Dimensions and surface roughness of magnesium alloy tubes were evaluated to elucidate the flattening behavior of asperity on the outer surface and the optimal condition for achieving thin wall and ...high surface quality in the combined process of the hollow sinking after die-less mandrel drawing. ZM21 tubes were drawn using a die after die-less mandrel drawing. Dimensions and surface roughness were measured, and the stress state of the asperity during hollow sinking was evaluated using finite element and slab methods. The results indicated that in hollow sinking after die-less mandrel drawing when the speed ratio was large, both a thin wall and high inner and outer surface qualities can be achieved under low die reduction, where the outer surface roughness was the least, owing to relatively large die pressure and drawing stress. Additionally, enhancing the flattening of the asperity due to bulk plastic deformation in tension was difficult even if drawing stress increased when the speed ratio in die-less mandrel drawing was large. Therefore, when the speed ratio of the die-less mandrel drawing was large before hollow sinking, the largest outer surface flattening was achieved under low die reduction, wherein die pressure was large.
Although sphingomyelin and cholesterol are major lipids of mammalian cells, the detailed distribution of these lipids in cellular membranes remains still obscure. However, the recent development of ...protein probes that specifically bind sphingomyelin and/or cholesterol provides new information about the landscape of the lipid domains that are enriched with sphingomyelin or cholesterol or both. Here, we critically summarize the tools to study distribution and dynamics of sphingomyelin and cholesterol. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.
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•Small molecules and proteins that bind specific lipids are employed as lipid probes.•Sphingomyelin (SM) and cholesterol (Chol) form characteristic lipid domains.•Lipid probes reveal the distribution of endogenous SM-and Chol-rich domains.•Fluorescent analogs of SM and Chol are useful to study dynamics of these lipids.
In eukaryotic cells, phospholipid flippases translocate phospholipids from the exoplasmic to the cytoplasmic leaflet of the lipid bilayer. Budding yeast contains five flippases, of which Cdc50p-Drs2p ...and Neo1p are primarily involved in membrane trafficking in endosomes and Golgi membranes. The ANY1/CFS1 gene was identified as a suppressor of growth defects in the neo1Δ and cdc50Δ mutants. Cfs1p is a membrane protein of the PQ-loop family and is localized to endosomal/Golgi membranes, but its relationship to phospholipid asymmetry remains unknown. The neo1Δ cfs1Δ mutant appears to function normally in membrane trafficking but may function abnormally in the regulation of phospholipid asymmetry. To identify a gene that is functionally relevant to NEO1 and CFS1, we isolated a mutation that is synthetically lethal with neo1Δ cfs1Δ and identified ERD1. Erd1p is a Golgi membrane protein that is involved in the transport of phosphate (Pi) from the Golgi lumen to the cytoplasm. The Neo1p-depleted cfs1Δ erd1Δ mutant accumulated plasma membrane proteins in the Golgi, perhaps due to a lack of phosphatidylinositol 4-phosphate. The Neo1p-depleted cfs1Δ erd1Δ mutant also exhibited abnormal structure of the endoplasmic reticulum (ER) and induced an unfolded protein response, likely due to defects in the retrieval pathway from the cis-Golgi region to the ER. Genetic analyses suggest that accumulation of Pi in the Golgi lumen is responsible for defects in Golgi functions in the Neo1p-depleted cfs1Δ erd1Δ mutant. Thus, the luminal ionic environment is functionally relevant to phospholipid asymmetry. Our results suggest that flippase-mediated phospholipid redistribution and luminal Pi concentration coordinately regulate Golgi membrane functions.
This study aimed to explore novel microRNAs in plasma for predicting chemoresistance in adjuvant chemotherapy for patients with gastric cancer (GC). We used the Toray 3D-Gene microRNA array-based ...approach to compare preoperative plasma microRNA levels between GC patients with and without recurrences after curative gastrectomy. All patients underwent adjuvant chemotherapy with S-1, an oral fluoropyrimidine. Of 2566 candidates, six candidate microRNAs (miR-1229-3p, 1249-5p, 762, 711, 1268a and 1260b), which were highly expressed in the preoperative plasma of patients with subsequent recurrences, were selected. In a large-scale validation analysis by quantitative RT-PCR, we focused on high plasma levels of miR-1229-3p, which was an independent poor prognostic factor for recurrence free survival (P = 0.009, HR = 3.71). Overexpression of miR-1229-3p in GC cells induced significant chemoresistance to 5-fluorouracil (5-FU), up-regulation of thymidylate synthase (TS) and dihydroprimidine dehydrogenase (DPD) and down-regulation of SLC22A7 both in vitro and in vivo. Intraperitoneal injection of miR-1229-3p in mice induced significant chemoresistance to 5-FU, accompanied by high levels of miR-1229-3p in plasma and tumor tissue. These findings suggest that plasma miR-1229-3p might be a clinically useful biomarker for predicting chemoresistance to S-1 and selecting other or combined intensive chemotherapy regimens in GC patients.
Epithelial cells provide cell-cell adhesion that is essential to maintain the integrity of multicellular organisms. Epithelial cell-characterizing proteins, such as epithelial junctional proteins and ...transcription factors are well defined. However, the role of lipids in epithelial characterization remains poorly understood. Here we show that the phospholipid phosphatidylinositol (4,5)-bisphosphate PI(4,5)P
is enriched in the plasma membrane (PM) of epithelial cells. Epithelial cells lose their characteristics upon depletion of PM PI(4,5)P
, and synthesis of PI(4,5)P
in the PM results in the development of epithelial-like morphology in osteosarcoma cells. PM localization of PARD3 is impaired by depletion of PM PI(4,5)P
in epithelial cells, whereas expression of the PM-targeting exocyst-docking region of PARD3 induces osteosarcoma cells to show epithelial-like morphological changes, suggesting that PI(4,5)P
regulates epithelial characteristics by recruiting PARD3 to the PM. These results indicate that a high level of PM PI(4,5)P
plays a crucial role in the maintenance of epithelial characteristics.
