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A myosin-Va tail fragment sequesters dynein light chains leading to apoptosis in melanoma cells

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T. C. Izidoro-Toledo
T. C. Izidoro-Toledo
T. C. Izidoro-Toledo
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Antonio Carlos Borges at Brazilian Center for Research in Energy and Materials
Antonio Carlos Borges
  • Brazilian Center for Research in Energy and Materials
Daniela Dover Araujo at Centro Universitário da Fundação Educacional Guaxupé
Daniela Dover Araujo
D P S Leitão Mazzi
D P S Leitão Mazzi
D P S Leitão Mazzi
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Abstract and Figures

Previous studies proposed that myosin-Va regulates apoptosis by sequestering pro-apoptotic Bmf to the actin cytoskeleton through dynein light chain-2 (DLC2). Adhesion loss or other cytoskeletal perturbations would unleash Bmf, allowing it to bind and inhibit pro-survival Bcl2 proteins. Here, we demonstrated that overexpression of a myosin-Va medial tail fragment (MVaf) harboring the binding site for DLC2 dramatically decreased melanoma cell viability. Morphological and molecular changes, including surface blebbing, mitochondrial outer membrane permeabilization, cytochrome-c and Smac release, as well as caspase-9/-3 activation and DNA fragmentation indicated that melanoma cells died of apoptosis. Immobilized MVaf interacted directly with DLCs, but complexed MVaf/DLCs did not interact with Bmf. Overexpression of DLC2 attenuated MVaf-induced apoptosis. Thus, we suggest that, MVaf induces apoptosis by sequestering DLC2 and DLC1, thereby unleashing the pair of sensitizer and activator BH3-only proteins Bmf and Bim. Murine embryonic fibroblasts (MEFs) lacking Bim and Bmf or Bax and Bak were less sensitive to apoptosis caused by MVaf expression than wild-type MEFs, strengthening the putative role of the intrinsic apoptotic pathway in this response. Finally, MVaf expression attenuated B16-F10 solid tumor growth in mice, suggesting that this peptide may be useful as an apoptosis-inducing tool for basic and translational studies.
MVaf expression reduces melanoma cell viability in an exon B-dependent manner. (a) Schematic diagram representing the primary structure of myosin-Va heavy chain shows the positions of the structure sub-domains (H, head; N, neck; PT, proximal tail; MT, medial tail; GT, globular tail) and the DLC2 binding site in the MT domain, with exon combination A, B, C, D, E and F. Exons B, D, and F are subject to alternative splicing. Detail: schematic diagram of selected segments for recombinant constructs. EGFP–MYO5A(1276–1328[+B]) fragment (MVaf1), and variants, MVaf2(1255–1320[+B]) and MVaf3(1255–1320[-B]). EGFP–MYO5C(1179–1232) construct was used as a control and comprised a region with low sequence conservation between the two paralogs (myosin-Va and -Vc). (b) Representative images of B16-F10 cell cultures, 18 and 72 h post transfection with pEGFP, pEGFP-MVcf and pEGFP-MVaf1. Images were acquired under a fluorescence stereomicroscope. (c) Proliferation rates were determined as the average number of fluorescent cells per area of growth (mm2) after 8, 18, 48 and 72 h post transfection. ***Statistic significance (P<0.001). Kinetics of recombinant protein expression levels: (d) EFGP expression in B16-F10 cell lysates was quantified through ImageJ software analysis of western blots generated with an anti-EGFP (bottom panel) relative to γ-tubulin, a loading control (top panel). (e) Viability of cells expressing EGFP-MVaf1 or EGFP was determined by PI staining of dead cells at indicated times post transfection. ***Statistically significant mean difference between EGFP and EGFP-MVaf1 groups (P<0.001). (f) Representative images of cultures taken 18 and 96 h post transfection with pEGFP (control), pEGFP-MVaf1, pEGFP-MVaf2 or pEGFP-MVaf3. (g) Proliferation rates of B16-F10 cell cultures expressing either EGFP (control), EGFP-MVaf1, EGFP-MVaf2 or EGFP-MVaf3 were determined as the average number of fluorescent cells per area of growth (mm2), per dish (n=3) at 8, 18, 48, 72 and 96 h post transfection. **Statistically significant mean difference between control (EGFP) and test groups (EGFP-MVaf1 and EGFP-MVaf2) (P<0.01)
MVaf expression reduces melanoma cell viability in an exon B-dependent manner. (a) Schematic diagram representing the primary structure of myosin-Va heavy chain shows the positions of the structure sub-domains (H, head; N, neck; PT, proximal tail; MT, medial tail; GT, globular tail) and the DLC2 binding site in the MT domain, with exon combination A, B, C, D, E and F. Exons B, D, and F are subject to alternative splicing. Detail: schematic diagram of selected segments for recombinant constructs. EGFP–MYO5A(1276–1328[+B]) fragment (MVaf1), and variants, MVaf2(1255–1320[+B]) and MVaf3(1255–1320[-B]). EGFP–MYO5C(1179–1232) construct was used as a control and comprised a region with low sequence conservation between the two paralogs (myosin-Va and -Vc). (b) Representative images of B16-F10 cell cultures, 18 and 72 h post transfection with pEGFP, pEGFP-MVcf and pEGFP-MVaf1. Images were acquired under a fluorescence stereomicroscope. (c) Proliferation rates were determined as the average number of fluorescent cells per area of growth (mm2) after 8, 18, 48 and 72 h post transfection. ***Statistic significance (P<0.001). Kinetics of recombinant protein expression levels: (d) EFGP expression in B16-F10 cell lysates was quantified through ImageJ software analysis of western blots generated with an anti-EGFP (bottom panel) relative to γ-tubulin, a loading control (top panel). (e) Viability of cells expressing EGFP-MVaf1 or EGFP was determined by PI staining of dead cells at indicated times post transfection. ***Statistically significant mean difference between EGFP and EGFP-MVaf1 groups (P<0.001). (f) Representative images of cultures taken 18 and 96 h post transfection with pEGFP (control), pEGFP-MVaf1, pEGFP-MVaf2 or pEGFP-MVaf3. (g) Proliferation rates of B16-F10 cell cultures expressing either EGFP (control), EGFP-MVaf1, EGFP-MVaf2 or EGFP-MVaf3 were determined as the average number of fluorescent cells per area of growth (mm2), per dish (n=3) at 8, 18, 48, 72 and 96 h post transfection. **Statistically significant mean difference between control (EGFP) and test groups (EGFP-MVaf1 and EGFP-MVaf2) (P<0.01)
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Cells expressing MVaf1 developed morphological features of apoptosis. Fluorescence and correlative light microscopy (LM)/SEM images are shown of B16-F10 cells, expressing either EGFP-MVaf1 or EGFP (control). (a) Fluorescence images show EGFP, F-actin (Phalloidin-Rhodamine) and nuclei (DAPI) staining. EGFP-MVaf1-expressing cells (*) lack stress fibers, prominent in control cells (>, <), and display a peripheral F-actin ring, as seen in the single section image (insert, arrow). (b) Time-lapse. B16-F10 cells grown on glass-bottom microwell dishes (Φ 35 mm) and transfected with pEGFP-MVaf1 or pEGFP (control). (c) SEM images of B16-F10 cells transfected with pEGFP-MVaf1. B16-F10 cells grown on CELLocate-microgrid glass coverslips were imaged by LM, 24 h post transfection. SEM images of the corresponding cells were taken in two subsequent magnifications. Transfected cells exhibit apoptotic features typical of late execution phase, such as loss of attachment and plasma membrane blebs. (d) Apoptotic response to EGFP-MVaf1 involves DNA fragmentation. Detection of DNA fragmentation in suspended B16-F10 cells was assessed by the TUNEL assay. Scatter dot plot with mean±S.E.M. showing the ratio of TUNEL-positive nuclei to total nuclei counts in EGFP- and EGFP-MVaf1-expressing cultures (n=10 fields). Each data point represents a pair of images, i.e., one field. ***Statistically significant mean difference between EGFP and EGFP-MVaf1 groups (P<0.001). Right panel shows representative differential interference contrast (DIC) and TUNEL stain (red) images of a single field from either EGFP and EGFP-MVaf1-expressing cultures
