AMPK is a mechano-metabolic sensor linking cell adhesion and mitochondrial dynamics to Myosin-dependent cell migration.

Cell migration is crucial for cancer dissemination. We find that AMP-activated protein kinase (AMPK) controls cell migration by acting as an adhesion sensing molecular hub. In 3-dimensional matrices, fast-migrating amoeboid cancer cells exert low adhesion/low traction linked to low ATP/AMP, leading to AMPK activation. In turn, AMPK plays a dual role controlling mitochondrial dynamics and cytoskeletal remodelling. High AMPK activity in low adhering migratory cells, induces mitochondrial fission, resulting in lower oxidative phosphorylation and lower mitochondrial ATP. Concurrently, AMPK inactivates Myosin Phosphatase, increasing Myosin II-dependent amoeboid migration. Reducing adhesion or mitochondrial fusion or activating AMPK induces efficient rounded-amoeboid migration. AMPK inhibition suppresses metastatic potential of amoeboid cancer cells in vivo, while a mitochondrial/AMPK-driven switch is observed in regions of human tumours where amoeboid cells are disseminating. We unveil how mitochondrial dynamics control cell migration and suggest that AMPK is a mechano-metabolic sensor linking energetics and the cytoskeleton.

Individual Limb Muscle Bundles Are Formed through Progressive Steps Orchestrated by Adjacent Connective Tissue Cells during Primary Myogenesis.

Although the factors regulating muscle cell differentiation are well described, we know very little about how differentiating muscle fibers are organized into individual muscle tissue bundles. Disruption of these processes leads to muscle hypoplasia or dysplasia, and replicating these events is vital in tissue engineering approaches. We describe the progressive cellular events that orchestrate the formation of individual limb muscle bundles and directly demonstrate the role of the connective tissue cells that surround muscle precursors in controlling these events. We show how disruption of gene activity within or genetic ablation of connective tissue cells impacts muscle precursors causing disruption of muscle bundle formation and subsequent muscle dysplasia and hypoplasia. We identify several markers of the populations of connective tissue cells that surround muscle precursors and provide a model for how matrix-modifying proteoglycans secreted by these cells may influence muscle bundle formation by effects on the local extracellular matrix (ECM) environment.

Trauma and Orthopedic Surgery Curriculum Concordance: An Operative Learning Curve Trajectory Perspective.

INTRODUCTION: Certification of completion of training in Trauma and Orthopedic (T&O) surgery in the UK requires the demonstration of operative competence in 12 index procedures, achieved through attaining a level 4 consultant-validated procedure-based assessment (PBA). The aim of this study was to evaluate the trajectory of operative learning curves related to PBA performance with respect to operative caseload and training time. DESIGN: Logbook data from consecutive 24 higher T&O trainees were compared with PBA evaluations to determine the relationship between PBA level, operative experience, training time, and indicative numbers. Learning curve gradients were calculated using trigonometry related to operative experience and training time. SETTING: A higher surgical orthopedic training program serving a single UK (Wales) Deanery. PARTICIPANTS: Twenty-four consecutive higher T&O surgery trainees. RESULTS: Median caseload to achieve level 4 competences ranged from 9 (interquartile range 6-12) for tension band wiring (olecranon or patella) to 101 (61-127) for arthroscopy, with significant variation between all 12 procedures (p < 0.001). Median number of PBAs to reach level 4 competences was 4 (2-6) with significant variation between procedures (p < 0.001). Median learning curve gradients to achieve level 4 competence for tension band wiring were 68.2° and 33.7° by caseload and training time respectively, compared with 12.2° and 45° for arthroscopy, with significant learning curve variation for all procedures related to caseload between first level 3 and first level 4 PBA (p < 0.001). Competence ratios were <1 (median 0.99, range 0.70-2.53) for 6 of the 12 indicative procedures. CONCLUSIONS: Significant learning curve trajectory variance was observed, with discrepancies between indicative operative numbers and the point at which competence was judged achieved. Numbers of index operations to achieve certification of completion of training warrant further examination.

FRCS first pass variance: deanery and specialty contrariety.

