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

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.

Figure 1

BICDR-1 is mainly expressed in kidney and neural tissues during development. (A) Schematic structure of BICD2 and BICDR proteins (CC, coiled-coil region). Antibodies were raised against GST-tagged BICDR-1 fusion proteins containing amino acids 1–146 (antibody #12) and 161–387 (antibody #13). (B) Expression of BICDR-1 mRNA in various rat tissues. (C) BICDR-1 antibodies #12 and #13 specifically recognize GFP-tagged BICDR-1 not the homologous proteins BICDR-2, BICD1 or BICD2. (D) Western blot analysis of BICDR-1, Rab6A and Rab6B in various mouse tissues. (E) Developmental expression patterns of BICDR-1, Rab6A, Rab6B, p150^glued and BICD2 in E10.5 (whole embryo), E13.5, E16, E18 and P1 (head only) mouse. (F–H) Lateral view of E10 mouse embryo hybridized with a BICDR-1-specific ribo-probe. Indicated structures: dorsal root ganglion (DRG); mesencephalon (Mes); rhombencephalon (Rhom); telencephalon (Tel). Scale bar in (F) 1 mm in (G–H) 100 μm. (I–L) Transverse cryosections (10 μm) of an E13.5 mouse kidney, hybridized with a BICDR-1 sense (I, K) or anti-sense (J, L) ribo-probe. Indicated structures: cortical region of metanephros (Ctx); medullary region of metanephros (Med); and metanephric tubule (Tub). Scale bars, 100 μm. (M–R) Transverse cryosections (10 μm) of an E13.5 mouse head, hybridized with a BICDR-1 sense (M, O, Q) or anti-sense (N, P, R) ribo-probe. Indicated structures: fourth ventricle (4v); third ventricle (3v); choroid plexus (Chor); myencephalon (My); metencephalon (Met); diencephalon (Di); trigeminal (V) ganglion (Trig); olfactory epithelium (Olf ep); ventricular zone (VZ); intermediate zone (IZ); neural layer of retina (NLR). Scale bars, 100 μm.

Figure 2

BICDR-1 has a critical role in zebrafish neural and eye development. (A) Dorsal and lateral views of zebrafish fixed and in situ hybridized with zBICDR-1-specific probe at 48, 72 or 96 hpf. (B) Lateral views of wild type (WT) and zBICDR-1 morphant fish (MO) at 48, 72 or 96 hpf. (C) Lateral views of wild type (WT) and morphant fish (MO) labelled with phospho-H3 or caspase-specific antibodies or acridine orange.

Figure 3

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.

Figure 4

BICDR-1 interacts with the dynein/dynactin motor complex. (A) Immunoprecipitations from extracts of Hela cells transfected with the indicated constructs and probed for DHC, p150^glued or dynein intermediate chain 74 (IC74). (B) Ratio of pericentrosomal versus cytoplasmic p150^glued or IC74 fluorescence intensity in cells with and without GFP-BICDR-1 overexpression (average ±s.e.m.; p150^glued control, n=29; p150^glued+BICDR-1, n=30; IC74 control, n=32; IC74+BICDR-1, n=19 cells). (C) Percentage of cells, transfected with indicated siRNAs, with endogenous pericentrosomal BICDR-1 (average ±s.e.m.; control, n=3040; DHC siRNA, n=533; p150^glued, n=792 cells). (D) Representative image of a Vero cell stained for endogenous BICDR-1 (green) and for p150^glued (red). (E) Representative image of Hela cells with and without GFP-BICDR-1 (green) overexpression stained for p150^glued (red). (F) Vero cells transfected with p150^glued-specific siRNA (right) and untransfected (left). Stained for BICDR-1 (green) and p150^glued (red). (D–F) Solid lines indicate the cell edge and dashed lines indicate the nucleus. The insets show magnifications of boxed areas. Scale bars, 10 μm. ^***P<0.001.

