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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 controls the trafficking of secretory vesicles in neurons. (A) Representative image of hippocampal neurons transfected with GFP-BICDR-1 (green) and stained for Rab6A (red) and α-tubulin (blue). (B) Quantification of Rab6A and Rab6B immunostaining intensities in hippocampal neurons transfected with GFP-BICDR-1 (average ±s.e.m.; Rab6A control, n=21; Rab6A+BICDR-1, n=10; Rab6B control, n=31; Rab6B+BICDR-1, n=16 cells). (C) DIV2+2 hippocampal neurons transfected with either NPY-GFP (green) and β-Gal (blue; top row) or NPY-GFP (green), mCherry-BICDR-1 (red) and β-Gal (blue; bottom row). (D) Quantification of the number of GFP-labelled vesicles per 10 μm neurite in DIV2+2 hippocampal neurons transfected with indicated constructs (average ±s.e.m.; Sem3A control, n=10; Sem3A+BICDR-1, n=10; BDNF control, n=5; BDNF+BICDR-1, n=5; NPY control, n=5; NPY+BICDR-1, n=5). (E) Time-lapse images of DIV4+1 hippocampal neurons transfected with NPY-GFP and mCherry (control, axon) or NPY-GFP, mCherry and HA-BICDR-1 (+BICDR-1, cell body) before and after stimulation by addition of KCl (60 mM final concentration). Scale bar, 1 μm; arrows indicate secreting vesicles; solid lines indicate the cell edge. (F) Fluorescence intensity traces of secretory events in the axon (control) or cell body (+BICDR-1) during KCl addition. ***P<0.001.
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f6: BICDR-1 controls the trafficking of secretory vesicles in neurons. (A) Representative image of hippocampal neurons transfected with GFP-BICDR-1 (green) and stained for Rab6A (red) and α-tubulin (blue). (B) Quantification of Rab6A and Rab6B immunostaining intensities in hippocampal neurons transfected with GFP-BICDR-1 (average ±s.e.m.; Rab6A control, n=21; Rab6A+BICDR-1, n=10; Rab6B control, n=31; Rab6B+BICDR-1, n=16 cells). (C) DIV2+2 hippocampal neurons transfected with either NPY-GFP (green) and β-Gal (blue; top row) or NPY-GFP (green), mCherry-BICDR-1 (red) and β-Gal (blue; bottom row). (D) Quantification of the number of GFP-labelled vesicles per 10 μm neurite in DIV2+2 hippocampal neurons transfected with indicated constructs (average ±s.e.m.; Sem3A control, n=10; Sem3A+BICDR-1, n=10; BDNF control, n=5; BDNF+BICDR-1, n=5; NPY control, n=5; NPY+BICDR-1, n=5). (E) Time-lapse images of DIV4+1 hippocampal neurons transfected with NPY-GFP and mCherry (control, axon) or NPY-GFP, mCherry and HA-BICDR-1 (+BICDR-1, cell body) before and after stimulation by addition of KCl (60 mM final concentration). Scale bar, 1 μm; arrows indicate secreting vesicles; solid lines indicate the cell edge. (F) Fluorescence intensity traces of secretory events in the axon (control) or cell body (+BICDR-1) during KCl addition. ***P<0.001.

Mentions: In young neurons, BICDR-1 coincides with NPY-GFP-positive vesicles in the cell body and neurites (Figure 5E) and endogenous Rab6 (Rab6A and Rab6B) co-localize with NPY-GFP in a similar manner (Figure 5F). Consistent with the Hela cell data, expression of GFP-BICDR-1 in DIV3 neurons causes a strong accumulation of dynein/dynactin (Supplementary Figure S7A and B) and Rab6A and Rab6B-positive vesicles in the soma (Figure 6A; Supplementary Figure S7C and D), whereas other trafficking markers, such as endosomes were unaffected (Supplementary Figure S7E). Quantification showed an ∼3-fold increase in Rab6A and Rab6B fluorescent staining intensity in the cell bodies of BICDR-1-transfected neurons compared with surrounding non-transfected control neurons (Figure 6B). Moreover, expression of BICDR-1 strongly accumulates NPY-GFP-positive secretory vesicles in the soma and caused a marked decrease in the number of NPY-GFP vesicles in developing neurites, compared with control cells (Figure 6C and D). Similar results were obtained with other neuronal secretory vesicle markers, such as GFP-Sema3A and BDNF-GFP (Figure 6D; Supplementary Figure S7F and G). Together these data suggest that BICDR-1 controls the trafficking of secretory vesicles most likely by shifting the transport balance towards the retrograde direction. As a result, Rab6/BICDR-1 secretory vesicles concentrate in the cell body and trafficking into growing neurites is blocked.


