C. elegans. In lin-44/wnt and lin-17/frizzled mutant animals, the PDB neurites often failed to prune and grew towards the lin-44-expressing cells. Surprisingly, membrane-tethered lin-44 is sufficient to induce proper neurite pruning in PDB, suggesting that neurite pruning does not require a Wnt gradient. LIN-17 and DSH-1/Dishevelled proteins were recruited to the pruning neurites in lin-44-dependent manners. Our results revealed the novel gradient-independent role of Wnt signaling in instructing neurite pruning.During development, the nervous system undergoes a refinement process by which neurons initially extend an excess number of neurites, the majority of which will be eliminated by the mechanism called neurite pruning. Some neurites undergo stereotyped and developmentally regulated pruning. However, the signaling cues that instruct stereotyped neurite pruning are yet to be fully elucidated. Here we show that Wnt morphogen instructs stereotyped neurite pruning for proper neurite projection patterning of the cholinergic motor neuron called PDB in  Development of the nervous system is a highly dynamic process which involves neurogenesis, cell migration, neuronal polarization and outgrowth of neuronal processes. It is also well known that neurons initially extend an excess number of neurites most of which will later be eliminated by the mechanism called neurite pruning . FailureDuring neuronal refinement, neurites often compete with each other for their target cells in an activity-dependent manner, and neurites that are outcompeted are eliminated by pruning. For example, each muscle fiber in the mouse trapezius muscle is initially innervated by multiple motor neurons in early postnatal stage, while axons with weaker synaptic strength are pruned by the mechanism called axosome shedding . The neuIn addition to activity-dependent pruning, many neurites undergo stereotyped pruning during development . SeveralNeurite pruning also plays a crucial role in sexually dimorphic neurocircuit formation. The expression of BDNF from the mammary mesenchyme is required for the innervation of the mammary gland sensory axons. In males but not in females, a truncated TrkB receptor isoform is expressed in the mammary mesenchyme which deprives BDNF availability for sensory axons thereby inducing male-specific axon pruning . Recent Caenorhabditis elegans, it has been shown that the transcription factor MBR-1/Mblk-1 is required for the pruning of the excess neurites of the AIM interneurons  that expresses a membrane-tethered GFPnovo2, a brighter GFP variant  and second larval (L2) stages. To understand how PDB neurite achieves V-shape turning, we first examined the structure of the developing PDB neurite using a population of semi-synchronized animals . Interesunc-13, which is required for synaptic vesicle fusion and neurotransmitter release  did not have PDB posterior neurites as observed in wildtype . Upon Wnt binding, these receptors execute distinct downstream cascades via the multi-domain protein, Dishevelled  expressed under the lin-44 promoter during PDB development. Strikingly, at the L2 stage when PDB posterior neurites undergo pruning, we found that the pruning posterior neurite was located in close proximity to the most posterior hypodermal cell, hyp10  and 6% (6/100) of the lin-44(n1792) null animals showed neurite guidance and cell fate determination defects (no PDB), respectively  of them showed ectopic posterior neurites in PDB at the L4 stage, suggesting the defective neurite pruning during PDB development   . This relin-17/fz also plays a role in neurite pruning in PDB. While lin-17(n671) null mutants showed significantly more severe cell fate or guidance defects than lin-44/wnt mutants (lin-44 mutants: only 17.0% of the posterior neurites (9/53) were pruned 24 hr after L2 stage  was not significantly different from lin-17 single mutants, suggesting that lin-44 and lin-17 act in the same genetic pathway  or the PDB promoter  significantly rescued the pruning defects of lin-17 mutants, suggesting that lin-17 acts cell-autonomously in PDB (LIN-44/Wnt utilizes LIN-17/Frizzled(Fz) as a primary receptor in many contexts . We then mutants , we obseL2 stage . The pru pathway . We alsoy in PDB . These rlin-44/wnt is expressed posteriorly to the PDB neurites, it is possible that LIN-44 acts as an instructive cue for PDB neurite pruning. Alternatively, it might also act as a permissive cue for PDB neurite pruning. In order to distinguish these two possibilities, we conducted the rescue experiment by expressing LIN-44 ectopically using the promoter of another wnt gene, egl-20. egl-20/wnt is expressed in the cells around preanal ganglions  (lin-44 null mutants (lin-44 from the lin-44 promoter (Ex[Plin-44::lin-44]) completely rescued the pruning defects to the wildtype level  and its receptors mig-1/fz and cam-1/Ror   was not  mutants . On the pe level . This rel events . We ther pruning . Consistam-1/Ror . Anotherthe worm . The PDBC. elegans , suggesting that anterior expression of lin-44 is not sufficient to induce anterior neurite pruning in PDB. Second, during PDB development, both growing anterior and pruning posterior neurites are located close to the lin-44-expressing hyp10 cell  and Wingless/Wnt (Nrt-Wg) is used to characterize the gradient-independent role of Wingless/Wnt  and BFP into the endogenous lin-44 locus by CRISPR/Cas9 (miz56[nrt-bfp-lin-44])  comparable to wildtype  . The BFPng cells . The briwildtype . This inen passant synapses onto dorsal body wall muscles  . It is t muscles . Previou muscles  . Similar to Frizzled receptors, Dsh proteins are often localized asymmetrically within the cells in Wnt-dependent manners . Nevertheless, DSH-1/Dsh::GFP puncta were localized in the posterior neurites in the wildtype and nrt-bfp-lin-44 but not in the lin-44 null animals (Dishevelled (Dsh/Dvl) proteins are the intracellular signaling components of both canonical and non-canonical Wnt signaling and are known to co-localize with active Frizzled receptors . There a manners . We did  animals . Consist animals . Taken t animals .C. elegans. Asymmetric Wnt signal from the posterior hypodermal cells specifically induces pruning of the posterior neurite to sculpt the V-shape projection pattern of the PDB neurite. The unique structure of PDB and its topographic arrangement relative to the signaling cues allowed us to uncover the unique function of gradient-independent Wnt signaling in neurite pruning.Developmental neurite pruning plays crucial roles in shaping functional neurocircuits, yet not much is known about the signaling cues that instruct stereotyped neurite pruning during neuronal development. Here, we found that Wnt signal from the hypodermal cells instructs stereotyped neurite pruning in the PDB cholinergic motorneuron in C. elegans, two Wnts, CWN-1 and CWN-2, act through CAM-1/Ror (receptor tyrosine kinase) to protect neurites from the mbr-1-mediated pruning in the AIM interneurons: the pruning defect in AIMs of mbr-1 mutants is suppressed by mutations in cam-1, while overexpression of cam-1 in AIMs inhibits neurite pruning   . CSDns u2+) in context-dependent manners. We have examined potential downstream signaling components such as bar-1/β-catenin, fmi-1/flamingo, vang-1/Van Gogh but none of them showed significant neurite pruning defects  are viable and exhibit no obvious morphological abnormalities. This implies that gradient-independent or contact-dependent Wnt signaling is largely sufficient for the normal tissue patterning during development . Further candidate and forward genetic screenings will reveal novel factors that are specifically required for the contact-dependent Wnt signaling.In this study, we showed that the membrane-tethered LIN-44 (Nrt-BFP-LIN-44) is sufficient for the PDB neurite pruning but not for the synapse patterning. Nrt-BFP-LIN-44 is non-diffusible since it did not replace the function of LIN-44 in DD6 axon termination and DA9 synapse patterning. How can cells distinguish gradient-dependent and independent Wnt signaling? One possible explanation is that each Wnt-dependent process requires a distinct threshold of the Wnt concentration to activate the signaling cascade. The processes that are gradient-dependent might require lower Wnt concentration to execute the process compared with the gradient-independent process. Alternatively, it is also possible that the gradient-independent or contact-dependent processes require transmembrane co-factors that mediate contact-dependent Wnt signaling. elopment , and sdnn-44/wnt . Howeverlin-44-expressing cells, can ignore the LIN-44/Wnt pruning cue. It also explains how the PDB neurite can initially grow posteriorly from its cell body toward the source of pruning cue (LIN-44-expressing cells). On the other hand, we do not exclude the possibility that the anterior neurite responds to the attractive guidance cues from the anterior cells so that it is protected from the Wnt-dependent neurite pruning. We have tested several candidates including sax-7/Neurofascin, which guides and stabilizes the dendritic arbor structure of the PVD sensory neuron  efficiently induced posterior neurite pruning, suggesting the contact with Wnt-expressing cells rather than the gradient distribution of LIN-44 triggers the PDB neurite pruning. The contact-dependent neurite pruning model can explain why the anterior neurite, which is also located in close proximity to the y neuron , unc-129 muscles , unc-6/Nreceptor , and cam pruning , but nonlin-44(n1792), lin-44(miz56), lin-17(n671), egl-20(n585), mig-1(e1787), cam-1(gm122), bar-1(ga80), fmi-1(tm306), vang-1(ok1142), unc-13(e450). Genotyping primers are listed in the supplemental material.Bristol N2 strain was used as wildtype reference. All strains were cultured in the nematode growth medium (NGM) as described previously  at 22°C.mizIs9 ; wyIs486 ; mizEx248, mizEx389 ; mizEx349, mizEx350 ; mizEx384, mizEx385 ; mizEx380, mizEx381 ; mizEx386, mizEx387 , mizEx374, mizEx376 ; mizEx366 ; mizEx271 ; mizEx295 ; mizEx291 . All rescuing constructs were injected at 10 ng/µl except Punc-129dm::lin-44 which was injected at 20 ng/µl.The transgenic lines were generated using standard microinjection method : mizIs9 C. elegans expression clones were made in a derivative of pPD49.26 (A. Fire), the pSM vector (a kind gift from S. McCarroll and C. I. Bargmann). dsh-1 cDNA clone was obtained by RT-PCR from N2 mRNA using Superscript III First-strand synthesis system and Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific). lin-17 cDNA clone was obtained from the plasmid used in the previous work  and cloned into the SphI and AscI sites of the pSM-GFPnovo2 vector . C. elegans codon-optimized BFP with three synthetic introns was inserted into the 3’ end of the Neurotactin-2xHA sequence to generate Neurotactin-2xHA-BFP construct. 