At the onset mitosis in higher eukaryotes, the nuclear envelope (NE) undergoes dramatic deconstruction to allow separation of duplicated chromosomes. Studies have shown that during this process of nuclear envelope breakdown (NEBD), the extensive protein networks of the nuclear lamina are disassembled through phosphorylation of lamins and several inner nuclear membrane (INM) proteins. The LINC complex, composed of SUN and nesprin proteins, is involved in multiple interactions at the NE and plays vital roles in nuclear and cellular mechanics by connecting the nucleus to the cytoskeleton. Here, we show that SUN1, located in the INM, undergoes mitosis-specific phosphorylation on at least 3 sites within its nucleoplasmic N-terminus. We further identify Cdk1 as the kinase responsible for serine 48 and 333 phosphorylation, while serine 138 is phosphorylated by Plk1. In mitotic cells, SUN1 loses its interaction with N-terminal domain binding partners lamin A/C, emerin, and short nesprin-2 isoforms. Furthermore, a triple phosphomimetic SUN1 mutant displays increased solubility and reduced retention at the NE. In contrast, the central LINC complex interaction between the SUN1 C-terminus and the KASH domain of nesprin-2 is maintained during mitosis. Together, these data support a model whereby mitotic phosphorylation of SUN1 disrupts interactions with nucleoplasmic binding partners, promoting disassembly of the nuclear lamina and, potentially, its chromatin interactions. At the same time, our data add to an emerging picture that the core LINC complex plays an active role in NEBD. The outer nuclear membrane (ONM) is contiguous with, and biochemically very similar to, the endoplasmic reticulum (ER). It is further connected to the inner nuclear membrane (INM) at the periphery of nuclear pore complexes (NPCs), the latter apparently restricting the passage of proteins from the ONM to the INM  is induced by partial disassembly of NPCs and the nuclear lamina in prophase.-S. pombe and C. elegans, respectively. The SUN domain resides in the NE lumen where it interacts with the short C-terminal KASH domain of nesprins.Since the initial phosphorylation studies of nuclear lamina components were performed, the number of known INM proteins has increased dramatically,,-,The LINC complex directly connects the nuclear lamina to the cytoskeleton and is vital for a range of functions including cellular mechanics, nuclear positioning and migration  on both tryptic and AspN-digested peptides. Combined sequence coverage was 87%, with 86% coverage in the N-terminal nucleoplasmic domain (residues 1–362), which is the region exposed to mitotic kinases in the nucleoplasm. A total of 3 mitotic phosphorylation sites were identified, at S48, S138, and S333, all located within the N-terminal domain of the protein . In contC. elegans ortholog, UNC-84, supporting their functional importance. In contrast, S333 is poorly conserved and so its significance is less obvious.Comparison of the sequences surrounding each of the phosphoserines with consensus phosphorylation sites of mitotic kinases revealed S48 and S333 as potential Cdk1 phosphorylation sites (consensus S/T*-P-x-K/R) and S138 as a potential Plk1 phosphorylation site (consensus D/E-x-S/T*-Φ) . The seqTo test whether Cdk1 and Plk1 are capable of phosphorylating SUN1, we performed in vitro kinase assays using purified MBP-tagged SUN1 protein fragments. We found that both Cdk1 and Plk1 phosphorylated a SUN1 fragment encompassing residues 1–217, but not residues 456–916, consistent with the presence of phosphorylation sites for both kinases in this N-terminal fragment . A fragmTo test the importance of Cdk1 and Plk1 for SUN1 phosphorylation in vivo, we treated mitotically-arrested HeLa cells with Cdk1 or Plk1 inhibitors and assessed whether the band-shift of endogenous SUN1 previously observed in mitotic extracts was lost. As shown in To investigate the role of the 3 identified phosphoserines in SUN1 phosphorylation, we generated myc-tagged phospho-null mutants of SUN1 by alanine substitution, creating S48A, S138A, and S333A mutants. In addition, we generated a double mutant of the Cdk1 phosphoserines S48 and S333, termed SUN1–2A, and a triple mutant of all 3 serines, termed SUN1–3A. A partial reduction in the band-shift was observed for SUN1-S48A while a more complete loss of the bandshift was seen with the 2A and 3A mutants . These dWe generated a polyclonal phospho-antibody against one of the two highly conserved phosphorylation sites, phospho-S48 (pS48), in order to confirm the phosphorylation site in vivo. We initially verified that the antibody was able to detect bacterially expressed wild-type SUN1, but not an S48A mutant, following incubation with Cdk1 in vitro . The antSUN1 participates in multiple interactions at the NE, most of which occur via its nucleoplasmic N-terminal domain at the INM. We therefore sought to determine whether SUN1 interactions with its INM binding partners are disrupted in mitotic cells. SUN1 was immunoprecipitated from either asynchronous or mitotic HeLa cells and probing of immunoprecipitates revealed that interaction with lamin A/C and emerin was disrupted in mitotic cells .To investigate changes in SUN1 interaction with nesprins, cells were transiently transfected with GFP-nesprin-2α, a short nesprin isoform that is thought to reside partly at the INM, prior to synchronization and immunoprecipitation of SUN1. Nesprin-2α was co-precipitated with SUN1 in both asynchronous and synchronized cells . Since SThese data suggested that the LINC complex remains intact during mitosis and we investigated this further by examining SUN1 self-interaction. SUN1 has been reported to oligomerize via the coiled-coil domain located in the luminal C-terminal regionWe next wished to determine whether the triple phosphomimetic SUN1 mutant was capable of recapitulating in asynchronous cells the loss of interaction with lamin A/C and emerin previously observed with endogenous SUN1 in mitotic cells. Cells were transfected with GFP-tagged constructs encoding WT SUN1, SUN1–3A, or SUN1–3D and asynchronous cultures subjected to GFP-Trap. Emerin and lamin A/C were co-precipitated equally well by all 3 forms of SUN1, suggesting that phosphorylation of emerin and lamin A/C may also be required for efficient disruption of their interaction with SUN1 .,Samp1 was recently identified as a human protein that interacts with SUN2.To further address the role of the identified SUN1 phosphorylation sites in mitotic events, we examined the subcellular localization of myc-SUN1 single, double, and triple phosphomimetic mutants in interphase U2OS cells and compared this with the equivalent phospho-null mutants. Whereas wild-type SUN1 and all phospho-null mutants localized predominantly at the NE, SUN1-S48D was frequently distributed also within the cytoplasm . In contTo extend these findings, we assessed the solubility of the SUN1 triple phospho-null and phosphomimetic mutants, SUN1–3A and SUN1–3D, by extraction in low and high salt Triton buffers. While SUN1–3A displayed similar solubility to that of wild-type SUN1, the solubility of SUN1–3D was increased 2-fold in both low and high salt buffers . Thus, w,In this study, we have demonstrated that SUN1 undergoes mitosis-specific phosphorylation on at least 3 sites. Furthermore, we have identified S48 and S333 as targets of Cdk1, whist S138 is a target of Plk1 phosphorylation. In agreement with our findings, S138 and S333 have also been identified as sites of mitotic phosphorylation in several phosphoproteomics studies.,All 3 identified phosphosites lie within the N-terminal domain of SUN1, which is located in the nucleoplasm and therefore exposed to mitotic kinases after their entry into the nucleus during prophase. In contrast, no phosphosites were identified in the C-terminal domain, which is located in the NE lumen and therefore not accessible to these kinases. The nucleoplasmic domain of SUN1 has binding sites for several nuclear lamina and INM components, including lamin A/C, emerin, and short nesprin isoforms,,,Despite no loss of interactions with the triple phosphomimetic SUN1 mutant, we observed a significant increase in its solubility compared with both wild-type SUN1 and a triple phospho-null mutant, together with a reduced association with the NE. This effect appeared to be largely controlled by phosphorylation of S48 since the S48D single phosphomimetic mutation had a similar effect on SUN1 localization to the NE compared with the triple mutation. Similar changes in other NE proteins have been shown to occur in response to mitotic phosphorylation: phosphorylation of lamins results in their solubilization and accumulation in the nucleoplasm during prophase,Lmna−/− cells.,Together, these findings suggest that disruption of the lamin A/C and emerin interaction may not be the major determinant of SUN1 solubilization. Indeed, lamin A/C and emerin are not the key binding partners responsible for anchorage of SUN1 at the NE since SUN1 maintains localization at the NE in cells ,In contrast to interactions mediated by the N-terminal domain of SUN1, we found that the central LINC complex SUN-KASH interaction remains intact in mitotic cells, suggesting that it plays a functional role during mitosis. In support of this, a recent study demonstrated that SUN1 and SUN2 are required for the formation of prophase nuclear invaginations (PNEIs) and for the separation of nuclear membranes from chromatin after NEBD.We also observed a novel interaction between SUN1 and NET5/Samp1 that was maintained during mitosis. The long isoforms of Samp1 have previously been shown to interact with SUN2 and are likely to contribute to nuclear movement in migrating fibroblasts.Thus, our findings indicate multiple roles for SUN1 during mitosis. We propose a model whereby phosphorylation of SUN1 on entry into mitosis disrupts interactions with nucleoplasmic binding partners, thereby promoting nuclear lamina disassembly and chromatin condensation. Loss of these interactions further enables nuclear membranes to be separated from the condensed chromatin after NEBD. On the other hand, maintenance of the LINC complex may promote dynein association with the NE in prophase, which in turn promotes timely NEBD through the action of NE-associated microtubules. This model suggests that the LINC complex, along with the NPC component Nup133,The human SUN1 (hSUN1) cDNA, encoding the 916 residue full-length protein, was generated by ligating together cDNA sequences obtained from two different sources. The sequence encoding residues 1–362 was PCR-amplified using IMAGE clone 40148216 as a template and introducing a BglII site at codon 356–357. The region encoding residues 349–916 was generated by RT-PCR amplification from U2OS cell RNA, again incorporating a BglII site at codon 356–357. The BglII site was then used to