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LIM Kinase 1: Evidence for a Role in the Regulation of Intracellular Vesicle Trafficking of Lysosomes and Endosomes in Human Breast Cancer Cells

Posted on: Saturday, 13 November 2004, 03:00 CST

LIM kinase 1; Rho/ROCK; Lysosomes; Endosomes; Endocytosis; Tumor invasion

LIM kinase (LIMK) plays a critical role in stimulus-induced remodeling of the actin cytoskeleton by linking signals from the Rho family GTPases to changes in cofilin activity. Recent studies have shown an important role for LIMK1 signaling in tumor cell invasion through regulating actin dynamics. In this study, we investigate the role of LIMK1 in intracellular vesicle trafficking of lysosomes/ endosomes. We analyzed by confocal immunofluorescence microscopy the cellular distribution of lysosomal proteins and the endocytosis of an endocytic tracer, epidermal growth factor (EGF), in LIMK1- transfected cells. We found in these cells an abnormal dispersed translocation of lysosomes stained for LIMPII and cathepsin D throughout the cytoplasm. The small punctate structures that stained for these lysosomal proteins were redistributed to the periphery of the cell. Computational 3D-image analysis of confocal immunofluorescence micrographs further demonstrated that these vesicles did not colocalize with the transferrin receptor, an early endosomal marker. Furthermore, LIMPII-positive lysosomes did not colocalize with early endosomes labeled with endocytosed Texas red- transferrin. These results indicate that there is no mixing between dispersed lysosomes and early endosomes in the LIMK1-transfected cells. Moreover, LIMK1 overexpression resulted in a marked retardation in the receptor-mediated internalization of Texas red- labeled EGF in comparison with mock-transfected cells. At 30 min after internalization, most of the Texas red-EGF staining overlapped with LIMPII-positive late endosomes/lysosonies in mock-transfected cells, whereas in LIMK1 transfectants only a small fraction of internalized EGF colocalized with LIMPII-positive structures in the perinuclear region. Taken together, the findings presented in this paper suggest that LIMK1 has a role in regulating vesicle trafficking of lysosomes and endosomes in invasive tumor cells.

Introduction

Invasion and metastasis are multistep processes that require the coordinated expression of a number of proteins by the tumor cells resulting in altered cell-cell and cell-substratum adhesion (Liotta and Kohn, 1990; Timar et al., 2001, 2002, 2003). In a variety of invasive tumor cells, increased expression of lysosomal cathepsins and an altered redistribution of lysosomes in the plasma membrane were observed in comparison with noninvasive tumors (Rochefort et al., 1990; Sloane et al., 1994). These abnormalities in the intracellular localization of lysosomes may reflect an important process for tumor invasion (Kageshita et al., 1995;Taniguchi et al., 1990). Indeed, we have previously demonstrated that the expression of the mutated small GTPase c-Ha-Ras oncogene in human breast epithelial cell line MCF10A resulted in high invasivcness and metastalic ability as well as redistribution of vesicles stained for lysosomal cathcpsin D and lamp-1 (lysosome-associated membrane protein) toward the cell surface. We therefore suggest that a Ras signaling pathway regulates the intracellular lysosomal membrane trafficking (Nishimura el al., 1998).

The small GTPase protein Rho is a molecular switch that regulates cellular events related to cell motility, cytoskeletal dynamics, and tumor progression (Hall, 1994,1998; Kishi et al., 1993; Paterson et al., 1990; Perona et al, 1993; Prendergast et al., 1995; Takaishi et al, 1993). It was demonstrated that expression of a dominant active form (Vall4) of RhoA in rat hepatoma cells (MM1) caused an increase in the invasive ability of these cells in vitro and in vivo, and enhanced the phosphorylation of myosin light chain MLC20 (Yoshioka et al., 1998, 1999). Therefore, it was suggested that there is a close correlation between the expression level of active RhoA and the invasiveness of these cells. Among the Rho targets are members of the family of Rho-associated serine-threonine protein kinases (ROCK) (Amano et al., 1997; Ishizaki et al, 1997; Leung et al., 1995; Matsui et al., 1996; Nakagawa et al., 1996) which also participate in cell-to-substrate adhesions, stress-fiber formation, and stimulation of actomyosin-based cellular contractility. It has been demonstrated that ROCK, like Rho, is involved in tumor invasion (Itoh et al., 1999). We have recently demonstrated that overexpression of RhoA and ROCK in human epithelial cell lines increases their invasiveness, and causes a selective redistribution of lysosomes (Nishimura et al., 2000, 2002, 2003). Since oncogenic Ras requires both RhoA and Rac-1 expression in order to establish a fully transformed state (Khosravi-Far etal., 1995), we expected that RhoA-ROCK signaling could possibly play a major role in regulating the intracellular vesicle trafficking of lysosomes.