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 controls the trafficking of secretory vesicles in neurons. (A) Representative image of hippocampal neurons transfected with GFP-BICDR-1 (green) and stained for Rab6A (red) and α-tubulin (blue). (B) Quantification of Rab6A and Rab6B immunostaining intensities in hippocampal neurons transfected with GFP-BICDR-1 (average ±s.e.m.; Rab6A control, n=21; Rab6A+BICDR-1, n=10; Rab6B control, n=31; Rab6B+BICDR-1, n=16 cells). (C) DIV2+2 hippocampal neurons transfected with either NPY-GFP (green) and β-Gal (blue; top row) or NPY-GFP (green), mCherry-BICDR-1 (red) and β-Gal (blue; bottom row). (D) Quantification of the number of GFP-labelled vesicles per 10 μm neurite in DIV2+2 hippocampal neurons transfected with indicated constructs (average ±s.e.m.; Sem3A control, n=10; Sem3A+BICDR-1, n=10; BDNF control, n=5; BDNF+BICDR-1, n=5; NPY control, n=5; NPY+BICDR-1, n=5). (E) Time-lapse images of DIV4+1 hippocampal neurons transfected with NPY-GFP and mCherry (control, axon) or NPY-GFP, mCherry and HA-BICDR-1 (+BICDR-1, cell body) before and after stimulation by addition of KCl (60 mM final concentration). Scale bar, 1 μm; arrows indicate secreting vesicles; solid lines indicate the cell edge. (F) Fluorescence intensity traces of secretory events in the axon (control) or cell body (+BICDR-1) during KCl addition. ***P<0.001.
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f6: BICDR-1 controls the trafficking of secretory vesicles in neurons. (A) Representative image of hippocampal neurons transfected with GFP-BICDR-1 (green) and stained for Rab6A (red) and α-tubulin (blue). (B) Quantification of Rab6A and Rab6B immunostaining intensities in hippocampal neurons transfected with GFP-BICDR-1 (average ±s.e.m.; Rab6A control, n=21; Rab6A+BICDR-1, n=10; Rab6B control, n=31; Rab6B+BICDR-1, n=16 cells). (C) DIV2+2 hippocampal neurons transfected with either NPY-GFP (green) and β-Gal (blue; top row) or NPY-GFP (green), mCherry-BICDR-1 (red) and β-Gal (blue; bottom row). (D) Quantification of the number of GFP-labelled vesicles per 10 μm neurite in DIV2+2 hippocampal neurons transfected with indicated constructs (average ±s.e.m.; Sem3A control, n=10; Sem3A+BICDR-1, n=10; BDNF control, n=5; BDNF+BICDR-1, n=5; NPY control, n=5; NPY+BICDR-1, n=5). (E) Time-lapse images of DIV4+1 hippocampal neurons transfected with NPY-GFP and mCherry (control, axon) or NPY-GFP, mCherry and HA-BICDR-1 (+BICDR-1, cell body) before and after stimulation by addition of KCl (60 mM final concentration). Scale bar, 1 μm; arrows indicate secreting vesicles; solid lines indicate the cell edge. (F) Fluorescence intensity traces of secretory events in the axon (control) or cell body (+BICDR-1) during KCl addition. ***P<0.001.
Mentions: In young neurons, BICDR-1 coincides with NPY-GFP-positive vesicles in the cell body and neurites (Figure 5E) and endogenous Rab6 (Rab6A and Rab6B) co-localize with NPY-GFP in a similar manner (Figure 5F). Consistent with the Hela cell data, expression of GFP-BICDR-1 in DIV3 neurons causes a strong accumulation of dynein/dynactin (Supplementary Figure S7A and B) and Rab6A and Rab6B-positive vesicles in the soma (Figure 6A; Supplementary Figure S7C and D), whereas other trafficking markers, such as endosomes were unaffected (Supplementary Figure S7E). Quantification showed an ∼3-fold increase in Rab6A and Rab6B fluorescent staining intensity in the cell bodies of BICDR-1-transfected neurons compared with surrounding non-transfected control neurons (Figure 6B). Moreover, expression of BICDR-1 strongly accumulates NPY-GFP-positive secretory vesicles in the soma and caused a marked decrease in the number of NPY-GFP vesicles in developing neurites, compared with control cells (Figure 6C and D). Similar results were obtained with other neuronal secretory vesicle markers, such as GFP-Sema3A and BDNF-GFP (Figure 6D; Supplementary Figure S7F and G). Together these data suggest that BICDR-1 controls the trafficking of secretory vesicles most likely by shifting the transport balance towards the retrograde direction. As a result, Rab6/BICDR-1 secretory vesicles concentrate in the cell body and trafficking into growing neurites is blocked.

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