Imaging the spatial dynamics of calmodulin activation during mitosis

Török, Katalin; Wilding, Martin; Groigno, Laurence; Patel, Rajnikant; Whitaker, Michael

doi:10.1016/s0960-9822(98)70275-1

Cited by 72 publications

(47 citation statements)

References 42 publications

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“…Characterization of the 6ROX-CaM, 6ROX-CaM1234 and mTrp probes Human liver CaM expressed in E. coli was labeled with 6-ROX, in conditions that had previously been used to generate Lys75-labeled TA-CaM and FL-CaM (Török and Trentham, 1994;Török et al, 1998a;Török et al, 2002). The reagent 6ROX was chosen to simultaneously image changes in intracellular [Ca 2+ ] by fluo-4 and CaM.…”

Section: Resultsmentioning

confidence: 99%

“…A cell permeant CaM-binding peptide based on the acetylated Trp peptide (Ac-RRKWQKTGHAVRAIGRL-CONH 2 ) (Török and CaM nuclear translocation Trentham, 1994;Török et al, 1998a;Török et al, 1998b) was generated by N-terminal myristoylation instead of acetylation. This myristoylated peptide (mTrp) was HPLC-purified to homogeneity and its K d for 2-chloro-(ε-amino-Lys75) [6-4-(N,N-diethhylamino)phenyl]-1,3,5-triazin-4-yl-CaM (TA-CaM) was determined by stopped-flow kinetic experiments in a Hi-Tech PQ/SF-51MX multimixing stoppedflow spectrofluorimeter (Hi-Tech Scientific, UK) in the solution conditions above with the exception of EGTA being replaced by 100 µM CaCl 2 .…”

Section: Peptide Inhibitorsmentioning

confidence: 99%

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

Thorogate

Török

2004

Journal of Cell Science

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Translocation from the cytosol to the nucleus is a major response by calmodulin (CaM) to stimulation of cells by Ca2+. However, the mechanisms involved in this process are still controversial and both passive and facilitated diffusion have been put forward. We tested nuclear translocation mechanisms in electroporated HeLa cells, rat cortical neurons and glial cells using novel calmodulin and inhibitor peptide probes and confocal microscopy. Passive diffusion of calmodulin across the nuclear membrane was measured in conditions in which facilitated transport was blocked and was compared to that of a similarly sized fluorescein-labeled dextran. Wheat germ agglutinin, which blocks facilitated transport but not passive diffusion, inhibited the nuclear entry of both wild-type and Ca2+-binding-deficient mutant calmodulin both in low and elevated [Ca2+]. Ca2+-dependent nuclear translocation was prevented by a membrane-permeant CaM inhibitor, the mTrp peptide, which indicated that it was specific to Ca2+/CaM. Diffusion of free CaM and Ca2+/CaM was considerably slower than the observed nuclear translocation by facilitated transport. Our data show that the majority of CaM nuclear entry occurred by facilitated mechanisms in all cell types examined, in part by a Ca2+-independent and in part by a Ca2+-dependent translocation mechanism.

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Section: Resultsmentioning

confidence: 99%

Section: Peptide Inhibitorsmentioning

confidence: 99%

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

Thorogate

Török

2004

Journal of Cell Science

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“…DISCUSSION A calcium increase in the perinuclear area where a variety of Ca 2ϩ stores are present (30) is required to trigger entry into the mitosis in early sea urchin embryos (31,32). Activation of calmodulin (CaM) occurs near the nuclear envelope just before mitosis entry (33). Ca 2ϩ transients are also associated with the progression of the meiotic cycle (34).…”

Section: Modulation Of the Actin Cytoskeleton By Mpf Is Responsible Fmentioning

confidence: 99%

The M-phase-promoting Factor Modulates the Sensitivity of the Ca2+ Stores to Inositol 1,4,5-Trisphosphate via the Actin Cytoskeleton

