Rochlin MW and Peng HB (1989), Localization of intracellular proteins at acety...

XB-ART-26559

J Cell Sci 1989 Sep 01;94 ( Pt 1):73-83.

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Localization of intracellular proteins at acetylcholine receptor clusters induced by electric fields in Xenopus muscle cells.

Rochlin MW , Peng HB .

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Electric fields cause acetylcholine receptor (AChR) patches to form on the cathodal sides of cultured muscle cells. These patches are stable for several hours following cessation of an electric field treatment, indicating that the receptors are anchored to the cluster sites. Furthermore, at the ultrastructural level, AChR patches induced by electric fields are marked by an accumulation of extracellular matrix material and a sarcolemmal density. Thus, these AChR patches are similar to those induced by other stimuli, including nerve, polycation-coated beads, and the tissue culture substratum. Proteins that may be involved in anchoring AChRs have been colocalized with AChR patches induced by the latter three stimuli, but not at AChR patches induced by electric fields. In this study, we demonstrate that three putative anchoring proteins, 43K (K = 10(3) Mr) protein, 58K protein and talin, are associated with field-induced AChR patches. We also show that these proteins persist at field-induced AChR patches following removal of the field, indicating that they are stabilized at the AChR patch. Our data are consistent with the possibility that these proteins contribute to the stabilization of AChRs at patches induced by the electric field. Since 43K, 58K and talin are intracellular proteins, and therefore could not undergo field-induced lateral electrophoresis, our observations support the notion that the electric field triggers the formation of an AChR-stabilizing specialization.

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	Fig. 1. Control for pre-existing AChR clusters on the cathode-facing edge of the muscle cell, and an evaluation of field-induced hotspot reduction. A,A'. The percentage of untreated cells with hotspots on an edge was recorded. Only those edge hotspots that resembled FIRPs were counted (see Materials and methods). The unweighted average (±S.E.M.) of the percentages obtained from different experiments was determined, and then halved to account for those that would have faced the cathode. B,B'. The percentage of 4 Vcm"1 field-treated 1-day-old cells with cathodal clusters. C.C. The percentage of untreated cells that had hotspots. D.D'. The percentage of 7-5 Vcm" field-treated 2-day-old cells that had hotspots. On field-treated cells, AChR staining away from the cathode-facing edge constituted hotspots. Data base (no. expts, no. cells): A,C, (3, 162); B, (9, 578); D, (4, 279); A',C\ (2 ,90); B', (3, 121); D', (3, 109).
	Fig. 2. FIRP formation on a formerly hotspot-free cathodal edge. A. Digitized phase-contrast image showing absence of debris from cathodal (left) edge. B. Hotspot-free cathodal edge prior to field treatment. C. FIRPs on same edge during 4Vcm~' field application, 25 min after onset. Note that the AChR staining is elevated in patches, rather than uniformly along the cathode facing edge of the cell (arrowheads). The bright staining of spherical structures in the middle of the cell and outside the cell is due to the autofluorescence of yolk platelets. Bar, 10 /im.
	Fig. 3. Control for primary antibody staining at FIRPs. In this example, 1-day-old cells were treated with the 1-h field, 2-h PFR protocol (see text). A. R-BTX staining showing the FIRP. B. Fluorescein optics showing donkey anti-rabbit staining following an exposure suitable for rabbit anti-talin staining. Very little nonspecific staining is evident in B. Cathode is towards the bottom in this micrograph. Bar, 10jUm.
	Fig. 4. 43K protein at hotspots and FIRPs. A,D,G, R-BTX staining; B,E,H, the corresponding 43K staining; C,F,I, the corresponding phase-contrast views. The top row shows a muscle cell from an untreated culture. The staining pattern at the ventral hotspot (A) matches the pattern of 43K staining (B) identically. Following the 1-h field, 2-h PFR (row 2) or the 3-h field, no PFR on 1-day-old cells (row 3), 43K (E,H arrows) was elevated at all FIRPs (D,G arrows). Cathode is to the left in this and all subsequent micrographs. Bar, 10/tfn.
	Fig. 5. Talin at hotspots and FIRPs. A,D,G, R-BTX staining; B,E,H, the corresponding talin staining; C,F,I, the corresponding phase-contrast views. The top row shows an example of a typical AChR (A) and talin (B) distribution in a 2-dayold untreated culture. Talin is always elevated at the ends of the myotomal muscle cells in vitro, independent of hotspot presence (A,B open arrows). Although there is overlap between the AChR and talin staining patterns (small arrows), there are regions of AChR staining that do not manifest a similar accumulation of talin (A,B arrowheads). After the 1-h field, 2-h PFR protocol, talin (E, arrows) was elevated at this FIRP (D, arrows). A particularly robust elevation of talin (H, arrows) was evident at the FIRP (G, arrows) induced by the 3-h field, no PFR treatment in this 2-day-old cell. A-F, bar, lO^im; G-I, bar = 6 Jim.
	Fig. 6. 58K protein at hotspots and FIRPs. A,D,G, R-BTX staining; B,E,H, the corresponding 58K staining; C,F,I, the corresponding phase-contrast views. The top row shows the colocalization between 58K (B) and a ventral hotspot (A) in an untreated 1-day-old cell (C). Unlike talin, 58K was typically well colocalized with AChR domains in hotspots. S8K proteins (E,H arrows) was elevated at virtually all FIRPs (D,G arrows) induced by both the 1 h field, 2-h PFR protocol (row 2) and the 3-h field, no PFR protocol in 1-day-old cells (row 3). Bar, 10 fun.