ligate the two fragments together and the complete cDNA was cloned by recombination into pLEICS-20  or pLEICS-21  mammalian expression vectors, using the University of Leicester Protex cloning service. MBP-hSUN1 fusion vectors were generated by PCR-amplification of the relevant regions of the SUN1 cDNA and cloning by recombination into pLEICS-10 . Site-directed mutagenesis, to produce phospho-null or phosphomimetic SUN1 mutants, was performed by the Protex cloning service. The pEGFP-nesprin 2α, pEGFP-nesprin 2α ΔTM, and pEGFP-nesprin 2α KASH constructs, provided by C Shanahan, have been described previously . The pEGFP-NET construct was kindly provided by E. Schirmer .Rabbit anti-human SUN1 (Atlas) and mouse monoclonal β-actin antibodies were obtained from Sigma. Mouse anti-Myc antibody was purchased from Zymed Laboratories Inc Mouse anti-cyclin B1, mouse anti-cyclin A and goat anti-lamin A/C (6215) antibodies were purchased from Santa Cruz Biotechnologies. Rabbit anti-GFP antibody was obtained from Abcam. Rabbit anti-emerin antibody was kindly provided by G. Morris . Anti-human SUN1 2383 has been reported previously.SUN1 serine 48 (pS48) phospho-antibodies were generated by immunization of rabbits with a peptide corresponding to residues 42–55 of human SUN1, phosphorylated at serine 48. Immunizations and antibody purification, using peptide columns, were performed by Eurogentec.2. Cells were transiently transfected using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. For synchronization experiments, cells were first pre-arrested in S phase by incubation for 16 h in growth medium supplemented with 1.6 µg/ml aphidicolin (Sigma). Cells were then washed with PBS and incubated with growth medium for 4 h. To arrest in mitosis, 500 ng/ml nocodazole (Sigma) was then added to the medium and the cells incubated for a further 16 h at 37 °C. Mitotic cells were collected by shake-off. Synchronization was confirmed by flow cytometry. To arrest in G2, 10 µM RO-3306  was added to the medium 4 h after release from aphidicolin and cells incubated for up to 4 h at 37 °C. For kinase inhibitor studies, the inhibitors were added to nocodazole-arrested cells 4 h prior to harvesting at the following concentrations: 100 µM roscovitine , 1 µM MLN 8054 (SelleckChem), 100 nM BI2536 (Axon Medchem) and 10 µM U0126 , along with 10 µM MG132  to prevent protein degradation.HeLa and U2OS cells were routinely grown in Dulbecco’s modified Eagle medium supplemented with 10% FBS, 1% Glutamax, and antibiotics (growth medium) at 37 °C and 5% COCells were permeabilized and fixed in ice-cold 70% ethanol then stored at -20 °C for a minimum of 24 h. Cells were then washed and stained with propidium iodide  staining solution . The cells were transferred to round-bottom tubes and incubated for 1 h at 37 °C. The DNA profile for each sample was obtained by FACS (BD Biosciences FACSCanto II) using FACSDiva 6.0 software (BD Biosciences) for acquisition and analysis.Total cell extracts for immunoblotting were prepared by resuspending trypsinized, pelleted cells in phosphate-buffered saline (PBS), followed by boiling in an equal volume of 2 × Laemmli buffer.Alternatively, soluble cell lysates were prepared by incubating the scraped and pelleted cells in lysis buffer , 2 mM AEBSF (Melford), 5 mM NaF, 50 mM β-glycerophosphate) for 30 min on ice. Samples were sonicated for 3 cycles of 15 s and 2 µm amplitude in an MSE Soniprep 150, then centrifuged at 10 000 g (20 000 g for GFP-Trap) for 10 min at 4 °C. The soluble supernatant fraction was further processed for immunoprecipitation or GFP-Trap.In some instances, soluble cell extracts were treated with lambda protein phosphatase (Sigma), according to the manufacturer’s instructions, prior to immunoblotting.2, 1% Triton X-100, 1 × complete protease inhibitor cocktail (Roche), 1 × PMSF; supplemented with either 50 [low] or 500 [high] mM NaCl). Samples were incubated on ice for 15 min and then centrifuged at 10 000 g and 4 °C. The supernatants were transferred to new tubes and boiled in an equal volume of Laemmli buffer. Pellets were resuspended in a double volume of 2 × Laemmli buffer and also boiled.To examine solubility of transiently expressed myc-SUN1 mutants, whole cell pellets were resuspended in high or low salt concentration buffers  as described previously,For co-immunoprecipitation studies, soluble lysates were prepared from untransfected HeLa cells, or those transfected with the appropriate GFP-hSUN1, myc-hSUN1, GFP-nesprin, or GFP-NET5 constructs, and immunoprecipitated as described previously  using 2 µg of hSUN1 2383, myc, or GFP antibodies. 5% of the initial lysate was retained for immunoblot analysis.SUN1 bandshifts were resolved on 6% polyacrylamide gels, while all other samples were resolved on 7.5% or 10% gels. This was followed by semi-dry transfer onto nitrocellulose membrane. Membranes were probed using the appropriate primary antibodies and dilutions: hSUN1 Atlas (1:500), lamin A/C (1:2000), emerin (1:2000), cyclin B1 (1:200), cyclin A (1:200), pS48 (1:50), myc (1:500), GFP (1:6000), and β-actin (1:50 000). Primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies (Sigma), and visualized using ECL reagents (Geneflow).t test.U20S cells were grown on coverslips, transfected with the appropriate myc-SUN1 construct and then fixed directly in ice-cold methanol for a minimum period of 10 min at -20 °C. Samples were then analyzed by immunofluorescence microscopy as previously describedProteomics was performed by the University of Leicester Proteomics Facility (PNACL), as described previously.E. coli BL21 and purification with amylose resin (New England Biolabs). For radiolabelled kinase assays, equal amounts (1–2 µg) of each MBP fusion protein, bound to amylose resin, or purified casein (Sigma) were incubated with 1 μCi of [γ-32P]-ATP in kinase buffer  and 5 μg of purified Cdk1/cyclin B or Plk1 (Merck Millipore) for 30 min at 30 °C. For non-radiolabelled kinase assays destined for MS analysis, [γ-32P]-ATP was omitted and reactions were incubated for 4 h at 30 °C. Reactions were stopped by addition of 25 μl of Laemmli buffer and analyzed by SDS-PAGE and autoradiography or further processed for LC-MS/MS analysis.MBP fusion proteins were produced by expression of the appropriate pLEICS10-hSUN1 plasmids in Protein bands from the dried Coomassie-stained gels were excised and placed in scintillation vials along with OptiPhase HiSafe scintillation fluid (Perkin Elmer). Radioactivity was measured in counts per minute (cpm) on an LS 6500 multipurpose scintillation counter (Beckman Coulter). Percentage phosphorylation for each hSUN1 variant was calculated relative to casein.Additional material


Artocarpus heterophyllous (AH) potently inhibited tyrosinase activity.Tyrosinase is an oxidoreductase that is very important in medicine and cosmetics because the excessive production of melanin causes hyperpigmentation. The development of novel, effective tyrosinase inhibitors has long been pursued. In preliminary tests, we found that an extract of the wood of 1) and artocaepin F (2), were isolated from the wood of AH, together with norartocarpetin (3), artocarpanone (4), liquiritigenin (5), steppogenin (6), and dihydromorin (7). Their structures were elucidated using one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) and mass spectrometry. The absolute configuration of 2 was determined from the circular dichroism (CD) spectrum. Artocarpanone (4) had the most potent tyrosinase inhibitory effect, with an IC50 of 2.0 ± 0.1 μM, followed by artocaepin E (1) and steppogenin (6), with IC50 values of 6.7 ± 0.8 and 7.5 ± 0.5 μM, respectively. A kinetic investigation indicated that 1 showed competitive inhibition, with an inhibition constant (Ki) of 6.23 μM.Two new flavonoids, artocaepin E  contains supplementary material, which is available to authorized users. This o-quinone is a highly reactive compound that can polymerize spontaneously to form melanin + , consistent with the molecular formula C29H24O9. The infrared (IR) spectrum suggested the presence of hydroxy (3410 cm−1), phenyl , and carbonyl  groups. The 1H NMR spectrum of 1 included signals due to two sets of ortho-coupled aromatic protons at δH 7.53  and 6.78 , three aromatic protons of the typical ABX system at δH 6.49 , 7.73 , and 6.44 , an isolated aromatic proton at δH 6.51 , two trans olefinic protons at δH 7.54  and 6.39 , and two isolated olefinic protons at δH 6.98  and 5.55 , together with one methyl, one methylene, one oxymethylene, and a characteristic signal of a hydrogen-bonded hydroxyl proton at δH 13.34   and heteronuclear multiple bond correlation (HMBC) spectra  with the C-9′″ (δC 166.5) ester carbonyl carbon of the trans-p-coumaroyl group. The nuclear Overhauser effect spectroscopy (NOESY) correlations of H-1″ with H-5″ and of H-2″ with H-4″ indicated an E-configuration for the C-2″/C-3″ double bond of 1. Therefore, the structure of artocaepin E was concluded to be 1.Compound 2) was isolated as a yellow amorphous solid. The molecular formula of 2 was determined to be C20H20O5 by HR-ESI–MS. The IR spectrum showed absorption bands of hydroxy (3400 cm−1), phenyl , and ketone (1705 cm−1) groups. The 1H NMR spectra of 2  and 6.90 , and others at δ 7.59  and 6.63 , together with two methylene signals at δH 2.70  and 3.00 , and one oxymethine at δH 5.44 , which are typical of the flavanone skeleton D25 −10.0° ; IR νmax (CHCl3) 3365, 1630, 1600, 1510 cm−1; 1H and 13C NMR (acetone-d6 500 MHz) see Table m/z 363.1224 .Artocaepin F -1 µg/mL (or µM for pure compounds). The tyrosinase inhibitory activity assay was performed as previously described by Arung et al. [l-DOPA in 0.1 M phosphate buffer pH 6.8). The assay mixture was incubated at room temperature for 7 min, and the absorbance at 475 nm was measured with a Shimadzu UV-1800 spectrophotometer. Kojic acid, a known tyrosinase inhibitor, was used as positive control. Tyrosinase inhibitory activity was expressed as the percentage inhibitory of enzyme tyrosinase in the above assay system, calculated as (1 − B/A) × 100, where A and B are the activities of the enzyme without and with test material. IC50 values were calculated from the mean values of data from four determinations.All the samples were first dissolved in DMSO and used for the actual experiment at concentrations of 100g et al. . The ass50, except that uninhibited and inhibited reactions were observed for three different concentrations of l-DOPA  at 30 °C in 0.1 M phosphate buffer pH 6.8. The dependence of absorbance (475 nm) on time was measured, and the reaction rate was calculated for all reactions (uninhibited and inhibited). Then, a Lineweaver–Burk plot was constructed, and Km and Vm values were calculated. Each measurement was performed in duplicate.The procedure for determination of the inhibition mechanism was similar to that for determination of IC1) and artocaepin F (2), together with five known compounds: norartocarpetin (3), artocarpanone (4), liquiritigenin (5), steppogenin (6), and dihydromorin (7). Regarding tyrosinase inhibition, artocarpanone (4) had the greatest inhibitory effect, followed by artocaepin E (1) and steppogenin (6). Liquiritigenin (5) also showed significant concentration-dependent inhibition. Kinetic studies indicated that the new active compound artocaepin E (1) displayed competitive inhibition. These results suggest that these compounds may serve as structural templates for the design and development of novel tyrosinase inhibitors as effective anti-browning agents in cosmetics.In this study, we identified two new flavonoids from the wood of AH, artocaepin E (


