The MYO6 interactome reveals adaptor complexes coordinating early endosome and cytoskeletal dynamics.

The intracellular functions of myosin motors requires a number of adaptor molecules, which control cargo attachment, but also fine-tune motor activity in time and space. These motor-adaptor-cargo interactions are often weak, transient or highly regulated. To overcome these problems, we use a proximity labelling-based proteomics strategy to map the interactome of the unique minus end-directed actin motor MYO6. Detailed biochemical and functional analysis identified several distinct MYO6-adaptor modules including two complexes containing RhoGEFs: the LIFT (LARG-Induced F-actin for Tethering) complex that controls endosome positioning and motility through RHO-driven actin polymerisation; and the DISP (DOCK7-Induced Septin disPlacement) complex, a novel regulator of the septin cytoskeleton. These complexes emphasise the role of MYO6 in coordinating endosome dynamics and cytoskeletal architecture. This study provides the first in vivo interactome of a myosin motor protein and highlights the power of this approach in uncovering dynamic and functionally diverse myosin motor complexes.

Filopodia formation and endosome clustering induced by mutant plus-end-directed myosin VI.

Myosin VI (MYO6) is the only myosin known to move toward the minus end of actin filaments. It has roles in numerous cellular processes, including maintenance of stereocilia structure, endocytosis, and autophagosome maturation. However, the functional necessity of minus-end-directed movement along actin is unclear as the underlying architecture of the local actin network is often unknown. To address this question, we engineered a mutant of MYO6, MYO6+, which undergoes plus-end-directed movement while retaining physiological cargo interactions in the tail. Expression of this mutant motor in HeLa cells led to a dramatic reorganization of cortical actin filaments and the formation of actin-rich filopodia. MYO6 is present on peripheral adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) signaling endosomes and MYO6+ expression causes a dramatic relocalization and clustering of this endocytic compartment in the cell cortex. MYO6+ and its adaptor GAIP interacting protein, C terminus (GIPC) accumulate at the tips of these filopodia, while APPL1 endosomes accumulate at the base. A combination of MYO6+ mutagenesis and siRNA-mediated depletion of MYO6 binding partners demonstrates that motor activity and binding to endosomal membranes mediated by GIPC and PI(4,5)P2 are crucial for filopodia formation. A similar reorganization of actin is induced by a constitutive dimer of MYO6+, indicating that multimerization of MYO6 on endosomes through binding to GIPC is required for this cellular activity and regulation of actin network structure. This unique engineered MYO6+ offers insights into both filopodia formation and MYO6 motor function at endosomes and at the plasma membrane.

Hyperbaric oxygen therapy for the prevention of arterial gas embolism in food grade hydrogen peroxide ingestion.

Food grade hydrogen peroxide ingestion is a relatively rare presentation to the emergency department. There are no defined guidelines at this time regarding the treatment of such exposures, and providers may not be familiar with the potential complications associated with high concentration hydrogen peroxide ingestions. In this case series, we describe four patients who consumed 35% hydrogen peroxide, presented to the emergency department, and were treated with hyperbaric oxygen therapy. Two of the four patients were critically ill requiring intubation. All four patients had evidence on CT or ultrasound of venous gas emboli and intubated patients were treated as if they had an arterial gas embolism since an exam could not be followed. After hyperbaric oxygen therapy each patient was discharged from the hospital neurologically intact with no other associated organ injuries related to vascular gas emboli. Hyperbaric oxygen therapy is an effective treatment for patients with vascular gas emboli after high concentration hydrogen peroxide ingestion. It is the treatment of choice for any impending, suspected, or diagnosed arterial gas embolism. Further research is needed to determine which patients with portal venous gas emboli should be treated with hyperbaric oxygen therapy.

Myosins: Domain Organisation, Motor Properties, Physiological Roles and Cellular Functions.

Myosins are cytoskeletal motor proteins that use energy derived from ATP hydrolysis to generate force and movement along actin filaments. Humans express 38 myosin genes belonging to 12 classes that participate in a diverse range of crucial activities, including muscle contraction, intracellular trafficking, cell division, motility, actin cytoskeletal organisation and cell signalling. Myosin malfunction has been implicated a variety of disorders including deafness, hypertrophic cardiomyopathy, Usher syndrome, Griscelli syndrome and cancer. In this chapter, we will first discuss the key structural and kinetic features that are conserved across the myosin family. Thereafter, we summarise for each member in turn its unique functional and structural adaptations, cellular roles and associated pathologies. Finally, we address the broad therapeutic potential for pharmacological interventions that target myosin family members.