700 bp of the 5’ homology arm of the lin-44 promoter region, Neurotactin-2xHA-BFP (5’ portion until middle of intron 1), BFP (3’ portion from intron 1), 700 bp of the 3’ homology arm spanning lin-44 coding region are cloned into the SacII and NotI sites of a dual-marker selection cassette (loxP + Pmyo-2::GFP::unc-54 3′UTR + Prps-27::neoR::unc-54 3′UTR + loxP vector) (The plasmid containing a codon-optimized  vector) .CGATCAGTGGTGCACCTGC, gRNA4-TATTTCCGTCTTCAGCCAA) near the start codon of the lin-44 gene were selected using CRISPR guide RNA selection tool (http://genome.sfu.ca/crispr/) and were cloned into RsaI site of the sgRNA (F+E) vector, pTK73    were co-eviously . The selEggs at various stages were collected by bleaching the gravid adults with a bleaching solution (2.5% sodium hypochlorite and 0.5N Sodium Chloride) for 5 min. The eggs were washed twice with M9 buffer and seeded onto an NGM plate. For observing the pruning event, the animal was rescued from the slide after the first imaging at the L2 stage (approximately 26 hr after seeding eggs onto the NGM plates) and placed it in an individual NGM plate to resume the development for 24 hr before re-imaging at early L4 stages.C. elegans using a Zeiss LSM800 Airyscan confocal microscope . Worms were immobilized on 2% agarose pad using a mixture of 7.5 mM levamisole (Sigma-Aldrich) and 0.225M BDM  (Sigma-Aldrich). Images were analyzed with Zen software (Carl Zeiss) or Image J .Images of fluorescently tagged fusion proteins were captured in live For time-lapse imaging, animals were mounted onto 5% agarose pads and immobilized using 0.5 µl of 0.10 µmpolystyrene latex microsphere (Alfa Aesar # 427124Y). The coverslip was sealed with Vaseline , to avoid dehydration of the animals and agarose pad during imaging.For quantification of LIN-17::GFP and DSH-1::GFP signal at the growth cones, 1.34 µm × 0.37 µm (LIN-17::GFP) or 1.54 µm × 0.46 µm (DSH-1::GFP) region of interest (ROI) were set at the tip of anterior and posterior neurites. The signals of the GFP and BFP channels from the adjacent region with the same size of ROI was used to subtract the background signal. The GFP signal was normalized by the BFP signal of the same ROI.Data were processed using Prism7 . We applied the Chi-square test (with Yates’ continuity corrected) for comparison between two binary data groups, and one-way ANOVA method for comparison among more than three parallel groups with multiple plotting points. Data were plotted with error bars representing standard errors of the mean (SEM) or standard error of the proportion (SEP). *, ** and *** represent p-value<0.05,<0.01 and<0.001 respectively. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Your study very nicely describes the molecular basis of an intriguing, developmental neurite pruning process, a topic of broad and general interest in the developmental neuroscience community.Decision letter after peer review:C. elegans" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor (Oliver Hobert) and Marianne Bronner as the Senior Editor. The reviewers have opted to remain anonymous. The reviewers have discussed the reviews with one another and with the Reviewing Editor.Thank you for submitting your article "Gradient-independent Wnt signaling instructs asymmetric neurite pruning in There is general agreement about the overall interest of this story and the novelty of insights provided. However, everyone agreed that a small and well-defined number of very straight-forward and simple experiments need to be done that will solidify the conclusions of the manuscript. The experiments are detailed below in the reviewer's reports.Reviewer #1:C. elegans. Interestingly, they find that this is dependent on close contact between the outgrowing neuronal process and the Wnt producing cells, and using membrane-tethered Wnt, they show that short-range signaling is sufficient for pruning. Moreover, they show that this local Wnt activity induces clustering of Frizzled and Dishevelled at the ectopic neurite. The role of Wnt signaling in neurite pruning is still poorly understood and this study provides important new insight into this process.The authors describe a role of Wnt signaling in the pruning of ectopic neurites of a developing neuron in lin-44/Wnt in PDB cell fate specification. In lin-44 mutants, 32.3% of animals show defects in PDB development, ranging from absence of the cell to neuronal polarity and axon guidance defects. It is therefore important to show that the effect on pruning is distinct from these functions in cell fate specification. One way to show this would be to examine these other aspects of PDB development in the membrane-tethered LIN-44 strain. If membrane-tethered LIN-44 only rescues the pruning phenotype, this would be a strong argument that this is indeed a separate (short-range) function of LIN-44.I have one major concern that the authors should address, and that is the role of Reviewer #2:C. elegans" characterizes the function of the Wnt ligands and Frizzled receptors in regulating neurite pruning. In particular, the authors first identify a novel pruning event on the axon of the posteriorly located PDB neuron, occurring during C. elegans larval stages. Then, they identify Wnt/LIN-44 as a key ligand for correct pruning of posterior extra branches of the PDB neurite. Moreover, the authors reveal a PDB-cell-autonomous function for LIN-17/Frizzled, which functions as the receptor for LIN-44 in this process. Interestingly, they also show that in this context LIN-44 can function when membrane-bound in the secreting epidermal cells, revealing a short-distance role in this process. Finally, they show an effect for LIN-44 in recruiting LIN-17 and Dishevelled to the posterior pruned sections of PDB. The significance of the manuscript relies on the identification of a novel pruning event on a specific neuron, and on the characterization of the molecular mechanisms regulating it. The manuscript is generally well written with a nice flow of the rationale of the experiments, and the discovery itself is quite compelling. However, the enthusiasm is reduced because a number of key experiments are underdeveloped to fully support the conclusions . Here below is a detailed description of these considerations. When completed, these experiments will strengthen the conclusions of this manuscript.The manuscript by Lu and Mizumoto entitled "Gradient-independent Wnt signaling instructs asymmetric neurite pruning in 1) Figure 1B, and D. The number of animals examined is too low. In general, for morphological studies, at least 50 animals are examined . The n should be added to the individual bars.2) In Figure 1—figure supplement 1 (time lapse) there is an additional branching more anterior to the one from which the posterior pruned sections extend. This branch, visible in every section, gets resolved at the 80 min time-point. This is not discussed anywhere in the manuscript. Is it a normal occurrence, or only observed in this representative animal? If common, is it affected by the Wnt and Frizzled mutations?3) Figure 2E. n are low and some of the genes tested here have not been discussed in the paper.lin-44 and lin-17, as well as any significant effect of the double mutants. This is important, because a difference between lin-17 and lin-44 might indicate that another molecule, possibly another Wnt, is involved. As this regard, have the author tested cwn-1, which is also expressed in the posterior region of the animal at early stages of development?4) Figure 3D. n too low to determine any difference between kal-1? If so, is it possible that there is also additional, non-cell-autonomous function of LIN-17?5) In Figure 3—figure supplement 1B, n should be indicated in each bar. How many transgenic strains were analyzed here? What DNA concentrations? Is there a significant difference in the rescue obtained with the rgef-1 promoter versus that provided by egl-20::lin-44 does not rescue the lin-44 lack of pruning is weak: only one transgenic strain was tested with an n = 22. To support this conclusion, the authors should show the absence of rescue for at least three independent extrachromosomal array transgenic strains, and for different concentrations of the transgene. Similarly, the data supporting the functional divergence between LIN-44 and EGL-20 is weak. Results showing that Plin-44::egl-20 does not rescue the lin-44 lack of pruning are only for a very small sample size (n = 14) and for only one transgenic strain. This hypothesis should be further tested.6) In Figure 4. The data showing that Pnrt-bfp-lin-44 animals should be strengthen by increasing the sample size to at least 50 animals. The difference in pruning rate between nrt-bfp-lin-44 and wild-type animals (65.6% vs. 82.1%) could indicate that the membrane-tethered version cannot fully replace the secreted version.7) Figure 5. The data showing that there is "normal" pruning in unc-129 promoter to express LIN-44 in a region in which the anterior/dorsal PDB neurite encounters the dorsal muscle?8) The idea of an instructive, highly concentrated source of LIN-44 is valid. It will be important to test if other transgenic strains expressing LIN-44 under the Pegl-20 promoter do present pruning of the anterior neurites (see point 6 above). Did they try to use an  Reviewer #1:[…] I have one major concern that the authors should address, and that is the role of lin-44/Wnt in PDB cell fate specification. In lin-44 mutants, 32.3% of animals show defects in PDB development, ranging from absence of the cell to neuronal polarity and axon guidance defects. It is therefore important to show that the effect on pruning is distinct from these functions in cell fate specification. One way to show this would be to examine these other aspects of PDB development in the membrane-tethered LIN-44 strain. If membrane-tethered LIN-44 only rescues the pruning phenotype, this would be a strong argument that this is indeed a separate (short-range) function of LIN-44.nrt-bfp-lin-44 exhibited cell fate determination and axon guidance defects at a similar frequency as lin-44 null mutants . The quantification is now included in Figure 2—figure supplement 1. In addition, we also found that the localization of the presynaptic marker, mCherry::RAB-3, in PDB is equally affected in lin-44 null and nrt-bfp-lin-44 animals. mCherry::RAB-3 puncta are mislocalized in the posterior dorsal neurite of the PDB neuron. This phenotype is very similar to the synapse localization defects observed in the DA9 neuron of lin-44 null and nrt-bfp-lin-44 animals. We have included this observation in Figure 6—figure supplement 1, and added the following sentences in the text.We appreciate the reviewer for raising this point. It is very important to test if the membrane-tethered LIN-44 exclusively rescues the pruning defects of the posterior neurites in PDB but not the other gradient-dependent phenotypes. “We further tested if NRT-BFP-LIN-44 is sufficient for replacing the function of diffusible LIN-44 in other aspects of PDB development. […] This result suggests that PDB can respond to diffusible and non-diffusible Wnt signal in a context-dependent manner: it utilizes LIN-44 gradient signal to determine its cell fate, neurite guidance and the subcellular localization of the presynaptic vesicles, while its neurite pruning is induced by gradient-independent LIN-44 signal.”Reviewer #2:[…] The manuscript is generally well written with a nice flow of the rationale of the experiments, and the discovery itself is quite compelling. However, the enthusiasm is reduced because a number of key experiments are underdeveloped to fully support the conclusions . Here below is a detailed description of these considerations. When completed, these experiments will strengthen the conclusions of this manuscript.We thank the reviewer for the criticism. We agree that the sample number for some of the experiments were low to make solid conclusions. We describe the details below.1) Figure 1B, and D. The number of animals examined is too low. In general, for morphological studies, at least 50 animals are examined . The n should be added to the individual bars.We appreciate your criticism. We did our best to increase the sample size. We would like to mention that unlike quantifications in other papers we cite, which mostly use a single snapshot of the animals, our quantification requires a lot more steps, which made the quantification low throughput. For examining the pruning event of single animal, we need to first look for an L2 animal with posterior neurites, take confocal image, rescue the animal from the slide, let it grow for a day and re-take confocal image 24 hours later. During this process, we lose more than half of the animals as they often do not recover or do not mount on the slide in proper orientation.2) In Figure 1—figure supplement 1 (time lapse) there is an additional branching more anterior to the one from which the posterior pruned sections extend. This branch, visible in every section, gets resolved at the 80 min time-point. This is not discussed anywhere in the manuscript. Is it a normal occurrence, or only observed in this representative animal? If common, is it affected by the Wnt and Frizzled mutations?lin-44/wnt-dependent since we often observe multiple neurites at L4 stage of the lin-44 mutant but not in the wildtype animals. However, we did not conduct detailed analyses of the pruning of these neurites since their position and the timing of the pruning are not as stereotyped as the posterior neurites. We have added arrowheads to indicate these transient neurites in the figure and the description in the figure legend.Yes, there are neurites that emerge from the anterior neurites that also undergo pruning. We think their pruning is also 3) Figure 2E. n are low and some of the genes tested here have not been discussed in the paper.We have examined at least 100 animals per genotype, and removed the genotypes we did not mention in the text. The total sample number including the animals with axon guidance and cell fate defects is included in Figure 2—figure supplement 1 B.4) Figure 3D. n too low to determine any difference between lin-44 and lin-17, as well as any significant effect of the double mutants. This is important, because a difference between lin-17 and lin-44 might indicate that another molecule, possibly another Wnt, is involved. As this regard, have the author tested cwn-1, which is also expressed in the posterior region of the animal at early stages of development?lin-44; lin-17, lin-44 and lin-17 mutants.We have increased the sample number as far as we could, and still observed no significant differences among lin-44; lin-17 double mutants (14.3%: 6/42 of the posterior neurites were pruned) was not significantly different from lin-17 single mutants, suggesting that lin-44 and lin-17 act in the same genetic pathway .”