Figure 5

BICDR-1 is expressed during early stages of neuronal development and is associated with Rab6-positive secretory vesicles in neurons. (A) Developmental expression of BICDR-1, Rab6A, Rab6B, p150^glued and actin in cultured hippocampal neurons at DIV1, DIV3, DIV5 and DIV26. (B) Immunofluorescent images of BICDR-1 in hippocampal neurons at DIV1 (left) and DIV3 (right). (C) DIV1 neuron fixed and stained for BICDR-1 (green) and Rab6A (red). (D) Time-lapse images of a DIV2 neuron transfected with mCherry-BICDR-1 (red) and GFP-Rab6A. Time indicated in seconds. (E) DIV2+2 hippocampal neuron transfected with mCherry-BICDR-1 (red) and NPY-GFP (green) and stained for Tuj1 (blue). The insets show magnifications of the boxed areas, arrows indicate co-localization (Pearson's coefficient, r_p=0.8). (F) Detail of DIV1+3 hippocampal neuron transfected with NPY-GFP (green) and stained for Rab6A (blue) and Rab6B (red). Merged images show co-localization of NPY-GFP/Rab6A (r_p=0.9), NPY-GFP/Rab6B (r_p=0.8) and Rab6A/Rab6B (r_p=0.8). (B, C, E) Solid lines indicate the cell edge; the insets show magnifications of boxed areas and arrows indicate co-localization. Scale bars, 10 μm. (F) Scale bar, 5 μm.

Figure 6

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.

Figure 7

BICDR-1 interacts with Kif1C. (A) Yeast two-hybrid analysis. Kif1C (356–888), (670–1090), (670–888) or (811–1090) 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 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. (B) Immunoprecipitations from extracts of Hela cells transfected with the indicated constructs and probed for Kif1C (^* indicates that cells were treated with 1 μM staurosporin for 1 h before immunoprecipitation). (C) Representative image of Hela cells with and without GFP-BICDR-1 overexpression stained for Kif1C and Rab6A. (D) DIV2+two hippocampal neuron transfected with GFP-BICDR-1 (green) and stained for Kif1C (red). (E) DIV1+three hippocampal neurons transfected with either NPY-GFP (green), Kif1C shRNA and β-Gal (blue). (F) Quantification of the number of GFP-labelled vesicles per 10 μm neurite in DIV1+three hippocampal neurons transfected with indicated constructs (average ±s.e.m.; control, n=5; NPY+Kif1C shRNA, n=6). (C–E) Solid lines indicate the cell edge and dashed lines indicate the nucleus. The insets show magnifications of boxed areas. Scale bars, 10 μm. ^***P<0.001.

Figure 8

BICDR-1 regulates neurite outgrowth. (A, B) Hippocampal neurons co-transfected at DIV0.25 with indicated constructs and β-Gal to visualize morphology, fixed at DIV3. Scale bar, 100 μm. (C) Total neurite length after 2-, 3- or 4-day overexpression of GFP-BICDR-1 or an empty pGW1-HA vector as control (DIV2 control n=9, BICDR-1 n=14; DIV3 control n=11, BICDR-1 n=12; DIV4 control n=14, BICDR-1 n=16). (D) Total neurite length after 3-day overexpression of GFP-BICDR-1, GFP-BICDR-1C or GFP-BICDR-1C-K512M. Control cells were transfected with an empty pGW1-HA vector (control n=37; BICDR-1 n=23; BICDR-1C n=15; BICDR-1C-K512M n=11). (E) Axon length after 3-day overexpression of GFP-BICDR-1 or an empty pGW1-HA vector as control (control n=9; BICDR-1 n=9). (F) Dendrite length after 3-day overexpression of GFP-BICDR-1 or an empty pGW1-HA vector as control (control n=14; BICDR-1 n=9). (G) Total neurite length after 3-day overexpression of GFP-BICDR-1 or GFP-BICD2. Control cells were transfected with an empty pGW1-HA vector (control n=37; BICDR-1 n=23; BICD1 n=12; BICD2 n=19). (H) Total neurite length after 3-day overexpression of GFP-BICDR-1, GFP-p50 or GFP-p150CC1. Control cells were transfected with an empty pGW1-HA vector (control n=37; BICDR-1 n=23; p50 n=15; p150CC1 n=17). (I) Total neurite length after 3-day overexpression of shRNA specific for Rab6A, Rab6B, Rab6A and Rab6B or Kif1C. Control cells were transfected with an empty pSuper vector (control n=14; Rab6A shRNA n=21; Rab6B shRNA n=10; Rab6A/B shRNA n=16; Kif1C shRNA n=13). (C–I) Average ±s.e.m., ^*P<0.05. ^**P<0.01. ^***P<0.001.