Dynamic remodeling of TRPC5 channel-caveolin-1-eNOS protein assembly potentiates the positive feedback interaction between Ca2+ and NO signals.

The cell signaling molecules nitric oxide (NO) and Ca2+ regulate diverse biological processes through their closely coordinated activities directed by signaling protein complexes. However, it remains unclear how dynamically the multi-component protein assemblies behave within the signaling complexes upon the interplay between NO and Ca2+ signals. Here we demonstrate that TRPC5 channels activated by stimulation of G-protein-coupled ATP receptors mediate Ca2+ influx, that triggers NO production from endothelial NO synthase (eNOS), inducing secondary activation of TRPC5 via cysteine S-nitrosylation and eNOS in vascular endothelial cells. Mutations in the caveolin-1-binding domains of TRPC5 disrupt its association with caveolin-1 and impair Ca2+ influx and NO production, suggesting that caveolin-1 serves primarily as the scaffold for TRPC5 and eNOS to assemble into the signal complex. Interestingly, during ATP receptor activation, eNOS is dissociated from caveolin-1 and in turn directly associates with TRPC5, which accumulates at the plasma membrane dependently on Ca2+ influx and calmodulin (CaM). This protein reassembly likely results in a relief of eNOS from the inhibitory action of caveolin-1 and an enhanced TRPC5 S-nitrosylation by eNOS localized in the proximity, thereby facilitating the secondary activation of Ca2+ influx and NO production. In isolated rat aorta, vasodilation induced by acetylcholine was significantly suppressed by the TRPC5 inhibitor AC1903. Thus, our study provides evidence that dynamic remodeling of the protein assemblies among TRPC5, eNOS, caveolin-1, and CaM determines the ensemble of Ca2+ mobilization and NO production in vascular endothelial cells.

N-terminal domain on dystroglycan enables LARGE1 to extend matriglycan on α-dystroglycan and prevents muscular dystrophy.

Dystroglycan (DG) requires extensive post-translational processing and O-glycosylation to function as a receptor for extracellular matrix (ECM) proteins containing laminin-G (LG) domains. Matriglycan is an elongated polysaccharide of alternating xylose (Xyl) and glucuronic acid (GlcA) that binds with high affinity to ECM proteins with LG domains and is uniquely synthesized on α-dystroglycan (α-DG) by like-acetylglucosaminyltransferase-1 (LARGE1). Defects in the post-translational processing or O-glycosylation of α-DG that result in a shorter form of matriglycan reduce the size of α-DG and decrease laminin binding, leading to various forms of muscular dystrophy. Previously, we demonstrated that protein O-mannose kinase (POMK) is required for LARGE1 to generate full-length matriglycan on α-DG (~150-250 kDa) (Walimbe et al., 2020). Here, we show that LARGE1 can only synthesize a short, non-elongated form of matriglycan in mouse skeletal muscle that lacks the DG N-terminus (α-DGN), resulting in an ~100-125 kDa α-DG. This smaller form of α-DG binds laminin and maintains specific force but does not prevent muscle pathophysiology, including reduced force production after eccentric contractions (ECs) or abnormalities in the neuromuscular junctions. Collectively, our study demonstrates that α-DGN, like POMK, is required for LARGE1 to extend matriglycan to its full mature length on α-DG and thus prevent muscle pathophysiology.

The mechanosensitive ion channel PIEZO1 promotes satellite cell function in muscle regeneration.

Muscle satellite cells (MuSCs), myogenic stem cells in skeletal muscles, play an essential role in muscle regeneration. After skeletal muscle injury, quiescent MuSCs are activated to enter the cell cycle and proliferate, thereby initiating regeneration; however, the mechanisms that ensure successful MuSC division, including chromosome segregation, remain unclear. Here, we show that PIEZO1, a calcium ion (Ca2+)-permeable cation channel activated by membrane tension, mediates spontaneous Ca2+ influx to control the regenerative function of MuSCs. Our genetic engineering approach in mice revealed that PIEZO1 is functionally expressed in MuSCs and that Piezo1 deletion in these cells delays myofibre regeneration after injury. These results are, at least in part, due to a mitotic defect in MuSCs. Mechanistically, this phenotype is caused by impaired PIEZO1-Rho signalling during myogenesis. Thus, we provide the first concrete evidence that PIEZO1, a bona fide mechanosensitive ion channel, promotes proliferation and regenerative functions of MuSCs through precise control of cell division.