Cells expressing MVaf1 developed morphological features of apoptosis. Fluorescence and correlative light microscopy (LM)/SEM images are shown of B16-F10 cells, expressing either EGFP-MVaf1 or EGFP (control). (a) Fluorescence images show EGFP, F-actin (Phalloidin-Rhodamine) and nuclei (DAPI) staining. EGFP-MVaf1-expressing cells (*) lack stress fibers, prominent in control cells (>, <), and display a peripheral F-actin ring, as seen in the single section image (insert, arrow). (b) Time-lapse. B16-F10 cells grown on glass-bottom microwell dishes (Φ 35 mm) and transfected with pEGFP-MVaf1 or pEGFP (control). (c) SEM images of B16-F10 cells transfected with pEGFP-MVaf1. B16-F10 cells grown on CELLocate-microgrid glass coverslips were imaged by LM, 24 h post transfection. SEM images of the corresponding cells were taken in two subsequent magnifications. Transfected cells exhibit apoptotic features typical of late execution phase, such as loss of attachment and plasma membrane blebs. (d) Apoptotic response to EGFP-MVaf1 involves DNA fragmentation. Detection of DNA fragmentation in suspended B16-F10 cells was assessed by the TUNEL assay. Scatter dot plot with mean±S.E.M. showing the ratio of TUNEL-positive nuclei to total nuclei counts in EGFP- and EGFP-MVaf1-expressing cultures (n=10 fields). Each data point represents a pair of images, i.e., one field. ***Statistically significant mean difference between EGFP and EGFP-MVaf1 groups (P<0.001). Right panel shows representative differential interference contrast (DIC) and TUNEL stain (red) images of a single field from either EGFP and EGFP-MVaf1-expressing cultures
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Protein interaction and co-expression assays. (a) MVaf1, but not MVcf, interacts with DLC1 and DLC2 in vitro. Bacterially expressed and purified GST or GST fusion proteins immobilized on Glutathione-Sepharose 4B beads were incubated with 200 ng (*) and 800 ng (**) of purified MBP-DLC2 proteins or MBP alone. Full-length myosin-Va MT fused to GST (GST-MT) was used as a positive control for interaction with DLC2. The purified proteins were separated on 5–20% SDS-PAGE gel and stained by Coomassie blue (top panel). The migration positions of the molecular mass markers (kDa) are indicated on the left hand side. Pulled down proteins were blotted with anti-DLC2 and anti-PIN antibodies (bottom panel). Black arrowheads indicate the migration position of MBP-DLC2. (b) GST pull-down experiments for MVaf1 and DLC1 or DLC2. Bacterially expressed and purified GST-MVaf1 and GST-MT fusion proteins immobilized on Glutathione-Sepharose 4B beads were incubated with cell lysates from HEK-293T, expressing either EGFP-DLC1, EGFP-DLC2 or EGFP alone. Proteins from pull-down interactions or post-pull-down supernatants were separated on 5–20% SDS-PAGE gel and stained by Coomassie blue (top panel). Western blots were probed with anti-EGFP and anti-PIN antibodies (bottom panels). Black arrowheads indicates EGFP-DLC 1 or 2, and white arrowhead indicates EGFP. (c) DsRed-MVaf1 co-localizes with EGFP-DLC2 in the cell cytoplasm. B16-F10 cells expressing either (top panel), or both (central panel) EGFP-DLC2 and DsRed-MVaf1, were fixed and imaged by confocal microscopy. Colocalization was analyzed using Leica LAS-AF software. (d) Co-expression of DLC2 with MVaf1 attenuates MVaf1-induced cell death. Proliferation rates of B16-F10 cell cultures expressing either EGFP (control), EGFP-MVaf1, EGFP-DLC2 or co-expressing EGFP-MVaf1 and EGFP-DLC2 were determined as the average number of fluorescent cells per area of growth (mm2), per dish (n=4), after 8, 18, 48 and 72 h post transfection. (e-g) GST pull-down experiments for investigating the interactions between MVaf1, DLCs, Bmf and Bcl2. Bacterially expressed and purified GST fusion proteins (as indicated) were immobilized on Glutathione-sepharose 4B beads and incubated with cell lysates from HEK-293T expressing EGFP-Bmf alone (e), or the combination of EGFP-Bmf plus EGFP-Bcl2 (f), or EGFP-Bmf plus either EGFP-DLC1 or EGFP-DLC2 (g). Proteins from pull-down interactions were separated on 5–20% SDS-PAGE gel and stained by Coomassie blue (top panels). Western blots were probed with anti-EGFP (bottom panels) or anti-Bcl2, as indicated. Note strong interaction of Bmf with DLC2 and only a poor interaction with DLC1. Also note that immobilized DLC2 bound Bmf/Bcl2, whereas immobilized myosin-Va tail fragments pulled-down DLCs but left behind Bmf in the supernatant, indicating that DLC2 was unable to play an adaptor function
Protein interaction and co-expression assays. (a) MVaf1, but not MVcf, interacts with DLC1 and DLC2 in vitro. Bacterially expressed and purified GST or GST fusion proteins immobilized on Glutathione-Sepharose 4B beads were incubated with 200 ng (*) and 800 ng (**) of purified MBP-DLC2 proteins or MBP alone. Full-length myosin-Va MT fused to GST (GST-MT) was used as a positive control for interaction with DLC2. The purified proteins were separated on 5–20% SDS-PAGE gel and stained by Coomassie blue (top panel). The migration positions of the molecular mass markers (kDa) are indicated on the left hand side. Pulled down proteins were blotted with anti-DLC2 and anti-PIN antibodies (bottom panel). Black arrowheads indicate the migration position of MBP-DLC2. (b) GST pull-down experiments for MVaf1 and DLC1 or DLC2. Bacterially expressed and purified GST-MVaf1 and GST-MT fusion proteins immobilized on Glutathione-Sepharose 4B beads were incubated with cell lysates from HEK-293T, expressing either EGFP-DLC1, EGFP-DLC2 or EGFP alone. Proteins from pull-down interactions or post-pull-down supernatants were separated on 5–20% SDS-PAGE gel and stained by Coomassie blue (top panel). Western blots were probed with anti-EGFP and anti-PIN antibodies (bottom panels). Black arrowheads indicates EGFP-DLC 1 or 2, and white arrowhead indicates EGFP. (c) DsRed-MVaf1 co-localizes with EGFP-DLC2 in the cell cytoplasm. B16-F10 cells expressing either (top panel), or both (central panel) EGFP-DLC2 and DsRed-MVaf1, were fixed and imaged by confocal microscopy. Colocalization was analyzed using Leica LAS-AF software. (d) Co-expression of DLC2 with MVaf1 attenuates MVaf1-induced cell death. Proliferation rates of B16-F10 cell cultures expressing either EGFP (control), EGFP-MVaf1, EGFP-DLC2 or co-expressing EGFP-MVaf1 and EGFP-DLC2 were determined as the average number of fluorescent cells per area of growth (mm2), per dish (n=4), after 8, 18, 48 and 72 h post transfection. (e-g) GST pull-down experiments for investigating the interactions between MVaf1, DLCs, Bmf and Bcl2. Bacterially expressed and purified GST fusion proteins (as indicated) were immobilized on Glutathione-sepharose 4B beads and incubated with cell lysates from HEK-293T expressing EGFP-Bmf alone (e), or the combination of EGFP-Bmf plus EGFP-Bcl2 (f), or EGFP-Bmf plus either EGFP-DLC1 or EGFP-DLC2 (g). Proteins from pull-down interactions were separated on 5–20% SDS-PAGE gel and stained by Coomassie blue (top panels). Western blots were probed with anti-EGFP (bottom panels) or anti-Bcl2, as indicated. Note strong interaction of Bmf with DLC2 and only a poor interaction with DLC1. Also note that immobilized DLC2 bound Bmf/Bcl2, whereas immobilized myosin-Va tail fragments pulled-down DLCs but left behind Bmf in the supernatant, indicating that DLC2 was unable to play an adaptor function
