using four different groups of sequence-specific DNA binding proteins. The first group was the entire set of sequence-specific DNA binding proteins defined on SGD . All results from analyses of the next three selected groups were normalized to this group. The first selected group contained the DNA binding proteins predicted to bind the partitioning motifs we identified in the 26 nonsilencer origins . If a particular sequence-specific DNA binding protein is associated with a specific phenotypic category, it means that experimental data provide evidence that the wild-type function of this protein contributes to the wild-type version of this phenotype. For example, Fkh1 and Fkh2 binding sites were among the collection of high frequency partitioning motifs (n = 10). Deletions of FKH1 and FKH2, while viable, alter cell cycle progression, normal cell shape and colony morphology, and other phenotypes particularly sensitive to normal G2/M regulation, and therefore cause pseudohyphal-like growth (We used the same strategy to assess Phenotypic Ontology terms (PO) as annotated in SGD except that, in contrast to the GO analysis, only selected phenotypes  PO terms were queried within six general categories: mitotic cell cycle, intracellular transport, development, morphology, colony appearance, and chromosome/plasmid maintenance , and centromeres. Adaptation of next-generation sequencing strategies to plasmid-based assays provides the opportunity to obtain information about these and other elements at levels of depth and breadth not previously possible. In this study, we used a competitive growth strategy coupled to high throughput sequencing to assay the competitive fitness of thousands of individual miniARS fragments simultaneously. We used this approach to try and disambiguate two fundamental contributors to efficient plasmid propagation: replication fitness and partitioning fitness. By comparing the competitive fitness behavior of fragments from a miniARS library cloned in two different plasmid backbones, one that contained a centromere (Cen) and one that lacked a centromere (Acen), we demonstrated that 26 of 112 yeast miniARS fragments contained previously undocumented putative plasmid-partitioning elements. We mapped chromosomal regions responsible for this ability and found that they contained 74 distinct motifs. Prior to this study, only one partitioning element had been defined—specifically the RAP1 site present in transcriptional silencers and telomeres. Thus, this study describes an effective high throughput approach that can help distinguish between partitioning and replication functions that influence plasmid propagation, reveals that a large fraction of ARS fragments likely contain uncharacterized mechanisms for partitioning, and identifies a large number of candidate factors that might be capable of regulating partitioning.The ability of small plasmids to propagate efficiently in the model eukaryote i.e., is essentially a by-product of) its chromosomal role in transcriptional silencing  silencers ARS317 (HMR-E) and ARS301 (HML-E). While it is possible that the reported “rereplication” ability of ARS317 might contribute to this element’s exceptional behavior in the Acen miniARS competition, we propose that plasmid partitioning was likely a larger, more general factor for two reasons , indicating that they lost competitive fitness in the Acen miniARS experiment when they lost Rap1-mediated plasmid partitioning ability. Therefore, the Acen mini ARS competition selected for retention of a chromosomal element known to influence plasmid partitioning.In the Acen miniARS competition experiment, many of the most competitive miniARS fragments mapped to the  reasons . First, HMR-I (ARS318) silencer was also identified as a partitioning ARS in this study, with the partitioning ability mapping to the HMR-I silencer ABF1 site. Though the silencer-ABF1 site is not as recognized as the RAP1 site for its silencer-related partitioning ability, early studies of HMR-E indicated it could influence Cen-independent inheritance and a later study also connected it to partitioning  is a weaker silencer than either HMR-E (ARS317) or HML-E (ARS301), and in this study the miniARS318 fragments with the highest competitive fitness value in the Acen-miniARS still showed less competitive fitness than the most competitive miniARS317 or miniARS301 fragments. The miniARS318 partitioning probably works through the same Sir4-Esc1-mediated mechanism, but HMR-I