Ikuo Ashikawa

Electroporation of cell membrane visualized under a pulsed-laser fluorescence microscope

Controlled permeability can be conferred to cell membranes by exposing cells to a microsecond electric pulse of sufficient intensity (electroporation). By constructing a fluorescence microimaging system with a submicrosecond time resolution we have been able to resolve temporally and spatially the events in a single cell under a microsecond electric pulse. An enormous membrane conductance, corresponding to a loss of 0.01-0.1% of the membrane area, was observed in those membrane regions where the transmembrane potential induced by the electric pulse exceeded a critical value. The conductance decreased to a low level in a submillisecond after the pulse, leaving a moderately electroporated cell.

Dynamics of Z-form DNA.

The torsional and bending rigidities of Z-form DNA have been studied by nanosecond fluorescence anisotropy measurements of intercalated ethidium. The results suggested that Z-form DNA was considerably more flexible than B-form DNA. We have investigated the temperature dependence of the rigidity of B- and Z-form DNA and found that the temperature dependence of the torsional rigidity of Z-form DNA was remarkably lower than that of B-form DNA.

Submicrosecond Imaging Under A Pulsed-Laser Fluorescence Microscope

A microscope system has been constructed that enables digital imaging of a fluorescent cell under pulsed illumination. Each image is produced by a single laser pulse of duration less than 0.3 11 s. With this system, microsecond responses of a single cell to an externally applied electric field have been resolved temporally and spatially. The cell membrane was stained with a voltage-sensitive fluorescent dye. The induction of transsmembrane potential by the applied field, and the perforation (electroporation) of the cell membrane under an intense field, were seen as successive images. The major finding was a transient increase, at the moment of perforation, in the membrane permeability to an enormous level in localized regions of the cell membrane. Possible roles in cell technology, as well as other applications of the microscope system, are discussed.

Changes of the DNA packaging mode during boar sperm maturation.

Changes in the mode of DNA packaging in nuclei during spermatogenesis were studied by measuring of the fluorescence anisotropy decay of an ethidium dye intercalated in the DNA in whole nuclei. The nuclei were isolated from boar spermatid or sperm cells at three distinct stages of spermatogenesis: just before the completion of a maturation process in the testis (late spermatid), immediately after a subsequent transformation into spermatozoa (caput spermatozoon), and after full maturation (cauda spermatozoon). Although these three kinds of nucleus were morphologically indistinguishable from each other, the anisotropy decay detected a clear difference. In the late spermatid nuclei, in which the replacement of histones by protamine was still in progress, the anisotropy decayed extensively. The decay suggested that the DNA in the spermatid nuclei contained very flexible regions, in which the interaction of the DNA and proteins may be weak. The rapid and extensive anisotropy decay was absent in the caput and cauda nuclei. The flexible portions must have turned into very rigid structure during transformation from the late spermatid into the caput spermatozoon.