Recent studies showed that ROCK can phosphorylale and aclivate LIM kinase 1 and 2 (LIMK1 and LIMK2) (Amano et al., 2001; Ohashi et al., 2000) as well as myosin light chain 20 (MLC20) (Amano et al., 1996). LIMKl regulates actin dynamics by inhibiting the activity of the actin-depolymerizing factor cofilin (Arber et al., 1998; Yang et al., 1998). Phosphorylated cofilin no longer binds to actin, resulting in net actin polymerization. Thus, LIMKs play a critical role in remodeling of the aclin cyloskeleton by linking the signal from lhe Rho family of small GTPases to lhe change in cofilin aclivily. Indeed, a recent study showed an important role for LIMK1 signaling in tumor cell invasion through regulating actin dynamics (Yoshioka el al., 2003).

In the present study, we investigate by confocal immunofluorescence microscopy the possibility that LIMK1 activity is involved in the regulation of vesicle trafficking of lysosomes and endosomes in MCF-7 cells, a human breast cancer cell line permanently expressing LIMK1. We examined the intracellular distribulion of lysosomes by using specific antibodies to LIMPII/ LGP85 or the lysosomal aspartic prolease, calhepsin D. These proteins are distributed within the endocytic organelles at the highest concentration in late endosome/lysosomes as observed for the other lysosomal glycoproteins, LAMP-1 and LAMP-2 (Kornfeld and Mellman, 1989; Okazaki et al., 1992, Sandoval et al., 1994; Tabuchi et al., 2000).

We found evidence that the overexpression of LIMK1 in MCF-7 cells resulted in an aberrant redistribution of lysosomes. We dcmonslrale that the dispersed lysosomes at the cell periphery do not colocalize with early endosome markers and are not accessible to the endocylic tracer, Texas red-transferrin. We also found that overexpression of LIMK1 resulted in a marked retardation of the receptor-mediated internalization of the endocytic tracer, Texas red-EGF. We propose that LIMK1 signaling plays an important role in the regulation of vesicle traffic of lysosomes and endosomes by reorganizing and influencing actin-filament dynamics in invasive tumor cells.

Materials and methods

Materials

MCF-7 human breast cancer cells (JCRB0134) were obtained from Health Science Research Resources Bank (Osaka, Japan). Texas redlabeled transferrin, Texas red-labeled EGF, Texas red-labeled phalloidin, and SlowFade anti-fade reagent were purchased from Molecular Probes (Eugene, OR, USA). Wortmannin was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The molecular weight markers for immunoblotting were from Amersham Pharmacia Biotech (Piscataway, NJ, USA). Other chemicals were of reagent grade and were obtained from commercial sources.

Transfection of LIMK1 cDNA

Myc-tagged DNA constructs encoding mouse LIMK1, as described elsewhere (Arber et al., 1998), were subcloned into FLAG-tagged pcDNA3 vector (Invitrogen; Groningen, The Netherlands). The Flag- tagged LIMK1 construct was transfccted into MCF-7 human breast cancer cells (JCRB0134) using Lipofectamine Plus reagent (Invitrogen), as described (Yoshioka et al., 2003). Stable cell lines expressing LIMK1 were selected in the presence of 800 g/ml G418 for 4 weeks and cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL; Grand Island, NY, USA) with non-essential amino acids (Invitrogen), 0.1 mm Na pyruvate (Invitrogen), 10 g/ml insulin (Invitrogen) and 10% fetal calf serum (FCS; Equitech-bio, TX, USA).

Antibodies

Alexa 488-labeled goat anti-mouse and goat anti-rabbit secondary antibodies were obtained from Molecular Probes. Normal goat serum was purchased from Sigma. Antisera were raised in rabbits (New Zealand white male) against the mature form of rat liver lysosomal cathepsin D (Nishimura et al., 1987, 1988) and the native form of LIMPII/LGP85 (Okazaki et al., 1992) as described previously. AnIicathepsin D or anti-LIMPII IgG was affinity-purified using protein A Sepharose CL-4B (Pharmacia Biotech, Uppsala, Sweden), followed by immunoaffinity chromatography using antigen-conjugated Sepharose 4B. The rat anti-LIMK1 monoclonal antibody has been described previously (Foletta et al., 2004). Anti-vinculin and anti- transferrin receptor monoclonal antibodies were obtained from Sigma and Immunotech (France), respectively.

