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Pericentrosomal targeting of Rab6 secretory vesicles by Bicaudal-D-related protein 1 (BICDR-1) regulates neuritogenesis.

Schlager MA, Kapitein LC, Grigoriev I, Burzynski GM, Wulf PS, Keijzer N, de Graaff E, Fukuda M, Shepherd IT, Akhmanova A, Hoogenraad CC - EMBO J. (2010)

Bottom Line: BICDR-1 expression is high during early neuronal development and strongly declines during neurite outgrowth.Later during development, BICDR-1 expression is strongly reduced, which permits anterograde secretory transport required for neurite outgrowth.These results indicate an important role for BICDR-1 as temporal regulator of secretory trafficking during the early phase of neuronal differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Erasmus Medical Center, Rotterdam, The Netherlands.

ABSTRACT
Membrane and secretory trafficking are essential for proper neuronal development. However, the molecular mechanisms that organize secretory trafficking are poorly understood. Here, we identify Bicaudal-D-related protein 1 (BICDR-1) as an effector of the small GTPase Rab6 and key component of the molecular machinery that controls secretory vesicle transport in developing neurons. BICDR-1 interacts with kinesin motor Kif1C, the dynein/dynactin retrograde motor complex, regulates the pericentrosomal localization of Rab6-positive secretory vesicles and is required for neural development in zebrafish. BICDR-1 expression is high during early neuronal development and strongly declines during neurite outgrowth. In young neurons, BICDR-1 accumulates Rab6 secretory vesicles around the centrosome, restricts anterograde secretory transport and inhibits neuritogenesis. Later during development, BICDR-1 expression is strongly reduced, which permits anterograde secretory transport required for neurite outgrowth. These results indicate an important role for BICDR-1 as temporal regulator of secretory trafficking during the early phase of neuronal differentiation.

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BICDR-1 binds Rab6A/B and is localized pericentrosomally. (A) GST pull-down assay with GST-Rabs (Rab1–43) and extracts of Cos7 cells expressing Flag-BICDR-1 or Flag-BICDR-2. Flag-tagged proteins were detected by western blotting with antibodies against Flag; GST proteins were visualized using Amido Black. (B) Yeast two-hybrid analysis. Rab6A-Q72R was linked to LexA and BICDR-1 (1–577), (1–353) or (382–577) was fused to a GAL4 activation domain. Interaction strength was scored according to the time needed for a β-galactosidase reporter to generate visible blue-coloured yeast colonies on X-Gal containing filters in a colony filter lift assay: +++0–30 min, ++30–60 min, +60–180 min and − no β-galactosidase activity. (C) In vitro binding assay using purified GST-Rab6A/B bound to beads and purified His-BICDR-1C or His-BICDR-1C-K512M. His-tagged proteins were detected by western blotting with antibodies against His; GST-Rab6A/B was visualized by Coomassie staining. (D) GST pull-down assay with GST-Rab6A and extracts of Cos7 cells expressing Flag-BICDR-1, Flag-BICD2, Flag-BICDR-1-K512M or Flag-BICD2-K785M. Flag-tagged proteins were detected by western blotting with antibodies against Flag; GST-Rab6A was visualized using Amido Black. (E) Sequence alignment of BICD1, BICD2 and BICDR-1 Rab6-binding region (* indicates site of BICD2-K785M and BICDR-1-K512M mutations). (F) Co-staining of BICDR-1 (green) and γ-tubulin (red) in a Vero cell. (G) Representative image of a Vero cell co-stained for endogenous BICDR-1 (green) and Rab6A (red). (F–G) Solid lines indicate the cell edge and dashed lines indicate the nucleus. The insets show magnifications of boxed areas. Scale bars, 10 μm.
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f3: BICDR-1 binds Rab6A/B and is localized pericentrosomally. (A) GST pull-down assay with GST-Rabs (Rab1–43) and extracts of Cos7 cells expressing Flag-BICDR-1 or Flag-BICDR-2. Flag-tagged proteins were detected by western blotting with antibodies against Flag; GST proteins were visualized using Amido Black. (B) Yeast two-hybrid analysis. Rab6A-Q72R was linked to LexA and BICDR-1 (1–577), (1–353) or (382–577) was fused to a GAL4 activation domain. Interaction strength was scored according to the time needed for a β-galactosidase reporter to generate visible blue-coloured yeast colonies on X-Gal containing filters in a colony filter lift assay: +++0–30 min, ++30–60 min, +60–180 min and − no β-galactosidase activity. (C) In vitro binding assay using purified GST-Rab6A/B bound to beads and purified His-BICDR-1C or His-BICDR-1C-K512M. His-tagged proteins were detected by western blotting with antibodies against His; GST-Rab6A/B was visualized by Coomassie staining. (D) GST pull-down assay with GST-Rab6A and extracts of Cos7 cells expressing Flag-BICDR-1, Flag-BICD2, Flag-BICDR-1-K512M or Flag-BICD2-K785M. Flag-tagged proteins were detected by western blotting with antibodies against Flag; GST-Rab6A was visualized using Amido Black. (E) Sequence alignment of BICD1, BICD2 and BICDR-1 Rab6-binding region (* indicates site of BICD2-K785M and BICDR-1-K512M mutations). (F) Co-staining of BICDR-1 (green) and γ-tubulin (red) in a Vero cell. (G) Representative image of a Vero cell co-stained for endogenous BICDR-1 (green) and Rab6A (red). (F–G) Solid lines indicate the cell edge and dashed lines indicate the nucleus. The insets show magnifications of boxed areas. Scale bars, 10 μm.

