AMBRA1 is a crucial factor for nervous system development, and its function has been mainly associated with autophagy. It has been also linked to cell proliferation control, through its ability to regulate c-Myc and D-type cyclins protein levels, thus regulating G1-S transition. However, it remains still unknown whether AMBRA1 is differentially regulated during the cell cycle, and if this pro-autophagy protein exerts a direct role in controlling mitosis too. Here we show that AMBRA1 is phosphorylated during mitosis on multiple sites by CDK1 and PLK1, two mitotic kinases. Moreover, we demonstrate that AMBRA1 phosphorylation at mitosis is required for a proper spindle function and orientation, driven by NUMA1 protein. Indeed, we show that the localization and/or dynamics of NUMA1 are strictly dependent on AMBRA1 presence, phosphorylation and binding ability. Since spindle orientation is critical for tissue morphogenesis and differentiation, our findings could account for an additional role of AMBRA1 in development and cancer ontogenesis.The online version contains supplementary material available at 10.1007/s00018-023-04878-6. Autophagy and Beclin 1 regulator 1 (AMBRA1) has been initially discovered as a key factor for nervous system development, and its deficiency has been mainly associated with a defect of autophagy –8. AMBRAA key step during cell cycle is cell division that is accomplished during M phase. Since any defects at this stage could cause genetic abnormalities in daughter cells, mitosis is a tightly regulated process, and several families of mitotic kinases ensure the fidelity of the entire process . Among ti complex, and is able to bind both astral microtubules, to anchor mitotic spindle, and Dynein/Dynactin motor, to generate pulling-force and to position mitotic spindle [Kinase activity is required for mitotic progression, which relies on the proper function of mitotic spindle machinery, that physically segregate chromosomes at opposite poles , 27. In  spindle –37. NUMA spindle , 38. Int spindle –42.In this work, we show that AMBRA1 is sequentially phosphorylated at mitosis by CDK1 and PLK1 on multiple sites. In particular, CDK1 is responsible for the early phosphorylations on T1209 and S1223, and it promotes additional late phosphorylation events by PLK1 on AMBRA1. Altogether, these phosphorylation events are critical for proper spindle function and orientation. Indeed, phosphorylated AMBRA1 can interact with NUMA1 and is responsible for NUMA1 proper localization at the cell cortex. Moreover, we observe that loss of AMBRA1 leads to PLK1 protein stabilization and to an increase in phospho-NUMA1 levels which, in turn, contributes to spindle orientation defects.In order to analyze the expression pattern of AMBRA1 throughout the cell cycle, we decided to perform synchronization experiments in HeLa cells, by using some pharmacological and physical methods. By using Thymidine, we synchronized cells at the G1/S boundary, and then we released cells from the block, allowing them to go through S, G2 and M phases in a synchronous way. As shown in Fig. 1209/1223) completely abrogates the Nocodazole-induced mobility shift  and ELM (http://elm.eu.org/), in addition to literature data [32P radioisotope-labeled ATP, on immunoprecipitated WT and AA1209/1223 AMBRA1, using the recombinant CDK1/Cyclin B1 complex. As shown in Fig. 1209/1223. Notably, AMBRA1 exhibits a basal phosphorylation in the absence of CDK1 that is most likely due to the activity of kinases co-precipitated with AMBRA1, e.g. the Unc-51 like kinase-1 (ULK1) [In order to identify a candidate kinase for AMBRA1 phosphorylation at mitosis, we used online available bioinformatics tools, as Scansite , and we found several potential sites for PLK1 binding. Among these sites, the CDK1-phosphorylated S1223 was predicted to be bound by the PLK1 substrate binding domain , and its surrounding sequence is also in line with the motif identified by Elia et al. [1209/1223), has also a reduced ability to bind PLK1 . These results demonstrate that PLK1 is able to bind phospho-AMBRA1 through its PBD domain, and that phospho-S1223 is a key residue, but most likely not the only one, for this interaction.Mitotic entry is marked by a peak of protein phosphorylation that is driven by several mitotic kinases . Among oa et al.   and in AMBRA1 knock-out (KO) 2FTGH  and HeLa cells generated with the CRISPR/Cas9 technology , in which we observed an increase in the number of cells showing a multipolar spindle  and chromosomes misalignment , together with a significant increase in mitotic spindle angle . Notably, also in these cells, WT AMBRA1 overexpression rescues the normal spindle angle value, while the phosphosilent mutant AA1209/1223 is unable to achieve a similar rescue . Moreover, AA1209/1223 MYC-AMBRA1 overexpression in control cells does not affect the spindle angle. This, indeed, suggests that the phosphosilent mutant does not exert a dominant negative effect . In addition, the role of AMBRA1 in spindle orientation was further confirmed by spinning-disk confocal live cell imaging of AMBRA1 KO cells  and AMBRA1 RNAi-treated HeLa cells , showing that metaphase spindles were tilted under these conditions . Of note, no difference was detected in metaphase spindle length of AMBRA1 KO cells (15.89 ± 0.86 in control compared to 15.54 ± 1.15 in AMBRA1 KO cells) . On the other hand, AMBRA1 phosphorylation had no effect on autophagy regulation during mitosis, as evidenced by ATG14 Ser29 phosphorylation  and LC3 II accumulation . Altogether, these data support the hypothesis that AMBRA1 phosphorylation is important for mitotic spindle function, independently from its role on autophagy.In the attempt to uncover AMBRA1 phosphorylation function, we reconstituted RA1 Fig. A, in seadle Fig. B and chrdle Fig. C. Import1209/1223 AMBRA1 mutant . Interestingly, NUMA1 de-localization from cortex goes along with NUMA1 accumulation at spindle poles .To identify the molecular partners of AMBRA1 in mitotic spindle regulation, we next took advantage of a Stable isotope labeling with amino acids in cell culture (SILAC) mass spectrometry analysis, performed on AMBRA1-overexpressing cells, comparing an asynchronous population with the mitotic-enriched one. Among the proteins whose interaction with AMBRA1 was significantly increased in Nocodazole-treated cells, mass spectrometry analysis suggested some crucial players for mitotic spindle progression, such as NUMA1, AURKA and PLK1 Fig. A. Among ant Fig. C. NotablRA1 Fig. D and E. 1209/1223 with AURKA was not significantly changed when compared with the WT protein . This implies that the phosphorylation state of AMBRA1 affects its capability to bind NUMA1, but not AURKA.In order to explore the molecular mechanism, by which AMBRA1 regulates NUMA1 localization, we first analyzed the expression of NUMA1 upstream regulators LGN and Gαi, responsible for NUMA1 anchorage at the plasma membrane . As showAMBRA1 KO asynchronous and mitotic cells, due to an increased protein stability  mutant, unable to bind PP2A, in siAMBRA1 cells  was not able to reproduce the anaphase defect induced by AMBRA1 depletion  and to promote c-Myc proto-oncogene de-phosphorylation and proteasomal degradation [Since its discovery in 2007, AMBRA1 has been mainly associated with autophagy and several efforts have been done to dissect its role at a molecular level , 46–51. radation , 15. Altradation . Severalradation , 12, andBy biochemical and molecular approaches, we thus identified T1209 and S1223 as key residues for AMBRA1 phosphorylation at mitosis. As these two residues lie in a CDK1-consensus motif, we investigated the involvement of this kinase in AMBRA1 phosphorylation, and we were able to demonstrate that AMBRA1 is a substrate of CDK1. Moreover, we found that phospho-AMBRA1 is recognized and further phosphorylated by PLK1, another kinase playing a key role in mitotic entry, progression and exit , 19, 21.In recent years, autophagy regulation at mitosis has been debated with different studies supporting the hypothesis that autophagy could be inhibited or induced during this cell cycle phase , 53–57. To add molecular insights to our findings, we decided to identify by mass spectrometry AMBRA1 dynamic interactions at mitosis. By using this approach, we observed a significant increase in AMBRA1 interaction with cell cycle-related proteins, and intriguingly with several proteins involved in spindle orientation. Among these, NUMA1 protein caught our attention for its key role in mitotic spindle function; NUMA1 localizes at the mitotic spindle, where it drives microtubule bundling to centrosomes, and at the cell cortex, where it is responsible for mitotic spindle anchoring and proper positioning, together with the Dynein/Dynactin complex , 35–37. AMBRA1 KO cells may result in an increased PLK1 phosphorylation of NUMA1 and consequent spindle pole retention. Moreover, we found that AMBRA1 loss also causes an increase in T2055 phosphorylation, which is not dependent on AMBRA1-PP2A interaction. In sum, our findings suggest that AMBRA1 impacts NUMA1 phosphorylation status, with consequences on its localization and spindle orientation. Last, AMBRA1 effect on NUMA1 localization most likely affects also NUMA1 function at spindle poles, as demonstrated by the occurrence of multipolar spindles and chromosome misalignments in the absence of AMBRA1 and its phosphorylation. Indeed, NUMA1 