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Human melanoma cell lines are prone to cell death triggered by MVaf1 and levels of myosin-Va/DLC2 appears to influence cell death sensitivity. (a). Proliferation rates of WM35 and WM902 cells expressing either EGFP (control) or EGFP-MVaf1 were determined as the average number of fluorescent cells per area of growth (20 random fields of 1.6 mm2 per dish; n=4), after 8, 18, 48, 72 and 96 h of transfection. Rates are expressed as a percentage increase or decrease in culture proliferation from 18–96 h (WM35) or 48–96 h (WM902) post transfection (on the right-hand side of proliferation curves). (b) Northern blot analysis. MYO5A and DLC2 gene expression profiles in human melanoma cell lines WM35 and WM902. Densitometry of the specific bands was done measured by the ratio of pixel intensity (relative signal) using ImageJ gel analysis software; β-actin mRNA was used as the loading control. (c) Proliferation rate of Cloudman S91 mouse melanoma cells (bearing a MYO5A loss-of-function mutation) expressing either EGFP (control) or EGFP-MVaf1. Rates expressed as percentage increase or decrease in proliferation from 18 to 72 h post transfection are indicated. (d) Cell death rates for Cloudman S91 cells expressing EGFP-MVaf1 and EGFP were determined by PI staining at 24, 48, 72 and 96 h post transfection. After imaging 20 random fields per dish (n=5), the percentage of unviable cells was calculated relative to the total number of green fluorescent cells. (e) Detection of DLCs in B16-F10 and S91 cell lysates. Cell lysates from B16-F10 and Cloudman S91 were collected, equivalent amounts of total protein was loaded and analyzed by electrophoresis on a 5–20% SDS-PAGE, stained with Coomassie blue (top panel). Anti-DLC/PIN, which recognizes both DLC 1 and 2, was used as detection antibody (bottom panel). bMVa, purified fraction of native myosin-Va from chick brain, was used as positive control for the detection of the light chain DLC2 (as indicated)
Human melanoma cell lines are prone to cell death triggered by MVaf1 and levels of myosin-Va/DLC2 appears to influence cell death sensitivity. (a). Proliferation rates of WM35 and WM902 cells expressing either EGFP (control) or EGFP-MVaf1 were determined as the average number of fluorescent cells per area of growth (20 random fields of 1.6 mm2 per dish; n=4), after 8, 18, 48, 72 and 96 h of transfection. Rates are expressed as a percentage increase or decrease in culture proliferation from 18–96 h (WM35) or 48–96 h (WM902) post transfection (on the right-hand side of proliferation curves). (b) Northern blot analysis. MYO5A and DLC2 gene expression profiles in human melanoma cell lines WM35 and WM902. Densitometry of the specific bands was done measured by the ratio of pixel intensity (relative signal) using ImageJ gel analysis software; β-actin mRNA was used as the loading control. (c) Proliferation rate of Cloudman S91 mouse melanoma cells (bearing a MYO5A loss-of-function mutation) expressing either EGFP (control) or EGFP-MVaf1. Rates expressed as percentage increase or decrease in proliferation from 18 to 72 h post transfection are indicated. (d) Cell death rates for Cloudman S91 cells expressing EGFP-MVaf1 and EGFP were determined by PI staining at 24, 48, 72 and 96 h post transfection. After imaging 20 random fields per dish (n=5), the percentage of unviable cells was calculated relative to the total number of green fluorescent cells. (e) Detection of DLCs in B16-F10 and S91 cell lysates. Cell lysates from B16-F10 and Cloudman S91 were collected, equivalent amounts of total protein was loaded and analyzed by electrophoresis on a 5–20% SDS-PAGE, stained with Coomassie blue (top panel). Anti-DLC/PIN, which recognizes both DLC 1 and 2, was used as detection antibody (bottom panel). bMVa, purified fraction of native myosin-Va from chick brain, was used as positive control for the detection of the light chain DLC2 (as indicated)
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Cells expressing MVaf1 undergo mitochondria-mediated apoptosis. (a) Immunolocalization of Cyt-c. B16-F10 cells expressing either EGFP-MVaf1 or EGFP (control) were stained by immunocytochemistry with anti-Cyt-c antibody. Cells displaying a diffuse cytosolic Cyt-c staining were counted among 10 random fields per coverslip, and the percentage of cells with released Cyt-c was calculated relative to the total number of green fluorescent cells. Right panel shows representative images of cells, expressing either EGFP or EGFP-MVaf1, 33 h post transfection. In most cells expressing EGFP (control), Cyt-c staining showed an abundant and granular perinuclear pattern indicating mitochondrial compartmentalization. EGFP-MVaf1-expressing cells showed more diffuse red fluorescence pattern indicating their release into cytosol (scale bar: 10 μm). (b) Smac-Cherry time-lapse. B16-F10 cells were co-transfected with pEGFP-MVaf1 and pSmac-Cherry. In the example shown, at 27 h post transfection, Smac-Cherry (red) is still compartmentalized in mitochondria. At 27 h and 20 min, Smac-cherry displays a widespread distribution within the cell. At 28 h, cells lost their fluorescence. (c) Western blot for caspase-9. Cell lysates from B16-F10 cells expressing either EGFP-MVaf1 or EGFP (control) were collected at 24, 36, 48 and 60 h post transfection, combined and analyzed by western blotting with anti-caspase-9, as detection antibody. Duplicates were run for each sample. (c, top) Bands corresponding to pro-caspase-9 (47 kDa) and caspase-9 (37 kDa), were identified in all samples; black arrowhead indicates a nonspecific cross-reacting band picked up by caspase-9 antibody, and served as loading control. (c, bottom) Graphical representation of the relative intensity of pro-caspase- and caspase-9 bands measured by the ratio of pixel intensity using ImageJ gel analysis software. (d) Caspase-3 activation. Frames selected from video acquired by time-lapse confocal microscopy. At 24 h post transfection with pEGFP-MVaf1 or pEGFP (control), B16-F10 cells were treated with caspase-3 Fluorogenic Substrate IX (Calbiochem) and (Z-DEVD)2–Rh110 for 10 min in order to monitor caspase-3 activity. (e) Scoring of apoptotic MEFs. Representative sequence of a 2.5-h exposure time-lapse of MEFWT, MEFBax−/−,Bak−/− and MEFBim−/−,Bmf−/− transiently transfected with pEGFP-MVaf1 and imaged 24 h later. Data is represented as percentage of dead cells relative to the total number of green fluorescent cells. Ten independent time-lapse recordings were acquired for each cell line. (f) Smac–Cherry release from MEFBax−/−, Bak−/− mitochondria. Bax/Bak double-knockout murine embryonic cells were co-transfected with pEGFP-MVaf1 and pSmac-Cherry. Images represent excerpts from DIC and fluorescence microscopy videos of a representative cell expressing EGFP-MVaf1. In the example shown, at 31 h and 20 min post transfection, Smac-Cherry (red) is still compartmentalized in mitochondria of the dying cell (observed as a punctate staining pattern). Wild-type MEFs underwent permeabilization of the mitochondrial outer membrane similar to B16-F10 melanoma cells (not shown). So MOMP is involved but ultimately does not appear to be crucial for the occurrence of cell death
