Cholesterol Dependence of Collagen and Echovirus 1 Trafficking along the Novel alpha 2 beta 1 Integrin Internalization Pathway

Peer reviewe

Nuuskaviesti vietiin välitunneille : toiminnallinen terveyden edistäminen tuottaa tuloksia

Terveydenhoitajaopiskelijat suunnittelivat ja toteuttivat ala-asteikäisille lapsille nuuskan vastaisen välituntiaktiviteetin osana terveydenhoitotyön kehittämistyötään. Lähtökohtana oli suunnitella toimintaa, jota on mahdollista toteuttaa myös myöhemmin esimerkiksi opettajien toimesta osana lasten ja nuorten terveyskasvatusta. Opiskelijoiden tuottama Nuuskaviesti-vihkonen ohjaa konkreettisesti opettajia ja muuta koulun henkilökuntaa toteuttamaan vastaavanlainen aktiviteetti

Evidence for the mechanosensor function of filamin in tissue development

Cells integrate mechanical properties of their surroundings to form multicellular, three-dimensional tissues of appropriate size and spatial organisation. Actin cytoskeleton-linked proteins such as talin, vinculin and filamin function as mechanosensors in cells, but it has yet to be tested whether the mechanosensitivity is important for their function in intact tissues. Here we tested, how filamin mechanosensing contributes to oogenesis in Drosophila. Mutations that require more or less force to open the mechanosensor region demonstrate that filamin mechanosensitivity is important for the maturation of actin-rich ring canals that are essential for Drosophila egg development. The open mutant was more tightly bound to the ring canal structure while the closed mutant dissociated more frequently. Thus, our results show that an appropriate level of mechanical sensitivity is required for filamins’ function and dynamics during Drosophila egg growth and support the structure-based model in which the opening and closing of the mechanosensor region regulates filamin binding to cellular components.peerReviewe

Calpains promote α2β1 integrin turnover in nonrecycling integrin pathway

A novel virus- and integrin clustering–specific pathway diverts integrin from its normal endo/exocytic traffic to a nonrecycling degradative endosomal route. Clustering of α2β1 integrin causes redistribution of the integrin to perinuclear endosomes, leading to enhanced integrin turnover promoted by calpains

Inhibition of cholesterol synthesis disrupts integrin internalization and EV1 infection.

<p>A) Thin layer chromatography analysis of sterols in SAOS-α2β1 cells. B) Representative pictures of internalization of α2 integrin after ketoconazole, 5% LPDS or 10% FCS treatments. Internalized integrin is seen as green labeling and surface-bound integrin as red or yellow dye. The ratio of voxels between surface and internalized integrin was quantified with internalization algorithm embedded in BioImageXD software. Higher ratio means higher amount of integrin in plasma membrane. Results are averages from together 33 cells from 3 independent tests+SE. C) Electron microscopic example images of integrin structures after antibody clustering in 5% LPDS+ketoconazole cells at 0.5 and 3.5 h time points. D) Proportional change of EV1 infectivity in cells treated with 10% FCS DMEM, 5% LDPS DMEM or 5% LPDS DMEM with ketoconazole. Results are averages of four independent tests (+SE). **P<0.05, ***P<0.001. E) EV1 infectivity in cells treated with 10% FCS DMEM, 5% LDPS DMEM or 5% LPDS+mβCD-cholesterol. Results are averages of three independent tests (+SE), together over 750 cells were counted. F) The effect of U18666A (3 µg/ml) on EV1 infection percentage. EV1 infectivity was calculated together from 750 cells from three individual tests (+SE). Cholesterol labeling with filipin and lysosomal labeling with Lamp-1 was performed to confirm the efficacy of the drug. Bars 10 µm.</p

Collagen uptake is disturbed by ketoconazole treatment.

<p>A) Percentage of cells showing internalized collagen vesicles vs. cell surface-enriched collagen was counted from 77 to 80 cells cultivated in 10% DMEM or 5% LPDS DMEM with ketoconazole, respectively. Typical images used in calculations are shown. Bars 10 µm. B) Ratio of cell surface-enriched vs. internalized collagen was calculated from confocal sections from 20 cells cultivated for 6 h in 10% DMEM, 5% LPDS or 5% LPDS with ketoconazole. Surface-labeled collagen is seen as green or yellow and internalized collagen as red staining. Representative images are shown. Higher ratio means higher collagen label at plasma membrane. Results are mean values from together 20 cells of 2 independent tests (+ SE). Bars 10 µm.</p

The effects of cholesterol aggregating drugs on EV1 uncoating.

<p>A) EV1 infectivity of control cells and cells treated with filipin and nystatin after 5 min p.i. (P<0.001, binomial <i>t</i>-test). Confocal images show labeled EV1 capsid proteins at 7 h p.i. Bars, 10 µm. B) EV1 infection percentage of cells treated with nystatin or filipin that were added at different time points p.i. The results are mean values of 3 independent experiments ± SE (more than 700 cells counted). C) Infection percentage of neutral-red labeled EV1 (NR-EV1) with light treatments in different times p.i. The control cells were not exposed to light reaction. Results are averages of 2 independent tests (+ SE) and over 800 cells were counted at the minimum. D) Sucrose gradient sedimentation assay of uncoating with [³⁵S]methionine-labeled EV1 at 4 h p.i. RNA containing virus sediments at 160S, whereas the 80S represents empty capsids from which the viral RNA genome is released. E-F) As in D except filipin or nystatin was added to the cells 15 min p.i. G) Cointernalized Fluospheres and clustered α2 integrin colocalize in endosomes. Total intensity of FluoSpheres in endosomes was analysed from confocal sections by using the intensity algorithm in BioImageXD. Together 20 cells from 2 separate tests were analysed (+ SE). *P<0.01. Bars, 10 µm.</p