CK1 evolves a lineage expansion, resulting in a unique branch of members exclusive to the kingdom. Among them, Arabidopsis Mut9p-LIKE KINASEs (MLKs) target diverse substrates including histones and the key regulatory proteins involving in physiological processes of light signaling, circadian rhythms, phytohormone and plant defense. Deregulation of the kinase activity by mutating the enzyme or the phosphorylation sites of substrates causes developmental disorders and susceptibility to adverse environmental conditions. Recent findings suggest that MLKs have evolved as a general kinase that modifies transcription factors or primary regulatory proteins in a dynamic way. Here, we summarize the current knowledge of the roles of MLKs and MLK orthologs in several commercially important crops.Casein kinase I (CK1), a ubiquitous serine/threonine (Ser/Thr) protein kinase in eukaryotes, plays pivotal roles in a wide spectrum of cellular functions including metabolism, cell cycle progression, developmental control and stress response. Plant  Casein kinase I CK1), a highly conserved serine/threonine (Ser/Thr) protein kinase, has been identified ubiquitously in eukaryotes ranging from yeast to human since the 1970s , a highl,3,4. ThePlant CK1 evolved an ancient lineage duplication event, resulting in the division of CK1 into two subsets, i.e., casein kinase 1 like (CKL) and a unique group with members exclusively from plant species . The latCK1, especially the MLK family members in growth and development. We summarize the regulation of CK1 expression and the kinase activity, and the phosphorylation target sites, as far as the available literature permits. In addition, multiple CK1-modulated pathways are discussed with a focus on light signaling, phytohormone, circadian clock and stress response based on the recent research achievements of CK1 in model plants Arabidopsis and rice. The purpose of this review is to provide an overview of the roles of plant CK1-like (CKL) cluster and a plant-specific CK1 cluster containing members exclusively from plants [Mut9p in the green alga Chlamydomonas reinhardtii [MLK1-4) and six rice CK1s belong to the cluster [MLK homologs in angiosperm including amborellales, chlorophyta, lycopodiophyta, monocot and dicot showed a tendency to increase from lower to higher plants  and the region of homology is largely limited to the catalytic motif containing the canonical domains of eukaryotic protein kinases . The SerMLKs and rice homologs showed that these genes were ubiquitously expressed in all tissues at different developmental stages (http://bbc.botany.utoronto.ca/efp) [in vitro catalytic activity of MLK4/PPK1 [in vitro kinase activity in the green alga and Arabidopsis, respectively [Although MLKs split from CKLs ,25, they.ca/efp) ,26. The .ca/efp) . The str.ca/efp) . MutatioLK4/PPK1 . MoreoveLK4/PPK1 , was indectively ,16. It iChlamydomonas [CK1 activity is modulated by inhibitors and the effectors affecting the kinase localization and compartmentalization. Several ATP-competitive small molecules have been identified and characterized as CK1-specific inhibitors in animals . Among tydomonas ,30,31. Pydomonas ,32. Receydomonas ,33. Inteydomonas , suggestydomonas , implyinydomonas , Mut9p, ydomonas ,22,36,37in vivo and in vitro methods are limited in precisely localizing the phosphorylation sites on the proteins. Several databases such as PhosPhAt [http://phosphat.uni-hohenheim.de/statistics.html). Further verification of these phosphorylation sites would be beneficial for the functional characterization of the phosphorylation events. With the rapid development of methodology for protein phosphorylation site(s) identification, a new generation MS instrument, such as quadrupole Orbitrap high-resolution Mass spectrometry, has been used to target hundreds of peptides within one single LC-MS/MS experiment and generated full MS/MS spectra containing sufficient fragmentations for precise localization of the phosphorylated residues [Considerable efforts have been made to uncover plant phosphoproteomes, but the phosphorylation events provided are far from complete due to the fact that phosphoproteomic approaches including PhosPhAt  and P3DBPhosPhAt  provide PhosPhAt   linked by histone H1 . In addiIn vitro assay showed that Chlamydomonus Mut9p and Arabidopsis MLK1 and MLK4 phosphorylated histones including H3, H2A and H4, albeit at different intensity [mut9 mutant strain and Arabidopsis mlk1mlk2 double mutant [in vivo H3T3 phosphorylation in the green alga and Arabidopsis, respectively. The existence of other kinases, especially the paralogos, may explain the incomplete loss of H3T3ph in the mutants. Additionally, MLK4 was reported to phosphorylate H2AS95 residue in vitro, while the global level of phosphorylated H2AS95 did not alter clearly in the loss-of-function mlk4 mutant [Chlamydomonus and Arabidopsis. Identification of upstream kinases and the target site(s) of individual enzyme is complicated due to the fact that there are numerous candidate kinases responsible for histone phosphorylation and that multiple sites of plant histones are potential phosphorylation targets. In plants, histone phosphorylation modulated by the canonical kinases, such as AtAurora3 and AtHaspin, has been recently reviewed ,46,49. Intensity ,22,37. Pe mutant ,22, confin vitro in blue-light dependent manner [In addition to the conserved histone residues, plant CK1 including CKL and MLK family members have been predicted, based on structural analysis, to have a broad range of substrates. For example, a rice lipase and an integral negative regulatory domain (NRD) of dehydration-responsive element-binding protein 2A (DREB2A) in Arabidopsis were potential phosphorylation targets of CKL protein kinases ,51. Arabt manner , and CKLt manner . Recentlt manner . In sesat manner  and GhTCt manner , respectPhosphorylation of an amino acid residue can have multiple consequences, including change of enzyme activity, subcellular localization, or protein stability, which affect the interaction with other molecules  and eventually alter the biological functions. In non-plant eukaryotes, the CK1-dependent phosphorylation has been shown to play diverse biological roles ,11, but MLKs act as a general kinase in a myriad of cellular processes of plant growth and development. Indeed, mlk quadruple mutant is lethal [MLKs in Arabidopsis. The role of the canonical plant CK1 in phosphorylation-dependent regulation of chromatin and cell cycle has been reviewed [CK1, especially MLK family members in light signaling, circadian rhythm, phytohormone and stress response. The information provides a connection among the molecular events mediated by plant CK1 through direct/indirect modification or interaction with the target proteins. An increasing number of proteins involving in plant growth and stress response have been confirmed as MLK targets in Arabidopsis via mutating the kinase activity site or the phosphosites of the target proteins . s lethal ,60, suppreviewed ,46,49. Wppk124 triple mutants, which showed a reduced level of light-induced PIF3 phosphorylation and degradation, more robust reduction was detected in PPKs quadruple-knockdown transgenic lines [PPKs/MLKs in Arabidopsis collectively promote light-induced degradation of PIF3 by phosphorylation. The triple mutants of ppk123/124/134 also displayed hypersensitivity to red light, which was strongly suppressed by mutating phyB, suggesting the hypersensitivity is attributed to the reduced degradation of phyB [ppk triple mutants with photo-hypersensitivity to blue light. PPKs were also shown to interact with and catalyze the blue light-dependent phosphorylation of CRY2, although individual kinase preferred different phosphosties. Substitution of several Ser/Thr residues at the phosphosites of CRY2 resulted in abolished phosphorylation [Plants perceive and response to light signal, a major developmental cue, using multiple families of photoreceptors including phytochromes and cryptochromes, which absorb red/far-red light and blue light, respectively. It has been documented that the stability of CRY2 was negatively regulated by CKL-mediated phosphorylation ,61 and lic lines , suggest of phyB . In an arylation . Thus, Prylation . Howeverrylation . It is tCK1 in circadian clock was found in a unicellular marine algal species Ostreococcus tauri [CK1 with diurnal fluctuation. Overexpression O. tauri CK1 caused a long-period phenotype and treatment with chemical inhibitor altered circadian period [Like most organisms, plants have evolved an internal timing mechanism, the circadian clock, which is an autoregulatory, endogenous biological cycle with a period of approximately 24 h. A functional and accurate clock-dependent synchronization of physiology with the environment allows for efficient allocation of resources and optimum fitness. CK1 has been known to affect timekeeping across metazoans and fungi ,62. Earlus tauri . The basn period . CK1-deficient mutants. For example, mutation of rice EL1/Hd16 (heading date 16) caused earlier flowering [Oryza sativa pseudo-response regulator 37), an ortholog of Arabidopsis circadian clock components PRRs, and act upstream of flowering regulators Ghd7  and Hd2 [mlk4 and the higher order of mlk4- combined mutant(s) flowered late with retarded elongation of hypocotyl [DWF4 (DWARF4) [GIGANTEA (GI), a positive flowering regulator [CKLs [prr5 toc1 mutants the period-lengthening effect was attenuated by inhibition of CK1 [In higher plants, the clock output processes, such as flowering time and hypocotyl elongation, are clearly affected in lowering ,4. Hd16  and Hd2 . In contypocotyl ,63. The ypocotyl . Among typocotyl . MLK1 anor [CKLs . In prr5n of CK1 .Therefore, plant CK1 plays a critical role in regulating the circadian clock via posttranslational modification of the central clock regulators CCA1 and PRRs (including TOC1), as it does in animal systems. Future studies aiming to decipher the interacting network between the circadian components targeted by plant CK1 should broaden our knowledge of the kinase in the epigenetic regulation of circadian rhythm.Phytohormones are generally small organic molecules that regulate both the physiological and developmental processes of plants through a complex network of signal transduction pathways. This section described the recent findings on the CK1-mediated phosphorylation of signal transduction components and the downstream proteins of phytohormone gibberellin (GA), auxin and abscisic acid (ABA). CK1 in GA signaling pathway. Mutation of rice EL1 caused enhanced GA response with taller plant height. EL1 phosphorylated rice DELLA protein SLR1 (SLENDER RICE 1) and mutation at its phosphorylation site altered GA signaling [mlk1mlk2 double mutant with shorter hypocotyl was hyposensitive to GA. Although the phosphorylation target(s) of MLK1/MLK2 are not known, both kinases were found to interact with RGA, a DELLA protein REPRESSOR OF ga1-3, to regulate hypocotyl elongation [CK1 plays a critical role in regulating the activity or turnover of GA-responsive transcription factors. An increasing body of experimental findings has demonstrated the involvement of ignaling . In contongation . These oCK1 has been implicated to regulate auxin synthesis or signaling pathways in plants. For example, rice OsCKI1 knockdown mutants displayed abnormal root development with altered auxin content [ltg1), representing dominant LTG1 (Low Temperature Growth 1) allele with functional CKL and the ltg1 allele, respectively, revealed that the latter possessed higher content of IAA (indole-3-acetic acid) than the former. Consistently, the shoot growth of NIL (ltg1) was less sensitive to NAA (1-naphthylacetic acid) treatment and was less affected by the auxin transport inhibitor NPA  relative to Asominori [LTG1 affects plant growth through an auxin-dependent process. Research on cotton CKL homolog showed that compared with Col-0, the IAA content in GhCK1 overexpressing Arabidopsis explants was higher, while lower in GhCKI RNAi line, suggesting a correlation between GhCK1 expression level and IAA content [GhPIF4 [ content . Comparisominori , suggest content . GhTCP15 [GhPIF4 . FurtherSeCK1 transcription in