Symptomatic extensor tendon irritation is a frequent complication in volar plate osteosynthesis of distal radius fractures. It is typically caused by dorsal screw protrusion and overdrilling of the dorsal cortex. The use of self-drilling locking screws (SDLS) could overcome both causes. The practical applicability of SDLS depends on two prerequisites: (1) the feasibility of preoperative distal screw length determination, and (2) sufficient primary biomechanical stability of SDLS compared to standard locking screws (SLS).p < 0.0125).We first assessed the feasibility of preoperative screw length determination (1): Distal radius width, depth and distal screw lengths were measured in 38 human radii. Correlations between distal radius width and depth were assessed, a cluster analysis (Ward’s method and squared Euclidean distance) for distal radius width conducted, and intra-cluster screw lengths analyzed (ANOVA). The biomechanical performance of SDLS (2) was assessed by comparison to SLS in a distal radius fracture model (AO-23 A3). 75 % distal screw length was chosen for both groups to simulate a worst-case scenario. Uniaxial compression tests were conducted to measure stiffness, elastic limit, maximum force and residual tilt. Statistics comprised of independent sample t-tests and a Bonferroni correction . Three distal radius width clusters could be identified: small <34 mm; medium 34–36.9 mm; large >36.9 mm. ANOVA and Tukey post-hoc analysis revealed significantly different volar-dorsal depths (p < 0.05) for nearly all screws. (2) To assess biomechanical stability nine specimens were tested each; no significant differences were found between the SDLS and SLS groups.(1) Distal radius width and depth showed a high correlation (This feasibility study demonstrates that (1) distal radius width can be used as a predictor for distal screw length and (2) that SDLS provides mechanical stability equivalent to SLS. These results highlight the feasibility of applying SDLS screws in volar plate osteosynthesis at least in extraarticular fractures. Volar locking plate osteosynthesis is the current standard treatment for unstable distal radius fractures (DRF) , 2. AlthDorsal screw protrusion typically results from incorrect intraoperative screw length measurement. Screw length measurements are hampered by the irregular shape of the dorsal cortex and the dorsal soft tissue. A recent ultrasound study suggests that dorsal screw protrusion might occur even more often than reported. Sügün et al.  found 26However, no study has yet tried to overcome the problem of primary extensor tendon damage due to overdrilling. While the use of self-drilling locking screws (SDLS) would eliminate the necessity of pre-drilling, they require the ability to preoperatively determine distal screw length. Additionally, their biomechanical stability has to be comparable to standard locking screws (SLS).When using SDLS pre-drilling is not required and hence screw length cannot be measured directly during the operation. Therefore, distal screw length has to be determined prior to surgery. The authors are not aware of any study assessing screw length preoperatively. Preoperative screw length assessment based on posterior-anterior (pa) radiographs appears possible, if the following two prerequisites are met: First, radius width (measured from pa radiographs) correlates to radius depth, i.e. distal screw length. Second, the distal screw orientation is known. Ljungquist et al.  have shoWhile there is experience with SDLS in other medical fields , 19 theyOverall, extensor tendon irritations can only be eliminated if both dorsal screw protrusion and overdrilling of the dorsal cortex are avoided. As outlined above, preoperative distal screw length determination seems possible. If SDLS prove to have similar stability to SLS, their application could be possible and would utterly eliminate both causes of extensor tendon irritation. A further advantage of this approach would be the reduction of operation time and a possible increase in overall patient satisfaction.Part 1: Is the distal radius width a reliable predictor for distal screw length?Part 2: Do SDLS provide sufficient mechanical stability for volar plate osteosynthesis of DRFs?Therefore, we have divided this work into two parts, to test the prerequisites for the use of SDLS in DRF outlined above independently:The ethics committee of the University Hospital Munich, LMU, approved the study (LMU #409-13). 22 pairs of fresh-frozen radii were obtained from the Centre of Anatomy and Cell Biology, Medical University of Vienna, Austria. Following thawing, the radii were cut to 14 cm lengths. High-resolution peripheral computer tomography (HRpQCT) scans  were performed and bone mineral density (BMD) and bone mineral content (BMC) were calculated . SpecimeWe evaluated whether the distal radius width can serve as a reliable predictor of distal screw length if the screw orientation is known. Maximum distal radius width and depth Fig.  were meaIntra-bone linear dependency between distal radius width and depthCluster analysis for distal width to identify distal radius width groupsDescriptive screw length statistics for each screw hole within each size clusterUsing the above data, statistical analyses were performed to determine whether correlations exist between geometric dimensions and whether these can be clustered into size groups:Dorsal screw protrusion must be avoided. Therefore, the shortest screw per hole was chosen as standard length within each cluster.Sufficient biomechanical stability must be achieved. Based on previous studies a screw length of 75 % of the volar dorsal distance was deemed sufficient .As a next step we evaluated whether a standard screw length could be defined per hole and within each size cluster. The evaluation was based on two aspects:Statistics were calculated using SPSS 21.0 (IBM Company). Tests performed include the Kolmogorov-Smirnov Test, standard descriptives, Pearson correlations and a hierarchical cluster analysis using the Ward’s method and the squared Euclidean distance. Group differences were calculated using ANOVA and Tukey post hoc test (level of significance α = 0.05).In the second part of this study, we aimed to evaluate the biomechanical performance of SDLS in volar plate osteosynthesis. Therefore the primary stability of SDLS was compared to SDL using a biomechanical worst-case scenario.For biomechanical testing eleven pairs of radii were randomly picked from the original 22. These were randomized pair-wise and side-alternating into the SLS and the SDLS group respectively. The pair-wise study design intended to ensure similar age and bone quality on both groups. 75 % distal screw length was chosen for both groups simulating a worst-case screw length scenario. Based on the initial screw length measurements, 75 % distal screws length Fig. /2b; for The herein applied, standardized, best evidence biomechanical fracture model has been descried in detail previously . In shorThe embedded specimens were mounted to the material testing machine . A CMS20S ultrasound motion tracking system  was installed to assess residual fragment tilt to quantify plastic deformation. The final setup is outlined in Fig. Primary stability was assessed based on maximum force, elastic limit, stiffness, and residual distal fragment tilt. Residual tilt was computed based on the initial and final marker positions of the motion tracking system using a rigid registration according to Veldpaus et al. . The remp < 0.0125). Effect size was calculated using Cohen’s d  [Statistics were calculated using SPSS 21.0 (IBM Company). Independent sample t-tests were conducted following Shapiro-Wilk and Levene tests to verify sample normality and variance equality. Due to multiple testing, a Bonferroni correction was applied p < 0.01. Effect R2 = 0.79; p < 0.001); standard descriptive statistics for distal width, depth and distal screw length are presented in Table 38 radii with a mean age of 79 ± 12 years  were included in the final analysis. Three pair of radii had to be excluded due to previous fractures. The prerequisite of normality was met. Distal radius width and depth were highly correlated  and BMC  did not differ significantly between the two groups. Table d = 0.58) [Of the eleven pairs of radii selected for biomechanical testing two pairs had to be excluded due to previous fractures. The remaining 9 pairs with a mean age of 71 ± 8 years  were tested successfully. Normality and variance equality criteria were met. BMD (237 ± 72 vs. 233 ± 76 mgHA/cm = 0.58) .Table 2GFor the first time, this study demonstrated the feasibility of SDLS volar plate osteosynthesis for DRF. Both prerequisites, i.e. screw length estimation based on distal radius width and primary stability of SDLS were fulfilled. First, three distal radius width clusters were identified and the definition of a standard screw length for each screw hole in each group appeared possible. Second, SDLS provided equal primary stability in volar locking plate osteosynthesis compared to SLS.Based on the data presented, the definition of group- and screw hole specific standard screw lengths appears feasible. When applying the “safe screw length corridor” concept, only seven out of 228 distal screws would have fallen short of the 75 % lower bound  may oversimplify the biomechanical situation; however, it is the method typically employed in biomechanical testing and eliminates confounding factors , 32, 33.Out tests show that SDLS provide sufficient primary mechanical stability in volar locking plate osteosynthesis. Additionally, distal radius width appears to be a valid predictor for screw hole specific standard distal screw length. While a larger sample size might be necessary to refine the distal radius width groups, the results obtained from this limited set give a first orientation. The results of this study highlight the feasibility of applying SDLS in volar plate osteosynthesis for the distal radius at least for extraarticular fractures saving not only operation time, but also eliminating the causes of extensor tendon irritations.


