1. Implication of Geranylgeranyltransferase I in Synapse Formation
Zhen G Luo, Hyun-Soo Je, Qiang Wang, Feng Yang, G.Clem Dobbins, Zhi-Hua Yang, Wen C Xiong, Bai Lu, Lin Mei 
Neuron 
Volume 40, Issue 4, Pages (November 2003)
DOI: /S (03)

2. Figure 1 MuSK Interaction with αG/F
(A) MuSK interaction with αG/F but not βG or βF. Y190 yeast cells were cotransformed with pGBT9-MuSKic and αG/F, βG, or βF in pACT2 or pGAD424 or with pGBT9-ErbB4ic and pACT2-αG/F. Transformed yeast cells were seeded in Leu−Trp−His− plates and scored for β-Gal activity: (−) no blue after 8 hr, (+) blue after 2 hr, (++) blue within 1 hr.
(B) αG/F interaction with MuSK deletion mutants. The indicated constructs were cotransformed with pACT2-αG/F or PICK1. Interaction was determined as in (A).
(C) Direct interaction between MuSK and αG/F. [35S]-labeled MuSK and Myc-αG/F were incubated in the binding buffer. The αG/F-bound MuSK was revealed by autoradiogram.
(D) Interaction between endogenous MuSK and αG/F in muscle cells. C2C12 myotube lysates (500 μg of protein) were incubated with rabbit anti-MuSK antibody, anti-ErbB4 antibody, normal rabbit serum (NRS), or no serum (No Ab) and subsequently with protein A-agarose beads. Resulting immunocomplexes were subjected to immunoblotting with mouse anti-αG/F antibody. Input, 5% of lysates.
(E) Quantification of the interaction between MuSK and αG/F in muscle cells. Immunoprecipitation was performed as in (D) with indicated antibodies. The amount of immunoprecipitated proteins was quantified by the respective standard curves (% of lysate inputs). NS, normal serum as control.
(F) No effect of Agrin on MuSK-αG/F interaction in C2C12 muscle cells. C2C12 myotubes were stimulated with Agrin for the indicated times. Immunoprecipitation was performed as in (D). Tyrosine phosphorylation of MuSK was also examined by immunoprecipitation with anti-phosphotyrosine (p-tyr) antibody followed by blotting with anti-MuSK antibody.
(G) Colocalization of αG/F with the AChR in skeletal muscles. Adult rat diaphragm sections were incubated with an anti-αG/F antibody (top panel) or the antibody that had been preincubated with the blocking peptide (bottom panel). Immunoreactivity was visualized by a FITC-conjugated secondary antibody. R-BTX was added to label the AChR.
Neuron  , DOI: ( /S (03) )

