Activity-Dependent Regulation of Distinct Transport and Cytoskeletal Remodeling Functions of the Dendritic Kinesin KIF21B - PubMed (original) (raw)
Activity-Dependent Regulation of Distinct Transport and Cytoskeletal Remodeling Functions of the Dendritic Kinesin KIF21B
Amy E Ghiretti et al. Neuron. 2016.
Abstract
The dendritic arbor is subject to continual activity-dependent remodeling, requiring a balance between directed cargo trafficking and dynamic restructuring of the underlying microtubule tracks. How cytoskeletal components are able to dynamically regulate these processes to maintain this balance remains largely unknown. By combining single-molecule assays and live imaging in rat hippocampal neurons, we have identified the kinesin-4 KIF21B as a molecular regulator of activity-dependent trafficking and microtubule dynamicity in dendrites. We find that KIF21B contributes to the retrograde trafficking of brain-derived neurotrophic factor (BDNF)-TrkB complexes and also regulates microtubule dynamics through a separable, non-motor microtubule-binding domain. Neuronal activity enhances the motility of KIF21B at the expense of its role in cytoskeletal remodeling, the first example of a kinesin whose function is directly tuned to neuronal activity state. These studies suggest a model in which KIF21B navigates the complex cytoskeletal environment of dendrites by compartmentalizing functions in an activity-dependent manner.
Keywords: BDNF; KIF21B; TrkB; dendrite; kinesin; microtubules.
Copyright © 2016 Elsevier Inc. All rights reserved.
Conflict of interest statement
The authors declare no competing financial interests.
Figures
Figure 1. KIF21B is motile and exhibits a retrograde bias in hippocampal dendrites
A: Schematic of KIF21B, with 1-657 and 657-1624 and additional constructs indicated. B: Representative images of 10 DIV hippocampal neurons co-expressing GFP and TMR-labeled HaloTag-KIF21B, full-length (FL; left) or 1-657 (right). Scale bar = 25 μm. C: Time-lapse images of dendritic segments indicated by white boxes in B, showing FL (left) and 1-657 (right) motility (colored arrows). Scale bar = 10 μm. D: Kymographs of KIF21B motility shown in C. Line colors correspond to arrows in C. Scale bar = 10 μm, 30 sec. E: Fraction of HaloTag-KIF21B observed in both dendrites and axons or dendrites only. n = 10 neurons/condition. F: Average speed and, G: average run length of motile KIF21B puncta. ** = p < 0.01 from FL, by ANOVA with Tukey posthoc test. **H:** Frequency distribution of average velocity of net anterograde or retrograde FL KIF21B puncta. **I**: Fraction of anterograde, stationary, or retrograde KIF21B puncta. Anterograde/retrograde = >5 μm in corresponding direction during the imaging period. For F–I: n = 18–20 neurons per condition. Error bars = S.E.M. See also Figure S1.
Figure 2. Characterization of single molecule motility and microtubule binding of KIF21B in vitro
A: Time-lapse images of FL, 1-657, and 657-1624 HaloTag-KIF21B (red) binding and motility along microtubules (MT; green). Motile particles indicated by arrows, nonmotile by arrowheads. Scale bar = 5 μm. B: Photobleaching of HaloTag-KIF21B particles; sample trace from a 1-657 particle (top) and distribution of fluorophores for both FL and 1-657 (bottom). n = 30 particles/condition. C: Average number of particles per μm MT, D: average dwell time on MT, E: average velocity, F: and average run length for HaloTag-KIF21B particles. ** = p < 0.01, *** = p < 0.001 from FL, by ANOVA with Tukey posthoc test. G: Frequency distribution for run length of motile HaloTag-KIF21B particles with exponential decay fit. H: Frequency distribution for velocity of motile HaloTag-KIF21B particles with Gaussian fit. Both run length and velocity distributions significantly different between FL and 1-657 (KS test; p < 0.001). For C–H: n = 416 (FL), 125 (1-657), 76 (657-1624) particles; N = 90 MTs/condition. I: Western blot (anti-HaloTag, 1:1000) of supernatant (S) and pellet (P) fractions from MT pelleting assay; 5 μM MTs, 10 mM AMPPNP. J: Western blot (anti-HaloTag, 1:1000) of supernatant (S) and pellet (P) fractions from MT pelleting assay; 5 μM MTs, 10 mM AMPPNP, MTs in right lanes digested with 200 μg/mL subtilisin before pelleting. Error bars = S.E.M. See also Figure S2 and S3.