Varroa mites are a serious pest of honey bees and the leading cause of colony losses. Varroa have relatively low reproductive rates, so populations should not increase rapidly, but often they do. Other factors might contribute to the growth of varroa populations including mite migration into colonies on foragers from other hives. We measured the proportion of foragers carrying mites on their bodies while entering and leaving hives, and determined its relationship to the growth of varroa populations in those hives at two apiary sites. We also compared the estimates of mite population growth with predictions from a varroa population dynamics model that generates estimates of mite population growth based on mite reproduction. Samples of capped brood and adult bees indicated that the proportion of brood cells infested with mites and adult bees with phoretic mites was low through the summer but increased sharply in the fall especially at site 1. The frequency of capturing foragers with mites on their bodies while entering or leaving hives also increased in the fall. The growth of varroa populations at both sites was not significantly related to our colony estimates of successful mite reproduction, but instead to the total number of foragers with mites (entering and leaving the colony). There were more foragers with mites at site 1 than site 2, and mite populations at site 1 were larger especially in the fall. The model accurately estimated phoretic mite populations and infested brood cells until November when predictions were much lower than those measured in colonies. The rapid growth of mite populations particularly in the fall being a product of mite migration rather than mite reproduction only is discussed. Varroa destructor Anderson & Trueman, is a serious pest to honey bees and a major cause of colony losses worldwide  substantially contributes to the mite population in colonies, there should be significant correlations between FWM and both the proportion of infested brood cells and adult bees with phoretic mites. Furthermore, if actual estimates of infested brood cells and adult bees with phoretic mites are significantly higher than predictions generated by the honey bee colony-varroa population dynamics model this would suggest that FWM are entering hives from other colonies and contributing to the unexpected increases in mite populations.Colonies were established from package bees in April of 2014 at two locations: the University of Arizona West Agriculture Facility, Tucson, AZ, USA (site 1) and the Red Rock Agricultural Facility, Red Rock, AZ, USA (site 2). Twenty hives were established at site 1 and 21 hives at site 2. The sites were 48 km apart. These areas are in mid-altitude desert climates  where temperatures are conducive for honey bee flight until November (average November min–max: 8 and 23 °C). All colonies initially contained approximately 9000 adult bees and a laying queen. There were managed colonies in nearby apiaries at both sites. Varroa were controlled in nearby apiaries with various commercial miticide treatments to prevent colony loss.Frames of bees and brood were measured monthly in all hives at both sites from May until November using methods described in DeGrandi-Hoffman et al. , 2014. AInitial populations of phoretic mites were estimated in all hives 5 days after the colonies were established and then monthly afterwards until November. The initial estimate occurred before there was sealed brood in the colonies. Phoretic varroa populations were estimated using the ‘sugar shake’ method. We brushed approximately 300 bees into glass jars with wire screen lids. Powdered sugar (0.5–1.0 tablespoons) was added to each jar through the wire screen. The jar was rolled gently to cover the bees and then set aside for 2–3 min. The jars were inverted and shaken vigorously over a white aluminum pan containing 2.5 cm of water until there was no sugar left in the jars. The mites were counted in the pan, and the bees were placed back in each colony. The mite counts were converted to ‘mites per 100 bees’ , and recorded the proportion with a foundress mite . These were classified as ‘infested cells’. We recorded the number of infested cells that had a male mite and adult females that were fully developed but not completely pigmented to estimate the proportion of infested cells with successful reproduction. Cells containing deutonymphs or protonymphs were not included because the mites would not have been mature when the bee emerged. The number of fully formed female offspring in each cell was totaled as were the number of foundress mites to estimate the number of offspring per foundress.Colony entrances were modified so that foragers exited and entered the hive through a 4.76-cm diameter PVC tube. The tube was 61 cm long. A slit was cut across the tube at the midpoint. During each sampling interval, a screen was dropped into the slit within the tube to separate foragers entering and leaving the hive . An additional repeated measures analysis was used to compare the populations of FWM between sites. In all cases, repeated measures analyses were run using JMP . Multiple regressions were conducted to test for relationships between phoretic mite population growth and colony size (frames of bees and brood), proportion of infested cells, and the proportion of incoming and outgoing FWM (incoming FWM + outgoing FWM). Separate Pearson correlation analyses were used to test for relationships between FWM (incoming + outgoing) and phoretic mites and infested cells at each site. The null hypothesis in all cases was that the correlation coefficient was zero. Actual and predicted phoretic mites per 100 bees and the proportion of infested cells were compared for each sample interval using Student’s t tests. Regression and correlation analyses, t tests and all descriptive statistics were run using Minitab . All reported means are ±SE.Multivariate repeated measures analyses were used to compare colony population sizes (adult bees and frames of brood), average numbers of phoretic mites per 100 bees and the percentage of infested brood cells in colonies between sites. Data were non-linear and were log1,24 = 1.97, p = 0.17) though the interaction term (time × site) was significant . Colonies at the two sites differed in frames of brood . The interaction term (time × site) also was significant .Colonies at site 1 grew to populations of about 19,000 ± 1377 adults (7.8 frames of bees) and 5.5 ± 0.3 frames of brood in September Fig. . PopulatAt site 1, we detected phoretic mites in 2 of the 20 colonies at the start of the study. In each case, the sample contained 0.33 mites per 100 bees. From June to September, mite numbers remained at <1.0 mite per 100 bees . At the start of the study, we detected varroa in four of the 21 colonies. Each of the four colonies had one mite per 100 bees. We continued to find one mite per 100 bees in less than half the colonies until October when we found mites in all but one hive. In October, colonies averaged 0.48 ± 0.09 mites per 100 bees or 128 ± 30.8 phoretic mites per colony. By November, we detected mites in all colonies with an average of 2.0 ± 0.6 mites per 100 bees or 418 ± 122 phoretic mites per colony.Estimates of mites per 100 bees were significantly lower at site 2 compared with site 1 . Mites successfully reproduced a fully developed daughter mite in 16 % of the infested cells at site 1 and 8 % at site 2. We did not find any cells where the foundress produced more than a single fully developed daughter mite in the infested cell. During each sampling interval, we examined on average 1300 cells among the colonies at site 1 and 1024 at site 2.Worker cells containing foundress mites were not detected in samples at either site in May or June Fig. . In July1,65 = 0.115, p = 0.73) or between morning and afternoon samples . Thus, the likelihood of collecting FWM was not due to differences in the number of foragers we sampled.At site 1, there was an average of 30–70 foragers captured either entering or leaving colonies during each sampling interval. There was no significant difference in the number of incoming and outgoing foragers during sampling intervals at either site . However, more FWM were captured in afternoon than morning samples . At site 1, we collected significantly more FWM in October and November than in August or September . The likelihood of capturing FWM at site 2 was slightly but not significantly greater in fall months than in summer . Overall, there were fewer FWM at site 2 than at site 1 .We rarely collected FWM in August at either site Fig. . In Septp) for FWM and phoretic mites per 100 bees were: rp = 0.81, p < 0.0001 for site 1, and rp = 0.61, p < 0.0001 for site 2. The proportion of cells infested with mites estimated at each sampling interval also was significantly correlated with FWM at site 1 , but not at site 2 .Separate analyses were conducted using data from each site to identify factors affecting mite population growth . Mites per 100 bees were significantly related to colony growth (frames of adult bees and brood) and the total incoming and outgoing FWM Table . Pearson19 = 4.24, p < 0.0001) . November predictions also were lower than actual infestation levels, though the differences were not significant . During October and November, predicted and actual colony sizes (adult bees and frames of brood) did not differ significantly  Fig. . Predict10 = 1.57, p = 0.15). Predictions of infested cells at site 2 were similar to observed for the entire study period. As at site 1, predicted and actual colony sizes did not differ in October and November.Predicted mites per 100 bees at site 2 were similar to observed until November when predictions were lower than observed. However, the differences were not significant  were much lower than observed in colonies. The combination of field data and simulations suggest that FWM were drifting in from other colonies and contributing to the growth of varroa populations in our colonies particularly in the fall.In our study, the proportion of adult bees with phoretic mites was not related to cell infestation levels, but instead to the proportion of FWM. The levels of infested cells and successful reproduction we measured were low, and if they were underestimated, we also would underestimate the relationship between reproduction and mite population growth. Therefore, the data analysis should be interpreted cautiously. Reproductive rates might have been underestimated because we did not examine enough cells per colony. We counted mites in 80–100 cells during each sampling interval except in November when brood of suitable age was limited and we averaged about 60 cells per colony. Others recommend 200 cells per colony  and numbers of offspring (1.5 fully developed daughter mites). At site 1 where FWM were consistently higher than site 2, actual estimates of the percentage of infested cells were similar to predictions until September. After this, actual infestation levels were much greater than predicted even though the predictions were based on higher rates of reproductive success. At site 2, there were fewer FWM, and estimates of infestation levels and phoretic mite populations were similar to the predicted for the entire study period. The contrast between results at the two sites might be related to differences in the frequency of capturing FWM and their contribution to mite population growth.The proportion of FWM was similar between those entering and leaving the hive. These results differ from Kralj and Fuchs  where thWe cannot be certain if the FWM we collected were from the same colony where the foragers originated or were from other colonies. However, if the FWM originated from the same colony we sampled and numbers of FWM entering and leaving the hive were not significantly different, we should not have seen an increase in the overall mite population or discrepancies between observed and predicted varroa populations in the fall. Furthermore, when mite populations are low as they were in our colonies (<10 % adult workers with mites), mites attach to nurse bees that they can distinguish from foragers by different cuticular chemical signatures and remain within the colony (Cervo et al. Increases in phoretic mite populations in the fall were expected since brood rearing is declining at this time and fewer brood cells are available to infest. The model also generates an inverse relationship between phoretic mites and brood rearing in the fall. Adding to the phoretic mite population can be an influx of mites on foragers robbing colonies that are collapsing due to high mite populations (Sakofski et al. The transfer of mites to foragers is a shift in the mite behavior from attaching to nurse bees for reproduction Kraus  to forag