Mentions: We next wanted to understand the cellular pathway in which BICDR-1 is involved. Previous studies have shown that members of BICD family interact with the small GTPase Rab6 (Matanis et al, 2002; Grigoriev et al, 2007). Therefore, we searched for possible interactions of BICDR-1 with Rab-GTPases by glutathione S-transferase (GST) pull-down assays with cell lysates expressing FLAG-tagged BICDR-1 using 60 different recombinant Rab proteins (including 43 subfamilies) immobilized on GST beads. BICDR-1 strongly interacts with only one Rab subfamily, Rab6A/B (Figure 3A, square) and marginally with Rab11B and Rab12, but not with any of the other Rabs tested (Figure 3A). In contrast, BICDR-2 interacts most strongly with Rab13 (Figure 3A). Moreover, both full-length BICDR-1 and a C-terminal region of BICDR-1, amino acids 382–577, interact with constitutively active GTPase-deficient Rab6A-Q72R and not with GDP-locked inactive Rab6A-T27N in the yeast two-hybrid assay (Figure 3B; Supplementary Figure S2A). Binding of BICDR-1 to Rab6A is direct because the purified His-tagged C-terminus of BICDR-1 (amino acids 382–577) interacts with purified GST-Rab6A and GST-Rab6B and not with GST only (Figure 3C). Interestingly, BICDR-1 binds most strongly to the neuron-specific isoform Rab6B (Figure 3A and C). The Rab6-binding region of BICD is well conserved in the C-terminal domain of BICDR-1 and contains a lysine residue (Figure 3E, asterisk) that is mutated to methionine in a loss of function Drosophila BicD allele (Ran et al, 1994). We next tested whether this mutation would disrupt Rab6 binding. GST-Rab6 pull-down assays with lysates of cells expressing mutant BICDR-1-K512M and an in vitro binding assay using purified His-BICDR-1C-K512M showed that lysine 512 is required for binding to Rab6 (Figure 3C). Similarly, BICD2-K785M could not be precipitated by GST-Rab6 (Figure 3D). Moreover, BICDR-1 and BICD2 compete for Rab6 binding in in vitro pull-down assays (Supplementary Figure S2B). Together these data show that the conserved C-terminal domain of BICDR-1 directly binds to Rab6A and Rab6B.