+2
Cells expressing MVaf1 undergo mitochondria-mediated apoptosis. (a) Immunolocalization of Cyt-c. B16-F10 cells expressing either EGFP-MVaf1 or EGFP (control) were stained by immunocytochemistry with anti-Cyt-c antibody. Cells displaying a diffuse cytosolic Cyt-c staining were counted among 10 random fields per coverslip, and the percentage of cells with released Cyt-c was calculated relative to the total number of green fluorescent cells. Right panel shows representative images of cells, expressing either EGFP or EGFP-MVaf1, 33 h post transfection. In most cells expressing EGFP (control), Cyt-c staining showed an abundant and granular perinuclear pattern indicating mitochondrial compartmentalization. EGFP-MVaf1-expressing cells showed more diffuse red fluorescence pattern indicating their release into cytosol (scale bar: 10 μm). (b) Smac-Cherry time-lapse. B16-F10 cells were co-transfected with pEGFP-MVaf1 and pSmac-Cherry. In the example shown, at 27 h post transfection, Smac-Cherry (red) is still compartmentalized in mitochondria. At 27 h and 20 min, Smac-cherry displays a widespread distribution within the cell. At 28 h, cells lost their fluorescence. (c) Western blot for caspase-9. Cell lysates from B16-F10 cells expressing either EGFP-MVaf1 or EGFP (control) were collected at 24, 36, 48 and 60 h post transfection, combined and analyzed by western blotting with anti-caspase-9, as detection antibody. Duplicates were run for each sample. (c, top) Bands corresponding to pro-caspase-9 (47 kDa) and caspase-9 (37 kDa), were identified in all samples; black arrowhead indicates a nonspecific cross-reacting band picked up by caspase-9 antibody, and served as loading control. (c, bottom) Graphical representation of the relative intensity of pro-caspase- and caspase-9 bands measured by the ratio of pixel intensity using ImageJ gel analysis software. (d) Caspase-3 activation. Frames selected from video acquired by time-lapse confocal microscopy. At 24 h post transfection with pEGFP-MVaf1 or pEGFP (control), B16-F10 cells were treated with caspase-3 Fluorogenic Substrate IX (Calbiochem) and (Z-DEVD)2–Rh110 for 10 min in order to monitor caspase-3 activity. (e) Scoring of apoptotic MEFs. Representative sequence of a 2.5-h exposure time-lapse of MEFWT, MEFBax−/−,Bak−/− and MEFBim−/−,Bmf−/− transiently transfected with pEGFP-MVaf1 and imaged 24 h later. Data is represented as percentage of dead cells relative to the total number of green fluorescent cells. Ten independent time-lapse recordings were acquired for each cell line. (f) Smac–Cherry release from MEFBax−/−, Bak−/− mitochondria. Bax/Bak double-knockout murine embryonic cells were co-transfected with pEGFP-MVaf1 and pSmac-Cherry. Images represent excerpts from DIC and fluorescence microscopy videos of a representative cell expressing EGFP-MVaf1. In the example shown, at 31 h and 20 min post transfection, Smac-Cherry (red) is still compartmentalized in mitochondria of the dying cell (observed as a punctate staining pattern). Wild-type MEFs underwent permeabilization of the mitochondrial outer membrane similar to B16-F10 melanoma cells (not shown). So MOMP is involved but ultimately does not appear to be crucial for the occurrence of cell death
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Supplementary resources (6)

Supplementary Video fig5d
Data
March 2013
T C Izidoro-Toledo · Antonio Carlos Borges · Daniela Dover Araujo · D P S Leitão Mazzi · Enilza M Espreafico
Supplementary Video fig2b EGFP
Data
March 2013
T C Izidoro-Toledo · Antonio Carlos Borges · Daniela Dover Araujo · D P S Leitão Mazzi · Enilza M Espreafico
Supplementary Video fig5b
Data
March 2013
T C Izidoro-Toledo · Antonio Carlos Borges · Daniela Dover Araujo · D P S Leitão Mazzi · Enilza M Espreafico
Supplementary Figure 1
Data
March 2013
T C Izidoro-Toledo · Antonio Carlos Borges · Daniela Dover Araujo · D P S Leitão Mazzi · Enilza M Espreafico
Supplementary Video fig2b EGFPMVaf
Data
March 2013
T C Izidoro-Toledo · Antonio Carlos Borges · Daniela Dover Araujo · D P S Leitão Mazzi · Enilza M Espreafico
Supplementary Video fig2c
Data
March 2013
T C Izidoro-Toledo · Antonio Carlos Borges · Daniela Dover Araujo · D P S Leitão Mazzi · Enilza M Espreafico
... Initial research of MYO5A concentrated on its role in neurological diseases [35][36][37][38]. As research progressed, MYO5A was also found to perform a critical effect in malignant melanoma [39][40][41][42]. Subsequently, it was disclosed that MYO5A was associated with metastasis in a variety of cancers. ...
circFNDC3B promotes esophageal squamous cell carcinoma progression by targeting MYO5A via miR-370-3p/miR-136-5p
Article
Full-text available
  • Sep 2023
  • BMC CANCER
Background Esophageal squamous cell carcinoma (ESCC) is a prevalent malignant tumor worldwide. Circular RNA (circRNA) is of great value in tumorigenesis progression. However, the mechanism of circFNDC3B in ESCC remains to be clarified. Methods Firstly, the circular characteristics of circFNDC3B were evaluated by Actinomycin D and RNase R measurements. The functions of circFNDC3B in ESCC cells were examined by CCK-8, EdU and flow cytometry. Subsequently, the molecular mechanism of circFNDC3B was explained using luciferase reporter gene detection. Finally, we constructed xenograft model to prove the role of circFNDC3B in vivo. Results Our study revealed that circFNDC3B was more stable than its linear RNA and prominently upregulated in ESCC. Functional findings suggested that silencing of circFNDC3B reduced the proliferation and enhanced apoptosis of ESCC cells in vitro. Meanwhile, knockdown of circFNDC3B attenuated tumor progression in vivo. Next, miR-370-3p/miR-136-5p was discovered to bind circFNDC3B. miR-370-3p/miR-136-5p reversed the promotive effect on cell proliferation and the inhibitory effect on cell apoptosis of circFNDC3B. MYO5A was a downstream target of miR-370-3p/miR-136-5p. CircFNDC3B served as a sponge for miR-370-3p/miR-136-5p and alleviated the prohibitory effect of miR-370-3p/miR-136-5p on MYO5A, which accelerated ESCC progression. Conclusion circFNDC3B positively adjusted the MYO5A expression via spongy miR-370-3p/miR-136-5p, hence achieving the cancer-promoting effect on ESCC. circFNDC3B was a prospective diagnosis marker for ESCC.