Figure 3. KIF21 is required for proper retrograde trafficking of TrkB+ signaling endosomes in dendrites
A: Kymographs of TrkB-RFP motility in 10 DIV Control, KIF21B RNAi, Full-Length (FL) Rescue, 1-657 Rescue, and 657-1624 Rescue hippocampal neuron dendrites, with sample events traced below. Scale bar = 10 μm, 30 sec. B: Ratio of retrograde to anterograde TrkB-RFP puncta. ** = p < 0.01 from Control, by ANOVA with Tukey posthoc test. C: Fraction of anterograde, stationary, and retrograde TrkB-RFP puncta. ** = p < 0.01 from appropriate Control, by ANOVA with Tukey posthoc test. D: Average run length of motile TrkB-RFP puncta. ** = p < 0.01, by ANOVA with Tukey posthoc test. n.s. = not significant. For A–D: n = 15–22 neurons/condition. E: Western blot of co-immunoprecipitation samples from 8 DIV hippocampal neuronal lysates (anti-TrkB,1:200; anti-KIF21B, 1:100; anti-DHC, 1:250). 10% input loaded. F: Average fraction of anterograde, stationary, or retrograde TrkB-RFP puncta colocalized with HaloTag-KIF21B. n = 10 neurons/condition. Error bars = S.E.M. See also Figure S4.
Figure 4. KIF21B regulates trafficking of functional BDNF-bound TrkB
A: Kymographs of TrkB-RFP motility in 10 DIV Control and KIF21B RNAi hippocampal neuron dendrites, with or without 100 nM BDNF, with sample events traced below. Scale bar = 10 μm, 30 sec. B: Ratio of retrograde to anterograde TrkB-RFP puncta. ** = p < 0.01, by ANOVA with Tukey posthoc test. C: Fraction of anterograde, stationary, and retrograde TrkB-RFP puncta. ** = p < 0.01 from appropriate Control, by ANOVA with Tukey posthoc test. D: Average run length of motile TrkB-RFP puncta. ** = p < 0.01 from Control, by ANOVA with Tukey posthoc test. n.s. = not significant. For A–D: n = 25–28 neurons/condition. E: Kymographs of BDNF-quantum dot (Qdot) motility in 10 DIV Control and KIF21B RNAi hippocampal neuron dendrites, with sample events traced below. Scale bar = 10 μm, 30 sec. F: Ratio of retrograde to anterograde BDNF-Qdot puncta. *** = p < 0.001 from Control, by ANOVA with Tukey posthoc test. G: Fraction of anterograde, stationary, and retrograde BDNF-Qdots. *** = p < 0.001 from appropriate Control, by ANOVA with Tukey posthoc test. H: Average run length of motile BDNF-Qdots. ** = p < 0.01 from Control, by ANOVA with Tukey posthoc test. n.s. = not significant. For E–H: n = 28–30 neurons/condition. I: Western blot (top; anti-pCREB and anti-CREB, 1:1000) and quantification (below) of lysates from 22 DIV wild-type (+/+) or KIF21B knockout (−/−) hippocampal neurons, with or without 100 ng/mL BDNF. J: Western blot (top) and quantification (below) of lysates from wild-type (+/+) or KIF21B knockout (−/−) acute hippocampal slices, with or without 100 ng/mL BDNF. * = p < 0.05, ** = p < 0.01, *** = p < 0.001 by student’s T-test. n.s. = not significant. Error bars = S.E.M. See also Figure S5.
Figure 5. KIF21B regulates microtubule dynamics through its C terminus in hippocampal neuron dendrites
A: Kymographs of EB3-GFP comets (green) and HaloTag-KIF21B (red) motility for 10 DIV Control, KIF21B RNAi, FL Rescue, 1-657 Rescue, and 657-1624 Rescue hippocampal neurons. Scale bar = 1 μm, 30 sec. B: Average lifetime, C: and average speed of anterograde (left) and retrograde (right) EB3-GFP comets. * = p < 0.05, ** = p < 0.01, *** = p < 0.001 from Control, by ANOVA with Tukey posthoc test. n = 500–754 comets/condition; N = 20–23 neurons/condition. Error bars = 95% confidence intervals.