“The pruning defect in the cwn-1 mutants and we did not observe obvious pruning defects. We included this data in Figure 2D and added the following description in the text.We also examined the PDB morphology in cwn-1, is also expressed in the posterior region of the worm . The PDB morphology was indistinguishable from wildtype in the cwn-1 mutants . While we do not completely exclude the involvement of other Wnts, our results indicate that LIN-44 is the major instructive cue for PDB neurite pruning.”“Another Wnt gene, 5) In Figure 3—figure supplement 1B, n should be indicated in each bar. How many transgenic strains were analyzed here? What DNA concentrations? Is there a significant difference in the rescue obtained with the rgef-1 promoter versus that provided by kal-1? If so, is it possible that there is also additional, non-cell-autonomous function of LIN-17?We now included the second transgenic line for each rescue experiment, and confirmed that they showed similar to the same rescue activity compared with the first line. We also observed multiple independent transgenic lines for all transgenic lines at L4 stage to make sure the consistency of the experiments (data not shown).lin-17 in PDB neurite pruning. The slightly weaker rescue activity of Pkal-1::lin-17 could be due to the late onset of the kal-1 promoter in the PDB neuron. However, together with the specific and lin-44-dependent subcellular localization of LIN-17 at the pruning neurite of PDB, we believe that lin-17 functions cell autonomously in PDB.Since neither promoter is PDB specific, we cannot completely exclude the possible non-cell autonomous roles of Regarding DNA concentration of the rescuing plasmids, we injected at 10ng/µl. This information is now included in the ‘Transgenes’ section in the Materials and methods as follows:unc-129dm::lin-44 which was injected at 20ng/µl.”“All rescuing constructs were injected at 10ng/µl except P6) In Figure 4. The data showing that Pegl-20::lin-44 does not rescue the lin-44 lack of pruning is weak: only one transgenic strain was tested with an n = 22. To support this conclusion, the authors should show the absence of rescue for at least three independent extrachromosomal array transgenic strains, and for different concentrations of the transgene. Similarly, the data supporting the functional divergence between LIN-44 and EGL-20 is weak. Results showing that Plin-44::egl-20 does not rescue the lin-44 lack of pruning are only for a very small sample size (n = 14) and for only one transgenic strain. This hypothesis should be further tested.egl-20::lin-44 nor Plin-44::egl-20 rescued the pruning defect of lin-44 mutants. While we could not conduct three independent lines due to the time-consuming nature of the experiment, we obtained more than three independent lines for each construct, and lin-44 animals carrying these transgenes looked like lin-44 mutants at L4 stage (data not shown). We also conducted another experiment to express lin-44 from the unc-129dm dorsal body wall muscles-specific promoter. Neither of two independent lines of lin-44 mutants with Punc-129dm::lin-44 rescued the pruning defects of the PDB posterior neurites. The data is included in Figure 4 and we have added the following sentence in the text:We have included the second independent transgenic lines and confirmed that neither Plin-44 from the dorsal body wall muscles using the truncated unc-129 promoter (Punc-129dm)  was not sufficient to rescue the PDB pruning defect in lin-44 null mutants .”“Similarly, ectopic anterior expression of lin-44 is not sufficient to rescue the posterior neurite pruning of PDB.With these data, we conclude that the anterior expression of We could not inject these Wnt constructs at high concentrations since they cause severe morphological defects which indirectly affect the PDB neurite structure.7) Figure 5. The data showing that there is "normal" pruning in nrt-bfp-lin-44 animals should be strengthen by increasing the sample size to at least 50 animals. The difference in pruning rate between nrt-bfp-lin-44 and wild-type animals (65.6% vs. 82.1%) could indicate that the membrane-tethered version cannot fully replace the secreted version.nrt-bfp-lin-44 mutants (82.6% and 71.2%). The slight, non-significant reduction in pruning rate in the nrt-bfp-lin-44 animal could be due to the slight axon guidance defects we could not detect.We have increased the sample size and saw no difference between wildtype and 8) The idea of an instructive, highly concentrated source of LIN-44 is valid. It will be important to test if other transgenic strains expressing LIN-44 under the Pegl-20 promoter do present pruning of the anterior neurites (see point 6 above). Did they try to use an unc-129 promoter to express LIN-44 in a region in which the anterior/dorsal PDB neurite encounters the dorsal muscle?lin-44 from the unc-129dm promoter in the wildtype background, and observed no ectopic pruning of the anterior neurite . Interestingly, the PDB morphology was indistinguishable from wildtype: we did not even observe axon guidance defects in the PDB neuron in these animals. The normal PDB morphology or lack of ectopic pruning of the anterior neurite could be due to the ectopic expression of LIN-44 in the body wall muscles. As the body wall muscles don’t normally express LIN-44, they might not secrete enough amount LIN-44 to induce neurite pruning. It is also important to note that muscle cells do not physically in contact with the neurites in the dorsal nerve cord until muscle arms (gigantic postsynaptic spine-like protrusions) reach the neurites to form neuromuscular junctions. The growth cone might not contact with these muscle cells to induce pruning. Since there can be many reasons for why such an artificial condition did not cause expected gain-of-function phenotype (ectopic anterior neurite pruning), we decided not to include this data in the manuscript.We have expressed


Plasmodium falciparum malaria are associated with cytoadhesion of parasite-infected erythrocytes (IE). The main parasite ligands for cytoadhesion are members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. Interactions of different host receptor-ligand pairs may lead to various pathological outcomes, like placental or cerebral malaria. It has been shown previously that IE can bind integrin αVβ3. Using bead-immobilized PfEMP1 constructs, we have identified that the PFL2665c DBLδ1_D4 domain binds to αVβ3 and αVβ6. A parasite line expressing PFL2665c binds to surface-immobilized αVβ3 and αVβ6; both are RGD motif-binding integrins. Interactions can be inhibited by cyloRGDFV peptide, an antagonist of RGD-binding integrins. This is a first, to the best of our knowledge, implication of a specific PfEMP1 domain for binding to integrins. These host receptors have important physiological functions in endothelial and immune cells; therefore, these results will contribute to future studies and a better understanding, at the molecular level, of the physiological outcome of interactions between IE and integrin receptors on the surface of host cells.Major complications and mortality from  Plasmodium falciparum malaria1. At present, more than a dozen human receptors have been identified that bind to IE. Most of them are expressed on endothelia and include CD36 (cluster of differentiation 36); ICAM-1 (intercellular adhesion molecule-1); EPCR ; CSA ; PECAM-1 ; TSP-1 (thrombospondin-1); non-immune IgM (in plasma); VCAM-1 (vascular cell adhesion molecule-1); integrin αVβ3; CR1 (complement receptor 1); heparan sulfate; E-selectin; and P-selectin (reviewed in4). The main parasite ligands for cytoadhesion are members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. However, detailed characterization of PfEMP1 domain-receptor pairs including the association of adhesion with specific domain sub-classes has been performed for only CD366, ICAM-18, CSA10, IgM12, and EPCR14 (reviewed in15). This gap in knowledge significantly restricts our understanding of malaria pathophysiology and the development of treatments against severe malaria. The recent discovery of EPCR as a novel malaria receptor as well as the identification and characterization of EPCR-binding PfEMP1 interacting domains13 and their involvement in severe malaria16 clearly demonstrate how identification of novel host receptors may influence studies on malaria pathology. Characterization of other IE-host receptor interactions at the molecular level will create a more complete picture of malaria parasite-host interactions and may have an impact on understanding malaria pathophysiology.Cytoadhesion of parasite-infected erythrocytes (IE) to endothelium and to immune cells plays a major role in complications and mortality from 17. In line with this finding, analysis of seven P. falciparum genomes revealed potential binding sites for Arg-Glu-Asp (RGD)-binding integrins in PfEMP1 proteins, pointing to the DBLα0 class of domains as the most likely targets18. Twenty-three RGD motifs in the NF54 genome are present in 21 DBL domain and 1 interdomain region; only one domain, PFL2665c DBLδ1_D4, contains two RGD sequences. We decided to test this delta sub-class domain18, along with ten domains not containing a RGD sequence, and four DBL domains containing a single RGD sequence (3 of which were of the α0 class) for αVβ3 integrin binding. Since all domains with RGD sequences were DBL domains followed by CIDR domains, which may organize a structural-functional unit, we initially tested these domains as DBL-CIDR tandem constructs. Only PFL2665c DBLδ1_D4-CIDRβ1_D5 domain tandem demonstrated integrin binding and was further studied using various in vitro approaches including live IE expressing PFL2665c protein. As a result, this work identified several individual DBL domains as integrin αVβ3- and αVβ6-binding functional units with the results confirmed with live IE expressing PFL2665c protein.It has been shown previously that IE can bind integrin αVβ313, including potential importance of EPCR- and ICAM1-binding PfEMP1 domain tandem presence within the same protein for cerebral malaria14; b) discovery that another variable surface protein of P. falciparum, a member of the rifin family, interacts with leucocyte immunoglobulin-like receptor B1 and downregulates activation of immune cells19. These examples clearly demonstrate that our knowledge