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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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Dynamitin is necessary to extend the microtubule cytoskeleton to the cell periphery and AJs and establish strong cell–cell adhesion. (A) Schematic model of a lentiviral shRNA vector used for the generation of dynamitin KD cells. CMV, cytomegalovirus; LTR, long terminal repeat. (B) Total protein extracts from wild-type (WT) and α–E-catenin−/− (KO) cells transduced with β-galactosidase (β-gal; control) and dynamitin (Dyn; constructs 1 and 2) shRNA lentiviruses analyzed by blotting with antidynamitin (Dyn) and anti–β-actin antibodies. Numbers represent relative levels of dynamitin. (C and D) Dynamitin is necessary to localize microtubules to AJs. Wild-type keratinocytes transduced with β-galactosidase shRNA (β-galsh; C) or dynamitin shRNA-2 (Dynsh; D) were analyzed by immunostaining 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‴. Note the prominent localization of microtubules to AJs in β-galactosidase shRNA cells (arrows) but not in dynamitin KD cells. (E) Quantitation of junctional localization of microtubules in control (WT + β-gal shRNA) and dynamitin KD (WT + Dyn shRNA) cells. Quantitation was performed as described in Fig. 2 E. n = 50. (F) dynamitin KD cells display cell–cell adhesion defects. Wild-type and α–E-catenin−/− keratinocytes expressing β-galactosidase shRNA or dynamitin shRNA-2 were allowed to aggregate for 1 h with and without Ca2+, and the total number of particles was counted. The degree of Ca2+-dependent cell aggregation (NCa+/NCa− percentage) was measured as a percentage of the decrease in the particle numbers in Ca2+-containing versus Ca2+-free conditions. Bars represent mean values; n = 3. The p-value was determined by t test. The error bars represent standard deviation. Bars: (C–C″ and D–D″) 28 μm; (C‴ and D‴) 11.2 μm.
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fig3: Dynamitin is necessary to extend the microtubule cytoskeleton to the cell periphery and AJs and establish strong cell–cell adhesion. (A) Schematic model of a lentiviral shRNA vector used for the generation of dynamitin KD cells. CMV, cytomegalovirus; LTR, long terminal repeat. (B) Total protein extracts from wild-type (WT) and α–E-catenin−/− (KO) cells transduced with β-galactosidase (β-gal; control) and dynamitin (Dyn; constructs 1 and 2) shRNA lentiviruses analyzed by blotting with antidynamitin (Dyn) and anti–β-actin antibodies. Numbers represent relative levels of dynamitin. (C and D) Dynamitin is necessary to localize microtubules to AJs. Wild-type keratinocytes transduced with β-galactosidase shRNA (β-galsh; C) or dynamitin shRNA-2 (Dynsh; D) were analyzed by immunostaining 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‴. Note the prominent localization of microtubules to AJs in β-galactosidase shRNA cells (arrows) but not in dynamitin KD cells. (E) Quantitation of junctional localization of microtubules in control (WT + β-gal shRNA) and dynamitin KD (WT + Dyn shRNA) cells. Quantitation was performed as described in Fig. 2 E. n = 50. (F) dynamitin KD cells display cell–cell adhesion defects. Wild-type and α–E-catenin−/− keratinocytes expressing β-galactosidase shRNA or dynamitin shRNA-2 were allowed to aggregate for 1 h with and without Ca2+, and the total number of particles was counted. The degree of Ca2+-dependent cell aggregation (NCa+/NCa− percentage) was measured as a percentage of the decrease in the particle numbers in Ca2+-containing versus Ca2+-free conditions. Bars represent mean values; n = 3. The p-value was determined by t test. The error bars represent standard deviation. Bars: (C–C″ and D–D″) 28 μm; (C‴ and D‴) 11.2 μm.

Mentions: To determine whether loss of dynamitin may phenocopy the phenotype in α–E-catenin−/− cells, we used a short hairpin RNA (shRNA) knockdown (KD) approach. Because dynamitin is critical for cell mitosis, dynamitin KD cells displayed a decrease but not a complete loss of dynamitin (Fig. 3, A and B). Nevertheless, similar to α–E-catenin−/− cells, dynamitin KD cells failed to extend the microtubule cytoskeleton to the cell periphery and AJs (Fig. 3, C–E). In addition, dynamitin KD cells displayed a prominent defect in short-term Ca2+-mediated cell–cell adhesion (Fig. 3 F). Thus, dynamitin KD cells displayed the defects in organization of the microtubule cytoskeleton and strengthening of cell–cell adhesion, which were similar to the phenotypes of α–E-catenin−/− cells. We propose that interaction between α–E-catenin and dynactin may facilitate dynactin function in the extension of the microtubule cytoskeleton to the cell periphery, and this can promote cell–cell junction formation.


