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First published online 2 November 2004
doi: 10.1242/jcs.01510

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Ca²⁺-dependent and -independent mechanisms of calmodulin nuclear translocation

Department of Basic Medical Sciences, St Georges Hospital Medical School, Cranmer Terrace, London, SW17 0RE, UK

View larger version (47K): [in a new window]   Fig. 1. Characterization of 6ROX-CaM (A) HPLC analysis of 6ROX-CaM purified by a Vydac reverse-phase C8 column (4.6x150 mm). The chromatogram shows a single absorbance peak at 215 nm with a corresponding fluorescent peak measured at an excitation wavelength of 568 nm and an emission wavelength of 602 nm. (B) MALDI-TOF mass spectroscopy of undigested 6ROX-CaM. Obtained was an average mass of 17226.8 Da [average theoretical mass, 17223.1 Da (6ROX reacted =516.7 Da + 16706.4 Da for human liver CaM expressed in E. coli)]. (C,D) MALDI-TOF mass spectroscopy of the labeled peptides of 6ROX-CaM digested with trypsin. Peptide 75-77 (theoretical monoisotopic mass, 405.2 Da) bound to the 6ROX probe (monoisotopic mass, 922.2 Da; theoretical monoisotopic mass, 922.3 Da) (C). Peptide 75-86 (theoretical monoisotopic mass, 1479.7 Da) bound to the 6ROX probe (monoisotopic mass, 1995.6 Da; theoretical monoisotopic mass, 1997.3 Da) (D). (E,F) Activation of PDE by unlabeled CaM () and 6ROX-CaM (). Km was 6.5 (±0.7) nM for CaM and 8.3 (±0.9) nM for 6ROX-CaM. (G) Fluorescent properties of 6ROX-CaM. Ca2+-titration of 100 nM 6ROX-CaM was carried out in 5 mM EGTA, 100 mM KCl, 2 mM MgCl2 and 50 mM KMES, pH 7.0 at 21°C. Emission was corrected for buffer. Ca2+ concentrations were in the order of rising relative fluorescence: 0.4 nM, 0.29 µM, 0.652 µM and 1.74 µM. (H) The effect of Ca2+-binding on tyrosine fluorescence of WT-CaM (+) and CaM1234 (). Fluorescence was monitored at an excitation wavelength of 278 nm and an emission wavelength of 305 nm. (Ii-Iiii) Measurement of the Kd of mTrp peptide binding to TACaM by stopped-flow kinetics. (Ii) Association kinetics. 12 nM TA-CaM was rapidly mixed with 175 nM mTrp peptide (concentrations in mixing chamber are given) in the same solution conditions as the other fluorescent measurements were made. The biphasic fluorescence increase was fitted by two exponentials at 45 seconds–1 (0.75) and 3.8 seconds–1 (0.25). (Iii) Observed rates of association are plotted as a function of mTrp peptide concentration. The gradient () was 2.2x108 M–1 seconds–1. The rate of the slow step saturated at 3.8 seconds–1 (). (Iiii) Ca2+/TA-CaM.mTrp complex dissociation kinetics. Ca2+/TA-CaM was displaced from the complex by an excess of 2 µM unlabeled CaM. The rate constant of dissociation was 0.003 seconds–1.

View larger version (29K): [in a new window]   Fig. 2. Effect of electroporation on intracellular Ca2+ stores in HeLa cells. Graphs show normalized fluorescence (F/FO(cytoplasm). (A) Cells in 2 mM extracellular Ca2+ were loaded with fluo-4AM and stimulated with 50 µM histamine at t100 seconds. (B) Cells in 2 mM EGTA were incubated for 15 minutes and stimulated with 50 µM histamine at t100 seconds. (C) Cells in 2 mM EGTA were electroporated with fluo-4 and stimulated with 50 µM histamine at t100 seconds. (D) Cells electroporated in 2 mM EGTA were stimulated with 2.1 mM Ca2+ at t30 seconds and then stimulated with 50 µM histamine t1000 seconds. Arrows indicate addition of 50 µM histamine.

