Microtubule-dependent PKC-{alpha} localization in A7r5 smooth muscle cells

A. C. Dykes,1 M. E. Fultz,1 M. L. Norton,2 and G. L. Wright1

1Department of Physiology, Joan C. Edwards School of Medicine, and 2Department of Chemistry, Marshall University, Huntington, West Virginia 25704

Submitted 7 November 2002 ; accepted in final form 4 March 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Using laser scanning confocal, fluorescence resonance energy transfer (FRET), and atomic force (AFM) microscopy, we investigated association of protein kinase C (PKC)-{alpha} with microtubules during stimulus-induced relocalization in A7r5 smooth muscle cells. Confocal microscopy with standard immunostaining techniques confirmed earlier observations that colchicine disruption of microtubules blocked PKC-{alpha} localization in the perinuclear region of the cell caused by phorbol 12,13-dibutyrate (PDBu; 106M). Dual immunostaining suggested colocalization of PKC-{alpha} and {beta}-tubulin in both unstimulated and PDBu-treated cells. This finding was verified by FRET microscopy, which indicated that association of PKC-{alpha} was heterogeneous in distribution and confined primarily to microtubules in the perinuclear region. FRET analysis further showed that association between the molecules was not lost during colchicine-induced dissolution of microtubules, suggesting formation of tubulin-PKC-{alpha} complexes in the cytosol. Confocal imaging indicated that perinuclear microtubular structure was more highly sensitive to colchicine dissolution than other regions of the cell. Topographic imaging of fixed cells by AFM indicated a well-defined elevated structure surrounding the nucleus that was absent in colchicine-treated cells. It was calculated that the volume of the nuclear sleevelike structure of microtubules increased approximately fivefold in PDBu-treated cells, suggesting a probable increase in microtubular mass. In light of PKC-{alpha} localization, increased colchicine sensitivity, and their volume change in stimulated cells, the results suggest that perinuclear microtubules form a specialized structure that may be more dynamically robust than in other regions of the cell. PKC-{alpha} could contribute to this dynamic activity. Alternatively, perinuclear microtubules could act as a scaffold for regulatory molecule interaction at the cell center.

cytoskeleton; protein kinase C-{alpha}; translocation


THE PROTEIN KINASE C (PKC) family consists of a group of serine-threonine kinases separated into three subgroups on the basis of their structure and activation requirements. Cells are commonly found to express several PKC isoforms (4), and PKC may be involved in the regulation of multiple cell functions within a single cell type. This suggests that individual isoforms phosphorylate specific substrate(s) and that compartmentalization of isoform activity occurs under physiological conditions (19). One way this is thought to occur is through translocation to isoform-specific sites located adjacent to target substrate. The inactive form of PKC is diffusely distributed throughout the cytosol or may be localized to specific regions or structures in the cell. After stimulation of the cell, isoforms may relocate from inactive pools to their active cell loci. Hence, the intracellular translocation of PKC may represent an important mechanism for targeting specific substrates and control of isoform function.

There is evidence that the translocation of an individual PKC isoform may be varied in a single cell type through use of different stimulatory agents (18), whether highly differentiated (7, 10, 14) or less highly differentiated passaged (1) cells are used and depending on cell culture conditions (8). In addition, our laboratory reported (16) that PKC-{alpha} may be selectively relocated at the plasmalemma or the perinucleus of A7r5 smooth muscle cells, depending on the concentration of phorbol ester. The A7r5 clonal cell line was chosen for these studies because it exhibits an adult smooth muscle phenotype (5). This cell line was originally derived from embryonic rat aortic smooth muscle (15) and shows expression and promoter activity of several highly restricted smooth muscle cell markers (5). In addition to these indications of smooth muscle phenotype, A7r5 cells retain the ability to contract by both Ca2+-dependent and -independent mechanisms (6, 17, 20), which is lost in passaged smooth muscle cells. Interestingly, our studies (16) with A7r5 cells indicated that translocation of PKC-{alpha} to the perinucleus but not the subplasmalemma was blocked by the use of colchicine to disrupt cell microtubules. These studies indicated that concentrations of phorbol 12,13-dibutyrate (PDBu) <=107 M resulted in colchicine-insensitive translocation of PKC-{alpha} to the subplasmalemma, whereas PDBu concentrations >107 M caused colchicine-sensitive relocation of the enzyme in the perinuclear region. By comparison, concentrations of PDBu ranging from 109 to 105 M resulted only in colchicine-sensitive translocation of PKC-{alpha} to the perinuclear region of multipassaged cells derived from rat aortic smooth muscle (1). These observations suggest that in highly proliferative, dedifferentiated passaged smooth muscle cells, only the colchicine-sensitive mechanism for perinuclear translocation of PKC-{alpha} is operative or sensitive to PDBu activation, whereas high concentrations of PDBu are required to unmask this mechanism in A7r5 cells. Hence, it was concluded that multiple pathways are available for the redistribution of active PKC-{alpha} in A7r5 cells that could utilize different mechanisms for the movement or docking of the isoform at specific target sites. However, primarily because of interference from the intensity of PKC-{alpha} immunostaining, this early study was unable to show clear evidence for a direct association of PKC-{alpha} with microtubules. Hence, the role of the microtubular cytoskeleton in PKC-{alpha} localization at the perinucleus after phorbol ester stimulation remained unclear.