accumulation at spindle poles has been associated with centrosome clustering-dependent multipolarity in cancer cells with supernumerary centrosomes [Given the role of AMBRA1 as a scaffold protein , 10, it trosomes . AlthougIn sum, our results disclose a new autophagy-independent role for AMBRA1 in controlling spindle orientation during mitosis. Mitotic spindle orientation is critical for the proper morphogenesis of epithelial tissues, since it determines cells positioning within the tissue, as well as differentiation factors segregation at mitosis , 37, 72.2 in a humidified atmosphere.HeLa, Hek293, HCT-116 and 2FTGH cells were cultured in DMEM (Lonza) in presence of 10% FBS (Gibco Life Technologies) and 1% Penicillin/Streptomycin mix (Gibco Life Technologies). Cells were grown at 37 °C and 5% COFor transfections, cells were incubated with plasmids and Lipofectamine 2000 (Thermo Fisher Scientific) in Opti-MEM medium (Gibco Life Technologies) for 4–6 h and then were cultured in DMEM for 24 or 48 h. For RNA interference (RNAi) experiments, cells were transfected at 30–50% confluence using Lipofectamine RNAiMAX (Thermo Fisher Scientific) for 48 h or 72 h.For CDK1 kinase activity inhibition, cells were treated with Nocodazole (see cell synchronization methods section) overnight and then were treated with 9 μM RO-3306 (Sigma-Aldrich). For protein synthesis and degradation inhibition, cells were treated with 100 μg/mL cycloheximide (Sigma-Aldrich) or 5 μM MG132 (Sigma-Aldrich).AMBRA1 RNA interference, a lentiviral pLK0.1 plasmid targeting AMBRA1 mRNA in 3′UTR was used . The lentiviral production was obtained by co-transfecting pMDLg and psPAX2 plasmids in HEK293T cells and supernatants were collected 48 h post-transfection. Stable AMBRA1 downregulated cells were obtained by infecting twice (18 h and 4 h) and selecting them by adding Puromycin (Sigma-Aldrich) to a final concentration of 2.5 μg/μL. As control, a pLK0.1 plasmid that does not target any known mammalian gene was used.For stable 1209/1223-reconstituted cell lines we used AMBRA1-downregulated cells. Viruses were produced as previously described [For stable FLAG-AMBRA1 transfection we used cells available in our laboratory, while for stable MYC-AMBRA1 WT and AAescribed .HeLa GFP-NUMA1 cells were generated by GFP-NUMA1 plasmid transfection and subsequent selection with 600 μg/mL of Hygromycin B (Gibco Life Technologies). GFP-positive cells were enriched using BD FACSAria (BD Biosciences) cell sorter.AMBRA1 were designed using the public available tool gRNA-identifier ZiFiT Targeter [AMBRA1 sgRNA #1, 5′-GAACCATAATATCTATATTA-3′; AMBRA1 sgRNA #2, 5′-GCTGCCTAGATGGGGAGGTT-3′. The sgRNAs were cloned into the U6-driven Cas9 expression vector (pSpCas9(BB)-2APuro; 48,319, Addgene). CRISPR/Cas9 AMBRA1 KO clones of HeLa cells were generated by the Dual-fluorescent surrogate system as described in Zhou et al. [AMBRA1 KO cells were generated using CRISPR-Cas9 lentiviral vectors specific for AMBRA1  and control sgRNA . Transfected cells were selected with Puromycin (Sigma-Aldrich) and used as an heterogenous mixed population without clonal selection.Single-guide RNAs (sgRNAs) targeting exon 4 of human Targeter  and screTargeter : AMBRA1 u et al. . For 2FTFor double Thymidine block and release, cells were treated twice with 2 mM Thymidine (Sigma-Aldrich) for 16–18 h, with a release of 8–9 h between the two treatments, and then were released. 4 h post-release, cells reaching M-phase were blocked adding 100 ng/mL Nocodazole (Sigma-Aldrich) to the medium. Cells were also directly synchronized at mitosis using either 200 ng/mL Nocodazole or 100 μM Monastrol (Sigma-Aldrich) for 16–18 h. Naturally occurring mitosis were collected with mitotic shake off as previously described .1209/1223), was generated as described above, using as template MYC-AMBRA1 S1223A and primers for T1209 Alanine substitution (same primer as above for T1209A). mCherry-AMBRA1 AA1209/1223 and PXP were produced by subcloning AMBRA1 AA1209/1223 and PXP into mCherry-pLPCX vector. mCherry-PLPCX and mCherry-AMBRA1 WT were produced in the lab as previously described [MYC-β-Galactosidase, FLAG-AMBRA1, and MYC-AMBRA1 WT were cloned in a modified pLPCX vector (Clonetech), as previously described [escribed .AMBRA1 (sequence: 5′-GGCCUAUGGUACUAACAAA-3′) or siAMBRA1 (5’UTR) (sequence: 5′-GGA CAA CUU ACA AGG ACC-3′).Transient RNA interference were achieved using non-targeting control siRNA  (sequence: 5′-UGGUUUACAUGUCGACUAA-3′), and custom-designed sil-stable Glutamine, 0.398 mM l-Arginine unlabeled (Arg0) or 13C15N labeled (Arg6), and 0.798 mM l-Lysine unlabeled (Lys0) or 13C labeled (Lys10). Unlabeled cells (Arg0–Lys0) were DMSO treated as control, while labeled cells (Arg6–Lys10) were treated with Nocodazole, as described in cell synchronization methods section, to have a mitotic population of cells. Protein extracts were then immunoprecipitated, as described in immunoprecipitation section, and analyzed by mass spectrometry, as described in mass spectrometry analysis section.For SILAC mass spectrometry, stably FLAG-AMBRA1 transfected cells were cultured in SILAC medium (Silantes) composed of DMEM without stable Glutamine and without Arginine and Lysine, supplemented with 10% FBS, 2 mM 2, 0.1 mM EDTA pH 8.0, 10% Glycerol, 100 mM NaCl, 0.5% Triton X-100) or CHAPS buffer . Lysis was performed with selected buffer plus protease inhibitors  and phosphatases inhibitors . After quantification with DC protein assay (BIO-RAD), 1–2 mg of protein extracts were incubated with 1–2 μg of primary antibodies for MYC (tag), AMBRA1, CDK1, or PLK1 (see antibodies section) overnight with rotation at 4 °C. After antibodies binding, immuno-complexes were purified with 20 μL of Protein-G sepharose (Roche), for monoclonal primary antibodies, or Protein-A sepharose (Roche), for polyclonal primary antibodies, for 1 h with rotation at 4 °C. Purified complexes were washed 4–5 times with the buffer with 5 min centrifugation at 500 g, and samples were used for kinase assay or for western blot (WB) analysis (see WB section). Equal amounts of total proteins were used for every experiment. Where a graph with quantification is present the amount of prey protein is always normalized over the amount of bait protein immunoprecipitated.Immunoprecipitation was performed on HeLa protein extracts using either HEMG buffer . Samples were then subjected to mass spectrometry analysis for phosphosites or significant changing interaction detection.For mass spectrometry Tandem affinity purification (TAP) buffer  was used. Cells transfected with FLAG-AMBRA1 WT or F3 were lysed with TAP buffer plus protease and phosphatase inhibitors as above, plus 1 mM PMSF (Sigma-Aldrich). Between 6 and 8 mg of proteins were immunoprecipitated with 300 μL of anti-FLAG M2 affinity gel (Sigma-Aldrich) for 3 h with rotation at 4 °C. After binding, immune-complexes were washed 4–5 times with TAP buffer with 5 min centrifugation at 500 g. Protein extracts, obtained by cell lysis or immunoprecipitation, were denatured with Laemmli Sample Buffer 4X  plus 12% β-mercaptoethanol and with boiling of samples for 10 min at 95 °C. Denatured samples were then subjected to SDS-PAGE electrophoresis on acrylamide gel (BIO-RAD) and subsequent electro blotted to a Nitrocellulose (Amersham biosciences) or Polyvinylidene difluoride  membrane. Then, the membrane was blocked with 5% nonfat dry milk or BSA (Sigma-Aldrich), and was incubated with primary antibodies overnight, with rotation at 4 °C. Detection was possible thanks to horseradish peroxidase-conjugated secondary antibodies. Chemiluminescent reaction was induced with ECL plus (Millipore) and images were acquired with a digital camera . Background adjustment and cropping of images were done with ImageJ and Photoshop softwares.Cells were lysed with CelLytic M (Sigma-Aldrich) or RIPA buffer plus protease and phosphatase inhibitors, as described in immunoprecipitation section, and cellular debris was removed by 15 min centrifugation at 10,0002. Then Phos-Tag gel was subjected to conventional SDS-PAGE. Prior to electro blotting the gel was washed twice in 1 mM EDTA dissolved in water and then in 1 mM EDTA dissolved in Blotting Buffer. Then WB was performed as described in the WB section.Phos-Tag Acrylamide gel was realized adding to resolving gel 50 μM Phos-Tag™ AAL  and 10 mM MnClThe primary antibodies that were used are: mouse anti-AMBRA1 antibody (Santa Cruz Biotechnology); mouse anti-Cyclin B1 antibody (Santa Cruz Biotechnology); rabbit anti-HSP 90 α/β antibody (Santa Cruz Biotechnology); rabbit anti-ACTIN antibody (Sigma-Aldrich); mouse anti-MYC(tag) antibody (Santa Cruz Biotechnology); rabbit anti-CDK1 antibody (Santa Cruz Biotechnology); rabbit anti-P Thr159 PIK3C3 antibody (kindly provided by prof. Yuan ); rabbitlambda protein phosphatase (λPP) from New England Biolabs and with its specific buffer . λPP activity was inhibited with Halt™ Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Reaction mix was incubated for 30 min at 30 °C and protein extracts were denatured and analyzed by WB, as described in WB section.Phosphatase assay was performed on immunoprecipitated AMBRA1 with ® Kinase from Sigma-Aldrich) and recombinant PLK1 (Millipore or PRECISIO® Kinase from Sigma-Aldrich), in presence of 1X NEBuffer for PK (when using CDK1 from New England Biolabs) or PRECISIO® Kinase Assay Buffer (when using kinases from PRECISIO® Kinase) plus 