©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Divergent Insulin and Platelet-derived Growth Factor Regulation of Focal Adhesion Kinase (pp125) Tyrosine Phosphorylation, and Rearrangement of Actin Stress Fibers (*)

John B. Knight(§)(¶) , Keishi Yamauchi (¶) , Jeffrey E. Pessin (**)

From the (1)

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Insulin treatment of Chinese hamster ovary cells expressing high levels of the human insulin receptor resulted in the tyrosine dephosphorylation of the 125-kDa focal adhesion kinase (pp125). The decrease in pp125 tyrosine phosphorylation paralleled a decrease in the cellular content of actin stress fibers, and these changes were independent of the extracellular matrix on which the cells were grown. The reduction in both pp125 tyrosine phosphorylation and actin stress fibers occurred in an insulin concentration-dependent manner, with significant effects at approximately 0.3 nM and a maximal effect at 3 nM. However, in the continuous presence of insulin, the decreases in the tyrosine phosphorylation state of pp125 and actin stress fiber content were transient. Maximal reduction of pp125 tyrosine phosphorylation was observed following 15 min of insulin treatment, with a return to unstimulated control levels by 60 min. Similarly, actin stress fiber content was maximally reduced by 15 min of insulin treatment and fully recovered by 60 min. In contrast to insulin, platelet-derived growth factor stimulation increased actin stress fiber content and enhanced pp125 tyrosine phosphorylation. These data demonstrate a novel signaling role for insulin in inducing the tyrosine dephosphorylation of pp125 and a concomitant reorganization of actin stress fibers, which underlies at least one aspect of signaling divergence between the insulin and platelet-derived growth factor receptor tyrosine kinases.


INTRODUCTION

Focal adhesions are regions of a cell in direct contact with the extracellular matrix, providing the anchorage site for actin stress fibers and forming a link between the extracellular matrix and the actin cytoskeleton (for review, see Refs. 1-3). Several proteins present in focal adhesion contacts have been shown to undergo tyrosine phosphorylation (for review, see Refs. 3, 4). One such protein, the focal adhesion kinase (pp125),() is a 125-kDa protein-tyrosine kinase that is itself tyrosine phosphorylated in response to a number of stimuli (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) . pp125, in conjunction with another tyrosine-phosphorylated focal adhesion protein, paxillin, appears to play an important role in localizing several Src homology 2 (SH2) domain-containing proteins such as Src, Fyn, phosphatidylinositol 3-kinase and the carboxyl-terminal Src kinase to focal adhesion contacts (17, 18, 19, 20, 21, 22) . In addition to the potential role of pp125 and paxillin as substrates for the Src-family member kinases and/or docking sites for the phosphatidylinositol 3-kinase, these proteins may play a role in promoting the formation of filamentous actin, since the extent of pp125 and paxillin tyrosine phosphorylation occurs in direct proportion to the cellular content of actin stress fibers (11, 16, 23) .

Recent studies have demonstrated that pp125 provides an important integration site for various extracellular signals including integrin receptor family members, G protein-coupled receptors, and both receptor and nonreceptor tyrosine kinases (for review, see Refs. 3 and 24). These signals appear to increase pp125 tyrosine phosphorylation and enhance the formation of actin stress fibers (11, 16, 23, 25, 26) . Previous studies have reported changes in cytoskeletal organization in KB and Rat1 fibroblasts following insulin treatment (27, 28) . To investigate the role of insulin in the regulation of cytoskeletal structure, we have examined the effect of insulin on pp125 tyrosine phosphorylation and actin stress fiber content. In this manuscript we demonstrate that in direct contrast to platelet-derived growth factor (PDGF), insulin stimulation of Chinese hamster ovary cells expressing the human insulin receptor (CHO/IR) resulted in the tyrosine-specific dephosphorylation of pp125 with a concomitant decrease in the length and number of actin stress fibers. This effect was transient with a complete recovery of pp125 tyrosine phosphorylation and actin stress fiber content within 1 h of continuous insulin stimulation.