Drosophila and mammals. Initially, we provide a general overview of the cellular functions of NDR kinases in cell cycle progression, centrosome biology, apoptosis, autophagy, DNA damage signalling, immunology and neurobiology. Finally, we put particular emphasis on discussing NDR1/2 as YAP kinases downstream of MST1/2 and MOB1 signalling in Hippo signalling.The Hippo tumour suppressor pathway has emerged as a critical regulator of tissue growth through controlling cellular processes such as cell proliferation, death, differentiation and stemness. Traditionally, the core cassette of the Hippo pathway includes the MST1/2 protein kinases, the LATS1/2 protein kinases, and the MOB1 scaffold signal transducer, which together regulate the transcriptional co-activator functions of the proto-oncoproteins YAP and TAZ through LATS1/2-mediated phosphorylation of YAP/TAZ. Recent research has identified additional kinases, such as NDR1/2  and MAP4Ks, which should be considered as novel members of the Hippo core cassette. While these efforts helped to expand our understanding of Hippo core signalling, they also began to provide insights into the complexity and redundancy of the Hippo signalling network. Here, we focus on summarising our current knowledge of the regulation and functions of mammalian NDR kinases, discussing parallels between the NDR pathways in  From proaches . CumulatNeurospora crassa [Trypanosoma brucei, the causative agent of human African trypanosomiasis [Ndr1/2 double knockout mouse embryos display multiple phenotypes including defective somitogenesis and cardiac looping, resulting in a developmental delay from embryonic Day 8.5 (E8.5) onwards, followed by embryonic lethality around E10 [The first NDR serine/threonine kinase, termed Dbf2p, was discovered in budding yeast , followea crassa  and humaa crassa , while Ta crassa . Notewora crassa ,15. Basea crassa . In thissomiasis , and gensomiasis . Furtheround E10 . Therefoin vivo functions of mammalian NDR1/2 kinases, but extensive biochemical studies have been carried out to understand the molecular regulation of NDR1/2 kinases, which up to recently [www.phosphosite.org). However, the functional significance of these additional post-translational modifications is yet to be established.Initially, little was known about the recently ,19 had trecently . In summrecently ,20,21). recently , the mutrecently , membranrecently , and thrrecently ,26. Therrecently  and ubiqExperiments using tissue culture cells indicate that NDR1/2 can play diverse roles in processes associated with the mammalian cell cycle. Through the regulation of c-myc and p21/Cip1 protein levels, NDR1/2 have been linked to the regulation of G1/S cell cycle progression ,29,30,31Current reports suggest that NDR1/2 can function as pro-apoptotic kinases downstream of Ste20-like kinases ,50,51,52βTrCP E3 ligase [By competing with MOB1 for binding to NDR1/2, MOB2 can interfere with NDR1/2 activation . Since M3 ligase , the reg3 ligase ,64, a pr3 ligase  and the 3 ligase .Ndr1 knockout mice are prone to develop T cell lymphomas, possibly due to defective apoptotic signalling in T lymphocytes [Ndr1 knockout mice further found that NDR1 contributes to the innate immune response of macrophages [Ndr1/2, Tang et al. found that NDR1/2 play important roles in thymocyte egress and migration [Initially, it was observed that the HIV-1 virus associates with NDR1/2 , howeverphocytes . An inderophages . In addiigration .Drosophila sensory neurons [et al. could demonstrate that NDR1/2 are also important for dendrite growth and spine development in mammals by phosphorylating AAK1 and Rabin8 [Members of the NDR family play key roles in the morphogenesis of polarised structures in yeast, worms and flies , suggest neurons , functio neurons  and TORC neurons . Subsequd Rabin8   and Mats are the orthologues of mammalian YAP/TAZ and MOB1, respectively. Since Trc/Warts double mutant cells displayed additive phenotypes combining the overgrowth phenotype  and the wing hair phenotype , it was proposed that the Trc and Warts pathways function in parallel in fly wing cells [diap1, cyclin E and expanded [et al. concluded that Trc does not regulate Yki in the fly wing [In ng cells . Neverthng cells . A seconng cells ,81, a phng cells . A thirdng cells . Althougng cells , Trc mutexpanded . Thus, Ffly wing , althouget al. addressed this possible connection experimentally, resulting in the discovery that mammalian NDR1/2 can function as YAP kinases [bona fide upstream kinases of YAP in vitro. Significantly, NDR1/2-mediated phosphorylation of YAP on Ser127 resulted in the inactivation of YAP by cytoplasmic retention [Ndr1 and Ndr2 in the murine intestinal epithelium resulted in elevated total YAP protein levels accompanied by decreased Ser127 phosphorylation without any alterations in LATS1/2 phosphorylation (either on the T-loop or hydrophobic motifs) and total LATS1/2 protein levels [in vivo as judged by nuclear accumulation of YAP and elevated YAP target gene expression [Yap in Ndr1/2 double knockout tissue resulted in decreased tumour incidences upon chemically-induced colon carcinogenesis [In mammalian cells, unlike LATS1/2 overexpression, the overexpression of NDR1/2 did not result in a phospho-shift of YAP . Thus, i kinases . They fo kinases ,82, hencetention , as repoetention ,77, thern levels . Signifipression . Moreoveogenesis , suggestogenesis  establisLats1/2 and Ndr1/2 knockout cell lines. Currently we know that Ser127 phosphorylation of YAP is abolished, while total YAP phosphorylation still occurs to a significant extent in Lats1/2 double knockout mouse embryonic fibroblasts [Lats1/2 double knockout HEK293 cells Ser127 and total YAP phosphorylation is dramatically diminished in different tissue culture conditions [Considering that the regulation of YAP by LATS1/2 has also been established on the biochemical, cellular and organismal levels ,77,82,83roblasts . In humanditions . Howevernditions . In othenditions , but whenditions , the depin vivo, while current evidence already suggest that MST1/2 function as upstream kinases of NDR1/2 in vivo in murine T cells [Ndr1/2 results in intestinal epithelium hyperplasia [Mst1/2 knockout mice [Ndr1/2 double null mouse embryos display multiple phenotypes [Mst1/2 double null mouse embryos [Lats1/2 and Mst1/2 knockout mice [in vivo.As already mentioned, Hippo, the fly MST1/2 kinase, can act upstream of Trc and Warts in fly neurons . Specifi T cells . Moreoveerplasia  as obserout mice ,89, and enotypes  that are embryos ,91,92. Tout mice ,91,92,93Drosophila, respectively [Map4k4 deletion in HEK293 cells does not affect YAP phosphorylation [Map4k1/2/3/4/6/7 and Mst1/2 genes), suggesting that another upstream kinase of LATS1/2 is yet to be identified [Intriguingly, NDR1/2 and LATS1/2 are also regulated by other members of the Ste20-like kinase family besides MST1/2. First, MST3 can act as a hydrophobic motif kinase of NDR1/2 ,94, whilectively ,8,9. Morectively  and MAP4ectively . Howeverrylation , hence Grylation . Neverthi.e., multiple wing hair cells) and of Warts loss-of-function , strongly suggesting that in flies Mats is required for Trc as well as Warts signalling [In flies, both Trc and Warts are regulated by Hippo, a key member of the Hippo core cassette see  above. Ignalling .Mob1a and Mob1b genes [et al. found that Warts/Mats complex formation is essential for Warts activation, while the stable interaction with Hippo appears to be dispensable. In this context, one should further note that the MST1/2-mediated phosphorylation of MOB1 on Thr12 and Thr35 is required to support the formation of NDR/MOB1 and LATS/MOB1 complexes [In mammalian cells, MOB1, encoded by the distinct 1b genes , can int1b genes ,99,100, 1b genes ,101,102.1b genes ,47 and M1b genes ,18, alth1b genes  that chaomplexes . In addiomplexes ,87, an eomplexes ,106.Mob1a/b knockout mice have revealed that MOB1 loss-of-function results in the most severe deregulation of the Hippo pathway in vivo [Mob1a/1b knockout mice. Therefore, future investigations are warranted to dissect the biological importance of MOB1 interactions with NDR1/2 and LATS1/2 in Hippo signalling.Recent studies of  in vivo ,108, henAs summarised in As already summarised above, the NDR and LATS pathways are regulated through molecular mechanisms involving different members of the Hippo core cassette, which influence the phosphorylation status of NDR1/2 and LATS1/2. Interestingly, these similarities can be further extended to additional regulators of NDR1/2 and LATS1/2. For example, the RASSF1A (RAS association domain family 1A) tumour suppressor can trigger apoptotic signalling through MST1/2-mediated activation of LATS1/2 ,111,112 Neurospora crassa, the NDR kinase COT1 is also regulated by interacting with a MOB co-activator that supports T-loop auto-phosphorylation and by hydrophobic motif phosphorylation mediated the Ste20-like kinase POD6 [Taken together, based on the current evidence, one should consider the mammalian NDR1/2 kinases as novel members of the Hippo core cassette . A key remaining question is to investigate in which other cell types and contexts NDR1/2 may function as YAP kinases downstream of Hippo core components such as MST1/2 and MOB1. In this regard, it might also be worth assessing whether Trc functions as a Yki regulator in specific fly tissues. Another interesting question will be to understand whether the regulation of the Hippo core cassette involving NDR1/2 is conserved throughout all eukaryotes. For example, like human NDR1/2 ,25,116, ase POD6 . HoweverAn additional key question should be the investigation of the potential regulatory crosstalk between the NDR and LATS pathways. Intriguingly, fly genetics could show that heterozygosity of a Warts mutant can enhance the Trc dominant negative phenotype in fly wings . Moreove


Substrate dephosphorylation by the cyclin-dependent kinase (Cdk)-opposing phosphatase, Cdc14, is vital for many events during budding yeast mitotic exit. Cdc14 is sequestered in the nucleolus through inhibitory binding to Net1, from which it is released in anaphase following Net1 phosphorylation. Initial Net1 phosphorylation depends on Cdk itself, in conjunction with proteins of the Cdc14 Early Anaphase Release (FEAR) network. Later on, the Mitotic Exit Network (MEN) signaling cascade maintains Cdc14 release. An important unresolved question is how Cdc14 activity can increase in early anaphase, while Cdk activity, that is required for Net1 phosphorylation, decreases and the MEN is not yet active. Here we show that the nuclear rim protein Nur1 interacts with Net1 and, in its Cdk phosphorylated form, inhibits Cdc14 release. Nur1 is dephosphorylated by Cdc14 in early anaphase, relieving the inhibition and promoting further Cdc14 release. Nur1 dephosphorylation thus describes a positive feedback loop in Cdc14 phosphatase activation during mitotic exit, required for faithful chromosome segregation and completion of the cell division cycle. During the cell cycle, a specific sequence of events leads to the formation of two daughter cells from one mother cell. Progression through the cell cycle is tightly controlled, with events occurring in the right place at the right time. Exactly how this is achieved is still being elucidated. In budding yeast, the events occurring during the final cell cycle phase – “mitotic exit” – are controlled by the phosphatase Cdc14. It is kept sequestered and inactive until it is needed for mitotic exit, at which time it is rapidly released. In this study, we have identified a new regulator of Cdc14 activity, the protein Nur1. In a series of experiments, we saw that Nur1 acts both upstream and downstream of Cdc14 activation, thereby creating a positive feedback loop. On the one hand, Nur1 contributes to inhibiting Cdc14 until the start of mitotic exit. On the other hand, through the actions of Cdc14 itself, Nur1 is disabled as an opponent of the phosphatase. This creates a robust system, rapidly switching between two opposing states and thus driving forward the mitotic exit transition. Saccharomyces cerevisiae, the Cdk subunit Cdc28 associates with a series of cell cycle stage-specific cyclins to bring about Cdk activity. It is opposed by the main Cdk-counteracting phosphatase Cdc14, which reverses Cdk phosphorylation events during mitotic exit Cellular reproduction is a highly regulated process that is controlled on a multiplicity of levels, ensuring orderly progression through the different phases of the cell cycle and accurate partitioning of the genome. At the heart of eukaryotic cell cycle control lie cyclin-dependent kinases (Cdks) and their opposing phosphatases Cdc55, which keeps Net1 