developing sesame seeds [CKL2 caused stomatal closure less sensitive to ABA and phosphorylation of ADF4 (actin depolymerizing factor 4) by CKL2 inhibited actin filament disassembly [mlk triple mutants to ABA treatment [CK1-defective mutants to ABA is supportive of the implication of CK1 in ABA signal transduction although how these ABA-involving processes are connected has yet to be explored.ABA is an important phytohormone in plant response to adverse environmental conditions by regulating stomata opening and closure. CK1-mediated phosphorylation of the transcriptional factors involving in ABA signaling appears to play a role in plant stress response or adaption. Early research found that application of ABA induced me seeds . Deletioassembly . RecentlCK1 altered plant response to multiple relevant phytohormones. Rice OsCKI1 knockdown mutant displayed abnormal root development with altered auxin content and was also hyposensitive to brassinosteroid (BR) and ABA treatment during germination. Consequently, in OsCKI1-deficient plants, the expression level of the genes related to hormone metabolism and signal transduction was altered [As phytohormones act through a complex network of signal transduction pathways, it is not surprising that mutation of the ubiquitous plant  altered . Support altered . Furthermlk1mlk2 double mutant with diminished level of H3T3 phosphorylation was hypersensitive to osmotic stress including salt and drought. mlk1mlk2 exhibited partial decondensation in pericentromeric/knob heterochromatin, and about one third of MLK1/MLK2-dependent genes were stress-related according to the annotation [Due to the sessile lifestyle, plant establishes its stress defense at the level of protection and adaption. Chromatin alterations in chromatin composition and histone modification are emerging as integral and complex elements of stress responses in plants ,68. Postnotation . These fmlk1, 3, 4, implying that MLKs affect plant immunity via transcriptional regulation of SA signaling [In addition to global phosphorylation, CK1 has been reported to be potentially responsible for phosphorylation of the negative regulatory domain (NRD) of DREB2A under normal growth conditions, which in turn facilitates the degradation of DREB2A. Upon heat stress, phosphorylation NRD of DREB2A was decreased, and application of a highly CK1-selective inhibitor caused the accumulation of DREB2A in a dose-dependent manner , suggestignaling . CKL genes from several species are involved in response to environmental cues, especially extreme temperature. In a high temperature (HT)-sensitive cotton line, for instance, GhCK1 was induced by HT and overexpression of GhCK1 resulted in elevated ABA levels [AtCKL2 and AtCKL7 were HT-induced due to the binding of AtMYB24 at the promoters [LTG1, which encodes a CKL protein, conferred low temperature (LT) tolerance, while the recessive ltg1 allele was hypersensitive to LT [Plant A levels . At the romoters . In contve to LT . It woulCK1 family members and the CK1s unique to the kingdom. The latter ones play fundamental roles in various cellular, physiological and developmental processes by phosphorylating or interacting with target proteins. During the last decade, research has been mainly focused on the regulatory mechanisms of CK1 in model plants. The findings have considerably advanced our understanding of the genetic regulation network mediated by CK1, however, there are still many questions to answer in order to address how plant CK1 functions. For example, what specifies the plant-specific CK1 from animal CK1? It would be promising to explore the roles of the C-terminus, which is distinct from that of the animals. As some nuclear protein CK1s also reside in the cytoplasm, identification of the non-nuclear targets would be likely to provide insights on CK1 functions in cytoplasm. CK1s, especially the MLK homologs, are implicated in multiple signaling pathways, but how they orchestrate these pathways is poorly understood. Further investigation of CK1 substrates and interacting partners would help to unveil the biological roles of CK1 in plant growth and development. Plants, owing to the sessile nature of their lifestyle, have a large number of CK1 encoding genes including both the canonical


Through the comparison of three-dimensional CT reconstruction between the supine position and the prone position, the relative position of thoracolumbar great vessels and vertebral body was studied, and the shortest safe distance between them was measured to improve the safety of bicortical pedicle screw insertion and reduce the risk of vascular injury.Forty adults were selected to participate the research. Three-dimensional reconstruction of thoracolumbar (T9-L3) CT was performed in the prone position and the supine position. The relative distance between the Aorta/Inferior Vena Cava (IVC) and vertebral body was obtained as AVD/VVD respectively. The relative angle of the Aorta/ IVC and the vertebral body was calculated as ∠AOY/∠VOY. Self-controlled experiments were carried out in the prone and the supine positions, and the data obtained were analyzed using SPSS 22.0 statistical software.P < 0.05), and the aorta in the prone position was more anteromedial than that of supine position.The AVD of the prone position and the supine position was the shortest at T12 (3.18 ± 0.68 mm), but the difference was not statistically significant. The aorta of the T9-L3 segment was shifted from the anterolateral to the anteromedial. The ∠AOY of the other groups differed significantly between the prone and supine positions in all vertebrae except T12 and L1 (P ≥ 0.05), and the minimum VVD of L3 segment is greater than 5.4 mm. The IVC has no obvious mobility and is fixed in the range of 20 ° ~ 30 ° near the midline.With regard to VVD/∠VOY, there was no significant difference between the prone and supine positions (When using bicortical anchoring of pedicle screws, it is safe to ensure that the protruding tips of the screw is less than 3 mm. Due to the mobility of the aorta in different postures and individual differences in anatomy, the prone position CT can help doctors to make better preoperative plans and decisions. Pedicle screw fixation is currently the first choice for the treatment of thoracolumbar spine diseases . In the However, in treating elderly patients, the pedicle screw bicortical fixation is commonly used. The deeper the screw is placed, the larger the contact area between the screw and the bone is, and the stress is dispersed throughout the hard anterior cortex, thus strengthening the screw fixation. The enhanced internal fixation will reduce the screw loosening, displacement and pullout, and improves the success rate of the operation –9.However, this technique is at risk of damaging the blood vessels in the anterior part of the vertebral body –12. To eForty adults were selected to participate in the study: twenty men and twenty women between ages of 21 and 76 with a mean age of 53.4 years old. All participants had no thoracolumbar deformities, major vascular malformations, and anterior thoracolumbar vascular lesions, and none of them had any history of retroperitoneal surgery or thoracolumbar surgery.Computed tomography was carried out by using PHILIPS brilliance iCT 256-row spiral CT, with PHILIPS image post-processing system. All subjects were injected with iodine contrast agent, and statistics were analyzed by using SPSS 22.0 statistical software.The thoracolumbar region was reconstructed in three dimensions CT, with the patients in the prone position and the supine position respectively. In order to obtain a clearer image, iodine contrast agent was injected during the scanning process for angiography. The collected images were reconstructed by Philips ICT image post-processing workstation.Fig.The experiment was designed to obtain an axial image of the optimal pedicle screw trajectory plane, which was perpendicular to the posterior plane of the vertebral body. In order to facilitate observation and measurement, the reference line was marked as axis X, axis Y, axis Z, and origin O. The X and Z axes passed through the posterior plane of the vertebral body, X axis passed through the center of the pedicle, and the Y axis passed through the midline of the vertebral body. Fig., Fig.2.On the axial plane, the Y axis was marked as 0°. The ∠AOY/∠VOY of the great vessel refers to the average angle between the tangent point on both sides of the vessel and the origin O relative to the Y axis. The AVD/VVD was measured in the direction of the angle of ∠AOY/∠VOY. Fig..Fig. 3SP value was less than 0.05, the difference was defined as statistically significant. The experimental data were shown by the table of mean ± standard deviation. Intra-observer reliability was assessed by calculating the spearman correlation coefficient for repeated measurements.Self-controlled experiments were carried out on the changes of posture in the prone and the supine positions. The data obtained were analyzed by using SPSS 22.0 statistical software. When the P < 0.05; Table In the prone and the supine positions, the distance from the aorta to the vertebral body of the T9 ~ L3 segment decreased at first and then increased, as shown in Fig.P < 0.05)  Table .Fig. 5TP ≥ 0.05; Table In the prone and the supine positions, the distance between IVA and vertebral body of L1 ~ L3 segment decreased gradually Fig.. HoweverIn the prone and the supine positions, the IVA of the L1-L3 segment was located in front of the right anterior side of the vertebral body Fig. . HoweverThe intra-observer reliability analysis on measurements of the distance and angle showed that the correlation coefficients of AVD/VVD and∠AOY/∠VOY are 0.95 and 0.93 respectively, representing excellent reliability.Bicortical fixation requires precise screw placement, as protruding screw tips can damage blood vessels , 14. TheIt has been the focus of scholars to enhance the pullout force and internal fixation stability of pedicle screw. Clinicians wisely use bone cement augmentation, cortical bone channels, expandable pedicle and novel screw fixation. These new techniques have achieved satisfactory clinical results for the osteoporosis patient , 15–18. Studies have shown that when the screw is inserted in the anterior cortex of the vertebral body but not penetrated, the fixation strength can be increased by 16%, and the anterior cortex can be broken through, which can increase the pedicle screw pullout force by 60% and fixation strength by 20–25% –24. BicoHuitema et al.  proved aSarwahi et al.  claimed When evaluating misplaced screws in contact with blood vessels, the protruding tips of some of the screws are too long, which puts more degrees of impingement on the blood vessels and is more likely to cause chronic vascular injury . There wDue to individual differences, some segmental vertebrae are close to blood vessels, and there is no obvious safe distance. However, simulating screw placement on preoperative supine and prone CT images can find the appropriate transverse screw angle (TSA), and the screw direction can avoid great vessels completely. As shown in Fig. This study also had certain limitations, such as a small sample size and inevitable measurement error, although we have chosen angiography CT to minimize errors. We observed that at the level of T8-T12, the boundary between the IVC and liver tissue passing through the hepatic vena cava sulcus is not obvious. However, there is a sufficient safe distance between it and the vertebral body, which will not affect the bicortical anchorage at all.(Fig.The present data show that it is safe to ensure that the protruding tips of the screw is less than 3 mm in the treatment of thoracolumbar spinal diseases with pedicle screw bicortical anchorage. When judging the shortest distance, the measurement in the supine position is safer, but the prone position CT should be referred to when choosing the implantation direction TSA. Due to the mobility of the aorta in different postures and individual differences in anatomy, the prone position CT can help doctors to make better preoperative plans and decisions, which is of great significance for safe implementation of the bi-cortical fixation.