In the present study, we investigated the association of PKC-{alpha} with microtubular structure in A7r5 smooth muscle cells stimulated with 106 M PDBu, the concentration previously demonstrated to cause colchicine-sensitive PKC-{alpha} translocation to the perinucleus. The results confirm earlier observations that perinuclear translocation of PKC-{alpha} requires an intact microtubular cytoskeleton. The results further indicate at least a partial association of PKC-{alpha} with microtubules in the unstimulated and PDBu-stimulated cell.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture. A7r5 cells, originally derived from embryonic rat aorta and exhibiting an adult smooth muscle phenotype (5), were obtained from American Type Culture Collection (Manassas, VA) and cultured in Dulbecco's modified Eagle's medium (DMEM) that was supplemented with 10% fetal bovine serum, 100 U/ml penicillin G, and 100 µg/ml streptomycin. Cells were plated onto 75-mm2 culture flasks and grown to ~85% confluence. Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 in air. The medium was changed every 2 days, and the cells were passaged at least once a week. Cells were detached from the culture flask by the addition of a 1:10, 0.5% 5.3 mM trypsin-EDTA solution in phosphate-buffered saline (PBS).

Cell plating and treatment. A7r5 cells were plated onto 22 mm x 22 mm no. 1 glass coverslips placed in six-well culture plates and returned to the incubator for a minimum of 24 h to allow cell attachment and spreading. Cells were treated before fixation according to one of five protocols: 1) control cells received vehicle but no treatment before fixation; 2) PDBu cells were treated with 106 M PDBu (Sigma, St. Louis, MO) for up to 30 min; 3) control-colchicine cells were treated with 40 µg/ml colchicine (Sigma) for 30 min; 4) PDBu-colchicine cells were treated with 106 M PDBu for 30 min followed by treatment with 40 µg/ml colchicine for 30 min; and 5) colchicine-PDBu cells were treated with 40 µg/ml colchicine for 30 min followed by treatment with 106 M PDBu for 30 min. Cells were fixed and permeabilized by the addition of ice-cold acetone for 1 min. Fixed cells were preserved in 1x PBS buffer.

Confocal and fluorescence resonance energy transfer microscopy. In preparation for experiments using laser scanning confocal microscopy, A7r5 cells were plated and fixed as described in Cell plating and treatment. After fixation the cells were washed three times with PBS containing 0.5% Tween 20 (PBS-T), pH 7.5, and incubated for 30 min in a blocking solution (5% nonfat dry milk in PBS). Cells were stained for {beta}-tubulin by incubation in a 1:500 dilution of monoclonal anti-{beta}-tubulin clone TUB 2.1 FITC-labeled antibody (Sigma) for a minimum of 2 h at room temperature. For {alpha}-actin staining, fixed cells were incubated with a 1:500 dilution of monoclonal anti-{alpha}-smooth muscle actin clone 1A4 FITC-labeled antibody (Sigma) for 30 min at room temperature. For total F-actin staining, cells were incubated with 2.5 x 106 M tetramethylrhodamine isothiocyanate (TRITC)-labeled phalloidin (Sigma) for 30 min at room temperature. Cells were stained for vimentin via incubation with a 1:300 dilution of monoclonal anti-vimentin clone V9 (Sigma) at room temperature for 2 h. Fixed cells were stained for PKC-{alpha} with a 1:500 dilution of anti-PKC-{alpha}-clone M4 (Upstate Biotechnology, Lake Placid, NY). Treatment with anti-vimentin and anti-PKC-{alpha} solutions was followed by the addition of Alexa Fluor 488-labeled secondary antibody (Molecular Probes, Eugene, OR). In studies using fluorescence resonance energy transfer (FRET) microscopy, Alexa Fluor 546 (Molecular Probes) was the secondary antibody to anti-PKC-{alpha}.

