Cohen MW , Jacobson C , Yurchenco PD , Morris GE , Carbonetto S .

DK 36425 NIDDK NIH HHS , R01 DK036425 NIDDK NIH HHS , R37 DK036425 NIDDK NIH HHS

	Figure 1 Western blot analysis of rabbit and Xenopus βDG. Nitrocellulose transfers of heavy microsomes from rabbit (R) and Xenopus (frog, F) muscle, which had been separated by SDSPAGE, were stained with mAb 43DAG1/8D5 raised to 15 of the last 16 amino acids at the extreme COOH terminus of the human dystroglycan sequence. It recognizes a band of roughly 43 kD in rabbit corresponding to the size of βDG (Ibraghimov-Beskrovnaya et al., 1992). A similar though somewhat larger band of 44–46 kD is visible in Xenopus. Molecular mass markers (in kD) are shown to the right.
	Figure 2 Western blot analysis of human and Xenopus utrophin (A) and dystrophin (B). The Xenopus (frog, F) tissues analyzed were skeletal muscle (m), liver (lr), and heart (h), and the human (H) tissues were skeletal muscle and lung (lg). Positions of molecular mass markers at 180, 116, and 84 kD are shown, while utrophin and dystrophin migrate at ∼400 kD. Lower molecular mass bands at ∼50 and 120 kD are nonspecific and due to cross reactions of the second antibody system.
	Figure 3 Distribution of DG and LBS on the dorsal surface of muscle cells in control cultures. (A) αDG immunofluorescence, revealing microclusters and a few macroclusters. (B1) βDG immunofluorescence. Part of a megacluster is seen. Microclusters are also apparent but are relatively faint. (B2) LBS immunofluorescence in same field as B1. Microclusters, as well as the megacluster, are well resolved. (C) Laminin immunofluorescence in the absence of pretreatment with laminin, revealing a megacluster but very few microclusters. (D1 and D2) αDG and LBS immunofluorescence, obtained by exposing the culture to 6 nM laminin for 10 min at low temperature, followed by staining for αDG and laminin. The brief pretreatment with laminin inhibited the staining for αDG (compare D1 with A). Bar, 5 μm.
	Figure 4 Sizes of individual αDG and LBS clusters. (A) αDG clusters (n = 4,989) in control cultures not exposed to laminin. (B) LBS clusters (n = 7,930) in control cultures exposed briefly (10–30 min) to laminin (0.6–6 nM) at low temperature immediately before staining. (C) LBS clusters (n = 3,621) after treatment with 6 mM laminin for 1 d at room temperature. In control cultures (A and B) there were very few macroclusters (>1 μm2), and most of the microclusters were <0.1 μm2. After treatment with laminin for 1 d (C) the incidence of macroclusters was increased whereas the incidence of the smallest microclusters was decreased.
	Figure 5 Laminin-induced clusters of βDG and LBS. (A1 and A2) βDG and LBS immunofluorescence after treatment with 2.4 nM laminin for 3 d, revealing large numbers of macroclusters and a greatly reduced density of microclusters (compare with Fig. 3 B). (B1 and B2) βDG immunofluorescence in the absence of permeabilization and LBS immunofluorescence after the same laminin treatment as in A. Only some slight “bleedthrough” of the bright LBS immunofluorescence is seen in B1, thereby confirming that the immunofluorescence in A1 involved the binding of the antiβDG mAb to an intracellular epitope. (C) βDG immunofluorescence on the ventral surface of a cell after the same laminin treatment as in A. (D) βDG immunofluorescence on the ventral surface of a cell in a culture which was not treated with laminin. Comparison of C and D reveals that the laminin treatment induced an extensive accumulation of βDG at the cell periphery but did not induce clustering over the rest of the ventral surface which is inaccessible to laminin. The laminin-induced clustering at the cell periphery is out of focus in A and B2. Bar, 5 μm.
	Figure 6 Effect of laminin treatment on (A) the percentage of surface area occupied by LBS macroclusters and (B) the density of LBS microclusters. Laminin concentrations (in nM) and exposure times are indicated at the bottom of B. The cultures were treated with laminin either at low temperature (open columns) or at room temperature (shaded columns). In some cases the laminin treatment was carried out in the presence of RG50864 (striped columns). On average the means and standard errors are based on 26 cells (range, 9–79).
	Figure 7 Relationship between the density of microclusters and the percentage of surface area occupied by macroclusters. From the data of Fig. 6.
	Figure 8 E3 lacks clustering activity and inhibits laminin-induced clustering. (A) E3 binding sites after treatment with 3.3 μM E3 for 2 d. Note the relatively high density of microclusters and the paucity of macroclusters. (B) LBS after treatment with 0.6 nM laminin for 1 d. (C) Laminin and E3 binding sites after treatment with 0.6 nM laminin and 3.3 μM E3 for 1 d. E3 inhibited the laminininduced clustering. Bar, 5 μm.