Endogenous circadian rhythms are biological processes generated by an internal body clock. They are self-sustaining, and they govern biochemical and physiological processes. However, circadian rhythms are influenced by many external stimuli to reprogram the phase in response to environmental change. Through their adaptability to environmental changes, they synchronize physiological responses to environmental challenges that occur within a sidereal day. The precision of this circadian system is assured by many post-translational modifications (PTMs) that occur on the protein components of the circadian clock mechanism. The most ancient example of circadian rhythmicity driven by phosphorylation of clock proteins was observed in cyanobacteria. The influence of phosphorylation on the circadian system is observed through different kingdoms, from plants to humans. Here, we discuss how phosphorylation modulates the mammalian circadian clock, and we give a detailed overview of the most critical discoveries in the field. E-boxes present on promoters of other clock genes such as Per  and Cry  genes. Per and Cry mRNAs are translated into proteins in the cytoplasm, and subsequently, they go back into the nucleus as PER homodimers , such as light, temperature, and nutrients, can entrain organisms to the 24-h cycle   of the hypothalamus. The SCN coordinates, comparable to an orchestra's conductor, the “tempo” of the clocks placed all over the body  promotes CLOCK nuclear localization. Here, modifications at Ser-446 and Ser-440/441 increase CLOCK transactivation activity , ZT0-12,PFK, a serine/threonine-protein kinase later identified as Casein Kinase 2α catalytic subunit (CK2α), was able to phosphorylate BMAL1 at Serine 90 (Ser-90), which displayed circadian oscillation, promoting cytoplasm/nuclear translocation , could regulate circadian proteins  that leads to the inhibition of the transactivation activity , which functions as repressor machinery of the circadian clock . In this specific case, the mutation S-G did not affect CRY1, whereas the mutation S-D impaired the function of inhibiting CLOCK: BMAL1 transcriptional activity. Since this phosphorylation is placed near FAD-contacting amino acid residues, these results would suggest that the amino acid charge of this site is more critical than the phosphorylation Additionally, the same MAPK Kinase can phosphorylate CRY2 at Serine-265 (Ser-265) with the same effect on the regulation of the catalytic activity  was the first kinase observed to have a dramatic impact on circadian rhythmicity  were tested using real-time bioluminescence recording to quantify the circadian period at the cellular level. The results showed a more extended period in CKI δ−/− explants compared to both WT and CKIε null mutants. These results suggested a hierarchical organization where CKIδ might be dominant over Ck1ε  was discussed before (see section The Negative Loop). Additionally, due to the -not clear- redundant activity of CK1ε and δ  can re-entrain circadian liver gene expression in arrhythmic SCN-lesioned mice ;- Phosphorylation mediated by kinases activated by time-restricted feeding- Phosphorylation of clock proteins that are proven to be functional only in the liver, but not in the SCN- Phosphorylation mediated by kinases which affect the food intake and feeding schedule- Phosphorylation mediated by kinases that are involved in the whole-body lipid/glucose homeostasis.It has been demonstrated that AKT can be activated upon the increased level of insulin through the PI3K/AKT pathway (see section Phosphorylation Clock: From the Cellular to the Systemic Level), and therefore phosphorylate CLOCK at Serine-845.This phosphorylation site was originally identified as an ASK kinase-dependent and cellular stress-responsive phosphorylation site , promoting its dissociation from the chromatin and cytoplasmic confinement driven by 14-3-3  ZT12-16 ad libidum, in mice kept under 12:12 light/dark conditions. PKCγ-mediated phosphorylation of BMAL1 seems dispensable during the normal light: dark entrainment, but it seems to be an essential regulator of BMAL1 under restricted feeding. AKT (which is activated by insulin increase) can modulate GSK3β through the pathway AKT- GSK3β, and this can counteract PKCγ activity on BMAL1. These observations suggest that food can synchronize the circadian clock through the axis GSK3β-BMAL1-PKCγ.On the other hand, under restricted feeding conditions (access to food only 4-h during the light phase), Protein Kinase Cγ (PKCγ) can phosphorylate BMAL1. This modification reduces the ubiquitination and stabilizes the protein, causing a circadian phase shift . This protein is located on a cytoplasmic membrane surface of tight intercellular junctions, where it traps PER1. Once the mTOR pathway is active, the heterodimer TJP1: PER1 is disassembled, promoting PER1 nuclear localization. Here, the clock factor can inhibit CLOCK: BMAL1 transcriptional activity  in the liver is associated with the regulation of feeding rhythms and food intake , in the liver, at Serine-71 (Ser-71) and, to a lesser extent, at Serine-280 (Ser-280) are involved in the regulation of CRY1 stability  regulates food intake in mice . This kind of regulation is balanced by the positively acting orphan nuclear receptors RORa  by Cyclin-Dependent-Kinase 1 (CDK1) in the mouse liver  4–6 when the light is on, and the second around CT 15–17 when the light is off. These peaks were in opposition to the ones observed for the total proteome –activated protein kinase (AMPK) is a serine/threonine kinase, which is activated by ATP exhaustion that leads to an increase of AMP, a usual signal of cellular stress Hardie, . The AMPAs a consequence, the Threonine-172 (T-172) is exposed, and it can be phosphorylated by Liver Kinase B1 (LKB1), which itself is sensitive to changes in AMP/ATP levels. This specific modification activates AMPK, which subsequently phosphorylates many targets. This mechanism is a direct readout of diurnal metabolic changes . This form of TSC2 can inhibit the conversion of Ras homolog enriched in the brain  from GDP to GTP-bound. The RHEB-GDP is not able to activate the mTOR pathway.On the other hand, the mTOR pathway is positively regulated at night by AKT. AKT, also known as PKB, is a cytosolic kinase involved in the Phosphoinositide 3-kinase (PI3K)-AKT pathway. In the presence of specific ligands, for instance, an increased level of insulin, phosphoinositide 3-kinase (PI3K) is activated. The PI3K cascade promotes AKT phosphorylation, and therefore activation. Active AKT phosphorylates TSC2 at different sites . This modified form of TSC2 inhibits the conversion of RHEB-GTP to RHEB-GDP. Thus RHEB in its GTP-bound form can trigger the mTOR pathway to promote ribosome biogenesis, which couples circadian phosphorylation and energy stress  Auger, . The FamAs a consequence, it causes an earlier onset and offset, which impacts the social life of these individuals. The first clock-gene mutation causing FASP discovered was the hPER2 S662G (mPER2 S659), which destabilizes the protein causing a shortening of the period length  (Kawauchi, Many forms of cancer are connected to circadian aberrations. For instance, alteration in the pathway of CRY1 Thr-300 phosphorylation within the phosphodegron is associated with the chemoresistance of colorectal cancer (Fang et al., Neurospora (Larrondo et al., Here we described the role of phosphorylation on the circadian clock and vice versa. Although the transcriptional-translational feedback loop is considered as a central dogma for the circadian clock regulation (Hurley et al., The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.AB and UA conceived and wrote the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


When the layer freezes, most of the group’s waves increase their losses, essentially forming an acoustic response towards water-to-ice transformation. Partial contributions to the responses originating from wave propagation, electro-mechanical transduction, and wave scattering were estimated and compared with the coupling constants, and the vertical displacements of the waves were calculated numerically at the water–plate and ice–plate interfaces. The maximum values of the responses (20–30 dB at 10–100 MHz) are attributed to the total water-to-ice transformation. Time variations in the responses at intermediate temperatures were interpreted in terms of a two-phase system containing both water and ice simultaneously. The results of the paper may turn out to be useful for some applications where the control of ice formation is an important problem .It is shown that, in spite of the wave radiation into the adjacent liquid, a large group of Lamb waves are able to propagate along piezoelectric plates (quartz, LiNbO Water is one of the most interesting liquids in nature. One of its peculiarities is the presence of the water-to-ice phase transition. This phase transition has been studied both theoretically and experimentally in a number of papers ,2,3,4,5.In general, the detection of water-to-ice transition and ice formation is an important problem for many applications, including aircraft, ships, roads, rotating elements, etc. . In receThe goal of the present paper is to study new facilities of the ultrasonic technique based on different piezoelectric crystals and various acoustic plate waves, whose sensing properties may be varied with mode order n, plate thickness h, and acoustic wavelength λ.3 , Y,Z+ 90°-LiNbO3 , ST,X-quartz , ST,X+ 90°-quartz , 360Y,X-LiTaO3 , and 36°Y,X+ 90°-LiTaO3  . The plates had a 500 µm thickness with one grinded and one polished surface. The grinded surface  had an average horizontal and vertical roughness of 0.16 and 0.8 μm, respectively. The polished surface  had an average horizontal and vertical roughness of 0.01 and 0.05 μm, respectively. The grinded surface was coated with the test sample (water or ice), 600 mg in mass, deposited on the propagation path directly, without using a special cell. The absence of the cell avoids the ice sample falling off the plate surface at low temperatures, providing rigid ice/plate contact. The thickness of the ice layer was about 0.5 mm.The measurements were carried out at atmospheric pressure using delay lines  implemenλ = 300 μm patterned from 100 nm-thick Cr and 1000 nm-thick Al. The large number of electrodes provided a narrow (5%) transducer pass band and good frequency resolutions from the modes with close velocities nV. The value of the normalized plate thickness h/λ = 500 μm/300 μm = 1.67  ensured a large variety of excited modes.The polished surface of the plates contained two pairs of input and output interdigital transducers (IDTs) with periodical structures aligned perpendicular to each other in order to generate two families of Lamb waves in the same substrate. Each transducer was comprised of 40 finger electrodes followed with period T) from −60 °C to +150 °C with a step of <0.1 °C. The time of the temperature variation from one value to the other depended on the interval between them. For example, cooling from +20 °C to −15 °C took 1100 s. The temperature of the plate was additionally controlled using an iron–constantan thermocouple.Acoustic delay lines were placed in a