2 mM phosphatases inhibitors (Sodium fluoride and Sodium orthovanadate) and 10 μCi/mL 32P-ATP (Perkin Elmer). Cold kinase assay was performed with 200 μM ATP (Sigma-Aldrich). The two kinases were used alone or in combination and the mix was incubated for 30 min a 30 °C. For combined kinase assay, MYC-AMBRA1 was at first incubated with CDK1 for 30 min and then, after centrifugation for 2 min at 500 g to discard the old mix, was incubated for other 30 min with PLK1 in presence of 1 μM Roscovitine, to keep CDK1 inactive (modified from [Kinase assay was performed after MYC-AMBRA1 immunoprecipitation with recombinant CDK1/Cyclin B1 (New England Biolabs or PRECISIOied from ). After ® 500) and washed twice with 200 µL kinase buffer containing 50 mM Tris/HCl pH 7.5, 150 mM NaCl, 2 mM EGTA pH 8.5, 5 mM MgCl2, 2 mM DTT. The liquid was removed by centrifugation at 10,000 g for 15 min. On the filter, the sample was incubated with 2 μL CDK1/Cyclin B1 (New England Biolabs) or PLK1 (Millipore) in kinase buffer with 500 µM ATP-Mg2+ salt at 30 °C, 900 rpm for 1 h. In the combined kinase experiments samples were first treated with CDK1, which was inhibited after 30 min by adding Roscovitine (Sigma-Aldrich) to final a concentration of 1 µM. Then samples were incubated with PLK1 for another 30 min. The reaction was quenched by adding 200 µL of 8 M Urea within 0.1 M Tris/HCl, pH 8.5. Protein digestion was performed overnight according to the filter-aided sample preparation (FASP) protocol [4HCO3. The eluate was acidified with Trifluoroacetic acid (TFA) to final concentration 1% prior Liquid chromatography-Mass spectrometry (LC–MS/MS) analysis.20 µL immunoprecipitated FLAG-AMBRA1 F3 was loaded on a 50 kD MWCO centrifugal Filter . The tips were washed with 10% Acetonitrile in 0.1% TFA, 80% Acetonitrile in 0.1% TFA and LC–MS grade water. Phosphopeptides were eluted with 50 μl of 1% Ammonia in 20% Acetonitrile and 50 µl of 1% Ammonia in 40% Acetonitrile. The eluate was mixed with 20 μl of 10% Formic acid. The Ammonium formate was removed by vacuum concentration. The dried sample was resuspended with 20 µl of 0.5% Acetic acid for LC–MS/MS analysis. The tip flow-through was stored at − 80 °C for non-phosphopeptide analysis.Titanium dioxide  beads were pretreated with 300 mg/mL Lactic acid in 80% Acetonitrile with 0.1% TFA. The sample was incubated with 2 mg TiOMass spectrometric (MS) analysis and measurements were performed, as previously described , on an LAll raw files were analyzed with the software MaxQuant . Cysteine carbamidomethylation was selected as fixed modification; Methionine oxidation, protein N-terminal acetylation, and phosphorylation on Serine, Threonine, and Tyrosine were selected as variable modifications. In case of SILAC labeled cells, respective labels were also set as quantification mode. A false discovery rate (FDR) of 1% and a minimum length of six amino acids were used for phosphopeptide and site identifications.http://www.matrixscience.com/). A UniProt Human database version July 2014 containing additional sequences of known typical contaminants  was used for searching. For all searches the peptide mass tolerance was ± 10 ppm, minimum Mascot score was 25.All raw files were searched with Mascot 2.2 . After 30 min of incubation at 4 °C and in dark, samples were read at Flow cytometer  with red channel (wavelength between 535 and 617 nm) and with a linear scale (FL2-H). 5000–10,000 events were counted for each sample. Data were collected and analyzed with CellQuest software (BD Transduction Laboratories).2, 10 mM EGTA, 1 mM PMSF, pH 6.9), then were incubated with Phem-0,5% Triton X-100, fixed with 4% Paraformaldehyde (Sigma-Aldrich), permeabilized with 0.3% Triton X-100 and then quenched and blocked as described above. Primary antibodies were incubated overnight at 4 °C or for 45 min at RT, and were visualized with the respective fluorescent dyes-conjugated secondary antibodies. Nuclei were stained with 500 ng/mL of Hoechst 33,342 (Thermo Fisher Scientific) or with 0.1 μg/mL DAPI (Sigma-Aldrich).Cells were grown on poly-Lysine or Fibronectin coated coverslips (Sigma-Aldrich) and immobilized with 4% Paraformaldehyde (Sigma-Aldrich) or 3.7% Formaldehyde (Sigma-Aldrich) plus 30 mM Sucrose (Sigma-Aldrich). Cell permeabilization was performed with 0.1% Triton X-100, then coverslips were blocked with 3% BSA—0.1% Triton X-100 or were quenched with 0.1 M Glycine and blocked with 3% BSA—0.05% Tween-20. For NUMA1 cortical staining cells were washed with Phem Buffer  objective and a Qicam Fast 1394 CCD camera (QImaging) or with Inverted microscope  equipped with 60X  objective and the Clara camera (ANDOR technology). Images were acquired and analyzed with Nis-Elements AR 3.2 (Nikon) or Nis-Elements H.C. 5.11 softwares using the JOBS module for automated acquisitions.For NUMA1 localization analysis, images were visualized with a Leica confocal microscope , equipped with a 63X 1.40–0.60 NA HCX Plan Apo oil BL objective at RT, using LasX software for image acquisition and processing.For PLK1 high-content imaging analysis, cells were grown on fibronectin-coated 96 well microplates  and stained as coverslips. Images were acquired with Operetta imaging system (Perkin-Elmer), and analyzed with Harmony software (Perkin-Elmer).For P-Ser10 H3 analysis images were visualized with ZEISS AXIO Observer 7 inverted LED microscope (ZEISS), equipped with AXIO cam 360. Images were acquired with ZEN 2.6 software and processed with Fiji software.For mitotic spindle orientation analysis, cells were fixed and stained as described in the immunocytochemistry section. Only images of cells at metaphase stage were acquired. Mitotic spindle angle was calculated as the angle between the mitotic spindle axis, built using the two centrosomes marked with anti-Pericentrin antibody, and cell growth surface. For mitotic spindle angle measure images were acquired with 100X  or 60X objectives along the z-axis every 0.4 μm for a total range varying from cell to cell, so to include both spindle poles, detected by pericentrin. Angle measure was obtained with mathematical formula α = Arctan(z/xy), where xy is the distance between centrosomes in maximum intensity projection, and z is the distance between the two planes in which centrosomes are in focus along z-axis, as previously described .2 supply (5%), using a Plan-Apochromat DIC 63X/1.4NA oil objective mounted on an inverted Zeiss Axio Observer Z1 microscope (Marianas Imaging Workstation from Intelligent Imaging and Innovations Inc. (3i), Denver, CO, USA), equipped with a CSU-X1 spinning-disk confocal head (Yokogawa Corporation of America) and four laser lines . Images were detected using an iXon Ultra 888 EM-CCD camera (Andor Technology).Time-lapse imaging of cells cultured in 35 mm glass-bottomed dishes  was performed in a heated incubation chamber (37 °C) with controlled humidity and COFor quantification of spindle orientation defects and spindle length, HeLa cells were treated with 5 nM SiR-tubulin (Spirochrome) for 1 h prior to imaging. Fifteen 1 µm-separated z-planes were collected every 2 min. The spindle length was quantified by measuring the three-dimensional distance between the spindle poles as described before . BrieflyQuantification of NUMA1 at the cell cortex was performed as described before . BrieflyFor analysis of spindle elongation during anaphase, HeLa cells stably expressing GFP-NUMA1 were treated with 10 nM SiR-tubulin for 1 h before imaging. Anaphase spindle elongation increase was quantified as the difference in pole-pole distance at 10 min after anaphase onset and metaphase spindle length. Anaphase onset was defined as the time point in which the spindle poles marked by GFP-NUMA1 and SiR-tubulin started to move apart and the time-point before anaphase onset was defined as metaphase. In rescue experiments, quantifications were performed only from cells expressing AMBRA1 identified by mCherry signal.n is indicated in figure legends and densitometric analysis was performed using ImageJ software. Histograms show mean ± s.e.m. or s.d., as indicated in figure legends. Statistical significance was assessed using GraphPad Prism software and using ordinary one-way or two-way ANOVA, as indicated in figure legends. For Flow cytometry analysis in supplementary figures, graphs are representative of the experiment shown and were realized with Microsoft Office Excel software. For mitotic spindle angle measure, between 20 and 30 cells for each condition in each experiment were acquired, and the measure of the angle was done by applying the specific formula  with Microsoft Office Excel software. For live cell imaging statistical analysis and graphs were generated in GraphPad Prism and MatLab softwares. Details of statistical tests used and its significance are indicated in the figure legends. The data points were tested for normality using Shapiro–Wilk test. Accordingly, statistical significance was determined by Student’s T test  or Mann–Whitney U test . For parametric tests, the F test was used to compare variances and Welch’s correction was performed accordingly.All the experiments were performed at least three times. For those experiments flanked by a graph, Supplementary file1 (DOCX 13026 KB)Supplementary file2 (XLSX 15 KB)Supplementary file3 (XLSX 18 KB)Supplementary file4 (AVI 20571 KB)Supplementary file5 (AVI 20571 KB)Below is the link to the electronic supplementary material.