Immunofluorescence microscopy

Immunofluorescence microscopy was described previously (Nishimura et al., 2000, 2002, 2003). In brief, cells were grown for 2 days on glass coverslips in 6-well plat\es in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Cells were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS), pH 7.4, and permeabilized in PBS containing 0.1% saponin. After washing with PBS, cells were blocked with PBS-10% normal goat serum. All subsequent antibody and wash solutions contained 0.1% saponin. The mock or the LIMK1 transfectants were incubated with specific primary antibodies (rabbit anti-LIMPII and anti-cathepsin D IgG, rat anti- LIMK1 mAb, mouse anti-vinculin mAb, or mouse anti-transferrin receptor IgG2), for 1 hour, followed by washes with PBS containing 0.1% saponin and incubation for 1 hour with the secondary antibodies at 20 g/ml. For F-actin staining, cells were incubated with Texas red-phalloidin (1 U/ml) for 30 min at room temperature followed by three washes with PBS containing 0.1% saponin. To label early endosomes, cells were treated with or without 100 nM wortmannin for 30 min at 37C followed by 20 rain incubation in culture medium containing Texas red-conjugated transferrin. The cells were fixed and stained for LIMPII or cathepsin D. Controls for antibody specificity were either preimmune serum (rabbit or mouse) or omission of the primary antibodies. To follow the endocytic pathway and determine the intracellular fate of internalized labeled ligand, the uptake of Texas red-conjugated EGF was measured. To minimize the contribution of recycling and/or lysosomal degradation of internalized EGF receptor (EGFR), we quantified the Texas red-EGF uptake for relatively brief time periods (5, 15, or 30 min).

The distribution of the labeled proteins was analyzed by confocal immunofluorcscence microscopy of the fixed cells. Slides were mounted with SlowFacle anti-fade reagent and observed on a Zeiss LSM 510 META confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany), equipped with krypton/argon laser sources. To obtain 3D- imaging data sets or to generate optical sections of the cells by confocal laser scanning microscopy, 30 optical sections along the Z- axis were acquired with increments of 0.35 m using a 100 oil immersion objective (NA 1.4), and the fluorescent data sets were analyzed using LSM 510 software. The multitrack standard FITC/ rhodamine or singletrack rhodamine configuration was selected. Pinhole diameter was maintained as 1 Airy Unit.

In order to confirm the colocalization of internalized Texas red- EGF with LIMPII-positive structures in the cells, the immunofluorescence intensity profiles of the green channel (Ch2-T1) and red channel (Ch3-T2) were analyzed in a 2D-histogram function using LSM 510 META software installed in a Zeiss laser scanning confocal microscope. The cells were incubated with Texas red-EGF for 30 min, and intracellular localization of the internalized Texas red- EGF and Alexa 488-labelled LIMPII-positive late endosomes/lysosomes was analyzed by confocal immunofluorescence microscopy after fixation of the cells. A significantly overlapping area (m^sup 2^) in the 2D-histogram was selected by using the colocalized area button, and thus selected colocalization appeared as white pseudo- color image in the superimposed data (see Fig. 7A, B). Images were converted to TIFF format, and contrast levels of the images were adjusted using Adobe Photoshop 6.0 software (Adobe Co.) on Power Macintosh G4 computers (Apple, Tokyo, Japan).

Immunoblot analysis

For detection of LIMK1 protein, cells (5 10^sup 4^) were lysed, separated by 7% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Richmond, CA, USA) using the semi- dry method. The blot was probed with rat anti-LIMK1 mAb (1:3000), followed by washing and incubation with goat anti-rat IgG alkaline phosphatase conjugate (Promega, Japan) (1:2500). The signal was developed using nitro blue tetrazolium and 5-bromo-4-chloro-3- indolyl-phosphate (Promega) as substrate. The membrane was scanned with a GT8200-UFflat scanner (Epson, Tokyo, Japan) and analyzed with NIH image software using a Power Macintosh G4 computer (Apple, Tokyo, Japan).

