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Alpha-E-catenin binds to dynamitin and regulates dynactin-mediated intracellular traffic.

Lien WH, Gelfand VI, Vasioukhin V - J. Cell Biol. (2008)

Bottom Line: Dynactin-mediated organelle trafficking is increased in alpha-E-catenin(-/-) keratinocytes, an effect that is reversed by expression of exogenous alpha-E-catenin.Although neither the integrity of dynactin-dynein complexes nor their association with vesicles is affected by alpha-E-catenin, alpha-E-catenin is necessary for the attenuation of microtubule-dependent trafficking by the actin cytoskeleton.Because the actin-binding domain of alpha-E-catenin is necessary for this regulation, we hypothesize that alpha-E-catenin functions as a dynamic link between the dynactin complex and actin and, thus, integrates the microtubule and actin cytoskeleton during intracellular trafficking.

View Article: PubMed Central - PubMed

Affiliation: Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

ABSTRACT
Alpha-epithelial catenin (E-catenin) is an important cell-cell adhesion protein. In this study, we show that alpha-E-catenin also regulates intracellular traffic by binding to the dynactin complex component dynamitin. Dynactin-mediated organelle trafficking is increased in alpha-E-catenin(-/-) keratinocytes, an effect that is reversed by expression of exogenous alpha-E-catenin. Disruption of adherens junctions in low-calcium media does not affect dynactin-mediated traffic, indicating that alpha-E-catenin regulates traffic independently from its function in cell-cell adhesion. Although neither the integrity of dynactin-dynein complexes nor their association with vesicles is affected by alpha-E-catenin, alpha-E-catenin is necessary for the attenuation of microtubule-dependent trafficking by the actin cytoskeleton. Because the actin-binding domain of alpha-E-catenin is necessary for this regulation, we hypothesize that alpha-E-catenin functions as a dynamic link between the dynactin complex and actin and, thus, integrates the microtubule and actin cytoskeleton during intracellular trafficking.

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α–E-catenin is necessary to extend dynactin and the microtubule cytoskeleton to the cell periphery and localize microtubules to the AJs. (A and B) Immunofluorescence staining of wild-type (WT) and α–E-catenin−/− (KO) cells with antidynamitin (Dyn) and anti–α-catenin (α-cat) antibodies. Regions in dashed boxes are shown at higher magnifications in A‴ and B‴. The cell edges are outlined with white dashed lines. (C and D) Immunofluorescence staining of wild-type and α–E-catenin−/− cells with anti–E-cadherin (E-cad) and anti–β-tubulin (β-tub) antibodies. Regions containing cell–cell junctions (dashed boxes) are shown at higher magnifications in C‴ and D‴. (E) Quantitation of microtubule accumulation at cell–cell junctions. Pairs of contacting cells displaying accumulation of E-cadherin at cell–cell borders were randomly selected. The levels of cell border accumulation of microtubules are expressed as ratios of the mean β-tubulin staining intensity at cell–cell junctions over the mean total β-tubulin intensity within two contacting cells. Each bar represents the mean value; n = 50. The p-value was determined by a t test. The error bars represent standard deviation. (F and G) β-Catenin localizes to AJs in α–E-catenin−/− cells. Immunofluorescence staining of wild-type and α–E-catenin−/− cells with anti–E-cadherin and anti–β-catenin (β-cat) antibodies. Regions containing cell–cell junctions (dashed boxes) are shown at higher magnifications in F‴ and G‴. Arrows denote the positions of the AJs. Bars: (A–A″ and B–B″) 16 μm; (A‴ and B‴) 8 μm; (C–C″, D–D″, F–F″, and G–G″) 30 μm; (C‴, D‴, F‴, and G‴) 13 μm.
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fig2: α–E-catenin is necessary to extend dynactin and the microtubule cytoskeleton to the cell periphery and localize microtubules to the AJs. (A and B) Immunofluorescence staining of wild-type (WT) and α–E-catenin−/− (KO) cells with antidynamitin (Dyn) and anti–α-catenin (α-cat) antibodies. Regions in dashed boxes are shown at higher magnifications in A‴ and B‴. The cell edges are outlined with white dashed lines. (C and D) Immunofluorescence staining of wild-type and α–E-catenin−/− cells with anti–E-cadherin (E-cad) and anti–β-tubulin (β-tub) antibodies. Regions containing cell–cell junctions (dashed boxes) are shown at higher magnifications in C‴ and D‴. (E) Quantitation of microtubule accumulation at cell–cell junctions. Pairs of contacting cells displaying accumulation of E-cadherin at cell–cell borders were randomly selected. The levels of cell border accumulation of microtubules are expressed as ratios of the mean β-tubulin staining intensity at cell–cell junctions over the mean total β-tubulin intensity within two contacting cells. Each bar represents the mean value; n = 50. The p-value was determined by a t test. The error bars represent standard deviation. (F and G) β-Catenin localizes to AJs in α–E-catenin−/− cells. Immunofluorescence staining of wild-type and α–E-catenin−/− cells with anti–E-cadherin and anti–β-catenin (β-cat) antibodies. Regions containing cell–cell junctions (dashed boxes) are shown at higher magnifications in F‴ and G‴. Arrows denote the positions of the AJs. Bars: (A–A″ and B–B″) 16 μm; (A‴ and B‴) 8 μm; (C–C″, D–D″, F–F″, and G–G″) 30 μm; (C‴, D‴, F‴, and G‴) 13 μm.