EXPERIMENTAL PROCEDURES

Cell Culture

CHO/IR cells expressing 3 10 human insulin receptors/cell were obtained as described previously (29) . These cells were maintained in minimal Eagle's medium containing nucleotides plus 10% fetal bovine serum. CHO/IR cells were grown to confluency on either uncoated or collagen IV-coated tissue culture plates (Collaborative Biomedical Products, Becton-Dickinson Laboratories) as indicated in the figure legends. To prevent any signaling events induced by the presence of other growth factors in serum, the cells were cultured in serum-free medium for 6 h prior to the addition of insulin or PDGF.

Immunoprecipitation of the pp125

Whole cell extracts were prepared by detergent solubilization in a lysis buffer (20 mM Hepes, pH 7.4, 1% Triton X-100, 3 mM MgCl, 2 mM EDTA, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 10 µg/ml aprotinin, and 1.5 µM pepstatin) for 1 h at 4 °C. The cell extracts (5 mg/ml in 150 µl) were then heated at 100 °C for 5 min in the presence of 1% SDS. The samples were diluted 10-fold (10 mM Tris, pH 7.4, 1.0% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 2 mM EDTA, 0.2 mM sodium vanadate, 0.2 mM phenylmethylsulfonyl fluoride) and immunoprecipitated with 4 µg of a FAK monoclonal antibody (FAK, Transduction Laboratories). The immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting (Enhanced Chemiluminescence detection kit, Amersham Corp.) using either the 4G10 monoclonal phosphotyrosine antibody (Upstate Biotechnology Inc.), the PY20-HRP monoclonal phosphotyrosine antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Inc.) or the FAK monoclonal antibody (Transduction Laboratories).

Fluorescent Labeling of Filamentous Actin

Confluent cultures of CHO/IR cells following treatments indicated in the figure legends were washed twice with ice-cold phosphate-buffered saline (PBS; 6.6 mM KHPO, 1.5 mM KHPO, pH 7.4, 2.7 mM KCl, 137 mM NaCl, 1 mM EGTA, and 2 mM MgCl). The cells were then fixed in 4% paraformaldehyde (in PBS) for 20 min at 4 °C followed by quenching for 15 min at 25 °C in a solution containing 0.1 M glycine and 0.2 M Tris-HCl, pH 7.4. The fixed cells were rinsed twice in PBS and permeabilized by incubation for 5 min at 25 °C in 0.5% Triton X-100. The cells were then rinsed twice again with PBS and blocked with a solution containing 0.1% bovine serum albumin in PBS for 15 min at 25 °C. Immunofluorescent labeling of filamentous actin was carried out by incubation for 30 min at 37 °C with 0.1 mg/ml of fluorescein isothiocyanate (FITC) or carboxytetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin (Sigma). Following incubation with the fluorochrome-conjugated phalloidin, the cells were rinsed three additional times with PBS, and the samples were mounted for viewing on a Bio-Rad MRC 600 confocal microscope.


RESULTS

Previous studies have reported that insulin stimulation resulted in increased membrane ruffling in both KB and Rat1 fibroblasts (27, 28) . To investigate the molecular basis for the insulin-mediated alteration in the actin cytoskeletal structure, we examined the effect of insulin in CHO/IR cells. In contrast to KB and Rat1 fibroblasts, acute insulin treatment of CHO/IR cells (100 nM for 15 min) had no effect on membrane ruffling but markedly reduced the number and length of actin stress fibers as determined by TRITC-phalloidin staining (Fig. 1, A and B). Since the cytoskeletal structure of cells may differ depending upon the extracellular matrix in contact with the cell surface membrane (1, 30) , we also determined the effect of insulin on the organization of filamentous actin in cells grown on collagen IV (Fig. 1, C and D). CHO/IR cells grown on collagen IV-coated plates displayed increased numbers of actin stress fibers (Fig. 1 C), which were thicker and shorter than those observed in cells grown on uncoated plates (Fig. 1 A). Nevertheless, insulin induced a similar decrease in actin stress fibers in the CHO/IR cells grown on collagen IV (Fig. 1 D) compared with cells grown on uncoated tissue culture plates (Fig. 1 B). This insulin-induced reduction in stress fibers was also evident in cells grown on collagen I, laminin, and fibronectin (data not shown).


