Mol. Cells 2018; 41(2): 103-109
Published online January 29, 2018
https://doi.org/10.14348/molcells.2018.2170
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: tjeon@chosun.ac.kr
Calcium ions are involved in the regulation of diverse cellular processes. Fourteen genes encoding calcium binding proteins have been identified in
Keywords calcium binding proteins, cell migration, development, dictyostelium
Calcium ions are involved in the regulation of diverse cellular processes such as chemotaxis, cell adhesion, and muticellular development (Clapham, 2007; Lusche et al., 2009; Siu et al., 2011). Calcium ions regulate cellular processes though their interactions with calcium-binding proteins (CBPs). Calcium-binding proteins function as calcium buffers to control the intracellular concentration of calcium ions or as calcium sensors to transduce signals to a series of downstream effectors (Chin and Means, 2000; Clapham, 2007).
Fourteen calcium-binding proteins (CBP) have been identified in
Cell adhesion assay was performed as described previously (Mun et al., 2014). Log-phase growing cells on the plates were washed and resuspended at a density of 2 × 106 cells/ml in 12 mM Na/K phosphate buffer. 200 μl of the cells were placed and attached on the 6-well culture dishes. Before shaking the plates, the cells were photographed and counted for calculating the total cell number. To detach the cells from the plates, the plates were constantly shaken at 150 rpm for 1 h, and then the attached cells were photographed and counted (attached cells) after the medium containing the detached cells was removed. Cell adhesion was presented as a percentage of attached cells compared with total cells.
Development was performed as described previously (Jeon et al., 2009). Exponentially growing cells were harvested and washed twice with 12 mM Na/K phosphate buffer (pH 6.1) and resuspended at a density of 3.5 × 107 cells/ml. 50 μl of the cells were placed on Na/K phosphate agar plates and developed for 24 h. For development of the cells under submerged conditions, exponentially growing cells (2 × 106 cells) were placed and developed in 12-well plates containing Na/K phosphate buffer. The muticellular developmental organisms was photographed and examined with a phase-contrast microscope at the indicated times in the figures.
Chemotaxis towards cAMP was examined as described previously (Jeon et al., 2007b; Mun et al., 2014). The aggregation-competent cells were prepared by incubating the cells at a density of 5 × 106 cells/ml in Na/K phosphate buffer for 10 h. Cell migration was analyzed using a Dunn Chemotaxis Chamber (Hawksley). The images of chemotaxing cells were taken at time-lapse intervals of 6 s for 30 min using an inverted microscope (IX71; Olympus). The data were analyzed using the NIS-Elements software (Nikon) and Image J software (National Institures of Health). For examining cell migration in the aggregation stage of development, 2.5% of RFP-labeled wild-type cells and 2.5% of cells expressing GFP-CBP7 were mixed with 95% of unlabeled wild-type cells and developed on Na/K phosphate agar plates. The fluorescence images of moving cells at the aggregation step of development were captured by the NIS-Elements software, and the movement of fluorescent cells was traced and analyzed using the Image J software. ‘Trajectory speed’ was used to quantify motility of the cells. The trajectory speed is the total distance travelled of a cell divided by time. ‘Directionality’ is a measure of how straight the cells move. Cells moving in a straight line have a directionality of 1.0. It was calculated as the distance moved over the linear distance between the start and the finish.
Fluo-4 AM, a fluo calcium indicator, was obtained from Molecular Probes, and the cells were labeled with fluo-4 AM according to the protocol provided by manufactures.
The total RNAs from wild-type cells and
The results were expressed as the mean ± standard deviation (SD) (at least three independent experiments). Data were analyzed using Student’s two-tailed
There are 14 genes encoding CBP proteins in the genomes of
To investigate the functions of CBP7, we prepared
We first examined the morphology of
Upon starvation,
To further investigate impairment of the aggregation stage in GFP-CBP7 cells, we examined the aggregation abilities of cells by placing them on 12-well plates containing developmental buffers instead of agar plates (Fig. 2B). In developmental buffer, wild-type cells and
In contrast with wild-type and
These results related to cAMP-dependent chemotaxis were further confirmed using a cell migration assay with chimeric cells containing 95% unlabeled wild-type cells, 2.5% RFP-labeled wild-type cells, and 2.5% GFP-CBP7 expressing cells. All cells were simultaneously starved of nutrients, and the migration speeds of the labeled cells were measured during the aggregation stage of development (Fig. 4). Wild-type cells exhibited a moderate moving speed (4.53 μm/min) that was significantly higher than GFP–CBP7 cells (2.03 μm/min) during aggregation at the 6 h time point (Fig. 4), indicating that GFP-CBP7 cells have a defect in forming aggregates by cAMP-dependent chemotaxis to the center of aggregation.
Influx of Ca2+ and elevation of its level by cAMP are important in aggregation of
Our results demonstrate that CBP7 is required for cell spreading and cell-substrate adhesion and has a negative impact on multicellular development, possibly through inhibition of chemoattractant-mediated cell migration in the initial aggregation stage of development.
