Nuclear calcium flux in Trypanosoma brucei can be quantified with targeted aequorin.
The following study was undertaken to determine if calcium ions move from the plasma membrane to the nucleus of Trypanosoma brucei. Nuclear and cytosolic calcium flux was measured with the calcium sensitive photoprotein, aequorin which was targeted to various locations in stably transformed procyclic cells. Immunoblots revealed that the recombinant proteins, CYT-AEQ and NUC-AEQ were translated in transformants, and that CYT-AEQ was contained in a soluble fraction. Immunolocalization demonstrated that NUC-AEQ was contained within the trypanosome nucleus. To evaluate calcium movement from the plasma membrane to the nucleus in live trypanosomes, aequorin was reconstituted in vivo with coelenterazine and luminescence was recorded. The resting levels of [Ca2+]cyt and [Ca2+]nuc were similar (314 +/- 43 and 287 +/- 28 nM, respectively). When calcium influx across the plasma membrane was initiated with 2 microM ionomycin, [Ca2+]cyt and [Ca2+]nuc each became elevated in parallel to a new steady state which was approximately 2-fold above the resting level. Compound 48/80 initiated a calcium flux across the plasma membrane by a different mechanism from ionomycin, and in a manner that was inhibited by the calcium channel antagonist, La3+. Compound 48/80 (8 micrograms/ml) transiently elevated [Ca2+]cyt to 1.73 +/- 0.3 microM over the course of 20 s, and also generated a transient rise in [Ca2+]nuc which peaked at 1.32 + 0.29 microM over the same time course. Overall, these data demonstrate that calcium moves into and out of the trypanosome nucleus in a manner which closely parallels changes in [Ca2+]cyt. A small calcium ion gradient between nucleus and cytoplasm was also observed.
Xiong
Z H
ZH
Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275, USA.
Ruben
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D000331:Aequorin / Q000096:biosynthesis / Q000187:drug effects* / Q000235:genetics; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D008163:Luminescent Measurements; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000187:drug effects; D014346:Trypanosoma brucei brucei / Q000378:metabolism*
D011994:Recombinant Proteins; D000331:Aequorin; D002118:Calcium