Dynamics of the CapG actin-binding protein in the cell nucleus studied by FRAP and FCS.
FRAP (fluorescence recovery after photobleaching) and FCS (fluorescence correlation spectroscopy) are spectroscopic methods for monitoring the dynamic distribution of proteins inside the nucleus of living cells. As an example we report our studies on the intracellular mobility of the actin-binding protein CapG in live breast cancer cells. This Gelsolin-related protein is a putative oncogene. It appears to be overexpressed especially in metastasizing breast cancer. Furthermore, the CapG protein is known to be involved in the motility control of non-muscle benign cells. Its increased expression triggers an increase in cell motility of benign cells. Thus it can be expected that in cancer cells overexpressing the CapG protein, motility, invasiveness and metastasis might be particularly promoted. Since the nuclear CapG fraction seems to be pivotal to the increase in cell motility, we focused our studies on the CapG mobility in cell nuclei of live breast cancer cells. Using FCS and FRAP we showed that the eGFP-tagged CapG is monomeric and characterized its diffusional properties on the microsecond to minute timescale. This information about the mobility and compartmentalization of CapG might help to provide insight into its function within the cell nucleus and give clues about its altered cellular function in malignant dedifferentiation.
Renz
Malte
M
German Cancer Research Center, Division Biophysics of Macromolecules, Im Neuenheimer Feld 580, D-69120, Heidelberg, Germany.
Langowski
Jörg
J
D001693:Biological Transport, Active; D001943:Breast Neoplasms / Q000378:metabolism / Q000503:physiopathology / Q000648:ultrastructure; D045744:Cell Line, Tumor; D002465:Cell Movement; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure*; D005260:Female; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D009361:Neoplasm Invasiveness; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*
D008840:Microfilament Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C066575:CAPG protein, human