Stained cells were surveyed by mounting on a Nikon Diaphot microscope. Confocal microscopy was performed with a Bio-Rad model 1024 scanning system equipped with a krypton/argon laser. Cells stained with Alex Fluor 488 or FITC conjugates were excited with the 488-nm laser line at 10% power. The resulting emission was visualized with a 522 DF 32 band-pass filter. For colocalization studies, the Bio-Rad scanning system's multichannel capacity was engaged. When both an FITC conjugate and Alexa Fluor 594 were present, a HQ 598/40 band-pass filter was used to observe the secondary fluorophore simultaneously on a second channel. Similarly, multiple channels were necessary in FRET microscopy. Within a FRET system two fluorophores with overlapping emission and excitation spectra are implemented. Here FITC (excitation, 488 nm; emission, 520 nm) and Alexa Fluor 546 (excitation, 546 nm; emission, 580 nm) were the donor-acceptor pair used. The donor molecule (FITC) was directly excited, and the resulting emission was visualized with the 522 DF 32 band-pass filter. However, a portion of energy given off by the fluorophore is not released as light but is transferred to a neighboring fluorophore that can be excited at that wavelength. Thus the FITC emission induced the Alexa Fluor 546 molecule to release energy as light at its characteristic emission wavelength, which was visualized on a second channel with an HQ 598/40 band-pass filter. The resonance energy transfer can only occur if the donor and acceptor molecules are close enough to each other for the transfer to occur efficiently. The Forster distance (R0) is the distance at which fluorescence resonance energy transfer from the donor dye to the acceptor dye is 50%. Distances >R0 will result in less intense emission, whereas distances <R0 will yield higher intensities. The R0 calculated for this pair is 64 Å. Resultant images were analyzed with Lasersharp and Confocal Assistant software (Bio-Rad, Hercules, CA).

To calculate the distance between PKC-{alpha} and {beta}-tubulin, images of cells were acquired with laser scanning confocal microscopy with excitation at the 488-nm laser line. To obtain an internal control, the imaging area was then excited at the 568-nm laser line at 100% power to photobleach the acceptor molecule (PKC-{alpha}). Subsequently, a second set of images was acquired again with 488-nm laser line excitation and the appropriate multichannel filter was set to measure {beta}-tubulin FITC emission (522 DF 32) and to verify the absence of anti-PKC-{alpha} secondary antibody Alexa Fluor 546 emission (HQ598/40). The images were analyzed by two methods, the determination of whole cell emission intensity and a single line scan emission intensity measurement. By selecting the entire scan area, an intensity profile was generated for each whole cell image [Image J Software, National Institutes of Health (NIH)] and the resulting plot was analyzed with Peakfit v4.11 software (SPSS Science, Chicago, IL) to obtain the area under the curve. These values were then used in calculating the efficiency of energy transfer (E) and the FRET distance between the donor and acceptor pair (r) with the following equations (13)

where ID is the intensity of the donor molecule in the presence of acceptor molecule and IDNA is the intensity of the donor molecule in the absence of acceptor (after photobleaching).

The resulting distances were analyzed by Sigma Stat 2.03 (SPSS Science) in comparisons of values obtained for control cells, those stimulated with PDBu, and colchicine-treated cells. Images analyzed by line scan emission intensity were also evaluated with Image J. For each set of images (ID, before photobleaching; IDNA, after photobleaching), a line was arbitrarily drawn to bisect the cell nucleus, extending to the visible peripheral borders of the cell. The resulting plot profile was then used to evaluate two aspects of the intracellular regional distribution of the association between {beta}-tubulin and PKC-{alpha}. Initially, we compared the FRET distance in the perinuclear vs. the peripheral region of the cell. This was accomplished by averaging the two highest values within the 20-pixel distance of the nuclear mass and the cell peripheral border. Second, we identified the point of maximum efficiency of energy transfer (E) on the line scan as a focal area in which PKC-{alpha} interaction with tubulin might approach saturating conditions.

Atomic force microscopy. In experiments using atomic force microscopy (AFM), fixed cells were rinsed with deionized water and dried with compressed air before study. Coverslips were mounted to 20-mm AFM metal specimen disks (TMMicroscopes, Sunnyvale, CA) with 12-mm carbon conductive tabs (Ted Pella, Redding, CA). Cells were imaged in contact mode with a TMMicroscopes Explorer atomic force microscope (2). All images were acquired with either TMMicroscopes model 1520-00 or model 1930-00 cantilevers. Subsequent analysis of images was performed with SPMLab version 5.01 analysis software (TMMicroscopes). On the basis of the whole cell scan of each cell, a line scan was performed bisecting the nucleus in an axis arbitrarily chosen to provide measurement outside the visible perimeter of the cell. The reference point for each cell height measurement was the default point assigned as zero by the SPMLab analysis software. Because the height of the nuclear sleeve was the object of study, and to avoid sampling bias, the measurement of cell height (untreated) or ground substance (colchicine treated) was obtained by using the whole cell scan to visually set measurement points on the sleeve rim or immediately adjacent to the nuclear mass, which were then identified for analysis on the line scan. The two measurements on opposite sides of the nucleus were then averaged to give a single value for height.