Pericentrosomal targeting of Rab6 secretory vesicles by Bicaudal-D-related protein 1 (BICDR-1) regulates neuritogenesis.

Schlager MA, Kapitein LC, Grigoriev I, Burzynski GM, Wulf PS, Keijzer N, de Graaff E, Fukuda M, Shepherd IT, Akhmanova A, Hoogenraad CC - EMBO J. (2010)

BICDR-1 binds Rab6A/B and is localized pericentrosomally. (A) GST pull-down assay with GST-Rabs (Rab1–43) and extracts of Cos7 cells expressing Flag-BICDR-1 or Flag-BICDR-2. Flag-tagged proteins were detected by western blotting with antibodies against Flag; GST proteins were visualized using Amido Black. (B) Yeast two-hybrid analysis. Rab6A-Q72R was linked to LexA and BICDR-1 (1–577), (1–353) or (382–577) was fused to a GAL4 activation domain. Interaction strength was scored according to the time needed for a β-galactosidase reporter to generate visible blue-coloured yeast colonies on X-Gal containing filters in a colony filter lift assay: +++0–30 min, ++30–60 min, +60–180 min and − no β-galactosidase activity. (C) In vitro binding assay using purified GST-Rab6A/B bound to beads and purified His-BICDR-1C or His-BICDR-1C-K512M. His-tagged proteins were detected by western blotting with antibodies against His; GST-Rab6A/B was visualized by Coomassie staining. (D) GST pull-down assay with GST-Rab6A and extracts of Cos7 cells expressing Flag-BICDR-1, Flag-BICD2, Flag-BICDR-1-K512M or Flag-BICD2-K785M. Flag-tagged proteins were detected by western blotting with antibodies against Flag; GST-Rab6A was visualized using Amido Black. (E) Sequence alignment of BICD1, BICD2 and BICDR-1 Rab6-binding region (* indicates site of BICD2-K785M and BICDR-1-K512M mutations). (F) Co-staining of BICDR-1 (green) and γ-tubulin (red) in a Vero cell. (G) Representative image of a Vero cell co-stained for endogenous BICDR-1 (green) and Rab6A (red). (F–G) Solid lines indicate the cell edge and dashed lines indicate the nucleus. The insets show magnifications of boxed areas. Scale bars, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2876961&req=5