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... Moreover, it has been proposed that DYNLL2 negatively regulates the BH3-only class of Bcl-2 family proteins, thereby inhibiting the pro-apoptotic activity of these proteins [17]. DYNLL2 is involved in many cellular processes [18], but so far, there is no evidence for its role in chicken or muscle cells. ...
Weighted gene co-expression network indicates that the DYNLL2 is an important regulator of chicken breast muscle development and is regulated by miR-148a-3p
Article
Full-text available
  • Apr 2022
  • BMC GENOMICS
Background The characteristics of muscle fibers determine the growth and meat quality of poultry. In this study, we performed a weighted gene co-expression network analysis (WGCNA) on the muscle fiber characteristics and transcriptome profile of the breast muscle tissue of Gushi chicken at 6, 14, 22, and 30 weeks. Results A total of 27 coexpressed biological functional modules were identified, of which the midnight blue module had the strongest correlation with muscle fiber and diameter. In addition, 7 hub genes were found from the midnight blue module, including LC8 dynein light chain 2 ( DYNLL2 ). Combined with miRNA transcriptome data, miR-148a-3p was found to be a potential target miRNA of DYNLL2 . Experiments on chicken primary myoblasts (CPMs) demonstrated that miR-148a-3p promotes the expression of myosin heavy chain (MYHC) protein by targeting DYNLL2 , proving that it can promote differentiation of myoblasts. Conclusions This study proved that the hub gene DYNLL2 and its target miR-148-3p are important regulators in chicken myogenesis. These results provide novel insights for understanding the molecular regulation mechanisms related to the development of chicken breast muscle.
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... Dynein light chains have since been shown to bind to a plethora of cellular proteins and have been linked to a large number of cellular functions. Of note, the Drosophila homolog, Cut up, has been proposed to be implicated in dynein-mediated transport of neuronal proteasomes [47], and human DYNLL2-in conjunction with myosin Va-in sequestration of pro-apoptotic factors, suggesting a role for these last two proteins as prosurvival molecules [84]. ...
Exploration of binary protein–protein interactions between tick-borne flaviviruses and Ixodes ricinus
Article
Full-text available
  • Mar 2021
Background Louping ill virus (LIV) and tick-borne encephalitis virus (TBEV) are tick-borne flaviviruses that are both transmitted by the major European tick, Ixodes ricinus. Despite the importance of I. ricinus as an arthropod vector, its capacity to acquire and subsequently transmit viruses, known as vector competence, is poorly understood. At the molecular scale, vector competence is governed in part by binary interactions established between viral and cellular proteins within infected tick cells. Methods To investigate virus-vector protein–protein interactions (PPIs), the entire set of open reading frames for LIV and TBEV was screened against an I. ricinus cDNA library established from three embryonic tick cell lines using yeast two-hybrid methodology (Y2H). PPIs revealed for each viral bait were retested in yeast by applying a gap repair (GR) strategy, and notably against the cognate protein of both viruses, to determine whether the PPIs were specific for a single virus or common to both. The interacting tick proteins were identified by automatic BLASTX, and in silico analyses were performed to expose the biological processes targeted by LIV and TBEV. Results For each virus, we identified 24 different PPIs involving six viral proteins and 22 unique tick proteins, with all PPIs being common to both viruses. According to our data, several viral proteins (pM, M, NS2A, NS4A, 2K and NS5) target multiple tick protein modules implicated in critical biological pathways. Of note, the NS5 and pM viral proteins establish PPI with several tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins, which are essential adaptor proteins at the nexus of multiple signal transduction pathways. Conclusion We provide the first description of the TBEV/LIV-I. ricinus PPI network, and indeed of any PPI network involving a tick-borne virus and its tick vector. While further investigation will be needed to elucidate the role of each tick protein in the replication cycle of tick-borne flaviviruses, our study provides a foundation for understanding the vector competence of I. ricinus at the molecular level. Indeed, certain PPIs may represent molecular determinants of vector competence of I. ricinus for TBEV and LIV, and potentially for other tick-borne flaviviruses.
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... Dynein light chains have since been shown to bind to a plethora of cellular proteins and been linked to a large number of cellular functions. Of note, the Drosophila homolog, Cut up, has been proposed to be implicated in dynein-mediated transport of neuronal proteasomes [47], and human DYNLL2 -in conjunction with myosin Va -in sequestration of pro-apoptotic factors, suggesting a role for these last two proteins as pro-survival molecules [83]. ...
Exploration of Binary Protein-Protein Interactions Between Tick-Borne Flaviviruses and Ixodes Ricinus
Preprint
Full-text available
  • Dec 2020
Background Louping ill virus (LIV) and tick-borne encephalitis virus (TBEV) are tick-borne flaviviruses that are both transmitted by the major European tick, Ixodes ricinus . Despite the importance of I. ricinus as an arthropod vector, its capacity to acquire and subsequently transmit viruses, known as vector competence, is poorly understood. At the molecular scale, vector competence is governed in part by binary interactions established between viral and cellular proteins within infected tick cells.Methods To investigate virus-vector protein-protein interactions (PPIs), the entire set of open reading frames for LIV and TBEV was screened against an I. ricinus cDNA library established from three embryonic tick cell lines using yeast two-hybrid methodology (Y2H). PPIs revealed for each viral bait were retested in yeast by applying a gap repair (GR) strategy and notably against the cognate protein of both viruses, to determine whether the PPIs were specific for a single virus or common to both. The interacting tick proteins were identified by automatic BLASTX and in silico analyses were performed to expose the biological processes targeted by LIV and TBEV.ResultsFor each virus, we identified 24 different PPIs involving six viral proteins and 22 unique tick proteins, with all PPIs being common to both viruses. According to our data, several viral proteins (pM, M, NS2A, NS4A, 2K and NS5) target multiple tick protein modules implicated in critical biological pathways. Of note, the NS5 and pM viral proteins establish PPI with several tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins, which are essential adaptor proteins at the nexus of multiple signal transduction pathways.Conclusion We provide the first description of the TBEV/LIV- I. ricinus PPI network, and indeed of any PPI network involving a tick-borne virus and its tick vector. While further investigation will be needed to elucidate the role of each tick protein in the replication cycle of tick-borne flaviviruses, our study provides a foundation for understanding the vector competence of I. ricinus at the molecular level. Indeed, certain PPIs may represent molecular determinants of vector competence of I. ricinus for TBEV and LIV, and potentially for other tick-borne flaviviruses.
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... Beach et al. reported that in breast cancers the switch between myosin IIC to myosin IIB is crucial to EMT which contributes to invasiveness, by affecting the cell contractility [258]. Myo Va acts as a pro-apoptotic protein when actin is depolymerized, or cell adhesion is disrupted by releasing the scavenger Bmf, which led to increased survival [259]. Increased expression of myosin Va positively correlated with the expression snail. ...