AFM measurements showed a well-defined, elevated structure surrounding the nucleus (see Figs. 9 and 11) that was absent in colchicine-treated cells (see Figs. 10 and 12). In light of these observations it was concluded that the central network of microtubules formed a sleevelike structure surrounding the nucleus. The sleeve diameter and thickness were directly measured, and sleeve height was obtained by subtracting the average cell height of colchicine-treated cells (ground substance) from the cell height of individual untreated cells (see Fig. 13). With these measurements the average volume (V) of the microtubular sleeve was then determined with the formula for calculation of the volume of a hollow cylinder

where V is the volume of the microtubular sleeve, h is the sleeve height, r0 is the outside radius, and r{iota} is the inside radius of the cylinder.



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Fig. 9. Topographic evaluation of the dense network of microtubules surrounding the nucleus in an untreated control cell with atomic force microscopy (AFM). Top: 3-dimensional view of a contact AFM image obtained from an unstimulated, fixed A7r5 cell. Bottom: line scan of the cell indicating cross-sectional structure and representing cell height in the nuclear area. The image is representative of those obtained in 3 individual experiments in which a total of 10 cells were evaluated.

 


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Fig. 11. Topographic evaluation of the perinuclear microtubular sleeve in a PDBu-contracted A7r5 cell with AFM. The cell was incubated with 106 M PDBu before fixation. Top: 3-dimensional view of a contact AFM image of the cell after PDBu treatment. Bottom: line scan of the image indicating cross-sectional structure and representing cell height in the nuclear area. The image is representative of those obtained in 3 individual experiments in which a total of 10 cells were evaluated.

 


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Fig. 10. Topographic evaluation of a colchicine-treated control cell with AFM. Top: 3-dimensional view of a contact AFM image of an unstimulated control cell incubated with 40 µg/ml colchicine before fixation. Bottom: line scan of the cell indicating cross-sectional structure and representing cell height in the nuclear area. The image is representative of those obtained in 3 individual experiments in which a total of 10 cells were evaluated.

 


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Fig. 12. Topographic evaluation of the perinuclear microtubule sleeve in a PDBu-contracted A7r5 cell incubated with colchicine. The cell was incubated with 106 M PDBu, after which 40 µg/ml colchicine was added for an additional 30 min before fixation. Top: 3-dimensional view of a contact AFM image of the cell. Bottom: line scan of the image indicating cross-sectional structure and representing cell height in the nuclear area. The image is representative of those obtained in 3 individual experiments in which a total of 10 cells were evaluated.

 


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Fig. 13. A model developed from measurements of control and colchicine-treated cells describing the 3-dimensional structure of the perinuclear microtubule sleeve. The cartoon depicts untreated (A, B) and colchicine-treated (C, D) cells, indicating the absence of the microtubular structure after colchicine. The table provides a summary comparison of cell dimensions in control and PDBu-activated cells. Direct measurements include cell height in untreated (control) (1) and colchicine-treated (5) cells, sleeve diameter (4), and sleeve thickness (3). The sleeve height (2) was obtained by subtracting the average ground substance height of colchicine-treated cells (5) from the height of individual control cells (1). Sleeve volume was calculated with the averaged cell measurements assuming that the shape of the structure was similar to a hollow cylinder. With the exception of sleeve volume, the values presented represent the means ± SE of 10 cells.

 

Statistics. Differences in FRET distances and AFM-obtained topographic measurements were analyzed by one- or two-way ANOVA followed by Student's t-test (Sigma Stat 2.03, SPSS Science). Differences were considered significant if P < 0.05 in all cases. Data are presented as means ± SE throughout the text.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous work indicated that stimulation of A7r5 cells with PDBu (>107 M) results in the perinuclear translocation of PKC-{alpha}, with evidence that this movement is blocked by treatment with colchicine (16). PKC-{alpha} appears to be diffusely distributed in the unstimulated cell (Fig. 1a). However, the addition of 106 M PDBu resulted in intense staining for PKC-{alpha} in the perinuclear/nuclear region of the cell (Fig. 1b). Addition of colchicine (40 µg/ml) before PDBu blocked the translocation of PKC-{alpha} (Fig. 1c), whereas its addition to the medium after stimulation with PDBu caused a punctate, diffuse redistribution of PKC-{alpha} back into the cytosol (Fig. 1d). These results suggest that activated PKC-{alpha} is associated in some fashion with perinuclear microtubules or other microtubule-dependent cell structure.



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Fig. 1. Effect of colchicine on the translocation of protein kinase C (PKC)-{alpha} in A7r5 smooth muscle cells. PKC-{alpha} appeared diffusely distributed throughout the cell before stimulation (a). Within 30 min after the addition of phorbol 12,13-dibutyrate (PDBu; 106 M), PKC-{alpha} translocated to the perinuclear region of the cell (b). The addition of colchicine (40 µg/ml) to the medium 20 min before PDBu blocked PKC-{alpha} translocation (c). The addition of colchicine 30 min after PDBu resulted in the dispersal of PKC-{alpha} from the perinuclear region back into the cytosol (d). Cells were fixed with acetone and stained with anti-PKC-{alpha} clone M4 primary antibody followed by Alexa Fluor 488-labeled secondary antibody before confocal imaging. Images represent those obtained in 3 individual experiments in which a total of >120 cells were evaluated from each treatment group.