under-phosphorylated. At anaphase onset, the protease separase is activated after APC-mediated destruction of its inhibitor securin. Separase now cleaves the chromosomal cohesin complex to trigger sister chromatid segregation. At the same time, separase uses a non-proteolytic activity to downregulate PP2ACdc55Cdc14 activity is stringently regulated. During most cell cycle phases, Cdc14 is sequestered in the nucleolus, and thus inactive, through inhibitory binding to Net1 While Cdc14 release during the early stages of anaphase depends on mitotic Cdk activity cdc14 mutants display rDNA segregation failure despite unobstructed cohesin cleavage. How Cdc14 promotes rDNA segregation is still being debated. Condensin is recruited to the rDNA in anaphase in a Cdc14-dependent manner, where it appears to promote decatenation of the locus, making the condensin complex a prime candidate for Cdc14 regulation Timely Cdc14 activation in early anaphase is important for successful chromosome segregation. It is required to stabilize the anaphase spindle and is also required for completion of chromosome segregation, in particular telomeres and the late segregating rDNA locus nuclear rim protein Nur1 as a Cdc14 substrate. Failure to dephosphorylate Nur1 causes rDNA missegregation, however, this turns out to be the consequence of compromised Cdc14 activation rather than a specific rDNA segregation defect. This leads us to discover that Nur1 has a previously uncharacterized role in Cdc14 inhibition, and that its inhibitory activity is phosphorylation-dependent. Constitutive Nur1 phosphorylation delays Cdc14 activation, while non-phosphorylatable Nur1 causes premature Cdc14 activation. Thus, Cdc14-dependent Nur1 dephosphorylation in early anaphase forms a positive feedback loop to promote further Cdc14 release, with important implications for faithful chromosome segregation.In this study, we take advantage of our recent phosphoproteome analysis of budding yeast mitotic exit In the search for Cdc14 targets that promote rDNA condensation and segregation during anaphase, we reviewed the phosphoproteome of budding yeast mitotic exit. Cells were arrested in metaphase by depletion of the APC coactivator Cdc20, then synchronous mitotic exit progression was induced by ectopic expression of the Cdc14 phosphatase. Mass spectrometry was used to survey the disappearance of phosphopeptides over the course of mitotic exit, with the original intention to identify proteins whose dephosphorylation controls cytokinesis Among the proteins that were dephosphorylated in response to Cdc14 expression, in addition to cytokinesis regulators, we identified the nuclear rim protein Nur1 . Along wcdc14-1 allele cdc14-1 strain, dephosphorylation was no longer observed and Nur1 accumulated in slow migrating forms in the late mitotic arrest. To confirm that the mobility shift observed on the Phos-tag gels is indeed due to Nur1 phosphorylation, Nur1 was immunoprecipitated from cells arrested in mitosis by nocodazole treatment and incubated in a control buffer, or in the presence of either λ-phosphatase or purified recombinant Cdc14 To confirm cell cycle-dependent Nur1 phosphorylation, and the role of Cdc14 in its dephosphorylation, we monitored the electrophoretic mobility of Nur1 using Phos-tag gels during synchronous cell cycle progression. Cells were arrested in G1 by pheromone α-factor treatment, before release to progress through the cell cycle at 35.5°C and either rearrest in the next G1 phase by readdition of α-factor, or arrest in late mitosis following Cdc14 inactivation using the temperature sensitive NUR1 gene at its genomic locus with a mitotic cyclin Clb2 tagging cassette. Clb2 is modified to lack localization and destruction signals, so as not to interfere with Nur1 function. After gene targeting, NUR1 remains initially separated from the cyclin tag by a selectable marker, flanked by loxP recognition sites for the Cre recombinase. The marker is then removed through the action of β-estradiol-activatable Cre-ER recombinase, following hormone addition to the growth medium. As a control, a similar tag was created in which Clb2 harbors three additional point mutations that prevent it from interacting with and recruiting the Cdc28 kinase subunit -Clb2 strain, rDNA segregation started at a spindle length of 5–6 µm and was complete by the time spindles reached 8 µm in length. In contrast, in Nur1-Clb2 cells, rDNA segregation only started at spindle lengths of 7–8 µm and never reached completion even when spindles were fully elongated . CytologADE2 marker within the rDNA repeats ADE2 loss causes accumulation of a red intermediate metabolite in the adenine biosynthesis pathway, thus causing the colony color to turn red. ADE2 loss during the first cell division after plating on agar medium will generate half red-sectored colonies. We therefore counted the fraction of half red-sectored colonies, among all colonies, as a measure for the ADE2 loss rate from the rDNA. The loss rate was approximately five-fold elevated in the Nur1-Clb2 strain at 25°C, compared to a wild type and Nur1-Clb2ΔCdk controls  cells. Strikingly, Cdc14 was released in over half of nur1Δ and nur1(9A) cells in early anaphase at spindle lengths below 3 µm, when Cdc14 release is almost never seen in wild type cells (nur1Δ and nur1(9A) cells displayed released Cdc14  alleles into a spo12Δ strain, lacking a key component of the FEAR network spo12Δ background, Cdc14 release is delayed until spindles reach about 6 µm in length. Deletion of nur1, or its replacement with nur1(9A), restored Cdc14 early anaphase release (nur1Δ spo12Δ and nur1(9A) spo12Δ double mutant strains approached that of wild type cells. However, at short spindle lengths, nur1Δ spo12Δ and nur1(9A) spo12Δ cells still showed premature Cdc14 release, as compared to wild type, while at longer spindle lengths the rescue did not fully match wild type release levels. These findings confirm that phosphorylated Nur1 is a potent inhibitor of Cdc14 release in early anaphase and that the FEAR network acts to overcome Cdc14 inhibition by Nur1. However, the incomplete rescue of Cdc14 release in spo12Δ cells by Nur1 ablation suggests that the FEAR network acts at least in part by a mechanism different from inactivating Nur1.Cdc14 activation is promoted by the FEAR network in early anaphase. To further study the impact of Nur1 on Cdc14 release at this time, we introduced the  release . The Cdcnur1Δ and nur1(9A) strains. Despite the advanced Cdc14 release in the latter strains, the timing of progression through mitosis, as measured by the fractions of cells displaying metaphase and anaphase spindles at each time point, was indistinguishable from wild type  restored the kinetics of mitotic progression close to wild type  that govern rDNA condensation, resolution and segregation in anaphase therefore remain elusive, we have uncovered a previously unknown Cdc14 substrate that acts to sustain Cdc14 activation, thus creating positive feedback of Cdc14 release in early anaphase .While inhibition of Cdc14 release by Nur1 is normally restricted to early anaphase while Nur1 is phosphorylated, our Nur1-Clb2 fusion likely extends that period, potentially throughout mitotic exit. Nur1-Clb2 may thus elicit phenotypic consequences beyond the delayed Cdc14 release characteristic of FEAR network mutants. An example of a difference is the temperature sensitive growth of Nur1-Clb2 cells, which is not typically shared by FEAR network mutants. A higher temperature causes cells to progress faster through the cell cycle, presumably rendering them less tolerant to delays or alterations to cell cycle signaling pathways. Phenotypes of late mitotic exit mutants, in particular those involved in cytokinesis, are known to be exacerbated at high temperatures Intriguingly, defective Cdc14 activation in Nur1-Clb2 cells caused not only a mitotic exit delay, but also a delay at the metaphase to anaphase transition. A small amount of Cdc14 is likely to be active by the time of metaphase, when it contributes to dephosphorylation of proteins like Dsn1 and possibly Spo12 Our results demonstrate that Nur1, through regulating Cdc14, plays an important role in promoting the accurate timing of mitotic exit events. Nur1 was previously characterized as chromosome linkage protein, connecting chromatin and the inner nuclear membrane, with functions in the maintenance of genome stability and replicative life span nur1Δ cells Constitutive Nur1 phosphorylation, caused by the Nur1-Clb2 fusion, increased marker loss at the rDNA, likely due to unequal sister chromatid exchange. A similar level of marker loss at the rDNA was previously reported in net1-1 mutants is a high rate of chromosome loss Nur1 interacts with Net1 and, in addition to Net1's role as a Cdc14 inhibitor, one of the phenotypes reported for nur1Δ), or its Cdk phosphorylation sites (nur1(9A)), resulted in premature Cdc14 release. Phosphorylated Nur1 therefore fulfills a role in attenuating Cdc14 activation in early anaphase. In a quantitative model in which Cdc14 substrate dephosphorylation timing is determined by the ratio of phosphatase activity versus Cdk kinase activity Removing Nur1  mutant was engineered by endogenous gene replacement using an integrative plasmid, based on a synthetic DNA construct . The conditional Nur1-Clb2 and Nur1-Clb2ΔCdk fusions were created as described All strains were of the W303 background and are listed in cdc14-1 or NUR1-CLB2 backgrounds, cultures were shifted to the higher temperatures at the time of release from α-factor arrest.Yeast cultures were grown in rich YP medium supplemented with 2% glucose 2 according to the manufacturer's instructions.Protein extracts for Western blotting were prepared following cell fixation using trichloroacetic acid, as described 2, 10% glycerol, 0.25% Triton X-100, 1 mM DTT, protease inhibitors) using glass bead breakage in a Multi Bead Shocker (Yasui Kikai). Extracts were precleared, incubated with antibody and finally adsorbed to Protein A Dynabeads. Beads were washed and elution was carried out in SDS-PAGE loading buffer. For the in vitro Nur1 dephosphorylation assay, immunoprecipitation was performed as above, then beads were resuspended in phosphatase buffer and 1 µg λ phosphatase (New England Biolabs), or 8 µg purified recombinant Cdc14 For immunoprecipitation, cell extracts were prepared in EBXG buffer , α-Tub1  and FITC and Cy3-dye labeled secondary antibodies . Cells were counterstained with the DNA binding dye 4',6-diamidino-2-phenylindole (DAPI). Fluorescent images were acquired using an Axioplan 2 imaging microscope (Zeiss) equipped with a 100x (NA = 1.45) Plan-Neofluar objective and an ORCA-ER camera (Hamamatsu). Spindle length measurements were carried out in ImageJ.S1 FigA. rDNA segregation is delayed in Nur1-Clb2, as compared to Nur1-Clb2ΔCdk cells. Completion of rDNA segregation is plotted as a function of spindle length. B. Progression through mitosis is delayed in Nur1-Clb2 cells, as seen by FACS analysis of DNA content of cultures passing through a synchronous cell cycle following α-factor arrest and release at 25°C. C. Cells from the timecourse shown in B were processed to visualize spindle microtubules by indirect immunofluorescence using an antibody against α-tubulin. A mitotic exit delay in Nur1-Clb2 cells is manifest by the longer persistence of cells containing elongated anaphase spindles.An rDNA segregation and mitotic exit delay in Nur1-Clb2 cells at a permissive temperature of 25°C. (TIF)Click here for additional data file.S2 FigA. cell cycle progression by FACS analysis of DNA content. B. levels of the cell cycle markers Clb2, Sic1 and Orc6 by Western blot analysis, and C. the percentages of cells displaying metaphase (1–3 µm) or anaphase (>3 µm) spindles. 100 cells were counted at each time point.Nur1 phosphorylation is responsible for the mitotic exit delay seen in Nur1-Clb2 cells. Nur1-Clb2 and Nur1(9A)-Clb2 cells were synchronized in G1 by α-factor treatment and released to progress through the cell cycle at 36°C, before being rearrested in the following G1. At time points throughout the cell cycle we monitored, (TIF)Click here for additional data file.S3 FigA. Progression through mitosis of wild type, nur1Δ, net1-6cdk and nur1Δ net1-6cdk cells was measured by counting percentages of cells displaying metaphase (1–3 µm) or anaphase (>3 µm) spindles during synchronous cell cycle progression following α-factor arrest and release. B. Quantification of Cdc14 release versus spindle length, as in Nur1's effect on Cdc14 is dependent on Net1's phosphorylation status. (TIF)Click here for additional data file.S1 TableList of yeast strains used in this study.(DOCX)Click here for additional data file.