Results

Overexpression of LIMK1 results in formation of actin stress fibres, membrane blebs, and focal adhesions

We generated an MCF-7 cell line stably expressing myc-tagged LIMK1 and examined the level of endogenous and overexpressed LIMK1 proteins by immunoblotting. As shown in Figure 1, the overexpressed LIMK1 protein migrates as a single band with a mobility slower than that of endogenous LIMK1 with an apparent molecular mass of approximately 66.5 kDa. The expression level of the overexpressed LIMK1 was 7.4-fold higher than the endogenous protein. A recent study has demonstrated that MCF-7 clones expressing high levels of LIMK1 (6 to 10-fold more than the endogenous protein) have an increased invasiveness in the Matrigel invasion assay (Yoshioka et al., 2003). However, it has also been verified that the overexpression of LIMK1 has no effect on the other signaling pathways of ROCK because the level of phosphorylated MLC-20 in LIMK1 transfectants was similar to that in mock transfectants (Yoshioka et al., 2003).

Fig. 1. Immunoblot analysis of LIMK1 expression in MCF-7 cells. Immunoblot of cell lysates prepared from mock or LfMKl transfectants probed with rat anti-LIMK1 mAb (upper panel) or anti-β-actin antibodies, as loading control (lower panel). The fold change in the level of overexpression of LIMK1 protein is indicated.

Next, we carried out a confocal immunofluorescencc analysis to determine the intracellular distribution of F-actin by staining with Texas red-labeled phalloidin, and the focal adhesion complexes visualized with an anti-vinculin antibody (Fig. 2). MCF-7 cells transfected with LIMK1 have an elongated polar cell shape with pointed edges and F-actin was associated with the peripheral plasma membrane. The morphology of the LIMK1 transfectants appears to be similar to that of transfected MCF-7 cells stably expressing active ROCK. ROCK expression markedly enhanced the formation of stress fibres terminating at the pointed edges of the cells (Nishimura et al., 2003). The size and number of focal adhesion complexes was increased in the LIMK1-transfected cells. 3D image analysis of LIMK1- MCF7 cells demonstrated that vinculin is localized at the pointed edges of the basal part of the plasma membrane (0-0.35 m from the bottom of the cell) where distinct actin aggregates were clearly seen (data not shown). Therefore, these results indicate that promotion and growth of actin aggregates is enhanced by the transfection of LIMK1 (Fig. 2A). When Y-27632, a selective ROCK inhibitor (Uehata et al., 1997), was added to the LIMK1 transfectants, the accumulation of focal adhesion complexes was not observed and actin stress fibres were absent (data not shown), indicating - as previously shown - that ROCK activates LIMK1 which subsequently is influencing the reorganization of the actin cytoskcleton and the assembly of focal adhesion complexes (Ohashi et al., 2000).

When the cells were double-stained for LIMKl and F-actin, LIMK1 staining in the mock transfectants appeared to be localized in small vesicular structures throughout the cytoplasm, and little staining was associated with the plasma membrane. In contrast, in the LIMK1 transfectants increased amounts of LIMK1 staining were seen at pointed edges in the peripheral area of the cell overlapping with accumulated aclin stress fibres (Fig. 2B).

LIMK1 overexpression induces selective redistribution of lysosomes

To determine the intracellular localization of late endosomes/ lysosomes, cells were double-labeled with Texas red-phalloidin and specific antibodies to LIMPII or cathepsin D (Fig. 3). Mock- transfectants did not show formation of distinct stress fibres, and LIMPII and cathepsin D were present primarily in small punctate structures in the perinuclear region (Fig. 3a-c, g-i), which corresponds to a typical pattern of lysosomal vesicles (Nishimura et al., 2000, 2002, 2003). In contrast, in the cells expressing LIMK1 cDNA, LIMPII- or cathepsin D-stained small punctate vesicles were widely redistributed throughout the cytoplasm (Fig. 3d-f, j-1). The dispersed punctate structures stained for LIMPII or cathepsin D mostly colocalized with LAMP1 (data not shown). The distribution of lysosomes and endosomes in the LIMK1 transfectants is similar to that observed in MCF-7 cells expressing active ROCK (Nishimura et al., 2003).

Fig. 2. Formation of focal adhesion complexes and distribution of LIMK1 and F-actin in mock and LIMK1 transfectants. A. Mock- (a-c) or LIMK1-transfected (d-f) MCF-7 cells were fixed and double-stained with anti-vinculin antibody (green) and for F-actin with Texas red- phalloidin (red). Confocal images were obtained as described in Materials and methods. The white arrows indicate the induced formation of focal adhesion complexes as visualized by vinculin staining. B. Mock (a-c) or LIMK1 (d-f) transfcctants were double- stained with anti-LIMK1 raAb (green) and for F-actin with Texas redphalloidin (red). Note that the induced actin stress fibres are associated with the plasma membrane where the increased focal adhesion complexes, revealed by vinculin staining, are clearly seen in the LIMK1 transfectants. Bars, 10 m.