 

Figure 2 shows the structural distribution of the microtubular ({beta}-tubulin), actin ({alpha}-actin, phalloidin), and intermediate filament (vimentin) components of the cytoskeleton in unstimulated (control) and PDBu-activated A7r5 cells. Unlike the distribution of actin filaments, the dense network of microtubular and intermediate filament structure surrounding the nucleus provided an early suggestion of these two filament types as candidates for a role in PKC-{alpha} localization. Dual immunostaining suggested a significant degree of colocalization of microtubules and PKC-{alpha} in both control and PDBu-stimulated cells but particularly in the perinuclear region of the PDBu-treated cells (Fig. 3). The conclusion of tubulin-PKC-{alpha} interaction was strengthened by FRET microscopic evaluation of whole cell scans that indicated an association between microtubules and PKC-{alpha} in both stimulated and unstimulated cells as well as colchicine-treated control and PDBu-stimulated cells (Fig. 4). These images further suggested that the tubulin-PKC-{alpha} association was largely confined to the central region of cells having an intact microtubular system.



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Fig. 2. Confocal images showing microtubular ({beta}-tubulin), microfilament ({alpha}-actin, phalloidin), and intermediate filament (vimentin) cytoskeletal structure in unstimulated (control) and PDBu-activated A7r5 smooth muscle cells. Microtubules were visualized with a monoclonal anti-{beta}-tubulin clone TUB2.1 FITC-labeled antibody. Actin was visualized by use of a monoclonal anti-{alpha}-smooth muscle actin clone 1A4 FITC-labeled antibody or tetramethylrhodamine isothiocyanate (TRITC)-labeled phalloidin. Intermediate filaments were imaged through use of a monoclonal anti-vimentin clone V9 primary antibody followed by Alexa Fluor 488-labeled secondary antibody. Images are representative of those obtained in 3 individual experiments in which a total of >120 cells were evaluated in each group.

 


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Fig. 3. Dual immunostaining of microtubules and PKC-{alpha} in unstimulated (control) and PDBu-activated A7r5 cells. Thirty minutes after the addition of PDBu (106 M) to the medium cells were fixed with acetone and prepared for confocal imaging. Microtubules were visualized with a monoclonal anti-{beta}-tubulin clone TUB2.1 FITC-labeled antibody. PKC-{alpha} was visualized by staining with monoclonal anti-PKC-{alpha} clone M4 primary antibody followed by Alexa Fluor 594-labeled secondary antibody. Yellow color indicates colocalization of the 2 proteins. Images shown are exemplary examples of those obtained in 4 individual experiments in which a total of 173 cells were evaluated.

 


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Fig. 4. Evaluation of microtubule and PKC-{alpha} colocalization by fluorescence resonance energy transfer (FRET) microscopy in unstimulated (control), PDBu-activated, and colchicine-treated A7r5 cells. Cells received vehicle or 106 M PDBu for 30 min and were fixed with acetone for FRET imaging. Colchicine (40 µg/ml) was added to the medium 30 min after the injection of PDBu. Cells were stained for {beta}-tubulin with monoclonal anti-{beta}-tubulin clone TUB2.1 FITC-labeled antibody and for PKC-{alpha} with monoclonal anti-PKC-{alpha} clone M4 primary antibody followed by Alexa Fluor 546-labeled secondary antibody. The FRET effect was observed by excitation at 488 nm only. At this wavelength FITC ({beta}-tubulin) was directly excited to fluoresce, with emission capture at 522 nm. The excitation of Alexa Fluor 546 (emission capture at 598 nm) and PKC-{alpha} visualization therefore indicate protein-protein interaction. Images represent those obtained in 4 or 5 individual experiments in which a total of 15 cells were evaluated.

 

Consistent with results from whole cell emission scans, FRET analysis of line scans from control (Fig. 5) and PDBu-stimulated (Fig. 6) cells indicated a heterogeneous distribution of tubulin-PKC-{alpha} association with evidence of high levels of efficiency of energy transfer (E) in the perinuclear region compared with that at the periphery of the cell.



