KIF1Bβ transports dendritically localized mRNPs in neurons and is recruited to synapses in an activity-dependent manner

Abstract

KIF1Bβ is a kinesin-like, microtubule-based molecular motor protein involved in anterograde axonal vesicular transport in vertebrate and invertebrate neurons. Certain KIF1Bβ isoforms have been implicated in different forms of human neurodegenerative disease, with characterization of their functional integration and regulation in the context of synaptic signaling still ongoing. Here, we characterize human KIF1Bβ (isoform NM015074), whose expression we show to be developmentally regulated and elevated in cortical areas of the CNS (including the motor cortex), in the hippocampus, and in spinal motor neurons. KIF1Bβ localizes to the cell body, axon, and dendrites, overlapping with synaptic-vesicle and postsynaptic-density structures. Correspondingly, in purified cortical synaptoneurosomes, KIF1Bβ is enriched in both pre- and postsynaptic structures, forming detergent-resistant complexes. Interestingly, KIF1Bβ forms RNA-protein complexes, containing the dendritically localized Arc and Calmodulin mRNAs, proteins previously shown to be part of RNA transport granules such as Purα, FMRP and FXR2P, and motor protein KIF3A, as well as Calmodulin. The interaction between KIF1Bβ and Calmodulin is Ca(+2)-dependent and takes place through a domain mapped at the carboxy-terminal tail of the motor. Live imaging of cortical neurons reveals active movement by KIF1Bβ at dendritic processes, suggesting that it mediates the transport of dendritically localized mRNAs. Finally, we show that synaptic recruitment of KIF1Bβ is activity-dependent and increased by stimulation of metabotropic or ionotropic glutamate receptors. The activity-dependent synaptic recruitment of KIF1Bβ, its interaction with Ca(2+) sensor Calmodulin, and its new role as a dendritic motor of ribonucleoprotein complexes provide a novel basis for understanding the concerted co-ordination of motor protein mobilization and synaptic signaling pathways.

Fig. 1

Human KIF1Bβ isoforms. Schematic comparison of isoforms NM015074, AX039604 and AB088213 [the latter being representative of the AB088210–13 subgroup (AB)]. NM015074 (NM) and AX039604 (AX) share a characteristic C-terminus (red) to which an anti-peptide antibody was raised (indicated at the top). The AB series have an alternative C-terminus (denoted by the dark green box) not recognized by the antibody. AX and AB have an additional 6-aa stretch (black box) close to the motor domain, and AX also has a unique additional 40-aa stretch (gray box). The positions of the two sets of oligonucleotides that are each specific to NM or AX only, and can thus discriminate between NM and AX by PCR (Fig. 2b, d–f), are indicated by arrows over the areas to which they hybridize. The sequences of these oligonucleotide pairs are given in Online Resource Table S1. The motor (yellow), FHA (light blue), and PH domains (light green), as well as the two stretches of amino acids (black and gray boxes), unique in certain isoforms, are highlighted. Numbers denote aa positions

Fig. 2

a KIF1Bβ (NM015074) mRNA expression pattern in human tissues. Equivalent RT-PCR reactions from various human tissues, including a mock reverse-transcription reaction as negative control (−RT). Equivalent reactions for the mRNA of ribosomal protein L19 were used as internal standards (bottom panel). b–d mRNA expression of KIF1Bβ NM015074 versus AX039604 isoforms in the CNS. Equivalent RT-PCR reactions specific to KIF1Bβ isoforms NM015074 (top panels) and AX039604 (central panels), and housekeeping gene L19 (bottom panels) for b areas of human cortex; c mouse neural tissue, including spinal motor neurons; d mouse stage-2 or stage-5 hippocampal neurons in vitro [18] or the hippocampus of E13, E18 and juvenile mice, and from proliferating astroglial cultures. e KIF1Bβ (NM015074) mRNA expression in human and mouse cell lines. Equivalent RT-PCR reactions reveal a pronounced expression level of KIF1Bβ mRNA in the mouse neuroblastoma NSC-34 line, lower expression levels in mouse NIH 3T3 fibroblasts, and expression below detection levels for other cell lines. Equivalent reactions for L19 (bottom panel). f Protein expression of KIF1Bβ in different brain areas by immunoblot (top panels). Total protein extracts from mouse cultured primary cortical neurons, NSC-34 or NIH 3T3 cells were analyzed by western blotting, using the anti-peptide KIF1Bβ antibody. A signal, consistent with the predicted M _r of 199.01 × 10³ for KIF1Bβ was detected (e). Also consistent with mRNA level for these cell lines, protein expression of KIF1Bβ in NSC-34 is higher than in NIH 3T3 cells (bottom panels). Expression of KIF1Bβ protein in equivalent samples from different parts of the mouse CNS (left); these signals are abolished if the antibody is pre-absorbed with the antigenic peptide (right). Immunoreactivity for GAPDH serves as internal loading control