climatic chamber UC-20CE  with 20 L in volume and a variable temperature (S12) versus frequency f was measured with a network analyzer E5061B  KEYSIGHT 5061B, operating in the amplitude–frequency format [S12 (f) was converted to the amplitude–time format S12 (τ), where the leakage was rejected using a proper gate start (just after leakage) and gate stop . Then, the time format S12 (τ) was converted back to the frequency format S12 (f) to obtain the measurements without leakage.The insertion loss of the delay lines . Then, the same loss h/λ = 500 μm/300 μm = 1.67 were compared with one another, and the mode (frequency) with the largest ice response (maximum response to icing) was determined for this group of materials at a fixed plate thickness of h/λ = 1.67 for all of them. Different modes n and various plate materials  were compared with each other and the best mode/material combination was taken for detailed investigation.The procedure of the measurements was as follows: First, for each mode In the beginning, the best combination was studied when the acoustic delay line with the test liquid was cooled without steps from +20 °C to −15 °C (liquid–ice transition) and then heated from −15 °C to +20 °C (ice–liquid transition). This procedure allowed the comparison of the final stages of the phase transitions for different plate materials. After that, the best combination was examined when the acoustic delay line with the test liquid was fluently cooled from +20 °C to −15 °C. In this case, the temperature changed in intervals of 5 °C with pauses of 60 s at each temperature. This procedure allowed the study of different steps of the transition processes at intermediate temperatures.-For delay lines with water loadings:-airTL = 0.5 × lqTL and iceTL in dB are the transduction losses of the input and output transducers  measured, respectively, without loading (air), with water (lq), and with ice (ice); lqSt and iceSt in dB are the losses produced by scattering the wave at the plate/liquid and plate/ice steps ; lqα and iceα in dB/mm are the attenuation coefficients of a mode with water and ice loadings (assumed to be much larger than for air), respectively; the propagation paths coated with water or ice in the delay lines, as shown in lqTL, iceTL, lqSt, iceSt, lqα, iceα} are equal to 6, so the solution of the system (1)–(6) is unambiguous. The precision of the solutions was about ±10%.For delay lines with ice loadings:Detection mechanisms responsible for ice-sensing with Lamb waves were analyzed using the delay line structures shown in S12lq a–c and S (S12ice a–c are mEquations (1)–(6) allow estimate partial contributions to the ice responses 3 plate, water layer, and ice layer of the same normalized thickness h/λ, as an example.An analysis of the Equations (1)–(6) from the measured 3 plate at room temperature were taken from [h/λ = 1.67). The orders of the modes in different structures are thoroughly controlled by small variations of h/λ from 0 (uncoated plate) to final value h/λ = 1.67 (test structure) resulting to step by step variations in phase velocities, coupling constants, and elastic displacements of the modes.Finally, in order to clarify the experimental results, the changes in the mode characteristics were numerically calculated for plates without loading (free faces) and for the same plates with water or ice loadings on one face. Calculations were accomplished using well-approved software  and theoken from . NormaliT were written as [3 were taken from [−4 C−1) and the thermal variations of the ice dielectric constant (11 × 10−4 C−1) at T > −35 °C were taken from [It should be remembered that for numerical calculations at different temperatures the temperature variations of the material constants of the plate, water, and ice should be taken into account. The elastic, piezoelectric, and dielectric constants and the density of the piezoelectric material at actual temperature itten as ,44. The ken from . Materiaken from ,46,47,48ken from . In our nV and shorted The results of the measurements are presented in S12 for acoustic plate modes detected at different frequencies in the plate with free faces (thin solid), with water loading (thick solid), and with ice loading (dotted). Most modes belong to the Lamb family, but some of them may be quasi-longitudinal  or quasi-shear-horizontal . As usual, the frequencies of the modes nf were determined by the phase velocity nV and wavelength λ (n = Vn/λf); the amplitudes of the modes depend on the electromechanical coupling coefficients 3 plate with a normalized thickness of h/λ = 1.67 has d plates .S12 loss are very small (<1 dB), as expected. On the other hand, when the plate is coated with water, the same variations are small again, but only at the beginning, when the water is still liquid. As the temperature drops , the liquid starts its transformation to a solid phase, and the 12S value drops sharply. Maximal change 12S at its maximum  cooled fluently from +20 °C to −15 °C in intervals of 5 °C with pauses of 60 s at each temperature. It is seen that, in addition to a stable part at the beginning and a fast decrease as the temperature decreases . This peculiarity, in our minds, indicates that the icing process is not instantaneous. We believe that the appearance of the picks originates from the simultaneous existence of two water/ice phases with small ice crystallites in the liquid broth.However, it is still unclear why the start of the ice formation (−5 °C), the existence of the two-phase system (−5 °C to −15 °C), and the formation of homogeneous ice (−15 °C) were detected at lower temperatures than may be expected. This fact demands additional investigations using, for example, the visual control of a liquid–solid transformation together with its acoustic analysis.lqα (line 4); the more coupling constants there are for lqTL (line 5) and iceTL (line 7). Furthermore, (i) for mode 11.4 MHz the integral value of the insertion loss and the partial contributions to the value, which are attributed to attenuation α, transduction TL, and steps St, are almost the same both for water and ice. Therefore, the mode is insensitive towards ice formation according to this experiment shown in f = 11.4 MHz, (ii) on the contrary, for mode 38.4 MHz, the dominant contribution is attributed to attenuation and the attenuation coefficient for ice iceα (line 6) is four times higher than it is for water lqα (line 4). Therefore, the mode 38.4 MHz has high water-to-ice sensitivity, in agreement with the experiment shown in f = 38.4 MHz, (iii) and in contrast to mode 38.4 MHz, the dominant response for mode 18.05 MHz originated from transduction loss, increasing from 6.8 dB for water (line 5) to 12.2 dB for ice (line 7), in accordance with decreasing the relevant coupling constants Analysis Equations (1)–(6) based on the measured  example . As expeS12air, S12lq, S12iceare the insertion losses of the delay lines measured without loadings for air, and with loadings for water and ice; lqα, iceα, lqTL, iceTL are the attenuation coefficients and transduction losses estimated from Equations (1)–(6); nk2 (air), nk2 (lq), nk2 (ice) are the coupling constants of the modes calculated numerically for the plate without loading, with water, and with ice for metallization on the free bottom of the plate  the difference in velocities of the neighbor modes is about 1000 m/s in uncoated plate, while it is about 200 m/s in the plate coated with water or ice. Velocities and electro-mechanical coupling constants of the modes in layered structures reduce with thickness of the layers. The values of these constants for most modes in the structures are very small except for the modes originated from relevant modes in uncoated plate.In general, the calculations show that amount of modes existing in water/plate and ice/plate structures are much larger than that is in uncoated plate of the same thickness. For example, for the thickness of the plate, water, and ice equal to x3 = α × h/λ of uncoated plate, the plate coated with water, and the same plate coated with ice are presented in The profiles of the normalized mechanical displacements of the modes vs thickness U3 displacement in water. As a result, the radiation losses of the modes are 4.9, 13.7, and 19 dB, respectively, (ii) in contrast to mode 11.4 MHz the modes 18.05 MHz and 38.4 MHz have larger displacements U1 and U3 in ice. As a results, the icing responses of the modes S12ice − lqS are 0.8, 13.7, and 19.7 dB, respectively, (iii) all displacements and, hence, elastic energy of the mode 38.4 MHz are strongly concentrated into the ice layer. As a result, the mode has largest icing response as compared with other two modes.Some results of the numerical calculations  correlatOn the whole, the calculations showed that the number of acoustic waves existing in the ice–plate double-material structure is much higher than for uncoated plates in the same frequency range. This result may be attributed to supplementary wave reflections from additional boundaries in the ice–plate double-material structure.Compared to the uncoated plate, the phase velocities of the Lamb waves are not decreased too much due to water and ice loadings. This may be explained by the small acoustic impedance of the loading materials.3 plate and in the plate–ice layered structure are increased. This result is explained by a stronger increase in effective elastic modulus and a lower increase in the density of the plate and ice at lower temperatures.When the temperature is decreased, velocities of the acoustic waves both in the uncoated Y,Z LiNbOOn the contrary, the phase velocities of the acoustic waves in the plate–water layered structure are decreased with decreasing temperature. Here, the temperature decrease of the velocity in water is much higher than the temperature increase of the velocity in the plate.Most Lamb waves are able to propagate along piezoelectric plates coated with liquid (water) and solid (ice) layers, though the layers produce additional propagation, transduction, and radiation losses. As compared with uncoated plates, the number of waves existing in their coated counterparts is increased, the velocities of the waves are decreased, and the wave losses for water are usually less than those for ice. An increase in the losses allows the detection of the water-to-ice transformation using acoustic wave propagation in plates. When the water/plate structure is cooled and the losses are increased, the stabilization of the losses at their maximum value may be attributed to the total transformation of water substance to ice. When the ice/plate structure is heated and the ice is melted, the decrease in the losses may be interpreted as the appearance of a thin water layer at the ice–plate interface. When losses move up and down at intermediate temperatures, the existence of two (water and ice) phases may be supposed. As a result, in order to transform water to ice completely, the water sample should not simply be cooled below a definite temperature (as for most liquids) but must be cooled within a certain temperature interval from about −5 to −15 °C. All of these details may turn out to be useful for some applications where the control of ice formation is an important problem .