Mentions: To determine localization of α–E-catenin and dynamitin in wild-type and α–E-catenin−/− keratinocytes, we performed immunofluorescence stainings. α–E-catenin prominently localized around the nuclei and at AJs in wild-type keratinocytes (Fig. 2 A′). Dynamitin colocalized with α–E-catenin around cell nuclei, and small amounts of dynamitin were found at the cell periphery and AJs (Fig. 2 A). In contrast, dynamitin was localized almost exclusively around the nucleus in α–E-catenin−/− cells, with very little amounts of dynamitin present at the cell periphery (Fig. 2, B and B‴). These data suggest that α–E-catenin is necessary to localize dynamitin to cell edges.


Alpha-E-catenin binds to dynamitin and regulates dynactin-mediated intracellular traffic.

Lien WH, Gelfand VI, Vasioukhin V - J. Cell Biol. (2008)

α–E-catenin is necessary to extend dynactin and the microtubule cytoskeleton to the cell periphery and localize microtubules to the AJs. (A and B) Immunofluorescence staining of wild-type (WT) and α–E-catenin−/− (KO) cells with antidynamitin (Dyn) and anti–α-catenin (α-cat) antibodies. Regions in dashed boxes are shown at higher magnifications in A‴ and B‴. The cell edges are outlined with white dashed lines. (C and D) Immunofluorescence staining of wild-type and α–E-catenin−/− cells with anti–E-cadherin (E-cad) and anti–β-tubulin (β-tub) antibodies. Regions containing cell–cell junctions (dashed boxes) are shown at higher magnifications in C‴ and D‴. (E) Quantitation of microtubule accumulation at cell–cell junctions. Pairs of contacting cells displaying accumulation of E-cadherin at cell–cell borders were randomly selected. The levels of cell border accumulation of microtubules are expressed as ratios of the mean β-tubulin staining intensity at cell–cell junctions over the mean total β-tubulin intensity within two contacting cells. Each bar represents the mean value; n = 50. The p-value was determined by a t test. The error bars represent standard deviation. (F and G) β-Catenin localizes to AJs in α–E-catenin−/− cells. Immunofluorescence staining of wild-type and α–E-catenin−/− cells with anti–E-cadherin and anti–β-catenin (β-cat) antibodies. Regions containing cell–cell junctions (dashed boxes) are shown at higher magnifications in F‴ and G‴. Arrows denote the positions of the AJs. Bars: (A–A″ and B–B″) 16 μm; (A‴ and B‴) 8 μm; (C–C″, D–D″, F–F″, and G–G″) 30 μm; (C‴, D‴, F‴, and G‴) 13 μm.
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fig2: α–E-catenin is necessary to extend dynactin and the microtubule cytoskeleton to the cell periphery and localize microtubules to the AJs. (A and B) Immunofluorescence staining of wild-type (WT) and α–E-catenin−/− (KO) cells with antidynamitin (Dyn) and anti–α-catenin (α-cat) antibodies. Regions in dashed boxes are shown at higher magnifications in A‴ and B‴. The cell edges are outlined with white dashed lines. (C and D) Immunofluorescence staining of wild-type and α–E-catenin−/− cells with anti–E-cadherin (E-cad) and anti–β-tubulin (β-tub) antibodies. Regions containing cell–cell junctions (dashed boxes) are shown at higher magnifications in C‴ and D‴. (E) Quantitation of microtubule accumulation at cell–cell junctions. Pairs of contacting cells displaying accumulation of E-cadherin at cell–cell borders were randomly selected. The levels of cell border accumulation of microtubules are expressed as ratios of the mean β-tubulin staining intensity at cell–cell junctions over the mean total β-tubulin intensity within two contacting cells. Each bar represents the mean value; n = 50. The p-value was determined by a t test. The error bars represent standard deviation. (F and G) β-Catenin localizes to AJs in α–E-catenin−/− cells. Immunofluorescence staining of wild-type and α–E-catenin−/− cells with anti–E-cadherin and anti–β-catenin (β-cat) antibodies. Regions containing cell–cell junctions (dashed boxes) are shown at higher magnifications in F‴ and G‴. Arrows denote the positions of the AJs. Bars: (A–A″ and B–B″) 16 μm; (A‴ and B‴) 8 μm; (C–C″, D–D″, F–F″, and G–G″) 30 μm; (C‴, D‴, F‴, and G‴) 13 μm.
Mentions: To determine localization of α–E-catenin and dynamitin in wild-type and α–E-catenin−/− keratinocytes, we performed immunofluorescence stainings. α–E-catenin prominently localized around the nuclei and at AJs in wild-type keratinocytes (Fig. 2 A′). Dynamitin colocalized with α–E-catenin around cell nuclei, and small amounts of dynamitin were found at the cell periphery and AJs (Fig. 2 A). In contrast, dynamitin was localized almost exclusively around the nucleus in α–E-catenin−/− cells, with very little amounts of dynamitin present at the cell periphery (Fig. 2, B and B‴). These data suggest that α–E-catenin is necessary to localize dynamitin to cell edges.

Bottom Line: Dynactin-mediated organelle trafficking is increased in alpha-E-catenin(-/-) keratinocytes, an effect that is reversed by expression of exogenous alpha-E-catenin.Although neither the integrity of dynactin-dynein complexes nor their association with vesicles is affected by alpha-E-catenin, alpha-E-catenin is necessary for the attenuation of microtubule-dependent trafficking by the actin cytoskeleton.Because the actin-binding domain of alpha-E-catenin is necessary for this regulation, we hypothesize that alpha-E-catenin functions as a dynamic link between the dynactin complex and actin and, thus, integrates the microtubule and actin cytoskeleton during intracellular trafficking.

View Article: PubMed Central - PubMed

Affiliation: Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

ABSTRACT
Alpha-epithelial catenin (E-catenin) is an important cell-cell adhesion protein. In this study, we show that alpha-E-catenin also regulates intracellular traffic by binding to the dynactin complex component dynamitin. Dynactin-mediated organelle trafficking is increased in alpha-E-catenin(-/-) keratinocytes, an effect that is reversed by expression of exogenous alpha-E-catenin. Disruption of adherens junctions in low-calcium media does not affect dynactin-mediated traffic, indicating that alpha-E-catenin regulates traffic independently from its function in cell-cell adhesion. Although neither the integrity of dynactin-dynein complexes nor their association with vesicles is affected by alpha-E-catenin, alpha-E-catenin is necessary for the attenuation of microtubule-dependent trafficking by the actin cytoskeleton. Because the actin-binding domain of alpha-E-catenin is necessary for this regulation, we hypothesize that alpha-E-catenin functions as a dynamic link between the dynactin complex and actin and, thus, integrates the microtubule and actin cytoskeleton during intracellular trafficking.

Show MeSH
Related in: MedlinePlus