Calcium Rises Abruptly and Briefly Throughout the Cell at the Onset of Anaphase

Abstract

Continuous measurement and imaging of the intracellular free calcium ion concentration ([Ca2+]i) of mitotic and interphase PtK1 cells was accomplished with the new fluorescent Ca2+ indicator fura-2. No statistically significant difference between basal [Ca2+]i of interphase and mitotic cells was detected. However, mitotic cells showed a rapid elevation of [Ca2+]i from basal levels of 130 nM to 500 to 800 nM at the metaphase-anaphase transition. The [Ca2+]i transient was brief, lasting approximately 20 seconds and the elevated [Ca2+]i appeared uniformly distributed over the entire spindle and central region of the cell. The close temporal association of the [Ca2+]i transient with the onset of anaphase suggests that calcium may have a signaling role in this event.

Effects of lemakalim on changes in Ca2+concentration and mechanical activity induced by noradrenaline in the rabbit mesenteric artery

“…Under these conditions, the background fluorescence intensity was 10-15% of the Fura 2 signals in smooth muscle strips at either excitation wavelength. Cytosolic Ca2+ concentrations were calculated with the formula described by Grynkiewicz et al (1985) and using in vitro calibration (Poenie et al, 1986;Becker et al, 1989). The ratio of maximum (FmaJ to minimum fluorescence (Fmin) was determined in the calibration solution after subtraction of background excited by either 340 or 380 nm and the 380 signals of Fura 2 were assumed to decrease by 15% in the cell due to the possible intracellular viscosity effects of the dye (Becker et al, 1989).…”

Intracellular free calcium and mitosis in mammalian cells: anaphase onset is calcium modulated, but is not triggered by a brief transient.

“…Injection buffer was composed of 115 mM KCI, 20 mM NaCl, 1.2 mM MgCl2, 10 mM NaHepes, pH 7.0. 50-/zg aliquots of dry fura-2 AM were dissolved in 10/xl dry DMSO, 10/H pluronic F-IT/in DMSO and 8/~1 FBS on the day of use according to published methods (Poenie et al, 1986). FBS was obtained from Hyclone Laboratories (Logan, UT).…”

“…A single transient rise in intracellular free calcium (Ca0 ~ has been proposed to serve as the trigger of anaphase onset (Poenie et al, 1986). Such a trigger is plausible since intracellular calcium regulation is extensive and calcium can potentially interact with many components of the mitotic spindle.…”

Evaluation of algorithms for ratio imaging in fluorescence microscopy

“…Both uniformly fluorescing fields and fluorescing cell images were employed to provide data for use in confirming our calculations and illustrating both the magnitude and spatial incidence of errors. It is shown that, through proper design of lookup tables, a significant reduction can be made in the errors generated in comparison with common methods available in most image processors.Key terms: Image processing, inversion method, log subtraction method Ratio imaging in fluorescence microscopy (16) is used in measuring the temporal and spatial dynamics of cellular parameters including pH (1-4,12) and pCa (8,9,13,15,17,18), as well as local relative concentration of fluorescent analogs (6,ll) and the local porosity of cytoplasm (10) in single living cells. The fundamental physical and chemical issues in the application of ratio imaging to fluorescence microscopy were addressed previously (1,2), including the effects of excitation light level, lamp operation, intracellular probe concentration, and spatial signal-to-noise ratio.…”

“…Ratio imaging in fluorescence microscopy (16) is used in measuring the temporal and spatial dynamics of cellular parameters including pH (1-4,12) and pCa (8,9,13,15,17,18), as well as local relative concentration of fluorescent analogs (6,ll) and the local porosity of cytoplasm (10) in single living cells. The fundamental physical and chemical issues in the application of ratio imaging to fluorescence microscopy were addressed previously (1,2), including the effects of excitation light level, lamp operation, intracellular probe concentration, and spatial signal-to-noise ratio.…”