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|Title:||Development of new tools for studying nuclear lamin function||Authors:||DUCKERS, Connie||Issue Date:||2006||Abstract:||Lamins are nuclear intermediate filament proteins encoded by the LMNA, LMNB1, and LMNB2 genes. They form a filamentous meshwork underneath the inner nuclear membrane (INM) and intranuclear structures. Lamins bind to many nuclear proteins and participate in various intranuclear activities. They can be divided in two subgroups; A- and B-type lamins. While A-type lamins are differentially expressed, B-type lamins are expressed in all cells and are essential for cell survival. Mutations in LMNA were found to cause an array of seemingly unrelated diseases. However, no such mutations have been found in LMNB1 or LMNB2. To study lamin A/C and lamin B2 function, two different model systems were developed. To study the functions of A-type lamins an in vitro model that allows controllable down- and upregulation of A-type lamin expression according to the ‘Tet-On’ system was developed. This system permits the study of these proteins in genetically identical cells and probably reduces the effects of inter-cell line variability. Controllable lamin A/C expression was achieved in HCT116 colon cancer cells. Lamin A/C knockdown had several effects on these cells; increase in proliferation, altered morphology, and reappearance of the gap-junction protein connexin 43. These findings support the previously suggested role of lamins in regulating cell proliferation. Altered cell-cell adhesion may be caused by an effect of A-type lamins on gene regulation. However, these differences were not confirmed in cells with tetracycline controllable lamin expression. Since cell biological experiments were only started recently these results need further research. The two main B-type lamins are lamin B1 and B2. Relatively little is known about lamin B2. To investigate lamin B2 function, a lamin B2-enhanced green fluorescent protein (EGFP) expression vector was constructed and transfected into mammalian cells. High lamin B2- EGFP expression resulted in nuclear growth and numerous nuclear abnormalities. Lamin B2 dynamics during mitosis were studied by time-lapse imaging with a fluorescence confocal laser scanning microscope. Interestingly, lamin B2 only reassembled after cytokinesis has completed. This emphasizes differences in lamin B1 and lamin B2 function during mitosis, since lamin B1 reassembles at late anaphase to early telophase. Lamin B2 even seems to relocate into the nucleus after A-type lamins have reassembled.||Document URI:||http://hdl.handle.net/1942/1051||Category:||T2||Type:||Theses and Dissertations|
|Appears in Collections:||Master theses|
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checked on May 25, 2022
checked on May 25, 2022
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