about host-parasite interactions is still limited and identification of novel receptor-domain pairs is essential for studies of malaria pathophysiology.Identification of novel host receptors that serve as ligands for IE is an important area of malaria studies. Recent examples include: a) identification of a novel interaction between EPCR and specific PfEMP1 domains and implication of this interaction in severe malaria20. Previously, it was shown that IE may interact with αVβ3 integrin17, but no IE ligand or details of this process have been described. The Arg-Glu-Asp (RGD) motif, found in many proteins, is the integrin-binding sequence for a third of integrins including αVβ3. A computational search in seven genomes for the RGD sequence in PfEMP1 proteins, including the NF54 line, found that only DBL, and not CIDR, domains contain this motif18. We have found that in NF54 line only one DBL domain, DBLδ1_D4 from PFL2665c, contains two of RGD motifs. The two RGD sequences are located in the loops of the predicted domain secondary structure , other tandem domains containing only one RGD sequence, and a variety of domains from different domain classes without an RGD sequence for αVβ3 integrin binding. Constructs were selected based on their robust expression in our mammalian expression system21 and on CD36 binding by those tandems that contain CD36-binding domains, which indicates their correct folding , making a perfect loop with RGD sequence, Cys-RGDKV-Cys. Whether presence of two RGD sequences in one domain or just one RGD in the right context provide measured high avidity to αVβ3 and αVβ6 is not clear at this time.Further, to confirm that IE expressing PFL2665c protein can bind αVβ3 integrin, we used the PFL2665c-expressing E9 parasite line, previously isolated from parent NF54 by limiting dilutionsn24 Fig.  inset, tn24 Fig. . BindingVβ6 Fig. , another30) and ICAM-1 (3G8 line expressing VAR1 PfEMP131), do not bind to surface-immobilized αVβ3  and not in the DBL class domain, a common domain class for RGD motif presence in PfEMP1 proteins. This might be the reason why IE expressing this protein does not bind to αVβ3.Two other lines, selected for binding to CSA  of the first, to the best of our knowledge, αVβ3 and αVβ6 integrin-binding PfEMP1 domain (DBLδ1_D4) may further contribute to better understanding of malaria-host interactions.These data clearly indicate that αVβ3 and αVβ6 integrins and the DBLδ_D4 domain from PFL2665c may organize receptor-ligand pairs, through which IE can potentially interact with endothelial and immune cells expressing this receptor. Interestingly, this DBL domain belongs to the delta class of domains32, and αV-containing integrins may strongly bind to a P. falciparum sporozoite surface proteins TRAP33. Our identification of an IE ligand for the integrin receptors further expands our knowledge about malaria interactions with an important class of host receptors, integrins, and allows studying of these effects at the molecular level. Future experiments with host vascular and immune cells expressing these integrins may reveal further physiologically relevant details of PfEMP1::integrin binding process.As integrins are important players in the physiology of endothelial and immune cells, it is likely that specific cellular functions might be affected or disturbed by their interactions with IE. Interestingly, though irrelevant to PfEMP1 proteins, non-RGD-binding integrin αDβ2 may mediate experimental malaria-associated acute respiratory distress syndrome in mice14. Moreover, binding of IE to CD36 recruits αVβ1 integrin in microvascular endothelial cells to increase strength of binding34. If similar effects also occur with other integrins, for example αVβ3 and αVβ6, this may potentially contribute to the level of virulence (positively or negatively) of some CD36-binding parasite lines with double binding specificity.In our example, PFL2665c molecules can bind two different receptors (CD36 and integrin) on the surface of the same cell, as both these receptors are normally expressed in endothelial cells and cells of the immune system, including monocyte/macrophages. PFL2665c resembles, in this sense, Group A PfEMP1 proteins that have binding ability to two different receptors, for example ICAM-1 and EPCR by PF11_0521, through two neighboring domains, CIDRα1.4_D3 and DBLβ3_D4Our work also assigns a functional role for DBL domains from the C-terminal half of the most numerous 4-domain class of PfEMP1 proteins. It is logical to suggest that other domains of the C-terminal half of PfEMP1 protein may have similar function interacting with other, not yet identified receptors.PFL2665c PfEMP1 domain pairs that may contribute to IE cytoadhesion to endothelial and immune cells and affect the physiological state of these cells, which, in turn, may play a role in malaria pathology.In conclusion, we have identified a novel αVβ3 and αVβ6 integrin::DBLδ1_D4var gene, PFL2665, has been described earlier23. The aliquots of this line (expanded in 10–15 growth cycles and frozen) were thawed and grown in human O+ erythrocytes at 2% hematocrit in complete RPMI 1640 medium supplemented with 10% human serum and 40 μg/mL gentamicin sulfate for about 2–5 growth cycles (as described in31) before binding experiments. Parasite lines CS2, NF54 and 3G8 (kindly provided by Dr J. Smith) were cultured similarly. Cultures were maintained at 37 °C in a gas mixture of 5% CO2, 5% O2 and 90% N2.Selection of the NF54 line E9 by limiting dilution, which expresses the single 35: DBLα1.2_D2-CIDRα1.1_D3PFD0020c, DBLα0.7_D2-CIDRα2.2_D3PFB0010w, DBLα0.19_D2-CIDRα2.3_D3PFL2665c, DBLδ1_D4-CIDRβ1_D5PFL2665c, DBLα0.16_D2-CIDRα3.4_D3PFL1955w, DBLβ3_D3PF11_0521, DBLα0.9_D2-CIDRα2.2_D3PFL0005w, DBLβ3_D3PF13_0003, DBLα2_D2-CIDRα1.6_D3PF08_0140, CIDRα3.1_D3PF08_0106, DBLζ5_D6PF08_0141, DBLα0.17_D2-CIDRα3.1_D3PF07_0049, DBLα0.24_D2-CIDRα3.4_D3PFD1015c, DBLα0.20_D2MAL7P1.56, DBLβ5_D4PFL0020w, DBLα0.7_D2PFB0010w, DBLδ1_D4PFL1955w, and DBLδ1_D4PFA0765c (domain classification according to18). PCR primers for each construct and protein domain boundaries are shown in Supplementary Fig. 23 using concentrations of the biotinylated receptor indicated in the text and figure legends. As the negative control in these experiments, beads with immobilized HisAdEx construct21, which contains all the same parts as recombinant domain constructs but short irrelevant 37-amino acid long peptide instead of PfEMP1 domain, were used. Experiments on receptor binding to bead-immobilized domains were repeated at least 3 times with qualitatively similar results. Results of representative experiments are shown.The following domains were cloned into pHisAdEx vector, expressed, and purified/immobilized on Bio-Plex beads using methods described earlier22 for ICAM1::PF11_0521 DBLβ3_D4 interactions with the following modifications. We used 1xTris-buffered saline (TBS) supplemented with 1 mM CaCl2 and 1 mM MgCl2 for all incubations and streptavidin-PE as the detection molecules. About 1000 to 2000 beads with immobilized PFL2665c DBLδ1_D4 domain were incubated with 3 concentrations  of biotinylated integrin at 0, 10, 30 min (αVβ3), and at 0, 5, 15 min (αVβ6) times in two independent experiments for each receptor. Each experiment was performed in duplicates for αVβ3 and in duplicate (exp. 1) and triplicate (exp. 2) for αVβ6. Binding was measured using BioPlex 200 machine (BioRad), corrected for the control construct HisAdEx21, initial velocities calculated, and plotted using Lineweaver-Burk plots to determine equilibrium dissociation constant KD. Molecular weight (MW) of soluble recombinant αVβ3 and αVβ6 was 191.3 kDa and 189.1 kDa, respectively, according to manufacturer information for molar concentration calculations. Linear regression lines, standard error of means (SEM) error bars, and p-values for each regression were calculated using GraphPad Prizm software.Measurements and calculations were performed as described in our earlier publication2 and 1 mM MgCl236. Before the adhesion assays, IE in trophozoite stage were enriched by magnetic LD columns  as described by manufacturer. Percentage of mature trophozoites in the elution varied from 22 to 95% and was adjusted using uninfected erythrocytes to the levels specified in the appropriate Figure legends. Cyclic peptide antagonist cyloRGDFV  was used to inhibit binding of IE to αVβ3 at concentrations specified in Figure legends. Enriched trophozoites were incubated with inhibitor peptide for 30 min at 37 °C, placed over the immobilized receptor, and incubated for 30 min at 37 °C. Non-adherent cells were washed by 1xTBS/1 mM CaCl2/1 mM MgCl2. Adherent cells were counted using microscopy in 20–40 randomly selected microscope fields. Each test was performed in duplicate spots of receptor. Binding was presented as averaged number of bound IE per filed and Standard Error of Mean (SEM) re-calculated for binding per mm2. Each binding experiment was repeated at least 2 times. Results of typical experiment in each group are presented.Binding of IE lines to various receptors  or control (BSA) immobilized on Petri dishes were performed using previously published methods and media supplemented with 1 mM CaClAnti-CD36 monoclonal antibody FA6-152  at 5 μg/ml was used to inhibit E9 binding to CD36 immobilized on Petri dish at 10 μg/ml using approach described above.All methods were carried out in accordance with relevant guidelines and regulations. Ethics approval was obtained from the Florida Atlantic University Institutional Review Board committee for using human erythrocytes for culturing malaria parasites. Blood was purchased from Valley Biomedical.Supplementary Information


Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine protein kinase that regulates brain development and neurodegeneration. Cdk5 is activated by p25 that is generated from calpain-dependent cleavage of p35. The generation of p25 is responsible for the aberrant hyper-activation of Cdk5, which causes neurodegeneration. Using in vitro assays, we discovered that F-box/WD repeat-containing protein 7 (Fbxw7) is a new substrate of Cdk5. Additionally, Cdk5-dependent phosphorylation of Fbxw7 was detected in the presence of p25, and two amino acid residues (S349 and S372) were determined to be major phosphorylation sites. This phosphorylation was eventually linked to decreased stability of Fbxw7. Using a culture model of cortical neurons challenged with glutamate, we confirmed that decreased stability of Fbxw7 was indeed Cdk5-dependent. Furthermore, diminished levels of Fbxw7 led to increased levels of transcription factor AP-1 (c-Jun), a known substrate of Fbxw7. Given that previous reports demonstrate that c-Jun plays a role in accelerating neuronal apoptosis in these pathological models, our data support the concepts of a molecular cascade in which Cdk5-mediated phosphorylation of Fbxw7 negatively regulates Fbxw7 expression, thereby contributing to neuronal cell death following glutamate-mediated excitotoxicity. One member of the CDK family, Cdk5, is primarily activated in the brain and plays a role in brain development, neuronal maturation, and synaptic plasticity4. This member has a unique physiological characteristic compared to other CDKs5. Specifically, Cdk5 is activated by neuron-specific regulatory proteins p35 or p39 instead of cyclin7. This distinctive property allows Cdk5 to play a unique role in neuronal cell physiology. Cdk5 phosphorylates specific substrates that are involved in axonal and dendritic growth as well as neuronal migration. In contrast to these beneficial effects, aberrant regulation of Cdk5 activity causes neurodegenerative disease8. Notably, the cleavage of p35 into p25 by calpain, a calcium-activated cysteine protease, leads to Cdk5 hyper-activation in response to neurotoxin treatment9. Under this condition, for example, Cdk5/p25 acts as a regulator of cell death by phosphorylating tau and p5311. Therefore, discovery of a novel Cdk5 substrate is necessary to expand our understanding of the primary