3. Figure 2
Agrin Regulation of αG/F Tyrosine Phosphorylation and Activity
(A) Agrin-induced tyrosine phosphorylation of αG/F in muscle cells. Lysates of Agrin-stimulated C2C12 myotubes were incubated with rabbit anti-αG/F or anti-MuSK antibodies, and the resulting immunocomplexes were subjected to blotting with anti-phosphotyrosine antibody. Lysates (1/20 of input for IP) were blotted to reveal equal protein levels of MuSK and αG/F.
(B) Requirement of MuSK for Agrin-induced tyrosine phosphorylation of αG/F. MuSK+/+ and MuSK−/− myotubes were stimulated with Agrin. αG/F tyrosine phosphorylation was analyzed as in (A).
(C) Enhanced GGT activity in Agrin-stimulated muscle cells. C2C12 myotubes, pretreated with GGTI286 or vehicle, were stimulated with Agrin or insulin and assayed for GGT activity. Data shown were means ± SEM of three independent experiments, each of which was performed in triplicate.
(D) Dependence of Agrin-increased GGT activity on MuSK. MuSK+/+ and MuSK−/− myotubes were stimulated with Agrin and assayed for GGT activity as in (C).
(E) Effect of Agrin on FT activity. C2C12 myotubes, pretreated with FTI277 (5 μM) or vehicle, were treated with Agrin or insulin for 15 min. FT activity was assayed as described in Experimental Procedures.
(F) Inhibition of Agrin-induced AChR clustering by GGTI286. C2C12 myotubes were treated with or without GGTI286 or FTI277 or vehicle (DMSO) prior to Agrin stimulation. Myotubes were fixed and stained with R-BTX. n = 15, **p < 0.01, ANOVA followed by post hoc tests.
(G) Effects of GGTI286 (5 μM) on surface AChR expression. Surface AChR in C2C12 myotubes were labeled with sulfo-NHS-LC-biotin, purified by streptavidin beads, and revealed by immunoblotting with anti-β subunit antibody.
(H) Inhibition of βG expression by βG-siRNA. C2C12 myoblasts were cotransfected with pBS/U6/βG or pBS/U6/βF (as control) and HA-tagged βG, Flag-MuSK, HA-Rapsyn, HA-JNK, or pEGFP. Thirty-six hours after transfection, cells were lysed for immunoblotting with indicated antibodies. NS, nonspecific protein.
(I) Agrin-induced AChR clusters were decreased in C2C12 myotubes expressing pBS/U6/βG. C2C12 myoblasts were cotransfected with pEGFP and pBS/U6/βG or pBS/U6 empty vector (as control) (1:20 plasmid DNA). Transfected myotubes were scored for AChR clusters labeled by R-BTX. Quantitative analyses of AChR clusters are shown in the histogram. pBS/U6/βG, n = 19; pBS/U6, n = 17, **p < 0.01, t test.
Neuron  , DOI: ( /S (03) )

4. Figure 3 Regulation of Rho GTPases by GGT in Response to Agrin
(A) Dependence of Agrin-induced Rac1 activation on MuSK. MuSK−/− and MuSK+/+ muscle cells were treated with or without Agrin for 15 min. Cell lysates were subjected to pulldown by GST-PBD immobilized on beads. Bound Rac1 was revealed by immunoblotting (IB) with anti-Rac1 antibody. Lysates (1/20 of input) were also probed with Rac1 antibody to indicate equal amount of Rac1. GST-PBD was revealed by immunoblotting with anti-GST antibody.
(B) Restoration of Rac1 activation in MuSK−/− muscle cells. Mutant myoblasts were cotransfected with Flag-MuSK and Myc-Rac1, and resulting myotubes were treated with Agrin for 15 min. Transfected MuSK and Rac1 were revealed by blotting with anti-Flag and anti-Myc antibodies, respectively. Active Rac1 was purified as in (A) followed by immunoblotting with anti-Myc antibody.
(C) Time-dependent Rac1 activation by Agrin.
(D) Time courses of Agrin activation of MuSK phosphorylation, GGT phosphorylation, GGT activity, and Rac1 activity. Data shown were means ± SEM of three or more independent experiments. SEM values were omitted for the clarity of the panel, which were smaller than 20% of corresponding data points.
(E) Inhibition of Rac1 activation by GGTI286. C2C12 myotubes were treated with GGTI286 (5 μM) or vehicle prior to stimulation with Agrin for 15 min. Active Rac1 was assayed as in (A). Quantitative analysis of GGTI286 inhibition is shown in the histogram. n = 3; *p < 0.05, t test.
(F) Inhibition of PAK activation by GGTI286 but not FTI277. C2C12 myotubes were treated with GGTI286 (5 μM), FTI277 (5 μM), or vehicle (DMSO, as control) prior to stimulation with Agrin. PAK1 was assayed as previously described (Luo et al., 2002). Radiolabeled MBP was revealed by autoradiogram. MBP and PAK were revealed by Coommasie staining and immunoblotting with anti-PAK antibody, respectively. Quantitative analysis is shown in the histogram. n = 3; *p < 0.05, t test.
(G) Blockade of Agrin-induced Rac1 activation by brief treatment with GGTI286. C2C12 myotubes were pretreated by GGTI286 (5 μM) for the indicated times and then stimulated with Agrin for 15 min. Active Rac1 was determined as in (A).
(H) Inhibition of Agrin-induced Rac1 activation by βG-siRNA. C2C12 myoblasts were transfected with Myc-Rac1 with pBS/U6/βG or pBS/U6. Myotubes were stimulated with Agrin for 15 min. Active Rac1 was purified as in (A) and revealed by immunoblotting by anti-Myc antibody. Transfected Rac1 and GST-PBD were revealed by immunoblotting with anti-Myc antibody and anti-GST antibody (bottom panel), respectively. Quantitative analysis is shown in the histogram. n = 3; **p < 0.01 in comparison with control.
(I) Rac1 membrane dissociation by βG-siRNA. Myoblasts were cotransfected with Myc-Rac1 and pBS/U6 or pBS/U6/βG. After Agrin stimulation, myotubes were homogenized and fractioned as described in Experimental Procedures. Homogenates (Total), S100, and P100 fractions were subjected to immunoblotting with the indicated antibodies.
Neuron  , DOI: ( /S (03) )