Figure 1: Insulin-induced decrease in actin stress fiber content in CHO/IR cells. CHO/IR cells were grown to confluency on uncoated ( A and B) or collagen type IV-coated ( C and D) tissue culture plates. The cells were incubated without ( A and C) or with 100 nM insulin ( B and D) for 15 min at 37 °C. The cells were then fixed, detergent permeabilized, and stained for filamentous actin using TRITC-phalloidin as described under ``Experimental Procedures.''



It has been well documented that the formation of actin stress fibers parallels focal adhesion formation and is accompanied by increased tyrosine phosphorylation of pp125 and paxillin (11, 16, 23, 26) . We therefore examined the tyrosine phosphorylation state of pp125 following insulin treatment of CHO/IR cells (Fig. 2 A). Compared with unstimulated cells (Fig. 2 A, lane1), or cells treated with 0.1 nM insulin (Fig. 2 A, lane2), there was a detectable decrease in pp125 tyrosine phosphorylation following treatment with 0.3 nM insulin (Fig. 2 A, lane3). The apparent lower level of tyrosine-phosphorylated pp125 observed in lane1 resulted from underloading of this particular sample as determined by FAK Western blotting (Fig. 2 B, lane1). Incubation of the cells with increasing concentrations of insulin from 1 nM (Fig. 2 A, lane4) to 3 nM (Fig. 2 A, lane5) resulted in a progressive decline in the extent of pp125 tyrosine phosphorylation. Maximal reduction of tyrosine phosphorylation occurred at 10 nM insulin (Fig. 2 A, lane6) and was not further affected by treatment with higher concentrations (30 nM) of insulin (Fig. 2 A, lane7). This decrease in phosphotyrosine content occurred with no significant alteration in the amount of pp125 protein immunoprecipitated under these conditions (Fig. 2 B).


Figure 2: Concentration dependence of insulin-stimulated pp125 tyrosine dephosphorylation. CHO/IR cells were grown to confluency on uncoated tisssue culture plates and incubated in the absence ( lane1) or presence of 0.1 ( lane2), 0.3 ( lane3), 1 ( lane4), 3 ( lane5), 10 ( lane6), and 30 nM ( lane7) insulin for 10 min at 37 °C. Cell extracts were prepared and subjected to immunoprecipitation with a monoclonal antibody ( FAK) directed against pp125 as described under ``Experimental Procedures.'' A, the FAK immunoprecipitates were then Western blotted with an antibody directed against phosphotyrosine ( 4G10). B, the Western blot transfer membrane that was probed with the 4G10 antibody in A was stripped and probed with the monoclonal antibody ( FAK) directed against pp125.



In agreement with the dose response relationship of pp125 tyrosine dephosphorylation, the decrease in TRITC-phalloidin-labeled actin stress fibers was also observed to be insulin concentration-dependent (Fig. 3), with a dose response paralleling that observed for tyrosine dephosphorylation of pp125. There was a small but significant reduction in the length and number of actin stress fibers at 0.1 nM insulin (Fig. 3 C) compared with untreated cells (Fig. 3 A) or cells treated with 0.03 nM insulin (Fig. 3 B). A marked decrease in filamentous actin was observed following incubation with 1 nM insulin, and maximal reduction occurred at 3 nM insulin (Fig. 3, D and E). Treatment of CHO/IR cells with 10 (data not shown), 30 (Fig. 3 F), or 100 nM insulin (data not shown) had no additional effect on the disassembly of actin stress fibers.


Figure 3: Insulin dose-dependent decrease of actin stress fiber content. CHO/IR cells were grown to confluency on uncoated tissue culture plates and incubated in the absence ( panelA) or in the presence of 0.03 ( panelB), 0.1 ( panelC), 1.0 ( panelD), 3 ( panelE), and 30 ( panelF) nM insulin for 15 min at 37 °C. The cells were then fixed, detergent permeabilized, and TRITC-phalloidin labeled for actin stress fibers as described under ``Experimental Procedures.''



