Cellular structures and compartmentalization is the result of a dynamic steady state exchange between its components. This thesis is focused in investigations of dynamic properties of green fluorescent protein (GFP)-labeled proteins in live cells using confocal laser microscopy in combination with bleaching techniques such as fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP).
Studies of dynamic properties of c-Myc in living cells showed that c-Myc is shuttling between the nucleus and the cytoplasm. c-Myc also enters the nucleoli during certain conditions. Nucleolar c-Myc is dynamically associated to structural component(s) of nucleoli, but can exchange with soluble pools in the nucleoplasm and cytoplasm.
Photobleaching experiments showed that a significant fraction of HIV-1 Vpr is dynamically associated with the NE and rapidly exchanges between the nucleoplasm and the cytoplasm. The yeast two-hybrid system, pull-down experiments and co-immunoprecipitating was used to show that Vpr interacts specifically and directly with a domain in the N-terminal portion of the NPC protein hCG1. The results suggest that the specific interaction of HIV-1 Vpr with the nucleoporin hCG1 results in the dynamic retention of Vpr at the nuclear envelope.
The distribution and dynamic properties of NPC proteins was investigated in NIH/3T3 cells, lacking the pore membrane protein gp210. Confocal laser scanning microscopy and FRAP experiments showed that the absence of gp210 from nuclear pores of NIH/3T3 cells did neither alter the distribution nor dynamic properties of POM121 and NUP107 (two NPC proteins stably integrated in the NPC).
Degradation of the integral nuclear pore membrane protein POM121 during apoptosis was investigated in relation to other apoptotic events. POM121 cleavage, which is the earliest sign of dismantling of the nuclear membrane, is due to caspase-3-dependent cleavage at aspartate-531. Loss of nuclear compartmentalization in live cells undergoing apoptosis was monitored as appearance of GFP-NLS in the cytoplasm. The time of appearance of cytoplasmic GFP-NLS correlated with caspase-3-dependent cleavage of POM121. Both events occured concomitantly with collapsing of chromatin against the nuclear periphery, but preceded the onset of nucleosomal DNA fragmentation.
Translocation ability of the cell-penetrating peptide, transportan, into living cells was investigated. Recombinantly expressed GFP was purified and conjugated to chemically synthesized transportan via a disulfide bond and added to tissues culture cells. Transportan was able to internalize a 27 kDa protein such as GFP in a native folded state into living cells.
Stockholm: Institutionen för neurokemi , 2004. , 41 p.
2004-05-28, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00