Fig. 3

Intracellular localization of KIF1Bβ in mouse primary motor neurons. a–d Red panels spinal motor neurons probed with antibodies against (a) non-phosphorylated neurofilament H, SM132; (b) MAP2; (c) PSD-95; (d) synaptophysin. Corresponding green panels represent labeling for KIF1Bβ. The red and green images are overlaid in the last column. E1–5. Segments of dendritic processes from mouse spinal cord motor neurons stained with AF647-phalloidin (blue, substituted pseudocolour to visualize infrared emission), anti-synaptophysin (green) or anti-KIF1Bβ antibodies (red). E1 A merged image displays an overlay of the three labels. E2–E5 A magnified detail (corresponding to the boxed area in panel E1) for each of the labels and their overlay image is shown in these panels. Examples of localization of KIF1Bβ at postsynaptic sites (co-localization with polymerized F-actin, as revealed by phalloidin staining) are indicated with white arrows (blue and red color merges to purple), while examples of co-localization of KIF1B and synaptophysin (a presynaptic marker) are indicated with yellow arrows (green and red color merges to yellow). Scale bars (E1) 10 μm, (E2–E5) 5 μm

Fig. 4

Intracellular localization of KIF1Bβ in mouse neuroblastoma NSC-34 cell line. NSC-34 cells stained with antibodies to a PSD-95 (red), b SV2 (red), with concurrent labeling of nuclei with Hoechst 33342 (blue). Green panels represent labeling with anti-KIF1Bβ. Scale bars (a) 20 μm, (b, c) 10 μm. c NSC-34, transiently transfected with pEYFP-KIF1Bβ, green). Anti-SV2 staining is in red, and DNA in blue. Of the two cells in the field, only the top cell is transfected: YFP-KIF1Bβ localization (green) overlaps extensively with that of SV2 (red), therefore merging to yellow in the overlay. Scale bar 10 μm. d Quantification of silencing efficiency by RT semi-quantitative PCR and immunoblot. Top two panels levels of KIF1Bβ mRNA at 120 h post-transfection in NSC-34 cells silenced with 320 pM of KIF1Bβ-specific siRNA cocktail are compared to control-silenced cells (negative control) and to mock-treated cells (no siRNA). RT-PCR detection of L19 mRNA levels serves as internal control. Bottom two panels KIF1Bβ protein levels are compared in corresponding samples. Detection of the intermediate chain of dynein serves as loading control in the three samples. e KIF1Bβ immunofluorescence in silenced cells versus negative control-silenced cells. NSC-34 cells were immunostained for KIF1Bβ (green) and counterstained for DNA (blue). KIF1Bβ-silenced cells display a marked reduction in the signal of KIF1Bβ-positive vesicular structures when imaged at the same exposure as negative control-silenced cells. Scale bar 10 μm

Fig. 5

a Detection of KIF1Bβ in purified mouse synaptoneurosomes. KIF1Bβ is detected in total lysates from the cortex (T; 5 μg total protein) and is enriched in a sample of synaptoneurosomes purified from the cortex (S; 5 μg). The level of detection of synaptophysin, a presynaptic marker, and PSD-95, a postsynaptic marker, are evidence of the enrichment of the synaptoneurosomal preparation, which was essentially devoid of non-neuronal proteins, such as myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP). b Detection of KIF1Bβ in further fractionated synaptoneurosomes. The synaptoneurosomal preparation from mouse cortex was further fractionated into its soluble, pre- and postsynaptic components and probed for KIF1Bβ in parallel with compartment-specific protein markers PSD-95 and synaptophysin. KIF1Bβ was detected in both the pre- and postsynaptic, but not in the soluble fraction. An amount of 15 μg of protein was loaded from each fraction