BACKGROUND: FRCS exit examination success may be interpreted as a surrogate marker for UK Deanery-related training quality. The aim of this study was to evaluate relative FRCS examination pass rates related to Deanery and Surgical Specialty. METHODS: Joint Committee on Surgical Training-published examination first attempt pass rates were scrutinised for type I higher surgical trainees and outcomes compared related to Deanery and Surgical Specialty. RESULTS: Of 9363 FRCS first attempts, 3974 were successful (42.4%). Median and mean pass rates related to Deanery were 42.1% and 30.7%, respectively, and ranged from 26.7% to 45.6%. Median (range) pass rates by specialty were urology 76.3% (60%-100%), trauma and orthopaedic surgery 74.7% (58.2%-100%), general surgery 70.0% (63.1%-86%), ENT 62.5% (50%-100%), cardiothoracic surgery 50.0% (25%-100%), oral and maxillofacial surgery 50% (40.0%-100%), neurosurgery 50% (22.7%-100%), plastic surgery 47.6% (30.0%-100%) and paediatric surgery 25% (16.7%-100%). Significant variance was observed across all specialties and deaneries (p=0.001). CONCLUSION: As much as threefold variance exists related to FRCS examination first attempt success, trainees should be aware of this spectrum when preferencing deaneries during national selection.

Rural Rotations at Core: Rarefied Exposure or Real Experience?

OBJECTIVE: Surgical rotations involving rural General Hospitals (rGH) are frequently associated with recruitment challenges, partly because of adverse perceptions regarding distances from social support networks and training opportunities. The aim of this study was to determine the outcomes of core surgical training rotations involving rGHs when compared with urban hospitals in a single UK Deanery. DESIGN: Online Intercollegiate Surgical Curriculum Programme portfolios from 163 core surgical trainees (CST) were examined related to postlocation, operative experience, workplace-based assessments, and academic achievement. Of the 163 CSTs, 27 had completed at least 50% of their 2-year training posts at rGHs and were compared with 136 control CSTs completing rotations in urban general and teaching hospitals (uGH). The primary outcome measures were MRCS pass rate and success at national ST3 selection. SETTING: A core surgical training program serving a single UK Deanery. PARTICIPANTS: Consecutive 177 CSTs appointed to a single UK Deanery between 2010 and 2016. RESULTS: Success at MRCS and national ST3 selection were similar for CSTs from rGH vs uGH rotations-MRCS success: 70.4 vs 72.8% (p = 0.816), and ST3 success: 22.2% vs 27.0% (p = 0.811). Median rGH vs uGH curriculum-based outcomes were operative case load: 378 vs 422 (p = 0.300); workplace-based assessments completed: 79 vs 94 (p = 0.499); audits performed: 4 vs 4 (p = 0.966); learned society communications: 1 vs 2 (p = 0.020); and scientific publications: 0 vs 0 (p = 0.478). CONCLUSION: CST rotations including rGHs produced a different spectrum of training experience compared with uGH rotations but overall primary outcomes were similar.

A novel role for atypical MAPK kinase ERK3 in regulating breast cancer cell morphology and migration.

ERK3 is an atypical Mitogen-activated protein kinase (MAPK6). Despite the fact that the Erk3 gene was originally identified in 1991, its function is still unknown. MK5 (MAP kinase- activated protein kinase 5) also called PRAK is the only known substrate for ERK3. Recently, it was found that group I p21 protein activated kinases (PAKs) are critical effectors of ERK3. PAKs link Rho family of GTPases to actin cytoskeletal dynamics and are known to be involved in the regulation of cell adhesion and migration. In this study we demonstrate that ERK3 protein levels are elevated as MDA-MB-231 breast cancer cells adhere to collagen I which is concomitant with changes in cellular morphology where cells become less well spread following nascent adhesion formation. During this early cellular adhesion event we observe that the cells retain protrusive activity while reducing overall cellular area. Interestingly exogenous expression of ERK3 delivers a comparable reduction in cell spread area, while depletion of ERK3 expression increases cell spread area. Importantly, we have detected a novel specific endogenous ERK3 localization at the cell periphery. Furthermore we find that ERK3 overexpressing cells exhibit a rounded morphology and increased cell migration speed. Surprisingly, exogenous expression of a kinase inactive mutant of ERK3 phenocopies ERK3 overexpression, suggesting a novel kinase independent function for ERK3. Taken together our data suggest that as cells initiate adhesion to matrix increasing levels of ERK3 at the cell periphery are required to orchestrate cell morphology changes which can then drive migratory behavior.