Lim

Ercolano

Kyozuka³

et al. 2003

Journal of Biological Chemistry

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The resumption of the meiotic cycle (maturation) induced by 1-methyladenine in prophase-arrested starfish oocytes is indicated by the breakdown of the germinal vesicle and is characterized by the increased sensitivity of the Ca 2؉ stores to inositol 1,4,5-trisphosphate (InsP 3 ) to InsP 3 starting at the animal hemisphere (where the germinal vesicle was originally located) and propagating along the animal/vegetal axis of the oocyte. This initiates Ca 2؉ signals around the germinal vesicle before nuclear envelope breakdown. Previous studies have suggested that the final activation of the maturation-promoting factor (MPF), a cyclin-dependent kinase, which is the major element controlling the entry of eukaryotic cells into the M phase, occurs in the nucleus. MPF is then exported to the cytoplasm where its activity is autocatalytically amplified following a similar animal/vegetal spatial pattern. We have investigated whether activated MPF was involved in the increased sensitivity of the Ca 2؉ response to InsP 3 . We have found that the development of increased sensitivity of the Ca 2؉ stores to InsP 3 receptors together with the Ca 2؉ signals in the perinuclear region was blocked in oocytes treated with the specific MPF inhibitor roscovitine. That the nuclear MPF activation is indeed required for changes of the InsP 3 receptors sensitivity was shown by enucleating or by dissecting oocytes into vegetal and animal hemispheres prior to the addition of 1-MA. MPF activity 50 min after 1-methyladenine addition was much lower in the enucleated oocytes and in the vegetal hemisphere, which did not contain the germinal vesicle, as compared with the animal hemisphere, which did contain it. The Ca 2؉ increase induced by InsP 3 under these experimental conditions correlated with the changes in actin cytoskeleton induced by MPF.

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“…CaM regulates the G2/M transition, spindle structure maintenance, furrow formation, cytokinesis completion, cell morphology and migration, etc, which are dependent upon the distribution of CaM in the nucleus, furrow and central, intracellular bridge and stress fibers (Li et al 1999;Yu et al 2004;Shen et al 2007;Yuan et al 2008).CaM itself has no enzymatic activity and it is believed that the spatiotemporal interaction of CaM and its targets is essential for CaM's regulatory function (Török et al 1998). CaM can alter the conformation of its partner when it binds to a relatively small region of the target protein with high affinity.…”

Section: Introductionmentioning

confidence: 99%

The association of CaM and Hsp70 regulates S-phase arrest and apoptosis in a spatially and temporally dependent manner in human cells

Huang

Wei

Peng

et al. 2009

Cell Stress and Chaperones

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The cell cycle is controlled by regulators functioning at the right time and at the right place. We have found that calmodulin (CaM) has specific distribution patterns during different cell-cycle stages. Here, we identify cell-cycle-specific binding proteins of CaM and examine their function during cell-cycle progression. We first applied immunoprecipitation methods to isolate CaMbinding proteins from cell lysates obtained at different cell-cycle phases and then identified these proteins using mass spectrometry methods. A total of 41 proteins were identified including zinc finger proteins, ribosomal proteins, and heat shock proteins operating in a Ca 2+ -dependent or independent manner. Fifteen proteins were shown to interact with CaM in a cell-phase-specific manner. The association of the selected proteins and CaM were confirmed with in vitro immunoprecipitation and immunostaining methods. One of the identified proteins, heat shock protein 70 (Hsp70), was further studied with respect to its cell-cycle-related function. In vivo fluorescence resonance energy transfer (FRET) analysis showed that the interaction of CaM and Hsp70 was found in the nucleus during the S phase. Overexpression of Hsp70 is shown to arrest cells at S phase and, thus, induce cell apoptosis. When we disrupted the CaM-Hsp70 association with HSP70 truncation without the CaM-binding domain, we found that S-phase arrest and apoptosis could be rescued. The results suggest that the spatial and temporal association of CaM and Hsp70 can regulate cell-cycle progression and cell apoptosis.

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Imaging the spatial dynamics of calmodulin activation during mitosis

Abstract: These data suggest that calmodulin regulates mitotic transitions and demonstrate the utility of fluorescent adducts for studying protein activation in living cells with good temporal and spatial resolution.

Cited by 72 publications

References 42 publications

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

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

The M-phase-promoting Factor Modulates the Sensitivity of the Ca2+ Stores to Inositol 1,4,5-Trisphosphate via the Actin Cytoskeleton

The association of CaM and Hsp70 regulates S-phase arrest and apoptosis in a spatially and temporally dependent manner in human cells

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