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.


METHODS
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.
National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina.
W E
WE
Bakewell
G J
GJ
Smith
B E
BE
Miller
C J
CJ
Viviano
G E
GE
Hook
D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D002467:Cell Nucleus / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D007106:Immune Sera / Q000276:immunology; D007834:Lasers; D008297:Male; D008715:Methionine / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D009685:Nuclear Envelope / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D011233:Precipitin Tests; D011510:Proteolipids / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology; D011650:Pulmonary Alveoli / Q000737:chemistry* / Q000166:cytology* / Q000648:ultrastructure; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D007106:Immune Sera; D011510:Proteolipids; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D008715:Methionine