Plasma membrane tension orchestrates membrane trafficking, cytoskeletal remodeling, and biochemical signaling during phagocytosis.

Phagocytes clear the body of undesirable particles such as infectious agents and debris. To extend pseudopods over the surface of targeted particles during engulfment, cells must change shape through extensive membrane and cytoskeleton remodeling. We observed that pseudopod extension occurred in two phases. In the first phase, pseudopods extended rapidly, with actin polymerization pushing the plasma membrane forward. The second phase occurred once the membrane area from preexisting reservoirs was depleted, leading to increased membrane tension. Increased tension directly altered the small Rho GTPase Rac1, 3'-phosphoinositide, and cytoskeletal organization. Furthermore, it activated exocytosis of vesicles containing GPI-anchored proteins, increasing membrane area and phagocytosis efficiency for large particles. We thus propose that, during phagocytosis, membrane remodeling, cytoskeletal organization, and biochemical signaling are orchestrated by the mechanical signal of membrane tension. These results put a simple mechanical signal at the heart of understanding immunological responses.

Primary Repair of Ear Avulsion with Adjuvant Hyperbaric Therapy and Nitroglycerin Ointment.

Traumatic auricular avulsion is a rare and deforming injury. Classically, repair has required microvascular anastomosis. In this publication, we report two separate cases of pediatric auricular avulsion from dog bites. In both cases, the ear was cleaned and surgically reattached. Adjunctive therapies included hyperbaric oxygen and nitroglycerin ointment. There was complete graft take for one patient and 90% graft take for the second, both achieving satisfactory aesthetic outcome. These unique cases highlight the benefits of surgical reattachment of the avulsed portion of the ear followed by hyperbaric oxygen therapy and nitroglycerin ointment.

Restricted state selection in fluorescent protein Förster resonance energy transfer.

The measurement of donor lifetime modification by Förster resonance energy transfer (FRET) is a widely used tool for detecting protein-protein interactions and protein conformation change. Such measurements can be compromised by the presence of a significant noninteracting fraction of molecules. Combining time-resolved intensity and anisotropy measurements gives access to both molecular distance and orientation. Fluorescent proteins frequently used to detect energy transfer in biological systems often exhibit decay characteristics indicative of more than one excited state. However, little attention has thus far been given to the specific modes of energy transfer, in particular, which states are predominantly coupled. Here, we use a previously characterized dimerization system to study energy transfer between EGFP and mCherry. Optically excited EGFP and mCherry both exhibit biexponential decays, and FRET should therefore involve dipole-dipole transfer between these four states. Analysis of the sensitized fluorescence anisotropy and intensity decays indicates that FRET transfer is predominantly from the shorter lived EGFP emitting state (2.43 ns) to the longer lived (ca. 2.77 ns) minority component (ca. 16%) of the optically excited mCherry emission. This high degree of state selection between these two widely used FRET pairs highlights the fundamental differences that can arise between direct optical excitation of an isotropic molecular population and dipole-dipole coupling in a far from isotropic interaction geometry and has consequences regarding the accurate interpretation of fluorescent protein FRET data.

Bedside Fluorescence Microangiography for Frostbite Diagnosis in the Emergency Department.

INTRODUCTION: Frostbite leads to progressive ischemia eventually causing tissue necrosis if not quickly reversed. Patients with frostbite tend to present to the emergency department (ED) for assessment and treatment. Acute management includes rewarming, pain management, and (when indicated) thrombolytic therapy. Thrombolytic therapy in severe frostbite injury may decrease rates of amputation and improve patient outcomes. Fluorescence microangiography (FMA) has been used to distinguish between perfused and non-perfused tissue. The purpose of this study was to evaluate the potential role of FMA in the acute care of patients with frostbite, specifically its role as a tool to identify perfusion deficit following severe frostbite injury, and to explore its role in time to tissue plasminogen activator (tPA). METHODS: This retrospective analysis included all patients from December 2020-March 2021 who received FMA in a single ED as part of their initial frostbite evaluation. In total, 42 patients presented to the ED with concern for frostbite and were evaluated using FMA. RESULTS: Mean time from arrival in the ED to FMA was 46.3 minutes. Of the 42 patients, 14 had clinically significant perfusion deficits noted on FMA and received tPA. Mean time to tPA (measured from ED arrival to administration of tPA) for these patients was 117.4 minutes. This is significantly faster than average historical times at our institution of 240-300 minutes. CONCLUSION: Bedside FMA provides objective information regarding perfusion deficits and allows for faster decision-making and improved times to tPA. Fluorescence microangiography shows promise for quick and efficient evaluation of perfusion deficits in frostbite-injured patients. This could lead to faster tPA administration and potentially greater rates of tissue salvage after severe frostbite injury.