	Figure 9 Agrin-induced clusters on the dorsal surface. (A1 and A2) βDG and LBS immunofluorescence, revealing several patterned clusters, excellent colocalization, and some decline in the density of microclusters in surrounding regions. (B1 and B2) AChR stain and utrophin immunofluorescence, revealing excellent colocalization. (C1 and C2) AChR stain and PY immunofluorescence, revealing excellent colocalization. The agrin treatment was 3 d in A and B and 2 d in C. Similar results were obtained when the agrin treatment was 1 d. Bar, 5 μm.
	Figure 10 Laminin-induced clusters contain dystrophin but lack AChRs, utrophin, and PY. (A1 and A2) LBS and dystrophin immunofluorescence, revealing excellent colocalization. (B1 and B2) Dystrophin immunofluorescence and AChR stain, revealing AChRs at a megacluster but not at laminin-induced macroclusters. (C1 and C2) LBS and utrophin immunofluorescence. Utrophin was not detected at laminin-induced clusters (but was always observed wherever AChRs were clustered). (D1 and D2) LBS and PY immunofluorescence, revealing PY at a megacluster but not at the laminin-induced macroclusters. The laminin treatment was 2.4 nM for 3 d in A and C, 6 nM for 2 d in B, and 6 nM for 1 d in D. Some slight bleedthrough of the bright LBS immunofluorescence is seen in C2 and D2. Bar, 5 μm.
	Figure 11 Effect of agrin and laminin on megaclusters. Cultures were stained for AChRs and photographed at low magnification. (A) In control cultures each muscle cell typically has one or two megaclusters. (B) After treatment with agrin for 1 d, the muscle cells have many agrin-induced macroclusters but often lack megaclusters. (C) After treatment with 6 nM laminin for 1 d, each muscle cell still has one or two megaclusters. Variable numbers of autofluorescent yolk granules are apparent in the central regions of the cells. The faint outlining of some of the cells in C is due to bleedthrough of the very bright LBS immunofluorescence (not shown) at cell peripheries. Bar, 25 μm.
	Figure 12 Schematic representation of some of the surface proteins clustered by laminin and agrin, both of which bind to αDG. Before clustering, DG complexes of αDG and βDG are considered to be mobile, like AChRs. Laminin-induced clusters of DG and dystrophin are considered to be linked by laminin–laminin binding. They do not contain utrophin, AChRs, or tyrosine phosphorylated molecules. Not shown is that the end of the vertical short arm of laminin also participates in self binding; that laminin may bind to other sarcolemma-associated molecules; that dystrophin binds to cytoskeletal actin; and that sarcoglycans and syntrophins may also be present at laminin-induced clusters. Agrin-induced clusters contain DG, sarcoglycans (unlabeled), utrophin, β2 syntrophin (syn), tyrosine phosphorylated AChRs, rapsyn (rn), and tyrosine phosphorylated muscle specific kinase (MuSK). Their formation unlike the formation of laminin-induced clusters, involves stimulation of tyrosine kinase activity.
	Figure 3. Distribution of DG and LBS on the dorsal surface of muscle cells in control cultures. (A) αDG immunofluorescence, revealing microclusters and a few macroclusters. (B1) βDG immunofluorescence. Part of a megacluster is seen. Microclusters are also apparent but are relatively faint. (B2) LBS immunofluorescence in same field as B1. Microclusters, as well as the megacluster, are well resolved. (C) Laminin immunofluorescence in the absence of pretreatment with laminin, revealing a megacluster but very few microclusters. (D1 and D2) αDG and LBS immunofluorescence, obtained by exposing the culture to 6 nM laminin for 10 min at low temperature, followed by staining for αDG and laminin. The brief pretreatment with laminin inhibited the staining for αDG (compare D1 with A). Bar, 5 μm.
	Figure 1. Western blot analysis of rabbit and Xenopus βDG. Nitrocellulose transfers of heavy microsomes from rabbit (R) and Xenopus (frog, F) muscle, which had been separated by SDSPAGE, were stained with mAb 43DAG1/8D5 raised to 15 of the last 16 amino acids at the extreme COOH terminus of the human dystroglycan sequence. It recognizes a band of roughly 43 kD in rabbit corresponding to the size of βDG (Ibraghimov-Beskrovnaya et al., 1992). A similar though somewhat larger band of 44–46 kD is visible in Xenopus. Molecular mass markers (in kD) are shown to the right.
	Figure 2. Western blot analysis of human and Xenopus utrophin (A) and dystrophin (B). The Xenopus (frog, F) tissues analyzed were skeletal muscle (m), liver (lr), and heart (h), and the human (H) tissues were skeletal muscle and lung (lg). Positions of molecular mass markers at 180, 116, and 84 kD are shown, while utrophin and dystrophin migrate at ∼400 kD. Lower molecular mass bands at ∼50 and 120 kD are nonspecific and due to cross reactions of the second antibody system.
