Fig. 5. Particle and squiggle motility contribute to fluorescence recovery after
photobleaching (FRAP). A bleach zone (areas denoted by red outlines) was made
along the length of a neurite in a GFP-peripherin-expressing PC12 cell
cultured in DM for 48 hours. Photobleaching required 
14 seconds (see A2,
C2). Fluorescence recovery was subsequently monitored by determining the
fluorescence intensity ratio (F.I.; see Materials and Methods) within this
region by capturing images at 60 seconds intervals for 800 seconds.
Using this ratio to determine the overall rate of fluorescence recovery, it
was observed that the t1/2 (see A) for peripherin in this cell is
400 seconds (also see A1-3, which represent the region prior to
photobleaching, immediately following photobleaching at 14 seconds and
800 seconds after bleaching). The F.I. ratio was also determined for two
subdivisions of the same region indicated by the large red box [B; B1-3
(subdivisions outlined in orange and green) at 333, 385 and 448 seconds].
Using this more detailed analysis of recovery, transient peaks in the F.I.
ratio were observed (see green and orange lines in B). These peaks were
attributable to the rapid movements of bright squiggles and particles seen
moving into and out of the bleach zone throughout the recovery period. In C,
the same neurite has been separated into 10 subdivisions, including those
depicted in B, each indicated by a different color on the graph (also see
C1-3). This resulted in the complex series of peaks detected within the bleach
zone during recovery. In addition, FRAP analysis was performed in a similar
manner on another neurite of a PC12 cell grown in DM for 48 hours and then in
DM containing 5 µg/ml colchicine for 30-45 minutes (D-1 to D-3). There was
very little recovery up to 930 seconds after photobleaching (compare A with
D). Interestingly, there was almost no fluctuation observed in the F.I. ratio
and no particles or squiggles were observed to move within the bleach zone.
Images E-H show GFP-peripherin particle movements through a photobleached area
of the neurite shown in the phase image (I). Images were taken at 5 second
intervals following photobleaching. The particle marked with an arrowhead
moved in an anterograde direction at rates that ranged from 0.31-1.0
µm/second (also see Movie 2, available at
jcs.biologists.org/supplemental).
Diagrams of three trajectories of GFP-peripherin squiggles were made from
another neurite (J). Black dots represent the beginning of squiggle tracks.
Reversals of particles and squiggles were very infrequent within neurites of
differentiated cells. E-H, Bar, 2 µm; I, J, Bars, 5 µm.