Particulate forms of APP in the extracellular milieu of cultured cells.
The principle externalized forms of amyloid precursor protein (APP) are soluble and well-characterized, but some evidence has suggested the additional presence of externalized APP in a nonsoluble form. To further assess this possibility, the current study has applied high resolution microscopy protocols in addition to immunoprecipitation to characterize externalized APP in three commonly used cell culture models (SH-SY5Y human neuroblastoma cells, fetal rat brain cells, and HEK 293 human embryonic kidney cells). Confocal immunofluorescence microscopy, using an antiserum against the c-terminal domain of APP, showed typical cell-associated APP, but hot spots of APP also were evident in cell-free areas, apparently associated with the culture substrata. These hot-spots were examined for evidence of cellular deterioration by whole mount transmission electron microscopy. Neither cell debris nor disrupted cells were present. Instead, the hot spots of substratum-bound APP comprised discrete microparticles, approximately 50-100 nm across. These microparticles also could be found near cells and in some cases were attached to cell surface fibrils. Substratum-bound APP also could be found clustered within the extracellular matrix made by primary cell cultures. Occurrence of APP in extracellular microparticles was verified by centrifugation-immunoprecipitation analysis of media conditioned by APP-transfected cells. Radiolabeling data showed that particulate APP was from metabolically active cells. Metabolic labeling of particle-associated APP, as well as the absence of cellular debris near the APP-containing particles, suggests that the occurrence of nonsoluble APP in the extracellular milieu derives from a physiologically active process.
Barber
K
K
Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60208, USA.
Enam
S A
SA
Bodovitz
S
S
Falduto
M
M
Frail
D
D
Klein
W L
WL
D016564:Amyloid beta-Protein Precursor / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000032:analysis; D001921:Brain / Q000378:metabolism / Q000648:ultrastructure; D002460:Cell Line; D005110:Extracellular Space / Q000378:metabolism*; D007668:Kidney / Q000196:embryology / Q000378:metabolism / Q000648:ultrastructure; D009447:Neuroblastoma / Q000378:metabolism / Q000648:ultrastructure; D051381:Rats; D014407:Tumor Cells, Cultured
D016564:Amyloid beta-Protein Precursor; D000906:Antibodies