CONFOCAL LASER SCANNING IMMUNOFLUORESCENCE MICROSCOPY OF THE PULMONARY SURFACTANT SYSTEM - ASSOCIATION OF SURFACTANT PROTEIN-A WITH THE NUCLEUS OF THE ALVEOLAR TYPE-II CELL

BACKGROUND: Localization of surfactant protein A (SP-A) to nucleus of type II cells isolated from the lungs of rats has been reported. Data suggested that most SP-A was located within lamellar bodies of the type II cell; however, some SP-A was found in other cytoplasmic regions of the cell and in particular in the nucleus. EXPERIMENTAL DESIGN: Type II cells and type II cell nuclei isolated from the lungs of rats were reacted with affinity-purified antibodies against SP-A. Location of SP-A was determined by using fluorescein isothiocyanate-labeled secondary antibodies and the distribution of fluorescence examined by using a laser scanning microscope fitted with a confocal aperture. Two-dimensional electrophoresis followed by Western blotting was used to separate and identify type II cell nuclear proteins. RESULTS: Nuclei were isolated from type II cells obtained from elastase-digested rat lungs and examined for the presence of SP-A. The nuclei contained both focal and diffuse deposits of SP-A. Some regions within the nuclear matrix (in particular the nucleolus) appeared to be relatively devoid of SP-A. The perinuclear membrane stained intensely for SP-A where optical sectioning showed its presence as a patchwork of punctate deposits. Analysis of the SP-A associated with the nucleus by two-dimensional electrophoresis revealed that it consisted of a family of proteins with molecular masses of 26, 32, and 36 kDa and pI 5.1. Biosynthesis of nuclear SP-A in primary cultures of type II cells was sensitive to inhibitors of glycosylation resulting in the presence of only the lowest molecular weight unprocessed form. CONCLUSIONS: These data indicate that three basic forms of SP-A are associated with the nucleus of the type II cell and are especially concentrated on the perinuclear membrane.