is simply less effective at binding Sir4, which would also explain why it functions as a weaker silencer.The ARS523, ARS1007, and ARS1018 each contained partitioning region(s) based on the regions’ importance in an Acen plasmid but their complete dispensability in a Cen plasmid. That is, these regions of these nonsilencer ARSs behaved like the RAP1 site within ARS317, in that they were not important for the intrinsic replication function of their associated ARSs. Motif analyses identified several distinct sequence motifs in the partitioning regions in these ARSs but none of these matched a RAP1 or ABF1 site, suggesting that silencer independent mechanisms for plasmid partitioning exist. Importantly, these ARSs’ partitioning regions functioned as well as the silencer ARS317Rap1-mediated partitioning ability, based on the PLRs determined from standard ARS assays. These locus-specific experiments, together with the silencer studies, formed the strong proof-of-principle foundation for the identification of 74 new candidate-partitioning motifs by comparing fragment competitive fitness in the Acen- and Cen-miniARS competition experiments.The silencer data raised the possibility that the Acen miniARS experiment had the potential to identify other as yet unknown partitioning elements in the yeast genome and, indeed, additional direct ARS assays showed that several other ARSs represented in the library likely contained as yet uncharacterized partitioning ability. In particular, i.e., ARS efficiency) in a Acen miniARS experiment, primarily because individual yeast cells can only tolerate so many copies of a given plasmid before the replication fitness of the plasmid is reduced . The assumption of such a penalty is supported by recent studies that show that the large fraction of origins present in the rDNA repeats  functionally compete with nonrDNA chromosomal origins for limiting origin activation factors   bypasses the blockade at the constricted bud neck and enhances a plasmid’s ability to get into the daughter cell. Thus, anchoring a plasmid to the INM provides an active mechanism to promote plasmid distribution to the daughter cell.The influence of INM association on plasmid behavior reflects a fundamental feature of yeast cell division (Rap1-INM tethering mechanism is part of a distinct, well-studied mechanism associated with a specific type of transcriptional repression, many links between transcriptional repression and the INM have been observed in multiple model organisms (Fkh1, Fkh2, Rlm1, Dig1, and Yap6 (Fkh1 and Fkh2 proteins activate transcription of CLB2-cluster genes in mid to late S-phase, but at other stages may repress or “antiactivate” target genes (Fkh1- and Fkh2- bound chromosomal regions become associated with the INM during G2- M-phase as part of the transcriptionally repressed state. INM-anchoring-mediated plasmid partitioning would be most critical during M-phase, when the nucleus is actually dividing (Azf1, Rim1, Dig1, and Yap6, physically interact with karyopherins and/or membrane proteins, suggesting links between these gene-regulatory proteins and the INM (While the silencer Fkh1 and Fkh2 proteins, identified here as high frequency partitioning factors, were recently implicated in establishing chromosome–chromosome interactions via 3D chromosome-capture assays (Dig1, Fkh1, and Yap6 physically associate with components of the kinetochore and or/spindle machinery (Linking a plasmid to the INM is only one general mechanism capable of enhancing plasmid partitioning. For example, artificially establishing an association between a plasmid and telomeres via a fusion protein also enhances plasmid partitioning (A deeper understanding of the mechanisms that modulate plasmid partitioning in yeast may provide novel insights into the cellular activities they reflect. Indeed, silencer-mediated partitioning has provided important tools and insights into the relationships between heterochromatin, telomere structure, and chromosomal architecture in the nucleus. An intriguing hypothesis concerning plasmid cell retention in mother cells is that mechanisms that favor this retention may act as a form of “genomic immunity,” which discourages the propagation of foreign DNA in the cell population (