However, motivated by the recently discovered regulation mechanism of activation loop cis-autophosphorylation by a kinase that is autoinhibited in trans, we here review the various mechanisms of autoregulation that have been proposed. We provide a framework for critically evaluating biochemical, kinetic, and structural evidence for protein kinase dimerization and autophosphorylation, and share some thoughts on the implications of these mechanisms within physiological signaling networks.Phosphorylation of proteins is a ubiquitous mechanism of regulating their function, localization, or activity. Protein kinases, enzymes that use ATP to phosphorylate protein substrates are, therefore, powerful signal transducers in eukaryotic cells. The mechanism of phosphoryl-transfer is universally conserved among protein kinases, which necessitates the tight regulation of kinase activity for the orchestration of cellular processes with high spatial and temporal fidelity. In response to a stimulus, many kinases enhance their own activity by autophosphorylating a conserved amino acid in their activation loop, but precisely how this reaction is performed is controversial. Classically, kinases that autophosphorylate their activation loop are thought to perform the reaction in  Additionally, their hydrogen bonding capacities are considerably different and, whilst the aspartate side chain is approximately the same length as phosphoserine, glutamate is one carbon longer and may not be capable of making the same interactions. This is particularly relevant in the case of tyrosine kinases, where it is not possible to faithfully mimic phosphotyrosine, although many studies have employed glutamate substitutions. In the same vein, alanine or phenylalanine substitutions used to prevent kinase activation may have unintended effects that are unrelated to the loss of phosphorylation, due to a deficit in hydrogen bonding intrinsic to the substituted amino acid. When designing site-specific mutations to derive mechanistic insight, consideration of protein structure and function is indispensable, irrespective of the target enzyme.cis) or from a second protein (trans). In the trans situation, this may be another protomer of the same molecule (trans-auto) or a different protein altogether. Within this framework, the following regulatory mechanisms and features can be combined to describe the regulation of all protein kinases.As intracellular transducers of cellular information, kinases need to be exquisitely responsive to upstream inputs that tune their activity accordingly. This regulatory potential is strongly related to the conformational flexibility previously described that enables kinases to interconvert between an inactive state and an active state, upon which substrate can be phosphorylated with high efficiency. A multitude of signaling inputs can trigger reversible switching between these states. This process of conformational switching requires activating and inhibitory forces, which may come from within the protein kinase itself  or by an upstream kinase (trans-phosphorylation). The requirements for autophosphorylation in cis and trans, as well as their functional implications, are different and will be discussed in more detail later.The conformation of the activation loop can be regulated by phosphorylation, as previously described . This phcis with an inhibitory domain or motif that is encoded on the same polypeptide chain, or in trans, either by a second protomer of the same kinase or by a different protein.Another regulatory mechanism is to block access of the substrate binding cleft. Sometimes referred to as steric occlusion, this can be achieved in cis and in trans. Since almost all kinases are controlled by one or more regulatory domains, it is worth emphasizing that the study of full-length kinases is necessary for a complete understanding of all aspects of kinase structure, function, and conformational dynamics.Finally, all kinases depend on the precise organization of the catalytic machinery for their activity . AllosteIn the next two sections, we will discuss well-characterized examples of each of the mechanisms described above, with the goal of illustrating conceptually different solutions to the control of kinase activity.Keeping kinases inactive in the absence of an activating stimulus is critical for high-fidelity, low-noise signal transduction. Nature has evolved a plethora of mechanisms by which to maintain protein kinases in their inactive conformations when their activity is not required. Relief of these inhibitory interactions is coupled to the acquisition of kinase activity at the right time and place in the cell. At the simplest level, a kinase may be autoinhibited by its own activation loop adopting an inactive conformation. This mechanism has been proposed to regulate a number of receptor tyrosine kinases (RTKs), including the insulin receptor kinase (IRK), in which the inactive conformation of the activation loop is incompatible with ATP binding . We willcis and trans. The Ca2+/calmodulin activated kinases (CAMK), for example, are maintained in an inactive conformation by a C-terminal cis-autoinhibitory helix that occupies the catalytic cleft  (The diversity of inhibitory mechanisms is further exemplified by members of the death-associated protein kinases (DAPKs), which form face-to-face dimers  that occtivation . Recentl D (PKD) . Finally D (PKD) . B-Raf i D (PKD) . This pr D (PKD) . In summcis and trans.The autoinhibition of protein kinases by their regulatory domains or other protein factors is mirrored by mechanisms that promote their activation. Like inhibition, activation can be accomplished both in cis-regulatory elements -bound dimer.The AGC kinases are characterized by an approximately 50 amino acid C-terminal extension to their kinase domain that contains a number of elements . The taielements . One sucelements . Structuelements , phosphoelements , additioelements , or sepaelements . Recentlelements . Here, htrans. In AurA, targeting protein for Xklp2 (TPX2) inserts a hydrophobic motif into the same pocket on the kinase domain that the AGC kinases use , inositol requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK), and NimA-related protein kinase 7 (NEK7), require back-to-back dimerization to induce their activation by autophosphorylation . Whetherf debate . As discf debate . The Raff debate . Althougrylation  and tranK by Raf  are stilK by Raf . FinallyK by Raf . ActivatK by Raf .In order to obtain a detailed mechanistic understanding of the autophosphorylation reaction, it is important to perform rigorously controlled biochemical experiments and to interpret the results according to the limitations of the chosen method. Here, we provide an overview of the strengths and weaknesses of the most commonly used readouts of protein phosphorylation .Intact mass spectrometry is a powerful tool for the precise and quantitative measurement of protein mass. Modern, well-calibrated instruments are precise to less than 1 Da in mass ranges up to 100 kDa and sometimes even up to 200 kDa . Site-diPhospho-peptide mapping of digested protein samples by tandem mass spectrometry (MS2) can be used to quantify the stoichiometry and specificity of protein phosphorylation. Combined with intact mass spectrometry, this permits an unbiased, site-specific, and quantitative description of the material that is used in biochemical experiments. Tandem mass spectrometry is limited by the ionization efficiency of the peptides resulting from proteolytic cleavage and care must be taken to ensure coverage of the relevant sites. While trypsin cleavage is most commonly employed in such studies, the sequence surrounding a phosphorylation site may not be amenable to trypsin cleavage. In these cases, proteases with different cleavage specificities can be effectively employed to provide coverage of those sites. The ionization properties of phosphorylated peptides can differ from the respective unphosphorylated peptide, so caution should be exercised in interpreting the absolute ratio of the two species. In addition, phosphorylation can hinder proteolytic cleavage, leading to erroneous conclusions regarding the degree of modification. In these situations, a protease with a more suitable cleavage pattern should be chosen.32P] ATP to measure the incorporation of the radioactive ɣ-phosphate into the kinase. By SDS-PAGE, the kinase can be separated from other reaction components or substrates and the kinase-specific autophosphorylation signal can be read out by autoradiography. Careful calibration and background subtraction allows the precise quantification of phosphate transferred during the reaction, but not however, the absolute phosphorylation state. If the absolute phosphorylation state of the starting material is determined, for example by intact mass spectrometry, the average phosphorylation state per kinase molecule can be calculated. Since the assay does not provide information about the phosphorylation site(s), validation of the specificity of the reaction with an unbiased approach like tandem mass spectrometry is required in order to draw robust conclusions.A conventional tool to monitor autophosphorylation reactions is a radionucleotide transfer assay that employs isotope labeled [ɣ- Autophosphorylation can also be read out with western blotting using phospho-specific antibodies. These antibodies are typically raised against a phospho-peptide of interest. In the case of multiple phosphorylation sites that overlap the binding epitope of the antibody, these antibodies may only recognize a specific subpopulation of the phosphorylated protein. Conversely, antibodies that have been raised against multiply-phosphorylated peptides may fail to recognize proteins singly phosphorylated on the canonical phospho-site . The priThe palette of phosphorylation read-outs is further complemented by a variety of semi-quantitative, site-agnostic SDS-PAGE-based approaches including phospho-tyrosine specific western blotting, electrophoretic mobility shift assays, and Phos-tag gels . Each ofAn example of the successful combination of complementary techniques is the characterization of FGFR1 autophosphorylation . The autModels of kinase autoregulation are commonly based on the dimerization dependence of the reaction . Dependitrans (trans (face-to-face), autophosphorylation in cis (back-to-back), or even trans-autophosphorylation via the face-to-face interaction of back-to-back dimers, as has been proposed for the UPR kinase IRE1 .The observation of a concentration-dependent increase in kinase activity is usually interpreted as a bi-molecular reaction in trans . Howeverase IRE1 . A dimer1  the association of the enzyme and the substrate, which is dictated by their interaction affinities and their respective concentrations, (b) on the intrinsic catalytic rate of the enzyme, and (c) on the dissociation of the product. In a closed system such as an in vitro kinase assay, the reaction conditions change over time as the substrate is turned into a product. Regardless, trans-autophosphorylation reactions can occur in mechanistically distinct ways that influence the kinetics of the reaction.The second case is autophosphorylation in cis-autophosphorylation reaction that is dependent on allosteric activation by back-to-back dimerization is a bi-molecular reaction, therefore, concentration-dependent, even if the enzyme and the substrate are the same molecules. We designate the reaction symmetric if the collision of two unphosphorylated