EQUILIBRATION AND EXCHANGE OF FLUORESCENTLY LABELED MOLECULES IN SKINNED SKELETAL-MUSCLE FIBERS VISUALIZED BY CONFOCAL MICROSCOPY

Confocal laser fluorescence microscopy was used to study in real time under nearly physiological conditions the equilibration and exchange c haracteristics of several different fluorescently labeled molecules in to chemically skinned, unfixed skeletal muscle fibers of rabbit psoas. The time required for equilibration was found to vary widely from a f ew minutes up to several days. Specific interactions of molecules with myofibrillar structures seem to slow down equilibration significantly . Time for equilibration, therefore, cannot simply be predicted from d iffusion parameters in solution. Specific interactions resulted in cha racteristic labeling patterns for molecules like creatine kinase (musc le type), pyruvate kinase, actin-binding IgG, and others. For the very slowly equilibrating Rh-NEM-S1, changes in affinity upon binding to a ctin in the absence of calcium and subsequent slow cooperative activat ion, beginning at the free end of the filament at the H-zone, were obs erved. In the presence of calcium, however, binding of Rh-NEM-S1 was h omogeneous along the whole actin filament from the very beginning of e quilibration, The dissociation properties of the dynamic interactions were analyzed using a chase protocol. Even molecules that bind with ra ther high affinity and that can be removed only by applying extreme ex perimental conditions like Rh-phalloidine or Rh-troponin could be disp laced easily by unlabeled homologous molecules.