We propose one possible mechanism by which CBP7 may inhibit cell migration and development, a hypothesis that should be addressed in future studies. Low levels of intracellular calcium in CBP7 overexpressing cells resulted in the loss of directional cell migration during the aggregation stage of development. CBP7 has four highly conserved EF-hand motifs for Ca2+ binding, which are rich in negatively charged amino acids such as glutamic acids and aspartic acids (Gifford et al., 2007) and has been demonstrated as a calcium binding protein through Ca2+-overlay experiments (Sakamoto et al., 2003). In this study, measurement of the free intracellular calcium levels revealed that overexpression of CBP7 resulted in significantly lower levels of calcium in the cytosol and that loss of CBP7 caused slightly increased levels of intracellular calcium compared to wild-type cells. Based on previously reported data and the results presented herein, CBP7 proteins appear to directly bind to free intracellular calcium and function as a calcium buffer to lower the levels of intracellular calcium. Low level of intracellular calcium in CBP7 overexpressing cells might affect chemoattractant-directed cell migration. In agreement with our results, many studies have demonstrated that calcium ions are involved in cell migration. In
Another possibility is that CBP7 is both a calcium sensor and a downstream effecter of calcium ions, as was illustrated for CBP3 (Lee et al., 2005; Mishig-Ochiriin et al., 2005). CBP3 has been shown to interact with the actin cytoskeleton and play important roles in cell aggregation and slug migration during development (Lee et al., 2005). Moreover, CBP3 undergoes conformational changes upon binding to Ca2+, which allows for interactions with binding partners (Mishig-Ochiriin et al., 2005). However, the roles of CBP7 seem to be opposite to those of CBP3. It was reported that cells overexpressing CBP3 showed accelerated cellular aggregation and increased numbers of small aggregates and fruiting body (Lee et al., 2005), whereas CBP7 overexpressing cells displayed no cell aggregation and complete loss of development. A large number of proteins have been identified as CBP protein-binding partners. CBP1 and CBP3 interact with the actin cytoskeleton and CBP1 also interacts with another calcium-binding protein, CBP4a, and the actin-binding proteins, protovillin and EF-1a, in yeast two-hybrid experiments (Dharamsi et al., 2000; Dorywalska et al., 2000). Nucelomorphin, a cell cycle checkpoint protein, is a known binding protein of CBP4a (Catalano and O’Day, 2013; Myre and O’Day, 2004). Further experiments are in progress to determine CBP7-binding proteins.
Mol. Cells 2018; 41(2): 103-109
Published online February 28, 2018 https://doi.org/10.14348/molcells.2018.2170
Copyright © The Korean Society for Molecular and Cellular Biology.
Byeonggyu Park, Dong-Yeop Shin, and Taeck Joong Jeon*
Department of Biology & BK21- Plus Research Team for Bioactive Control Technology, College of Natural Sciences, Chosun University, Gwangju 61452, Korea
Correspondence to:*Correspondence: tjeon@chosun.ac.kr
Calcium ions are involved in the regulation of diverse cellular processes. Fourteen genes encoding calcium binding proteins have been identified in
Keywords: calcium binding proteins, cell migration, development, dictyostelium
Calcium ions are involved in the regulation of diverse cellular processes such as chemotaxis, cell adhesion, and muticellular development (Clapham, 2007; Lusche et al., 2009; Siu et al., 2011). Calcium ions regulate cellular processes though their interactions with calcium-binding proteins (CBPs). Calcium-binding proteins function as calcium buffers to control the intracellular concentration of calcium ions or as calcium sensors to transduce signals to a series of downstream effectors (Chin and Means, 2000; Clapham, 2007).
Fourteen calcium-binding proteins (CBP) have been identified in
Cell adhesion assay was performed as described previously (Mun et al., 2014). Log-phase growing cells on the plates were washed and resuspended at a density of 2 × 106 cells/ml in 12 mM Na/K phosphate buffer. 200 μl of the cells were placed and attached on the 6-well culture dishes. Before shaking the plates, the cells were photographed and counted for calculating the total cell number. To detach the cells from the plates, the plates were constantly shaken at 150 rpm for 1 h, and then the attached cells were photographed and counted (attached cells) after the medium containing the detached cells was removed. Cell adhesion was presented as a percentage of attached cells compared with total cells.
Development was performed as described previously (Jeon et al., 2009). Exponentially growing cells were harvested and washed twice with 12 mM Na/K phosphate buffer (pH 6.1) and resuspended at a density of 3.5 × 107 cells/ml. 50 μl of the cells were placed on Na/K phosphate agar plates and developed for 24 h. For development of the cells under submerged conditions, exponentially growing cells (2 × 106 cells) were placed and developed in 12-well plates containing Na/K phosphate buffer. The muticellular developmental organisms was photographed and examined with a phase-contrast microscope at the indicated times in the figures.
