Cytoskeletal vimentin regulates cell size and autophagy through mTORC1 signaling

The nutrient-activated mTORC1 (mechanistic target of rapamycin kinase complex 1) signaling pathway determines cell size by controlling mRNA translation, ribosome biogenesis, protein synthesis, and autophagy. Here, we show that vimentin, a cytoskeletal intermediate filament protein that we have known to be important for wound healing and cancer progression, determines cell size through mTORC1 signaling, an effect that is also manifested at the organism level in mice. This vimentin-mediated regulation is manifested at all levels of mTOR downstream target activation and protein synthesis. We found that vimentin maintains normal cell size by supporting mTORC1 translocation and activation by regulating the activity of amino acid sensing Rag GTPase. We also show that vimentin inhibits the autophagic flux in the absence of growth factors and/or critical nutrients, demonstrating growth factor-independent inhibition of autophagy at the level of mTORC1. Our findings establish that vimentin couples cell size and autophagy through modulating Rag GTPase activity of the mTORC1 signaling pathway

Cytoskeletal vimentin regulates cell size and autophagy through mTORC1 signaling

The nutrient-activated mTORC1 (mechanistic target of rapamycin kinase complex 1) signaling pathway determines cell size by controlling mRNA translation, ribosome biogenesis, protein synthesis, and autophagy. Here, we show that vimentin, a cytoskeletal intermediate filament protein that we have known to be important for wound healing and cancer progression, determines cell size through mTORC1 signaling, an effect that is also manifested at the organism level in mice. This vimentin-mediated regulation is manifested at all levels of mTOR downstream target activation and protein synthesis. We found that vimentin maintains normal cell size by supporting mTORC1 translocation and activation by regulating the activity of amino acid sensing Rag GTPase. We also show that vimentin inhibits the autophagic flux in the absence of growth factors and/or critical nutrients, demonstrating growth factor-independent inhibition of autophagy at the level of mTORC1. Our findings establish that vimentin couples cell size and autophagy through modulating Rag GTPase activity of the mTORC1 signaling pathway. © 2022 Mohanasundaram et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</p

Density and function of actin-microdomains in healthy and NF1 deficient osteoclasts revealed by the combined use of atomic force and stimulated emission depletion microscopy

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Density and function of actin-microdomains in healthy and NF1 deficient osteoclasts revealed by the combined use of atomic force and stimulated emission depletion microscopy

Actin and myosins (IIA, IIB, and X) generate mechanical forces in osteoclasts that drive functions such as migration and membrane trafficking. In neurofibromatosis, these processes are perturbed due to a mutation in neurofibromatosis type 1 (NF1) gene. This mutation leads to generation of hyperactive bone-resorbing osteoclasts that increases incidence of skeletal dysplasia e.g. early-onset osteoporosis in patients suffering from neurofibromatosis. To study the density and function of actin clusters in mutated cells we introduce a new approach for combined use of a stimulated emission depletion (STED) microscope with an atomic force microscope (AFM). We resolved actin-cores within actin-microdomains at four typical structures (podosome-belt, podosome raft, actin patches, and sealing zone) for osteoclasts cultured on bone as well as on glass. Densities of actin-cores in these structures were higher on bone than on glass, and the nearest neighbor distances were shortest in sealing zones, where also an accumulation of vesicular material was observed at their center. In NF1 deficient osteoclasts, the clustering was tighter and there was also more vesicular material accumulated inside the sealing zone. Using the STED-AFM system, we measured the condensation of the actin structures in real-time after a bone-coated cantilever was placed in contact with a differentiated osteoclast and found that the condensation of actin was initiated at 40 min, after sufficient local actin concentration was reached. A functional implication of the less dense clustering in NF1 deficient cells was that the adhesion of these cells was less specific for bone. The data and new methodologies presented here build a foundation for establishing novel actomyosin dependent mechanisms during osteoclast migration and resorption.</p

Vimentin modulates focal adhesion dynamics in directionally migrating fibroblasts

Cell migration is an integral process in several physiological events essential for normal functioning of an organism. Cell migration is a mechanistic phenomenon that occurs in many steps including cell polarization, attachment to the substratum, extension of leading edge, cell body movement, and detachment of the cell rear. This process is mediated by the interaction of cytoskeletal proteins such as actin and microtubules with focal adhesion at the substratum and other signalling factors. Fibroblastic migration plays a significant role in macrophage mediated immune response, wound healing and metastasis, however the role of cytoskeletal protein vimentin is still poorly understood. It has been demonstrated that vimentin knockout fibroblasts show decreased wound healing and delayed migration. Here we are studying the effects of vimentin on the dynamics of focal adhesion proteins in fibroblasts in an in vitro wound healing assay. We demonstrate that vimentin influences the morphology, turnover, and behavioural dynamics of focal adhesions in directionally migrating fibroblasts. The data demonstrates that the disassembly rate of focal adhesions in vimentin knockout cells is significantly higher than their wild type counterpart. In addition, vimentin null cells form lower number of focal adhesions with smaller size, express lesser amount of focal adhesions proteins like paxillin, focal adhesion kinase, and vinculin. Vimentin filament disruption has been shown to dramatically affect the morphology of focal adhesions. There is a marked variation in the overall behaviour of focal adhesions in migrating cells in terms of size and shape over time in vimentin null mouse embryonic fibroblasts (MEFs). MEF vimentin null cells appear to have disorganized and disoriented adhesions in the direction of migration. These cumulative findings indicate the significance of vimentin intermediate filament in modulating focal adhesions during fibroblastic migration and help understand the interplay in a biologically relevant context

