Probing platelet factor 4 alpha-granule targeting.
The storage mechanism of endogenous secretory proteins in megakaryocyte alpha-granules is poorly understood. We have elected to study the granule storage of platelet factor 4 (PF4), a well-known platelet alpha-granule protein. The reporter protein green fluorescent protein (GFP), PF4, or PF4 fused to GFP (PF4-GFP), were transfected in the well-characterized mouse pituitary AtT20 cell line, and in the megakaryocytic leukemic DAMI cell line. These proteins were also transduced using a lentiviral vector, in human CD34+ cells differentiated into megakaryocytes in vitro. Intracellular localization of expressed proteins, and colocalization studies were achieved by laser scanning confocal microscopy and immuno-electronmicroscopy. In preliminary experiments, GFP, a non-secretory protein (no signal peptide), localized in the cytoplasm, while PF4-GFP colocalized with adrenocorticotropin hormone (ACTH)-containing granules in AtT20 cells. In the megakaryocytic DAMI cell line and in human megakaryocytes differentiated in vitro, PF4-GFP localized in alpha-granules along with the alpha granular protein von Willebrand factor (VWF). The signal peptide of PF4 was not sufficient to specify alpha-granule storage of PF4, since when PF4 signal peptide was fused to GFP (SP4-GFP), GFP was not stored into granules in spite of its efficient translocation to the ER-Golgi constitutive secretory pathway. We conclude that the PF4 storage pathway in alpha-granules is not a default pathway, but rather a regular granule storage pathway probably requiring specific sorting mechanisms. In addition PF4-GFP appears as an appropriate probe with which to analyze alpha-granule biogenesis and its alterations in the congenital defect gray platelet syndrome.
Briquet-Laugier
V
V
U348 INSERM, IFR6-Circulation, Hôpital Lariboisière, Paris, France.
Lavenu-Bombled
C
C
Schmitt
A
A
Leboeuf
M
M
Uzan
G
G
Dubart-Kupperschmitt
A
A
Rosa
J-P
JP
D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000818:Animals; D018952:Antigens, CD34 / Q000096:biosynthesis; D001791:Blood Platelet Disorders / Q000097:blood / Q000151:congenital; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D005312:Fetal Blood / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D047468:Immunoprecipitation; D016086:Lentivirus / Q000235:genetics; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D010978:Platelet Factor 4 / Q000378:metabolism*; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D014162:Transfection; D014841:von Willebrand Factor / Q000378:metabolism
D018952:Antigens, CD34; D017931:DNA Primers; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D049452:Green Fluorescent Proteins; D010978:Platelet Factor 4; D000324:Adrenocorticotropic Hormone; D013917:Thrombin