CLASP2-dependent microtubule capture at the neuromuscular junction membrane requires LL5β and actin for focal delivery of acetylcholine receptor vesicles

Abstract

A hallmark of the neuromuscular junction (NMJ) is the high density of acetylcholine receptors (AChRs) in the postsynaptic muscle membrane. The postsynaptic apparatus of the NMJ is organized by agrin secreted from motor neurons. The mechanisms that underlie the focal delivery of AChRs to the adult NMJ are not yet understood in detail. We previously showed that microtubule (MT) capture by the plus end-tracking protein CLASP2 regulates AChR density at agrin-induced AChR clusters in cultured myotubes via PI3 kinase acting through GSK3β. Here we show that knockdown of the CLASP2-interaction partner LL5β by RNAi and forced expression of a CLASP2 fragment blocking the CLASP2/LL5β interaction inhibit microtubule capture. The same treatments impair focal vesicle delivery to the clusters. Consistent with these findings, knockdown of LL5β at the NMJ in vivo reduces the density and insertion of AChRs into the postsynaptic membrane. MT capture and focal vesicle delivery to agrin-induced AChR clusters are also inhibited by microtubule- and actin-depolymerizing drugs, invoking both cytoskeletal systems in MT capture and in the fusion of AChR vesicles with the cluster membrane. Combined our data identify a transport system, organized by agrin through PI3 kinase, GSK3β, CLASP2, and LL5β, for precise delivery of AChR vesicles from the subsynaptic nuclei to the overlying synaptic membrane.

FIGURE 1:

LL5β colocalizes with CLASP2 at agrin-induced AChR clusters. (A) LL5β is enriched at agrin-induced AChR clusters. Primary myotubes were cultured on focal agrin deposits on a laminin substrate, and AChR clusters were stained with α-BTX–Alexa 488 (green) and for LL5β (red). Arrowheads mark uneven distribution of LL5β. Scale bar, 5 μm. (B) Regions of elevated LL5β (red) inside AChR clusters (blue) are enriched for CLASP2 (green, marked with arrowheads), consistent with an LL5β-CLASP2 interaction and CLASP2-dependent MT capture at synaptic membranes by LL5β. At 48 h postinfection, myotubes expressing adenovirus-delivered GFP-CLASP2 were stained for AChRs (blue), endogenous LL5β (red), and GFP with antibody. Bottom, magnification of boxed area at top. Scale bar, 10 μm (top), 5 μm (bottom). (C) Quantification of LL5β and CLASP2 inside AChR clusters. Bar graph shows that CLASP2 load/area increases with LL5β levels within the myotube region indicated on x-axis. Means ± SE; **p < 0.01; *p < 0.05; n = 6 myotubes with >10 comets/cell.

FIGURE 2:

Knockdown of LL5β expression and overexpression of CLASP2-C abolish capture of MT plus ends at agrin-induced AChR clusters. (A) Western blot of LL5β isolated from cultured primary myotubes expressing shLL5β and shScrambled (ctrl). Myotubes were infected with lentiviral constructs encoding shLL5β (hairpin no. 2, see Materials and Methods) or shScrambled for control. Two days later, LL5β expression levels were analyzed. (B) MT plus end densities visualized by staining for EB3 are reduced inside AChR clusters upon LL5β knockdown with shLL5β. Two days after infection with shLL5β or with shScrambled (control), respectively, myotubes were stained for AChRs with α-BTX–Alexa 488 (green) and EB3 (red), and densities of EB3-decorated comets inside vs. outside AChR clusters were compared. Boxes 1 (inside cluster) and 2 (outside cluster) are enlarged on the right. Scale bars, 10 μm; enlarged, 2.5 μm. Similar data were obtained with hairpin no. 4 (see Supplemental Methods). (C) Forced expression of GFP-CLASP2-C (green) abolishes increase in EB3 (red) comet density at agrin-induced AChR clusters compared with noncluster region, indicating impairment of MT capture. Boxes are enlarged at the bottom. Scale bar, 10 μm; enlarged, 2.5 μm. (D) Density of EB3 comets inside relative to outside AChR clusters in scrambled control vs. LL5β-knocked-down myotubes and in GFP control vs. GFP-CLASP2-C–transfected myotubes. Means ± SE. N = 12 ctrl, 12 shLL5β, 6 GFP-CLASP2-C, and 7 GFP alone. *p < 0.05. (E) Clustering of prelabeled AChRs by agrin is not affected by LL5β knockdown, indicating that LL5β does not act by trapping AChRs. Representative images of primary myotubes infected with control and shLL5β lentivirus and stained with α-BTX–Alexa 488, followed by addition of 10 nM mini-agrin for 18 h. Arrowheads point to examples of AChR clusters. Scale bar, 50 μm. (F) Quantification of cells from E. Number of clusters per myotube was quantified (left); N = 167 clusters for control and 138 for shLL5β knockdown cells. Quantification of relative AChR cluster density (middle) and area (right); N = 65 clusters in control and 74 clusters in shLL5β-knockdown cells. All bars depict means ± SE.