5. Figure 4 Specific Inhibition of Agrin-Induced AChR Clustering by K164A
(A) Insulin-induced Rac1 activation in muscle cells. C2C12 myotubes were stimulated with insulin (100 nM) for the indicated times. Rac1 activation was assayed as in Figure 3A.
(B) Inhibition of insulin-induced Rac1 activation by αG/F mutants. C2C12 myoblasts were transfected with Myc-Rac1 with or without Myc-K164A, Y200F, or αG/F. Myotubes were stimulated with insulin for 10 min. Active Rac1 was assayed as in Figure 3A.
(C) Differential interaction of αG/F mutants with MuSK. Y190 yeast cells were cotransformed with pGBT9-MuSKic and αG/F, K164A, or Y200F mutants in pACT2. Liquid β-Gal assay was performed to quantitatively analyze the interaction. The β-Gal activity of αG/F-MuSK transformants was set as 100%.
(D) Specific inhibition of Agrin-induced AChR clusters by K164A. C2C12 myoblasts (30 mm dish) were transfected with different amounts (0.2, 0.4, or 0.8 μg plasmid DNA) of αG/F, Myc-K164A, or Y200F. Myc-positive myotubes were scored for Agrin-induced AChR clusters. Images on the left were from myotubes transfected with 0.2 μg DNA. Histograms show quantitative analyses from three independent experiments. Control, nontransfected.
(E) Agrin-induced Rac1 activation was impaired in muscle cells expressing K164A. C2C12 myoblasts were transfected with Myc-Rac1 without or with K164A or Myc-αG/F. Myotubes were stimulated with Agrin for 15 min. Active Rac1 was isolated by GST-PBD as in Figure 3A and revealed by immunoblotting by anti-Myc antibody. Transfected αG/F and Rac1, which migrated at different molecular weights, were revealed by immunoblotting anti-Myc antibody. Right: densitometric analyses of active Rac1. n = 3; **p < 0.01, ANOVA followed by t test.
Neuron  , DOI: ( /S (03) )

6. Figure 5
Inhibition of GGT Attenuates AChR Clustering and Synaptic Transmission in Xenopus Spinal Neuron-Muscle Cocultures
(A) Nomarski and fluorescence images showing a triplet: a motoneuron (N) innervating two myocytes, one of which expresses K164A (M+), whereas the other does not (M−).
(B) R-BTX-labeled AChR clusters of the same field as in (A).
(C) Effects of αG/F (wt) or K164A on AChR clusters at the NMJ triplets. *p < 0.05, paired t test.
(D) Representative SSCs (downward deflections of varying amplitudes) of a triplet. M−, control; M+, K164A.
(E) Effects of postsynaptic expression of αG/F (M+, wt) or K164A (M+) on SSC properties. The numbers of synapses recorded were n = 12 (control) and n = 7 (wt); control (n = 11) and K164A (n = 9). *p < 0.001, t test.
Neuron  , DOI: ( /S (03) )

