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The localization of myosin VI at the golgi complex and leading edge of fibroblasts and its phosphorylation and recruitment into membrane ruffles of A431 cells after growth factor stimulation.

Buss F, Kendrick-Jones J, Lionne C, Knight AE, Côté GP, Paul Luzio J - J. Cell Biol. (1998)

Bottom Line: It was found that in NRK and A431 cells, myosin VI was associated with both the Golgi complex and the leading, ruffling edge of the cell as well as being present in a cytosolic pool.In vitro experiments suggested that a p21-activated kinase (PAK) might be the kinase responsible for phosphorylation in the motor domain.These results strongly support a role for myosin VI in membrane traffic on secretory and endocytic pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Cambridge, CB2 2QR, United Kingdom. fb1@mole.bio.cam.ac.uk

ABSTRACT
Myosin VI is an unconventional myosin that may play a role in vesicular membrane traffic through actin rich regions of the cytoplasm in eukaryotic cells. In this study we have cloned and sequenced a cDNA encoding a chicken intestinal brush border myosin VI. Polyclonal antisera were raised to bacterially expressed fragments of this myosin VI. The affinity purified antibodies were highly specific for myosin VI by immunoblotting and immunoprecipitation and were used to study the localization of the protein by immunofluorescence and immunoelectron microscopy. It was found that in NRK and A431 cells, myosin VI was associated with both the Golgi complex and the leading, ruffling edge of the cell as well as being present in a cytosolic pool. In A431 cells in which cell surface ruffling was stimulated by EGF, myosin VI was phosphorylated and recruited into the newly formed ruffles along with ezrin and myosin V. In vitro experiments suggested that a p21-activated kinase (PAK) might be the kinase responsible for phosphorylation in the motor domain. These results strongly support a role for myosin VI in membrane traffic on secretory and endocytic pathways.

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Localization of myosin VI, V, and ezrin in A431 cells  after stimulation with EGF. Cells were fixed at 0, 2, 5, and 15 min  after addition of EGF and stained with rabbit polyclonal antibodies to myosin VI (affinity-purified PGT ab), myosin V (affinity-purified tail domain ab), and ezrin (affinity-purified ab) followed  by fluorescently labeled anti–rabbit IgG. Extended focus projections of a z-series of images obtained by confocal microscopy are  shown. Bar, 20 μm.
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Figure 5: Localization of myosin VI, V, and ezrin in A431 cells after stimulation with EGF. Cells were fixed at 0, 2, 5, and 15 min after addition of EGF and stained with rabbit polyclonal antibodies to myosin VI (affinity-purified PGT ab), myosin V (affinity-purified tail domain ab), and ezrin (affinity-purified ab) followed by fluorescently labeled anti–rabbit IgG. Extended focus projections of a z-series of images obtained by confocal microscopy are shown. Bar, 20 μm.

Mentions: The observed localization of myosin VI in the leading edge of fibroblasts suggested a functional role in the dynamic formation of ruffles and other membrane protrusions at the plasma membrane. In human carcinoma A431 cells this ruffling can be induced by the addition of EGF (Chinkers et al., 1979), and since these cells contain a high number of surface receptors for this growth factor, the ruffling response is very fast and dramatic. Ruffling requires an intact actin cytoskeleton and after the addition of EGF, the actin-membrane linking protein ezrin becomes phosphorylated and is recruited into microvilli/filopodia and the newly formed ruffles (Bretscher 1989). As shown in Fig. 5 we found that in unstimulated cells, ezrin showed a uniform cytosolic distribution with no particular localization, but 2 min after EGF stimulation, ezrin became concentrated in small ruffles and microvilli-like structures covering the entire surface of the cell. Some minutes later (5–10 min) ruffling reached its peak around the edge of the cell, where ezrin became concentrated. After 15 min, the cells rounded up and ezrin was observed in the retraction fibers and in a very fine punctate pattern below the plasma membrane on the upper surface of the cells. We used the tail ab (PGT) to examine the involvement of myosin VI in the response of A431 cells to EGF. Since myosin V has been implicated in the transport of membrane vesicles along actin fibres to cellular extensions (reviewed in Titus, 1997) and has been shown to be involved in regulation of filopodia extensions from neuronal cell growth cones (Wang et al., 1996) we also examined its recruitment into ruffles of A431 cells using a specific antibody. In unstimulated cells, both myosins showed a cytosolic distribution with the myosin V being slightly enriched in preexisting small surface ruffles and myosin VI being concentrated in the juxtanuclear region. After EGF stimulation for 2 min, unlike ezrin, myosin V and VI were not recruited into surface structures on top of the cells, but along with ezrin they became very concentrated in the ruffles at the edge of the cell after 5 min stimulation. After 15 min stimulation with EGF, myosin VI was present in the contraction fibers, whereas myosin V displayed a more even distribution throughout the cell.


