Rab5ab-Mediated Yolk Cell Membrane Endocytosis Is Essential for Zebrafish Epiboly and Mechanical Equilibrium During Gastrulation

Morphogenesis in early embryos demands the coordinated distribution of cells and tissues to their final destination in a spatio-temporal controlled way. Spatial and scalar differences in adhesion and contractility are essential for these morphogenetic movements, while the role that membrane remodeling may play remains less clear. To evaluate how membrane turnover modulates tissue arrangements we studied the role of endocytosis in zebrafish epiboly. Experimental analyses and modeling have shown that the expansion of the blastoderm relies on an asymmetry of mechanical tension in the yolk cell generated as a result of actomyosin-dependent contraction and membrane removal. Here we show that the GTPase Rab5ab is essential for the endocytosis and the removal of the external yolk cell syncytial layer (E-YSL) membrane. Interfering in its expression exclusively in the yolk resulted in the reduction of yolk cell actomyosin contractility, the disruption of cortical and internal flows, a disequilibrium in force balance and epiboly impairment. We conclude that regulated membrane remodeling is crucial for directing cell and tissue mechanics, preserving embryo geometry and coordinating morphogenetic movements during epiboly.

In Silico Insights of Tunable Small Molecules With Regulatory Activity of Acid Sphingomyelinase-Protein Kinase Cδ Interaction

Acid sphingomyelinase demonstrates a housekeeping role and plays a key role in maintaining membrane turnover through sphingolipid metabolism. While deficiency of ASM housekeeping function leading to a lysosomal storage disorder, activation and translocation of ASM leads to AD pathology. Phosphorylation of ASM by PKCδ is critical for translocation of ASM to the plasma membrane. However, several strategies have been developed for the treatment of patients with AD, limitations of treatments and occurrence of AD by alternative signaling pathways become hurdle to the mortality of patients. In the present study, we report the interface of ASM- PKCδ interactions by performing molecular modeling, docking and dynamics simulation analysis. By considering the interacting site of ASM with PKCδ as an allosteric site, we screened a large number of small molecules by virtual screening and identified four lead molecules that show high binding affinity and interacting mode with residues of ASM that are critical to making interaction with PKCδ. The proven role of ASM in apoptosis, neurodegenerative diseases, and the existing functionality of leads in these diseases, together with strengthens the lead compounds, ZINC85551993, ZINC71928291, ZINC71773625 and ZINC85432419 as allosteric inhibitors for ASM. We believe that the lead molecules could be potential allosteric modulators for the translocation of ASM.

βA3/A1-crystallin regulates apical polarity and EGFR endocytosis in retinal pigmented epithelial cells

The retinal pigmented epithelium (RPE) is a monolayer of multifunctional cells located at the back of the eye. High membrane turnover and polarization, including formation of actin-based apical microvilli, are essential for RPE function and retinal health. Herein, we demonstrate an important role for βA3/A1-crystallin in RPE. βA3/A1-crystallin deficiency leads to clathrin-mediated epidermal growth factor receptor (EGFR) endocytosis abnormalities and actin network disruption at the apical side that result in RPE polarity disruption and degeneration. We found that βA3/A1-crystallin binds to phosphatidylinositol transfer protein (PITPβ) and that βA3/A1-crystallin deficiency diminishes phosphatidylinositol 4,5-biphosphate (PI(4,5)P2), thus probably decreasing ezrin phosphorylation, EGFR activation, internalization, and degradation. We propose that βA3/A1-crystallin acquired its RPE function before evolving as a structural element in the lens, and that in the RPE, it modulates the PI(4,5)P2 pool through PITPβ/PLC signaling axis, coordinates EGFR activation, regulates ezrin phosphorylation and ultimately the cell polarity.