Hepatoprotective and hypoglycemic effect of lactic acid fermented Indian Gooseberry-Amla beverage on chronic alcohol-induced liver damage and diabetes in rats

Background: Plant-based fermented foods rich in lactic acid bacterial metabolites, antioxidants, and phytochemicals, can promote recovery from ethanol-induced liver damage by restoring liver antioxidant levels and suppressing liver inflammation, and improving certain metabolic disorders such as diabetes mellitus. Methods: In the present study, the protective effects of nutraceutical-enriched lactic acid-fermented Amla beverage on chronic alcohol-induced biochemical modulations and diabetes in Wistar rats were investigated. Results: The hepatoprotective studies showed that the fermented beverage was able to reverse the damage caused to the liver with ethanol administration in terms of liver index, liver enzymes (AST, ALT), serum enzymes (g-GT), and serum TG, TCH, hepatic TG, LPO levels, antioxidants (GSH, TSOD, CAT, GSH-Px). Along similar lines, in the hypoglycemic studies, the fermented beverage evidently improved body weight, and fasting blood glucose levels, reducing fasting HbAlc levels, improving C-peptide and GLP-1 levels, and alleviating renal dysfunction and lipid metabolism compared with diabetic rats. All these outcomes were supported by histological observations within the liver and pancreas. Conclusion: The present study suggests that the consumption of fermented Amla beverage may have a protective effect against chronic alcohol-induced toxicity and diabetes. The effects of fermented Amla beverage may be attributed to antioxidant activity, flavonoids, bioactive compounds produced by LAB and their metabolites, which help to counteract free radicals induced by ethanol and in reducing glucagon levels, enhancing glucose utilization, leading to a decrease in blood glucose. The results show that fermented Amla beverage has positive effects in reducing the detrimental effect of alcohol and diabetes

Magnetic Resonance Imaging versus Computed Tomography in Transient Ischemic Attack and Minor Stroke: The More &#x03A5;ou See the More You Know

Background: Magnetic resonance imaging (MRI) is proposed as the preferred imaging modality to investigate patients with transient ischemic attack (TIA). This is mainly based on a higher yield of small acute ischemic lesions; however, direct prospective comparisons are lacking. In this study, we aimed to directly compare the yield of acute ischemic lesions on MRI and computed tomography (CT) in the emergency diagnosis of suspected TIA or minor stroke. Methods: Consecutive patients aged 18 years or older presenting with minor stroke (NIHSS Results: A total of 347 patients were included, 168 with TIAs, 147 with minor strokes and 32 with a final diagnosis of a mimic. Acute ischemic lesions were detected in 39% of TIAs by using MRI versus 8% by using CT (p Conclusion: MRI is superior to CT in detecting the small ischemic lesions occurring after TIA and minor stroke. Since these lesions are clinically relevant, MRI should be the preferred imaging modality in this setting

Magnetic Resonance Imaging versus Computed Tomography in Transient Ischemic Attack and Minor Stroke: The More Υou See the More You Know

BACKGROUND: Magnetic resonance imaging (MRI) is proposed as the preferred imaging modality to investigate patients with transient ischemic attack (TIA). This is mainly based on a higher yield of small acute ischemic lesions; however, direct prospective comparisons are lacking. In this study, we aimed to directly compare the yield of acute ischemic lesions on MRI and computed tomography (CT) in the emergency diagnosis of suspected TIA or minor stroke. METHODS: Consecutive patients aged 18 years or older presenting with minor stroke (NIHSS <4) or high-risk TIA and who were examined by a stroke neurologist within 24 h of symptom onset were prospectively enrolled in the CATCH study. Patients who had undergone both a baseline CT and an MRI within 24 h of symptom onset were included in this substudy. Baseline MRI and CT were interpreted independently to identify an acute ischemic lesion. The rates of acute ischemic lesions on CT and MRI were compared, and the volume of acute ischemic lesions was measured on MRI. In addition, the volume of acute ischemic lesions on MRI was compared between patients who had evidence of acute ischemia on CT and in those who did not. RESULTS: A total of 347 patients were included, 168 with TIAs, 147 with minor strokes and 32 with a final diagnosis of a mimic. Acute ischemic lesions were detected in 39% of TIAs by using MRI versus 8% by using CT (p < 0.0001) and in 86% of minor strokes by using MRI versus 18% by using CT (p < 0.0001). Compared to MRI, CT had a sensitivity of 20% and a specificity of 98% in identifying an acute ischemic lesion. The infarct volume on diffusion-weighted MRI was larger in cases where the CT also showed an acute ischemic lesion (median 5.07 ml, IQR 10) as compared to lesions seen only on MRI (median 0.68 ml, IQR 1.31, p < 0.0001). CONCLUSION: MRI is superior to CT in detecting the small ischemic lesions occurring after TIA and minor stroke. Since these lesions are clinically relevant, MRI should be the preferred imaging modality in this setting

Refinement of Imaging Predictors of Recurrent Events following Transient Ischemic Attack and Minor Stroke.

