Requirement of Dynactin p150Glued Subunit for the Functional Integrity of the Keratinocyte Microparasol

The keratinocyte microparasol, composed of a perinuclear microtubular/melano–phagolysosomal complex, protects the nucleus from UV-induced DNA damage. We have previously demonstrated that cytoplasmic dynein is the motor involved in the perinuclear-directed aggregation of phagocytosed melanosomes. Dynactin, of which p150Glued is the major subunit, can link directly to microtubules and links organelles to dynein at different domains. To further define the mechanism of the microparasol, we transfected siRNA targeted against p150Glued into human keratinocytes cultured with 0.5mm fluorescent microspheres and performed time-lapse analysis, confocal immunolocalization, and Western immunoblotting after 24 and 48 hours. Western blots revealed a significant knockdown of the p150Glued subunit. The knockdown decreased p150Glued colocalization with microtubules and decreased perinuclear positioning of the convergent microtubular framework. It also inhibited perinuclear aggregation of phagocytosed fluorescent microspheres and reduced mean centripetal microsphere displacement. The findings provide evidence that dynactin p150Glued plays an important role in the functional integrity of the keratinocyte microparasol

Mining Endonuclease Cleavage Determinants in Genomic Sequence Data*

Homing endonucleases have great potential as tools for targeted gene therapy and gene correction, but identifying variants of these enzymes capable of cleaving specific DNA targets of interest is necessary before the widespread use of such technologies is possible. We identified homologues of the LAGLIDADG homing endonuclease I-AniI and their putative target insertion sites by BLAST searches followed by examination of the sequences of the flanking genomic regions. Amino acid substitutions in these homologues that were located close to the target site DNA, and thus potentially conferring differences in target specificity, were grafted onto the I-AniI scaffold. Many of these grafts exhibited novel and unexpected specificities. These findings show that the information present in genomic data can be exploited for endonuclease specificity redesign