V567es), but not the full-length AR (AR-FL), reduced the sensitivities to taxanes in LNCaP cells. Treatment with taxanes inhibited the transcriptional activity of AR-FL, but not those of AR-Vs. This could be explained, at least in part, due to the inability of taxanes to block the nuclear translocation of AR-Vs. Through a series of deletion constructs, the microtubule-binding activity was mapped to the LBD of AR. Finally, taxane-induced cytoplasm sequestration of AR-FL was alleviated when AR-Vs were present. These findings provide evidence that constitutively active AR-Vs maintain the AR signaling axis by evading the inhibitory effects of microtubule-targeting agents, suggesting that these AR-Vs play a role in resistance to taxane chemotherapy.Docetaxel-based chemotherapy is established as a first-line treatment and standard of care for patients with metastatic castration-resistant prostate cancer. However, half of the patients do not respond to treatment and those do respond eventually become refractory. A better understanding of the resistance mechanisms to taxane chemotherapy is both urgent and clinical significant, as taxanes (docetaxel and cabazitaxel) are being used in various clinical settings. Sustained signaling through the androgen receptor (AR) has been established as a hallmark of CRPC. Recently, splicing variants of AR (AR-Vs) that lack the ligand-binding domain (LBD) have been identified. These variants are constitutively active and drive prostate cancer growth in a castration-resistant manner. In taxane-resistant cell lines, we found the expression of a major variant, AR-V7, was upregulated. Furthermore, ectopic expression of two clinically relevant AR-Vs (AR-V7 and AR Prostate cancer is the most common non-skin cancer and the second leading cause of cancer mortality in men in the United States. Androgen deprivation therapy, which disrupts androgen receptor (AR) signaling by reducing androgen levels through surgical or chemical castration, or by administration of anti-androgens that compete with androgens for binding to AR , is the Paclitaxel, docetaxel, and cabazitaxel belong to the taxane family of chemotherapeutic agents. Taxanes bind to the microtubules and prevent their disassembly, thereby suppressing microtubule dynamics, leading to mitotic arrest and apoptosis . This wav567es, have shorter cancer-specific survival than other CRPC patients [Sustained signaling through AR has been established as a hallmark of CRPC. Recently, alternative splicing variants of AR (AR-Vs) that lack the ligand-binding domain (LBD) have been identified –13. Thespatients . In addipatients –20.Recently, laboratory and clinical studies have suggested the existence of a cross-resistance mechanism between taxane-based chemotherapy and second-line hormonal therapies –25. In tWe first established taxane-resistant 22Rv1 and LNCaP95 lines by culturing cells in escalating doses of paclitaxel and docetaxel over a period of 2 months. The response to taxanes were determined by the MTT assay Fig. . Westernv567es into the AR-V-null LNCaP cells, and measured the responses to taxanes. As shown in Fig. v567es, but not in those overexpressing AR-FL, than in vector-transfected cells. Similar observations were made with paclitaxel and cabazitaxel  assays in COS-7 cells expressing fluorescence-tagged AR proteins. Following treatment with docetaxel, selected nuclei were photobleached and the cells were imaged at regular intervals. Nuclear translocation is indicated by recovery of the nuclear to cytoplasmic fluorescence ratio (Fn/c). As indicated by the confocal images Fig.  and the lls Fig. . To subs7es Fig. . Collectin vivo microtubule-binding assays in COS-7 cells ectopically expressing AR. Under the condition in which the microtubules were stabilized, the majority of AR-FL co-precipitated with the microtubules and was found in the pellet  than ADT alone [To date, docetaxel and cabazitaxel are the only chemotherapeutic agents that have been shown to offer survival benefits for patients with mCRPC. Even in today's rapidly evolving landscape of treatment options for mCRPC, taxane-based chemotherapy continues to be an important component of the treatment regimens. Recently, a randomized phase III trial supports the expansion of the indications of taxanes to earlier disease stages. The CHARRTED trial demonstrated that the addition of docetaxel to ADT in patients with high-volume, metastatic, hormonal-sensitive disease improves overall survival by 17 months , p53, Rb, and parathyroid hormone-related protein (PTHrP) . On the v567es have a significantly shorter survival than other CRPC patients [The clinical relevance of AR-Vs has been demonstrated by a myriad of studies. Higher