An Institutional Protocol for the Treatment of Severe Frostbite Injury-A 6-Year Retrospective Analysis.

The treatment of severe frostbite injury has undergone rapid development in the past 30 years with many different diagnostic and treatment options now available. However, there is currently no consensus on the best method for management of this disease process. At our institution, we have designed a protocol for severe frostbite injury that includes diagnosis, medical treatment, wound cares, therapy, and surgery. This study assess the efficacy of our treatment since its implementation six years ago. During this time, all patients with severe frostbite injury were included in prospective observational trial of the protocol. We found that this protocol results in significant tissue salvage with over 80.7% of previously ischemic tissue becoming viable and not requiring amputation. We also were able to improve our center's efficiency over the course of six years and now our current average time from rapid rewarming to delivery of thrombolytics is under six hours.

CASE SERIES OF HYPERBARIC OXYGEN THERAPY FOR CENTRAL RETINAL ARTERY OCCLUSION.

PURPOSE: To retrospectively report the outcomes of patients presenting to our facility with central retinal artery occlusion and receiving therapy with hyperbaric oxygen (HBO). METHODS: This was a retrospective, chart review at a single hospital center. Patients with diagnosed central retinal artery occlusion were treated with HBO twice daily for 5 days during their inpatient stay for a total of 10 HBO treatments. Main outcome was change from the documented presenting best-corrected visual acuity to discharge best-corrected visual acuity. Thirty-nine patients with central retinal artery occlusion were included in the analysis during a 30-month period. RESULTS: Twenty-eight of 39 patients (72%) had some improvement in acuity. There was a mean of 5.05 lines of improvement using a modified Snellen chart after completing their HBO treatment course. Patients treated within 12 hours of symptom onset showed the greatest improvement in their visual acuity (6.11 mean lines of improvement). Complications of therapy included middle ear barotrauma (10/39) and confinement anxiety (1/39) and did not interfere with the therapy regimen or hospital course. CONCLUSION: This retrospective case series supports the use of emergent HBO therapy as a viable treatment option for patients with central retinal artery occlusion. Hyperbaric oxygen therapy was safely administered and well tolerated.

Availability of Bedside and Laboratory Testing for Carbon Monoxide Poisoning in the Upper Midwestern United States.

INTRODUCTION: The objective of this study was to assess the ability to test patients for carbon monoxide (CO) exposure in all hospitals in three United States (U.S.) Midwestern states. METHODS: We surveyed hospitals in three states. Telephone queries assessed processes for measuring carboxyhemoglobin, including capacity for real-time vs send-out testing. Facilities were separated based on their location's population size for further analysis. Descriptive statistics are reported. RESULTS: Of the 250 hospitals queried, we ultimately excluded 25. Nearly all (220, 97.8%) reported a process in place to test for CO exposure. Over 40% (n=92) lacked real-time testing. Testing ability was positively associated with increasing population size quartile (range 32.6% - 100%). Hospitals in the lowest-quartile population centers were more likely to report that they were unable to test in real time than those in the largest-quartile population centers (67.4% vs 0%). CONCLUSION: In a large geographic region encompassing three states, hospital-based and real-time capacity to test for CO exposure is not universal. Hospitals in smaller population areas are more likely to lack real-time testing or any testing at all. This may have significant public health, triage, and referral implications for patients.

Esophageal Stricture Following Radiation, Concurrent Immunochemotherapy, Treated With Hyperbaric Oxygen and Dilation.