However, the exact physiological function of β2ARs in cardiomyocytes is unknown. In this study, we showed that neonatal mouse cardiomyocytes had different contraction and internalization responses to sustained or repeated, transient agonist stimulation. Specifically, short-time stimulation (10 min) with epinephrine or norepinephrine increased the cardiomyocyte contraction rate, reaching a maximum at 5 min, followed by a slow decline. When the agonist was re-added after a 60-min wash-out period, the increase in the cardiomyocyte contraction rate was similar to the initial response. In contrast, when cardiomyocytes were exposed continuously to epinephrine or norepinephrine for 60 min, the second agonist stimulation did not increase the contraction response. These results indicated that continuous β2AR stimulation caused functional desensitization. Phosphorylation of β2ARs at serine (Ser)355/356 GRK phosphorylation sites, but not at Ser345/346 PKA phosphorylation sites increased with continuous epinephrine stimulation for 60 min. Accordingly, β2AR internalization increased. Interestingly, β2AR internalization was blocked by mutations at the GRK phosphorylation sites, but not by mutations at the PKA phosphorylation sites. Furthermore, inhibition of β2AR dephosphorylation by okadaic acid, a phosphatase 2A inhibitor, impaired the recovery of internalized β2ARs and reduced the cardiomyocyte contraction rate in response to epinephrine. Finally, epinephrine treatment induced the physical interaction of β-arrestin with internalized β2ARs in cardiomyocytes. Together, these data revealed the essential role of the Ser355/356 phosphorylation status of β2ARs in regulating receptor internalization and physiological resensitization in neonatal cardiomyocytes to contraction functions.Previous studies have demonstrated that β Dysregulation of β2ARs is associated with heart failure, asthma, and other diseases [2ARs induces receptor binding to G proteins followed by dissociation of the Gα and Gβγ subunits. β2ARs couple to both Gs and Gi proteins that regulate adenylyl cyclase generating cAMP to activate protein kinase A (PKA), which phosphorylates downstream proteins, thus mediating various physiological functions [βdiseases –3. Activunctions .2ARs leads to rapid receptor phosphorylation by PKA and G protein-coupled receptor kinases (GRKs), which leads to receptor binding to β-arrestin, terminates G protein-mediated signaling, and facilitates receptor endocytosis resulting in receptor desensitization and internalization [2 agonists leads to a loss of their effect. Once β2ARs are dephosphorylated through protein phosphatase 2A (PP2A), the receptor becomes resensitized to agonist stimulation. β2ARs can be phosphorylated on their C termini and intracellular loops, such as Ser261/262/345/346 by PKA and Ser355/356/360/364 by GRKs, and the distinct phosphorylation sites mediate different intracellular signaling pathways and physiological functions [Activation of βlization . This is2AR phosphorylation, internalization, and resensitization has been performed in transiently transfected cells. There are few data from primary cells or animal models, which limits our understanding of the physiology of β2AR phosphorylation. At the same time, although the mechanism of β2AR desensitization is well characterized, less is known about β2AR resensitization, particularly the role of β2AR dephosphorylation in functional resensitization and internalization in primary cardiomyocytes. Recent studies have revealed that resensitization should be considered equally important as desensitization for the regulation of β2AR functions [Much of the work on βunctions .261/262 in cardiomyocytes has been previously demonstrated, and was shown to be involved in regulating the contraction rate response and Gi signaling coupling [360/364 in cardiomyocytes owing to the lack of phosphor-specific antibodies for these sites. Therefore, in the current study, we focused on Ser345/346 and Ser355/356. We found that the Ser355/356 phosphorylation sites on β2ARs have a crucial role in regulating β2AR internalization and resensitization in neonatal mouse cardiomyocytes.Phosphorylation of Sercoupling . At the All animal study protocols were approved by the Institutional Animal Care and Use Committee of the Wenzhou Medical University and complied with the regulations of the Ministry of Health, China, and the USA National Institutes of Health Guidelines for the use and care of laboratory animals. All efforts were made to minimize animal suffering and to reduce the number of animals used.β1/β2AR double knockout (DKO) mice (Adrb1tm1BkkAdrb2tm1Bkk/J) were purchased from the Jackson Laboratory  . β1AR kn2AR phosphorylation at the GRK phosphorylation sites Ser355 and Ser356, a pcDNA 3.1-FLAG-tagged murine β2AR was used as a template for mutagenesis with the QuickChange site-directed mutagenesis kit . Both serines were replaced with alanines according to the manufacturer’s instructions. In addition, the PKA phosphorylation sites (Ser345 and Ser346) on β2AR were mutated by replacing the two serines with alanines. The mutant plasmids were named GRK2A and PKA2A, respectively. Mutant β2AR constructs were confirmed by sequencing and transformed into adenovectors to produce adenoviruses using the AdEasy Adenoviral Vector System . Virus titers were assessed by determining the receptor expression levels by both western blot and ligand binding assays, as described previously [To determine the role of βeviously .5 cells) was placed in the center of 35-mm dishes and cultured for 24 h. Then, the medium was changed and the cells were cultured for another 24 h to obtain a uniformly beating syncytium. The cells were equilibrated in a chamber on the stage of an inverted microscope at 37°C for 10 min before monitoring of the contraction rate. For the desensitization/resensitization assay, the contraction rates of the syncytia were recorded every 2 min for 10 min after the addition of epinephrine  or norepinephrine  (Sigma-Aldrich). Then, the medium was changed or the cells were kept in the medium containing stimulator for 60 min, after which the contraction rates were recorded for another 10 min following a second stimulation. Data were recorded using the software on the computer. For measurements of the effect of repeated stimulation with agonist Epi or NE, the contraction response of mouse neonatal cardiomyocytes was recorded in real-time for 40 min.Measurements of the spontaneous contraction of cardiac myocytes were performed as described previously with modification . Briefly2ARs at a multiplicity of infection (MOI) of 100 for 24 h. After serum starvation for 2 h, the myocytes were stimulated with 10 μM Epi for different periods. For receptor recycling, the cells were stimulated with Epi for 10 min, rinsed, and refed with serum-free medium for different periods after removal of Epi for 1 h. The cells were then fixed, permeabilized, incubated with anti-FLAG antibody, and visualized using an Alexa 488-conjugated goat anti-mouse antibody . Fluorescence images were taken with a camera on a Zeiss Axioplan 2 microscope and analyzed with Metamorph software . To quantify the surface receptor level, a fluorescence-linked immunosorbent assay (FLISA) was applied as described previously [2AR adenovirus at a MOI of 100 for 24 h, stimulated with Epi under the indicated conditions, and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS). Without permeabilization, the cells were blocked directly with 2.5% goat serum in PBS, and then stained with Alexa 488-conjugated M1 antibody (Sigma) at a concentration of 1 μg/mL for 30 min at room temperature. The unbound antibody was removed by washing four times with PBS. The cells were harvested with 1% SDS in PBS, and the intensity of Alexa 488 emission (495 to 580 nm) was measured using a spectrofluorometer with an excitation wavelength of 485 nm and an integration time of 0.3 s/nm. The fluorescence intensity was normalized by subtracting the background from cells without M1 antibody.Neonatal cardiac myocytes isolated from DKO mice were transfected with plasmids encoding wild-type or phosphorylation-site mutant βeviously . Briefly2ARs at a MOI of 100 for 48 h. Then, the cells were serum-starved for 2 h and stimulated with 10 μM Epi for the indicated times. The cells were lysed in radioimmunoprecipitation assay lysis buffer  at 4°C. Samples were clarified by centrifugation at 16,100 × g for 10 min, and the protein concentration of the supernatant was measured by the bicinchoninic acid assay (Thermo Scientific) according to the manufacturer's instructions. Samples were resolved by SDS-PAGE for western blotting. Phosphorylation of β2AR by GRK at Ser355/356 or by PKA at Ser345/346 was detected with phospho-specific AR antibodies . IRDye 800CW-conjugated donkey anti-rabbit antibody  was used as a secondary antibody. Western blots were visualized with an Odyssey CLx infrared imager (LI-COR) and quantified after normalization against baseline levels.Neonatal cardiac myocytes were transfected with FLAG-tagged wild-type or mutant β2AR recombinant adenoviruses at a MOI of 100 for 24 h. The cells were serum-starved for 2 h before stimulation with 10 μM Epi for the indicated times. Then, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized, and incubated with an anti-FLAG antibody overnight at 4°C. After washing with PBS, the cells were incubated with an Alexa 488-conjugated goat anti-mouse secondary antibody for 1 h at room temperature. β2AR recycling was assessed by washing out the agonists after 10 min and allowing receptor recovery for an additional 60 min. Images were obtained using a Zeiss Axioplan 2 microscope with Metamorph software, or a Zeiss 710 confocal microscope.Cardiac myocytes were cultured on coverslips in 6-well plates and transfected with wild-type or mutant βt-test; a value of P < 0.05 was considered significant.Curve-fitting and statistical analyses were performed using Prism . All experiments were performed independently at least three times. Data are presented as the mean ± standard deviation (SD). Statistical significance was determined using ANOVA or Student’s 2AR stimulation caused functional desensitization of cardiac myocytes, which prevented contraction rate increases in response to a second agonist challenge. In contrast, removal of the β2AR agonist after 10 min allowed recovery of the contraction response, indicating functional resensitization of the receptor. This suggested that these treatment scenarios provided excellent in vitro cell models for the β2AR desensitization/resensitization response in cardiac myocytes.Short-time stimulation (10 min) with Epi or NE rapidly increased the neonatal cardiomyocyte contraction rate, reaching a maximum at 5 min, followed by a slow decline. When the agonist was removed for 60 min and then re-added at the same concentration, the increase in the cardiomyocyte contraction rate was similar to the initial response . However2ARs is related to receptor internalization and recycling. We assessed these processes in our in vitro cardiac myocyte model using the same contraction response conditions as for Epi stimulation. DKO cardiac myocytes transfected with FLAG-β2AR adenovirus were treated with Epi for 10 min. Internalization of FLAG-β2ARs was observed clearly as punctate intracellular staining (2ARs were again rapidly (10 min) internalized  or Ser345/346 (PKA2A) to alanines to generate β2ARs with defective phosphorylation sites. When these mutant β2ARs were expressed in DKO cardiac myocytes and stimulated with Epi, the PKA2A mutant receptors generated punctate intracellular staining similar to that in wild-type cells. In contrast, the GRK2A mutant receptors showed impaired internalization , a PP2A inhibitor, prior to Epi stimulation. Epi induced rapid (10 min) receptor phosphorylation at Ser355/356 (2ARs underwent dephosphorylation over 60 min. Pretreatment with OA attenuated β2AR dephosphorylation (2AR dephosphorylation by OA resulted in the accumulation of phosphorylated β2ARs in the cytoplasm after withdrawal of Epi for 60 min (To confirm whether phosphorylation of βr355/356 . After rrylation . In addir 60 min . Interesr 60 min .2ARs and the cytoplasmic protein β-arrestin 2 after