	Figure 10. Laminin-induced clusters contain dystrophin but lack AChRs, utrophin, and PY. (A1 and A2) LBS and dystrophin immunofluorescence, revealing excellent colocalization. (B1 and B2) Dystrophin immunofluorescence and AChR stain, revealing AChRs at a megacluster but not at laminin-induced macroclusters. (C1 and C2) LBS and utrophin immunofluorescence. Utrophin was not detected at laminin-induced clusters (but was always observed wherever AChRs were clustered). (D1 and D2) LBS and PY immunofluorescence, revealing PY at a megacluster but not at the laminin-induced macroclusters. The laminin treatment was 2.4 nM for 3 d in A and C, 6 nM for 2 d in B, and 6 nM for 1 d in D. Some slight bleedthrough of the bright LBS immunofluorescence is seen in C2 and D2. Bar, 5 μm.
	Figure 11. Effect of agrin and laminin on megaclusters. Cultures were stained for AChRs and photographed at low magnification. (A) In control cultures each muscle cell typically has one or two megaclusters. (B) After treatment with agrin for 1 d, the muscle cells have many agrin-induced macroclusters but often lack megaclusters. (C) After treatment with 6 nM laminin for 1 d, each muscle cell still has one or two megaclusters. Variable numbers of autofluorescent yolk granules are apparent in the central regions of the cells. The faint outlining of some of the cells in C is due to bleedthrough of the very bright LBS immunofluorescence (not shown) at cell peripheries. Bar, 25 μm.
	Figure 4. Sizes of individual αDG and LBS clusters. (A) αDG clusters (n = 4,989) in control cultures not exposed to laminin. (B) LBS clusters (n = 7,930) in control cultures exposed briefly (10–30 min) to laminin (0.6–6 nM) at low temperature immediately before staining. (C) LBS clusters (n = 3,621) after treatment with 6 mM laminin for 1 d at room temperature. In control cultures (A and B) there were very few macroclusters (>1 μm2), and most of the microclusters were <0.1 μm2. After treatment with laminin for 1 d (C) the incidence of macroclusters was increased whereas the incidence of the smallest microclusters was decreased.
	Figure 6. Effect of laminin treatment on (A) the percentage of surface area occupied by LBS macroclusters and (B) the density of LBS microclusters. Laminin concentrations (in nM) and exposure times are indicated at the bottom of B. The cultures were treated with laminin either at low temperature (open columns) or at room temperature (shaded columns). In some cases the laminin treatment was carried out in the presence of RG50864 (striped columns). On average the means and standard errors are based on 26 cells (range, 9–79).
	Figure 7. Relationship between the density of microclusters and the percentage of surface area occupied by macroclusters. From the data of Fig. 6.
	Figure 8. E3 lacks clustering activity and inhibits laminin-induced clustering. (A) E3 binding sites after treatment with 3.3 μM E3 for 2 d. Note the relatively high density of microclusters and the paucity of macroclusters. (B) LBS after treatment with 0.6 nM laminin for 1 d. (C) Laminin and E3 binding sites after treatment with 0.6 nM laminin and 3.3 μM E3 for 1 d. E3 inhibited the laminininduced clustering. Bar, 5 μm.
	Figure 9. Agrin-induced clusters on the dorsal surface. (A1 and A2) βDG and LBS immunofluorescence, revealing several patterned clusters, excellent colocalization, and some decline in the density of microclusters in surrounding regions. (B1 and B2) AChR stain and utrophin immunofluorescence, revealing excellent colocalization. (C1 and C2) AChR stain and PY immunofluorescence, revealing excellent colocalization. The agrin treatment was 3 d in A and B and 2 d in C. Similar results were obtained when the agrin treatment was 1 d. Bar, 5 μm.
	Figure 12. Schematic representation of some of the surface proteins clustered by laminin and agrin, both of which bind to αDG. Before clustering, DG complexes of αDG and βDG are considered to be mobile, like AChRs. Laminin-induced clusters of DG and dystrophin are considered to be linked by laminin–laminin binding. They do not contain utrophin, AChRs, or tyrosine phosphorylated molecules. Not shown is that the end of the vertical short arm of laminin also participates in self binding; that laminin may bind to other sarcolemma-associated molecules; that dystrophin binds to cytoskeletal actin; and that sarcoglycans and syntrophins may also be present at laminin-induced clusters. Agrin-induced clusters contain DG, sarcoglycans (unlabeled), utrophin, β2 syntrophin (syn), tyrosine phosphorylated AChRs, rapsyn (rn), and tyrosine phosphorylated muscle specific kinase (MuSK). Their formation unlike the formation of laminin-induced clusters, involves stimulation of tyrosine kinase activity.

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