Regulation of thermoregulatory behavior by commensal bacteria in Drosophila.

Commensal bacteria affect many aspects of host physiology. In this study, we focused on the role of commensal bacteria in the thermoregulatory behavior of Drosophila melanogaster. We demonstrated that the elimination of commensal bacteria caused an increase in the preferred temperature of Drosophila third-instar larvae without affecting the activity of transient receptor potential ankyrin 1 (TRPA1)-expressing thermosensitive neurons. We isolated eight bacterial strains from the gut and culture medium of conventionally reared larvae and found that the preferred temperature of the larvae was decreased by mono-association with Lactobacillus plantarum or Corynebacterium nuruki. Mono-association with these bacteria did not affect the indices of energy metabolism such as ATP and glucose levels of larvae, which are closely linked to thermoregulation in animals. Thus, we show a novel role for commensal bacteria in host thermoregulation and identify two bacterial species that affect thermoregulatory behavior in Drosophila.

Piezo1 activation using Yoda1 inhibits macropinocytosis in A431 human epidermoid carcinoma cells.

Macropinocytosis is a type of endocytosis accompanied by actin rearrangement-driven membrane deformation, such as lamellipodia formation and membrane ruffling, followed by the formation of large vesicles, macropinosomes. Ras-transformed cancer cells efficiently acquire exogenous amino acids for their survival through macropinocytosis. Thus, inhibition of macropinocytosis is a promising strategy for cancer therapy. To date, few specific agents that inhibit macropinocytosis have been developed. Here, focusing on the mechanosensitive ion channel Piezo1, we found that Yoda1, a Piezo1 agonist, potently inhibits macropinocytosis induced by epidermal growth factor (EGF). The inhibition of ruffle formation by Yoda1 was dependent on the extracellular Ca2+ influx through Piezo1 and on the activation of the calcium-activated potassium channel KCa3.1. This suggests that Ca2+ ions can regulate EGF-stimulated macropinocytosis. We propose the potential for macropinocytosis inhibition through the regulation of a mechanosensitive channel activity using chemical tools.

Cell-autonomous control of intracellular temperature by unsaturation of phospholipid acyl chains.

Intracellular temperature affects a wide range of cellular functions in living organisms. However, it remains unclear whether temperature in individual animal cells is controlled autonomously as a response to fluctuations in environmental temperature. Using two distinct intracellular thermometers, we find that the intracellular temperature of steady-state Drosophila S2 cells is maintained in a manner dependent on Δ9-fatty acid desaturase DESAT1, which introduces a double bond at the Δ9 position of the acyl moiety of acyl-CoA. The DESAT1-mediated increase of intracellular temperature is caused by the enhancement of F1Fo-ATPase-dependent mitochondrial respiration, which is coupled with thermogenesis. We also reveal that F1Fo-ATPase-dependent mitochondrial respiration is potentiated by cold exposure through the remodeling of mitochondrial cristae structures via DESAT1-dependent unsaturation of mitochondrial phospholipid acyl chains. Based on these findings, we propose a cell-autonomous mechanism for intracellular temperature control during environmental temperature changes.

A Piezo1/KLF15/IL-6 axis mediates immobilization-induced muscle atrophy.

Although immobility is a common cause of muscle atrophy, the mechanism underlying this causality is unclear. We here show that Krüppel-like factor 15 (KLF15) and IL-6 are upregulated in skeletal muscle of limb-immobilized mice and that mice with KLF15 deficiency in skeletal muscle or with systemic IL-6 deficiency are protected from immobility-induced muscle atrophy. A newly developed Ca2+ bioimaging revealed that the cytosolic Ca2+ concentration ([Ca2+]i) of skeletal muscle is reduced to below the basal level by immobilization, which is associated with the downregulation of Piezo1. Acute disruption of Piezo1 in skeletal muscle induced Klf15 and Il6 expression as well as muscle atrophy, which was prevented by antibodies against IL-6. A role for the Piezo1/KLF15/IL-6 axis in immobility-induced muscle atrophy was validated in human samples. Our results thus uncover a paradigm for Ca2+ signaling in that a decrease in [Ca2+]i from the basal level triggers a defined biological event.