molecules results in the phosphorylation of both. If the effect of the allosteric activator is independent of its phosphorylation, the concentration of the activator remains constant, while the substrate is depleted over time. In this case, the reaction is limited by both the intrinsic catalytic rate constant and the substrate concentration. The reaction rate, therefore, decays over time  the asymmetric unit, which is the minimum non-symmetric unit of the lattice that, with the application of appropriate symmetry operations, can be copied to make (ii) the unit cell, a rhomboid-shaped unit that can be stacked in a repeating array in 3-dimensions. The formation of a crystal lattice requires contacts between (a) the molecules that make up the unit cell and (b) neighboring unit cells. These contacts do not form accidentally, but rely on an empirical combination of solvent molecules that promote the regular packing of molecules together. Since a crystal lattice is entropically unfavorable, the loss of entropy is offset by a combination of enthalpic free energy gain and the gain in entropy caused by the de-solvation of protein surfaces involved in lattice contacts . As suchtrans-autophosphorylation reaction requires the exchange of activation loop segments between opposing protomers in a dimer. By definition, the requirement for phosphorylation dictates that these segments are flexible in their unphosphorylated state and, by extension, that the surface to which they dock in their phosphorylated conformation is not occupied. As such, the activation loop is an exchangeable element that can be used to promote interactions with conserved surfaces on neighboring molecules compatible with crystal lattice growth. There are essentially two flavors of such interactions: one is the docking of the activation loop in trans (instead of in cis) with the kinase domain of another protomer, and the second is the binding of the activation loop to the substrate binding surface of a neighbor (trans). In the absence of conformational restraints imposed by regulatory domains that are often missing, the physiological relevance of these interactions can be hard to deduce from the arrangement of molecules in the crystal lattice. This is further complicated by the fact that activation loop exchange, in which the free energy of binding of the activation loop to its own kinase domain (cis) is close to equivalent to that with a neighboring molecule (trans), can be used to drive lattice formation by creating a dyad axis of symmetry. Such packing interactions are illustrated by the structure of the kinase domain of Chk2  in contact with the activation loop of each protomer was modeled in this structure, while a second magnesium ion (green) was found in a non-physiological location in the N-lobe  domain , depictslization . A back-lization . Howevered dimer , which bcis , size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), small-angle X-ray scattering (SAXS), mass photometry (MP), cross-linking mass spectrometry (XL-MS) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) . HoweverThe interpretation of crystal structures has been particularly influential in efforts to understand the mechanism of activation loop autophosphorylation. However, before we begin to unpack some of those structures, let us first consider what has come to be known as the ‘enigma of autophosphorylation’ . Simply cis-autophosphorylation, symmetric trans-autophosphorylation, or asymmetric trans-autophosphorylation  and Interleukin 1 (IL-1) receptor-associated kinase 4 (IRAK4), based on their crystal structures (Early kinetic data indicated that autophosphorylation of IRK could occur in both cis  and trans (trans . Howevers (trans . Indeed,rotomers  (symmetrrotomers  or just rotomers  (asymmetrotomers  is trapprotomers  and FGFRrotomers  are veryrotomers . Whetherructures . Interesructures .cis conformation of the activation loop has also been observed in IRK (Examination of the activation loop trajectories of IGFR1 and FGFR3, shows that they can sample a large conformational space . Both thd in IRK  (magentad in IRK . This std in IRK . Since tcis, trans symmetric, trans asymmetric, and trans substrate structures reveals that the activation loop adopts the same conformation in each case, with the tyrosine poised for transfer of phosphate from ATP to its hydroxyl group (cis, trans symmetric, trans asymmetric, and trans substrate reactions are all stereochemically possible. It is perhaps also not surprising that what are essentially identical interactions can all be observed in the context of a crystal lattice. Does that mean that they can all happen in the cell? The answer to this question is more complicated and depends on the factors that limit each of these reactions, including local concentration and the available conformational space that can be sampled. Many of these mechanisms are surely rooted in strong biochemical evidence, but, once again, we should be careful not to let our eyes deceive us: what we see in a crystal lattice and what we can observe in a test tube do not necessarily reflect reality in the cell.Why is this important? Zooming in on the conformation of the activation loop surrounding the phosphorylated tyrosine in the yl group . We can,The regulation of protein kinases by activation loop autophosphorylation is an elegant mechanism by which ligand-induced conformational changes can be coupled to kinase activation. However, despite decades of work on numerous kinases, much is still unknown about this seemingly simple reaction. Fundamental barriers to progress include both the difficulty of studying these reactions quantitatively and mechanistically in the complex environment of the cell and, conversely, the pitfalls of the reductionist, in vitro approaches that characterize interactions and biochemistry that are physically and chemically possible, but that may not occur in the context of the native, cellular environment. Spatial confinement, restrictions on the conformational space that can be sampled by a kinase, and local concentration are all tightly regulated in the cell, but challenging to recapitulate in vitro. New in silico tools in structural biology promise much in terms of modeling transient, but specific interactions, while technological improvements in cryogenic electron microscopy are continually lowering the size of structures that can be determined at high resolution. However, we have a collective responsibility to report more quantitatively and more specifically. The reproducibility crisis is particularly grave in the case of antibodies, many of which are poorly validated or have been shown to recognize different targets. While DNA sequencing is nowadays an obligation, we have yet to make the validation of recombinant proteins by mass spectrometry a routine procedure or require its reporting. This is especially important in the context of reversible post-translational modifications, such as phosphorylation, which regulate the conformation, localization, and activity of proteins. Finally, new tools for the spatial arrangement of molecules in vitro on scaffolds that more faithfully mimic the cellular environment are desperately needed. Advances in protein design , DNA nan


The mechanical behavior of arena surfaces has been identified as a contributor to injuries of performance horses. Evidence of excessive fetlock extension in association with stiff surfaces has propelled the installation of synthetic surfaces in performance horse arenas to reduce injury risk. However, the effect of arena surface properties on hoof slide during show jumping has not been widely studied. Therefore, this study measured the forelimb hoof motion of horses during takeoff and landing from a 1.1 m jump using a high-speed video motion capture system on five dirt and seven synthetic surfaces. Hoof slide was not significantly different between dirt and synthetic surfaces, but it was greater at takeoff than at landing and greater for the leading limb than for the trailing limb. These results indicate that horses are able to compensate for the effect of surface differences on hoof slide at a moderate jump height.p < 0.05), while the relationships of hoof movement with surface mechanical properties were examined with correlation. Slide time (p = 0.032), horizontal velocity of the hoof (p < 0.001), and deceleration (p < 0.001) were greater in the leading limb, suggesting a higher risk of injury to the leading limb when braking. However, surface type and jump phase did not significantly affect deceleration during braking.During the stance phase of equine locomotion, ground reaction forces are exerted on the hoof, leading first to rapid deceleration (“braking”) and later to acceleration (“propulsion”) as the hoof leaves the ground. Excessive hoof deceleration has been identified as a risk factor for musculoskeletal injury and may be influenced by arena surface properties. Therefore, our objective was to evaluate the effect of arena surface type  on hoof translation of the leading and trailing forelimbs during jump takeoff and landing. Solar hoof angle, displacement, velocity, and deceleration were captured using kinematic markers and high-speed video for four horses jumping over a 1.1 m oxer at 12 different arenas . Surface vertical impact and horizontal shear properties were measured simultaneously. The effects of surface type , jump phase , and limb  on hoof movement were assessed using ANOVA ( The mechanical properties of equine arena surfaces have been proposed as extrinsic risk factors for musculoskeletal injury . During The support, or midstance, phase follows the impact and slide phases and is characterized by relatively little hoof movement and peak vertical loads. If hoof displacement during support is large, the horse may be required to apply more muscular force to maintain speed, making the horse more susceptible to fatigue . FinallyDesired surface characteristics of arena surfaces, as reported by riders, significantly differ by discipline . Show juHoof movement during locomotion has been previously recorded with accelerometers ,14,15,16The effect of surface type on hoof movement during a gallop has been previously characterized for the hindlimb, where dirt surfaces exhibited significantly higher horizontal motion of the heel during slide than synthetic surfaces . AdditioTherefore, our first objective was to characterize the angle, displacement, velocity, and deceleration of the forelimb hoof during jumping to evaluate the effect of limb , jump phase , and surface type . We hypothesized that horizontal hoof deceleration would be greater for the leading limb, in line with a previous study . FurtherOur second objective was to identify relationships between hoof movement parameters and measured properties of the arena surface . We hypothesized that hoof horizontal motion would be negatively correlated with shear properties  and vertical hoof motion would be positively correlated with the vertical displacement of the surface tester and negatively correlated with other vertical impact properties such as surface stiffness and vertical deceleration at impact.A repeated measures study design was used to track solar hoof angle, position , velocity, and acceleration for four horses  jumping three times over a 1.1 m oxer at twelve arenas in northern California . The weight of the rider and tack used for all horses was 84 kg. Horses were visually observed for orthopedic pain and discomfort by a licensed veterinarian at the beginning of each testing