View larger version (36K): [in a new window]   Fig. 3. FL-dextran distribution in electroporated cells. (A-G) HeLa cells. Passive diffusion rates of 19.5 kDa FL-dextran. Cells were electroporated with 19.5 kDa FL-dextran and nucleoplasmic to cytoplasmic fluorescence ratio (Fnp/Fcytoplasm) was monitored for 60 min in 2 mM EGTA () or 2.1 mM Ca2+ containing electroporation buffer (). (B) Graphs show Fnp/Fcytoplasm ratios of cells in the specified conditions. Order of groups of columns from left to right. Cells in 2 mM EGTA for 15 minutes; cells in 2 mM EGTA and 20 µg/ml WGA for 15 minutes; addition of 2 mM Ca2+ for 1 hour; addition of 2 mM Ca2+ for 1 hour with 20 µg/ml WGA electroporated; addition of 10 µM BAPTA-AM before electroporation in 2 mM EGTA. (C-G) Confocal images of 4.3-, 9.5-, 19.5-, 40- and 77-kDa FL-dextrans, respectively, in HeLa cells in 2 mM EGTA imaged 15 minutes after electroporation. (H,I) Dextran distributions in cortical glial cells (H) and cortical neurons (I). Order of groups of columns from left to right: cells in 2 mM EGTA for 15 minutes; cells in 2 mM EGTA and 20 µg/ml WGA for 15 minutes; addition of 2 mM Ca2+ for 1 hour; addition of 2 mM Ca2+ for 1 hour with 20 µg/ml WGA electroporated.

View larger version (23K): [in a new window]   Fig. 4. Cellular distribution of CaM in conditions of low Ca2+. Bar graphs show the ratio of nucleoplasmic to cytoplasmic fluorescence (Fnp/Fcytoplasm). (A) CaM distributions in glial, neuronal and HeLa cells in conditions of low Ca2+ concentration. Cells electroporated with 6ROX-CaM with or without 20 µg/ml WGA and 2 mM EGTA. (B) HeLa cells, electroporated with 6ROX-CaM and 6ROX-CaM1234 in 2 mM EGTA with or without 20 µg/ml WGA. 6ROX-CaM was also measured after electroporation of 10 mM EGTA instead of 2 mM EGTA. The distribution of 6ROX-CaM is shown in cells incubated with 10 µM mTrp peptide before electroporation (single bar at the right). (C) 6ROX-CaM diffusion rates in low intracellular Ca2+ concentration (<10 nM). HeLa cells were electroporated with 6ROX-CaM and with 20 µg/ml WGA in 2 mM EGTA and the Fnp/Fcytoplasm ratio for 6ROX-CaM was recorded for 1 hour.

View larger version (55K): [in a new window]   Fig. 5. Ca2+-dependent nuclear translocation of 6ROX-CaM in HeLa cells. (A) Cells electroporated with 6ROX-CaM and fluo-4 in 2 mM EGTA (t0) were stimulated (at t43) with 2.1 mM Ca2+. t1/2 f the translocation of 6ROX-CaM into the nucleus was 85 seconds. At t500, the concentration of 6ROX-CaM that was associated with nucleoli and was present in the nucleoplasm had increased 3.9-fold and 1.7-fold, respectively. Nuclear translocation of 6ROX-CaM was measured at 0, 100, 200, 300, 400 and 500 seconds (t0 to t500) and the data are shown in (B). Colored boxes indicate areas that have been measured for nuclear translocation of 6ROX-CaM. (B) Time course of the normalized 6ROX-CaM fluorescence (F/F(O)cytoplasm) in the () nucleolus, () nucleoplasm and (*) cytoplasm. The arrow indicates the addition of 2.1 mM Ca2+.

View larger version (32K): [in a new window]   Fig. 8. Inhibition of Ca2+-dependent nuclear translocation of 6ROX-CaM by mTrp peptide in HeLa cells. (A,B) Fluorescence of (A) 6ROX-CaM and (B) fluo-4 in HeLa cells in 2 mM EGTA incubated with 10 µM mTrp-peptide for 15 minutes (t0). At t50 cells were stimulated with 2.1 mM Ca2+. (C,D) Time course of normalized fluorescence (F/F(O)cytoplasm) of 6ROX-CaM (C) and normalized fluorescence (F/F(O)cytoplasm) of fluo-4 (D) in the nucleolus (), nucleoplasm () and cytoplasm (). Arrow in C indicates the addition of 2.1 mM Ca2+. (E) Relative fluorescence of TA-CaM with increasing concentrations of AIP. A solution of 21 nM TA-CaM, 100 mM KCl, 2 mM MgCl2 and 50 mM K-MES, pH 7.0 at 21°C was titrated with up to 140 nM mAIP in 100 µM Ca2+ (). (F) 6ROX-CaM diffusion rate at elevated levels of intracellular Ca2+. HeLa cells were electroporated with 6ROX-CaM and stimulated with 2.1 mM Ca2+ in the presence of 10 µM mTrp peptide for 30 minutes. The Fnp/Fcytoplasm ratio over a period of 30 minutes was monitored.