Fig. 3. The sub-cellular localization of lysosomal proteins and F- actin in mock and LIMK1 transfectants. Mock (a-c, g-i), or LIMK1 (d- f, j-1) transfectants were fixed and double-stained for LIMPII1 or cathepsin D (green) and F-actin (red) as described in Materials and methods. Superimposed images of lysosomal proteins and F-actin are shown in (c, f, i, and 1). Note that in mock-transfected cells, lysosomal LIMPII and cathepsin D are mainly localized in the perinuclear region. By contrast, in LIMK1 transfectants a dispersed localization of lysosomal proteins is observed. Furthermore, actin stress fiber formation is clearly seen in LIMK1-transfected cells. Bars, 10 m.

Endocytosed transferrin is not found inthe dispersed lysosomes of LIMK1 transfectants

Because the dispersed small vesicles that stained for LIMPII were punctate, round in shape, and peripherally distributed, we speculated that they might be of endocytic origin such as early endosomes, recycling endosomes, late endosomes or lysosomes, the content of which is transported in vivo and in vitro in a microtubule-dependent fashion (Mattconi and Kreis, 1987). To confirm the identity of the LIMPII-immunoreactive dispersed vesicular structures seen in the LIMKl transfectants, the cells were co- stained for LIMPII and the transferrin receptor, or for EEA1 (early endosomal antigen) which serve as markers for early endosomal structures (Mu et al., 1995). We found that in the LIMK1 transfcctants the dispersed LIMPII-containing vesicular structures did not colocalize with the immunostained transferrin receptor or EEA1 (data not shown).

Fig. 4. Texas red-transferrin is not delivered to the dispersed LIMPII-positive lysosomes. Mock or LIMK1 transfectants preincubated at 37 C for 30 min in the absence (A) or presence (B) of wortmannin were further incubated for 20 min with Texas red-labeled transferrin (Tf). The distribution of the internalized Texas red-transferrin and late endosomes/lysosomes stained with antiLIMPII antibody was analyzed by confocal immunofluorescence microscopy after fixation of the cells. Superimposed images of LIMPII and Texas red-transferrin are shown as indicated. Each optical section along the Z-axis was acquired with increments of 0.35 m as described in Materials and methods (B, lower panels). An orthographic section (at 0.35 m from the cell bottom) is shown in (C). Note that internalized Texas red- transferrin can reach early endosomes after 20 min incubation, but does not colocalize with the dispersed LIMPII-positive structures in LIMK1 transfectants. Bars, 10 m.

Fig. 5. Texas red-EGF is endocytosed efficiently into the early endosomes and late endosomcs/lysosomes in mock-transfected cells. Mock-transfccted MCF-7 cells were incubated at 37C with Texas red- EGF for 5 min (A), 15 min (B), or 30 min (C). The distribution of internalized Texas red-EGF in early endosomes (stained with anti- transferrin receptor antibody) and late endosomes/lysosomes (stained with anti-LIMPII antibody) was studied by confocal immunofluorescence microscopy after fixation of the cells. Superimposed images of EGF and transferrin receptor (TfR) or EGF and LIMPII are shown as indicated. Optical sections along the Z-axis were acquired with increments of 0.35 m as described in Materials and methods. X-Y optical sections (0.35 m) are shown in (D) and numbers in the lower left corner of each panel indicate the distance of the sections to the cell bottom. Each orthographic section of the cell is shown in (D), and colocalizations are indicated by white arrows. The top horizontal window of each panel in (D) indicates X- Z section (green horizontal line), and the right vertical window indicates Y-Z section (red horizontal line). Note that significant amounts of internalized Texas red-EGF overlap with transferrin receptor at 5 min internalization (A). After 15 min incubation, some of the EGF staining already colocalizes with LIMPII, indicating that the internalized EGF is efficiently transported through early endosomes to late endosomes/lysosomes within 15 min, and that the cndocytic pathway is functioning normally in mock-transfected MCF-7 cells.

Fig. 6. Overexpression of LIMK1 in MCF-7 cells impedes uptake and delivery of Texas red-EGF into early endosomes and late endosomes/ lysosomes. The LIMK1 transfectants were incubated and treated as described for Figure 5. Note that internalization of Texas red-EGF is not observed in the first 5 min (A). After 15 min internalization, only a small fraction of internalized EGF colocalizes with transferrin receptor-positive early endosomes, and there is no overlap of EGF staining with LIMPII-positive late endosomes/lysosomes (B). Even after 30 min internalization the small vesicles containing internalized EGF are clearly distinct from the LIMPII-positivc lysosomes in the pcrinuclear region (C).

