mTORC1 activity is supported by spatial association with focal adhesions

The mammalian target of rapamycin complex 1 (mTORC1) integrates mitogenic and stress signals to control growth and metabolism. Activation of mTORC1 by amino acids and growth factors involves recruitment of the complex to the lysosomal membrane and is further supported by lysosome distribution to the cell periphery. Here, we show that translocation of lysosomes toward the cell periphery brings mTORC1 into proximity with focal adhesions (FAs). We demonstrate that FAs constitute discrete plasma membrane hubs mediating growth factor signaling and amino acid input into the cell. FAs, as well as the translocation of lysosome-bound mTORC1 to their vicinity, contribute to both peripheral and intracellular mTORC1 activity. Conversely, lysosomal distribution to the cell periphery is dispensable for the activation of mTORC1 constitutively targeted to FAs. This study advances our understanding of spatial mTORC1 regulation by demonstrating that the localization of mTORC1 to FAs is both necessary and sufficient for its activation by growth-promoting stimuli

A Mammalian Target of Rapamycin-Perilipin 3 (mTORC1-Plin3) Pathway is essential to Activate Lipophagy and Protects Against Hepatosteatosis

[Background and Aims] NAFLD is the most common hepatic pathology in western countries and no treatment is currently available. NAFLD is characterized by the aberrant hepatocellular accumulation of fatty acids in the form of lipid droplets (LDs). Recently, it was shown that liver LD degradation occurs through a process termed lipophagy, a form of autophagy. However, the molecular mechanisms governing liver lipophagy are elusive. Here, we aimed to ascertain the key molecular players that regulate hepatic lipophagy and their importance in NAFLD.[Approach and Results] We analyzed the formation and degradation of LD in vitro (fibroblasts and primary mouse hepatocytes), in vivo and ex vivo (mouse and human liver slices) and focused on the role of the autophagy master regulator mammalian target of rapamycin complex (mTORC) 1 and the LD coating protein perilipin (Plin) 3 in these processes. We show that the autophagy machinery is recruited to the LD on hepatic overload of oleic acid in all experimental settings. This led to activation of lipophagy, a process that was abolished by Plin3 knockdown using RNA interference. Furthermore, Plin3 directly interacted with the autophagy proteins focal adhesion interaction protein 200 KDa and autophagy-related 16L, suggesting that Plin3 functions as a docking protein or is involved in autophagosome formation to activate lipophagy. Finally, we show that mTORC1 phosphorylated Plin3 to promote LD degradation.[Conclusions] These results reveal that mTORC1 regulates liver lipophagy through a mechanism dependent on Plin3 phosphorylation. We propose that stimulating this pathway can enhance lipophagy in hepatocytes to help protect the liver from lipid-mediated toxicity, thus offering a therapeutic strategy in NAFLD.Supported by C0120R3166, C0245R4032, and BH182173 from Newcastle University. M. G.-M. is a Sara Borrell Postdoctoral fellow (CD18/00203) from the Ministerio de Ciencia, Innovación y Universidades (Spain). J. P. B. is funded by the Agencia Estatal de Investigación, grants PID2019-105699RB-I00/AEI/10.13039/501100011033 and RED2018-102576-T, Instituto de Salud Carlos III (CB16/10/00282), Junta de Castilla y León (Escalera de Excelencia CLU-2017-03), Ayudas Equipos Investigación Biomedicina 2017 Fundación BBVA, and Fundación Ramón Areces. V. I. K. acknowledges support from Biotechnology and Biological Sciences Research Council (BB/M023389/1, BB/R008167/1, BBPeer reviewe

Oncogenic PIK3CA corrupts growth factor signaling specificity

Pathological activation of the PI3K/AKT pathway is among the most frequent defects in human cancer and is also the cause of rare overgrowth disorders. Yet, there is currently no systematic understanding of the quantitative flow of information within PI3K/AKT signaling and how it is perturbed by disease-causing mutations. Here, we develop scalable, single-cell approaches for systematic analyses of signal processing within the PI3K pathway, enabling precise calculations of its information transfer for different growth factors. Using genetically-engineered human cell models with allele dose-dependent expression of PIK3CAH1047R, we show that this oncogene is not a simple, constitutive pathway activator but a context-dependent modulator of extracellular signal transfer. PIK3CAH1047Rreduces information transmission downstream of IGF1 while selectively enhancing EGF-induced signaling and transcriptional responses. This leads to a gross reduction in signaling specificity, akin to “blurred” signal perception. The associated increase in signaling heterogeneity promotes phenotypic diversity in a human cervical cancer cell line model and in human induced pluripotent stem cells. Collectively, these findings and the accompanying methodological advances lay the foundations for a systematic mapping of the quantitative mechanisms of PI3K/AKT-dependent signal processing and phenotypic control in health and disease

Severe White Matter Astrocytopathy in CADASIL.