FIGURE 3:

Knockdown of LL5β expression levels in muscle fibers reduces the stability and insertion rate of AChRs at the neuromuscular junction in vivo. (A–C) Stability and insertion rate of synaptic AChRs are reduced at NMJs upon knockdown of LL5β. Histone2B-RFP–expressing pLKO.1-shLL5β or pLKO.1-shScrambled was electroporated into sternomastoid muscles. (A) Schematic depiction of the experimental protocol. (B) Examples of synapses imaged (in pseudocolor) and nuclei marked by RFP in their subsynaptic nuclei used for identification of successfully electroporated fibers. Scale bar, 10 μm. (C) Bar graphs summarizing the effect of LL5β knockdown on AChR stability and insertion. Numbers in columns give numbers of synapses analyzed. Bars indicate mean fluorescence intensities (± SE) relative to that at t = 0. ***p <0.0001; **p < 0.005; two sided t test. (D–F) The stability and insertion rate of both recycled and newly inserted AChRs at NMJs are reduced upon knockdown of LL5β. Electroporation of constructs was as in A). (D) Schematic depiction of the experimental protocol. (E) Examples of synapses in pseudocolor. Scale bar, 10 μm. (F) Graphs summarizing the effect of LL5β knockdown on stability and insertion of recycled and newly inserted AChRs. All other indications are as in B and C. Numbers on columns give numbers of synapses analyzed. Bars depict means ± SE. ***p <0.0001; two sided t test.

FIGURE 4:

LL5β and actin are required for MT-dependent AChR delivery to agrin-induced AChR clusters in cultured myotubes. (A–D) Primary myotubes grown on laminin substrates with local deposits of neural agrin were infected with adenovirus expressing the AChR–γ-GFP subunit, and GFP-AChR clusters were visualized by TIRF microscopy. Photobleaching was begun 90 min after addition of drugs and carried out in their presence. (A) Examples of GFP-AChR clusters in control and nocodazole- (10 μM, >90 min) and CytoD-treated myotubes (2 μM, >90 min) before, immediately at, and 20 min after bleaching. Images were acquired every minute for 45 min after bleaching, when monitor bleaching began to interfere. Scale bars, 10 μm. (B) Fluorescence recovery is reduced in myotubes upon LL5β knockdown upon treatment with nocodazole or with CytoD. Note that suppression of fluorescence recovery is almost identical for all treatments. Values of mean fluorescence ± SE as a function of time. Clusters containing bleached regions were imaged every minute for a total of 45 min; background fluorescence outside cells was subtracted; the fluorescence values for each time point were processed to correct for monitor bleaching, and residual fluorescence at t = 0 was normalized to zero as described in Materials and Methods. Means ± SE from eight untreated, six nocodazole-treated, six CytoD treated, and seven shLL5β-treated cells. (C) Scheme used for testing whether fluorescence recovery was due to diffusion of GFP-AChRs from unbleached to the bleached regions. To this end, total fluorescence intensity per area in an annulus (I_r) at the periphery of the bleached region was compared, during recovery, with that in a circle (I_c) at its center. Their radii were as follows: entire FRAP region, 6 μm; inner circle, 2.3 μm; inner edge of annulus, 4.0 μm (resulting in a distance between the outer edge of the circle and the inner edge of the annulus of 1.7 μm); and outer edge of annulus, 4.7 μm (resulting in width of the annulus of 0.70 μm). (D) Graph of the ratio I_c/I_r as a function of time. A value of 1, reached within the first 10 min after bleaching, indicates no gradient from the periphery to the center and, thus, negligible diffusion, consistent with FRAP due to insertion of new GFP-AChRs. Means ± SE from N = 5 control cells. (E) CytoD treatment used for experiments in A and B is effective in breaking down actin cables, as revealed by phalloidin staining. Primary myotubes cultured on agrin substrate were stained with α-BTX–Alexa 488 (green) and phalloidin–Alexa 594 (red) 90 min after CytoD treatment. Scale bar, 10 μm.

