The molecular organization of GFP chimeras. The dynamics of K119TmATS-GFP (A-J) expressed in 3D7 (left panels) or D10 (right panels) was compared with that of KAHRP1-119-GFP in 3D7 transfectants (L) and fluorescein-BSA in resealed RBCs (K). In each case, the first panel shows the differential interference contrast image. The fluorescence images (green) comprise prebleach (pre) and postbleach images at the times (s) indicated following the bleach pulse. (The first postbleach image is defined as zero time.) The position of the bleach pulse is indicated by white arrows. In some images of D10 transfectants, bright “particles” that move position between successive images are marked with blue arrows. Scale bars = 2 μm. Panels A and B show bleaching of Maurer cleft–associated K119TmATS-GFP with 100- and 200-ms bleach pulses, respectively. Panels C-F show FLIP measurements of K119TmATS-GFP where the region indicated by the arrow was subjected to 5 or ten 1- to 2-second bleach pulses separated by 10 to 20 seconds. The 2 prebleach images shown for the cell in panel C were taken at different photomultiplier gains to illustrate the faint nature of the fluorescence associated with the RBC cytosol. Note that the FLIP images have had their brightness and contrast enhanced relative to the prebleach images to permit direct comparisons. Panels G and H illustrate the localized nature of bleaching of RBC cytosol obtained with 0.5- and 1.0-second bleach pulses, respectively, and subsequent recovery. The B image was calculated from the prebleach and postbleach images. Panels I and J show photobleaching measurements of K119TmATS-GFP, illustrating recovery on a time scale of 5 to 10 seconds. Panel K shows photobleaching of a resealed ghost containing fluorescein-BSA with a bleach pulse of 25 ms, indicating very rapid diffusion. Panel L shows photobleaching of a 3D7-KAHRP1-119-GFP with a bleach pulse of 1 second and recovery on a time scale of a few hundred milliseconds. In the case of panels I-L, only the regions indicated by the dotted lines in the differential interference contrast (DIC) image were imaged in the photobleaching measurements. The graphs shown in panels I-L show the temporal dependence of fluorescence intensity in the bleached region following the bleach event, relative to prebleach intensity.