regulating function of Cdk5/p25 in neuronal death.Cyclin-dependent kinases (CDKs) comprise a family of protein kinases that regulates the cell cycleFBXW7) E3 ligase complex that regulates degradation of proteins involved in cell division and growth13. Fbxw7 is associated with tumor suppression because most of its substrates are proto-oncogene proteins, including c-Jun14, cyclin E15, c-Myc16, Notch17, and Mcl-118. Most Fbxw7 substrates contain Cdc4 degron motifs phosphorylated by glycogen synthase kinase 319. Downregulation or mutation of Fbxw7 leads to the development of various cancers in humans. Many Fbxw7-related studies have therefore focused primarily on understanding of its role in cancer metabolism. Interestingly, Fbxw7 has also been reported to play an important role during brain development and differentiation by regulating Notch and c-Jun21. Since the publication of a report demonstrating the importance of maintaining the stability of Fbxw7 to retain its biological function22, several studies have focused on determining upstream molecules that directly govern Fbxw7 expression and degradation; for examples, polo-like kinase 2 (Plk2) phosphorylates and regulates the degradation of Fbxw723, and COP9 signalosome subunit 6 (CSN6) promotes ubiquitination and degradation of Fbxw724. However, despite the widespread expression of Fbxw7 in the brain, its regulatory mechanism and pathophysiological role during neurodegeneration have been less studied.F-box/WD repeat-containing protein 7 (Fbxw7) is a substrate recognition component of the SKP1–Cul1–F-box protein  into HEK293 cells and subsequently analyzed Fbxw7 phosphorylation levels. Phosphorylation of Fbxw7 was completely diminished in combination with Cdk5 DN compared to Cdk5 wild-type (Cdk5 WT)  cells, was observed in the presence of Cdk5/p25 Fig.  and to aWT) Fig. . SimilarWT) Fig. . Direct WT) Fig. .Fig. 1CdImmunoprecipitation assay results for the physical interaction between Fbxw7 and Cdk5 in transfected HEK293 cells showed that Fbxw7 physically bonded to Cdk5, but not to p35 or p25 activators Fig. . InteresNext, we explored the specific Cdk5/p25 phosphorylation site(s) of Fbxw7. Bioinformatics analysis revealed four putative (Ser/Thr)-Pro Cdk5 phosphorylation motifs of Fbxw7 Fig. . We gene23 and CSN624. For example, Plk2 phosphorylates Fbxw7 on Ser25, Ser176, and Ser349, and this event leads to Fbxw7 degradation. Therefore, we specifically investigated whether Cdk5/p25-mediated phosphorylation of Fbxw7 affected Fbxw7 stability. When HEK293 cells were co-transfected with Fbxw7 and Cdk5/p25, we observed decreased expression levels of Fbxw7  chase assays to confirm that site-specific phosphorylation of Fbxw7 by Cdk5 is linked to decreased stability of Fbxw7. When HEK293 cells were transfected with Fbxw7 and Cdk5/p25, the basal level of Fbxw7 was less than that in HEK293 cells transfected only with Fbxw7 Fig. . Further4. Mechanistically, calpain is activated by disruption of calcium homeostasis resulting from oxidative stress, which can be induced by a neurotoxin29. Activated calpain cleaves p35 to p25, ultimately leading to Cdk5 hyper-activation8. Therefore, we examined whether Cdk5 hyper-activation is linked to decreased expression of Fbxw7 in pathological models of excitotoxicity30. We chose a primary culture of cortical neurons that were treated with glutamate to induce Cdk5 hyper-activation and to subsequently determine endogenous levels of Fbxw7. As shown in Fig. +)   Fig. . We nextxw7 Fig. , confirm31, the regulation and functional consequence of Fbxw7 in neurons has been less examined. Here, we proposed that the novel mechanism underlying glutamate-mediated excitotoxicity, which is phosphorylation of Fbxw7 by hyper-activated Cdk5, may be a critical step to determine neuronal death. Specifically, we demonstrated that Fbxw7 is a novel substrate of Cdk5 and activated Cdk5 phosphorylates S349 and S372 residues of Fbxw7. We found that Cdk5-mediated phosphorylation of Fbxw7 is linked to destabilization of Fbxw7. Using a cortical neuronal culture model of excitotoxicity, we demonstrated an inverse relationship between expression of Fbxw7 and its substrate, c-Jun14. As such, accumulation of c-Jun was a consequence of its escaping from the Fbxw7-dependent degradation as a result of Cdk5-dependent decreased expression of Fbxw7. Recently, the phosphorylation of Fbxw7 was discovered32 and its decreased level was observed during oxidative stress33. As c-Jun is regarded as a pro-apoptotic protein34, we propose a temporal neuronal death pathway in which decreased expression of Fbxw7 via Cdk5 hyper-activation eventually leads to accumulation of c-Jun. This result may play an important cytotoxic role during excitotoxicity  targeting of Cdk5 reduces tau phosphorylation and decreases the number of neurofibrillary tangles in the hippocampus of triple-transgenic Alzheimer’s disease (AD) mice35. Luo et al. demonstrated that Cdk5 phosphorylates huntingtin, and this phosphorylation reduces its cleavage by caspases and results in attenuation of aggregate formation and neurotoxicity36. Similarly, it has been shown that phosphorylation by Cdk5 decreases E3 ligase activity of Parkin and modulates the formation of inclusion bodies37, demonstrating the cases in which Cdk5-mediated phosphorylation of the disease-related proteins is involved in pathogenesis of several neurodegenerative diseases. On the other hand, accumulating evidence has indicated that Cdk5 acts as an important link between disease-initiating factors and cell-death effector38. Along this line of investigations, several recent studies, including reports from our laboratory, have raised the possibility that Cdk5-mediated phosphorylation of the E3 ligase complex is critically involved in pathogenesis40. In support of this notion, the present study provides an additional regulation of pro-apoptotic protein by Cdk5-mediated phosphorylation of E3 ligase component, in which accumulation of c-Jun is achieved by decreased expression and stability of the phosphorylated Fbxw7. Although we have not examined whether Cdk5 directly phosphorylates c-Jun41 and Mcl-142 after excitotoxicity, our results support a notion that accumulation of c-Jun comprises a specific execution phase in the temporal cell-death sequences of excitotoxicity. In consideration of a previous report42 demonstrating that hyper-activated Cdk5 phosphorylates and triggers Mcl-1 ubiquitination after excitotoxicity, we propose that Cdk5/p25-mediated Fbxw7 destabilization could accelerate cell death via two pathways; namely, accumulation of pro-apoptotic c-Jun and destabilization of anti-apoptotic Mcl-1. Surge of intracellular free Ca2+ level caused by disturbance of Ca2+ homeostasis has been considered as one of the major changes that occurs following ischemic stroke. Interestingly, previous study has demonstrated that ischemic stroke injury is mediated by aberrant Cdk5 activation25. Consequently, they have demonstrated that pharmacological inhibition or conditional knockout of Cdk5 prevents neuronal death in response to ischemic injury. Based on this and other related studies, we speculate that ischemia-mediated activation of Cdk5 may lead to decreased expression of Fbxw7 and increased expression of c-Jun. Therefore, we infer that regulation of Cdk5 activity may be the potential therapeutic target for ischemic injury.Cdk5 is a well-known Ser/Thr kinase and a member of the highly conserved cyclin-dependent kinase family, but it is not involved in cell cycle progression. Although it is ubiquitously expressed, its co-activator proteins (p35 and p39) are exclusively expressed in neurons. Cdk5 kinase activity is prominently confined into the nervous system and, therefore, is involved in neuronal differentiation and neuronal death44. Protein turnover is important to maintain homeostasis of post-mitotic neurons, and UPS plays a major role in controlling protein quality to ensure healthy neuronal function45. Many neurodegenerative diseases are characterized by the presence of aggregate-prone neurotoxic proteins that are believed to perturb neuronal function. Therefore, malfunction of the UPS has been believed to be one of the prime culprits for generation of abnormal protein aggregates46. In this regard, several E3 ligases have been implicated in the pathogenesis of neurodegenerative diseases. For example, decreased activity of Parkin found in an AD mouse model and overexpression of Parkin ameliorates beta-amyloid load and restores behavioral abnormalities47. Similarly, loss-of function mutations in Parkin is shown to impair its E3 ligase activity and leads to the accumulation of protease-resistant alpha-synuclein and the formation of Lewy bodies49. Dysfunction of the SCF complex has been indicated in the pathogenesis of poly-glutamine diseases like Huntington’s disease (HD)50. Also, reduced levels of Cul1 and Skp1 were found in HD mice brain and the cellular HD model. Consequently, silencing of Cul1 results in increased aggregate load and enhanced poly-glutamine-induced neurotoxicity, arguing that reduced levels of SCF complex contribute to the pathogenesis of poly-glutamine disease. Fbxw7, a well-characterized SCF component, contributes to substrate specificity and mediates ubiquitination of proteins targeted for degradation by the UPS. Therefore, F-box protein is essential in the regulation of SCF activity. So far, studies of Fbxw7 have been mainly focused on its tumor suppression. This study provided an intriguing pathway in which phosphorylation-stimulated, ubiquitin-dependent degradation of target protein comprises a critical loop of determining neuronal survival and death. This study is quite in line with our study demonstrating a novel cell-death axis of Cdk5-Fbxw7-c-Jun in which Cdk5-stimulated phosphorylation of Fbxw7 contributes to regulation of cell death by causing accumulation of its substrate c-Jun during excitotoxicity. This agreement of results demonstrates that the E3 ligase are key player in determining neuronal death in experimental models of excitotoxicity51. Taken together, these data support a notion that the ubiquitin E3 ligase pathway might be regarded as a potentially novel and critical component in the regulation of neurodegeneration.Here, we raised the possibility that the Cdk5-Fbxw7-c-Jun axis plays a critical role in promoting neuronal death. To date, dysfunction of E3 ligase and its associated ubiquitin-proteasome system (UPS) has been observed in neurodegenerative diseases and is an important therapeutic target52. Our study provides evidence that Cdk5/p25 plays the role of another negative regulator of Fbxw7. More specifically, phosphorylation of Fbxw7 by Cdk5/p25 is a novel mechanism for the control of Fbxw7 protein stability. Furthermore, Cdk5/p25 hyper-activity leads to neuronal death via decreased Fbxw7 level and concomitant accumulation of c-Jun. Here, we addressed the idea that homeostatic maintenance of Fbxw7 protein level is not limited to tumorigenesis. Our findings allow us to speculate that with proper regulation, Fbxw7 is a potential conceivable therapeutic concept for mitigation of neuronal death53.Previously it has been demonstrated that oxidative stress-activated Plk2 act as a negative regulator of Fbxw7Nhe1 site of a pCI-neo mammalian expression vector . Flag-tagged human Fbxw7 were sub-cloned into the pCI-neo vector. Multiple Fbxw7 mutants were generated using a site-directed mutagenesis kit . Generation of V5-, and HA-tagged wild-type (WT) mouse Cdk5 and dominant-negative Cdk5 (DN), as well as HA- and Myc-tagged mouse p25 and p35 were described previously54. All plasmids were purified using endotoxin-free plasmid DNA isolation kits .GST-tagged human Fbxw7 was generated by sub-cloning into the pGEX4T-3 bacterial expression vector. Flag, V5, HA, and Myc sequences were integrated into the 2. For transient expression, HEK293 and HEK293T cells were transfected with a pre-determined amount of the indicated DNA using polyethylenimine . Establishment of primary cultures of cortical