To further characterize the tyrosine dephosphorylation of pp125, we examined the time-dependence of this effect following insulin treatment (Fig. 4). At saturating insulin concentrations (100 nM), pp125 tyrosine dephosphorylation was initially detected by 2 min (Fig. 4 A, lane2). There was a progressive decline in pp125 tyrosine phosphorylation that reached a maximum at 10-15 min of insulin treatment (Fig. 4 A, lanes4 and 5). However, the insulin-mediated decrease in pp125 tyrosine phosphorylation was transient and began to recover 30 min subsequent to the addition of insulin (Fig. 4 A, lane6). Following 60 min of insulin treatment, the tyrosine phosphorylation state of pp125 was similar to that observed for the unstimulated control CHO/IR cells (Fig. 4 A, compare lanes1 and 7). To insure that the observed changes in phosphotyrosine blotting of pp125 did not result from alterations in pp125 expression or differential immunoprecipitation, the FAK immunoprecipitates were subjected to Western blotting with the FAK antibody (Fig. 4 B). During this period of insulin treatment (0-60 min), there was no significant alteration in the amount of immunoprecipitated pp125. Thus, the insulin-mediated decrease and subsequent recovery of pp125 phosphorylation observed in the phosphotyrosine immunoblot was a direct result of changes in the tyrosine phosphorylation state of pp125.


Figure 4: Time dependence of insulin-stimulated pp125 tyrosine dephosphorylation. CHO/IR cells were grown to confluency on uncoated tissue culture plates and incubated in the absence ( lane1) or presence of 100 nM insulin for 2 ( lane2), 5 ( lane3), 10 ( lane4), 15 ( lane5), 30 ( lane6), and 60 ( lane7) min at 37 °C. Cell extracts were prepared and subjected to immunoprecipitation with a monoclonal antibody ( FAK) directed against pp125. A, the immunoprecipitates were then Western blotted with an antibody directed against phosphotyrosine ( 4G10) as described under ``Experimental Procedures.'' B, the Western blot transfer membrane that was probed with the 4G10 antibody in panelA was stripped and probed with the monoclonal antibody ( FAK) directed against pp125.



Consistent with the time-dependent change in pp125 tyrosine phosphorylation (Fig. 4), there was a concomitant decrease in the number and length of actin stress fibers, which was followed by recovery to normal levels (Fig. 5, A-F). We consistently observed a small but significant decline in stress fibers following 3 min of insulin treatment (Fig. 5 B) compared with unstimulated control cells (Fig. 5 A). However, there was a marked reduction in FITC-phalloidin labeling following 5 min of insulin treatment (Fig. 5 C). A maximal decrease in actin stress fibers was routinely detected after 15 min of insulin exposure (Fig. 5 D). In addition, in the continuous presence of insulin, restoration of the actin stress fiber network was apparent within 30-60 min (Fig. 5, E and F). These time-dependent alterations in actin stress fibers closely paralleled the changes in the tyrosine phosphorylation state of pp125 (Fig. 4). To insure that the transient nature of actin stress fiber rearrangement and pp125 tyrosine phosphorylation was not due to depletion of the exogenously added insulin, we performed radioimmunoassay analyses of the culture media. These data demonstrated that insulin levels decreased from 100 to 50 nM following the 1-h incubation time (data not shown). Thus, the concentration of insulin remaining in the media at 60 min was more than sufficient to maximally stimulate acute tyrosine dephosphorylation of pp125 and reduce the cellular content of actin stress fibers (Figs. 2 and 3).


Figure 5: Time course of insulin treatment on actin stress fiber content. CHO/IR cells were grown to confluency on collagen IV-coated tissue culture plates and incubated in the absence ( panelA) or in the presence of 100 nM insulin for 3 ( panelB), 5 ( panelC), 15 ( panelD), 30 ( panelE), and 60 ( panelF) min at 37 °C. The cells were then fixed, detergent permeabilized, and FITC-phalloidin-labeled for actin stress fibers as described under ``Experimental Procedures.''