To examine whether the LIMK1-induced dispersed lysosomes are stained with endocytic markers, the LIMK1 transfectants were allowed to internalize Texas red-labeled transferrin for 20 min. Then cells were fixed and stained for LIMPII (Fig. 4). Texas red-transferrin was efficiently endocytosed and was found in early endosomes in the peripheral region. After 20 min incubation, some of the transferrin was also seen in the recycling endosomes in the perinuclear region, suggesting that LIMK1 overexprcssion has no effect on the intracellular endocytic trafficking of transferrin during a 20-min incubation. Importantly, LIMPII-positive lysosomes did not overlap with early endosomes in the LIMK1 transfectants (Fig. 4A). The spatial relationship between the distribution of LIMPII-positive lysosomes and Texas red-labeled transferrin in the LIMK1 transfectants was further studied by confocal immunofluorescence microscopy. The optical sections acquired along the Z-axis showed that the redistributed lysosomes were found at the bottom part of the cell (0.33 m), in the same area as the early endosomes labeled with Texas red-transferrin. However, there was no colocalization (Fig. 4C), indicating that the lysosomes redistributed toward the cell surface do not enter the endocylic pathway and do not fuse with early endosomes in the LIMK1-transfected cells.

Recently, we have demonstrated that wortmannin, an inhibitor of phosphatidylinositol 3-kinase, known to mediate membrane trafficking at several distinct steps of the early endocytic pathway (Brown et al., 1995; Davidson, 1995; Reaves ct al., 1996), caused the induction of large swollen vacuoles in the cytoplasm of the active RhoA or ROCK transfectants. These enlarged vacuoles were positive for LIMPII or cathepsin D but did not stain with anti-transferrin receptor antibodies. Therefore, we suggested that the large vacuoles were derived from late endosomes/lysosomes (Nishimura et al., 2002, 2003). These findings suggest that inhibition of phosphatidylinositol 3-kinase by wortmannin selectively enhances the enlargement of late endosomes/lysosomes, but does not cause the mixing of early endosomes and late endosomes/lysosomes.

In order to further analyze whether the dispersed lysosomes in the LIMK1 transfectants are accessible to the early endosomes, we examined the effect of wortmannin on the redistribution of lysosomes. The LIMK1 transfectants were treated with 100 nM wortmannin for 30 min before incubation with Texas red-transferrin for 20 min and subsequent staining for LIMPII. Wortmannin treatment resulted in an increased number of enlarged swollen vacuoles in the perinuclear region and the disappearance of the LIMPII-positive lysosomes scattered throughout the cytoplasm (Fig. 4B). Furthermore, all the LIMPII staining was localised in these swollen vacuoles. However, these LIMPII-positive swollen vacuoles were devoid of Texas red-transferrin. Analysis of the optical sections throughout the Z- axis further demonstrated that LIMPII-positive large swollen vacuoles were accumulated in the perinuclear region and were not doubly positive for LIMPII and endocytoscd transferrin (Fig. 4C). Taken together, these results confirm that the small punctate vesicles immunostained for LIMPII or cathepsin D dispersed throughout the cytoplasm are indeed lysosomes, thereby implying that increased ROCK-LIMK1 signaling induces the selective dispersion of lysosomes in the cytoplasm. These dispersed lysosomes exclude the early endocytic compartments.

Endocytosis of EGF is suppressed in the LIMK1 transfectants

To clarify the role of overexpressed LIMK1 in EGFR intemalization, we followed the uptake of Texas red-conjugated EGF with time by mock and LIMK1 transfectants. To minimixe the contribution of recycling and/or lysosomal degradation of the internalized EGFR, we quantified the Texas red-EGF uptake in the LIMK1 transfectants for time periods no longer than 30 min. The distribution of internalized Texas rcd-EGF, early endosomes (stained with anti-transferrin receptor antibody) and late endosomes/ lysosomes (stained with anti-LIMPII antibody) was then studied by confocal immunofluorescence microscopy.