Objectives Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is characterised by strategic white matter (WM) hyperintensities on MRI. Pathological features include WM degeneration, arteriolosclerosis, lacunar infarcts and the deposition of granular osmiophilic material. Based on the hypothesis that the gliovascular unit is compromised, we assessed the nature of astrocyte damage in the deep WM of CADASIL subjects. Methods We evaluated post‐mortem brains from CADASIL, cerebral small vessel disease, similar age cognitively normal and older control subjects. Standard immunohistochemical, immunofluorescent and unbiased stereological methods were used to evaluate the distribution of astrocytes, microvessels and autophagy markers in five different brain regions. Results Compared to the controls, the deep WM of CADASIL subjects overall showed increased numbers of glial fibrillary acidic protein (GFAP)‐positive clasmatodendritic astrocytes (P=0.037) and a decrease in the percentage of normal appearing astrocytes (P=0.025). In accord with confluent WM hyperintensities , the anterior temporal pole contained abundant clasmatodendritic astrocytes with displaced aquaporin 4 immunoreactivity. Remarkably, we also found strong evidence for the immunolocalisation of autophagy markers including microtubule associated protein 1, light chain 3 (LC3) and sequestosome 1/p62 and Caspase‐3 in GFAP‐positive clasmatodendritic cells, particularly within perivascular regions of the deep WM. LC3 was co‐localised in more than 90% of the GFAP‐positive clasmatodendrocytes. Conclusions Our novel findings show astrocytes undergo autophagy‐like cell death in CADASIL, with the anterior temporal pole being highly vulnerable. We propose astrocytes transform from normal appearing type A to hypertrophic type B and eventually to clasmatodendritic type C cells. These observations also suggest the gliovascular unit of the deep WM is severely impaired in CADASIL

Oxidation of SQSTM1/p62 mediates the link between redox state and protein homeostasis

Cellular homoeostatic pathways such as macroautophagy (hereinafter autophagy) are regulated by basic mechanisms that are conserved throughout the eukaryotic kingdom. However, it remains poorly understood how these mechanisms further evolved in higher organisms. Here we describe a modification in the autophagy pathway in vertebrates, which promotes its activity in response to oxidative stress. We have identified two oxidation-sensitive cysteine residues in a prototypic autophagy receptor SQSTM1/p62, which allow activation of pro-survival autophagy in stress conditions. The Drosophila p62 homologue, Ref(2)P, lacks these oxidation-sensitive cysteine residues and their introduction into the protein increases protein turnover and stress resistance of flies, whereas perturbation of p62 oxidation in humans may result in age-related pathology. We propose that the redox-sensitivity of p62 may have evolved in vertebrates as a mechanism that allows activation of autophagy in response to oxidative stress to maintain cellular homoeostasis and increase cell survival.Peer reviewe

Oxidation of SQSTM1/p62 mediates the link between redox state and protein homeostasis

Cellular homoeostatic pathways such as macroautophagy (hereinafter autophagy) are regulated by basic mechanisms that are conserved throughout the eukaryotic kingdom. However, it remains poorly understood how these mechanisms further evolved in higher organisms. Here we describe a modification in the autophagy pathway in vertebrates, which promotes its activity in response to oxidative stress. We have identified two oxidation-sensitive cysteine residues in a prototypic autophagy receptor SQSTM1/p62, which allow activation of pro-survival autophagy in stress conditions. The Drosophila p62 homologue, Ref(2)P, lacks these oxidation-sensitive cysteine residues and their introduction into the protein increases protein turnover and stress resistance of flies, whereas perturbation of p62 oxidation in humans may result in age-related pathology. We propose that the redox-sensitivity of p62 may have evolved in vertebrates as a mechanism that allows activation of autophagy in response to oxidative stress to maintain cellular homoeostasis and increase cell survival

Влияние смесей полиэтиленгликолей в качестве мазевой основы на физико-химические свойства лавсановых атравматичных раневых повязок

