Of these, the C-terminal β-NLS mediates nuclear transport conventionally through binding the nuclear transport molecule importin (Imp) β1, whereas the second N-terminal Ca M-NLS facilitates nuclear transport through direct binding to the calcium binding protein Ca M (3).
The dual nuclear import mechanisms of SRY contribute approximately equally to the overall nuclear accumulation of SRY (2 as a switch between the two (3).
Experiments were carried out in 5 μl containing 10 μ GFP-fusion protein, a 70-k Da Texas red dextran to assess nuclear integrity, untreated rabbit reticulocyte lysate (45 μg/μl; Promega, Madison, WI), and an ATP regenerating system (0.125 g/ml creatine phosphokinase; 30 m hsc70 (Stressgen Biotechnologies) and/or Ca M (Calbiochem, La Jolla, CA) purified protein.
Image analysis was performed using NIH Image J software as described above.
Following si RNA treatment, He La cells were transiently transfected and imaged live on a heated stage using an Olympus Fluoview 1000 microscope and an 100× oil immersion lens (Nikon) 20 h post-transfection.We find that direct binding of hsc70 to SRY·Ca M dependent on Ca Plasmids encoding the GFP-SRY full length and HMG wild type and mutant derivatives of the Ca M-NLS (M64T and R76P) and the β-NLS (Y127C and R133W) for bacterial and mammalian expression, as well as GFP-TRF1, GFP-T-ag-NLS (amino acids 111–135), and GFP alone, have all been described (3, 13, 14).-humidified atmosphere at 37 °C, in Dulbecco's modified Eagle's medium (DMEM; ICN Biomedicals, Costa Mesa, CA), supplemented with 10% heat-inactivated fetal calf serum (FCS; CSL, Ltd., Parkville, Victoria, Australia), 1 m hsc70 Stealth si RNA duplexes (Invitrogen) with the sense sequence 5′-UAAUUCUAAGUACAUUGAGACCAGC-3′) (7) or plasmid DNA expression constructs respectively, according to the manufacturer's specifications.Images were collected prior to photobleaching using 3% total laser power with excitation at 488 nm, scanning at a rate of 12.5 μs/pixel.Bleaching was performed in an area corresponding to ∼50% of the nucleus, scanning the area 40 times at a rate of 10 μs/pixel at 100% laser power.
Cells were imaged live by confocal laser scanning microscopy (CLSM, Nikon C1) 20h post-transfection and quantitative analysis of digitised images performed using Image J (National Institutes of Health, Bethesda, MD) for the nuclear (Fn) and cytoplasmic (Fc) fluorescence subsequent to subtraction of autofluorescence to derive the nuclear to cytoplasmic ratio (Fn/c) as described previously (3, 15).