The localization of myosin VI at the golgi complex and leading edge of fibroblasts and its phosphorylation and recruitment into membrane ruffles of A431 cells after growth factor stimulation.

Buss F, Kendrick-Jones J, Lionne C, Knight AE, Côté GP, Paul Luzio J - J. Cell Biol. (1998)

Localization of myosin VI, V, and ezrin in A431 cells  after stimulation with EGF. Cells were fixed at 0, 2, 5, and 15 min  after addition of EGF and stained with rabbit polyclonal antibodies to myosin VI (affinity-purified PGT ab), myosin V (affinity-purified tail domain ab), and ezrin (affinity-purified ab) followed  by fluorescently labeled anti–rabbit IgG. Extended focus projections of a z-series of images obtained by confocal microscopy are  shown. Bar, 20 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132970&req=5

Figure 5: Localization of myosin VI, V, and ezrin in A431 cells after stimulation with EGF. Cells were fixed at 0, 2, 5, and 15 min after addition of EGF and stained with rabbit polyclonal antibodies to myosin VI (affinity-purified PGT ab), myosin V (affinity-purified tail domain ab), and ezrin (affinity-purified ab) followed by fluorescently labeled anti–rabbit IgG. Extended focus projections of a z-series of images obtained by confocal microscopy are shown. Bar, 20 μm.
Mentions: The observed localization of myosin VI in the leading edge of fibroblasts suggested a functional role in the dynamic formation of ruffles and other membrane protrusions at the plasma membrane. In human carcinoma A431 cells this ruffling can be induced by the addition of EGF (Chinkers et al., 1979), and since these cells contain a high number of surface receptors for this growth factor, the ruffling response is very fast and dramatic. Ruffling requires an intact actin cytoskeleton and after the addition of EGF, the actin-membrane linking protein ezrin becomes phosphorylated and is recruited into microvilli/filopodia and the newly formed ruffles (Bretscher 1989). As shown in Fig. 5 we found that in unstimulated cells, ezrin showed a uniform cytosolic distribution with no particular localization, but 2 min after EGF stimulation, ezrin became concentrated in small ruffles and microvilli-like structures covering the entire surface of the cell. Some minutes later (5–10 min) ruffling reached its peak around the edge of the cell, where ezrin became concentrated. After 15 min, the cells rounded up and ezrin was observed in the retraction fibers and in a very fine punctate pattern below the plasma membrane on the upper surface of the cells. We used the tail ab (PGT) to examine the involvement of myosin VI in the response of A431 cells to EGF. Since myosin V has been implicated in the transport of membrane vesicles along actin fibres to cellular extensions (reviewed in Titus, 1997) and has been shown to be involved in regulation of filopodia extensions from neuronal cell growth cones (Wang et al., 1996) we also examined its recruitment into ruffles of A431 cells using a specific antibody. In unstimulated cells, both myosins showed a cytosolic distribution with the myosin V being slightly enriched in preexisting small surface ruffles and myosin VI being concentrated in the juxtanuclear region. After EGF stimulation for 2 min, unlike ezrin, myosin V and VI were not recruited into surface structures on top of the cells, but along with ezrin they became very concentrated in the ruffles at the edge of the cell after 5 min stimulation. After 15 min stimulation with EGF, myosin VI was present in the contraction fibers, whereas myosin V displayed a more even distribution throughout the cell.

Bottom Line: It was found that in NRK and A431 cells, myosin VI was associated with both the Golgi complex and the leading, ruffling edge of the cell as well as being present in a cytosolic pool.In vitro experiments suggested that a p21-activated kinase (PAK) might be the kinase responsible for phosphorylation in the motor domain.These results strongly support a role for myosin VI in membrane traffic on secretory and endocytic pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Cambridge, CB2 2QR, United Kingdom. fb1@mole.bio.cam.ac.uk

ABSTRACT
Myosin VI is an unconventional myosin that may play a role in vesicular membrane traffic through actin rich regions of the cytoplasm in eukaryotic cells. In this study we have cloned and sequenced a cDNA encoding a chicken intestinal brush border myosin VI. Polyclonal antisera were raised to bacterially expressed fragments of this myosin VI. The affinity purified antibodies were highly specific for myosin VI by immunoblotting and immunoprecipitation and were used to study the localization of the protein by immunofluorescence and immunoelectron microscopy. It was found that in NRK and A431 cells, myosin VI was associated with both the Golgi complex and the leading, ruffling edge of the cell as well as being present in a cytosolic pool. In A431 cells in which cell surface ruffling was stimulated by EGF, myosin VI was phosphorylated and recruited into the newly formed ruffles along with ezrin and myosin V. In vitro experiments suggested that a p21-activated kinase (PAK) might be the kinase responsible for phosphorylation in the motor domain. These results strongly support a role for myosin VI in membrane traffic on secretory and endocytic pathways.

Show MeSH