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Fig. 5. Line scan analysis showing {beta}-tubulin emission intensity before (A, a) and after (B, b) photobleaching of PKC-{alpha} in an unstimulated control cell. The line scan was performed in an axis arbitrarily chosen to bisect the nucleus, extending to the visible borders of the cell (A and B); the results are shown in a and b. {beta}-Tubulin was visualized with a monoclonal anti-{beta}-tubulin clone TUB2.1 FITC-labeled antibody. PKC-{alpha} was visualized with a monoclonal anti-PKC-{alpha} clone M4 primary antibody followed by Alexa Fluor 546-labeled secondary antibody. The basal image of {beta}-tubulin emission was obtained by excitation with the 488-nm laser line at 10% power (A). The cell and surrounding area were then excited with the 568-nm laser line at 100% power to photobleach all traces of PKC-{alpha}-Alexa Fluor 546 emission. Immediately after photobleaching, a second {beta}-tubulin image was captured with the 488-nm laser line (B) at the exact settings used in basal imaging. According to FRET theory, donor molecule ({beta}-tubulin) emission is expected to increase in the absence of the acceptor molecule (PKC-{alpha}). The images represent results obtained in 3 individual experiments in which a total of 20 cells were evaluated.

 


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Fig. 6. Line scan analysis showing {beta}-tubulin emission intensity before (A, a) and after (B, b) photobleaching of PKC-{alpha} in a PDBu-stimulated cell. The line scan was performed in an axis arbitrarily chosen to bisect the nucleus, extending to the visible borders of the cell (A and B); the results are shown in a and b. {beta}-Tubulin was visualized with a monoclonal anti-{beta}-tubulin clone TUB2.1 FITC-labeled antibody. PKC-{alpha} was visualized with a monoclonal anti-PKC-{alpha} clone M4 primary antibody followed by Alexa Fluor 546-labeled secondary antibody. The basal image of {beta}-tubulin emission was obtained by excitation with the 488-nm laser line at 10% power (A). The cell and surrounding area were then excited with the 568-nm laser line at 100% power to photobleach all traces of PKC-{alpha}-Alexa Fluor 546 emission. Immediately after photobleaching, a second {beta}-tubulin image was captured with the 488-nm laser line (B) at the exact settings used in basal imaging. According to FRET theory, donor molecule ({beta}-tubulin) emission is expected to increase in the absence of the acceptor molecule (PKC-{alpha}). The images represent results obtained in 3 individual experiments in which a total of 20 cells were evaluated.

 

The calculation of FRET distances from whole cell emission scans yielded similar values among control, PDBu-stimulated, and colchicine-treated groups (Table 1), supporting the conclusion of significant tubulin-PKC-{alpha} association in cells both before and after disruption of microtubular structure. Furthermore, FRET distances were significantly increased in the periphery compared with the perinucleus in both control and PDBu-stimulated cells, verifying visual observations of differences in the regional distribution of tubulin-PKC-{alpha} interaction within the cell. Interestingly, the FRET distances calculated for points of peak E obtained from each line scan (Table 1) were very similar among the different groups (60.1 ± 0.4 Å) and fell at the upper end of the range of measurements reported between known loci on actin and myosin (22). These latter findings further suggest the occurrence of discrete "hot spots" of tubulin-PKC-{alpha} interaction within the cell.


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Table 1. FRET distances calculated from whole cell emission scans and line scans of selected regions in unstimulated, PDBu-stimulated, and colchicine-treated cells

 

Because colchicine caused dispersal of PKC-{alpha} from the perinuclear region in cells preactivated with PDBu (Fig. 1), it was of interest to evaluate the effect of the drug on the major cytoskeletal elements. Colchicine treatment had no detectable effect on actin microfilaments or on intermediate filaments (Fig. 7), greatly reducing the likelihood of their involvement in PKC-{alpha} localization. By comparison, the drug caused the complete disruption of all but the very peripheral microtubular structure within 20 min after addition to the medium (Fig. 8). Interestingly, the colchicine-induced dissolution of microtubules was clearly observed to occur at variable rates in different regions of the cell. The organization of microtubules was consistent among the great majority of A7r5 cells imaged, forming a dense network about the nucleus from which cables of microtubules radiated to a second dense network at the subplasmalemma. On treatment with colchicine, we invariably noted initial dissolution of the central network, which was generally complete before loss in structural integrity of radiating cables or peripheral structure was noticeable.



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Fig. 7. The effect of colchicine on microfilaments and intermediate filaments. Unstimulated A7r5 cells were incubated with vehicle (control) or 40 µg/ml colchicine for 20 min before fixation and staining. Actin stress fibers were visualized by staining with a monoclonal anti-{alpha}-smooth muscle actin clone 1A4 FITC-labeled antibody. Intermediate filaments were imaged with a monoclonal anti-vimentin clone V9 primary antibody followed by Alexa Fluor 488-labeled secondary antibody. The results indicate that colchicine had no significant effect on actin or intermediate filament structure. Images are representative of those obtained in 3 individual experiments in which a total of 150–175 colchicine-treated cells were evaluated.

