In vivo calcineurin crystals formed using the baculovirus expression system.
Calcineurin is a heterodimeric phosphatase involved in the signal transduction of antigen-activated T cells. Coexpression of its two subunits, the regulatory subunit from human and the catalytic subunit from Neurospora crassa in cultured insect cells using the baculovirus expression system results in the formation of very large crystals in the cytoplasm. The crystals are formed initially in vesicles, but their subsequent growth appears to be uninhibited and continues without the need of an enclosing membrane until the host cell lyses. Although these in vivo crystals are low in population, ranging only 0-3 per cell, they are extremely large, over 10 mu m in some cases. Biochemical assays confirm their calcineurin origin, with the regulatory subunit incorporated being myristoylated, although both the myristoylated and unmyristoylated forms are expressed. The lattice structure of the in vivo crystals, with a spacing of 5.5 nm, is preserved with the regular electron microscopic (EM) specimen preparation procedure.
Fan
G Y
GY
National Center for Microscopy and Imaging Research, University of California at San Diego, La Jolla 92093-0608, USA.
Maldonado
F
F
Zhang
Y
Y
Kincaid
R
R
Ellisman
M H
MH
Gastinel
L N
LN
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D002467:Cell Nucleus / Q000648:ultrastructure; D002478:Cells, Cultured; D003460:Crystallization; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D006801:Humans; D007313:Insecta; D008854:Microscopy, Electron; D009492:Neurospora crassa / Q000235:genetics; D010749:Phosphoprotein Phosphatases / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic
D002148:Calmodulin-Binding Proteins; D011994:Recombinant Proteins; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases