A COMPARATIVE NUCLEAR-LOCALIZATION STUDY OF GALECTIN-1 WITH OTHER SPLICING COMPONENTS

Using both conventional and laser confocal fluorescence microscopy, th e intracellular distribution of galectin-l in HeLa cells was analyzed and compared with the localization of previously documented markers of the nucleus and cytoplasm. The Sm epitopes of the small nuclear ribon ucleoprotein complexes (snRNPs) and the non-snRNP splicing factor SC35 yielded only nuclear staining. On the other hand, the enzyme lactate dehydrogenase was cytoplasmic. In contrast to these patterns in which nuclear versus cytoplasmic :Localizations appeared to be mutually excl usive, galectin-l, as well as galectin-3, yielded simultaneous nuclear and cytoplasmic staining. Confocal microscopy showed galectin-l fluor escence throughout most of the sections from the top of the cell to th e bottom. Through the middle sections, as the plane of focus cuts thro ugh the nucleus, there was definite fluorescence staining in the nucle ar compartment. This nuclear localization was critically dependent on the type of detergent used to permeabilize the cell: cells treated wit h saponin or digitonin yielded exclusively cytoplasmic staining while Triton X-100-treated cells showed nuclear as well as cytoplasmic label ing. Finally, double-immunofluorescence analysis showed that, within t he nucleoplasm, the following pairs of nuclear antigens could be coloc alized in certain speckled structures: (a) SC35 versus Sm; (b) galecti n-l versus Sm; (c) galectin-3 versus Sm; and (d) galectin-l versus gal ectin-3. These results establish the presence of galectin-l in the nuc lei of HeLa cells, a conclusion consistent with the identification of the protein in nuclear extracts of the same cells and with its documen tation as a factor in pre-mRNA splicing. (C) 1998 Academic Press.