Coiled body heterogeneity induced by G(1) arrest with amiloride + bumetanide.
Ki67 is a nuclear protein expressed in proliferating cells, but not in quiescent or G(0)-arrested cells. Similar to the proliferating cell nuclear antigen and several other well-characterized molecules, Ki67 exhibits a repeating pattern of regulated expression and redistribution during the cell cycle, making it a useful marker for cell cycle phase. In addition to other structures labeled, concentrated foci may be observed in the nucleus and sometimes the cytoplasm. We observed that these Ki67 foci can be found at any stage of the endothelial cell cycle. They are not coincident with coiled bodies (CB), as determined in double-label immunofluorescence experiments with anti-Ki67 and antibodies to the CB marker protein pigpen. However, arrest of BPA47 endothelial cells in G(1) with amiloride + bumetanide induces colocalization of pigpen and Ki67 in 40% of cells exhibiting Ki67 foci. We conducted a series of experiments to examine the possibilities that pigpen was exported from CB and into unique, Ki67-containing foci or that Ki67 was imported into pigpen-containing CB. Our results showed us that although CB typically contain both coilin and pigpen, amiloride + bumetanide-induced G(1) arrest reconfigured the CB compartment into three populations of foci: one containing pigpen without coilin, the second containing coilin without pigpen, and a third containing both pigpen and coilin together. Furthermore, G(1) arrest resulted in Ki67 redistribution into both coilin- and pigpen-containing foci. The results suggest that under certain conditions, "resident" CB proteins can be differentially redistributed, and proteins not previously recognized as resident in CB can be driven into that compartment. Our observations underscore the fluid nature of CB. They demonstrate that previously reported heterogeneity in the CB compartment can be amplified by a specific experimental manipulation. This may be useful in future analyses of protein trafficking within the CB compartment and between CB and other cellular compartments. Finally, the redistribution of Ki67 into CB represents a new finding for a widely expressed but poorly understood molecule, one that may be useful in elucidating function.
Alliegro
Mark C
MC
Department of Cell Biology and anatomy, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA. mallie@lsuhsc.edu
D000584:Amiloride / Q000494:pharmacology*; D000818:Animals; D002034:Bumetanide / Q000494:pharmacology*; D002417:Cattle; D002453:Cell Cycle; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D020541:Coiled Bodies / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins; D004730:Endothelium, Vascular / Q000737:chemistry; D016193:G1 Phase / Q000187:drug effects; D019394:Ki-67 Antigen / Q000032:analysis / Q000378:metabolism; D009687:Nuclear Proteins / Q000032:analysis / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D016601:RNA-Binding Proteins / Q000032:analysis / Q000378:metabolism
D004268:DNA-Binding Proteins; D019394:Ki-67 Antigen; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; C099524:pigpen protein, bovine; D002034:Bumetanide; C068921:p80-coilin; D000584:Amiloride