Rapid recycling of glutamate transporters on the astroglial surface

Glutamate uptake by astroglial transporters confines excitatory transmission to the synaptic cleft. The efficiency of this mechanism depends on the transporter dynamics in the astrocyte membrane, which remains poorly understood. Here, we visualise the main glial glutamate transporter GLT1 by generating its pH-sensitive fluorescent analogue, GLT1-SEP. FRAP-based imaging shows that 70-75% of GLT1-SEP dwell on the surface of rat brain astroglia, recycling with a lifetime of ~22 s. Genetic deletion of the C-terminus accelerates GLT1-SEP membrane turnover while disrupting its surface pattern, as revealed by single-molecule localisation microscopy. Excitatory activity boosts surface mobility of GLT1-SEP, involving its C-terminus, metabotropic glutamate receptors, intracellular Ca2+ and calcineurin-phosphatase activity, but not the broad-range kinase activity. The results suggest that membrane turnover, rather than lateral diffusion, is the main 'redeployment' route for the immobile fraction (20-30%) of surface-expressed GLT1. This finding reveals an important mechanism helping to control extrasynaptic escape of glutamate.

Clinical Application of Magnetic Resonance Spectroscopy in Diagnosis of Intracranial Mass Lesions

Introduction. Conventional MR imaging provides highly detailed anatomic information with unrivalled soft tissue contrast making it the mainstay in the diagnosis of suspected brain lesions. Despite this, MRI alone at times cannot answer the diagnostic questions in quite a few patients. Proton MR Spectroscopy (H-MRS) provides information on the metabolic composition within an area under interrogation. By comparing the relative concentrations of specific metabolites, the neuroradiologist can deduce critical information regarding neuronal cell density and integrity, cell membrane turnover, metabolic fuel, and possible necrosis in the region of interest. This provides a biochemical picture of the underlying pathology and thus aids in the diagnosis. Methods. This was a cross-sectional comparative study. Results. Of the 63 patients examined by MRI and MRS for intracranial mass lesions, the radiologists were able to offer a single imaging diagnosis based on MRI alone in only 15 patients (23.8%) while when MRI imaging was combined with MR spectroscopy, a single imaging diagnosis was offered in 47 patients (74.6%). This was an overall statistically significant improvement. Conclusion. MRS aided the radiologist in offering a single diagnosis in high versus low-grade gliomas, high-grade gliomas versus tuberculomas, and recurrent tumours versus radiation necrosis.

A ‘Dynamic Adder Model’ For Cell Size Homeostasis in Dictyostelium Cells

After a cell divides into two daughter cells, the total cell surface area of the daughtercells should increase to the original size to maintain cell size homeostasis in a single cellcycle. Previously, three models have been proposed to explain the regulation of cell sizehomeostasis: sizer, timer, and adder models. Here, we precisely measured the total cellsurface area of Dictyostelium cells in a whole cell cycle by using the agar-overlaymethod, which eliminated the influence of surface membrane reservoirs, such asmicrovilli and membrane winkles. The total cell surface area linearly increased duringinterphase, slightly decreased at the metaphase, and then increased by approximately20% during cytokinesis. From the analysis of the added surface area, we concluded thatthe cell size was regulated by the near-adder model in interphase and by the timer modelin the mitotic phase. The adder model in the interphase is not caused by a simple cellmembrane addition, but is more dynamic due to the rapid cell membrane turnover. Wepropose a ‘dynamic adder model’ to explain cell size homeostasis in the interphase.

A novel roadmap connecting the 1H-MRS total choline resonance to all hallmarks of cancer following targeted therapy

This review describes a cellular adaptive stress signalling roadmap connecting the 1H magnetic resonance spectroscopy (MRS) total choline peak at 3.2 ppm (tCho) to cancer response after targeted therapy (TT). Recent research on cell signalling, tCho metabolism, and TT of cancer has been retrospectively re-examined. Signalling research describes how the unfolded protein response (UPR), a major stress signalling network, transduces, regulates, and rewires the total membrane turnover in different cancer hallmarks after a TT stress. In particular, the UPR signalling maintains or increases total membrane turnover in all pro-survival hallmarks, whilst dramatically decreases turnover during apoptosis, a pro-death hallmark. Recent research depicts the TT-induced stress as a crucial event responsible for interrupting UPR pro-survival pathways, leading to an UPR-mediated cell death. The 1H-MRS tCho resonance represents the total mobile precursors and products during the enzymatic modification of phosphatidylcholine membrane abundance. The tCho profile represents a biomarker that noninvasively monitors TT-induced enzymatic changes in total membrane turnover in a wide variety of existing and new anticancer treatments targeting specific layers of the UPR signalling network. Our overview strongly suggests further evaluating and validating the 1H-MRS tCho peak as a powerful noninvasive imaging biomarker of cancer response in TT clinical trials.

