Fig. 5. In vivo observation of muscle attachment failure and molecular analysis of detached free ends. A single fibre (A,B short arrows) viewed in vivo in the process of detaching myosepta, under differential interference contrast (A, lateral view, 5 days post-fertilisation) and labelled with EBD (B). A gap is visible between the separating posterior end of the fibre (right, short arrow) and the myoseptum (arrowhead). A narrowed retraction zone has formed where the contractile apparatus has withdrawn from the centre of the fibre (between the short arrows). The anterior end of the fibre (left, short arrow) is partly obscured by a second dye-positive detached cell (long arrow). Confocal microscopy of GFP revealed this example of a free end of a single detached fibre (D, bracket, lateral view, 6 days post-fertilisation). Stacked confocal images allow tracing of individual fibres to their insertion points at the muscle attachments. Fibres within sap homozygotes (D) exhibit a club-like or faceted appearance at their newly detached membranes, not evident in wild-type embryos (C). ßDG protein at the muscle attachments is not retained in the fibre membranes once detachment occurs. Two neighbouring detached and retracted mutant cells visible under DIC (arrowheads in E, 72 hours post-fertilisation, lateral view) are still attached to their posterior (right) myoseptum (arrows in E,F). Labelling with anti-ßDG (F) shows that this integral membrane protein has been lost from their free (left) ends upon detachment, possibly maintaining its binding to -dystroglycan and laminin at the myoseptum. Phosphorylated focal adhesion kinase is enriched in terminal cytoplasm at muscle attachments (arrows in G,H, lateral confocal images, 72 hours post-fertilisation). Unlike ßDG, however, p(tyr397)FAK remains visibly localised to the free end of detached cells, indicating the retention of terminal cytoplasm by detached and retracted fibres (arrowheads in H).

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