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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 negatively regulates dynactin-mediated intracellular traffic in an AJ-independent manner. (A) Quantitation of lysosome movements in wild-type (WT) and α–E-catenin−/− (KO) keratinocytes. The total length of lysosome movements within 5 min was determined using Imaris software analysis of the time-lapse videos. Bars represent the percentage of the vesicles that moved over the indicated distances; n = 315 for wild type and n = 342 for α–E-catenin−/−. Note the prominent increase in lysosome motility in α–E-catenin−/− cells. (B and C) Expression of exogenous α–E-catenin in α–E-catenin−/− cells. Keratinocytes were transduced with retroviruses expressing the HBT tag (HBT) or HBT-tagged α–E-catenin (HBT–α-cat) and analyzed by blotting (B) and immunostaining (C) with streptavidin or anti–α-catenin (α-cat), anti–β-actin, and anti–E-cadherin (E-cad) antibodies. (D) Reexpression of α–E-catenin in α–E-catenin−/− cells rescues lysosome motility defects. Quantitation of lysosome movements in α–E-catenin−/− keratinocytes expressing HBT (KO + HBT) or HBT-α–E-catenin (KO + HBT–α-cat). n = 354 for KO + HBT and n = 318 for KO + HBT–α-cat. (E) Disruption of AJs in keratinocytes cultured in low-calcium media. Immunofluorescence staining of wild-type and α–E-catenin−/− cells incubated in normal or low-calcium (LowCa) media with anti–E-cadherin (green) antibodies and phalloidin (actin; red). (F and G) Quantitation of lysosome movements in wild-type (F) and α–E-catenin−/− (G) keratinocytes incubated in normal or low-calcium media. n = 319 for WT, n = 326 for WT + LowCa, n = 307 for KO, and n = 347 for KO + LowCa. Bars: (C) 25 μm; (E) 33 μm.
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fig4: α–E-catenin negatively regulates dynactin-mediated intracellular traffic in an AJ-independent manner. (A) Quantitation of lysosome movements in wild-type (WT) and α–E-catenin−/− (KO) keratinocytes. The total length of lysosome movements within 5 min was determined using Imaris software analysis of the time-lapse videos. Bars represent the percentage of the vesicles that moved over the indicated distances; n = 315 for wild type and n = 342 for α–E-catenin−/−. Note the prominent increase in lysosome motility in α–E-catenin−/− cells. (B and C) Expression of exogenous α–E-catenin in α–E-catenin−/− cells. Keratinocytes were transduced with retroviruses expressing the HBT tag (HBT) or HBT-tagged α–E-catenin (HBT–α-cat) and analyzed by blotting (B) and immunostaining (C) with streptavidin or anti–α-catenin (α-cat), anti–β-actin, and anti–E-cadherin (E-cad) antibodies. (D) Reexpression of α–E-catenin in α–E-catenin−/− cells rescues lysosome motility defects. Quantitation of lysosome movements in α–E-catenin−/− keratinocytes expressing HBT (KO + HBT) or HBT-α–E-catenin (KO + HBT–α-cat). n = 354 for KO + HBT and n = 318 for KO + HBT–α-cat. (E) Disruption of AJs in keratinocytes cultured in low-calcium media. Immunofluorescence staining of wild-type and α–E-catenin−/− cells incubated in normal or low-calcium (LowCa) media with anti–E-cadherin (green) antibodies and phalloidin (actin; red). (F and G) Quantitation of lysosome movements in wild-type (F) and α–E-catenin−/− (G) keratinocytes incubated in normal or low-calcium media. n = 319 for WT, n = 326 for WT + LowCa, n = 307 for KO, and n = 347 for KO + LowCa. Bars: (C) 25 μm; (E) 33 μm.

Mentions: The primary function of dynactin is to facilitate the microtubule motor–mediated traffic of cell organelles and vesicles (Schroer, 2004). To examine whether α–E-catenin may be involved in regulation of dynactin-dependent traffic, we performed analyses of lysosome movement in live wild-type and α–E-catenin−/− cells. We labeled lysosomes with LysoTracker dye and followed their movements using time-lapse microscopy (n ≥ 10; Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200805041/DC1). Software-mediated quantitation of the total length of movement over 300 individual lysosomes from 15–20 randomly selected cells of each genotype showed a significant increase in the distances traveled by lysosomes in α–E-catenin−/− cells (Fig. 4 A). This defect was rescued by expression of exogenous α–E-catenin in α–E-catenin−/− cells (n ≥ 8; Fig. 4, B–D; and Videos 3 and 4). Thus, we conclude that α–E-catenin negatively impacts dynactin-dependent microtubule traffic and that organelle motility is increased in α–E-catenin−/− cells.