Objectives. Modern atraumatic wound dressings are based on polyethylene terephthalate, or Lavsan, which is shaped to form threads. The aim of the study was to determine the reasons for Lavsan woven nets’ hardening and becoming more trauma-prone during storage, and to find ways of eliminating these effects.Methods. We used differential scanning calorimetry, performed on a NETZSCH DSС 204 F1 Phoenix device, in a dynamic mode with a temperature range from 20 to 300 °C in argon flow to determine phase states, glass transition temperatures, and melting temperatures of Lavsan fibers (including those treated with polyethylene glycol mixtures). We performed rheoviscometry studies on a Brookfield DV2TLV rotational viscometer, with a SC4-16 thermostatic control unit, at the following temperatures: 25, 36.6, 40, 45, 50, and 55 °C, with shear rates ranging from 120 to 200 s–1 to determine dynamic viscosity and investigate the mixing characteristics of polyethylene glycols with different molecular weights.Results. We have established that samples of Lavsan woven nets, stored long-term in laboratory conditions (up to 2, 3, and 16 years), are in the crystalline state with a high degree of crystallinity. Upon heating these nets to 300 °C, it is possible to reduce the degree of crystallinity by 19–32%, but it does not completely eliminate the effect. Polyethylene glycols and their mixtures which exhibit non-Newtonian flow behavior and are used as an ointment base, have a significant effect on Lavsan’s crystallinity. We have determined that the optimal ratio of polyethylene glycols for the modification of Lavsan nets is PEG-400:PEG-1500 = 80:20 wt %. Upon storing Lavsan woven nets in this mixture at room temperature, the Lavsan’s crystallinity is greatly reduced, and upon heating the system, the crystallinity practically disappears.Conclusions. The effect of polyethylene glycol mixtures (the base for therapeutic ointments) with various molecular weights on the phase organization of Lavsan has been evaluated. As a result of this study, we can offer a new approach to reduce the injuring effect of synthetic (Lavsan) bases of atraumatic wound dressings.Цели. Основой современных атравматичных раневых повязок является полиэтилентерефталат (или лавсан), которому придают вид нитей. Целью работы являлось определение причин увеличения жесткости и травматичности лавсановых тканых сеток при хранении и поиск путей устранения этого эффекта.Методы. Для определения фазового состояния, температуры стеклования и плавления лавсановых волокон, в том числе после обработки смесями полиэтиленгликолей, использовали дифференциальную сканирующую калориметрию, которую проводили на приборе NETZSCH DSС 204 F1 Phoenix в динамическом режиме в диапазоне температур от 20 до 300 ºС в токе аргона. Для определения динамической вязкости и оценки характера смешения полиэтиленгликолей разной молекулярной массы применяли метод реовискозиметрии, которую осуществляли на ротационном вискозиметре Brookfield DV2TLV с термостатируемым рабочим узлом SC4-16 при температурах: 25, 36.6, 40, 45, 50 и 55 ºС в диапазоне скоростей сдвига от 120 до 200 с –1 .Результаты. Установлено, что длительно выдержанные в лабораторных условиях (до 2, 3 и 16 лет) образцы лавсановых тканых сеток находятся в кристаллическом состоянии с высокой степенью кристалличности. Прогрев этих сеток до 300 ºС позволяет снизить степень кристалличности на 19–32%, но не устраняет ее полностью. Полиэтиленгликоли и их смеси, которые используют в качестве мазевой основы, проявляющие неньютоновское поведение при течении, оказывают заметное влияние на степень кристалличности лавсана. Установлено оптимальное соотношение полиэтиленгликолей для модификации лавсановых сеток: ПЭГ-400:ПЭГ-1500 = 80:20 мас. ч. После выдерживания лавсановой тканой сетки в этой смеси при комнатной температуре степень кристалличности лавсана сильно снижается, а после прогрева такой системы кристалличность практически исчезает.Заключение. Оценено влияние на фазовую организацию лавсана смесей полиэтиленгликолей различной молекулярной массы, являющихся основой лечебных мазей. В результате проведенной работы предложен новый подход для уменьшения травматичности синтетических (лавсановых) основ атравматичных раневых повязок

Oxidation of SQSTM1/p62 mediates the link between redox state and protein homeostasis.

Cellular homoeostatic pathways such as macroautophagy (hereinafter autophagy) are regulated by basic mechanisms that are conserved throughout the eukaryotic kingdom. However, it remains poorly understood how these mechanisms further evolved in higher organisms. Here we describe a modification in the autophagy pathway in vertebrates, which promotes its activity in response to oxidative stress. We have identified two oxidation-sensitive cysteine residues in a prototypic autophagy receptor SQSTM1/p62, which allow activation of pro-survival autophagy in stress conditions. The Drosophila p62 homologue, Ref(2)P, lacks these oxidation-sensitive cysteine residues and their introduction into the protein increases protein turnover and stress resistance of flies, whereas perturbation of p62 oxidation in humans may result in age-related pathology. We propose that the redox-sensitivity of p62 may have evolved in vertebrates as a mechanism that allows activation of autophagy in response to oxidative stress to maintain cellular homoeostasis and increase cell survival