Low-dose palliative radiation may offer symptomatic relief in patients with spinal metastases from primary renal cell cancer and is unlikely to result in radiation injury. Patients with advanced malignancy requiring palliative radiation are often also receiving chemotherapy. Synergistic adverse effects resulting from combined palliative radiation and novel antiprogrammed cell death-1 (anti-PD 1) and/or multityrosine kinase inhibitors are rare. We report about a 60-year-old woman with metastatic clear-cell renal cancer, status post-left nephrectomy, with debilitating mid-back pain from metastatic tumor burden and foraminal nerve compression. Her chemotherapeutic regimen was repeatedly altered because of progression of disease until she was maintained on the anti-PD 1 checkpoint inhibitor, nivolumab. She received palliative radiation to her thoracic spine over a 2-week period, and nivolumab was then switched to cabozantinib midway through a course of palliative radiation. The patient rapidly developed severe esophagitis, progressing to esophageal stricture, and required placement of a percutaneous endoscopic gastrostomy tube. She was successfully treated with serial esophageal dilation and hyperbaric oxygen treatments to diminish inflammation and improve tissue vascularity. Concurrent use of anti-PD 1 and/or multityrosine kinase drugs may accelerate development of radiation injury regardless of radiation dosage. Radiation-induced esophageal stricture was managed successfully in this patient with serial esophageal dilation and adjuvant hyperbaric oxygen.

Microangiography: An Alternative Tool for Assessing Severe Frostbite Injury.

Assessment of frostbite injury typically relies on computed tomography, angiography, or nuclear medicine studies to detect perfusion deficits prior to thrombolytic therapy. The aim of this study was to evaluate the potential of a novel imaging method, microangiography, in the assessment of severe frostbite injury. Patients with severe frostbite were included if they received a post-thrombolytic Technetium 99 (Tc99) bone scan, a Tc99 bone scan without thrombolytic therapy, and/or post-thrombolytic microangiography (MA) study. We included all patients from the years 2006 to 2018 with severe frostbite injury who had received appropriate imaging for diagnosis: Tc99 scan alone (N = 82), microangiography alone (N = 22), and both Tc99 and microangiography (N = 26). The majority of patients received thrombolytic therapy (76.2%), and the average time to thrombolytics was 6.9 hours. Tc99 scans showed strong correlation with amputation level (r = .836, P < .001), and microangiography showed a slightly stronger positive correlation with amputation level (r = .870, P < .001). In the subset who received both Tc99 scan and microangiography (N = 26), we observed significant differences in the mean scores of perfusion deficit (z = 3.20, P < .001). In this subset, a moderate correlation was found between level of perfusion deficit on Tc99 bone scan and amputation level (r = .525, P = .006). A very strong positive correlation was found between the microangiography studies and the amputation level (r = .890, P < .001). These results demonstrate that microangiography is a reliable alternative method of assessing severe frostbite injury and predicting amputation level.

Microangiography to Monitor Treatment Outcomes Following Severe Frostbite Injury to the Hands.

Frostbite injury causes direct damage to tissues following exposure to temperatures below their freezing point causing tissue death potentially leading to serious amputations. After rewarming, a variety of treatment options have been employed to avoid amputation. This case report details the use of indocyanine green fluorescence microangiography to monitor the clinical progression of perfusion following hyperbaric oxygen therapy (HBOT) for severe frostbite injury. We present a case report of a man with deep frostbite of the bilateral hands treated with thrombolytics and HBOT. After rewarming, the patient received thrombolytics shortly after arrival and then went on to be treated with HBOT on hospital day 5. Patient's healing progress was monitored using serial microangiography. Microangiography evaluation was performed on day 6 and then weekly to track treatment progress. A more uniform brightness appears in his left hand by completion of his therapies, consistent with normal perfusion. The dark ischemic areas in the right hand receded in digits 1 to 3 and appeared normalized in the fourth digit. The patient received a total of 20 HBO treatments. After completion of therapy, the patient went on to have a partial amputation of his first, second, and third fingers on his right hand. Our case report demonstrates serial microangiography to monitor a frostbite patient's progress during HBOT and provided additional information allowing us to plan duration of treatments. Our case report describes the role that microangiography may serve in monitoring patient progress following severe frostbite injury.

Myosin VI-Dependent Actin Cages Encapsulate Parkin-Positive Damaged Mitochondria.

Mitochondrial quality control is essential to maintain cellular homeostasis and is achieved by removing damaged, ubiquitinated mitochondria via Parkin-mediated mitophagy. Here, we demonstrate that MYO6 (myosin VI), a unique myosin that moves toward the minus end of actin filaments, forms a complex with Parkin and is selectively recruited to damaged mitochondria via its ubiquitin-binding domain. This myosin motor initiates the assembly of F-actin cages to encapsulate damaged mitochondria by forming a physical barrier that prevents refusion with neighboring populations. Loss of MYO6 results in an accumulation of mitophagosomes and an increase in mitochondrial mass. In addition, we observe downstream mitochondrial dysfunction manifesting as reduced respiratory capacity and decreased ability to rely on oxidative phosphorylation for energy production. Our work uncovers a crucial step in mitochondrial quality control: the formation of MYO6-dependent actin cages that ensure isolation of damaged mitochondria from the network.