View larger version (42K): [in a new window]   Fig. 6. Ca2+-dependent nuclear translocation of 6ROX-CaM in glial cells (A) Glial cell electroporated with 6ROX-CaM and fluo-4 in 2 mM EGTA (t0). Cells were stimulated with 2.1 mM Ca2+ at t50. t1/2 of the translocation of 6ROX-CaM into the nucleus was 40 seconds. At t300 seconds, the concentration of 6ROX-CaM associated with nucleoli and in the nucleoplasm had increased 1.5-fold and 1.4-fold, respectively. Nuclear translocation of 6ROX-CaM and fluo-4 was measured at 0, 50, 150, 300, 600 and 900 seconds (t0 to t900) and the respective data are shown in (B and C) Colored boxes indicate areas that have been measured for nuclear translocation of 6ROX-CaM and fluo-4. (B) Time course of normalized 6ROX-CaM fluorescence (F/F(O)cytoplasm) in the () nucleolus, () nucleoplasm and (*) cytoplasm. The arrow indicates the addition of 2.1 mM Ca2+. (C) Time course of intracellular free Ca2+ concentration changes monitored by fluo-4 normalized fluorescence (F/F(O)cytoplasm) in the same area of the cell as 6ROX-CaM in B.

View larger version (34K): [in a new window]   Fig. 7. Ca2+-dependent nuclear translocation of 6ROX-CaM in rat cortical neurons. (A) Neurons electroporated with 6ROX-CaM and fluo-4 in 2 mM EGTA (first image, t0). The cell was stimulated with 2.1 mM Ca2+ (t50). t1/2 of the translocation of 6ROX-CaM into the nucleus was 90 seconds. At t300 seconds, the concentration of 6ROX-CaM associated in the nucleoplasm had increased 1.7-fold. Nuclear translocation of 6ROX-CaM and fluo-4 was measured at 0, 50, 270 and 400 seconds (t0 to t400) and the respective data are shown in (B and C) Colored boxes indicate areas that have been measured for nuclear translocation of 6ROX-CaM and fluo-4. (B) Time course of the Fnp/Fcytoplasm ratio of 6ROX-CaM and (C) and of the normalized fluorescence (F/F(O)cytoplasm) of fluo-4 in a representative record. Arrows indicate 2.1 mM Ca2+ addition.

View larger version (62K): [in a new window]   Fig. 9. Inhibition of nuclear translocation by WGA. (A-C) Inhibition of Ca2+-dependent nuclear translocation of 6ROX-CaM in HeLa, neuronal and glial cells by WGA. HeLa cells (A) were electroporated with 6ROX-CaM and 20 µg/ml WGA in 2 mM EGTA (t0). The cells was stimulated with 2.1 mM Ca2+ at t60. A further image, showing inhibition of nuclear translocation, was taken at t250. Neurons (B) were electroporated with 6ROX-CaM and 20 µg/ml WGA in 2 mM EGTA (t0). The cells were stimulated with 2.1 mM Ca2+ at t36. A further image, showing inhibition of nuclear translocation, was taken at t150. Glial cells (C) were electroporated with 6ROX-CaM and 20 µg/ml WGA in 2 mM EGTA (t0). The cells were stimulated with 2.1 mM Ca2+ at t43. A further image, showing inhibition of nuclear translocation, was taken at t179. (D) HeLa cell electroporated with 6ROX-CaM1234 and 20 µg/ml WGA in 2 mM EGTA (t0). The cell was stimulated with 2.1 mM Ca2+ at t79. A further image, showing inhibition of nuclear translocation, was taken at t200.

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