7. Figure 6 Generation of K164A Transgenic Mice
(A) Schematic structures of the αG/F gene and the transgene. The αG/F gene has nine exons, and K164 locates in the fifth exon. Myc-K164A was inserted into the NotI site of pBSX-HSAvpA. The upstream primer covers the initial ATG, and the downstream primer covers K164A. The size of PCR products is ∼8.9 kb for the wild-type and 516 bp for the mutant.
(B) Identification of a transgenic founder mouse. From nine pups, #2 and #9 contain the transgene. The AChR ϵ subunit genomic DNA was used as control.
(C) Specific expression of K164A in the skeletal muscle of transgenic mice. Tissues were isolated from K164A DNA+ transgenic mice (P30), homogenized, and analyzed for K164A protein expression using anti-Myc antibody.
(D) Time-dependent expression of K164A in transgenic mice. Skeletal muscles were isolated from transgenic or wild-type littermate mice at the indicated times. Expression of K164A was analyzed as in (C). Top two panels, immunoblots with antibodies against Myc or actin. Bottom panel, detection of the transgene by PCR.
(E) Normal expression of MuSK, AChRα, αG/F, Rac1, or β-actin in skeletal muscles of transgenic mice. Muscle homogenates of wild-type littermates and transgenic mice were immunoblotted with antibodies against indicated proteins.
(F) Agrin-induced Rac1 activation was impaired in muscle cells isolated from K164A mutant mice. Myotubes of wild-type or K164A mutant mice were stimulated with Agrin for 15 min. Active Rac1 was isolated by GST-PBD as in Figure 3A and revealed by immunoblotting by anti-Rac1 antibody. Endogenous Rac1 and Myc-K164A were revealed by immunoblotting with antibodies against Rac1 and Myc, respectively.
Neuron  , DOI: ( /S (03) )

8. Figure 7 Postsynaptic Defects at the NMJ in K164A Transgenic Mice
(A) Aneural AChR clusters in the wild-type and K164A mutant mice. Diaphragm muscles from E14.5 embryos were whole-mount immunostained with anti-synaptophysin and neurofilament and with R-BTX. Arrows indicated aneural AChR clusters. Control, wild-type littermates.
(B) Quantitative analyses of endplate band width and the length and area of AChR clusters in (A).
(C) Endplate regions in diaphragm muscles from P0 wild-type littermates (Control) and K164A transgenic mice. Diaphragms were whole-mount stained with R-BTX.
(D) Width of endplate bands in diaphragms in P0 control and transgenic mice. The width was quantitated by measuring the myotube length contained in the polygon that connects the most peripheral AChR clusters (Misgeld et al., 2002). Data shown are mean ± SEM; K164A, n = 15; control, n = 12. **p < 0.01, Student's t test.
(E) Comparison of the size and area of AChR clusters in diaphragms between control and K164A mutant mice (P0). Data shown are mean ± SEM; K164A, n = 96; control, n = 62. *p < 0.05; **p < 0.01, Student's t test.
(F) AChR plaques in sternomastoid muscles from P30 wild-type littermates.
(G) AChR plaques in sternomastoid muscles from P30 K164A mutant mice were smaller and less complex.
(H) Comparison of AChR plaques in sternomastoid muscles between control and K164A mutant mice (P30). Data shown are mean ± SEM. Control, n = 30; K164A, n = 35; **p < 0.01; *p < 0.05; Student's t test.
(I) In K164A mutants, some axons (10%) terminate on unspecialized areas of muscle. No
AChR clusters form.
Neuron  , DOI: ( /S (03) )