TIA and minor stroke have a high risk of recurrent stroke. Abnormalities on CT/CTA and MRI predict recurrent events in TIA and minor stroke. However there are many other imaging abnormalities that could potentially predict outcome that have not been assessed in this population. Also the definition of recurrent events used includes deterioration due to stroke progression or recurrent stroke and whether imaging is either of these is not known.To improve upon the clinical, CT/CTA and MRI parameters that predict recurrent events after TIA and minor stroke by assessing further imaging parameters. Secondary aim was to explore predictors of stroke progression versus recurrent stroke.510 consecutive TIA and minor stroke patients had CT/CTA and most had MRI. Primary outcome was recurrent events (stroke progression or recurrent stroke) within 90 days. Further imaging parameters were assessed for prediction of recurrent events (combined outcome of stroke progression and recurrent stroke). We also explored predictors of symptom progression versus recurrence individually.36 recurrent events (36/510, 7.1% (95% CI: 5.0-9.6)) including 19 progression and 17 recurrent strokes. On CT/CTA: white matter disease, prior stroke, aortic arch focal plaque≥4 mm, or intraluminal thrombus did not predict recurrent events (progression or recurrent stroke). On MRI: white matter disease, prior stroke, and microbleeds did not predict recurrent events. Parameters predicting the individual outcome of symptom progression included: ongoing symptoms at initial assessment, symptom fluctuation, intracranial occlusion, intracranial occlusion or stenosis, and the CT/CTA metric. No parameter was strongly predictive of a distinct recurrent stroke.There was no imaging parameter that could improve upon our original CT/CTA or MRI metrics to predict the combined outcome of stroke progression or a recurrent stroke after TIA and minor stroke. We are better at using imaging to predict stroke progression rather than recurrent stroke

Abstract 3595: Perfusion imaging predicts Outcome in TIA and Minor Stroke. A Prospective Derivation-Validation Study

Background: Patients presenting with transient or minor ischemic symptoms (TIA/MIS) are at risk for early deterioration. Identification of those at highest risk for progression may justify more aggressive acute reperfusion treatments. We tested the hypothesis that baseline perfusion (PWI)- diffusion (DWI) mismatch predicts clinical deterioration and infarct growth on follow-up imaging in this population. Methods: Patients with TIA/MIS (NIH Stroke Scale ≤ 3) were prospectively enrolled and imaged within 24 hours of symptom onset as part of two sequential prospective imaging studies. All patients had clinical follow-up. Baseline DWI and PWI (tmax+4s delay) and follow-up FLAIR infarct volumes (day 30 (derivation), day 90 (validation) cohort) were measured. Mismatch volumes were calculated as (Tmax+4s delay) - DWI lesion volume. Primary outcome was infarct growth on FLAIR imaging which was defined a priori as growth of at least 2.5 ml. Secondary outcome was clinical progression. Results: 137 patients were included in the derivation and 281 patients in the validation cohorts. The rates of DWI (54% vs 56%, p= 0.67) and PWI lesions (42% vs 34.5%, p=0.16) at baseline were similar between the cohorts. The median time between symptom onset and baseline imaging was significantly shorter in the derivation (9.2 h, IQR=9.4) relative to the validation sets (15.1h, IQR=12.5 p<0.001). More patients had follow-up imaging in the derivation (87%) compared to the validation (76%) cohort (p=0.021). Primary and secondary outcome occurred in 18.5% and 9.5% in the derivation and 5.5% and 4.6% in the validation cohort. In the derivation cohort, baseline mismatch volumes adjusting for age, sex and time from symptom onset to MRI significantly predicted radiographic progression (OR=1.06 [1.03-1.09], p<0.001). The optimal threshold for maximizing sensitivity (Sen) and specificity (Spec) in predicting infarct growth occurred at a mismatch volume of 10ml; which correctly predicted infarct expansion with 82% (Sen) and 91%(Spec) (Area under the curve (AUC)= 0.89 [0.80-0.98]). In the validation cohort, this threshold was highly predictive of radiological progression (p=0.011, McNemars test). Linear regression showed that for every 10ml of mismatch, there would be 2.5ml infarct growth on day 30 FLAIR [R=0.80, p<0.001] (derivation set) and 1.1 ml of growth on day 90 FLAIR (R=0.22, p<0.001) (validation set). Baseline mismatch showed a high discriminative value in predicting clinical deterioration in the derivation (AUC =0.81 [0.67-0.96]) and moderate value in the validation cohort (AUC=0.66 [0.46, 0.85]). Conclusion: In a population of patients with minor stroke and TIA, early MR perfusion-diffusion mismatch predicts infarct growth and clinical progression. These findings suggest that there may be a group of patients with minor symptoms in whom reperfusion strategies may be beneficial