In mock-transfected cells, a rapid intemalization of EGF was observed after 5 min, and part of the internalized EGF colocalized with tranferrin receptor (Fig. 5A). After 15min intemalization, considerable amounts of EGF already colocalized with LIMPII- positive punctate structures (Fig. 58 and D), and at 30 min, most of the EGF staining overlapped with LIMPII-positive lysosomes (Fig. 5C and D). These results indicate that EGF-EGFR complexes are efficiently internalized in mock-transfected cells after 5 min and that this complex is transported to late endosomes/lysosomes after passing through early endosomes within 15 min intemalization. In contrast, in the LIMK1 transfectants, no intemalization of Texas red- EGF was observed after 5 min (Fig. 6A). After 15 min intemalization, only a small fraction of the internalized Texas red-EGF was found to colocalizc with transferrin receptor-positive early endosomes, and no EGF staining was found in the LIMPII-positive late endosomes/ lysosomes (Fig. 6B and D). Even after 30 min internalization, the small punctate vesicles that positively stained for the internalized EGF were found in close proximity to the LIMPII-positive vesicular structures in the perinuclear region, but EGF staining was clearly distinct from the LIMPII-positive lysosomes (Fig. 6C and D). These data indicate that the endocytotic machinery necessary for intemalization of EGF is considerably suppressed in the LIMK1-MCF-7 cells.

In order to further study the effects of LIMK1 overexpression on the internalization of EGF, superimposed images of the internalized Texas red-EGF and Alexa 488-labelled \LIMPII were analyzed by confocal immunofluorescence microscopy at 30 min after internalization and compared by histogram LSM510 META software (Fig. 7). The colocalization between the internalized Texas red-EGF and Alexa 488-labelled LIMPII-positive late endosomes/lysosomes appeared as white pseudo-color in transfectants. In the mock transfectants most of the EGF staining overlapped with the LIMPII-positive late endosomes/lysosomes after 30 min internalization (Fig. 7A). In contrast, only part of the Texas red-EGF-positive vesicular structures colocalized with the LIMPII-positive vesicles in the perinuclear region of the LIMK1 transfectants (Fig. 7B), indicating that the mixing of these vesicles does not occur during the 30 min internalization. Therefore, we suggest that overexpression of LIMK1 has a suppressive effect on the endocytosis of EGF.

Discussion

To investigate the role of LIMK1 on the intracellular vesicle trafficking of lysosomes/cndosomes, we analyzed the cellular distribution and trafficking of lysosomal proteins and of endocytic tracers by confocal immunofluorescence microscopy in LIMK1- transfected cells. We have demonstrated that overexpression of LIMK1 protein in MCF-7 leads to an abnormal dispersed intracellular localization of LIMPII- and cathepsin D-positive lysosomes. As previously demonstrated (Yoshioka et al., 2003), it was shown that highly increased LIMK1 activity resulted in enhanced accumulation of F-actin aggregates mostly associated with the plasma membrane. In these cells LIMK1 co-localized with focal adhesion complexes as revealed by co-staining with anti-vinculin antibody. Indeed, endogenous LIMK1 was found to co-localize with focal adhesion complexes in mouse embryonic fibroblasts and MDA-MB-231 cells (Foletta et al, 2004).

A recent study has demonstrated that the expression level of LIMK1 and its activity are increased in highly invasive breast and prostate cancer cell lines in comparison with less invasive cells, suggesting that LIMK1, a critical regulator of cellular actin dynamics, plays an important role in tumor cell invasion (Yoshioka et al, 2003). The aberrant distribution of lysosomes and their translocation to the cell periphery may be a mechanism common to cells that are involved in the degradation of the extracellular matrix and the basement membrane, which is a critical step in tumor cell invasion. It is possible that overexpression of LIMK1 is involved in this abnormal translocation process of lysosomes, however, the molecular mechanism for this signaling pathway remains to be elucidated.

In the present study, to further delineate whether LIMK1-induced dispersed lysosomes are accessible to endocytic markers, the LIMK1 transfectants were allowed to internalize Texas red-labeled transfcrrin. Our 3D-image analysis revealed that LIMPII- or cathepsin D-positive lysosomes do not colocalize with early endosomes in transfected cells, further confirming that lysosomes redistributed toward the cell surface do not participate in the endocytic pathway.