Fig. 6

a DHPG stimulation increases the amount of KIF1Bβ in synaptoneurosomes. Immunoblot (representative of 8 independent experiments) of protein extracts of cortical synaptoneurosomes either resting (control) or treated with DHPG and probed for KIF1Bβ, PSD-95, synaptophysin, syntaxin1, SNAP-25, and also GAPDH (loading control). b Quantification of the experiments shows a significant increase of KIF1Bβ protein levels in synaptoneurosomes after DHPG stimulation (*p < 0.05; Student’s t test). c Quantification of PSD-95, synaptophysin, syntaxin1 and SNAP-25 levels in the same experiments as in (b) shows no significant change. d, e DHPG stimulation recruits KIF1Bβ to the synapses. d Cortical neurons resting (left panels) or treated with DHPG (right panels), fixed, and stained with phalloidin, anti-synaptophysin or anti-KIF1Bβ antibodies. Merged images show an overlay of the mask that marks vicinity to a synapse (green) and the KIF1Bβ signal in the control or after DHPG, respectively (red). Arrows point to strong KIF1Bβ signals in the vicinity of synapses. Scale bar 10 μm. e Quantification of KIF1Bβ fluorescence that fell into total synaptic areas, as defined by the mask described in “Materials and Methods” (n = 20 neurons; *p < 0.05; Student’s t test). f Quantification of KIF1Bβ signal from 6 independent experiments, showing increases of KIF1Bβ concentration in synaptoneurosomes after stimulation of different types of glutamate receptor (confirmation of PKC stimulation with PMA in Online Resource Fig. S4). The most pronounced increase corresponds to stimulation of the NMDA receptor (n = 20 neurons, 6 independent experiments, **p = 0.0014; Student’s t test)

Fig. 7

A1–A3 Partial co-localization of KIF1Bβ and CaM in spinal motor neurons. Double immunofluorescence for KIF1Bβ (green) and CaM (red) reveals their co-localization (yellow) in both the cell body and neuronal processes (detail at higher magnif.). Scale bars 20 μm, inset 10 μm. b Pull-down assays with cell extracts confirming the interaction between endogenous KIF1Bβ and CaM and demonstrating its Ca²⁺-dependence. CaM, immobilized on Sepharose beads, was incubated with equivalent lysates (10 μg total protein) from NSC-34 cells in the presence of increasing Ca²⁺concentrations. Bound (B; lanes 3, 5, 7, 9) and unbound proteins (U; lanes 2, 4, 6, 8) were probed by immunoblot with anti-KIF1Bβ (top panels). Sepharose beads without CaM were used as negative control (top panels, lanes 10–13; B and U). An anti-dynein antibody (bottom panels) was used as internal control for equal loading. Detection of endogenous KIF1Bβ is obtained only in the presence of CaM (lanes 3, 5, 7, 9) and is enhanced by increased Ca²⁺ concentration, peaking at about 0.5 mM CaCl₂ (lane 5). c Pull-down assays with recombinant proteins confirming a direct interaction between KIF1Bβ and CaM and mapping the interaction site on KIF1Bβ. CaM on Sepharose beads was incubated with equivalent extracts of E. coli expressing GST-tagged deletion constructs of KIF1Bβ (M, C1–C5; shown in schematic form), in the absence (lanes 1–5) or presence of 1 mM Ca²⁺ (lanes 6–9). Bound (B) and unbound (U) proteins were probed by anti-KIF1Bβ the KIF1Bβ band is indicated by arrows, a lower band appearing in some samples is a degradation product). Sepharose-only beads served as negative control at the same conditions (lanes 4, 5, 8, 9). GST-vector-only was an additional negative control to exclude interaction of the KIF1Bβ constructs and CaM via GST sequences (vector; bottom panels). F full-length KIF1Bβ, M motor-domain-containing construct, C1–C5 non-motor-domain-containing constructs. Constructs are also described in Table 1. Numbers accompanying the construct sketches denote amino-acid residues