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

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

Dynamitin is necessary to extend the microtubule cytoskeleton to the cell periphery and AJs and establish strong cell–cell adhesion. (A) Schematic model of a lentiviral shRNA vector used for the generation of dynamitin KD cells. CMV, cytomegalovirus; LTR, long terminal repeat. (B) Total protein extracts from wild-type (WT) and α–E-catenin−/− (KO) cells transduced with β-galactosidase (β-gal; control) and dynamitin (Dyn; constructs 1 and 2) shRNA lentiviruses analyzed by blotting with antidynamitin (Dyn) and anti–β-actin antibodies. Numbers represent relative levels of dynamitin. (C and D) Dynamitin is necessary to localize microtubules to AJs. Wild-type keratinocytes transduced with β-galactosidase shRNA (β-galsh; C) or dynamitin shRNA-2 (Dynsh; D) were analyzed by immunostaining 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‴. Note the prominent localization of microtubules to AJs in β-galactosidase shRNA cells (arrows) but not in dynamitin KD cells. (E) Quantitation of junctional localization of microtubules in control (WT + β-gal shRNA) and dynamitin KD (WT + Dyn shRNA) cells. Quantitation was performed as described in Fig. 2 E. n = 50. (F) dynamitin KD cells display cell–cell adhesion defects. Wild-type and α–E-catenin−/− keratinocytes expressing β-galactosidase shRNA or dynamitin shRNA-2 were allowed to aggregate for 1 h with and without Ca2+, and the total number of particles was counted. The degree of Ca2+-dependent cell aggregation (NCa+/NCa− percentage) was measured as a percentage of the decrease in the particle numbers in Ca2+-containing versus Ca2+-free conditions. Bars represent mean values; n = 3. The p-value was determined by t test. The error bars represent standard deviation. Bars: (C–C″ and D–D″) 28 μm; (C‴ and D‴) 11.2 μm.
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fig3: Dynamitin is necessary to extend the microtubule cytoskeleton to the cell periphery and AJs and establish strong cell–cell adhesion. (A) Schematic model of a lentiviral shRNA vector used for the generation of dynamitin KD cells. CMV, cytomegalovirus; LTR, long terminal repeat. (B) Total protein extracts from wild-type (WT) and α–E-catenin−/− (KO) cells transduced with β-galactosidase (β-gal; control) and dynamitin (Dyn; constructs 1 and 2) shRNA lentiviruses analyzed by blotting with antidynamitin (Dyn) and anti–β-actin antibodies. Numbers represent relative levels of dynamitin. (C and D) Dynamitin is necessary to localize microtubules to AJs. Wild-type keratinocytes transduced with β-galactosidase shRNA (β-galsh; C) or dynamitin shRNA-2 (Dynsh; D) were analyzed by immunostaining 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‴. Note the prominent localization of microtubules to AJs in β-galactosidase shRNA cells (arrows) but not in dynamitin KD cells. (E) Quantitation of junctional localization of microtubules in control (WT + β-gal shRNA) and dynamitin KD (WT + Dyn shRNA) cells. Quantitation was performed as described in Fig. 2 E. n = 50. (F) dynamitin KD cells display cell–cell adhesion defects. Wild-type and α–E-catenin−/− keratinocytes expressing β-galactosidase shRNA or dynamitin shRNA-2 were allowed to aggregate for 1 h with and without Ca2+, and the total number of particles was counted. The degree of Ca2+-dependent cell aggregation (NCa+/NCa− percentage) was measured as a percentage of the decrease in the particle numbers in Ca2+-containing versus Ca2+-free conditions. Bars represent mean values; n = 3. The p-value was determined by t test. The error bars represent standard deviation. Bars: (C–C″ and D–D″) 28 μm; (C‴ and D‴) 11.2 μm.
Mentions: To determine whether loss of dynamitin may phenocopy the phenotype in α–E-catenin−/− cells, we used a short hairpin RNA (shRNA) knockdown (KD) approach. Because dynamitin is critical for cell mitosis, dynamitin KD cells displayed a decrease but not a complete loss of dynamitin (Fig. 3, A and B). Nevertheless, similar to α–E-catenin−/− cells, dynamitin KD cells failed to extend the microtubule cytoskeleton to the cell periphery and AJs (Fig. 3, C–E). In addition, dynamitin KD cells displayed a prominent defect in short-term Ca2+-mediated cell–cell adhesion (Fig. 3 F). Thus, dynamitin KD cells displayed the defects in organization of the microtubule cytoskeleton and strengthening of cell–cell adhesion, which were similar to the phenotypes of α–E-catenin−/− cells. We propose that interaction between α–E-catenin and dynactin may facilitate dynactin function in the extension of the microtubule cytoskeleton to the cell periphery, and this can promote cell–cell junction formation.

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