Previous studies have demonstrated that various extracellular signals increase pp125 tyrosine phosphorylation and enhance the formation of actin stress fibers (11, 16, 23, 25, 26) . In particular, low concentrations of PDGF (0.2 nM) have been observed to increase the cellular content of actin stress fibers and pp125 tyrosine phosphorylation. Paradoxically, high concentrations of PDGF (>1.2 nM) have been reported to have no effect or to reduce the cellular levels of filamentous actin and tyrosine-phosphorylated pp125(16, 31) . We therefore directly compared the tyrosine phosphorylation state of pp125 in cells stimulated with insulin and PDGF (Fig. 6). As previously observed, stimulation of CHO/IR cells with 100 nM insulin for 15 min decreased in the extent of tyrosine-phosphorylated pp125 (Fig. 6 A, lanes1 and 2). In direct contrast, incubation with 0.2 nM (5 ng/ml) PDGF for 15 min resulted in an increase in pp125 tyrosine phosphorylation (Fig. 6 A, lane3), whereas 0.4 nM (10 ng/ml) and 1.2 nM (30 ng/ml) PDGF had little effect on the extent of tyrosine-phosphorylated pp125 (Fig. 6 A, lanes4 and 5). Although the relative increase in pp125 tyrosine phosphorylation at 0.2 nM (5 ng/ml) PDGF was small, it was highly reproducible (1.56 ± 0.04, p < 0.01 compared with control, n = 3). To control for the efficiency of pp125 immunoprecipitation, we also determined the amount of pp125 by FAK immunoblot analyses. Under these conditions, there were no significant differences in pp125 protein levels (Fig. 6 B, lanes1-5).


Figure 6: Comparison between insulin and PDGF stimulation of the tyrosine phosphorylation state of pp125. CHO/IR cells were grown to confluency on uncoated tisssue culture plates and incubated in the absence ( lane1) or presence of 100 nM insulin ( lane2), 0.2 nM PDGF (5 ng/ml, lane3), 0.4 nM PDGF (10 ng/ml, lane4), or 1.2 nM PDGF (30 ng/ml, lane5) for 15 min at 37 °C. Cell extracts were prepared and subjected to immunoprecipitation with a monoclonal antibody ( FAK) directed against pp125 as described under ``Experimental Procedures.'' A, the FAK immunoprecipitates were then Western blotted with an antibody directed against phosphotyrosine ( PY20- HRP). B, the Western blot transfer membrane that was probed with the PY20-HRP antibody in panelA was stripped and probed with the monoclonal antibody ( FAK) directed against pp125. Please note that the higher background observed in panelB compared with Figs. 2 and 4 resulted from use of the PY20-HRP antibody in the initial immunoblot. Although PY20-HRP resulted in a lower background for the phosphotyrosine immunoblot ( A), this antibody cannot be completely removed for the second FAK immunoblot ( B).



We next determined the effect of PDGF treatment on the cellular content of actin stress fibers in comparison with that of insulin (Fig. 7). As observed in Figs. 3 and 5, incubation with insulin for 15 min decreased the length and number of actin stress fibers (Fig. 7, A and B). In contrast, incubation with 0.2 nM (5 ng/ml) PDGF enhanced the actin stress fiber content of the CHO/IR cells (Fig. 7 C). Consistent with the inability of 1.2 nM (30 ng/ml) PDGF to effect pp125 tyrosine phosphorylation (Fig. 6), this concentration had no significant effect on the actin cytoskeleton compared with untreated cells (Fig. 7 D). These data directly demonstrated that the identical CHO/IR cell population responded to insulin and low concentrations of PDGF in an opposite manner with respect to pp125 tyrosine phosphorylation and actin stress fiber formation.


Figure 7: Effect of insulin and PDGF treatment on actin stress fiber content. CHO/IR cells were grown to confluency on uncoated tissue culture plates and incubated in the absence ( panelA) or in the presence of 100 nM insulin ( panelB), 0.2 nM PDGF (5 ng/ml, panelC), or 1.2 nM (30 ng/ml, panelD) for 15 min at 37 °C. The cells were then fixed, detergent permeabilized, and FITC-phalloidin-labeled for actin stress fibers as described under ``Experimental Procedures.''