Figure 6. C-terminal KIF21B is a positive regulator of microtubule dynamicity in vitro
A: Kymographs of microtubule dynamics in vitro. GMPCPP seeds (purple), tubulin (green), and KIF21B (red) above; cartoon representation of seeds and KIF21B below. White arrowheads = catastrophe. Scale bar = 2 μm, 3 min. B: Catastrophes per minute for Control and KIF21B (100 nM) microtubules. ** = p < 0.01 from Control, by ANOVA with Tukey posthoc test. C: Cumulative frequency plot of time to catastrophe, corresponding to B. Distributions for FL and 657-1624 significant from Control, by KS test (p < 0.01). D: Average growth rate for Control and KIF21B (100 nM) microtubules. ** = p < 0.01 from Control, by ANOVA with Tukey posthoc test. E: Cumulative frequency plot of average growth rate, corresponding to D. Distributions for FL and 657-1624 significant from Control, by KS test (p < 0.01). For A–E: n = 28–30 microtubules/condition. F: Catastrophes per minute with varying concentrations of KIF21B protein. * = p < 0.05, ** = p < 0.01 from 0 nM, # = p < 0.01 from 50 nM, by ANOVA with Tukey posthoc test. n = 12–15 microtubules/condition for 50 nM and 300 nM, 28–30 microtubules/condition for 100 nM. G: Fraction of catastrophes positive for KIF21B (100 nM). n = 28–30 microtubules. Error bars = S.E.M. H: Still frames and corresponding line scans of fluorescence intensity of microtubule dynamics 2 frames (20 seconds) prior to (“Before”), at (“Start”), during (“During”), and 10 frames (100 seconds) after (“After”) catastrophe, showing KIF21B localization (red) along microtubule (green). Scale bar = 2 μm. Yellow arrowheads = KIF21B, White arrowheads = plus-end. Line scans = normalized KIF21B fluorescence over the length of the microtubule at each time.
Figure 7. Neuronal activity enhances KIF21B motility
A: Average speed, B: and average run length of motile HaloTag-KIF21B (FL and 1-657) puncta in 10 DIV hippocampal neuron dendrites, with and without 50 mM KCl. ** = p < 0.01 from appropriate FL condition, by ANOVA with Tukey posthoc test. n = 20–23 neurons/condition. C: Time-lapse images of dendritic segments from 10 DIV hippocampal neurons expressing HaloTag-KIF21B (FL or 1-657), showing motility with and without 50 μM bicuculline/200 μM glycine (red arrows). Scale bar = 10 μm. D: Kymographs of KIF21B motility shown in C. Line colors correspond to arrows in C. Scale bar = 10 μm, 30 sec. E: Average speed, F: and average run length of motile HaloTag-KIF21B puncta. ** = p < 0.01 from appropriate FL condition, by ANOVA with Tukey posthoc test. n = 20–23 neurons/condition. Error bars = S.E.M. G: Model for KIF21B-dependent regulation of dendritic trafficking and microtubule dynamics. See also Figure S6.
Figure 8. Neuronal activity promotes the function of KIF21B in TrkB trafficking at the expense of microtubule dynamicity regulation
A: Kymographs of EB3-GFP comets for Control, KIF21B RNAi, and KIF21B-overexpressing (OE) 10 DIV hippocampal neurons, with and without 50 μM bicuculline/200 μM glycine. Scale bar = 1 μm, 30 sec. B: Average lifetime, C: and average speed of anterograde (left) and retrograde (right) EB3-GFP comets. * = p < 0.05, ** = p < 0.01, *** = p < 0.001 from Control, by ANOVA with Tukey posthoc test. n > 500 comets/condition; N = 27–32 neurons/condition. D: Kymographs of TrkB-RFP motility for conditions described in A, with sample events traced below. Scale bar = 10 μm, 30 sec. E: Ratio of retrograde to anterograde TrkB-RFP puncta. *** = p < 0.001 from Control, by ANOVA with Tukey posthoc test. F: Fraction of anterograde, stationary, and retrograde TrkB-RFP. ** = p < 0.01 from appropriate Control, by ANOVA with Tukey posthoc test. G: Average run length of motile TrkB-RFP puncta. * = p < 0.05, ** = p < 0.01, by ANOVA with Tukey posthoc test. n.s. = not significant. n = 27–32 neurons/condition. In B–C, error bars = 95% confidence interval; in E–G, error bars = S.E.M. H: KIF21B plays a dual role in dendrites (left): regulation of cargo trafficking, including the retrograde transport of BDNF/TrkB, and regulation of microtubule dynamicity. KIF21B moves along the microtubule, attached by both N-terminal (which determines motor activity) and C-terminal binding sites. At the plus-end, microtubule dynamics are mediated through the C-terminal binding site. With increased neuronal activity (right): KIF21B-dependent regulation of microtubule dynamicity is downregulated but motility is preserved. See also Figure S7.
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