 


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Fig. 8. Time course of colchicine-induced dissolution of microtubular structure in A7r5 cells. Cells were untreated (vehicle control; A) or were incubated with 40 µg/ml colchicine for 5 (B), 10 (C), or 20 (D) min before fixation and staining. Microtubules were imaged with a monoclonal anti-{beta}-tubulin clone TUB2.1 FITC-labeled antibody. Images are typical of those obtained in 4 separate experiments in demonstrating the dissolution of the dense network of microtubules surrounding the nucleus (arrows) in advance of significant losses in peripheral structure.

 

Topographic imaging of fixed cells by AFM revealed a well-defined, elevated structure surrounding the nucleus (Fig. 9), which was absent after treatment with colchicine (Fig. 10). A similar analysis of the PDBu-activated cell (Figs. 11 and 12) indicated that the contraction of the cell significantly altered the dimensions and increased the volume of this structure by roughly fivefold (Fig. 13). The results suggest that the dense network of microtubules at the center of the cell forms a sleevelike structure that, given the enhanced sensitivity to colchicine and the regional association with PKC-{alpha}, may represent a more dynamic and specialized subgroup of microtubules. The significant change in the dimensions and volume of the microtubular sleeve could reflect compression of the structure during cell contraction or an artifact of sample preparation. Alternatively, these results could be suggesting active polymerization and an increase of microtubular structure in the central region of the activated A7r5 cell.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our results confirm earlier findings that treatment of cells with colchicine to disrupt microtubules blocks the translocation of PKC-{alpha} to the perinucleus (1, 18, 23). In light of this finding, it was suggested that microtubules could influence both the movement of PKC-{alpha} to the perinucleus and/or the docking mechanism in this region of the cell (1). We further show that treatment of preactivated cells in which translocation is complete with colchicine causes dispersal of PKC-{alpha} back into the cytosol (Fig. 1). This suggests that the principal impact of the microtubules is not on the actual movement of PKC-{alpha} but that they act to stabilize the aggregation of the enzyme in different regions of the cell. In previous work using a PKC-{alpha}-green fluorescent protein (GFP) fusion protein expression, we (18) were not able to clearly demonstrate a direct relationship between PKC-{alpha} and microtubules in A7r5 cells. However, we now show that dual immunostaining for PKC-{alpha} and {beta}-tubulin with confocal microscopic imaging (Fig. 3) and analysis of overlapping emission/excitation spectra by FRET microscopy (Figs. 4, 5, 6, Table 1) indicate significant colocalization and close association of PKC-{alpha} with microtubules in both unstimulated control and PDBu-stimulated A7r5 cells. The present finding that association of PKC-{alpha} with tubulin does not require cell stimulation suggests that the activation of the enzyme is not necessary for interaction between the two molecules. Alternatively, these results may reflect a basal level of PKC-{alpha} activity in resting cells. Evidence that PKC-{alpha} is associated with tubulin in colchicine-treated cells (Fig. 4, Table 1) further indicates that PKC-{alpha} may complex with tubulin in the cytosol.

It is noteworthy that both confocal imaging (Fig. 3) and FRET analysis (Fig. 4) indicate a heterogeneous distribution of PKC-{alpha} in association with microtubules. Visual inspection of whole cell images suggests that the interaction is primarily confined to the central region, whereas the microtubular structure at the cell periphery appears devoid of PKC-{alpha}. FRET measurements (Table 1) verified a predominance of tubulin-PKC-{alpha} interaction in the region immediately adjacent to the nucleus and further indicated the existence of discrete focal points of intense resonance energy transfer. The FRET distances calculated for these hot spots of maximal tubulin-PKC-{alpha} interaction are within the range of values reported for other intermolecular interactions (22) and could be indicating the direct binding of PKC-{alpha} with tubulin. However, the magnitude of the FRET distance recorded could also be consistent with the association of {beta}-tubulin and PKC-{alpha} via a linker protein. In view of the highly restricted compartmentalization of the tubulin-PKC-{alpha} interaction, we consider it unlikely that PKC-{alpha} is constitutively bound to tubulin. It must be emphasized that the results do not allow quantitation of PKC-{alpha}-microtubule association, and the possibility remains that a portion of PKC-{alpha} is associated with other structures in the perinuclear region. Together, however, the results suggest that the microtubules could act as a scaffold for PKC-{alpha} binding. This function is primarily confined to the perinuclear microtubular structure and occurs through an interaction of PKC-{alpha} with microtubule-associated compounds.

In comparison with other cytoskeletal components, the microtubules of the A7r5 cell form a unique structural configuration, as seen with confocal microscopy (Fig. 2). A dense network about the nucleus is connected by thick cables radiating across the cell body to a dense network of microtubules lying under the plasmalemma. Interestingly, a highly reproducible sequence of colchicine-induced dissolution of microtubular structure was observed in these various regions of the cell (Fig. 8). There was initial loss of the central perinuclear network with complete disassembly realized by 5 min after colchicine addition. By comparison, significant loss of radiating cables occurred at 10–20 min, whereas the subplasmalemma network of microtubules was often at least partially intact after 20 min of colchicine treatment. Because colchicine is thought to prevent polymerization by binding tubulin subunits, the high level of colchicine sensitivity suggests that microtubular structure in the perinuclear region is more dynamically robust than in other regions of the cell.

