Agonist-stimulated free calcium in subcellular compartments. Delivery of recombinant aequorin to organelles using a replication deficient adenovirus vector.
Changes in the concentration of calcium ions ([Ca2+]) within cellular organelles play a central role in controlling cellular function. We have engineered the Ca2+ sensitive photoprotein aequorin to monitor selectively [Ca2+] within defined subcellular compartments, namely the cytosol, nucleus and endoplasmic reticulum. DNA encoding the engineered aequorins have been inserted into a replication deficient adenovirus (Ad) type 5 E1-vector, under control of the cytomegalovirus (CMV) major immediate early promoter. The Ad vector provides a simple and efficient method to express the photoproteins in a wide variety of mammalian cell types. Efficient targeting of the photoproteins to the appropriate cellular compartment was established immunocytochemically in COS7 cells, where it was expressed in up to 100% of the target population. Levels of expression could be controlled by virus dose and chemical agents which affect the activity of the CMV promoter. In HeLa cells expressing nuclear targeted aequorin or cytosolic aequorin, ATP or histamine induced immediate biphasic elevations of both nuclear and cytosolic [Ca2+]; subsequent challenge with agonist evoked similar responses. In addition to epithelial type adherent cell lines (COS7 and HeLa), aequorin expression was also readily detected in non-adherent cells of myeloid lineage (K562 and HL60) and non-adherent primary cells polymorphonuclear leucocytes (neutrophils). The Ad vectors can, therefore, be used to express targeted aequorin in a range of different cell types and represents a novel method to monitor changes in free [Ca2+] in cellular organelles.
Kendall
J M
JM
Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, UK.
Badminton
M N
MN
Sala-Newby
G B
GB
Wilkinson
G W
GW
Campbell
A K
AK
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000256:Adenoviridae / Q000235:genetics; D017498:Adenovirus E1 Proteins / Q000235:genetics; D000331:Aequorin / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002451:Cell Compartmentation / Q000187:drug effects* / Q000502:physiology*; D002467:Cell Nucleus / Q000187:drug effects / Q000235:genetics / Q000821:virology; D003587:Cytomegalovirus / Q000235:genetics; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000235:genetics / Q000821:virology; D005822:Genetic Vectors / Q000235:genetics; D000882:Haplorhini; D006367:HeLa Cells; D006632:Histamine / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000188:drug therapy / Q000473:pathology / Q000821:virology; D015473:Leukemia, Promyelocytic, Acute / Q000188:drug therapy / Q000473:pathology / Q000821:virology; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000187:drug effects; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014779:Virus Replication
D017498:Adenovirus E1 Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D000331:Aequorin; D006632:Histamine; D000255:Adenosine Triphosphate; D002118:Calcium