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 negatively regulates dynactin-mediated intracellular traffic in an AJ-independent manner. (A) Quantitation of lysosome movements in wild-type (WT) and α–E-catenin−/− (KO) keratinocytes. The total length of lysosome movements within 5 min was determined using Imaris software analysis of the time-lapse videos. Bars represent the percentage of the vesicles that moved over the indicated distances; n = 315 for wild type and n = 342 for α–E-catenin−/−. Note the prominent increase in lysosome motility in α–E-catenin−/− cells. (B and C) Expression of exogenous α–E-catenin in α–E-catenin−/− cells. Keratinocytes were transduced with retroviruses expressing the HBT tag (HBT) or HBT-tagged α–E-catenin (HBT–α-cat) and analyzed by blotting (B) and immunostaining (C) with streptavidin or anti–α-catenin (α-cat), anti–β-actin, and anti–E-cadherin (E-cad) antibodies. (D) Reexpression of α–E-catenin in α–E-catenin−/− cells rescues lysosome motility defects. Quantitation of lysosome movements in α–E-catenin−/− keratinocytes expressing HBT (KO + HBT) or HBT-α–E-catenin (KO + HBT–α-cat). n = 354 for KO + HBT and n = 318 for KO + HBT–α-cat. (E) Disruption of AJs in keratinocytes cultured in low-calcium media. Immunofluorescence staining of wild-type and α–E-catenin−/− cells incubated in normal or low-calcium (LowCa) media with anti–E-cadherin (green) antibodies and phalloidin (actin; red). (F and G) Quantitation of lysosome movements in wild-type (F) and α–E-catenin−/− (G) keratinocytes incubated in normal or low-calcium media. n = 319 for WT, n = 326 for WT + LowCa, n = 307 for KO, and n = 347 for KO + LowCa. Bars: (C) 25 μm; (E) 33 μm.
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fig4: α–E-catenin negatively regulates dynactin-mediated intracellular traffic in an AJ-independent manner. (A) Quantitation of lysosome movements in wild-type (WT) and α–E-catenin−/− (KO) keratinocytes. The total length of lysosome movements within 5 min was determined using Imaris software analysis of the time-lapse videos. Bars represent the percentage of the vesicles that moved over the indicated distances; n = 315 for wild type and n = 342 for α–E-catenin−/−. Note the prominent increase in lysosome motility in α–E-catenin−/− cells. (B and C) Expression of exogenous α–E-catenin in α–E-catenin−/− cells. Keratinocytes were transduced with retroviruses expressing the HBT tag (HBT) or HBT-tagged α–E-catenin (HBT–α-cat) and analyzed by blotting (B) and immunostaining (C) with streptavidin or anti–α-catenin (α-cat), anti–β-actin, and anti–E-cadherin (E-cad) antibodies. (D) Reexpression of α–E-catenin in α–E-catenin−/− cells rescues lysosome motility defects. Quantitation of lysosome movements in α–E-catenin−/− keratinocytes expressing HBT (KO + HBT) or HBT-α–E-catenin (KO + HBT–α-cat). n = 354 for KO + HBT and n = 318 for KO + HBT–α-cat. (E) Disruption of AJs in keratinocytes cultured in low-calcium media. Immunofluorescence staining of wild-type and α–E-catenin−/− cells incubated in normal or low-calcium (LowCa) media with anti–E-cadherin (green) antibodies and phalloidin (actin; red). (F and G) Quantitation of lysosome movements in wild-type (F) and α–E-catenin−/− (G) keratinocytes incubated in normal or low-calcium media. n = 319 for WT, n = 326 for WT + LowCa, n = 307 for KO, and n = 347 for KO + LowCa. Bars: (C) 25 μm; (E) 33 μm.
Mentions: The primary function of dynactin is to facilitate the microtubule motor–mediated traffic of cell organelles and vesicles (Schroer, 2004). To examine whether α–E-catenin may be involved in regulation of dynactin-dependent traffic, we performed analyses of lysosome movement in live wild-type and α–E-catenin−/− cells. We labeled lysosomes with LysoTracker dye and followed their movements using time-lapse microscopy (n ≥ 10; Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200805041/DC1). Software-mediated quantitation of the total length of movement over 300 individual lysosomes from 15–20 randomly selected cells of each genotype showed a significant increase in the distances traveled by lysosomes in α–E-catenin−/− cells (Fig. 4 A). This defect was rescued by expression of exogenous α–E-catenin in α–E-catenin−/− cells (n ≥ 8; Fig. 4, B–D; and Videos 3 and 4). Thus, we conclude that α–E-catenin negatively impacts dynactin-dependent microtubule traffic and that organelle motility is increased in α–E-catenin−/− cells.

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