Saccharomyces cerevisiae, a powerful model for studying evolutionarily conserved eukaryotic processes. Deletion of SET1 or SET2 causes an increase in serine-2 phosphorylation within the C-terminal domain of RNA polymerase II (RNAP II) and in the recruitment of the cleavage/polyadenylation complex, both of which could cause the observed switch in pA site usage. Chemical inhibition of TOR signaling, which causes nutritional stress, results in Set1- and Set2-dependent APA. In addition, Set1 and Set2 decrease efficiency of using single pA sites, and control nucleosome occupancy around pA sites. Overall, our study suggests that the methyltransferases Set1 and Set2 regulate APA induced by nutritional stress, affect the RNAP II C-terminal domain phosphorylation at Ser2, and control recruitment of the 3′ end processing machinery to the vicinity of pA sites.Adjusting DNA structure via epigenetic modifications, and altering polyadenylation (pA) sites at which precursor mRNA is cleaved and polyadenylated, allows cells to quickly respond to environmental stress. Since polyadenylation occurs co-transcriptionally, and specific patterns of nucleosome positioning and chromatin modifications correlate with pA site usage, epigenetic factors potentially affect alternative polyadenylation (APA). We report that the histone H3K4 methyltransferase Set1, and the histone H3K36 methyltransferase Set2, control choice of pA site in  The basic structural unit of chromatin is the nucleosome, consisting of a histone octamer, around which 147 base pairs of DNA are coiled . Epigenecis-regulatory elements, but also within introns and coding sequences. Choice of pA site determines the location at which the pre-mRNA is cleaved and polyadenylated leading to the production of different mRNA isoforms in a process called alternative polyadenylation (APA). Controlling 3′ UTR length may promote or repress mRNA degradation, nuclear export, and translation. Thus, APA leads to production of proteins with different expression levels, structure, function and subcellular localization so that the cell properly responds to different environmental stimuli  sites . Most hu stimuli . Recent  stimuli ,11,14–18 stimuli , DNA dam stimuli ,26, diff stimuli , metabol stimuli , heart f stimuli ,37, pulm stimuli ,39, neur stimuli ,41 and i stimuli ,32,42–46Because polyadenylation occurs co-transcriptionally, epigenetic factors are likely to affect APA. Indeed, slowing the transcriptional elongation rate of RNAP II favors utilization of upstream pA sites . FurtherSaccharomyces cerevisiae, a powerful model for studying evolutionarily conserved eukaryotic processes. In S. cerevisiae, Set1 co-transcriptionally methylates histone H3 on lysine 4  assay, we show that in set1Δ and set2Δ cells, the amount of 3′ end processing complex at pA sites preferred in the mutants is increased. In addition, set1Δ cells had increased RNAP II CTD Ser2 phosphorylation (Ser2-P) at pA sites, which enhances RNAP II’s association with polyadenylation factors , or in their 3′ UTRs (RAD53 and RPB2)  Figure . Switch s Figure –D, as meand RTG2 . The exp2Δ cells , while ts Figure  and F. Tthe same .set1Δ and set2Δ cells , Cleavage Factor IB (CF IB), and holo-CPF, which contains core processing subunits and the Associated with Pta1 (APT) factor  factor . To unden Figure . Howevers Figure –D, and as Figure –F. The ss Figure –F. TheseS. cerevisiae expresses two TOR proteins: Tor1 and Tor2, both of which are inhibited by rapamycin. Consistent with previous reports , rapamycin. TOR senses nutrients and regulates cell growth and aging. Mammalian TOR (mTOR) hyperactivation has been shown to lead to global mRNA 3′ UTR shortening in human cells, which upregulates translation of a subset of mRNAs . S. cere reports ,121, Set reports . Wild-ty1 Figure –D, and t3 Figure  and G. RA Figure .ISM1, FAT1, MDV1 and RRD2   , or RNAP1 (Ser2)  and Rtr11 (Ser2) ,132–135,ase Fcp1  alters tDecreased RNAP II processivity has also been implicated as a mechanism for switching to upstream pA sites . It coulSRG1 3′ end (KDM5B results in increased nucleosome occupancy at promoters in embryonic stem cells (Nucleosome positioning correlates with 3′ end formation , and the1 3′ end . Likewisem cells . MethylaHistone H3K4 and H3K36 residues and HMTs that modify them may also affect pA site choice via interaction with other epigenetic factors. For example, histone H3K36me3 can be bound by NuA3  and NuA4CAT1 pA site, which in turn results in increased expression of the human amino acid transporter Cat1 (HSP70.3 pA site, which results in increased translation of the heat shock protein HSP70.3 (APA and epigenetic modifications allow cells to quickly adjust their RNA and protein composition ,144,145.ter Cat1 . During ter Cat1 , while d HSP70.3 ,150. Str HSP70.3 . The APA HSP70.3 , while i HSP70.3 . Likewis HSP70.3 ,25, whil HSP70.3 .Set1 and Set2 are important for the cell's proper response to cellular cues and environmental stress. Set2 regulates the proper response to carbon source , DNA damTaken together, we present evidence that the epigenetic factors, Set1 and Set2, control choice of pA sites via modulation of RNAP II, and recruitment of the 3′ end processing machinery. As described above, changes in histone H3K4 and H3K36 methylation also affect APA during nutritional stress, and it will be interesting to see if they have similar roles in other cell responses. Set1 and Set2 increase nucleosome occupancy around pA sites, but it remains unknown whether this leads to changes in pA site selection. A better understanding of mechanisms regulating pA site choice, and APA’s role in human health, is required in order to manipulate it to affect disease outcomes.gkaa292_Supplemental_FileClick here for additional data file.


Syncope in setting of AV nodal re‐entrant tachycardia should prompt the clinician to perform further workup for comorbid conditions, including structural cardiac abnormalities. In complex cases, ICE may be helpful in delineating the anatomy to facilitate safe ablations. Syncope in setting of AV nodal re‐entrant tachycardia should prompt the clinician to perform further workup for comorbid conditions, including structural cardiac abnormalities. In complex cases, ICE may be helpful in delineating the anatomy to facilitate safe ablations. Dual AV nodal physiology, substrate required for AVNRT, is present in approximately 20%‐30% of the population though only a minority of such patients suffers from symptomatic tachycardia.2A 69‐year‐old man without prior cardiovascular events, presented with new‐onset SVT resulting in shortness of breath, dizziness, and multiple syncopal episodes over the course of 1‐year. Cardiovascular medicines included amlodipine, atenolol, and doxazosin for treatment of hypertension though adjustment of these medicines did not mitigate syncopal episodes. Other etiologies, including autonomic dysfunction, reflex syncope, orthostatic hypotension, were also considered but given the patient's history and description of the patient's symptomology, it deemed highly unlikely. Physical examination was grossly benign at presentation—nontoxic appearing with cardiovascular examination revealing regular rate and rhythm without any extra heart sounds. Workup with ECG was concerning for SVT Figure . Given m3Although there is a myriad of different causes of syncope,Accordingly, multiple case studies reporting syncope in setting of AVNRT with other comorbid conditions have been published.In our patient, we hypothesize that the syncopal episodes were a result of RA compression due to an enlarged aortic root, resulting in underfilling of the right ventricle during episodes of AVNRT Figure . AnotherGiven multiple prior noted anatomic challenges at the time of the initial EP study, we elected to use ICE during the follow‐up procedure. Adjunctive use of ICE with fluoroscopy and electroanatomic mapping allowed direct visualization of the surrounding structures, improving catheter guidance to the ablation target within the distorted triangle of Koch. The use of ICE revealed challenges noted in the initial ablation primarily centered around RA distortion from the prominent aortic root. In addition, ICE offers other advantages over traditional methods of fluoroscopy and electroanatomic mapping systems in that it allows for real‐time anatomic evaluation, assessment of the catheter contact with the endocardium and real‐time catheter location in relation to neighboring cardiac structures. Use of ICE also minimizes the risk of VA block, as demonstrated by the results from the study by Fisher and colleagues.Our decision to use cryoablation to treat our patient was supported by proximity of slow pathway fibers to the His bundle and the baseline first‐degree AV block, both of which increase potential for damage to the native conduction system. In summary, presentation of SVT in context of syncope or presyncope should alert clinicians to consider additional workup. Complementary to TTE, ICE can serve as a valuable tool to assist with evaluation of cardiac anatomy, in particular anatomy important in relationship to electrophysiological procedures, real‐time catheter positioning, and monitoring catheter‐tissue contact. Lastly, clinicians should consider the use of ICE to assist with challenging catheter ablation procedures attempting to treat AVNRT.4We present an uncommon case of recurrent syncope and presyncope secondary to AVNRT in the setting of structural cardiac abnormality that further compromised the cardiac output. Syncope should alert clinicians to perform further workup assessing for comorbid conditions or structural heart disease. In addition to facilitating safer ablation, ICE can be useful in delineating cardiac anomalies, at times not evident by routine surface echocardiography.None declared.Paul Lee: involved in writing the article and interpretation of data. Paul Varosy: involved in critical revision of the article. Amneet Sandhu: involved in conception and design, interpretation of data, critical revision of the article, and overall responsibility of article.