neurons was accomplished as previously described55. Briefly, primary cortical neurons were isolated from E14.5 mouse embryos. Dissociated cortical cells were treated with 1 mg/ml papain  at room temperature for 5 min. After enzyme inactivation, isolated neurons were homogenized in Hank’s balanced salt solution (Sigma–Aldrich) supplemented with 40 μl DNase (10 unit/μl) and passed through a 70-μm mesh cell-strainer . The singly dispersed neurons were plated on densities of either 1.25 × 106 cells per well of a 6-well plate or 7.5 × 106 cells per 10-cm plate that were pre-coated with 25 μg/ml poly-D-lysine (Sigma–Aldrich) and 1 μg/ml laminin (Invitrogen). Cultures were then cultivated in neurobasal medium (Invitrogen) supplemented with 2% B27 (Invitrogen) and 0.5 mM L-glutamine (Sigma–Aldrich). Half of the medium was replaced every 3–4 days. All experiments were conducted after 11 days in vitro. All mice were handled in accordance with the Yonsei University guidelines for animal care and use. All experimental procedures were approved (2017-10-647-01 and 2018-01-689-01). When appropriate, cells were treated for the indicated time periods with an empirically determined concentration of MG132 , cycloheximide , roscovitine (Sigma–Aldrich), glutamate (Sigma–Aldrich), hydrogen peroxide (Sigma–Aldrich), and MPP+ iodide (Sigma–Aldrich). The incubation periods of all drugs used in this study were also empirically determined.HEK293 and HEK293T cells were cultivated at 37 °C in Dulbecco’s Modified Eagle’s Medium supplemented with 10% heat-inactivated fetal bovine serum  in an atmosphere of 95% air and 5% COg for 15 min at 4 °C. The resulting supernatant was incubated overnight at 4 °C with GST beads . The protein samples were washed three times with 1 × PBS. GST-bound human Fbxw7 protein was incubated with 0.1 μg Cdk5/p35  or Cdk5/p25  in a reaction buffer containing 25 mM Tris (pH7.5), 100 mM NaCl, 1 mM DTT, 100 μM unlabeled ATP and 5 μCi [γ-32P] ATP at 30 °C for 30 min. Roscovitine (10 μM) was added 30 min prior to the kinase reaction. The samples were resolved by SDS-PAGE and visualized by autoradiography.GST-tagged WT and mutant variants of human Fbxw7 were expressed in BL21 bacteria with 0.1 mM isopropyl-β-D-thiogalactopyranoside (Sigma–Aldrich) and incubated at 37 °C for 4 h. Bacterial pellets were resuspended in a lysis buffer containing 30 mM Tris-HCl (pH 7.5), 0.1 mM NaCl, 1% Triton X-100 (Sigma–Aldrich), 1 mM DTT (Sigma–Aldrich), and 1 × protease inhibitor cocktail  and then sonicated on ice. Lysates were centrifuged at 12,000 × 3VO4 and 1 × protease inhibitor cocktail. For detecting phosphorylation status, RIPA lysis buffer containing 25 mM NaF, 2 mM, 2 mM Na3VO4 and 1 × protease inhibitor cocktail was used. After incubation on ice for 30 min, lysates were collected by centrifugation at 12,000 × g at 4 °C for 15 min. The supernatants were subjected to immunoprecipitation and immunoblot analysis using the indicated antibodies. For immunoprecipitation of Flag-tagged Fbxw7, cell lysates (1 mg) were incubated with 20 μl of anti-Flag M2 affinity gel beads (Sigma–Aldrich) overnight at 4 °C. The conjugates were collected by centrifugation at 800 × g for 2 min at 4 °C. Proteins were eluted from the beads by adding protein sample buffer and then denatured by boiling. Protein samples were then separated by SDS-PAGE and subjected to immunoblot analysis using the indicated antibodies. Antibodies used included: horseradish peroxidase (HRP)-conjugated anti-Flag antibody ; HRP-conjugated anti-HA ; anti-Myc-tag ; HRP-conjugated anti-V5 ; anti-Fbxw7 ; anti-GAPDH ; anti-phospho-Cdk5-substrate motif ; mouse anti-Cdk5 ; rabbit anti-Cdk5 ; anti-p35/p25 ; anti-GST ; anti-c-Jun ; anti-ub ; HRP-conjugated goat anti-rabbit antibody ; and HRP-conjugated goat anti-mouse antibody . Specific bands were detected with enhanced chemiluminescence .Cell lysates were prepared using RIPA lysis buffer containing 5 mM NaF, 2 mM Nag for 2 min at 4 °C. Proteins were eluted from the beads by adding protein sample buffer and then denatured by boiling. Protein samples were then separated by SDS-PAGE and subjected to immunoblot analysis using anti-Fbxw7  and anti-Cdk5  antibodies.Primary cultures of cortical neurons were maintained until 11 days in vitro for this purpose. For immunoprecipitation of Fbxw7 and Cdk5, lysates from cortical neurons were pre-incubated with protein A agarose beads (Millipore) for pre-clearing, and then further incubated with 1 μg of anti-Fbxw7  and 4 μg of anti-Cdk5 , respectively. For controls, both rabbit anti-IgG  and mouse anti-IgG  were used. After overnight incubation, lysates were further incubated with protein A agarose bead for conjugation. The conjugates were collected by centrifugation at 800 × 30. The lentivirus particles were generated by co-transfection into HEK293T cell with lentivirus vectors and three other vectors including pMDLg/pRRE, pMD2.G and pRSV-Rev , using polyethylenimine (PEI). Two days after transfection, the cell culture media were filtered using a 0.45-μm filter (Millipore). Infection to primary neuron was conducted after 3 days in vitro. After 24 h of infection at 37 °C, the culture media was replaced with fresh culture media. Cdk5 protein level was assessed by immunoblot analysis. All experimental procedures were approved by the Institutional Biosafety Committee of Yonsei University (IBC-A-201901-192-03).Cdk5 knockdown for RNA interference was achieved using Mission shRNA-encoding lentivirus directed to mouse Cdk5 mRNA  as recommended by the manufacturer’s protocols. Briefly, lentiviral vectors (in pLKO.1) containing Cdk5 shRNA sequences (TRCN0000009521) and non-target shRNA control vector  were purchased from SigmaTM Green PCR Master Mix (Fisher Scientific) with the CFX 384 Touch Real-Time PCR Detection System (Bio-Rad). To analyze human Fbxw7 mRNA expression levels, the following primer pairs were used; human Fbxw7, 5′-AAAGAGTTGTTAGCGGTTCTCG-3′ and ′5-CCACATGGATACCATCAAACTG-3′; human GAPDH, 5′-TGAACCACCAACTGCTTAGC-3′ and 5′-GGCATGGACTGTGGTCATGAG-3′.Total RNA was prepared using an RNAeasy micro kit (Qiagen) and cDNA was generated using a SuperScriptIII First-Strand System (Fisher Scientific) according to the manufacturer’s instructions. Quantitative real-time PCR analyses were performed using a Power SYBRt-tests or one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc tests were performed using Prism 6 software . Statistical significance of differences is indicated as follows: ***p < 0.001, **p < 0.01, and *p < 0.05.Data are expressed as the mean ± standard deviation (SD) for three independent experiments. To determine the significance of differences between groups, two-tailed Student’s Supplementary informations


A key question in immunology is whether antigen recognition and Fc receptor (FcR) binding are allosterically linked. This question is also relevant for therapeutic antibody design. Antibody Fab and Fc domains are connected by flexible unstructured hinge region. Fc chains have conserved glycosylation sites at Asn297, with each conjugated to a core heptasaccharide and forming biantennary Fc glycan. The glycans modulate the Fc conformations and functions. It is well known that the antibody Fab and Fc domains and glycan affect antibody activity, but whether these elements act independently or synergistically is still uncertain. We simulated four antibody complexes: free antibody, antigen-bound antibody, FcR-bound antibody, and an antigen-antibody-FcR complex. We found that, in the antibody’s “T/Y” conformation, the glycans, and the Fc domain all respond to antigen binding, with the antibody population shifting to two dominant clusters, both with the Fc-receptor binding site open. The simulations reveal that the Fc-glycan-receptor complexes also segregate into two conformational clusters, one corresponding to the antigen-free antibody-FcR baseline binding, and the other with an antigen-enhanced antibody-FcR interaction. Our study confirmed allosteric communications in antibody-antigen recognition and following FcR activation. Even though we observed allosteric communications through the IgG domains, the most important mechanism that we observed is the communication via population shift, stimulated by antigen binding and propagating to influence FcR recognition. These four chains fold into three domains, two Fab domains that bind antigen and one Fc domain that binds Fc receptors (FcRs).2 The Fab domains contain variable and constant domains. The variable domains, especially complementarity-determining regions (CDRs), are mainly responsible for specificity and affinity,3 while the constant domains modulate isotype/effector functions.4 The Fc domain contains CH2 and CH3 domains. The CH2 domain mainly interacts with FcRs, which are on the cell surface and play pivotal roles in humoral and cellular protection. The Fab and Fc domains are connected by a flexible unstructured hinge region. Fc chains have conserved glycosylation sites at Asn297. Each is conjugated to a core heptasaccharide. They form a biantennary Fc glycan. Thus, three structural elements  synergistically determine antibody activity.Immunoglobulin G (IgG) molecules bind to their cognate antigens. The resulting complexes interact either with type I or type II Fc receptors (FcRs) on effector cells and on B cells, modulating both humoral and innate immune processes.7 Recent studies suggested allosteric effects during antibody-antigen recognition 8, with both the variable and constant domains playing a role.14 For instance, our recent work on crenezumab suggested that antibodies with identical variable domains, but different constant domains, have significantly different affinities to amyloid beta (Aβ).15 Engineering CH and CL in trastuzumab and pertuzumab recombinant models also affect antigen-binding.16 A previous study based on over 100 crystal structures of antibody Fab domains in either unbound or bound form indicated a common behavior, with distant CH1-1 loops undergoing significant fluctuations upon antigen binding.17Antibody-antigen recognition is a complex event that involves antibody conformational transitions mediated by its inherent flexibility.19 One of the most important antibody activities involves killing target cells by triggering antibody-dependent cell-mediated cytotoxicity (ADCC). Fc-optimized antibodies can have higher binding affinity with FcRs and achieve a higher ADCC potency.24 For example, antibody Fc engineering promotes serial killing mediated by natural killer cells.21 Fc-optimized anti-CD25 22 and anti-CD13324 antibodies were reported to achieve certain success. Antigen presentation is also an important immune-response step. FcγRs efficiently internalize antigen-antibody (Ag-Ab) complexes, inducing processing of antigens into peptides presented by major histocompatibility complex (MHC) class II molecules. The recognition of p-MHC (peptide-MHC) complexes by T-cell receptors (TCR) triggers further immune reactions.IgGs are the most common template for antibody drugs. Antibody Fc-FcRs interactions are crucial in the design of therapeutic agents, as well as vaccines.26 While glycan truncation may affect antibody stability,27 defucosylation may enhance effector functions.28 N-Glycan optimization can also be used to maximize ADCC.29 Most intriguing, glycan may also regulate antigen recognition, and it has been reported that core fucosylation of IgG B cell receptor is required for antigen recognition and antibody production.30Fc glycans modulate Fc conformations and functions.31 indicating the intrinsic connection between antigen recognition and Fc receptor binding. However, the signals that dictate antigen binding, Fc conformational change, and IgG effector function during immune response development remain poorly understood. Whether intramolecular signaling occurs is still debated.33 While the associative hypothesis is attractive, since Fc receptor crosslinking could increase the affinity of antigen–antibody complexes, there is sufficient evidence to support the