day (SS). No horses showed signs of lameness during the testing period. However, one horse (gelding) was unavailable for the first testing day (dirt surface) and thus only jumped on eleven of the twelve arena surfaces. These data were collected under an IACUC protocol issued by the University of California Davis .Synthetic surfaces had greater than 1% fiber content by volume; none contained rubber, oil, or wax. Fibers were primarily polyester; however, polyethylene, polypropylene, and nylon fibers were also observed in some synthetic surfaces. One dirt surface had 0.6% fiber with large felt strips poorly integrated in the cushion layer. All other dirt surfaces contained 0% fiber content.The oxer was preceded by a ground pole and two cross rails (0.56 m from ground to center) to center and standardize the approach of the horse to the measured jump . Horses Two kinematic markers were placed on a 3D-printed extension bar and rigidly attached to the hoof wall to capture solar hoof angle, translation, and velocity while the hoof was submerged in the surface material during stance . The extThe hoof angle of the solar surface of the hoof (SHA) as well as horizontal and vertical translation, velocity, and acceleration of the toe and heel were determined from marker motion using custom software . Solar hoof angle was defined relative to the arena surface where an angle of 0 degrees indicates that the solar surface of the hoof was parallel with the arena surface. Positive SHAs represent a hoof orientation with the dorsal wall of the hoof rotated counterclockwise with respect to the arena surface (toe down orientation); negative SHAs represent a hoof orientation with the dorsal wall of the hoof rotated clockwise with respect to the arena surface (toe up orientation) . MinimumHoof translation data were also used to further divide the stance phase into three previously defined subphases  by a tramax = FN × µ + a) in conjunction with maximum shear force (Fmax) measured by the device and the normal force (FN) applied to the device. Adhesion and coefficient of friction describe shear properties of the surface–hoof/horseshoe interface. Surfaces with either high adhesion or high coefficient of friction are expected to be associated with high grip and relatively low hoof slide [Both shear and vertical ground reaction forces were measured immediately following kinematic data collection of each horse at locations parallel to the jumping grid with surface testing equipment. Shear and vertical properties of these surfaces have been previously reported ,20. Sheaof slide . Verticaof slide . From thCushion depth and surface temperature were also measured in conjunction with each horse, and samples (approximately 250 g) from the top two inches of the surface were collected in waterproof, airtight containers for moisture content and compositional analysis  according to previously described methods .p < 0.20, as determined from the univariate regression results, were used as inputs in a multivariate stepwise regression model. Statistical significance was p < 0.05. SAS software  was used to perform all statistical analyses.The central tendency (least squares mean/median) and variation (standard error/range) in all variables are described for normally or non-normally distributed data. The effect of jump phase , limb , and arena surface material  on SHA, displacement, velocity, and acceleration were all assessed in a single mixed-model ANOVA with horse included as a random effect and with jump height and horizontal velocity of the horse at takeoff included as covariates. Higher-level interactions between surface and phase and surface and lead were also analyzed. Normality of the ANOVA residuals was assessed using a Shapiro–Wilk test (W > 0.9). ANOVA on ranked data was used for variables with non-normally distributed residuals. The relationships of hoof motion measurements with surface composition, manageable factors, shear surface properties, and vertical impact properties were examined first using univariate regression see . For eacThe jump height at the girth was 1.27 ± 0.07 m (mean ± standard deviation) for all horses. At takeoff, the average horizontal velocity measured at the girth increased during stance in preparation for the jump . At landing, the average horizontal velocity measured at the girth was lower than that at takeoff but also increased during stance . The resultant velocity at takeoff was directed upward, toward the jump  and had a higher magnitude than the resultant velocity at landing, which was, on average, directed toward the ground and away from the jump .p < 0.001), and also significantly longer when the instrumented limb was the leading limb . Surface type  did not have a significant effect on contact time (p = 0.438). However, when evaluating combined effects, the effect of leading limb on contact time was greater on dirt surfaces than on synthetic surfaces (p = 0.028).The contact time for the hoof was significantly longer at takeoff than at landing  and landing (black) is depicted in Hoof angle was maximal (“toe down” orientation) at the end of the stance phase for all trials (100 ± 0%). Hoof angle was minimal (“toe up” orientation) near the beginning of the stance phase for takeoff (location of minimum at 1.8 ± 8.4% stance) as the hoof started “toe up” during the stance phase of takeoff in all but 2 of the 139 takeoff jump trials . However, at landing, there was wide variation in the location of the minimum hoof angle amongst trials (38.0 ± 23.7% stance). At landing, the hoof started stance “toe down” in 103 of the 142 jump trials , and the minimum SHA occurred during midstance rather than at hoof strike.p = 0.004). However, surface type, jump phase, and jump lead did not have a significant effect on the duration of slide  did not have a significant effect on horizontal hoof displacement and velocity during slide.p = 0.034).The horizontal deceleration of both the toe and heel was significantly greater when the instrumented limb was leading. The horizontal deceleration was not significantly different between takeoff and landing or between dirt and synthetic surface types; however, there was a significant combined effect of jump phase and surface type on the horizontal deceleration at the heel, where deceleration was higher at landing than takeoff on dirt surfaces but lower at landing than takeoff on synthetic surfaces (The toe and heel moved deeper into the surface during slide (reported as positive numbers). The vertical displacement and average vertical velocity of the toe during slide were greater for the leading limb, at takeoff, and on synthetic surfaces, while vertical displacement and average vertical velocity of the heel during slide were greater for the trailing limb at landing. The average vertical velocity of the heel during slide was also greater on synthetic than dirt surfaces.p = 0.025) and when the instrumented limb was the leading limb than the trailing limb . The hoof angle during support was significantly lower (more “toe up”) for the leading limb, at takeoff, and on dirt surfaces.On average, the support phase constituted the middle 55.0 ± 13.4% of the stance phase. The support phase was significantly longer and a greater percent of stance at takeoff than landing . The horizontal toe and heel displacement was significantly greater at landing than at takeoff , and horThe toe and heel also moved deeper into the surface during support (reported as positive numbers). The vertical displacement and vertical velocity of the toe during support were greater for the leading limb at takeoff, while the vertical displacement and vertical velocity of the heel during support were greater for the trailing limb at landing. Surface type  did not have a significant effect on the vertical hoof displacement and velocity during support.p = 0.023). The grab phase was also a significantly greater percent of stance for the trailing limb .On average, the grab phase constituted the last 20.0 ± 3.3% of the stance phase. The grab phase was significantly longer and a greater percent of stance on synthetic surfaces than dirt surfaces  SHAs observed during grab. The horizontal toe displacement during grab was significantly greater (more negative) at landing . The aveThe heel displaced out of the surface during grab, while the toe sometimes penetrated deeper into the surface (positive) and sometimes further out of the surface (negative) by the end of the grab phase. The toe displacement during grab was significantly related to the surface type, where the toe displaced further out of synthetic surfaces and further into dirt surfaces during grab. Additionally, the toe of the leading limb displaced into the surface and the toe on the trailing limb displaced out of the surface during grab. The heel displacement out of the surface was significantly greater for the trailing limb at landing. The average vertical velocity of the toe was significantly greater for the trailing limb and on synthetic surfaces with a direction out of the surface. The average vertical velocity of the heel was significantly greater for the trailing limb and at landing with a direction out of the surface.p = 0.006 displacement; p = 0.004 velocity).When considering the combined effects of surface type and leading limb, the toe moved further backward and had a larger horizontal velocity with the leading limb than the trailing limb on synthetic surfaces, while the toe moved further backward and had a larger horizontal velocity with the trailing limb than the leading limb on dirt surfaces , manageable properties , shear properties , and vertical impact properties  as reported by Rohlf et al. ,20 are dp = 0.022) and velocity  of the heel into the surface during slide. At landing, surfaces with higher clay content were significantly related to less vertical displacement  and velocity  of the toe into the surface during slide. During the grab phase of landing, surfaces with higher clay content exhibited a reduced SHA (less “toe down” orientation) , reduced horizontal heel displacement  and velocity , less vertical heel displacement , and less vertical toe velocity . Soils with more variable particle size distributions were also related to reduced horizontal heel displacement  and lower vertical toe velocity  during the grab phase at takeoff, and less vertical displacement of the toe out of the surface  during the grab phase at landing.The relationships between surface properties and hoof translation parameters were analyzed with a multivariate stepwise regression see . Clay cop = 0.005), while surfaces with higher temperatures exhibited less horizontal toe deceleration . At landing, surfaces with higher adhesion increased the deceleration of both the toe and heel .The horizontal deceleration of the hoof during slide was also related to surface characteristics. At takeoff, surfaces with greater variation in soil particle size exhibited less horizontal heel deceleration  and velocity  during slide. Additionally, higher surface temperatures were related to greater vertical displacement  and velocity  of the toe during support, but reduced vertical velocity  of the toe during slide. Surfaces with deeper cushion layers were also related to greater vertical displacement  and velocity  of the toe during support and a lower average SHA . Surfaces with higher vertical deceleration rates were related to less vertical displacement of the heel  and less horizontal displacement of the toe  during grab. Surfaces with greater impulse during vertical impact (factor of impact force and duration of impact) exhibited less vertical toe displacement  during grab. Finally, synthetic surfaces with longer fibers exhibited more vertical heel displacement into the surface during the slide phase  and more vertical heel displacement out of the surface during the grab phase .Several surface properties were only related to hoof movement variables at takeoff. Surfaces with greater soil rebound exhibited more vertical heel displacement  and velocity  during slide, as well as greater horizontal heel displacement opposite the forward momentum of the horse  during support and greater vertical velocity of the heel out of the surface during grab . Surfaces with greater dissipated energy at impact  exhibited less horizontal toe displacement and velocity (opposite the forward momentum of the horse) during support  and grab . Stiffer surfaces , surfaces with higher loading rates during vertical impact , and surfaces with less sand content  were related to less heel displacement out of the surface during grab. Finally, surfaces with larger soil particles exhibited less vertical velocity of the toe out of the surface during grab .Additionally, some surface properties were only related to hoof movement variables at landing. Surfaces with higher adhesion exhibited greater horizontal toe and heel displacement  were expected to be strongly and negatively correlated with horizontal displacement of the hoof, especially during the slide phase, adhesion was the only shear property correlated with hoof movement. This hypothesis was based on previous research which showed that high shear forces (traction) restricted the amount of hoof movement ,28. BothVertical hoof displacement was also expected to be strongly and positively correlated with vertical displacement of the vertical impact tester because the vertical impact testing device was designed to simulate the interaction of the hoof and the ground . HoweverThe counterintuitive and unexpected correlation results between hoof movement and surface properties measured with the mechanical testing devices suggest that further experimentation and development of these devices are required to better represent surface–hoof interactions during a jump. While these devices are not expected to replicate the magnitude of forces during equine stance, it is important that these devices are further validated to ensure that surface properties as measured by these devices are representative of true hoof–surface interactions. The benefit of mechanical testing devices lies in their ability to create a more standardized testing environment than is possible from live animal studies, and to reduce the number of confounding variables when comparing the behavior between different surfaces. However, even with further development, mechanical testing devices will have limitations and may not be able to represent hoof movement accurately or precisely. Therefore, it may be preferable to focus on the enhancement and development of non-invasive and low-profile equipment that can measure movement and forces directly at the hoof .Another limitation of this study is related to possible vibration of the hoof extension bar at impact which is not consistent with the sagittal plane motion of the hoof. However, large vibrations would likely have been observed while reviewing video capture, and small vibrations were removed during the high-pass filtering of the motion capture data. Additionally, dirt and synthetic arena surfaces evaluated in this study were limited to the northern California region, which may not capture the variation in arena properties worldwide. However, our sample size  was larger than that of previous studies which compared only one to two surfaces of each type, and it likely captures more variation than these previous studies. Finally, compositional and manageable properties were not varied within a particular arena surface in this study. Thus, other compositional and manageable properties may be confounding factors when trying to determine the individualized effects of surface parameters on joint motion. The confounding effect of surface temperature is especially noteworthy as dirt surfaces had significantly higher temperatures than synthetic surfaces. Although multivariate stepwise regression was chosen to statistically account for possible relationships between surface properties when reporting correlations, future research studies should evaluate the individualized effects of changing a single surface management or compositional parameter on hoof motion.Large surface property variation among synthetic and dirt surfaces precluded the ability for surface type, described categorically, to be a good predictor for hoof translation in the surface. However, hoof deceleration during slide was significantly dependent on the forelimb that was leading during the jump, where the leading leg had significantly greater deceleration than the trailing leg. This finding suggests that the leading leg may have a higher risk of musculoskeletal injury. To alleviate the increased risk of injury to the leading leg, it may be beneficial for trainers and riders to switch leads during training and competition to reduce the number of repetitions of high deceleration on a single forelimb.


The multitude of fixation options for radial neck fractures, such as pins, screws, biodegradable pins and screws, locking plates, and blade plates, has led to a lack of consensus on the optimal implant choice and associated biomechanical properties. This study aims to evaluate the biomechanical strength of various fixation constructs in axial, sagittal, and torsional loading directions. We included biomechanical studies comparing different interventions, such as cross/parallel screws, nonlocking plates with or without augmented screws, fixed angle devices (T or anatomic locking plates or blade plates), and cross pins. A systematic search of MEDLINE (Ovid), Embase, Scopus, and CINAHL EBSCO databases was conducted on September 26th, 2022. Data extraction was carried out by one author and verified by another. A network meta-analysis (NMA) was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. Primary outcomes encompassed axial, bending, and torsional stiffness, while the secondary outcome was bending load to failure. Effect sizes were calculated for continuous outcomes, and relative treatment ranking was measured using the surface under the cumulative ranking curve (SUCRA). Our analysis encompassed eight studies, incorporating 172 specimens. The findings indicated that fixed angle constructs, specifically the anatomic locking plate, demonstrated superior axial stiffness  in comparison to the cross screw. Additionally, the blade plate construct excelled in bending stiffness  relative to the cross screw construct, while the cross-screw construct proved to be the most robust in terms of bending load failure. The parallel screw construct performed optimally in torsional stiffness  when compared to the cross screw construct. Lastly, the nonlocking plate, locking T plate, and cross-pin constructs were found to be inferior in most respects to alternative interventions. The NMA indicated that fixed angle devices (blade plate and anatomic locking plate) and screw fixations may exhibit enhanced biomechanical strength in axial and bending directions, whereas cross screws demonstrated reduced torsional stability in comparison to parallel screws. It is imperative for clinicians to consider the application of these findings in constraining forces across various directions during early range of motion exercises, taking into account the distinct biomechanical properties of the respective implants. The radial head stabilizes a valgus stress and works as a weight-bearing structure in axial orientation and aids in maintaining elbow stability7. Stable surgical fixation of displaced RNFs is mandatory to permit early postoperative range of motion exercises, prevent elbow stiffness and restore elbow function9. Progressive fracture displacement, nonunion, hardware failure and loss of reduction are not uncommon due to inadequate fracture fixation13.Radial neck fractures (RNFs) account for 3–4% of all fractures and one-third of elbow fractures14, adult RNFs present a multitude of fixation options such as metal17 or biodegradable19 pins/screws, locking or nonlocking plates16, and blade plates13. Screw and pin fixation are less invasive approaches that offer low profile fixation and interfragmentary compression, enhancing construct stability and yielding satisfactory outcomes17. Plate fixation is also a popular treatment option16, but the lack of direction contact between the fracture site may lead to biomechanical inferiority and unfavorable outcomes20. Additionally, the biomechanical advantages of fixed-angle devices over nonlocking devices are also unknown for the fixation of RNFs22. Currently, there is no consensus on which fixation method provides better fixation strength for displaced radial neck fractures23.In contrast to pediatric RNFs, which typically employ pins or elastic nail fixation27, none have been performed for adult RNF, either biomechanically or clinically. Therefore, the aim of the present study is to perform a systematic review and network meta-analysis (NMA) with an up-to-date search of existing evidence for comparisons of biomechanical properties between different fixation constructs in terms of axial, sagittal and torsional loading.Although there have been several clinical meta-analyses evaluating outcomes of radial head fractures between arthroplasty, resection and interval fixation for adult radial head fractures28 and was registered at PROSPERO (CRD 42022323386). We searched Embase, Medline and Scopus databases without language restriction until September 26, 2022. The following Medical Subject Heading terms were used: radius, fracture fixation, cadaver, synthetic bone, artificial bone, biomechanic, or mechanic. The complete search strategy and algorithm are shown in Supplementary Table The NMA was performed according to the preferred reporting items for Systematic Reviews and Meta-analyses (PRISMA) guidelines , nonlocking plates (NLPs), NLPs with an augmented screw blade plate and cross pins, using either cadaveric or synthetic radii. Trials were excluded when studies were performed with pathologic, pediatric, and animal models or studies comparing the same techniques (pins or screws) with different designs or implant materials (Supplementary Table Two authors (YCS and YYW) independently screened all the titles and abstracts according to the selection criteria. Full texts were evaluated after proper screening. If disagreements were noted, a third author (CAS) was involved until the conclusion was made.29. Data extraction was confirmed by a second author (CAS).One author independently (YCS) extracted the following information: the first author’s name, publication year, design of the study, numbers and type of specimens (cadaveric radii or synthetic radii), fracture model , implant selection, mechanical testing protocol and biomechanical outcomes (stiffness and failure strength). In studies reporting only medians, we used the median as the means and interquartile ranges/1.35 as the standard deviations21. When a study measured stiffness in cyclic loading or failure loading, we extracted the stiffness value measured during cyclic loading since cyclic stiffness is used in most of the biomechanical studies or as the only measured stiffness value.When a study used different plate