We observed that phosphorylation of all five tyrosine residues, multiple N-terminal tyrosine residues , or specific phosphorylation only at residue Tyr-310 abolishes Tau aggregation and inhibits its microtubule- and lipid-binding properties. NMR experiments indicated that these effects are mediated by a local decrease in β-sheet propensity of Tau's PHF6 domain. Our findings underscore Tyr-310 phosphorylation has a unique role in the regulation of Tau aggregation, microtubule, and lipid interactions. These results also highlight the importance of conducting further studies to elucidate the role of Tyr-310 in the regulation of Tau's normal functions and pathogenic properties.The microtubule-associated protein Tau is implicated in the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease. Increasing evidence suggests that post-translational modifications play critical roles in regulating Tau's normal functions and its pathogenic properties in tauopathies. Very little is known about how phosphorylation of tyrosine residues influences the structure, aggregation, and microtubule- and lipid-binding properties of Tau. Here, we sought to determine the relative contributions of phosphorylation of one or several of the five tyrosine residues in Tau  to the regulation of its biophysical, aggregation, and functional properties. We used a combination of site-specific mutagenesis and  The misfolding and aggregation of the microtubule (MT)-associated protein Tau have been linked to the pathogenesis of several neurodegenerative disorders , includiTau is extensively post-translationally modified with phosphorylation being one of the predominant modifications . Althougin vitro, in cell culture and in vivo. For example, Fyn  are important elements for Tau binding to microtubules and constitute the minimal motif allowing fibrillization (A and B), there is only one tyrosine phosphorylation site (Tyr-310) within the MT-binding domain. This residue is located in the repeat peptide R3, which has also been shown to play key roles in regulating Tau aggregation, PHF formation, and membrane interactions , only a handful of studies have specifically investigated the roles of this residue in regulating Tau structure, aggregation, and toxicity. Importantly, none of the previous studies –42 speci–et al.  showed te et al. , 45, 46 e et al. , 47, thein vitro phosphorylation using the c-Abl kinase to produce milligram quantities of Tau phosphorylated at single or multiple tyrosine residues. Our findings show that phosphorylation of Tyr-310 is sufficient to inhibit Tau aggregation and plays a key role in regulating Tau microtubule-binding properties and interactions with the membrane, thus underscoring the importance of further studies to elucidate the role of phosphorylation at this residue in regulating Tau function under normal and pathological conditions.This knowledge gap combined with the location of Tyr-310 within the PHF6 domain of Tau prompted us to conduct more detailed investigations to: 1) determine the effects of tyrosine phosphorylation on the regulation of the normal function and pathogenic properties of full-length Tau; 2) elucidate the relative contribution of phosphorylation at each tyrosine residue; and 3) investigate the effect of phosphorylation at Tyr-310 on the structure, aggregation, and microtubule-binding of the full-length Tau and microtubule-binding domain-containing Tau K18 fragment. Toward achieving these goals, we utilized a combination of mutagenesis and in vitro. Toward this goal, we assessed the phosphorylation efficiency and specificity of c-Abl kinase for Tau. Recombinant full-length Tau was subjected to in vitro phosphorylation using recombinant c-Abl and the extent of phosphorylation and number of phosphorylation sites was systematically followed by LC/MS . Fig. 2A shows that c-Abl phosphorylated Tau at multiple sites, up to 5 sites after 4 h of incubation in reaction buffer. Increasing the amount of kinase or incubation time did not cause a shift toward species with a higher number of phosphorylated tyrosine residues or relative phosphorylation levels (data not shown). Although RP-HPLC allowed the separation of the kinase from the phosphorylated Tau proteins, it was not possible to achieve separation of the different phosphorylated Tau species, which eluted in one peak as observed by the UPLC and a single band by SDS-PAGE (B and C). To determine the exact tyrosine phosphorylation profile of Tau, the purified mixture of pTau was digested by trypsin and the resulting peptides were analyzed by LC/MS/MS. The analysis of the phosphorylated sites was performed using the Scaffold software (RRID:SCR_01435), which determines the abundance of phosphorylated peptides based on the spectral counting (SP). As shown in D and E, c-Abl phosphorylated Tau on Tyr-18, Tyr-29, Tyr-197, Tyr-310 and Tyr-394, with Tyr-310 and Tyr-394 being the major phosphorylation sites.To investigate the role of tyrosine phosphorylation in regulating Tau structure, aggregation, and MT-binding properties, we first sought to develop conditions that allow for efficient phosphorylation of Tau in vitro phosphorylation of Tau using the c-Abl kinase and purified the mixture of Tau-phosphorylated proteins using RP-HPLC. Next, we compared in vitro aggregation of the nonphosphorylated to c-Abl–phosphorylated Tau (m) over 2 days at 37 °C under shaking conditions, in the presence of 2.5 μm heparin. We found that phosphorylated Tau (pTau) was unable to form canonical β-sheet–containing fibrils under conditions where the unmodified Tau readily forms fibrils, as demonstrated by: 1) EM (A); 2) the absence of conversion of Tau conformation to β-sheet structure as discerned by circular dichroism (CD) (B); and 3) the fact that the thioflavin T (ThT) fluorescence remained at baseline levels (C). However, large oligomers were discernible by EM at 12 h, and longer fibrils were apparent only after 24–48 h incubation. These observations were further confirmed by the fact that the sedimentation assay showed an increase of the SDS-resistant oligomeric species over time in the pTau sample, but not for the unmodified protein (A and D).To determine the effect of tyrosine phosphorylation on Tau aggregation, we carried out ated Tau  by incuby: 1) EM A; 2) theism (CD) B; and 3)e levels C. Howeve protein , A and DD). These results demonstrate that phosphorylation of Tau at multiple tyrosine residues significantly delays its aggregation and fibril formation and results in the accumulation of phosphorylated Tau oligomers.It is likely that oligomeric species observed in the unmodified Tau sample were ON-pathway to fibril formation and represent intermediate species that disappeared by the end of the reaction through equilibrium shift toward fibrillar structures. In contrast, the oligomers formed by pTau might represent OFF-aggregation pathway species that did not proceed to form fibrils and remained in the sample at 96 h D. These A, open circles). These Tau variants were subjected to in vitro phosphorylation using c-Abl  and the phosphorylated proteins were purified and their aggregation properties were compared with that of the corresponding nonphosphorylated variants. For the 4F/Tyr-310 and 4F/Tyr-394 mutants, which could only be phosphorylated at Tyr-310 and Tyr-394, complete phosphorylation was achieved, whereas a mixture of up to four phosphorylation sites was observed for the single Y310F and Y394F mutants , whereas 4F/pTyr-394 retained its capacity to form β-sheet–containing species. These findings were supported by EM data , where only the 4F/pTyr-394 sample was found to contain Tau of fibrillar morphology, whereas all other phosphorylated mutants only showed the presence of oligomeric species . These results were further supported by the sedimentation assay, whereupon removal of fibrillar species by ultracentrifugation and quantification of unsedimentable Tau species showed a time-dependent decrease in soluble Tau in all nonphosphorylated mutants  that paralleled the increase in ThT fluorescence signal , signifying the recruitment of monomeric Tau into β-sheet–containing species. A time-dependent decrease in monomeric Tau over time was also observed for phosphorylated Tau mutants. However, in contrast to their nonphosphorylated counterparts or mutant 4F/pTyr-394, higher molecular weight Tau bands were apparent for phosphorylated Tau mutants 4F/pTyr-310, pTau-Y310F, and pTau-Y394F , suggesting the presence of Tau oligomeric species, which agrees with EM and ThT data. These findings suggest that phosphorylation at Tyr-310, but not Tyr-394 is sufficient to inhibit the fibrillization of Tau, as 4F/pTyr-394 was able to aggregate to the same extent as its nonphosphorylated version. Interestingly, phosphorylation of the Y310F mutant also suppressed Tau fibril formation, even though in this mutant the Tyr-310 residue was not available for phosphorylation, indicating that phosphorylation of multiple N-terminal tyrosine residues  may also play an important role in inhibiting Tau aggregation. These results show that although Tyr-310 phosphorylation is sufficient to suppress the aggregation of the full-length Tau, the interplay between phosphorylation of the N-terminal tyrosine residues is also of critical importance in modulating Tau oligomerization and fibril formation.To determine the relative contribution of each tyrosine residue to the c-Abl phosphorylation-mediated inhibition of Tau aggregation, we generated Tau proteins that were phosphorylated or not at one or a selected number of tyrosine residues focusing on Tyr-394 and Tyr-310. To allow site-specific phosphorylation of these residues, we mutated all tyrosine residues except Tyr-310 (4F/Tyr-310) or Tyr-394 (4F/Tyr-394) to phenylalanine. In addition, we generated Tyr-310 and Tyr-394 residue-specific phosphorylation-deficient Tau mutants, where each of these residues is mutated to phenylalanine to allow phosphorylation of all other tyrosine residues except at Tyr-310 (Y310F) or Tyr-394 (Y394F)  studies on the K18 peptide, which encompasses the MT-binding region (residues 244–368 of Tau-441 sequence) and includes the highly amyloidogenic PHF6 and PHF6* hexapeptides, the former of which contains Tyr-310 . K18 is in cells .13C,15N-labeled K18 and subjected it to in vitro phosphorylation using recombinant c-Abl under the same experimental conditions as full-length Tau. Given that there is only one tyrosine residue in K18, we were able to achieve complete K18 phosphorylation on Tyr-310 within 4 h and obtained highly pure Tyr-310 specifically modified pK18 (A–C). Next, to determine the effect of Tyr-310 phosphorylation on the structural properties of K18, we performed NMR analysis of 15N-labeled pK18. The proton-nitrogen HSQC spectrum of pK18 compared with that of K18 showed that phosphorylation at the Tyr-310 residue induced local effects on the structure of the peptide, as indicated by chemical shift changes for signals originating from residues surrounding Tyr-310 (D). Interestingly, signals that were affected in the spectrum of pK18 consistently moved toward lower 15N chemical shifts. Because increases/decreases in NMR 15N chemical shifts correlate with increases/decreases in local β-strand structure . This effect likely results from the added