f3: BICDR-1 binds Rab6A/B and is localized pericentrosomally. (A) GST pull-down assay with GST-Rabs (Rab1–43) and extracts of Cos7 cells expressing Flag-BICDR-1 or Flag-BICDR-2. Flag-tagged proteins were detected by western blotting with antibodies against Flag; GST proteins were visualized using Amido Black. (B) Yeast two-hybrid analysis. Rab6A-Q72R was linked to LexA and BICDR-1 (1–577), (1–353) or (382–577) was fused to a GAL4 activation domain. Interaction strength was scored according to the time needed for a β-galactosidase reporter to generate visible blue-coloured yeast colonies on X-Gal containing filters in a colony filter lift assay: +++0–30 min, ++30–60 min, +60–180 min and − no β-galactosidase activity. (C) In vitro binding assay using purified GST-Rab6A/B bound to beads and purified His-BICDR-1C or His-BICDR-1C-K512M. His-tagged proteins were detected by western blotting with antibodies against His; GST-Rab6A/B was visualized by Coomassie staining. (D) GST pull-down assay with GST-Rab6A and extracts of Cos7 cells expressing Flag-BICDR-1, Flag-BICD2, Flag-BICDR-1-K512M or Flag-BICD2-K785M. Flag-tagged proteins were detected by western blotting with antibodies against Flag; GST-Rab6A was visualized using Amido Black. (E) Sequence alignment of BICD1, BICD2 and BICDR-1 Rab6-binding region (* indicates site of BICD2-K785M and BICDR-1-K512M mutations). (F) Co-staining of BICDR-1 (green) and γ-tubulin (red) in a Vero cell. (G) Representative image of a Vero cell co-stained for endogenous BICDR-1 (green) and Rab6A (red). (F–G) Solid lines indicate the cell edge and dashed lines indicate the nucleus. The insets show magnifications of boxed areas. Scale bars, 10 μm.
Mentions: We next wanted to understand the cellular pathway in which BICDR-1 is involved. Previous studies have shown that members of BICD family interact with the small GTPase Rab6 (Matanis et al, 2002; Grigoriev et al, 2007). Therefore, we searched for possible interactions of BICDR-1 with Rab-GTPases by glutathione S-transferase (GST) pull-down assays with cell lysates expressing FLAG-tagged BICDR-1 using 60 different recombinant Rab proteins (including 43 subfamilies) immobilized on GST beads. BICDR-1 strongly interacts with only one Rab subfamily, Rab6A/B (Figure 3A, square) and marginally with Rab11B and Rab12, but not with any of the other Rabs tested (Figure 3A). In contrast, BICDR-2 interacts most strongly with Rab13 (Figure 3A). Moreover, both full-length BICDR-1 and a C-terminal region of BICDR-1, amino acids 382–577, interact with constitutively active GTPase-deficient Rab6A-Q72R and not with GDP-locked inactive Rab6A-T27N in the yeast two-hybrid assay (Figure 3B; Supplementary Figure S2A). Binding of BICDR-1 to Rab6A is direct because the purified His-tagged C-terminus of BICDR-1 (amino acids 382–577) interacts with purified GST-Rab6A and GST-Rab6B and not with GST only (Figure 3C). Interestingly, BICDR-1 binds most strongly to the neuron-specific isoform Rab6B (Figure 3A and C). The Rab6-binding region of BICD is well conserved in the C-terminal domain of BICDR-1 and contains a lysine residue (Figure 3E, asterisk) that is mutated to methionine in a loss of function Drosophila BicD allele (Ran et al, 1994). We next tested whether this mutation would disrupt Rab6 binding. GST-Rab6 pull-down assays with lysates of cells expressing mutant BICDR-1-K512M and an in vitro binding assay using purified His-BICDR-1C-K512M showed that lysine 512 is required for binding to Rab6 (Figure 3C). Similarly, BICD2-K785M could not be precipitated by GST-Rab6 (Figure 3D). Moreover, BICDR-1 and BICD2 compete for Rab6 binding in in vitro pull-down assays (Supplementary Figure S2B). Together these data show that the conserved C-terminal domain of BICDR-1 directly binds to Rab6A and Rab6B.

Bottom Line: BICDR-1 expression is high during early neuronal development and strongly declines during neurite outgrowth.Later during development, BICDR-1 expression is strongly reduced, which permits anterograde secretory transport required for neurite outgrowth.These results indicate an important role for BICDR-1 as temporal regulator of secretory trafficking during the early phase of neuronal differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Erasmus Medical Center, Rotterdam, The Netherlands.

ABSTRACT
Membrane and secretory trafficking are essential for proper neuronal development. However, the molecular mechanisms that organize secretory trafficking are poorly understood. Here, we identify Bicaudal-D-related protein 1 (BICDR-1) as an effector of the small GTPase Rab6 and key component of the molecular machinery that controls secretory vesicle transport in developing neurons. BICDR-1 interacts with kinesin motor Kif1C, the dynein/dynactin retrograde motor complex, regulates the pericentrosomal localization of Rab6-positive secretory vesicles and is required for neural development in zebrafish. BICDR-1 expression is high during early neuronal development and strongly declines during neurite outgrowth. In young neurons, BICDR-1 accumulates Rab6 secretory vesicles around the centrosome, restricts anterograde secretory transport and inhibits neuritogenesis. Later during development, BICDR-1 expression is strongly reduced, which permits anterograde secretory transport required for neurite outgrowth. These results indicate an important role for BICDR-1 as temporal regulator of secretory trafficking during the early phase of neuronal differentiation.

Show MeSH
Related in: MedlinePlus