Fig. 8

a Immunoprecipitation (IP) for KIF1Bβ from mouse brain. Analysis by western blot, using different antibodies, of the anti-KIF1Bβ-immunoprecipitated proteins revealed that KIF1Bβ and its interacting protein Calmodulin (CaM) are part of the same complex. Furthermore, the dendritically-localized RNA-binding proteins Purα, FMRP and FXR2P, as well as the motor protein KIF3A, were also detected in the same KIF1Bβ complex. RNA-binding protein ELAVs and the motor protein KIF5A were absent. Lane 1 represents 1/20 (25 μg) of the original extract used for immunoprecipitation (input), lanes 2 and 3 show the immunoprecipitated protein using, respectively, equal amounts of anti-KIF1Bβ antibody or rabbit IgGs (negative control). b Co-localization of KIF1Bβ and dendritic RNA-binding proteins in mouse cultured cortical neurons. Staining of cortical neurons (12–14 div.) with anti-KIF1Bβ (red panels) and concurrent labeling with antibodies to Purα; FMRP; FXR2P (green panels), reveal extensive pairwise co-localization in puncta (arrows) in a detail of a dendritic process. Scale bar 5 μm. c RT-PCR analysis of the RNA content of the KIF1Bβ IP material. Calmodulin, Arc, KIF1Bβ, TrkB, β-actin, eEIF1α, β-catenin, CamKIIα mRNAs were amplified, following KIF1Bβ immunoprecipitation. Prior to RNA extraction, each sample was spiked with 100 ng of exogenous human BC200 mRNA, which was then tested by PCR to show that RNA extraction, reverse transcription, and amplification efficiency was equal across samples. Lane 1 represents the RT-PCR reaction of 1/20 of the original extract used for immunoprecipitation (input), lanes 2 and 3 represent the RNA immunoprecipitated using equal amounts, respectively, of anti-KIF1Bβ antibody and rabbit IgGs. CaM and Arc mRNAs were detected in the KIF1Bβ complex

Fig. 9

Processive movement of YFP-KIF1Bβ granules in the dendrites of living cortical neurons. a Example of a mouse cultured cortical neuron transfected with plasmid pEYFP-KIF1Bβ (full-length construct in Fig. 7c), showing fluorescent granules. The dendrite in this cell is indicated with a red arrowhead and the axon with a yellow arrowhead. Scale bar 10 μm. b Histogram of the maximum speed of 30 granules (average ± SD: 0.060 ± 0.034 μm/s) (left panel); final distances these 30 granules covered in 180 s (average ± SD: 0.9 ± 2.7 μm) (right panel). c Upper panels a series of still images, taken at 20-s intervals, from a time-lapse movie of the rectangular area marked in (a), as an example of the movement of a granule (arrowheads) for a period of 180 s. Scale bar 2 μm. The full video of this sequence is given as an Online Resource video1. (The full video of a sequence derived from a neuron transfected with pEGFP-KIF1Bβ-C1 is given as an Online Resource video2). Bottom panels time course, during 180 s, of the distances covered by this representative granule from the start point, as traced by the arrowheads (upper panels). Corresponding time course of the speeds of the sample granule during the 180 s recording (right panel). d Equivalent example of cortical neuron transfected with pEGFP-KIF1Bβ-C1 (motor-less construct “C1” in Fig. 7c), showing diffuse fluorescent labeling. The dendrite in this cell is indicated with a red arrowhead and the axon with a yellow arrowhead. e Kymographs of neurons transfected with pEYFP-KIF1Bβ or pEGFP-KIF1Bβ-C1. Rectangular areas marked in (a) and (d) indicate, respectively, the region selected to generate the kymographs. Red dashed lines indicate the orthogonal plane represented by the kymographs. Scale bar 1 μm