We have recently shown that overexpression of active RhoA and ROCK in rat hepatoma and MCF-7 cells, respectively, resulted in a remarkable redistribution of lysosomal vesicles towards the cell periphery, and that these vesicles are not accessible to early endocytic tracers (Nishimura et al., 2002, 2003). The dispersed vesicles were found to retain their acidity and were also reclustered in the perinuclear region when the cells were incubated with Y-27632 (Nishimura et al., 2000, 2002, 2003). However, there was no apparent change in the fluid-phase endocytosis of dextran from the cell surface through early endosomes to lysosomes and in the receptor-mediated endocytic pathway. Therefore, we postulate that the RhoA-ROCK signaling pathway preferentially regulates the intracellular vesicle trafficking of lysosomes but has no effect on the early stage of the endocytic pathway. Because LIMK1 is one of the downstream effectors of ROCK, it is possible that ROCK-LIMK1 signaling exerts its effects on lysosome and endosome trafficking by affecting actin dynamics or association of actin-binding proteins. Alternatively, we cannot exclude the possibility that the organization of the actin cytoskeleton is involved in lysosome and endosome trafficking through regulating microtubule dynamics in the transformed cell lines, since changes in the actin cytoskeleton are often correlated with microtubule rearrangements. Indeed, it has previously been reported that the cross-talk of the Rho/ROCK signaling is sufficient to induce neurite retraction and cell rounding, which is accompanied by elevated phosphorylation of myosin light chains and the disassembly of microtubules and intermediate filaments in neuroblastoma cells (Hirose et al., 1998). On the other hand, it was reported that Rho proteins interact with kinectin, an anchoring protein of kinesin motor (Hotta et al., 1996). Moreover, it has been shown that RhoG interacts with kinectin that binds to conventional kinesin and activates its microtubule-stimulated ATPase activity (Vignal et al., 2001). A morphological study of active RhoG- transfected cells demonstrated an enhanced movement of lysosomes. The molecular mechanisms by which overexpressed RhoG influences microtubules remains unknown, and the biological significance of the interaction between RhoA and the microtubule motor protein is unclear.

Fig. 7. The delivery of internalized Texas red-EGF into LIMPII- positive lysosomes is significantly retarded in LIMK1-transfected MCF-7 cells. Mock (A) or LIMK1 (B) transfectants were incubated with Texas red-EGF for 30 min and the intracellular localization of the internalized Texas red-EGF and Alexa 488-labelled LIMPII-positive structures was analyzed by confocal immunofluoresccnce microscopy after fixation of the cells. The white arrows indicate colocalization. In (C), the immunofluorescence intensity profiles of the green (Ch2-T1) and red channels (Ch3-T2) were analyzed in a 2D- histogram as described in Materials and methods. An area of significant overlap (shaded box) was selected, resulting in white pseudo-color imaging of colocalizations in the merged micrographs (A, B).

We examined the effect of LIMK1 overexpression on the internalization of EGF, and found significant retardation in the internalization of labeled EGF in comparison with mock transfectants, suggesting that the endocytic machinery for internalization of EGF is considerably suppressed by the overexpression of LIMK1 in MCF-7cells.

In active ROCK transfectants, on the other hand, internalization of EGF was observed after 5 min, and the internalized EGF colocalized with the transferrin receptor (data not shown). At 15 min after internalization, most of the EGF staining co-localized with the transferrin receptor and some of the internalized EGF was already associated with LIMPII staining. Moreover, the ROCK inhibitor (Y-27632) did not have any inhibitory effect on the efficient internalization of EGF in active ROCK transfectants, and dextran beads, as a fluid-phase endocytosis marker, were efficiently internalized, reaching the LIMPII-positive lysosomes. In addition, wortmannin, which was reported to have an inhibitory effect on the receptor-mediated uptake of endocytic markers (Shpetner et al., 1996), has also an inhibitory effect on the internalization of dextran beads in RhoA transfectants. We therefore suggest that the expression of the RhoA-ROCK pathway does not perturb lysosomal/ endosomal function with regards to fluid-phase endocytosis. It is not clear at present why LIMK1 overexpression results in the retardation and suppressive effect on EGF endocytosis. Because the molecular mechanism by which LIMK1 regulates actin dymanics differs from that of the Rho-ROCK pathway, it is possible that LIMK1, independent of the ROCK-myosin light chain pathway, may be involved in the regulation of receptor-mediated endocytosis. Further studies will have to clarify the detailed mechanisms how the LIMK1 signaling pathway regulates intracellular vesicle trafficking of lysosomes and endosomes.

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Yukio Nishimura1)a, Kiyoko Yoshioka(b), Ora Bernard(c), Masaru Himenoa, Kazuyuki Itoh(b)

a Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

b Department of Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-2 Nakamichi, Higashinari-ku, Osaka 537- 8511, Japan

c The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia

Received November 7, 2003

Received in revised version April 7, 2004

Accepted May 4, 2004

1) Corresponding author: Dr. Yukio Nishimura, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan, e-mail: nishimur@bioc.phar.kyushuu.ac.jp, Fax: +81926426617.

Copyright Urban & Fischer Verlag Aug 2004

Source: European Journal of Cell Biology

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