FIGURE 5:

The actin cytoskeleton is required for MT capture at AChR clusters. (A–C) CytoD inhibits MT plus end capture at agrin-induced AChR cluster in live myotubes. Myotubes derived from GFP–CLIP170-KI mutant mice were cultured on agrin patches, and dynamics of GFP-labeled MT plus ends was recorded in TIRFM at 1 frame/s for 180 s with and without CytoD treatment. (A) First frames (left) and projection of all 180 frames superimposed in control (top) and CytoD-treated myotubes (bottom). The outlines of AChR clusters are shown by solid black lines. Note higher number of stable comets (dots in projection) in clusters in control than in CytoD-treated myotubes. Scale bar, 5 μm. (B) Examples of kymographs generated from stable and moving MT plus ends; kymographs were generated from time-lapse recordings as illustrated in A. (C) Graph summarizing the percentages of stable MT plus ends inside and outside AChR clusters. Note the lowered percentage of stable MT plus ends inside AChR clusters in CytoD-treated myotubes, whereas percentages outside clusters remained unchanged. Owing to differing MT plus end abundance between myotubes, percentage values of stable comets inside relative to outside AChR clusters were calculated for each myotube, and percentages were averaged. Bars give means ± SE (N = 4 cells each for control and CytoD-treated myotubes). (D, E) CytoD inhibits MT plus end capture at agrin-induced AChR clusters, as visualized in fixed myotubes by density of EB3-stained MT plus ends. (D) Myotubes were cultured on agrin patches, fixed after 90 min of CytoD treatment, and stained with α-BTX–Alexa 488 (AChRs, green) and an antibody to EB3 (red). Right, boxes 1 (inside) and 2 (outside clusters) enlarged to visualize EB3-positive plus end densities. Scale bar, 7.5 μm; boxes, 2.5 μm. (E) Bars summarizing densities of EB3-stained MT plus ends inside relative to outside clusters. Means ± SE; N = 18 cells for control, 19 cells for CytoD-treated myotubes; *p < 0.05.

FIGURE 6:

Analysis of AChR vesicle delivery to agrin-induced AChR clusters in myotubes expressing GFP–AChR-γ subunit. (A–F) GFP-AChR clusters were bleached and fluorescence recovery observed at 3 frames/s for 30 s in TIRFM. (A) AChR cluster before, immediately after, and 30 s after bleaching. Scale bar. 10 μm. (B) Example of a single frame containing a vesicle fusion event (red box) and its intensity profile during the fusion process as calculated using the SOS plug-in (see Materials and Methods). Scale bar, 2.5 μm. (C) Enlarged red square, showing the complete sequence of frames for the same vesicle as in B during the entire fusion process. (D) Double-fusion event occurring at the same site as in B at a later time point during the acquisition and its corresponding intensity profile. (E) Snapshot of a schematic three-dimensional projection showing the time lapse of the cell in A to illustrate the appearance of double-fusion events at the same position. The bleached region was cropped, individual events were marked as green circles, and double events corresponding to events 8 and 15 were marked as red circles using the MTrackJ plug-in. The time lapse was then projected in the Vaa3D software. The z-axis corresponds to the time scale. (F) GFP fluorescence intensities of all 15 fusion events in the cell shown in A. Bars denote mean ± SE of intensities sampled over the entire duration of the fusion event at 3 frames/s (see the example shown in B). The open triangles denote the number of frames used in the calculation of each such event intensity and SE. Note the uniform intensities of events. The fusions 8 and 13 each can be interpreted as events arising from two consecutive fusions at the same site partly overlapping in time, thus accounting for the higher SE.

FIGURE 7:

LL5β knockdown and CytoD treatment differentially affect frequency, duration, and GFP fluorescence intensity of AChR vesicle fusions. All methods and parameters as defined in Figure 6. Bars give means ± SE. (A–C) LL5β-knockdown myotubes. Note the reduction of fusion numbers during 30-s observation period upon LL5β knockdown without changes in duration and intensity of fusion events. The relative number of double fusions at the same sites was not affected (see text). N = 12 cells for control and 14 for cells for LL5β-knockdown myotubes. Means ± SE. *p < 0.05. (D–F) CytoD-treated myotubes. Note stronger reduction of fusion numbers during 30-s observation period upon CytoD treatment, with significant reduction in duration but not intensity of fusion events. Means ± SE. N = 14 cells for control and 14 cells for CytoD-treated myotubes. **p < 0.001, ***p < 0.0001.