Significant correlations between focal photopic negative response and focal visual sensitivity and ganglion cell complex thickness in glaucomatous eyes.

PURPOSE: To determine whether there are significant correlations between the focal photopic negative response (PhNR), the focal visual sensitivity and the ganglion cell complex (GCC) thickness in glaucomatous eyes. STUDY DESIGN: Single-center observational study. METHODS: Fifty-two eyes of 52 patients (71.4 ± 9.42 years) with clinically diagnosed open angle glaucoma were studied. Thirty-six age-matched normal subjects served as controls. The focal PhNR of the focal macular electroretinograms (fmERGs) were elicited by a 15° circular, a superior semicircular or an inferior semicircular stimulus centered on the fovea. The thickness of the GCC was measured in the corresponding retinal areas in the spectral-domain optical coherence tomographic images. The visual sensitivities (dB) were measured by microperimetry at the retinal area where the fmERGs were elicited and were converted to liner values (1/Lambert). RESULTS: The focal PhNR amplitudes were significantly correlated with the visual sensitivities of the full-circle (R = 0.532), the superior (R = 0.530) and inferior (R = 0.526) semicircular responses (P < 0.0001). The GCC thickness was correlated with the visual sensitivities in the same areas with stronger correlations (R = 0.700, 0.759 and 0.650, respectively; P < 0.0001). The focal PhNR amplitudes were proportionally reduced with the thinning of the GCC thickness (R = 0.494, 0.518 and 0.511, respectively; P < 0.0001). CONCLUSIONS: The significant correlations between the focal PhNR amplitudes, the focal visual sensitivities and the GCC thickness indicate that these may be good biomarkers to track the changes in the physiology and anatomy of the macular area in glaucomatous eyes.

Long-Term Observations of Thickness Changes of Each Retinal Layer following Macular Hole Surgery.

PURPOSE: To determine the long-term changes of the thickness of each retinal layer following macular hole (MH) surgery combined with internal limiting membrane (ILM) peeling. METHOD: The medical records of 42 eyes of 42 patients (41 to 86 years of age) who underwent MH surgery with ILM peeling between February 2016 and October 2018 were reviewed. A single surgeon operated on all patients, and all were followed for at least 24 months postoperatively. Spectral-domain optical coherence tomography (OCT) was performed to obtain retinal thickness maps of the parafoveal region corresponding approximately to the ILM peeled area. Each retinal layer was automatically segmented by the embedded software, and thickness maps were constructed for the total retinal layer (TRL), inner RL (IRL), middle RL (MRL), and outer RL (ORL). The averaged value of each retinal layer thickness was analyzed in the temporal/upper, temporal/lower, nasal/upper, and nasal lower quadrants. RESULTS: The TRL thickness was significantly decreased in the temporal areas postoperatively. The IRL thickness thinned progressively and significantly until 6 months without further thinning in the temporal quadrants. The MRL thickness of all areas was significantly thicker than the baseline values at 0.5 months and then gradually decreased in the temporal regions. However, the thickening in the nasal regions returned to the baseline values after 1.5 months. The ORL decreased transiently relative to the baseline values at 0.5 months in all areas. CONCLUSIONS: The ILM peeling does not affect only the thickness of the inner retina but also the middle and outer retinae in the parafoveal region. The chronological changes of the thickness after surgeries varied among the retinal layers and macular regions.

Extreme deformability of insect cell membranes is governed by phospholipid scrambling.

Organization of dynamic cellular structure is crucial for a variety of cellular functions. In this study, we report that Drosophila and Aedes have highly elastic cell membranes with extremely low membrane tension and high resistance to mechanical stress. In contrast to other eukaryotic cells, phospholipids are symmetrically distributed between the bilayer leaflets of the insect plasma membrane, where phospholipid scramblase (XKR) that disrupts the lipid asymmetry is constitutively active. We also demonstrate that XKR-facilitated phospholipid scrambling promotes the deformability of cell membranes by regulating both actin cortex dynamics and mechanical properties of the phospholipid bilayer. Moreover, XKR-mediated construction of elastic cell membranes is essential for hemocyte circulation in the Drosophila cardiovascular system. Deformation of mammalian cells is also enhanced by the expression of Aedes XKR, and thus phospholipid scrambling may contribute to formation of highly deformable cell membranes in a variety of living eukaryotic cells.