Cytoskeletal Remodeling in Cancer
Article
Full-text available
  • Nov 2020
Successful metastasis depends on cell invasion, migration, host immune escape, extravasation, and angiogenesis. The process of cell invasion and migration relies on the dynamic changes taking place in the cytoskeletal components; actin, tubulin and intermediate filaments. This is possible due to the plasticity of the cytoskeleton and coordinated action of all the three, is crucial for the process of metastasis from the primary site. Changes in cellular architecture by internal clues will affect the cell functions leading to the formation of different protrusions like lamellipodia, filopodia, and invadopodia that help in cell migration eventually leading to metastasis, which is life threatening than the formation of neoplasms. Understanding the signaling mechanisms involved, will give a better insight of the changes during metastasis, which will eventually help targeting proteins for treatment resulting in reduced mortality and longer survival.
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... Cell polarity is crucial for normal physiology and also plays a major role in development of tumor metastasis [45]. A link between the isoform MYO5A and cancer has already been established [29,46]. MYO5A show elevated expression in metastatic cancer cell lines derived from various tissue types, and is connected to tumor cell migration and metastasis in vitro and in vivo [29]. ...
MYO5B mutations in pheochromocytoma/paraganglioma promote cancer progression
Article
Full-text available
Identification of additional cancer-associated genes and secondary mutations driving the metastatic progression in pheochromocytoma and paraganglioma (PPGL) is important for subtyping, and may provide optimization of therapeutic regimens. We recently reported novel recurrent nonsynonymous mutations in the MYO5B gene in metastatic PPGL. Here, we explored the functional impact of these MYO5B mutations, and analyzed MYO5B expression in primary PPGL tumor cases in relation to mutation status. Immunohistochemistry and mRNA expression analysis in 30 primary PPGL tumors revealed an increased MYO5B expression in metastatic compared to non-metastatic tumor cases. In addition, subcellular localization of MYO5B protein was altered from cytoplasmic to membranous in some metastatic tumors, and the strongest and most abnormal expression pattern was observed in a paraganglioma case harboring a MYO5B:p.G1611S mutation. In addition to five previously discovered MYO5B mutations, the present mutation analysis of 30 PPGL also revealed two, and hence recurrent, mutations in the paralog MYO5A. The three MYO5B missense mutations with the highest prediction scores (p.L587P, p.G1611S and p.R1641C) were selected and functionally validated using site directed mutagenesis and stable transfection into human neuroblastoma cells (SK-N-AS) and embryonic kidney cells (HEK293 cell line). In vitro analysis showed a significant increased proliferation rate in all three MYO5B mutated clones. The two somatically derived mutations, p.L587P and p.G1611S, were also found to increase the migration rate. Expression analysis of MYO5B mutants compared to wild type clones, demonstrated a significant enrichment of genes involved in migration, proliferation, cell adhesion, glucose metabolism, and cellular homeostasis. Our study validates the functional role of novel MYO5B mutations in proliferation and migration, and suggest the MYO5-pathway to be involved in the malignant progression in some PPGL tumors.
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... In addition to actin, which was the most abundant protein in the final eluate, several known myosin Va binding partners were identified (Table II, bold italics). These included calcium/ calmodulin-dependent kinase type II (CaMKII; [Costa et al. 1999], Disks large homolog 4/PSD95 [Yoshii et al. 2013], LC8 dynein light chain [Espindola et al. 2000;Hodi et al. 2006;Izidoro-Toledo et al. 2013;Wagner et al. 2006], inositol 1,4,5-trisphosphate receptor type 1 (IP3R1; [Wada et al. 2016], neurofilament light peptide [Alami et al. 2009;Rao et al. 2002], Shank1 [Naisbitt et al. 2000], and tubulin [Cao et al. 2004]. Of note, the final eluate also contained myosin light chain 6B (Myl6b), previously identified as the light chain for IQ1 of chicken myosin Va [De La Cruz et al. 2000;Espindola et al. 2000]. ...
Creation of a myosin Va-TAP tagged mouse and identification of potential myosin Va-interacting proteins in the cerebellum
Article
  • Jul 2018
The actin‐based motor myosin Va transports numerous cargos, including the smooth endoplasmic reticulum (SER) in cerebellar Purkinje neurons (PNs) and melanosomes in melanocytes. Identifying proteins that interact with this myosin is key to understanding its cellular functions. Towards that end, we used recombineering to insert via homologous recombination a tandem affinity purification (TAP) tag composed of the IgG binding domain of Protein A, a TEV cleavage site, and a FLAG tag into the mouse MYO5A locus immediately after the initiation codon. Importantly, we provide evidence that the TAP‐tagged version of myosin Va (TAP‐MyoVa) functions normally in terms of SER transport in PNs and melanosome positioning in melanocytes. Given this and other evidence that TAP‐MyoVa is fully functional, we purified it together with associated proteins directly from juvenile mouse cerebella and subjected the samples to mass spectroscopic analyses. As expected, known myosin Va binding partners like dynein light chain were identified. Importantly, numerous novel interacting proteins were also tentatively identified, including Guanine nucleotide‐binding protein G(o) subunit alpha (Gnao1), a biomarker for schizophrenia. Consistently, an antibody to Gnao1 immunoprecipitates myosin Va, and Gnao1’s localization to PN dendritic spines depends on myosin Va. The mouse model created here should facilitate the identification of novel myosin Va binding partners, which in turn should advance our understanding of the roles played by this important myosin in vivo. This article is protected by copyright. All rights reserved.
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Dialogue moléculaire des virus de l'encéphalite à tique et louping ill avec leur vecteur tique Ixodes ricinus
Thesis
  • Jan 2021
A l’échelle mondiale, l’émergence de maladies virales transmises par les tiques est en nette augmentation depuis le début du XXIe siècle. En Europe, la tique est le premier arthropode capable de transmettre la plus large variété de pathogènes à l’homme et à l’animal. Ixodes ricinus, espèce de tique majoritairement représentée en Europe, transmet notamment les virus de l’encéphalite à tique (TBEV) et louping ill (LIV), responsables de méningo-encéphalites respectivement chez l’homme et les moutons. Bien que la transmission de ces virus par la tique soit connue depuis près d’une centaine d’années, les mécanismes permettant à la tique de répliquer et transmettre les virus à un hôte sont encore méconnus. Aussi, une meilleure compréhension du dialogue moléculaire entre le virus et son vecteur est un prérequis majeur dans la lutte contre les maladies vectorielles.Le premier objectif de ce projet de thèse a été d’identifier les interactions protéiques établies par TBEV et LIV avec leur vecteur I. ricinus. En ce sens, une cartographie des interactions entre les protéines de TBEV, de LIV et celles d’I. ricinus a été établie et a révélé que les protéines de TBEV et LIV interagissaient avec différents modules protéiques des cellules de tique tels que des voies de signalisation contrôlant la transduction de signaux, la transcription, la dégradation protéique et des fonctions du cytosquelette.La tique, quant à elle, met en place des moyens de lutte afin de limiter l’impact du virus sur ses traits de vie. Cette lutte se traduit notamment par l’initiation et le maintien d’une réponse immunitaire. Le second objectif était ainsi d’explorer la réponse ARN interférence (voie antivirale décrite comme majeure des arthropodes) d’I. ricinus lors de l’infection à TBEV et à LIV. Le séquençage des petits ARNs issus de cellules de tique infectées par TBEV et LIV et de tiques vivantes infectées par TBEV a montré qu’I. ricinus produisaient des petits ARNs de 22 nucléotides à partir des ARNs viraux.Le rôle des protéines de tiques interagissant avec TBEV et LIV, ainsi que celui d’acteurs de l’immunité antivirale, dans le cycle viral sera appréhendé par l’inactivation de leur expression.Ce sujet de thèse initie un programme de recherche qui vise à élucider les mécanismes moléculaires de la compétence vectorielle d’I. ricinus pour les virus qu’elle transmet, dans le but de prédire la transmission d’autres arbovirus par cette même tique afin d’anticiper leurs émergences en Europe.