expression of AR-V7 in hormone-naïve prostate tumors predicts increased risk of biochemical recurrence following radical prostatectomy , 12, andpatients , indicatpatients –19. Partpatients .While the roles of AR-Vs are well recognized in resistance to hormonal therapies, evidence has just started to accumulate to support their involvement in resistance to taxane chemotherapy. Thadani-Mulero and colleagues are the first to show evidence supporting a role of AR-V7 in resistance to taxane chemotherapy . In addiv567es is inhibited by the taxanes. In contrast to the data present herein, Thadani-Mulero and colleagues showed that ARv567es associates with the microtubules and that the nuclear translocation of ARv567es is inhibited by taxanes. In addition, the microtubule-binding activity is mapped to the DNA-binding and hinge domains of AR [in vitro assays in which cell lysates containing AR proteins tagged by GFP or hemagglutinin were incubated with purified tubulin in a cell-free system to allow microtubule polymerization and association. In contrast, we conducted in vivo microtubule-binding assays in which the microtubules and associated proteins were extracted from cells expressing untagged AR isoforms. Another major difference between the two studies is the dosage of taxanes. Docetaxel was applied at a concentration of 1 μM in the cell culture studies by Thadani-Mulero et al., in contrast to the clinically attainable [v567es.The main disparity between our study and that of Thadani-Mulero et al.  is on whns of AR . One postainable  nanomolav567es. These findings are consistent with our importazole data, suggesting that the nuclear import of the AR-Vs is mediated by the importin α/β pathway. They also found that unlike AR-FL, the nuclear localization of AR-V7 and ARv567es is not affected by an inhibitor for heat shock protein 90. Together, this study and our data present herein suggest a fundamental difference between AR-FL and AR-Vs in the events upstream of importin α/β-mediated nuclear entry.The canonical AR nuclear localization signal (NLS) is located in the hinge domain, encoded by exons 3 and 4. Sequence analysis predicted that this NLS is truncated in AR-V7. However, the study by Chan et al. demonstrated that splicing of exon 3 with cryptic exon 3 in AR-V7 reconstitutes this bipartite NLS, which mediates the nuclear import of AR-V7 . In addiv567es in attenuating the response to taxane-based chemotherapy. Mechanistically, we demonstrated that both variants translocate to the nucleus in a microtubule-independent manner. Additionally, these variants can reduce the microtubule-binding activity of AR-FL, thus circumventing its cytoplasm sequestration triggered by taxanes. These findings have important clinical implications. The expression status of these AR variants could potentially be used as a biomarker to aid treatment selection and sequencing. More importantly, targeting AR-Vs could be a fruitful direction to pursue to enhance the efficacy of taxane chemotherapy. To this end, several small molecule inhibitors at various stages of clinical development have shown promises against AR-Vs [In summary, our study provides support for the involvement of AR-V7 and ARst AR-Vs –43, openLNCaP, 22Rv1, and COS-7 cells were obtained from American Type Culture Collection. With the exception of drug-resistant lines, cells used in this study were within 20 passages (∼3 months of non-continuous culturing). All cell lines were tested and authenticated by the method of short tandem repeat profiling. Docetaxel, cabazitaxel, and paclitaxel were purchased from Selleck Chemicals . Nocodazole was from Sigma Aldrich, and KX-01 was provided by Kinex Pharmaceuticals. The following antibodies were used in Western blot analysis: anti-GAPDH, anti-AR , anti-importin β1, anti-β-actin (Santa Cruz), anti-p53 , anti-histone H3 , and anti-AR-V7 (Precision Antibody).22Rv1 cells were initially treated with 10 nM paclitaxel for 72 hours and the surviving cells were re-seeded and allowed to recover for 1 week. Paclitaxel-resistant cells were developed over a period of 2 months by stepwise increasing concentrations of paclitaxel (5–50 nM). Age-matched parental cells which did not receive treatments were maintained in parallel. Docetaxel-resistant 22Rv1 and LNCaP95 lines were generated in a similar manner, but with different doses of docetaxel . The resistant cells were continuously maintained in the highest concentration of the taxane in which they