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pended in diethyl-pyrocarbonate-treated water. ODNs (8 µM) were added to a cell medium three times at 4-h intervals (6).

Rhodamine-Actin-Based Detection of Barbed Ends of Actin Filaments. Stock rhodamine-labeled actin was thawed and diluted with 1 mM Hepes (pH 7.5), 0.2 mM MgCl2, and 0.2 mM ATP, sonicated, and clarified in Beckman centifuge at 95 krpm, for 20 min. Cells were permeabilized with 20 mM Hepes (pH 7.5), 138 mM KCl, 4 mM MgCl2, 9 mM EGTA, 0.25 mg/ml saponin, 1 mM ATP, 1% BSA containing 0.45 µm rhodamine-actin that was added to the lysis buffer just before application to cells. One to three minutes after incubation, cells were fixed for 5 min with 3.7% formaldehyde in PBS, incubated with 0.1 M glycine in PBS for 10 min, and washed with PBS. Cells were stained with 1 µM fluorescein phalloidin in buffer for 40 min in humidified chamber, washed, and mounted on 0.1 M N-propylgallate in 50% glycerol in PBS, pH 7.0.

Immunofluorescence. Cells were plated on coverslips, fixed in 3.7% formaldehyde, permeabilized with 0.5% Triton in PBS, and incubated with primary antibodies to ß-actin (a gift of Ira Herman, Tufts Medical School, Boston) and secondary fluorescein-labeled antibodies to rabbit IgG for 1 h and mounted as described (12).

In Situ Hybridization. Chicken ß-actin-specific 3' UTR probes (five probes of 50 nt each, with five amino linkers per probe spaced ˜10 nt apart) were synthesized on an Applied Biosystems 394 DNA/RNA Synthesizer. Chicken ß-actin probes were labeled with CY3. To detect ß-actin mRNAs, coverslips were rehydrated in PBS, permealized with 0.5% Triton in PBS for 10 min, and then hybridized for 3 h at 37°C with 5 ng of the mixture of five oligonucleotides. Each oligonucleotide can hybridize independently with ß-actin mRNA so as to increase the signal to noise when all five have hybridized to a single molecule (25 fluorochromes total; ref. 13). Coverslips were washed twice with 50% formamide in 2×SSC (300 mM NaCl, 30 mM sodium citrate, pH 7.0), then in 2×SSC, 1×SSC, and mounted.

High-Resolution Microscopy. An Olympus BX60 microscope was used with a×60 planapo objective numerical aperture 1.4. Digital images were captured by using a Photometries camera and CELLSCAN software.

Computer-Assisted Analysis of Cell Behavior. Cells were recorded with an Olympus microscope equipped with a charge-coupled device camera through a×10 objective with a 1-min time interval between image frames over 60 min. Images were processed with DIAS (Dynamic Image Analysis System) software (14). Cell motility data were displayed as an overlay of cell perimeters, i.e., as a stack of every fifth video frame (cell perimeter plot) and as a centroid plot showing the location of the geometrical center of the cell as a function of time.

Results

Antisense Treatment of Cells. It was shown previously that cisacting elements in the 3' UTR of chicken ß-actin mRNA were responsible for the localization of this mRNA. The 54 nt 3' of the stop codon were most potent in localizing ß-actin mRNA. This region is called the zip code and can be divided into A, B, and C regions (6). In this study, an antisense ODN, complementary to the 3' 18 nt of the zip code was used (C). For a control, the sense strand (C+) was used.

Effects of Antisense ODNs on Cell Motility. It was reported previously that the velocity of cell locomotion is reduced by treatment of cells with antisense-oligos directed against the zip code of ß-actin mRNA (2). To confirm and extend this observation we

Table 1. Percent of cells with ß-actin mRNA, ß-actin protein, and nucleation (barbed ends) localized to the leading edge as a function of zip code antisense (C) or sense (C+) oligonucleotide treatments

 

C % localization

C+ % localization

 

Leading edge

Diffuse

Leading edge

Diffuse

ß-actin

mRNA

(n)

33

(186)

67

(375)

58

(392)

42

(288)

Barbed

ends

(n)

30

(130)

70

(303)

70

(210)

30

(90)

ß-actin

protein

(n)

32

(70)

68

(149)

68

(128)

32

(60)

repeated these experiments. CEFs were treated with zip code antisense (C) or sense (C+) ODNs. The distribution of ß-actin mRNA was determined in each population by using fluorescence in situ hybridization. Cells treated with antisense showed decreased localization of ß-actin mRNA to the leading edge, whereas cells treated with sense ODNs (control) localized the mRNA to the leading edge to an extent that was statistically indistinguishable from untreated cells (Table 1, ß-actin mRNA).

The average path length migrated by antisense-treated and control cells was measured as a change in the nuclear position during a 60-min observation period. In Table 2, the average path lengths for 100 antisense-treated and 64 control cells are presented. The antisense-treated cells migrated shorter distances, and this difference was statistically significant. This result is similar to that reported previously by Kislauskis et al. (2). However, the underlying mechanism for this observation has not been investigated. Therefore, we subjected the cells to a more rigorous analysis of their motility to ascertain which of the components of cell motility was most affected. To do this we correlated various aspects of cell motility with ß-actin mRNA localization. Treated and control cells were monitored by using an inverted microscope supplemented with a heating chamber. Time-lapse movies were obtained over 60 min with 1-min intervals between frames. Fig. 1 demonstrates the difference in behavior between these two cell populations. In the presence of the zip code antisense, cells did not translocate appreciably, whereas in the presence of sense ODNs, the cells continued to migrate. The movies obtained in this way were analyzed by using the Dynamic Image Analysis System (Materials and Methods). Several cell motility parameters were determined: net path length, average speed, average instantaneous speed (protrusion velocity), directionality, and persistence (Table 3). Delocalization of ß-actin mRNA in CEFs correlated with a significant decrease in net path length and average speed. Total path length and average protrusive velocity were not statistically different from control cells (Table 3). These results are explained by a decrease in the directionality and persistence of movement

Table 2. Average path length migrated by CEFs (measured as a change in the nuclear position) during 60 min as a function of zip code antisense (C) or sense (C+) oligonucleotide treatment

 

C (n=100)

C+ (n=64)

Average net path length, µm

12.67

16.32

SE

0.87

1.56

t test

4.39%



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