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Nuclear myosins – roles for molecular transporters and anchors
Article
  • Jun 2020
The myosin family of molecular motors are well-characterised cytoskeletal proteins. However, myosins are also present in the nucleus, where they have been shown to have roles in transcription, DNA repair and viral infections. Despite their involvement in these fundamental cellular processes, our understanding of these functions and their regulation remains limited. Recently, research on nuclear myosins has been gathering pace, and this Review will evaluate the current state of the field. Here, we will focus on the variation in structure of nuclear myosins, their nuclear import and their roles within transcription, DNA damage, chromatin organisation and viral infections. We will also consider both the biochemical and biophysical properties and restraints that are placed on these multifunctional motors, and how they link to their cytoplasmic counterparts. By highlighting these properties and processes, we show just how integral nuclear myosins are for cellular survival.
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Association between aberrant dynein cytoplasmic�1 light intermediate chain�1 expression levels, mucins and chemosensitivity in colorectal cancer
Article
Full-text available
  • Apr 2020
Dynein transport along the cytoskeletal microtubules towards the minus end is essential for cell division, cell migration and other basic cellular functions. Dynein cytoplasmic 1 light intermediate chain 1 (DYNC1LI1) has been previously associated with pancreatic ductal adenocarcinoma, hepatocellular carcinoma and prostate cancer. Cytoskeletal structures are involved in the regulation of the mucosal barrier integrity. Thus, improving our understanding of the molecular mechanisms that regulate the mucosal barrier is critical for cancer management and treatment. The present study aimed to investigate DYNC1LI1 expression in colorectal cancer (CRC) tissues. The American Joint Committee on Cancer Stage II CRC cell line LS 174T was used to determine the association between the cellular expression levels of DYNC1LI1 and different types of mucin (MUC) by reverse transcription‑quantitative PCR. The role of DYNC1LI1 in cell chemosensitivity and proliferation was also evaluated in the presence of the DNA analog 5‑fluorouracil (5‑FU) or the platinum‑based drug, oxaliplatin by the MTT assay. LS 174T cells with decreased expression levels of DYNC1LI1 were discovered to be more sensitive to 5‑FU compared with LS 174T cells with endogenous DYNC1LI1 expression levels. Moreover, LS 174T cells transfected with short hairpin RNA targeting DYNC1LI1 were associated with low MUC1 and high MUC2, MUC4 and MUC5AC expression levels. Notably, the CRC cells with low MUC1 expression levels and high expression levels of the other MUCs (MUC2, MU4 and MUC5AC) were shown to benefit from 5‑FU treatment. In conclusion, the findings of the present study have suggested that DYNC1LI1 expression may be significantly associated with MUC expression levels and may be used to predict the chemotherapeutic efficiency. However, additional functional studies and clinical reports are required for an improved understanding of the significance of these molecular interactions in tumorigenesis.
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Rab10 and myosin-Va mediate insulin-stimulated GLUT4 storage vesicle translocation in adipocytes
Article
Full-text available
Rab proteins are important regulators of insulin-stimulated GLUT4 translocation to the plasma membrane (PM), but the precise steps in GLUT4 trafficking modulated by particular Rab proteins remain unclear. Here, we systematically investigate the involvement of Rab proteins in GLUT4 trafficking, focusing on Rab proteins directly mediating GLUT4 storage vesicle (GSV) delivery to the PM. Using dual-color total internal reflection fluorescence (TIRF) microscopy and an insulin-responsive aminopeptidase (IRAP)-pHluorin fusion assay, we demonstrated that Rab10 directly facilitated GSV translocation to and docking at the PM. Rab14 mediated GLUT4 delivery to the PM via endosomal compartments containing transferrin receptor (TfR), whereas Rab4A, Rab4B, and Rab8A recycled GLUT4 through the endosomal system. Myosin-Va associated with GSVs by interacting with Rab10, positioning peripherally recruited GSVs for ultimate fusion. Thus, multiple Rab proteins regulate the trafficking of GLUT4, with Rab10 coordinating with myosin-Va to mediate the final steps of insulin-stimulated GSV translocation to the PM.
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The light chain composition of chicken brain myosin-Va: Calmodulin, myosin-II essential light chains, and 8-kDa dynein light chain/PIN
Article
  • Dec 2000
  • CELL MOTIL CYTOSKEL
Class V myosins are a ubiquitously expressed family of actin-based molecular motors. Biochemical studies on myosin-Va from chick brain indicate that this myosin is a two-headed motor with multiple calmodulin light chains associated with the regulatory or neck domain of each heavy chain, a feature consistent with the regulatory effects of Ca²⁺ on this myosin. In this study, the identity of three additional low molecular weight proteins of 23-,17-, and 10 kDa associated with myosin-Va is established. The 23- and 17-kDa subunits are both members of the myosin-II essential light chain gene family, encoded by the chicken L23 and L17 light chain genes, respectively. The 10-kDa subunit is a protein originally identified as a light chain (DLC8) of flagellar and axonemal dynein. The 10-kDa subunit is associated with the tail domain of myosin-Va. Cell Motil. Cytoskeleton 47:269–281, 2000. © 2000 Wiley-Liss, Inc.
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Primary structure and cellular localization of chicken brain myosin-V (p190), an unconventional myosin with calmodulin light chains
Article
  • Dec 1992
Recent biochemical studies of p190, a calmodulin (CM)-binding protein purified from vertebrate brain, have demonstrated that this protein, purified as a complex with bound CM, shares a number of properties with myosins (Espindola, F. S., E. M. Espreafico, M. V. Coelho, A. R. Martins, F. R. C. Costa, M. S. Mooseker, and R. E. Larson. 1992. J. Cell Biol. 118:359-368). To determine whether or not p190 was a member of the myosin family of proteins, a set of overlapping cDNAs encoding the full-length protein sequence of chicken brain p190 was isolated and sequenced. Verification that the deduced primary structure was that of p190 was demonstrated through microsequence analysis of a cyanogen bromide peptide generated from chick brain p190. The deduced primary structure of chicken brain p190 revealed that this 1,830-amino acid (aa) 212,509-D) protein is a member of a novel structural class of unconventional myosins that includes the gene products encoded by the dilute locus of mouse and the MYO2 gene of Saccharomyces cerevisiae. We have named the p190-CM complex "myosin-V" based on the results of a detailed sequence comparison of the head domains of 29 myosin heavy chains (hc), which has revealed that this myosin, based on head structure, is the fifth of six distinct structural classes of myosin to be described thus far. Like the presumed products of the mouse dilute and yeast MYO2 genes, the head domain of chicken myosin-V hc (aa 1-764) is linked to a "neck" domain (aa 765-909) consisting of six tandem repeats of an approximately 23-aa "IQ-motif." All known myosins contain at least one such motif at their head-tail junctions; these IQ-motifs may function as calmodulin or light chain binding sites. The tail domain of chicken myosin-V consists of an initial 511 aa predicted to form several segments of coiled-coil alpha helix followed by a terminal 410-aa globular domain (aa, 1,421-1,830). Interestingly, a portion of the tail domain (aa, 1,094-1,830) shares 58% amino acid sequence identity with a 723-aa protein from mouse brain reported to be a glutamic acid decarboxylase. The neck region of chicken myosin-V, which contains the IQ-motifs, was demonstrated to contain the binding sites for CM by analyzing CM binding to bacterially expressed fusion proteins containing the head, neck, and tail domains. Immunolocalization of myosin-V in brain and in cultured cells revealed an unusual distribution for this myosin in both neurons and nonneuronal cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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Till Death Us Do Part
Article
  • Sep 2001
  • SCIENCE
Epithelial cells need to remain firmly attached to each other and to the extracellular matrix. In the event that these cells become detached, they undergo apoptosis. In a Perspective, Hunt and Evan discuss new work that identifies the trigger that sets in motion the apoptosis program following detachment ( Puthalakath et al.). The trigger turns out to be the pro-apoptotic protein Bmf. This protein is attached to the myosin V motor complex of the actin cytoskeleton under normal conditions, but is set free once cells become detached.