MYO6 Regulates Spatial Organization of Signaling Endosomes Driving AKT Activation and Actin Dynamics.

APPL1- and RAB5-positive signaling endosomes play a crucial role in the activation of AKT in response to extracellular stimuli. Myosin VI (MYO6) and two of its cargo adaptor proteins, GIPC and TOM1/TOM1L2, localize to these peripheral endosomes and mediate endosome association with cortical actin filaments. Loss of MYO6 leads to the displacement of these endosomes from the cell cortex and accumulation in the perinuclear space. Depletion of this myosin not only affects endosome positioning, but also induces actin and lipid remodeling consistent with endosome maturation, including accumulation of F-actin and the endosomal lipid PI(3)P. These processes acutely perturb endosome function, as both AKT phosphorylation and RAC-dependent membrane ruffling were markedly reduced by depletion of either APPL1 or MYO6. These results place MYO6 and its binding partners at a central nexus in cellular signaling linking actin dynamics at the cell surface and endosomal signaling in the cell cortex.

F-actin waves, actin cortex disassembly and focal exocytosis driven by actin-phosphoinositide positive feedback.

Actin polymerization is controlled by the phosphoinositide composition of the plasma membrane. However, the molecular mechanisms underlying the spatiotemporal regulation of actin network organization over extended length scales are still unclear. To observe phosphoinositide-dependent cytoskeletal dynamics we combined the model system of frustrated phagocytosis, total internal reflection microscopy and manipulation of the buffer tonicity. We found that macrophages interacting with IgG-coated glass substrates formed circular F-actin waves on their ventral surface enclosing a region of plasma membrane devoid of cortical actin. Plasma membrane free of actin cortex was strongly depleted of PI(4,5)P2 , but enriched in PI(3,4)P2 and displayed a fivefold increase in exocytosis. Wave formation could be promoted by application of a hypotonic shock. The actin waves were characteristic of a bistable wavefront at the boundary between the regions of membrane containing and lacking cortical actin. Phosphoinositide modifiers and RhoGTPase activities dramatically redistributed with respect to the wavefronts, which often exhibited spatial oscillations. Perturbation of either lipid or actin cytoskeleton-related pathways led to rapid loss of both the polarized lipid distribution and the wavefront. As waves travelled over the plasma membrane, wavefront actin was seen to rapidly polymerize and depolymerize at pre-existing clusters of FcγRIIA, coincident with rapid changes in lipid composition. Thus the potential of receptors to support rapid F-actin polymerization appears to depend acutely on the local concentrations of multiple lipid species. We propose that interdependence through positive feedback from the cytoskeleton to lipid modifiers leads to coordinated local cortex remodeling, focal exocytosis, and organizes extended actin networks.

Changes in the preserved heart that limit the length of preservation.

BACKGROUND: The variability in donor heart viability following preservation is well known and very dependent on the animal model used and the methods of preservation. Using hypothermia (2 to 4 degrees C) with the University of Wisconsin solution and microperfusion (3 microl/g/min) myocardial functional recovery after reperfusion is 50% to 60% after 18 hours of preservation. Many investigators have found the 18-hour preservation barrier for functional recovery of the heart with different experimental protocols. In previous studies, we have found in normothermic global ischemia that with necrosis there was a significant amount of apoptosis. It was therefore the purpose of this study to investigate the importance of apoptosis in heart preservation and determine if this cellular disruption plays a part in limiting heart viability during recovery from preservation. METHODS: Using a heterotopic heart transplant model, myocardial function (end-systolic elastance), high energy phosphate changes, metabolic substrate utilization, electron microscopy to determine degree of injury, and histologic changes that included apoptosis and lamin B(1) analyses were conducted on canine hearts preserved for 18 hours and reperfused for 6 hours. The changes were compared with a control group with no preservation. RESULTS: The 18-hour preserved heart regained approximately 50% to 60% of its original function, with significant decreases in adenosine triphosphate, no irreversible cellular changes (necrosis), and a 6% to 8% increase in apoptotic myocytes with a concomitant 8% decrease in lamin B(1). CONCLUSIONS: Preserved hearts can be maintained for approximately 12 hours with functional recovery near normal. However, approaching 18 hours, there is an attenuated functional response which may be related to the to development of apoptosis.