allosteric hypothesis.33 Elucidation of the allosteric hypothesis is important for understanding the mechanism of recognition, but, by shifting the focus from solely the variable region to the entire antibody molecule, it is also critical for antibody engineering.Antibody effectors can be antigen specific,Here, we investigate whether antigen binding induces conformational change in the Fc domain and hinge region, and whether an antigen-bound antibody populates conformations that facilitate or inhibit the binding of FcRs to Fc. We selected an Aγ peptide as antigen to minimize antigen size effects, since a larger antigen may introduce uncertainties in sampling antibody states. We focus on human FcγR I (hFcγRI), a major immune receptor expressed in immune cells, macrophages, neutrophils, and dendritic cells. hFcγRI binds IgG1, 3, and 4 with high affinity. hFcγRI contains three subunits, D1, D2, and D3. Structures of the unbound hFcγRI and hFcγRI-IgG1 complex show an asymmetric binding surface, as well as significant conformational change of both hFcγRI D3 and CH2(A) domains.34 These signals also propagate through the CL/CH1 domain as a bypass. These two pathways enhance the signaling from the antigen to FcR. We hypothesized that these allosteric events are entropy controlled. Antibody-antigen binding reduces the entropy of CDR loops. The entropy is transferred to the hinge region, leading to Fab re-orientation. Entropy may also be transferred to the Fc CH2 domains, leading to open CH2 domain conformations. CH2 transferred the entropy to the glycans, detached one heptasaccharide from the domain, facilitating hFcγRI recognition. Together, this work provides conceptual insight at the atomic level into the correlation of antibody-antigen recognition and effector function.Our molecular dynamics (MD) simulations showed that Aβ binding leads to large Fab re-orientation into two dominant conformational clusters, as well as open conformations of Fc CH2 domains. The conformations share properties with the dominant states of Aβ-solanezumab-hFcγRI complex. This suggested that Aβ binding shifts the antibody ensemble to promote hFcγRI binding. We further analyzed the cross-talk among subunits in the Aβ-solanezumab-hFcγRI complex. We found that Aβ binding and FcRs binding are highly correlated events. Not surprisingly, antigen binding signals are mainly transferred through the hinge region.35 A total of 160,000 structures were evaluated, and the distribution showed profiles similar to ET (ab) concentrated at between 65 to 85 Å.As a first step, we simulate the conformational distribution of a free antibody in solution. To enhance the sampling, we performed 12 independent MD simulations of an unbound antibody with 12 different initial conformations , includiar to ET . This suab) and between Fabs and Fc domain (Dac), whereas the simulation indicated that Dab has a narrower distribution than Dac, suggesting that two Fabs do not move independently. The averaged contact area between the two Fabs (1118.1 ± 317.6 Å2) is larger than the contact area between Fab and Fc (848.1 ± 319.7Å2). Therefore, while the antibody is highly flexible and the three subunits (two Fabs and Fc) form/break contacts dynamically, the domains’ movements are not random.Even though our simulations and experimental ET provided similar profiles of the domain conformational distributions, the simulations attain higher resolution. For example, ET showed almost overlapping COM distributions for the distance between two Fabs (Dac = Dab = 70 Å), which could be the reason why it is hard to distinguish between Dac and Dab experimentally. However, when Fab is loaded with antigen, the conformational distribution changes dramatically. The original (Dac = Dab = 70 Å) cluster becomes less populated, while two major clusters centered around  and  appear. These two clusters, cluster2 and cluster3, contain ~ 13% and ~ 19% of the population. Their potential energies are very similar, although cluster2 is ~ 50 kcal/mol lower than cluster3. In cluster2, the Fabs-Fc distance is larger than Fab-Fab (Y shape), while in cluster3, the Fab-Fc distance is smaller than Fab-Fab (T shape).We also explored whether the antibody can bind the Fc receptor without binding antigen. We simulated the antibody/hFcγRI complex . There aac = 65 Å; Dab = 80 Å). Cluster8 overlaps cluster3 in the antibody-Aβ system, close to cluster5 in the antibody/hFcγRI complex. Cluster8 could correspond to the antibody functional activation. Overall, the population shifts reveal that allosteric signaling can be transmitted through antibody conformational dynamics.For the antibody-FcR complex after antigen loading (antibody/antigen/hFcγRI), cluster8 dominates, with over 43% of the conformation concentrated around DWe superimposed the CH3 domain and calculated the root mean square deviation (RMSDs) of the Fc region from the conformers obtained in the simulations of the four complexes. Each of the 48,000 structures was compared with all others. We averaged the RMSDs from each of the four complexes and averaged the RMSDs from two different complexes . The 2D We clustered the Fc region using RSMD of 4 Å . For theThe open Fc conformation is accompanied by the change of twist between CH2/CH3 of the Fc domain. We calculated the relative angle and dihedral angle for CH2/CH3 of the left and right chain in the Fc domain . In the The increase in the open Fc conformations expanded the exposure of the Fc receptor-binding residues as well. We defined Fc interfacial residues with contact frequency larger than 20% as key binding residues in Fc receptor recognition. The accessible surface area (ASA) of the key binding residues in the unbound antibody is 2333.5 ± 195.3 Å2, whereas in the Aβ-bound antibody it is 2449.5 ± 188.4 Å2, an ~ 116 Å2 increase upon Aβ binding. This suggests that Aβ binding increases the accessible area of Fc, which facilitates hFcγRI binding. The dynamic cross-correlation matrix (DCCM) of the Fc region of the four complexes  indicateTo verify that antigen binding changes the antibody conformation and dynamics, rather than direct interactions between Fc domain and receptor, we examine the electrostatic and hydrophobic interactions and the hydrogen bonds that are crucial for Fc/FcR recognition. We firstly evaluated the interfacial residues/glycans within 3Å between hFcγRI and antibody. We found that the patterns are similar with/without Aβ binding . This su25 in which only the Fc domain of the antibody was studied. However, when Aβ binds to the antibody, the distance distribution differs for the two sides of the antibody. The 6 Å peak of the left chain was enhanced while the distribution of the right chain remained similar to the apo form. This suggested that when Aβ binds to the antibody, one N-glycan arm showed dominant bound state conformation while the other arm showed both free and bound states. When hFcγRI binds to the antibody without antigen, the distance distribution showed a similar pattern to the antibody-Aβ complex. When both Aβ antigen and hFcγRI bind to the antibody, this effect is clearer. The 6 Å peak dominates the left chain, but there are two major peaks at ~ 10 Å and ~ 14 Å for the right chain, suggesting that the left glycan binds tightly to the Fc domain and the right glycan disassociates. Thus, when both Aβ and hFcγRI bind to the antibody, one N-glycan arm showed dominant bound state conformation while the other arm showed dominant free states.In the complex with Aβ binding, the KHR motif  showed the largest contact area with one arm of the N-glycans with numerous hydrogen bonds . Asn1441The structural changes of the paratope and variable domain were analyzed . The VH/VL orientation fluctuation decreased after Aβ binding , with the RMSDs of the paratope , i.e., each individual CDR loop, showing lower structural flexibility. The root mean square fluctuations (RMSFs) of individual residues in the variable domain also suggested that the CDR loops diminished their flexibility. We also found that the RMSFs of non-CDR loops also decreased upon antigen binding. This suggests that the non-CDR loops of the variable domain, which is not directly in contact with the antigen, respond to the antigen binding.36 To evaluate all residues, including proline, we calculated the generalized order parameter S2 of the C = O bond of each individual residue of the antibodies , allosteric signaling from Aβ to hFcγRI directly through amino acid residue contact network could be more efficient.The hinge region, which connects the Fabs and the Fc region, showed the largest SWe examined if, upon Aβ binding, the Fab can directly transfer the signals to hFcγRI through the residue contact network. In the antibody- hFcγRI-Aβ complex, we found that the population converged to a dominant cluster (cluster10) with ~ 43.0% of the total population. We obtained all the structures in this cluster and analyzed the subdomain communication and signaling from Aβ to hFcγRI. Evaluation of the DCCM of the whole complex indicates that there is positive correlation between the Fab or Fc region or hFcγRI . In contWe also used the community network analysis, and considered the antibody as a network to evaluate the communication among the domains . Nodes wThe final question to be answered is if the Fc receptor conformational dynamics can show different signals from binding of antibody with and without antigen. To evaluate the motion and conformational change of hFcγRI, we superimposed the D1, D2 domain as they bind to the Fc domain. We measured the angles between neighboring domains . In the 37 and allosteric signaling can be transferred through protein conformational dynamics and population shift.38 Conformational dynamics permits both promiscuity and specificity.44 Protein complex formation redistributes the dynamics,46 allowing allosteric signaling through protein domains. Antibody-antigen recognition, which is associated with structural transitions through inherent conformational flexibility,7 involves conformational selection.47 To regulate the immune response, antibody-antigen interaction sends a signal for complement activation and Fc receptor binding. Signaling pathways depend on the sequences of the variable regions, through hydrogen bonding network, electrostatic interactions, and residue contacts. Through their changes, they result in population shift of dynamic conformations.48 However, intramolecular signaling is complex and exactly how it takes place in distinct structures and under certain conditions is still not entirely clear.33Allostery is an intrinsic protein property,50 However, recent studies showed cross-talk between the variable and constant domains. For example, different IgG subclasses with identical V domains exhibit different target-binding affinities and specificities.52 Several studies have shown that modifications of the constant domain  or altering the entire constant domain15 of a Fab can influence the antigen binding affinity. Antigen binding correlates with long range conformational change in the constant domain of Fabs, especially the CH1-1 loop.17 Our studies indicate that V domain recognition and C domain effector function are dependent on each other, which contradicts the classical “independent” theory.The Fab variable domain recognizes the antigen, followed by effector activation by the Fc domain. Classically, these two processes were thought to be independent. This led to the associative model in which antigen (largely)-mediated crosslinking of Fab domains increases the proximity of the Fc domain, leading to higher avidity for FcγR and C1q.53 Recent electron tomography of IgG1 antibody molecules showed similar flexibility, but the subdomains distance/angles were not evenly distributed.35 Coarse-grained modeling showed that the antigen binding process is highly related to the internal dynamics of the IgG.54 Our simulations generally agree with the Fab-Fab and Fab-Fc angles and distance55 of IgG1. Due to the different number of disulfide bonds and length of the hinge