Ultrastructural observations on the oncomiracidium epidermis and adult tegument of Discocotyle sagittata, a monogenean gill parasite of salmonids

During their different life stages, parasites undergo remarkable morphological, physiological, and behavioral “metamorphoses” to meet the needs of their changing habitats. This is even true for ectoparasites, such as the monogeneans, which typically have a free-swimming larval stage (oncomiracidium) that seeks out and attaches to the external surfaces of fish where they mature. Before any obvious changes occur, there are ultrastructural differences in the oncomiracidium’s outer surface that prepare it for a parasitic existence. The present findings suggest a distinct variation in timing of the switch from oncomiracidia epidermis to the syncytial structure of the adult tegument and so, to date, there are three such categories within the Monogenea: (1) Nuclei of both ciliated cells and interciliary cytoplasm are shed from the surface layer and the epidermis becomes a syncytial layer during the later stages of embryogenesis; (2) nuclei of both ciliated cells and interciliary syncytium remain distinct and the switch occurs later after the oncomiracidia hatch (as in the present study); and (3) the nuclei remain distinct in the ciliated epidermis but those of the interciliary epidermis are lost during embryonic development. Here we describe how the epidermis of the oncomiracidium of Discocotyle sagittata is differentiated into two regions, a ciliated cell layer and an interciliary, syncytial cytoplasm, both of which are nucleated. The interciliary syncytium extends in-between and underneath the ciliated cells and sometimes covers part of their apical surfaces, possibly the start of their shedding process. The presence of membranous whorls and pyknotic nuclei over the surface are indicative of membrane turnover suggesting that the switch in epidermis morphology is already initiated at this stage. The body tegument and associated putative sensory receptors of subadult and adult D. sagittata are similar to those in other monogeneans.

Deuterium Depletion Inhibits Cell Proliferation, RNA and Nuclear Membrane Turnover to Enhance Survival in Pancreatic Cancer

The effects of deuterium-depleted water (DDW) containing deuterium (D) at a concentration of 25 parts per million (ppm), 50 ppm, 105 ppm and the control at 150 ppm were monitored in MIA-PaCa-2 pancreatic cancer cells by the real-time cell impedance detection xCELLigence method. The data revealed that lower deuterium concentrations corresponded to lower MiA PaCa-2 growth rate. Nuclear membrane turnover and nucleic acid synthesis rate at different D-concentrations were determined by targeted [1,2-13C2]-D-glucose fate associations. The data showed severely decreased oxidative pentose cycling, RNA ribose 13C labeling from [1,2-13C2]-D-glucose and nuclear membrane lignoceric (C24:0) acid turnover. Here, we treated advanced pancreatic cancer patients with DDW as an extra-mitochondrial deuterium-depleting strategy and evaluated overall patient survival. Eighty-six (36 male and 50 female) pancreatic adenocarcinoma patients were treated with conventional chemotherapy and natural water (control, 30 patients) or 85 ppm DDW (56 patients), which was gradually decreased to preparations with 65 ppm and 45 ppm deuterium content for each 1 to 3 months treatment period. Patient survival curves were calculated by the Kaplan-Meier method and Pearson correlation was taken between medial survival time (MST) and DDW treatment in pancreatic cancer patients. The MST for patients consuming DDW treatment (n = 56) was 19.6 months in comparison with the 6.36 months’ MST achieved with chemotherapy alone (n = 30). There was a strong, statistically significant Pearson correlation (r = 0.504, p < 0.001) between survival time and length and frequency of DDW treatment.