Relationships between the vascular structure and neural function of the macula in patients with diabetes mellitus.

PURPOSE: To determine the significance of the correlation between the vascular structure and neural function of the macula in patients with diabetes mellitus. STUDY DESIGN: Single-center observational study. PATIENTS AND METHODS: Ninety eyes of 90 diabetic patients with an average (SD) age of 63.5 (3.8) years were studied. Fifty of the eyes had no clinically apparent diabetic retinopathy (non-DR), and 40 eyes had mild-to-moderate nonproliferative DR (NPDR). Thirty age-matched healthy individuals were also studied in the same way. Swept-source optical coherence tomography angiography (OCTA) was performed to obtain 3 × 3-mm en face images of the posterior pole of the eye. The vascular densities (VDs) of the superficial capillary plexus (SCP) and the deep capillary plexus (DCP) were determined. The focal macular electroretinograms (ERGs) elicited by a 15° circular stimulus centered on the fovea were recorded. The amplitudes of the a- and b-waves, sum of the oscillatory potentials (ΣOPs), photopic negative response (PhNR), and implicit times of the individual OPs (OP1-OP3) were measured. RESULTS: The VDs of the SCP and DCP were reduced in eyes with advanced DR (P < .01 for SCP). The implicit times of OP1-OP3 were significantly prolonged in eyes with a lower VD of the SCP and DCP in the non-DR group (P < .05). The amplitudes of the ΣOPs were significantly smaller in eyes with a reduced VD of the SCP and DCP in the NPDR group (P < .05). The correlation coefficients were higher for the OP implicit times than for the ΣOP amplitudes in the non-DR group. CONCLUSIONS: The OPs of the focal macular ERG are smaller with prolonged implicit times in association with capillary loss in the macula of diabetic patients. The implicit times are the most sensitive functional parameter that reflects the early changes of the microvasculature in the macula caused by diabetes.

Functional expression of Δ12 fatty acid desaturase modulates thermoregulatory behaviour in Drosophila.

Polyunsaturated fatty acids (PUFAs) play crucial roles in adaptation to cold environments in a wide variety of animals and plants. However, the mechanisms by which PUFAs affect thermoregulatory behaviour remain elusive. Thus, we investigated the roles of PUFAs in thermoregulatory behaviour of Drosophila melanogaster. To this end, we generated transgenic flies expressing Caenorhabditis elegans Δ12 fatty acid desaturase (FAT-2), which converts mono-unsaturated fatty acids to PUFAs such as linoleic acid [C18:2 (n-6)] and linolenic acid [C18:3 (n-3)]. Neuron-specific expression of FAT-2 using the GAL4/UAS expression system led to increased contents of C18:2 (n-6)-containing phospholipids in central nerve system (CNS) and caused significant decreases in preferred temperature of third instar larvae. In genetic screening and calcium imaging analyses of thermoreceptor-expressing neurons, we demonstrated that ectopic expression of FAT-2 in TRPA1-expressing neurons led to decreases in preferred temperature by modulating neuronal activity. We conclude that functional expression of FAT-2 in a subset of neurons changes the thermoregulatory behaviour of D. melanogaster, likely by modulating quantities of PUFA-containing phospholipids in neuronal cell membranes.

Changes of focal macular and full-field electroretinograms after intravitreal aflibercept in patients with central retinal vein occlusion.

PURPOSE: To evaluate the physiology of the macular and whole retina after intravitreal aflibercept (IVAs) injections in patients with macular edema associated with a central retinal vein occlusion (CRVO) by electroretinography (ERG). METHODS: We studied 20 eyes of 20 patients with non-ischemic CRVO (72.0 ± 9.2 years). All patients were treated with monthly injections of IVA for the initial 3 months and then treated by the treat-and-extend (TAE) regimen for 12 months. The best-corrected visual acuity (BCVA), optical coherence tomographic images, focal macular ERGs (fmERGs), and full-field ERGs recorded before and after the treatment were compared. The fmERGs were elicited by a 15° white stimulus spot centered on the fovea. The full-field ERGs were recorded by a protocol recommended by International Society for Clinical Electrophysiology of Vision. The amplitudes and implicit times determined before and after the IVA were compared. RESULTS: The foveal thickness was significantly reduced accompanied by improvement of the BCVA after the treatments, and the improvements were maintained for at least 12 months. The amplitudes and implicit times of the fmERGs improved continuously for the 12 months. On the other hand, the reduced amplitudes of the full-field ERG, summed oscillatory potentials, and the photopic negative responses remained unchanged for the 12-month period. However, the implicit times of the maximum and cone responses were significantly shortened after the IVA. CONCLUSIONS: IVA injections by the TAE regimen led to a continuous improvement of the macular function in patients with ME associated with a CRVO. However, the function of the whole retina changed differently than the macula after the treatment.

