ACCESSIBILITY OF T-TUBULE VACUOLES TO EXTRACELLULAR DEXTRAN AND DNA -MECHANISM AND POTENTIAL APPLICATION OF VACUOLATION

In addition to its function in excitation-contraction coupling, the ab ility of the T-system of skeletal muscle fibres to undergo reversible vacuolation indicates that it may play a role in volume regulation. Th e mechanism of reversible vacuolation has been investigated by confoca l microscopy using fluorescein dextran to probe the accessibility of T -tubules to the extracellular environment. Vacuolation was induced by loading the fibres with 60-100 mM glycerol for 30 minutes and then ret urning them to glycerol-free medium. Devacuolation was subsequently in duced by the reentry of glycerol. During their formation from T-tubule s, the vacuoles filled with fluorescent dextran from the extracellular medium. The inaccessibility of the vacuoles to extracellular ferritin observed in a previous study raised the possibility that the vacuoles may be detached from the surface membrane after their formation. Howe ver, it is apparent from the present work that, although the tubules o f the treated fibres are constricted, the vacuoles maintain an open co nnection with the external medium for smaller macromolecules. In the l ight of these experiments, it is proposed that vacuolation is caused b y water moving into T-tubules from the cytoplasm faster than it can ex it to the extracellular space during a decrease in fibre volume. Since T-tubules have been implicated in the transfection of skeletal muscle by direct injection, the accessibility of plasmid DNA to T-tubules ha s also been investigated. DNA penetrated into the vacuoles from the ex tracellular medium less well than dextran, but many vacuoles containin g fluorescent DNA were observed in the superficial layers of vacuolate d fibres, and it is suggested that T-tubule vacuolation might be used to improve the efficiency of the transfection of skeletal muscle by di rect injection. (C) Chapman & Hall Ltd.