negative charges of the phosphate group. Notably, the affected region adopts β-sheet conformations in all known Tau fibril structures. Thus, the local decrease in β-sheet propensity may have important implications in the context of Tau fibrillization, especially as the PHF6 hexapeptide exhibits the strongest intrinsic β-sheet propensity in the Tau sequence  and the sedimentation assays (B) showed a delay of about 2 h in the fluorescence increase and loss of soluble protein, respectively. Interestingly, the final absolute value of the plateau ThT signal of the phosphorylated K18 was consistently about two to three times higher than that of WT K18, suggesting a difference in fibril structure, packing of the fibrils or susceptibility to fragmentation (C), which may affect ThT binding capacity and thus the end-point ThT value.To further test whether Tyr-310 phosphorylation impacted K18 aggregation, we compared the f WT K18 . As show the ThT A and then assays B showed entation C, which A) but sedimented more rapidly than pK18 (B). In addition, K18 fully converted to β-sheet within 1 h, whereas pK18 clearly showed a markedly delayed conformational shift toward β-sheet spectra during 4 h of incubation (C). As shown in Fig. 6D, EM studies confirmed that fibril formation by pK18 was also delayed and showed significant accumulation of oligomeric amorphous aggregates at early and intermediate time points , and accumulation of fragmented and fully mature fibrillar morphologies, in addition to some oligomers at later time points . These findings suggest that Tyr-310 phosphorylation results in delayed formation of β-sheet–rich K18 fibrillar structures (A and B) and has an inhibitory role during the nucleation phase of K18 aggregation, as demonstrated by attenuation of the pK18 conformational change .To further investigate the effect of phosphorylation at Tyr-310 on the early formation of β-sheet–containing species of K18, we monitored changes in K18 secondary structure over time under aggregation conditions by CD spectroscopy. K18 displayed a significantly lower ThT fluorescence signal than pK18 A but sedhan pK18 B. In addcubation C. As shoD). We first assessed the binding propensity of pTau to the MTs by quantifying the percent of protein pelleted when incubated with pre-formed paclitaxel-stabilized MTs at a single concentration (250 μg/ml Tau and 100 μg/ml of tubulin) (A). We observed, as previously reported (A). These results were further confirmed by SDS-PAGE, where a band corresponding to the molecular weight of nonphosphorylated Tau was enriched in the pellet fraction , whereas pTau remained predominantly in the soluble fraction. EM data showed no morphological differences for MTs bound to Tau or pTau . Our results confirm the negative impact of tyrosine phosphorylation on the interaction of Tau with MTs, likely mediated by the large shift toward the negative electrostatic potential as well as structural rearrangements of the MT-binding region through the phosphorylation of C-terminal Tyr-310 and Tyr-394, with a possible contribution from other phosphorylated tyrosine residues requiring further investigation.In light of the prominent direct association of Tau with MTs, we next asked whether Tau tyrosine phosphorylation, especially on residues Tyr-310 and Tyr-394, influence Tau microtubule-binding propensity. To address this question, we used full-length Tau phosphorylated by c-Abl, taking advantage of the preferential phosphorylation of Tyr-310 and Tyr-394 among other residues, as demonstrated by MS SP counts D. We firtubulin) A. We obsreported , that Tareported A. These We have previously shown that both Tau and K18 interact with brain phosphatidylserine (BPS) vesicles resulting in vesicle disruption and the formation of highly stable oligomeric protein-phospholipid complexes . TherefoA, red box), signifying reduced Tau affinity and binding to lipids.To address this question, we incubated Tau and K18 and their respective tyrosine-phosphorylated forms with BPS vesicles at a molar Tau to phospholipid ratio of 1:20 and performed sedimentation assays. We found that Tau and K18 incubated with BPS vesicles co-sedimented with vesicles to a larger extent compared with their phosphorylated counterparts. This is demonstrated by the reduction of phosphorylated Tau and K18 in the pellet fractions , indicating no major differences in the absolute capacity of pTau and pK18 to form oligomeric structures compared with their nonphosphorylated counterparts. These findings were further supported by CD spectroscopy (C) that showed the conformational changes in proteins due to formation of oligomeric species by both nonphosphorylated and phosphorylated Tau and K18.Next, to confirm the formation of oligomeric Tau- and K18-phospholipid complexes, EM was performed, where oligomeric complexes were detected for all proteins without discernible morphological differences B, indicatroscopy C that shD, red) systematically eluted later compared with their nonphosphorylated counterparts . These data are well in line with biochemical data  and suggest that Tau-lipid complex formation is affected by tyrosine phosphorylation of Tau and K18, resulting in oligomeric Tau- and K18-phospholipid complexes of smaller size, either through the reduction of the number of protein and/or phospholipid molecules per complex, through complex compaction.Next, to verify the formation of the Tau/K18 phospholipid-oligomer complexes by the nonphosphorylated and phosphorylated Tau and K18, size exclusion chromatography (SEC) was performed. pTau and pK18 complexes peaks D, red syC).Tau and K18 were reported to undergo the structural transitions upon binding to lipid membranes , 53–55. 53–Fig. 8Our results demonstrate that phosphorylated Tau and K18 had reduced lipid vesicle binding affinity and formed oligomers that showed less pronounced structural conformation transitions and SEC elution profiles that were shifted toward smaller species compared with nonphosphorylated Tau and K18 in the presence of BPS vesicles. However, the capacity to associate with lipids was still preserved in the phosphorylated variants. These observations combined with the differences in the SEC and CD profiles of phosphorylated Tau and K18 compared with the unmodified proteins suggest that the oligomers formed by the phosphorylated proteins exhibit distinct conformational and size properties.in vitro.Next, we sought to determine the underlying structural basis for the reduced lipid-binding affinity of tyrosine-phosphorylated Tau. Previously, we showed that the core of the highly-stable oligomeric Tau-phospholipid complexes was comprised of both the PHF6* and PHF6 hexapeptide motifs, the latter in a β-sheet conformation . BecauseA). We observed a very rapid formation of large fibrils by R3, whereas the pR3 peptide exhibited a significant delay in fibril formation . Furthermore, whereas R3 formed long fibrils at a later time point, pR3 formed relatively shorter fibrils .First, we incubated R3 and pR3 peptides with BPS vesicles at a molar peptide to phospholipid ratio of 1:1 and monitored fibril formation using EM A. We obsormation A, 1 h. Fin vitro aggregation studies suggested that the phospholipids become incorporated within the fibrils during the fibrilization of R3. Therefore, we assessed the effects of Tyr-310 phosphorylation on R3 binding and incorporation of phospholipids into R3 fibrills. To this end, we co-incubated the R3 and pR3 peptides with vesicles containing 1% NBD-labeled fluorescent phospholipids (fPS). Under these conditions, we observed the formation of fluorescent fibrils in the case of R3, suggesting that R3 bound tofibrils in the case of R3, suggesting that R3 bound to and incorporated phospholipids within the fibril matrix. No fluorescent signal was observed in the case of the fibrils formed by pR3, suggesting that they were devoid of phospholipids (B).Our holipids B.C). In this assay, vesicles alone floated and remained in the supernatant  and did not pellet to a high extent. However, upon association with the R3 peptide, a fluorescent signal could be observed in the pellet fractions. The signal present in the pellet fraction of R3-fPS at ∼30 min was likely due to the high aggregation propensity of R3 and aggregation that occured during the short of period of time required to aliquot and process the sample. We observed that phospholipids in the R3 sample were solely present in the pellet fraction after 24 h incubation, but that only a small fraction of the phospholipids in the pR3 sample were found in the pellet . These results further confirm our EM and fluorescence microscopy data and indicate that the capacity of R3 to form fibrils that contain phospholipids in the presence of BPS vesicles was significantly diminished by phosphorylation of residue Tyr-310. These findings are in line with data obtained with the full-length Tau and K18 data , which is implicated in Tau secretion  and regu63–a) phosphorylation of Tau at multiple tyrosine residues inhibits Tau aggregation through cumulative effects of phosphorylation at multiple or all tyrosine residues, and that (b) phosphorylation at residue Tyr-310 is a key determinant of Tau aggregation in vitro. We show that Tyr-310 phosphorylation appears to be sufficient to significantly delay Tau aggregation in vitro and reduces the propensity of the microtubule-binding region of Tau to aggregate through attenuation of the PHF6 hexapeptide propensity to adopt β-sheet–rich structure in the context of K18 (Our results indicate that (t of K18  and potein vitro was found to bind to MTs with an efficiency similar to that of the nonphosphorylated protein. These findings suggest that tyrosine phosphorylation of Tau on either Tyr-18 (i.e. by Fyn) or Tyr-394 (by c-Abl or Arg) did not significantly alter its affinity for MTs. Our data point toward residue Tyr-310 as an important regulator of Tau-MTs interactions. One notable difference to previous reports is that our studies relied on the use of pTau proteins that are site-specifically phosphorylated at residues Tyr-310 or Tyr-394 (D). To the best of our knowledge, this is the first report demonstrating the role of tyrosine phosphorylation and Tyr-310 in particular in regulating Tau MT-binding domain.Tyr-310 is the only tyrosine residue located in the MT-binding region of Tau which suggest a potential role of Tyr-310 in regulating Tau affinity for MTs. Our work here shows that phosphorylation on tyrosine residues affects full-length Tau affinity for MTs, in contrast to previous reports, where Tau phosphorylated by Fyn , Arg 1919, or c- Tyr-394 D. To theThe fact that the MT-binding domain of Tau is also responsible for Tau-lipid interactions also points to the potential role of Tyr-310 in regulating Tau affinity for lipid membranes. We have previously demonstrated that the PHF6 hexapeptide located within R3 interacts with negatively charged vesicles, resulting in the formation of β-strand–rich mixed phospholipid/peptide fibrils . In thiset al.  and cloned into the pT7-7 for bacterial expression. Tau 2N4R in pCMV6-XL5 vectors were obtained from OriGene Technologies, Inc. and cloned in pET-15b for bacterial expression. Single-site mutagenesis was performed in the pET-15b using the QuikChange Site-directed Mutagenesis Kit (Stratagene), as per the manufacturer's instructions. pET21d SH2-CD c-Abl T231R and pCDFDuet-1 YopH for bacterial expression were kindly donated by Prof. Oliver Hantschel .306VQIVYKPVDLSKVTSKCGSLGNIHHK331 and the pY-R3 (306VQIV(pY)KPVDLSKVTSKCGSLGNIHHK331) corresponding to the third microtubule-binding repeat without/with phosphorylation at Tyr-310 were purchased from CS Bio Co.The R3 (Taxus brevifolia (purity of >99.5%) was used to inhibit microtubule depolymerization, was provided as a lyophilized powder and resuspended at 2 mm anhydrous DMSO purchased from Cytoskeleton Inc. GTP, provided as a lyophilized powder, was resolubilized at 100 mm with ice-cold MilliQ water purchased from Cytoskeleton Inc. MAPF, isolated from bovine brain, was used as positive control for the MT-binding assay, and supplied as lyophilized powder purchased from Cytoskeleton Inc. Phosphatidylserine isolated from porcine brain (BPS) and fluorescently labeled phospholipid 1-oleoyl-2-{6-[amino]hexanoyl-sn-glycero-3-phosphoserine (fPS) were purchased from Avanti Polar Lipids, Inc. Heparin sodium salt was from Applichem GmbH.Tubulin protein isolated from porcine brain (97 or >99% pure), provided as a lyophilized powder was purchased from Cytoskeleton Inc. Paclitaxel, from the pacific yew tree Escherichia coli strain BL21. The purification protocol of Tau 2N4R Tau was adapted from Ref. m urea in 10 mm MES, pH 6.5, 1 mm DTT, 1 mm EDTA, 1 mm PMSF). After centrifugation at 150,000 × g for 1 h at 4 °C, 1% (w/v) of streptomycin sulfate was added to the supernatant, and the solution was stirred for 90 min at 4 °C. After centrifugation at 27,000 × g for 1 h at 4 °C, the supernatant was dialyzed overnight at 4 °C in ion exchange (IEX) buffer A . The supernatant was filtered and loaded on a cation-exchange column  and the protein was eluted using a salt gradient (increasing the NaCl concentration of IEX buffer A from 20 mm to 1 m NaCl over 20 column volumes). Fractions containing the proteins were dialyzed overnight against acetic buffer (5% acetic acid in water) and loaded on a reverse-phase HPLC C4 column , and the protein was eluted using a gradient from 30 to 40% buffer B over 40 min (15 ml/min). K18 in pT7-7 was purified following a protocol adapted from Ref. m HEPES, pH 6.5, 1 mm MgCl2, 20 mm NaCl, 1 mm DTT, 1 mm PMSF). After centrifugation at 40,000 × g for 30 min, the supernatant was boiled until the solution became cloudy (∼5 min) and the supernatant was centrifuged again for 30 min. The supernatant was filtered and loaded on a cation-exchange column , and the protein was eluted using a salt gradient (increasing the NaCl concentration of IEX buffer B from 20 mm to 1 m NaCl over 20 column volumes). Fractions containing the K18 fragment were immediately loaded on a reverse-phase HPLC C4 column , and the protein was eluted using a gradient from 30 to 40% buffer B over 40 min (15 ml/min).Tau isoforms 2N4R in pET-15b and K18 in pT7-7 were expressed in E. coli as previously described  and centrifuged twice at high speed , before being injected in 5 ml of His-tag columns . Selected fractions were desalted using two HiPrep 26/10 desalting columns  in series , combined and further purified by anion-exchange chromatography using a MonoQ 5/50 GL column . The final concentration of the c-Abl kinase was determined using the UV absorbance at 280 nm .Recombinant SH2-CD c-Abl, T231R, and YopH were co-overexpressed and SH2-CD c-Abl was purified from escribed . Brieflym Tris, 5 mm MgCl2, 1 mm DTT, 20 mm Na3VO4 (phosphatase inhibitor) in the presence of 3 mm MgATP, pH 7.5, at 30 °C. We used c-Abl kinase at a mass to mass ratio of 1:20 (kinase:Tau). The reaction mixture was followed by ESI-MS to verify the completion of the phosphorylation. Additional kinase and MgATP were added when needed. Phosphorylated K18, Tau, and Tyr → Phe mutants were purified by a reverse-phase HPLC preparative C4 column  using a linear gradient of 30 to 40% of B in 40 min for Tau or 20 to 35% of B in 40 min for K18. The HPLC fractions were analyzed by LC-MS and once pure were pooled and lyophilized.The large-scale phosphorylation of 10 mg of K18, Tau, and Tau Tyr → Phe mutants were performed for 4 h in 50 mm phosphate, pH 7.4, 50 mm NaF, and 0.5 mm DTT with heparin sodium salt  at a molar heparin:protein ratio of 1:4 under constant orbital agitation  at 37 °C for up to 72 h.Fibrils of WT, mutants, and phosphorylated Tau and K18 were formed by incubating monomeric protein in 10 mm R3 or pY-R3 peptide were dissolved in 10 mm Tris, 50 mm NaF, 0.5 mm DTT and pH adjusted to 7.4. The solution was then vortexed, centrifuged for 20 min at 14,000 rpm, and the supernatant was incubated in the absence or presence of 25 μm heparin sodium salt (Applichem GmbH) at 37 °C, with constant orbital agitation .100 μm HEPES, pH 7.4, 100 mm NaCl, and 2.5 mm DTT to the desired final concentration by sonication. The solution was then passed through an Avestin LiposoFast extruder (Avestin Inc.) (membrane pore size: 0.1 μm), and the size and homogeneity of the resulting vesicles were assessed by EM. For the preparation of fluorescently labeled vesicles, fluorescent and nonfluorescent phospholipids in chloroform were mixed at a molar ratio of 1:99 prior to the initial drying step.Vesicles were prepared using the extrusion method. Briefly, individual phospholipids or phospholipid mixtures in chloroform were dried using an argon stream to form a thin film on the wall of a glass vial. Potentially remaining chloroform was removed by placing the vial under vacuum overnight. The phospholipids were resolubilized in 10 mm HEPES, pH 7.4, 100 mm NaCl, 2.5 mm DTT for 48 h at 37 °C. The resulting protein-phospholipid complexes were separated from the remaining vesicles and soluble protein by SEC using a Superose 6 column .Protein-phospholipid complexes were prepared by incubating BPS vesicles with Tau or K18 at a molar protein:phospholipid ratio of 1:20 in 10 mm R3 and pY-R3 peptides were incubated with BPS vesicles at a molar ratio of 1:1. For fluorescence microscopy, the peptides were incubated with vesicles containing 1% fluorescent NBD-phospholipids, imaged with confocal laser-scanning microscope , and analyzed using Zen software.100 μm PIPES, 0.5 mm EGTA, 2 mm MgCl2, pH 6.9) supplemented with 1 mm GTP and snap-frozen as 10-μl aliquots in liquid nitrogen and stored at −80 °C. Microtubules were assembled by defrosting an aliquot of 10 μl of tubulin stock in a room temperature water bath, immediately placed on ice, and incubated for 20 min at 35 °C in 100 μl of pre-warmed (35 °C) General tubulin buffer supplemented with 1 mm GTP and 5% glycerol. Then the solution was diluted with 100 μl of General tubulin buffer supplemented with 1 μl of paclitaxel , gently mixed, and kept at room temperature. Microtubule morphology was systematically verified by EM.Tubulin was dissolved at a concentration of 5 mg/ml in ice-cold General tubulin buffer . The samples were placed on top of a 100 μl of cushion buffer  in 1.5-ml tubes. The tubes were centrifuged at 100,000 × g for 40 min at room temperature. The top 50 μl of each tube was mixed with 10 μl of 5× Laemmli buffer (supernatant) and the pellet was resuspended in 50 μl of 1× Laemmli buffer (pellet). The samples were run on SDS-PAGE gels. The relative amounts of Tau, pTau, MAPF, or BSA in the supernatant and in the pellet were estimated by measuring the band intensity using Fiji software  over 5 repeats, and represented as mean ± S.D.Pre-formed paclitaxel-stabilized MTs were incubated for 30 min at room temperature with Tau, pTau, MAPF, or bovine serum albumin (BSA) (250 μg/ml of protein and 100 μg/ml of tubulin) in General tubulin buffer supplemented with 1 mm Tris, 5 mm MgCl2, 1 mm DTT, 20 mm Na3VO4 (phosphatase inhibitor), 3 mm MgATP, pH 7.5. Reactions were performed for 4 h at 30 °C without agitation. The reactions were followed using ESI-MS on a Thermo LTQ ion trap system.100 or 200 μg of WT Tau or K18 were incubated with 5 or 10 μg of recombinant c-Abl in 50 mm ammonium bicarbonate, pH 8.4, and dried. The proteins were digested in gel by covering gel pieces with a trypsin solution  overnight at 37 °C. The digested peptides were subjected to TiO2 phosphopeptide enrichment, concentrated to dryness, resuspended in acetonitrile, and dried again. After repeating this step, the dried peptides were resuspended in 20 μl of 20% formic acid, and 2% acetonitrile, desalted on C18 OMIX tips (Agilent Technologies), and analyzed by a capillary LC-ESI-MS system (Thermo Scientific LTQ Orbitrap instrument). The collected MS/MS spectra were matched against human and scored using Mascot and Sequest algorithms. The data were then analyzed using Scaffold version 4.0 .Tau phosphorylated with c-Abl as described above was run on SDS-PAGE and stained using Simply Blue Safe Stain (Life Technologies). The bands corresponding to Tau were excised, destained with 50% ethanol in 100 mSamples (3.5 μl) were applied onto glow-discharged Formvar/carbon-coated 200-mesh copper grids (Electron Microscopy Sciences) for 1 min. The grids were blotted with filter paper, washed twice with ultrapure water and once with staining solution ), and then stained with uranyl formate for 30 s. The grids were blotted and dried. Specimens were inspected with a Tecnai Spirit BioTWIN operated at 80 kV and equipped with an LaB6 filament and a 4K × 4K FEI Eagle CCD camera.m phosphate buffer, pH 7.4, with 50 mm NaF and 0.5 mm DTT. CD spectra were acquired from 195 to 250 nm in 0.2-nm increments at a 50 nm/min scan rate. For each sample, five spectra were averaged and smoothed using binomial approximation.CD spectra were recorded on a Jasco J-815 CD spectrometer operated at 20 °C. To minimize buffer absorption, the buffer was modified to 10 m1H-15N HSQC spectra were collected at 25 °C on a Bruker Avance 600 MHz spectrometer using samples containing 500 μm K18 or pK18 peptide in 10 mm Na2HPO4, 100 mm NaCl, 1 mm DTT, 10% D2O buffer at pH 7.4. Resonance assignments are based on the previously published assignments of the K18 spectrum  and averaged over at least three independent repeats.Taking advantage of the fact that high-speed centrifugation sediments fibrils but not monomeric or soluble oligomeric species, the sedimentation assay makes it possible to assess the ratio of fibrillized to soluble protein. 20 μl of WT Tau, K18, or Tyr → Phe mutants aggregation reactions were pelleted by ultracentrifugation for 2 h at 200,000 × m and peptide concentration of 60 μm or a protein concentration of 3 μm in 50 mm glycine, pH 8.5, using a Bucher Analyst AD plate reader without shaking.Thioflavin T (ThT) fluorescence reading  was performed in triplicate with a ThT concentration of 60 μm) were mixed with BPS vesicles (200 μm) to a total volume of 20 μl and immediately centrifuged for 2 h at 200,000 × g at 4 °C. At this speed, the vesicles pellet, and protein bound to them co-sediment with the vesicles. Both supernatant, containing unbound protein, and the resuspended pellet, containing vesicles with bound protein, were run on 15% SDS-PAGE gels.The co-sedimentation assay was used to assess initial binding of protein to vesicles. Tau or K18 WT, or tyrosine phosphorylated (10 μAll data are provided in manuscript and raw files are available upon request from the corresponding author.