DISCUSSION

Although the role of pp125 in growth factor signaling has not been elucidated to date, it appears to be an important integration point for the actions of G protein-coupled receptors, the non-tyrosine kinase integrin receptor family members, and both receptor and nonreceptor tyrosine kinases (3, 24) . The enhanced tyrosine phosphorylation of pp125 induced by these signaling pathways parallels an increase in the formation of filamentous actin structures (11, 16, 23, 26) . In particular, stimulation of Swiss 3T3 fibroblasts with low concentrations of PDGF (5 ng/ml) was observed to enhance pp125 tyrosine phosphorylation and actin stress fiber formation (16, 31, 32) . Interestingly, the effect of PDGF appeared to be biphasic with high concentrations of PDGF (>1.2 nM) having either no or an inhibitory effect (16, 32) . We have confirmed these observations in CHO/IR cells stimulated with both low and high concentrations of PDGF. Under our experimental conditions, CHO/IR cells incubated with 0.2 nM (5 ng/ml) PDGF for 15 min stimulated both pp125 tyrosine phosphorylation and actin stress fiber formation, whereas incubation of cells with 1.2 nM (30 ng/ml) PDGF had no detectable effect on pp125 tyrosine phosphorylation and little effect on actin stress fibers.

In contrast to the effect of PDGF, insulin treatment of CHO/IR cells resulted in the tyrosine dephosphorylation of pp125 that correlated with a decrease in the cellular content of actin stress fibers. Thus, activation of the insulin or PDGF receptor tyrosine kinases resulted in diametrically opposite signaling events in terms of pp125 tyrosine phosphorylation and the reorganization of filamentous actin.

In addition to these divergent responses to insulin and PDGF, the effect of insulin was highly sensitive and occurred in a typical linear dose-dependent manner, whereas PDGF displayed a biphasic dose-response curve. Furthermore, the effect of insulin was transient, with maximal tyrosine dephosphorylation of pp125 and reduction in actin stress fibers occurring at 15 min. However, following 60 min in the continuous presence of insulin, both pp125 tyrosine phosphorylation and actin stress fibers recovered to the unstimulated state. Although 0.2 nM (5 ng/ml) PDGF increased actin stress fibers, we have also observed that this response occurred in a transient manner with the near complete return of actin stress fiber content to the basal state following 60 min of PDGF addition (data not shown). These data demonstrate that the acute effects of insulin and PDGF were divergent in terms of pp125 tyrosine phosphorylation and assembly of actin stress fibers. However, in each case the cells ultimately recovered to steady-state levels that were similar to the unstimulated state.

The physiological role of this complex pattern of filamentous actin reorganization and pp125 tyrosine phosphorylation is completely unknown. In addition to the structural role for the actin cytoskeleton and its involvement in cellular adhesion, it may also provide important elements necessary for mediating metabolic and/or mitogenic actions of various growth factors. In this regard, it has recently been suggested that the actin cytoskeleton is an essential component necessary for the insulin-stimulated translocation of GLUT4 protein-containing intracellular vesicles (33) .

Since all receptor tyrosine kinases, including the insulin and PDGF receptors, appear to utilize a similar cadre of effector proteins to elicit biological responses, the molecular basis for signal specificity has remained obscure. The divergent, acute effects of insulin and PDGF on actin stress fibers and pp125 tyrosine phosphorylation is likely to underlie an important component of receptor tyrosine kinase signaling specificity. In future studies, analyses of the signaling pathways that couple to these events will not only elucidate the role of these events in biological responsiveness, but they will also provide important information defining the molecular basis of tyrosine kinase receptor signaling specificity.


FOOTNOTES

*
This work was supported by research Grants DK33823 and DK25295 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a research fellowship training award from The University of Iowa Cardiovascular Center.

Contributed equally to this study.

**
To whom correspondence should be addressed: Dept. of Physiology and Biophysics, The University of Iowa, Iowa City, IA 52242.

The abbreviations used are: pp125, 125-kDa focal adhesion kinase; PDGF, platelet-derived growth factor; CHO/IR, Chinese Hamster ovary cells expressing the human insulin receptor; SH2, Src homology 2 domain; PBS, phosphate-buffered saline; TRITC-phalloidin; car-boxytetramethylrhodamine isothiocyanate-conjugated phalloidin; FITC-phalloidin, fluorescein isothiocyanate-conjugated phalloidin.


ACKNOWLEDGEMENTS

We thank Tom Moninger and Randy Nessler for assistance with the fluorescent microscopic analysis and Paul Tompach and Dr. Alice Fulton for helpful advice on the labeling of filamentous actin.


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