Changes in cone-driven functions after intravitreal aflibercept injections in patients with age-related macular degeneration.

PURPOSE: To determine the changes in the cone-driven functions in patients with age-related macular degeneration (AMD) treated with intravitreal aflibercept. METHODS: We studied 44 eyes of 44 patients diagnosed with AMD whose mean age was 75 years. The contralateral unaffected eyes served as controls. All patients were initially treated with 3 consecutive monthly intravitreal aflibercept injections and thereafter with bimonthly injections for 12 months. Full-field cone electroretinograms (cone ERGs) were recorded at the baseline and at 3, 6, and 12 months after beginning the intravitreal aflibercept injections. The cone ERGs were elicited by red stimuli on a blue background. The focal macular ERGs (fmERGs) were elicited by 15 degrees white stimulus spot centered on the fovea. The amplitudes of the a- and b-waves, photopic negative response (PhNR), and sum of the oscillatory potentials (ΣOPs, sum of OP1-3 amplitudes) were analyzed. In addition, the implicit times of the a- and b-waves were also analyzed. RESULTS: The amplitudes and implicit times of all components of the fmERGs were significantly improved compared to the baseline at 3 months after beginning the intravitreal aflibercept injections (P < 0.0005-0.05). The amplitudes of the a-waves and PhNRs were further increased during the maintenance phase (P < 0.005-0.01). On the other hand, the amplitudes of the full-field a-waves and PhNR of the cone ERGs were significantly reduced at 6 and 12 months compared to the baseline. CONCLUSIONS: The macular function improved continuously during the maintenance phase of the intravitreal aflibercept injections. In contrast, the cone-driven functions of the more peripheral retina decreased with repeated injections suggesting adverse effects of the intravitreal aflibercept injections on the function of the more peripheral normal retina.

Comparisons of photopic negative responses elicited by different conditions from glaucomatous eyes.

PURPOSE: To compare the clinical significance of the photopic negative response (PhNRs) elicited by different stimuli from glaucomatous eyes. STUDY DESIGN: Single-center observational study METHOD: Eighty-four eyes of 84 patients with open angle glaucoma (OAG) and 40 eyes of 40 normal subjects were studied. Cone electroretinograms (ERGs) were elicited by white stimuli on a white background (W/W) or red stimuli on a blue background (R/B). The luminance of the stimuli was 0.5, 1.0, 2.0 or 3.0 cd-s/m2, and of the background light was 10 cd/m2. The first and second troughs of the ERGs that appeared following the b-wave were designated as PhNR1 and PhNR2, respectively. The thickness of the circumpapillary retinal nerve fiber layer (cpRNFL) was measured by spectral-domain optical coherence tomography. The mean deviation (MD) was determined by standard automated perimetry. The area under the receiver operating characteristic curves (AUCs) was created to determine the diagnostic ability of the PhNRs elicited by the different stimulus conditions. RESULTS: The correlation coefficients of the amplitudes of the PhNR1 elicited by W/W stimuli to the MDs and cpRNFL thickness were generally stronger, and the regression lines steeper than for the amplitudes of the PhNR1 elicited by R/B stimuli. In contrast, the correlation coefficients of the amplitudes of the PhNR2 elicited by R/B stimuli to the MDs and cpRNFL thickness were generally stronger, and the regression lines were steeper than the amplitudes of the PhNR2 elicited by W/W stimuli. With both types of stimuli, the slopes of the regression lines became steeper when the ERG recorded with higher stimulus intensities. The AUCs were significantly larger for the PhNR2 elicited by the R/B stimuli at 3.0 cd-s/m2 than for PhNR1 and PhNR2 elicited by W/W stimuli at the same intensity when the PhNRs were used for diagnosing advanced glaucoma. CONCLUSION: The PhNR1 and PhNR2 elicited by the W/W and R/B stimuli are suitable measures to assess the function of the RGCs in eyes with OAG. The PhNR2 elicited by R/B stimuli at higher stimulus intensities is most effective in detecting functional and structural changes of the RGCs with the highest diagnostic capacity in discriminating advanced glaucoma.