thicknesses for biomechanical comparison, the 2.7 mm-thick plate construct was extracted, which is the most common plate type among other studies30. A third author (CAS) was consulted for any disagreement.Methodologic quality was independently assessed by two reviewers (YCS and YYW) using the Cochrane risk of bias tool, including randomization, allocation concealment, blinding, incomplete outcomes, selective reporting, and other sources of biasStiffness measured in the axial, bending, and torsional directions was the primary outcome. Load to failure and torque to failure in different directions were the secondary outcomes.I2 and the Cochrane Q test were calculated in the pairwise meta-analysis for evaluation of heterogeneity. NMA was performed combining both direct and indirect evidence for multiple intervention comparisons. For the assumption of transitivity, we considered that any of the interventions in the network could have been given to any specimen in the network. Potential inconsistency was evaluated by a design-by-treatment model for assessing global inconsistency and loop inconsistency models and node-splitting models for local inconsistency31. Meta-regression analyses were tested for fracture models in terms of fracture pattern and fracture comminution. We calculated the surface under the cumulative ranking curve (SUCRA) to rank the treatment outcomes for different interventions. The publication bias was evaluated by funnel plots and Egger’s regression plots.We used spreadsheet software  for data extraction, and the statistical software STATA was later used  for statistical analysis. For direct comparisons and network meta-analysis, we conducted traditional pairwise meta-analysis to combine direct and indirect evidence. Fixed-effects models were used because of limited study numbers for random-effect model estimation. The 22. These included studies comparing different constructs published from 1900 to 2022 with sample sizes in each group that ranged from 2 to 12 specimens. Three studies involved radial head and neck fractures22, and 5 studies involved radial neck fractures21. Five studies conducted their experiment with cadaveric radii21, while 3 used synthetic radii22. Of the included studies, there were 6 trials using cross-screw fixation22, 3 trials using LP (T plate) fixation20, 3 trials using LP (anatomic plate) fixation22, 5 trials using non-LP fixation21, 1 trial using non-LP with augmented screw fixation16, 3 trials using blade plate fixation21, 1 trial using parallel screw fixation20, and 2 trials using cross-pin fixation17.We identified 338 studies during the study selection process Fig. . After tThe main domains for potential bias were the randomization process, allocation concealment, and blinding , the summary MD of axial stiffness was − 13.72 N/mm for the LP-T plate (95% CI − 20.93 to − 6.51), 23.59 N/mm for the LP-anatomic plate (95% CI 8.12 to 39.06), − 5.13 N/mm for the nonlocking plate (95% CI − 12.86 to 2.60), and 3.63 N/mm for the blade plate (95% − 5.13 to 12.40). The LP-anatomic plate (SUCRA = 99.9%) was most likely to be ranked the best in terms of axial stiffness.Four studies measured axial stiffness20. Compared with the control group (cross screw), the summary MD of bending stiffness was − 23.78 N/mm for LP-T plate (− 32.57 to − 14.98), − 24.09 N/mm for non-LP (95% CI − 33.87 to − 14.32), − 21.97 N/mm for non-LP with augmented screw (95% CI − 31.20 to − 12.74), 32.37 N/mm for blade plate (95% CI − 47.37 to 112.11), − 5.10 N/mm for parallel screw (95% CI − 14.64 to 4.44) and − 22.52 N/mm for cross pin (95% CI − 31.97 to − 13.07). The blade plate (SUCRA = 87.1%) or cross screw (SUCRA = 84.5%) was most likely to be ranked the best in terms of bending stiffness.Two studies measured bending stiffness20. Compared with the control group (cross screw), the summary MD of bending failure load was − 139.29 N for LP-T plate (95% CI − 210.89 to − 67.69), − 24.00 N for LP-Anatomic plate screw (95% CI − 121.29 to 73.29), − 86.60 N for non-LP (95% CI − 129.99 to − 43.20), − 5.71 N for blade plate (95% CI − 193.47 to 182.05), − 18.78 N for parallel screw (95% CI − 93.59 to 56.03), and − 43.74 N for cross pin (95% CI − 86.52 to − 0.96). The cross screw (SUCRA = 81.4%) was most likely to be ranked the best in terms of bending failure load.Four studies measured the bending failure load20. Compared with the control group (cross screw), the summary MD of torsional stiffness was 1.19 Nm/degree for LP-T plate (95% CI − 1.18 to 3.56), 92.72 Nm/degree for LP-anatomic plate screw (95% CI 66.08 to 119.35), 21.60 for non-LP (95% CI 12.16 to 31.03), 18.92 Nm/degree for non-LP with augmented screw (95% CI − 3.76 to 41.60), 51.57 Nm/degree for blade plate (95% CI 34.71 to 68.42), 139.39 Nm/degree for parallel screw (95% CI 0.79 to 277.98) and − 7.08 Nm/degree for cross pin (95% CI − 23.03 to 8.86). The parallel screw (SUCRA = 92.3%) or LP-anatomic plate screw (SUCRA = 89.0%) were most likely to be ranked the best in terms of torsional stiffness.Three studies had biomechanical measurements of torsional stiffnessOverall, for outcomes regarding axial stiffness, bending stiffness, and bending failure load, the funnel plots showed low publication bias, and Egger’s regression plots did not show any substantial asymmetry. However, there was symmetry, mainly resulting from the comparison between the cross screw and the LP-T plate groups, in the torsional stiffness in the adjusted funnel plots and Egger’s regression plot comparisons  and significantly higher for the nonlocking plate group  either when using the combined radial head and neck model rather than the isolated radial neck model or when using the comminuted RNF model rather than the noncomminuted model  or local inconsistency with the loop-specific approach (p = 0.4716) or the side-splitting method. For the measurement of bending stiffness, significant global inconsistency (p < 0.001) and local inconsistency with the loop-specific approach (p = 0.040) and the side-splitting methods (p < 0.001) were noted. Regarding the bending failure load measurement, although significant global inconsistency (p < 0.001) and local inconsistency using the loop approach (p = 0.008) were found, the local inconsistency with the side-splitting method revealed no significant inconsistency (p = 0.677). For the measurement of torsional stiffness, NMAs demonstrated significant global inconsistency (p < 0.001) and local inconsistency using the side-splitting method; however, local inconsistency using the loop approach revealed no significant inconsistency (p = 0.801).The measurement of axial stiffness revealed no significant global inconsistency  construct13, as shown in our study and other biomechanical studies21. In addition, the superiority of the plate may be related to the higher stability of rigid fixation and compression, reducing the risk of osteonecrosis and nonunion13. Although several advantages were noted, the main concerns of plate fixation were postoperative forearm rotation loss and a higher potential for hardware removal due to implant irritations34.Different plate designs may have different biomechanical properties for RNF fixation. In terms of plate fixation for RNFs, our results and those of others20. Although oblique/cross screw fixation was described in the fixation for comminuted RNFs before, it was more easily applicable in axially stable RNFs10. The NMA showed that crossed-screw fixation had the best ranking in the posterior bending direction and a lower ranking in the torsional directions, which is consistent with prior biomechanical studies showing that the cross-screw construct had better biomechanical performance in sagittal and axial loading than plate constructs23. However, in terms of torsional loading, care should be taken when small cross screws are applied (≤ 2 mm in diameter) or in the comminuted RNFs, as the fixation strength may be weaker and pose the risks for loss of fixation during torsional movement12. In contrast, Chen et al. suggested that the cross-screw fixation may resist higher torsional stress because two ends of the fracture were stressed in the cross-screw fixation20. Gutowski et al. suggested that crossed screws are more suitable for simple transverse RNF fixation11.Screw fixation has been a promising alternative technique for RNF fixation because screws can not only have rigid connections through the internal ends of the fracture but also decrease scarring and hardware irritation due to plate placement beneath the annular ligament20. The configuration was suggested to have advantages over the cross-screw fixation by either minimizing the soft tissue exposure from parallel trajectories or avoiding the need for forearm rotation that poses the risks of loss of reduction during the insertion of the screws from opposite entry points20. Future studies are needed to compare the clinical outcomes of parallel screw fixation with other fixation techniques.Our NMA results also showed that parallel fixation had better torsional stiffness than the others. However, parallel-screw fixation was less frequently used clinically and was evaluated and compared in only one biomechanical study35 or biodegradable37 pins are also possible options for RNF fixation. The clinical results of biodegradable pins in the treatment of comminuted radial head and neck fractures are promising36. However, our NMA showed that using metal crossed-pin fixation was biomechanically weaker, ranking the worst in torsional stiffness and ranking as inferior in bending loading. Poor performance of pins in torsional stiffness was also found in other biomechanical studies17. The biodegradable pins were not included in the present NMA. However, several biomechanical studies have shown that the biomechanical strength of biodegradable pins is inferior to that of metal screws18 and plates37 in RNF fixation. Thus, pinning fixation should be used with caution because the biomechanical strength is lower than that of plate and screw fixations.Clinically, crossed metalThe main strength of the current NMA is that it is the first study to perform multiple biomechanical comparisons between different fixation methods for RNFs regarding biomechanical strength. However, the study was subject to several limitations. First, the power of some conclusions regarding outcomes on biomechanical strength would be limited because we had a small number of included trials. Second, heterogeneity and inconsistencies existed in the present study, possibly due to the experimental designs and testing protocols, specimen types (cadaveric/synthetic radii specimens), fracture model type  and the presence of fracture comminution (comminuted/noncomminuted RNF models). However, we performed meta-regression analysis based on the specimen and fracture type, and the rankings were not changed after adjustment. Finally, we employed a fixed effect model for analysis because the trial numbers in the NMA were inadequate for random effects model estimation.The NMA indicated that fixed angle devices (blade plate and anatomic locking plate) and screw fixations may exhibit enhanced biomechanical strength in axial and bending directions, whereas cross screws demonstrated reduced torsional stability in comparison to parallel screws. It is imperative for clinicians to consider the application of these findings in constraining forces across various directions during early range of motion exercises, taking into account the distinct biomechanical properties of the respective implants.Supplementary Information.