internalization, we constructed two specifically labeled plasmids that expressed β2ARs and β-arrestin 2. Two days after transfection, DKO cardiac myocytes that highly expressed FLAG-mβ2AR and β-arrestin-GFP were stimulated with Epi for 5 to 60 min. Co-immunoprecipitation demonstrated that phosphorylated β2ARs underwent internalization and interacted with β-arrestin 2 (2ARs interacted and co-located with β-arrestin 2, and that the interacted proteins time-dependently assembled in the cytoplasm (To determine the interaction between βrestin 2 . Furtherytoplasm . This su2ARs as assessed by a neonatal cardiomyocyte contraction assay in vitro, as well as the receptor internalization response under repeated agonist stimulation. These responses involved β2AR phosphorylation on the Ser355/356 sites. Desensitization of GPCRs is an important physiological feedback mechanism that protects cells against acute and chronic receptor overstimulation [2AR functional desensitization by showing that a second β2AR stimulation did not increase the cardiomyocyte contraction rate after continuous stimulation (2AR as shown by a similar contraction response when compared to the initial stimulation with Epi (We report the functional resensitization of βmulation . Indeed,mulation . In contwith Epi .2ARs with Epi promoted receptor internalization and their retention in the cytoplasm (2ARs at Ser355/356 sites was critical to this physiological resensitization and internalization. Specifically, elimination of β2AR phosphorylation at Ser355/356 but not Ser345/346 by mutation to alanine completely blocked receptor internalization, even with sustained Epi stimulation (2AR at Ser355/356 by OA treatment decreased the contraction response in cardiomyocytes (Sustained stimulation of βytoplasm , while wytoplasm . Importamulation . At the myocytes .355/356 plays a pivotal role in desensitization and internalization of β2ARs [2ARs at Ser355/356 contributed to receptor internalization in primary neonatal cardiomyocytes. This finding is consistent with previous data from cardiomyocytes [2AR at Ser355/356 affecting β2AR trafficking and the contraction rate response in cardiomyocytes [2AR at Ser355/356, while GRK2 inhibits phosphorylation at these sites in HEK293 cells stimulated with isoproterenol [2AR desensitization and β-arrestin-mediated ERK activation, but not for internalization.In HEK293 cells, Serof β2ARs . In the myocytes . GRK2 anmyocytes . We, andmyocytes . It has 2ARs could be phosphorylated by different GRKs in various cells or tissues, thereby mediating distinct physiological functions [345/346 PKA phosphorylation sites, the internalization of β2ARs did not change, indicating that these phosphorylation sites are not necessary for receptor internalization in cardiomyocytes. With Epi continually present in the medium for 60 min, the phosphorylation state of β2AR at Ser355/356 in cardiomyocytes was sustained, and the receptor was retained in the cytoplasm rather than recycled (345/346 sites were not phosphorylated, confirming that phosphorylation of β2ARs at these sites might not be involved in receptor internalization.Previous observations suggested that βunctions ,16. Whenrecycled . In this2AR phosphorylation sites have been identified in HEK293 cells using isoproterenol treatment [2ARs in cardiomyocytes following Epi stimulation was unknown. In addition, whether all 13 phosphorylation sites were essential for receptor internalization and resensitization remained to be elucidated. In the present work, we found that dephosphorylation at Ser355/356 sites 60 min after agonist removal allowed a second functional response, including receptor re-internalization and an increased contraction rate. Therefore, while the total phosphorylation pattern was unclear in cardiac β2ARs with Epi stimulation, the β2AR Ser355/356 phosphorylation state definitely contributed to receptor desensitization and resensitization.Although 13 βreatment , the pho355/356 and recovery of the receptor and cell functions in response to a second agonist challenge. PP2A is important for regulating the dephosphorylation of β2AR in cardiomyocytes [2AR and affects different functions including internalization and resensitization. For example, β2AR resensitization is defective in severe asthma [2AR dephosphorylation at Ser355/356, leading to β2AR dysfunction. The molecular mechanisms of βAR resensitization have been reported to also involve the inhibition of phosphoinositide 3-kinase-γ in the heart [2AR dephosphorylation at Ser355/356 was involved in this process, which is consistent with our observations. In addition, some scaffolding A-kinase-anchoring proteins may regulate βAR resensitization by targeting the phosphatase-receptor complex [Our results also indicated that elimination of sustained agonist stimulation promoted receptor dephosphorylation at Sermyocytes . The bale asthma . In thishe heart . This in complex ,20. Howe2AR internalization is required for resensitization. Once internalized, receptors undergo dephosphorylation in the early endosomes by PP2A [2AR dephosphorylation and resensitization [2AR internalization, which targets receptors to the endosome for dephosphorylation and resensitization [355/356 phosphorylated β2ARs in cardiomyocytes. Additionally, we found that β-arrestin was bound to β2ARs for a longer time in the continuous presence of the agonist, slowing the dephosphorylation process, which might block the functional resensitization of the receptors.It is believed that β by PP2A  before t by PP2A . β-Arrestization . β-Arrestization . We conf355/356 phosphorylation status of β2ARs in neonatal cardiomyocytes is critical for receptor internalization and functional resensitization. This finding suggested that the physiological function of β2ARs not only depends on agonist stimulation, but also on specific conditions in cells or tissues, as well as the current receptor phosphorylation modification status.Together, these data revealed that the Ser


Schizosaccharomyces pombe cells, the DYRK-family kinase Pom1 gradients control cell division timing and placement. Upon dephosphorylation by a Tea4-phosphatase complex, Pom1 associates with the plasma membrane at cell poles, where it diffuses and detaches upon auto-phosphorylation. Here, we demonstrate that Pom1 auto-phosphorylates intermolecularly, both in vitro and in vivo, which confers robustness to the gradient. Quantitative imaging reveals this robustness through two system’s properties: The Pom1 gradient amplitude is inversely correlated with its decay length and is buffered against fluctuations in Tea4 levels. A theoretical model of Pom1 gradient formation through intermolecular auto-phosphorylation predicts both properties qualitatively and quantitatively. This provides a telling example where gradient robustness through super-linear decay, a principle hypothesized a decade ago, is achieved through autocatalysis. Concentration-dependent autocatalysis may be a widely used simple feedback to buffer biological activities.Concentration gradients provide spatial information for tissue patterning and cell organization, and their robustness under natural fluctuations is an evolutionary advantage. In rod-shaped  In the cis configuration, Pom1 auto-phosphorylation is independent of Pom1 local concentration. By contrast, if two Pom1 molecules phosphorylate in trans, then phosphorylation is directly linked to the local Pom1 concentration  hypothesis that concentration-dependent modulation of Pom1 auto-phosphorylation, and thus detachment rate, may explain both Pom1 gradient properties, that is, the negative correlation between the Pom1 gradient amplitude and decay length, and the negative correlation between Pom1/Tea4 ratio and Tea4 levels. Pom1 auto-phosphorylation may occur intramolecularly  by the Tea4-GFP signal. We note that the concentration of cytosolic Pom1  does not appear to strongly influence S because we observe no correlation between the amplitude of Pom1 at the pole and Pom1 concentration in the cytoplasm .To formally explore this hypothesis, we considered a mathematical model of Pom1 gradient formation through intermolecular phosphorylation. Previous experimental data showed that Pom1 auto-phosphorylates on multiple sites, at least 6 of which control its membrane affinity  is the cortical concentration of Pom1 phosphorylated i times, the total Pom1 concentration is given by S(x), diffuses along the cortex with a coefficient D, and phosphorylates intermolecularly with a rate β. Phosphorylated Pom1 molecules detach with increasing rate κi < κi+1, resulting in the following dynamics: If αγ is the effective detachment rate and 1.5 < γ ≤ 2. The above approximation is validated by numerical simulations. Theoretical considerations (see Supplementary Text S1) along with experimental data indicate that γ ≅ 2, resulting in the following model: Summing those equations yieldsx0 is proportional to the decay length of the profile, λ, and A is the gradient amplitude at the pole and can be computed as The solution at steady state is given by Ptot in the gradient is given by The total amount of Pom1 k-fold increase in the Pom1 attachment rate S, a position given by a threshold concentration of Pom1 is shifted by x0. See Supplementary Text S1 for more details.Upon a et al, et al, p is the slope of [Pom1] vs. λ in log–log space). This prediction matches well our experimental measurements (p = −0.52 ± 0.06 (SE); Figtrans-phosphorylation model also predicts a power law relationship with p = 2/3 between the Pom1 amplitude at the pole and Tea4 , FigsThis simple model can be solved analytically and leads to a gradient profile that decays as a power of the distance from the source   in vitro . Thus, Pom1 auto-phosphorylates in trans in vitro.To directly address whether Pom1 auto-phosphorylation occurs in ions Figs. Phosphotrans-phosphorylates in vivo. Indeed, inactive or non-phosphorylatable Pom1 alleles bind the plasma membrane more strongly and decorate the entire cell periphery because they do not rely on local dephosphorylation by the Tea4-PP1 complex for membrane binding  but not in cells expressing Pom1-GFP . These results are in agreement with the idea that Pom1-GFP phosphorylates Pom1as1-tdTomato to delay its delocalization, providing in vivo evidence for Pom1 trans-phosphorylation. We note that our in vivo data cannot exclude that Pom1 may also auto-phosphorylate in cis. Sustained drug treatment eventually led to Pom1as1-tdTomato redistribution over the entire cell periphery even in the presence of Pom1-GFP as observed in the case of Pom1KD . In conclusion, in vitro and in vivo data are consistent with the idea that Pom1 auto-phosphorylates intermolecularly.We used Pom1 localization as readout of Pom1 phosphorylation state to test whether Pom1 iddle Fig. This shIn vitro and in vivo evidence indicates that Pom1 trans-phosphorylates in vivo, and cell population analysis of Pom1 gradient shape exhibits the exact system properties expected from such a trans-phosphorylation-induced Pom1 detachment: a −1/2 power law between the gradient amplitude and decay length and a 2/3 power law between the Tea4 concentration and the Pom1 concentration at the pole. Such population-level properties are much more powerful for distinguishing between different models than the shape of individual gradient profiles . We conclude that the Pom1 gradient is buffered against fluctuation in attachment rate through intermolecular auto-phosphorylation. This, however, does not exclude that Pom1 also auto-phosphorylates intramolecularly. Similarly, although a previously proposed concentration-dependent effect on diffusion rate (Saunders et al, et al, et al, et al, trans-auto-phosphorylation of the myosin IIIa, an unconventional myosin possessing a kinase domain, to down-regulate its own localization at the tips of filopodia (Quintero et al, trans-auto-phosphorylation of PLK4, a major kinase for centriole biogenesis, to promote its degradation and prevent excessive centriole number (Cunha-Ferreira et al, trans-phosphorylation of Src to promote its inactivation (Osusky et al, Conceptually, similar nonlinear self-regulatory mechanisms have been shown to buffer morphogen gradients against variations in developing embryos and tissues (Eldar