Changes in the physicochemical properties of fish cell membranes during cellular senescence.

Fish cell lines are widely used for the studies of developmental biology, virology, biology of aging, and nutrition physiology. However, little is known about their physicochemical properties. Here, we report the phospholipid compositions and mechanical properties of cell membranes derived from freshwater, anadromous and marine fish species. Biophysical analyses revealed that fish cell lines have highly deformable cell membranes with significantly low membrane tensions and Young's moduli compared with those of mammalian cell lines. The induction of cellular senescence by DNA demethylation using 5-Aza-2'-deoxycytidine significantly reduced the deformability of fish cell membrane, but hydrogen peroxide-induced oxidative stress did not affect the deformability. Mass spectrometry analysis of phospholipids revealed that the level of phosphatidylethanolamine molecules containing polyunsaturated fatty acids significantly increased during the 5-Aza-2'-deoxycytidine-induced cellular senescence. Fish cell lines provide a useful model system for studying the changes in the physicochemical properties of cell membranes during cellular senescence.Abbreviations: 2D-TLC: two-dimensional thin layer chromatography; 5-Aza-dC: 5-Aza-2'-deoxycytidine; DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; FBS: fetal bovine serum; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; PS: phosphatidylserine; PUFA: polyunsaturated fatty acid; SA-ß-gal: senescence-associated beta-galactosidase; SM: sphingomyelin.

Different mechanisms for selective transport of fatty acids using a single class of lipoprotein in Drosophila.

In mammals, lipids are selectively transported to specific sites using multiple classes of lipoproteins. However, in Drosophila, a single class of lipoproteins, lipophorin, carries more than 95% of the lipids in the hemolymph. Although a unique ability of the insect lipoprotein system for cargo transport has been demonstrated, it remains unclear how this single class of lipoproteins selectively transports lipids. In this study, we carried out a comparative analysis of the fatty-acid composition among lipophorin, the CNS, and CNS-derived cell lines and investigated the transport mechanism of fatty acids, particularly focusing on the transport of PUFAs in Drosophila We showed that PUFAs are selectively incorporated into the acyl chains of lipophorin phospholipids and effectively transported to CNS through lipophorin receptor-mediated endocytosis of lipophorin. In addition, we demonstrated that C14 fatty acids are selectively incorporated into the diacylglycerols (DAGs) of lipophorin and that C14 fatty-acid-containing DAGs are spontaneously transferred from lipophorin to the phospholipid bilayer. These results suggest that PUFA-containing phospholipids and C14 fatty-acid-containing DAGs in lipophorin could be transferred to different sites by different mechanisms to selectively transport fatty acids using a single class of lipoproteins.

Protective role for the N-terminal domain of α-dystroglycan in Influenza A virus proliferation.

α-Dystroglycan (α-DG) is a highly glycosylated basement membrane receptor that is cleaved by the proprotein convertase furin, which releases its N-terminal domain (α-DGN). Before cleavage, α-DGN interacts with the glycosyltransferase LARGE1 and initiates functional O-glycosylation of the mucin-like domain of α-DG. Notably, α-DGN has been detected in a wide variety of human bodily fluids, but the physiological significance of secreted α-DGN remains unknown. Here, we show that mice lacking α-DGN exhibit significantly higher viral titers in the lungs after Influenza A virus (IAV) infection (strain A/Puerto Rico/8/1934 H1N1), suggesting an inability to control virus load. Consistent with this, overexpression of α-DGN before infection or intranasal treatment with recombinant α-DGN prior and during infection, significantly reduced IAV titers in the lungs of wild-type mice. Hemagglutination inhibition assays using recombinant α-DGN showed in vitro neutralization of IAV. Collectively, our results support a protective role for α-DGN in IAV proliferation.