Chemotaxis towards cAMP was examined as described previously (Jeon et al., 2007b; Mun et al., 2014). The aggregation-competent cells were prepared by incubating the cells at a density of 5 × 106 cells/ml in Na/K phosphate buffer for 10 h. Cell migration was analyzed using a Dunn Chemotaxis Chamber (Hawksley). The images of chemotaxing cells were taken at time-lapse intervals of 6 s for 30 min using an inverted microscope (IX71; Olympus). The data were analyzed using the NIS-Elements software (Nikon) and Image J software (National Institures of Health). For examining cell migration in the aggregation stage of development, 2.5% of RFP-labeled wild-type cells and 2.5% of cells expressing GFP-CBP7 were mixed with 95% of unlabeled wild-type cells and developed on Na/K phosphate agar plates. The fluorescence images of moving cells at the aggregation step of development were captured by the NIS-Elements software, and the movement of fluorescent cells was traced and analyzed using the Image J software. ‘Trajectory speed’ was used to quantify motility of the cells. The trajectory speed is the total distance travelled of a cell divided by time. ‘Directionality’ is a measure of how straight the cells move. Cells moving in a straight line have a directionality of 1.0. It was calculated as the distance moved over the linear distance between the start and the finish.
Fluo-4 AM, a fluo calcium indicator, was obtained from Molecular Probes, and the cells were labeled with fluo-4 AM according to the protocol provided by manufactures.
The total RNAs from wild-type cells and
The results were expressed as the mean ± standard deviation (SD) (at least three independent experiments). Data were analyzed using Student’s two-tailed
There are 14 genes encoding CBP proteins in the genomes of
To investigate the functions of CBP7, we prepared
We first examined the morphology of
Upon starvation,
To further investigate impairment of the aggregation stage in GFP-CBP7 cells, we examined the aggregation abilities of cells by placing them on 12-well plates containing developmental buffers instead of agar plates (Fig. 2B). In developmental buffer, wild-type cells and
In contrast with wild-type and
These results related to cAMP-dependent chemotaxis were further confirmed using a cell migration assay with chimeric cells containing 95% unlabeled wild-type cells, 2.5% RFP-labeled wild-type cells, and 2.5% GFP-CBP7 expressing cells. All cells were simultaneously starved of nutrients, and the migration speeds of the labeled cells were measured during the aggregation stage of development (Fig. 4). Wild-type cells exhibited a moderate moving speed (4.53 μm/min) that was significantly higher than GFP–CBP7 cells (2.03 μm/min) during aggregation at the 6 h time point (Fig. 4), indicating that GFP-CBP7 cells have a defect in forming aggregates by cAMP-dependent chemotaxis to the center of aggregation.
Influx of Ca2+ and elevation of its level by cAMP are important in aggregation of
Our results demonstrate that CBP7 is required for cell spreading and cell-substrate adhesion and has a negative impact on multicellular development, possibly through inhibition of chemoattractant-mediated cell migration in the initial aggregation stage of development.
We propose one possible mechanism by which CBP7 may inhibit cell migration and development, a hypothesis that should be addressed in future studies. Low levels of intracellular calcium in CBP7 overexpressing cells resulted in the loss of directional cell migration during the aggregation stage of development. CBP7 has four highly conserved EF-hand motifs for Ca2+ binding, which are rich in negatively charged amino acids such as glutamic acids and aspartic acids (Gifford et al., 2007) and has been demonstrated as a calcium binding protein through Ca2+-overlay experiments (Sakamoto et al., 2003). In this study, measurement of the free intracellular calcium levels revealed that overexpression of CBP7 resulted in significantly lower levels of calcium in the cytosol and that loss of CBP7 caused slightly increased levels of intracellular calcium compared to wild-type cells. Based on previously reported data and the results presented herein, CBP7 proteins appear to directly bind to free intracellular calcium and function as a calcium buffer to lower the levels of intracellular calcium. Low level of intracellular calcium in CBP7 overexpressing cells might affect chemoattractant-directed cell migration. In agreement with our results, many studies have demonstrated that calcium ions are involved in cell migration. In
Another possibility is that CBP7 is both a calcium sensor and a downstream effecter of calcium ions, as was illustrated for CBP3 (Lee et al., 2005; Mishig-Ochiriin et al., 2005). CBP3 has been shown to interact with the actin cytoskeleton and play important roles in cell aggregation and slug migration during development (Lee et al., 2005). Moreover, CBP3 undergoes conformational changes upon binding to Ca2+, which allows for interactions with binding partners (Mishig-Ochiriin et al., 2005). However, the roles of CBP7 seem to be opposite to those of CBP3. It was reported that cells overexpressing CBP3 showed accelerated cellular aggregation and increased numbers of small aggregates and fruiting body (Lee et al., 2005), whereas CBP7 overexpressing cells displayed no cell aggregation and complete loss of development. A large number of proteins have been identified as CBP protein-binding partners. CBP1 and CBP3 interact with the actin cytoskeleton and CBP1 also interacts with another calcium-binding protein, CBP4a, and the actin-binding proteins, protovillin and EF-1a, in yeast two-hybrid experiments (Dharamsi et al., 2000; Dorywalska et al., 2000). Nucelomorphin, a cell cycle checkpoint protein, is a known binding protein of CBP4a (Catalano and O’Day, 2013; Myre and O’Day, 2004). Further experiments are in progress to determine CBP7-binding proteins.
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