Topographic analysis of fixed cells by AFM indicated that the perinuclear network of microtubules formed an elevated sleevelike structure enveloping the nucleus (Figs. 9 and 10). A similar evaluation of PDBu-activated cells indicated that the dimensions of the perinuclear sleeve were significantly altered during cell shortening (Figs. 11 and 12). By using measurements of height, thickness, and diameter (Fig. 13), we calculated the volume of the sleeve assuming a form roughly the shape of a hollow cylinder. This estimate indicated that the volume of the microtubular sleeve was ~4.9 µm3 in control cells and 25.5 µm3 in cells treated with PDBu. Differences in microtubular volume between control and PDBu-stimulated cells could reflect an artifact of colchicine treatment or sample preparation for imaging. Alternatively, the fivefold increase observed in PDBu-treated cells could represent an increase in microtubular mass in the activated cells. As noted in RESULTS, the interaction between PKC-{alpha} and {beta}-tubulin was retained after treatment with colchicine, suggesting that PKC-{alpha} may associate with monomeric or small aggregates of tubulin in the cytosol. The possibility occurs that such a tubulin-PKC-{alpha} complex is translocated to the perinucleus during expansion of the microtubular structure in this region during cell activation.

Activation of PKCs has been shown to result in changes in cytoskeletal structure in association with the phosphorylation of an array of cytoskeletal proteins (12). It is well documented that PKCs may interact with each of the cytoskeletal components, with some evidence to suggest increased activity in the activated cell (9). Hence, there is a large literature on the role of PKCs in cytoskeletal regulation. However, the great bulk of research has focused on regulation of actin microfilaments (12) and relatively little is known of the role of PKC in microtubule dynamics. Kabir et al. (11) reported the interesting finding that activation of PKC by phorbol esters in Aplysia bag cell neurons in primary cell culture resulted in a doubling in the length of a typical microtubule growth episode while increasing and decreasing rescue and catastrophe frequencies, respectively. They concluded that PKC may play an important role in regulating cellular processes involving directed microtubular growth. This group subsequently showed (21) phorbol ester-induced translocation of PKCs to microtubules, suggesting a direct role of these kinases in the regulation of distal microtubular advance in neural growth cones. Their findings open the possibility that the translocation of PKC-{alpha} to the perinuclear microtubules in A7r5 cells could serve a regulatory function directed toward microtubular growth and stability in this region of the cell. This function, in turn, would appear consistent with increased dynamic activity evidenced by colchicine sensitivity and the increase in volume of perinuclear microtubular structure in the present study. Alternatively, the translocation of PKC-{alpha} could reflect a specialized role of perinuclear microtubules as a scaffold for interaction of regulatory molecules influencing a variety of cell functions in the central region of the cell. There is evidence from a variety of cell types suggesting a link between PKC-mediated function and the capacity for microtubule reorganization. For example, the prevention of microtubular depolymerization with taxol blocked PKC (phorbol ester)-induced NF-{kappa}B activation in murine NIH/3T3 cells (24) while causing hyperphosphorylation of vimentin and reorganization of the intermediate filament cytoskeleton via a PKC signaling pathway in 9L rat brain tumor cells (3). Others have proposed that microtubule catastrophe may be obligatory for PKC (phorbol ester)-mediated MCL1 gene expression in the MCL1 human myeloblastic leukemia cell line (25). As a matter of speculation, a dynamic microtubular scaffold could serve to compartmentalize regulatory interactions while providing for rapid and potentially directional expansion during growth of structure and reduction of interaction or compartmentalized release of compounds during catastrophe and loss of structure.

In summary, the results confirm earlier conclusions that the microtubular cytoskeleton is required for PDBu-induced localization of PKC-{alpha} at the perinuclear region in A7r5 smooth muscle cells. The results suggest that this is due to an association of activated PKC-{alpha} with microtubule-associated compounds confined to the microtubules in this region of the cell. In addition to the regional limitation of PKC-{alpha} localization, the distribution of microtubular structure in the cell and differences in colchicine sensitivity suggest that microtubules in the perinuclear region represent a distinct subpopulation that may differ in their dynamic growth characteristics from those in other regions. PKC could be an important factor in determining these characteristics. Alternatively, the perinuclear microtubules could serve as a scaffold for interaction of regulatory proteins at the cell center.


    FOOTNOTES
 

Address for reprint requests and other correspondence: G. L. Wright, Joan Edwards School of Medicine, Marshall Univ., 1542 Spring Valley Drive, Huntington, WV 25704 (E-mail: wrightg{at}marshall.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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