Phosphoregulation of the titin-cap protein telethonin in cardiac myocytes.

Telethonin (also known as titin-cap or t-cap) is a muscle-specific protein whose mutation is associated with cardiac and skeletal myopathies through unknown mechanisms. Our previous work identified cardiac telethonin as an interaction partner for the protein kinase D catalytic domain. In this study, kinase assays used in conjunction with MS and site-directed mutagenesis confirmed telethonin as a substrate for protein kinase D and Ca(2+)/calmodulin-dependent kinase II in vitro and identified Ser-157 and Ser-161 as the phosphorylation sites. Phosphate affinity electrophoresis and MS revealed endogenous telethonin to exist in a constitutively bis-phosphorylated form in isolated adult rat ventricular myocytes and in mouse and rat ventricular myocardium. Following heterologous expression in myocytes by adenoviral gene transfer, wild-type telethonin became bis-phosphorylated, whereas S157A/S161A telethonin remained non-phosphorylated. Nevertheless, both proteins localized predominantly to the sarcomeric Z-disc, where they partially replaced endogenous telethonin. Such partial replacement with S157A/S161A telethonin disrupted transverse tubule organization and prolonged the time to peak of the intracellular Ca(2+) transient and increased its variance. These data reveal, for the first time, that cardiac telethonin is constitutively bis-phosphorylated and suggest that such phosphorylation is critical for normal telethonin function, which may include maintenance of transverse tubule organization and intracellular Ca(2+) transients.

WIP regulates the stability and localization of WASP to podosomes in migrating dendritic cells.

The Wiskott-Aldrich Syndrome protein (WASP) is an adaptor protein that is essential for podosome formation in hematopoietic cells. Given that 80% of identified Wiskott-Aldrich Syndrome patients result from mutations in the binding site for WASP-interacting-protein (WIP), we examined the possible role of WIP in the regulation of podosome architecture and cell motility in dendritic cells (DCs). Our results show that WIP is essential both for the formation of actin cores containing WASP and cortactin and for the organization of integrin and integrin-associated proteins in circular arrays, specific characteristics of podosome structure. We also found that WIP is essential for the maintenance of the high turnover of adhesions and polarity in DCs. WIP exerts these functions by regulating calpain-mediated cleavage of WASP and by facilitating the localization of WASP to sites of actin polymerization at podosomes. Taken together, our results indicate that WIP is critical for the regulation of both the stability and localization of WASP in migrating DCs and suggest that WASP and WIP operate as a functional unit to control DC motility in response to changes in the extracellular environment.

Role of vinculin in regulating focal adhesion turnover.

Although vinculin (-/-) mouse embryo fibroblasts assemble focal adhesions (FAs), they spread more slowly, less extensively, and close a wound more rapidly than vinculin (+/+) cells. To investigate the structure and dynamics of FAs in these cells, we used real-time interference reflection microscopy (IRM) thus avoiding the need to express exogenous GFP-tagged FA proteins which may be misregulated. This showed that the FAs were smaller, less abundant and turned over more rapidly in vinculin null compared to wild-type cells. Expression of vinculin rescued the spreading defect and resulted in larger and more stable FAs. Phosphatidylinositol 4,5-bisphosphate (PIP2) is thought to play a role in vinculin activation by relieving an intramolecular association between the vinculin head (Vh) and tail (Vt) that masks the ligand binding sites in Vh and Vt. To investigate the role of the vinculin/PIP2 interaction in FA dynamics, we used a vinculin mutant lacking the C-terminal arm (residues 1053-1066) and referred to as the deltaC mutation. This mutation reduced PIP2 binding to a Vt deltaC polypeptide by >90% compared to wild type without affecting binding to Vh or F-actin. Interestingly, cells expressing the vinculin deltaC mutant assembled remarkably stable FAs. The results suggest that vinculin inhibits cell migration by stabilising FAs, and that binding of inositol phospholipids to Vt plays an important role in FA turnover.