region, the distribution may vary in other IgG subtypes.56How do these two processes take place synergistically? Molecular dynamics simulation and fluorescence anisotropy have shown that antibody molecules are highly flexible.57 After hFcγRI binding, the distance between the two CH2 domains increased to 9.1 Å. Our simulations showed that in the dominant conformation of the unbound antibody, the Fc CH2 subdomains are close to each other. In the dominant conformation upon Aβ binding, the Fc CH2 subdomains are more open. The structures become similar upon Fc binding to hFcγRI. Thus, antigen binding changes the Fc domain to an intermediate conformation between the unbound and hFcγRI-bound states. It has been reported that the hinge region and the CH2-CH3 interface residues are important for CH2-CH2 motion and conformation,25 and that modification of the human IgG1 hinge region can modulate its effector functions.58 The order parameters of the antibody hinge region suggested that the flexibility is reduced upon antigen binding. The change in flexibility in the hinge region further influences the CH2-CH2 motion, suggesting that the hinge region served as a linker as well as an “entropy transport cable” while transferring the antigen binding signals.We observed population changes with (sub)domain-(sub)domain distance when the antibody binds to antigen and/or hFcγRI. The entropy change from the antigen binding can be transferred to the hinge region, and then to the Fc CH2 domains, coupled with opening the Fc conformations to facilitate the Fc receptor binding. Conformational change of the Fc CH2 domains upon hFcγRI binding has been discovered by crystal structures.59 We have proposed a general antibody-antigen recognition mechanism based on the population shifts, as illustrated in In addition to the CH2 conformational shift, we observed that antigen binding shifts the relative Fab-Fab and Fab-Fc orientation into two main clusters, one “Y”-shaped, the other “T”-shaped . In the 60 and thus Fc-FcR recognition.64 Deglycosylation of IgG1 resulted in a 40-fold loss in FcγRI binding.61 The noncovalent interactions of multiple Fc domain residues with the N-glycan are necessary for optimal recognition of FcγRI.65 Single amino acid mutations of these Fc residues affect glycan processing.67 In the apo form, terminal carbohydrate N-glycans residues are flexible: α1-3Man-linked branch is usually unconstrained, while the α1-6Man-linked branch has two states, free and bound to nearby Fc domain polypeptides.69 N-glycans dynamics are crucial in Fc-receptor interactions and enzymatic glycan remodeling.68 The composition of N-glycans can modulate the binding affinity of IgG1 Fc to FcγRs.73 X-ray crystallography and NMR data indicated that the two arms of N-glycan are either in the bound state (attached to the Fc)74 or in the free state (detached to the Fc).68 N-glycans may directly interact with the hFcγRI D2 domain (PDB ID: 4X4M),61 but do not show direct contact with hFcγRI in a high resolution structure (PDB ID: 4W4O).57 Based on our 6 independent MD simulations of hFcγRI-antibody complexes built on the high resolution structure (PDB ID: 4W4O),57 we confirmed that hFcγRI D2 domain, especially the KHR motif formed extensive hydrogen bonds with the N-glycans. Lee et al reported that the C’E loop and the CH2-CH3 orientation are dynamic, and changes in N-glycan composition optimize the interface with the Fc receptor. 75 In this study, we showed that the N-glycans also respond to antigen binding and shift their conformations and the CH2 domain ensembles. Antigen binding shifted one N-glycan arm to the bound state and the other to the free state. The asymmetric distribution of the states is similar to the distribution when hFcγRI binds. Thus, binding shifts the N-glycans to an asymmetric ensemble, which is required for hFcγRI binding.The glycans at Asn-297 (N-glycan) help maintain the quaternary structure and Fc stability, 76 Structural  data has shown that, upon antigen binding, the C domains can affect the V region paratope conformation. Simulations and experiments showed that modification of the constant domain influences binding affinity82 and specificity84 of the antibody-antigen interaction. This may have important implications in antibody engineering and isotype choice. Lua et al. used antibody isotype swapping by grafting the VL and VH of trastuzumab and pertuzumab onto human CHs and CLs to minimize side effects.16 They showed that the LC constant region changes have no major effects on HER2 binding, while some IgM and IgD heavy chain isotypes can modulate it. Xia et al. showed that the constant region plays an important role in the nephritogenicity of anti-DNA antibodies by affecting immunoglobulin affinity and specificity,85 with the order of IgG3> IgG2a> IgG1> IgG2b> IgM. Not all C domain (isotype) changes cause V region changes,86 suggesting a possible dependence on antigen type. Engineering is often done to reduce antibody size; however, with reduced size, entropy dissipation may be limited. In the case of the unstable scFv, only the V portion exists with the VH and VL domains connected by a linker; thus, stabilizing any CDR loop in the VH domain triggers a destabilizing response in all CDR loops in the VL domain and vice versa.87 The entropy upon antigen binding cannot be released to the C portion and might result in instability, as observed for the gammabody heavy chain variable domain, 88 with a long grafted CDR3 loop stabilized upon antigen binding, but limited ability to dissipate entropy.89 Aβ peptide rigidifies the solanezumab Fab domain,15 implying a high entropy penalty. However, within the full antibody framework studied here, we do not see a similar Fab domain rigidification. Therefore, even though the lack of C domain might not hinder affinity/specificity directly, it may limit it.The population shift mechanism described here suggests two-way communication between the Fc and Fv domains, i.e., modifications of Fc can influence the Fv antigen recognition. In line with this, IgA Fc mutations have been reported to reduce binding to human epidermal growth factor receptor 2 (HER2).34 When bound to antigen, the relative Fab-Fab and Fab-Fc orientation shifts to dominant conformational clusters that may facilitate the FcR or C1q recognition, with the Fc CH2 domain becoming more open. We propose that population shift and the associated entropy redistribution is the major allosteric mechanism in antibody activation.In conclusion, antigen recognition and FcR binding result in conformational change and subdomain cross-talk. The apo antibody is highly flexible, and its motion is not random.90 To construct the Fc region, we performed sequence alignment of CH2/CH3 domains between solanezumab and 4W4O. There is only one residue difference . Thus, the Fc region was directly built from the structure of 4W4O with mutation from alanine to serine. The N-glycans of solanezumab Fc and glycans of FcγRI were modeled directly from the corresponding templates . Missing residues were modeled by template-based homology modeling using the SWISS-MODEL Server.91 To determine the relative positions of Fab and Fc within the full antibody, we used 1IGT as the template in which the distances between Fc COM and either Fab COM are roughly similar to avoid bias in initial configuration. The rebuilt systems  were submitted to CHARMM-GUI glycan reader for the input for the MD simulation. The antibody-Aβ, antibody-FcγRI complex, and antibody was generated by removing FcγRI, Aβ, and FcγRI/Aβ, respectively.The sequences of solanezumab, hFcγRI, and Aβ are listed in Table S1. As the non-sequential Kabat numbering scheme is used in the crystal structures, we renumber the residues for convenience (see Supplementary file). The structures of the Fab/peptide complex were built based on the crystal structure Protein Data Bank ID 4XXD.Initial antibody random conformations were generated by adjusting three sets of torsion angles. 231C-232N-232CA-232C, 232N-232CA-232C-233N, and 232CA-232C-233N-233CA (numbering in 1IGT), each step with 60° rotation. During the conformation randomization, the Fc domain was fixed and the Fab domains move freely, leading to 216 conformations. Excluding conformations with closed Fab domain or with Fc domain clashes, 12 conformations were selected as the starting points for the simulations. In the complexes between hFcγRI and antibody, 4 of 12 representative conformations are selected to avoid clashes between Fabs and hFcγRI.3+ and COO−, respectively. The systems were solvated by TIP3P water molecules, and sodium and chlorides were added to neutralize the system and to achieve a total concentration of ~ 150 mM. The systems were energy minimized for 5000 conjugate gradient steps, where the protein was fixed and water molecules and counterions could move, followed by additional 5000 conjugate gradient steps, where all atoms move. In the equilibration stage, each system was gradually relaxed by a series of dynamic cycles, in which the harmonic restraints on proteins were gradually removed to optimize the protein-water interactions. In the production stage, all simulations were performed using the NPT ensemble at 310 K. All MD simulations were performed using the NAMD software92 with CHARMM36 force field.93 MD trajectories were saved by every 2 ps for analysis. A summary of all simulation systems is listed in Table S2.Conserved disulfide bonds were constructed according to the specific IgG subtypes. The N-termini and C-termini were charged, NH94 The RMSD was averaged over all pairs of either apo or Aβ-bound structures from all 12 MD simulations. The RMSFs were evaluated by the internal module of CHARMM.To calculate the VH/VL orientation, the two antibody structures, apo and Aβ-bound, were superimposed according to the variable domain of the H/L chains, and the RMSD is calculated. As the full-length antibody has two Fab domains, the VH/VL orientation was evaluated separately. The six CDRs were defined as described by Ofran et al.To study the populations of the Fc domain, the trajectories of the four systems were aligned by the CH3 domain (residues 543 to 633 and 1207 to 1322), clustered by the Fc domain (443 to 633 and 1107 to 1322) using the clustering tool of VMD with cluster number of 5 and RMSD cutoff of 4 Å. To evaluate the conformational distribution of the full-length antibody, the trajectories of the four systems were aligned and the distance between the center of mass of two Fabs and between the center of mass of one Fab and Fc were measured and mapped onto a 2D plane.95 which can calculate the accumulated contact map by summing up all the frames during simulations.To identify the essential interactions between Fc and Fabs, all atoms within 3 Å between Fc and Fabs during the last 100 ns simulation were considered as input into PROTMAP2D,96 after 500 steps of energy minimization to relax the local geometries caused by the thermal fluctuations that occurred in the MD simulations. In the GBMV calculation, the dielectric constant of water is set to 80 and no distance cutoff is used.To evaluate the total potential energy of the system, the trajectory for each system was extracted from the last 20 ns of explicit solvent MD without water molecules and ions. The solvation energies of all systems were calculated using the generalized Born method with molecular volume (GBMV)100 ranging from −1 to 1. If two residues move in the same (opposite) direction in most frames, the motion is considered as (anti-)correlated, and the correlation value is close to −1 or 1. If the correlation value between two residues is close to zero, they are uncorrelated. The correlation evaluation was performed using CARMA.101 The weighted network, optimal/sub-optimal paths in Fab/peptide systems is analyzed using NetworkView 102 module in VMD.Correlations between the residues in the different clusters from the four systems were analyzed using the normalized covariance to characterize the correlation in motion of protein residues,