Sterols are important lipid components of the plasma membrane (PM) in eukaryotic cells, but it is unknown how the PM retains sterols at a high concentration. Phospholipids are asymmetrically ...distributed in the PM, and phospholipid flippases play an important role in generating this phospholipid asymmetry. Here, we provide evidence that phospholipid flippases are essential for retaining ergosterol in the PM of yeast. A mutant in three flippases, Dnf1-Lem3, Dnf2-Lem3, and Dnf3-Crf1, and a membrane protein, Sfk1, showed a severe growth defect. We recently identified Sfk1 as a PM protein involved in phospholipid asymmetry. The PM of this mutant showed high permeability and low density. Staining with the sterol probe filipin and the expression of a sterol biosensor revealed that ergosterol was not retained in the PM. Instead, ergosterol accumulated in an esterified form in lipid droplets. We propose that ergosterol is retained in the PM by the asymmetrical distribution of phospholipids and the action of Sfk1. Once phospholipid asymmetry is severely disrupted, sterols might be exposed on the cytoplasmic leaflet of the PM and actively transported to the endoplasmic reticulum by sterol transfer proteins.
P4‐ATPases translocate aminophospholipids, such as phosphatidylserine (PS), to the cytosolic leaflet of membranes. PS is highly enriched in recycling endosomes (REs) and is essential for endosomal ...membrane traffic. Here, we show that PS flipping by an RE‐localized P4‐ATPase is required for the recruitment of the membrane fission protein EHD1. Depletion of ATP8A1 impaired the asymmetric transbilayer distribution of PS in REs, dissociated EHD1 from REs, and generated aberrant endosomal tubules that appear resistant to fission. EHD1 did not show membrane localization in cells defective in PS synthesis. ATP8A2, a tissue‐specific ATP8A1 paralogue, is associated with a neurodegenerative disease (CAMRQ). ATP8A2, but not the disease‐causative ATP8A2 mutant, rescued the endosomal defects in ATP8A1‐depleted cells. Primary neurons from Atp8a2−/− mice showed a reduced level of transferrin receptors at the cell surface compared to Atp8a2+/+ mice. These findings demonstrate the role of P4‐ATPase in membrane fission and give insight into the molecular basis of CAMRQ.
Synopsis
The aminophospholipid translocase ATP8A1 enriches phosphatidylserine at the cytosolic leaflet of recycling endosomes, recruiting fission protein EHD1. Phosphatidylserine translocation is required for cargo sorting and membrane fission, a process perturbed in CAMRQ disease‐associated ATP8A2 mutant cells.
P4‐ATPase ATP8A1 flips phosphatidylserine (PS) to the cytosolic leaflet of recycling endosomes (REs).
PS recruits membrane fission protein EHD1 to REs.
The ATP8A1/PS/EHD1 axis regulates membrane traffic from REs.
Membrane traffic defects at REs may underlie a neurodegenerative disease associated with ATP8A2 mutations.
The aminophospholipid translocase ATP8A1 enriches phosphatidylserine at the cytosolic leaflet of recycling endosomes, recruiting fission protein EHD1. Phosphatidylserine translocation is required for cargo sorting and membrane fission, a process perturbed in CAMRQ disease‐associated ATP8A2 mutant cells.
The crystal deformation behavior of the micro tube was investigated to clarify the crystal orientation, which suppresses the development of the inner surface roughness during the hollow sinking. ...Stainless steel tubes with an outer diameter of 1.50 mm and a wall thickness of 0.045 mm were drawn without an inner tool. The inner surface roughness and crystal orientation were examined using the same measurement area. The results indicated that the crystal grains with the {102} crystal planes vertical to the normal direction to the inner surface (ND) suppressed the increase in the height of the unevenness of the surrounding crystal grains, including themselves. The height deviation of the unevenness of the wall-thickened tube was larger than that of the wall-thinned one. Meanwhile, the number of crystal grains with the {102} crystal plane vertical to ND of the wall-thinned tube was larger than that of the wall-thickened one. Therefore, it was considered that this crystal orientation suppressed the development of the inner surface roughness during the hollow sinking. The development of the inner surface roughness was suppressed with decreasing wall thickness because the crystal grains rotated so that the normal direction of {102} crystal plane was parallel to the ND: meanwhile the tensile and compressive stresses were applied to the inner surface of the micro tube in the drawing and transversal directions, respectively.
The distributions of the outer and inner radii of die-less drawn β titanium alloy tubes were evaluated in the drawing direction to clarify the effect of the deformation behavior of the tube on taper ...shape control in the die-less forming. Ti–22V–4Al DAT51 (β Ti alloy) tubes with an outer radius of 0.81 mm were die-less drawn under various heating temperature and taper slope conditions. The outer and inner radius distributions were measured, and the drawing stress was evaluated during the drawing. As a result, a maximum reduction in cross-sectional area of 81% was achieved in a single pass of the die-less forming under a heating temperature of 800 °C and taper slope of −0.005. The outer radius became larger than that with the linear contour theory, which achieved the desired taper slope when the outer radius became smaller than a certain value during drawing. This is because the large slope in the deformation zone caused non-uniaxial tensile deformation dominated by wall thickness reduction. Therefore, the length between the heating and cooling coils should be large such that the slope of the deformation zone becomes small to satisfy uniaxial deformation, which is an assumption of the linear contour theory.
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