PTEN couples Sema3A signalling to growth cone collapse.

Distinct changes in glycogen synthase kinase-3 (GSK-3) signalling can regulate neuronal morphogenesis including the determination and maintenance of axonal identity, and are required for neurotrophin-mediated axon elongation. In addition, we have previously shown a dependency on GSK-3 activation in the semaphorin 3A (Sema3A)-mediated growth-cone-collapse response of sensory neurons. Regulation of GSK-3 activity involves the intermediate signalling lipid phosphatidylinositol 3,4,5-trisphosphate, which can be modulated by phosphatidylinositol 3-kinase (PI3K) and the tumour suppressor PTEN. We report here the involvement of PTEN in the Sema3A-mediated growth cone collapse. Sema3A suppresses PI3K signalling concomitant with the activation of GSK-3, which depends on the phosphatase activity of PTEN. PTEN is highly enriched in the axonal compartment and the central domain of sensory growth cones during axonal extension, where it colocalises with microtubules. Following exposure to Sema3A, PTEN accumulates rapidly at the growth cone membrane suggesting a mechanism by which PTEN couples Sema3A signalling to growth cone collapse. These findings demonstrate a dependency on PTEN to regulate GSK-3 signalling in response to Sema3A and highlight the importance of subcellular distributions of PTEN to control growth cone behaviour.

Vinculin acts as a sensor in lipid regulation of adhesion-site turnover.

The dynamics of cell adhesion sites control cell morphology and motility. Adhesion-site turnover is thought to depend on the local availability of the acidic phospholipid phosphatidylinositol-4,5-bisphosphate (PIP(2)). PIP(2) can bind to many cell adhesion proteins such as vinculin and talin, but the consequences of this interaction are poorly understood. To study the significance of phospholipid binding to vinculin for adhesion-site turnover and cell motility, we constructed a mutant, vinculin-LD, deficient in acidic phospholipid binding yet with functional actin-binding sites. When expressed in cells, vinculin-LD was readily recruited to adhesion sites, as judged by fluorescence recovery after photobleaching (FRAP) analysis, but cell spreading and migration were strongly impaired, and PIP(2)-dependent disassembly of adhesions was suppressed. Thus, PIP(2) binding is not essential for vinculin activation and recruitment, as previously suggested. Instead, we propose that PIP(2) levels can regulate the uncoupling of adhesion sites from the actin cytoskeleton, with vinculin functioning as a sensor.

Coronary ligament rupture as a cause of medial knee pain.

Three cases of a meniscal injury variant are presented, the signs and symptoms of which imitate meniscal tear, but that required no definitive intervention and resolved with conservative management. We include a review of the literature on these injuries. Three patients attended clinic giving a history and exhibiting symptoms suggestive of medial meniscal injury. Symptoms were severe and of long enough duration to warrant arthroscopic examination of the knees. These patients were found to have coronary ligament ruptures. All the patients were treated conservatively. The pain resolved in all cases over a few months. No patient required a second arthroscopy. The patients were followed up for 9 months in 2 cases and 2 years in 1 case. By final follow-up examination, all patients were symptom free. Meniscal cartilage tear is the most common injury to the knee requiring surgery. Standard practice is to diagnose meniscal tear based on history and clinical evaluation, and to proceed to arthroscopy if severity of symptoms warrants intervention. Although coronary ligament rupture is reported in the literature, these reports have been, in the main, arthrographic diagnoses. Three case reports with arthroscopic illustration are presented.

Rapid actin transport during cell protrusion.

Transformed rat fibroblasts expressing two variants of green fluorescent protein, each fused to beta-actin, were used to study actin dynamics during cell protrusion. The recently developed FLAP (fluorescence localization after photobleaching) method permits the tracking of one fluorophore after localized photobleaching by using the other as a colocalized reference. Here, by visualizing the ratio of bleached to total molecules, we found that actin was delivered to protruding zones of the leading edge of the cell at speeds that exceeded 5 micrometers per second. Monte Carlo modeling confirmed that this flow cannot be explained by diffusion and may involve active transport.