selected.Cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed with 2X Cell Lysis Buffer  containing a phosphatase inhibitor and the protease inhibitor cocktail (Sigma). After incubating the cells on ice for 30 min, lysates were collected by centrifugation at 10, 000 rpm for 10 minutes. Protein concentrations were determined by the BCA Protein Assay kit (Pierce). The samples were separated on 10% SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking in TBS buffer  containing 5% nonfat milk, the blots were incubated with a primary antibody overnight at 4°C and a fluorescent-labeled secondary antibody for 1 h at room temperature. The fluorescent signals were obtained by the Odyssey Infrared Imaging System (LI-COR Bioscience).v567es. After incubating with the transfection mixture for 4 h, cells were re-plated in RPMI 1640 containing 10% charcoal-stripped fetal bovine serum (cs-FBS). Cells were allowed to recover overnight before treated with DTX (1 and 2.5 nM) or PTX (2.5 or 5 nM) in the presence or absence of 10 nM DHT. Dual-luciferase assay was performed at 24 h post treatment using the Dual-luciferase Reporter Assay System (Promega). The renilla luciferase activity was used to normalize that of firefly luciferase.COS-7 cells were seeded in 10-cm dishes at a density to reach 80–90% confluency at time of transfection. Transient transfection was performed by using the Lipofectamine and Plus reagents following the manufacturer's instructions (Invitrogen). Cells were co-transfected with ARR3-luc luciferase reporter contruct and pRL-TK, along with a plasmid encoding for AR-FL, AR-V7 or ARv567es plasmids were generated by cloning the cDNA fragments for AR-V7 and ARv567es, respectively, into the pCMV-TurboFP635 vector. COS-7 cells were transfected with indicated plasmids and cultured in phenol red-free RPMI-1640 supplemented with 10% cs-FBS. At 40 hr after transfection, cells were pre-treated with or without 10 nM docetaxel for 6 hr, followed by treatment with or without 1 nM R1881 for 4 hr. COS-7 cells were subsequently fixed with 2% paraformaldehyde, and the nuclei were stained with 2.5 μM DRAQ5 . Confocal images were obtained by using a Leica TCS SP2 system with a 63X oil-immersion objective on a Z-stage, and an average of 6 fields with ∼10 cells per field were captured for each group. Data quantitation was performed as described [Subcellular localization of AR proteins was analyzed by confocal fluorescence microscopy. The pTurboFP635-AR-V7 and pTurboFP635-ARescribed .FRAP assay was performed using a Leica TCS SP2 microscope equipped with 20X, 40X and 63X oil immersion lenses (Nikon) in combination with a heated stage , as described by Roth et al.  with modIn Vivo Assay Kit  following the manufacturer's instructions. Briefly, 3 × 106 cells were lysed in 4 mL pre-warmed (37°C) Lysis and Microtubule Stabilization 2 (LMS2) buffer  glycerol, 0.1% Nonidet P40, 0.1% Triton X-100, 0.1% Tween 20, 0.1% β-mercaptoethanol, 0.001% Antifoam, 100 μM GTP, 1 mM ATP, 1 × protease inhibitors cocktail) in a 10-cm cell culture dish. The lysates were collected and spun at 2,000 g for 10 min at 37°C to remove nuclei and unbroken cells. The supernatants were then subjected to ultracentrifugation at 100, 000 g for 30 min at 37°C to separate the microtubules from the soluble, unpolymerized tubulin. The pellet was washed with pre-warmed LMS2 buffer and centrifuged at 100, 000 g for 30 min at 37°C. For microtubule destabilization conditions, LMS2 buffer containing nocodazole (5 μg/ml) or CaCl2 (2 mM), or ice-cold LMS2 buffer were used in the above procedure. The pellets were resuspended in ice-cold 2 mM CaCl2 and incubated in room temperature for 15 min to depolymerize microtubules. The supernatant (S), wash solution (W), and resuspended pellet (P) were adjusted to equal volumes and analyzed by Western blotting.The AR deletion constructs were generated by inserting PCR products of the corresponding cDNA regions into the pcDNA3.1(−) vector. The resulting plasmids were sequenced to confirm sequence accuracy and in-frame reading. COS-7 cells were transfected with indicated plasmids and cultured in RPMI 1640 medium supplemented with 10% cs-FBS. Microtubule-binding assay was performed by using the Microtubule/Tubulin Student's two-tailed t-test was used to determine the difference in means between two groups. P < 0.05 is considered significant. Data are presented as mean ± standard error of men (SEM).Statistical analysis was performed using Microsoft Excel. The