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Glucose transporters in cancer metabolism
Article
  • Aug 2012
  • CURR OPIN ONCOL
Transformed cells exhibit a high rate of glucose consumption beyond that necessary for ATP synthesis. Glucose aids in the generation of biomass and regulates cellular signaling critical for oncogenic progression. A key rate-limiting step in glucose utilization is the transport of glucose across the plasma membrane. This review will highlight key glucose transporters (GLUTs) and current therapies targeting this class of proteins. GLUTs, enabling the facilitative entry of glucose into a cell, are increasingly found to be deregulated in cancer. Although cancer-specific expression patterns for GLUTs are being identified, comprehensive analyses substantiating a role for individual GLUTs are still required. Studies defining GLUTs as being rate-limiting in specific tumor contexts, the identification of GLUT1 inhibitors via synthetic lethality screens, novel engagement of the insulin-responsive GLUT4 in myeloma and identification of GLUT9 being a urate transporter, are key advances underscoring the need for continued investigation of this large and enigmatic class of proteins. Tumor cells exhibit elevated levels of glucose uptake, a phenomenon that has been capitalized upon for the prognostic and diagnostic imaging of a wide range of cancers using radio-labeled glucose analogs. We have, however, not yet been able to target glucose entry in a tumor cell-specific manner for therapy. GLUTs have been identified as rate-limiting in specific tumor contexts. The identification and targeting of tumor-specific GLUTs provide a promising approach to block glucose-regulated metabolism and signaling more comprehensively.
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BH3-only protein silencing contributes to acquired resistance to PLX4720 in human melanoma
Article
  • Aug 2012
B-RAF is mutated to a constitutively active form in 8% of human cancers including 50% of melanomas. In clinical trials, the RAF inhibitor, PLX4032 (vemurafenib), caused partial or complete responses in 48-81% of mutant B-RAF harboring melanoma patients. However, the average duration of response was 6-7 months before tumor regrowth, indicating the acquisition of resistance to PLX4032. To understand the mechanisms of resistance, we developed mutant B-RAF melanoma cells that displayed resistance to RAF inhibition through continuous culture with PLX4720 (the tool compound for PLX4032). Resistance was associated with a partial reactivation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, recovery of G1/S cell-cycle events, and suppression of the pro-apoptotic B-cell leukemia/lymphoma 2 (Bcl-2) homology domain 3 (BH3)-only proteins, Bcl-2-interacting mediator of cell death-extra large (Bim-EL) and Bcl-2 modifying factor (Bmf). Preventing ERK1/2 reactivation with MEK (mitogen-activated protein/extracellular signal-regulated kinase kinase) inhibitors blocked G1-S cell-cycle progression but failed to induce apoptosis or upregulate Bim-EL and Bmf. Treatment with the histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid, led to de-repression of Bim-EL and enhanced cell death in the presence of PLX4720 or AZD6244 in resistant cells. These data indicate that acquired resistance to PLX4032/4720 likely involves ERK1/2 pathway reactivation as well as ERK1/2-independent silencing of BH3-only proteins. Furthermore, combined treatment of HDAC inhibitors and MEK inhibitors may contribute to overcoming PLX4032 resistance.Cell Death and Differentiation advance online publication, 3 August 2012; doi:10.1038/cdd.2012.94.
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The Role of Bim, a Proapoptotic BH3‐Only Member of the Bcl‐2 Family, in Cell‐Death Control
Article
Apoptosis is an evolutionarily conserved process for killing unwanted cells. Genetic and biochemical experiments have indicated that three groups of proteins are necessary for activation of the cell-death effector machinery: cysteine proteases, their adaptors, and proapoptotic Bcl-2 family members. Antiapoptotic Bcl-2 family members are needed for cell survival. We have cloned Bim, a proapoptotic Bcl-2 family member that shares with the family only a 9-16 aa region of homology [Bcl-3 homology region (BH3)], but is otherwise unique. Bim requires its BH3 region for binding to Bcl-2 and activation of apoptosis. Analysis of Bim-deficient mice has shown that Bim is essential for the execution of some but not all apoptotic stimuli that can be antagonized by Bcl-2. Bim-deficient mice have increased numbers of lymphocytes, plasma cells, and myeloid cells, and most develop fatal autoimmune glomerulonephritis. In healthy cells, Bim is bound to the microtubule-associated dynein motor complex, and is thereby sequestered from Bcl-2. Certain apoptotic signals unleash Bim and allow it to translocate to intracellular membranes, where it interacts with Bcl-2 or its homologues. These results indicate that BH3-only proteins are essential inducers of apoptosis that can be unleashed by certain death signals. Unleashed BH3-only proteins neutralize the prosurvival function of Bcl-2-like molecules, and this is thought to liberate Apaf-l-like adapters to activate caspase zymogens, which then initiate cell degradation.
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The light chain composition of chicken brain myosin‐Va: Calmodulin, myosin‐II essential light chains, and 8‐kDa dynein light chain/PIN
Article
  • Dec 2000
  • CELL MOTIL CYTOSKEL
Class V myosins are a ubiquitously expressed family of actin-based molecular motors. Biochemical studies on myosin-Va from chick brain indicate that this myosin is a two-headed motor with multiple calmodulin light chains associated with the regulatory or neck domain of each heavy chain, a feature consistent with the regulatory effects of Ca2+ on this myosin. In this study, the identity of three additional low molecular weight proteins of 23-,17-, and 10 kDa associated with myosin-Va is established. The 23- and 17-kDa subunits are both members of the myosin-II essential light chain gene family, encoded by the chicken L23 and L17 light chain genes, respectively. The 10-kDa subunit is a protein originally identified as a light chain (DLC8) of flagellar and axonemal dynein. The 10-kDa subunit is associated with the tail domain of myosin-Va. Cell Motil. Cytoskeleton 47:269–281, 2000. © 2000 Wiley-Liss, Inc.
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BH3-only proteins in apoptosis at a glance
Article
  • Mar 2012
  • J CELL SCI
Apoptosis is a genetically programmed process for the elimination of damaged or redundant cells by activation of caspases (aspartate-specific cysteine proteases). Caspases cleave vital proteins, leading the cell to fragment into vesicles that are rapidly engulfed by phagocytes (for reviews, see Hotchkiss et al., 2009; Strasser et al., 2011). Defects in apoptosis contribute to many diseases, ranging from cancer and autoimmunity to degenerative disorders. This Poster focuses on key initiators of apoptosis: the BH3-only proteins. CertainBH3-onlyproteinshave also been implicated in non-apoptotic processes (see Elgendy et al., 2011; Yeretssian et al., 2011).
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