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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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Association of myosin VI with the Golgi complex.  NRK cells were double labeled by incubation with the affinity  purified tail ab (PGT) (a) together with an mAb to TGN38 (b).  Extended focus projections of a z-series of images obtained by  confocal microscopy are shown. (c) Immunoblot of 15 μg of purified rat liver Golgi membranes (LG) or rat kidney Golgi membranes (KG) probed with antibodies to TGN38 or myosin VI. (d)  Immunoblot of rat kidney factions. 10 μg each of cytosol (S,  100,000 g, 60 min supernatant), pellet (P, 100,000 g, 60 min pellet)  and purified rat kidney Golgi membranes (G) were probed with  the antibodies to TGN38 or myosin VI.
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Figure 4: Association of myosin VI with the Golgi complex. NRK cells were double labeled by incubation with the affinity purified tail ab (PGT) (a) together with an mAb to TGN38 (b). Extended focus projections of a z-series of images obtained by confocal microscopy are shown. (c) Immunoblot of 15 μg of purified rat liver Golgi membranes (LG) or rat kidney Golgi membranes (KG) probed with antibodies to TGN38 or myosin VI. (d) Immunoblot of rat kidney factions. 10 μg each of cytosol (S, 100,000 g, 60 min supernatant), pellet (P, 100,000 g, 60 min pellet) and purified rat kidney Golgi membranes (G) were probed with the antibodies to TGN38 or myosin VI.

Mentions: To investigate the juxtanuclear pool of myosin VI, NRK cells were double labeled for immunofluorescence and myosin VI was shown to colocalize with the trans Golgi network marker TGN 38 (Fig. 4, a and b). Colocalization with other Golgi markers (GM130 for the cis-Golgi, mannosidase II for the medial Golgi and galactosyltransferase for the trans-Golgi network [TGN]) was also observed (data not shown) consistent with myosin VI being present in the Golgi complex and the TGN.


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)

Association of myosin VI with the Golgi complex.  NRK cells were double labeled by incubation with the affinity  purified tail ab (PGT) (a) together with an mAb to TGN38 (b).  Extended focus projections of a z-series of images obtained by  confocal microscopy are shown. (c) Immunoblot of 15 μg of purified rat liver Golgi membranes (LG) or rat kidney Golgi membranes (KG) probed with antibodies to TGN38 or myosin VI. (d)  Immunoblot of rat kidney factions. 10 μg each of cytosol (S,  100,000 g, 60 min supernatant), pellet (P, 100,000 g, 60 min pellet)  and purified rat kidney Golgi membranes (G) were probed with  the antibodies to TGN38 or myosin VI.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132970&req=5

Figure 4: Association of myosin VI with the Golgi complex. NRK cells were double labeled by incubation with the affinity purified tail ab (PGT) (a) together with an mAb to TGN38 (b). Extended focus projections of a z-series of images obtained by confocal microscopy are shown. (c) Immunoblot of 15 μg of purified rat liver Golgi membranes (LG) or rat kidney Golgi membranes (KG) probed with antibodies to TGN38 or myosin VI. (d) Immunoblot of rat kidney factions. 10 μg each of cytosol (S, 100,000 g, 60 min supernatant), pellet (P, 100,000 g, 60 min pellet) and purified rat kidney Golgi membranes (G) were probed with the antibodies to TGN38 or myosin VI.
Mentions: To investigate the juxtanuclear pool of myosin VI, NRK cells were double labeled for immunofluorescence and myosin VI was shown to colocalize with the trans Golgi network marker TGN 38 (Fig. 4, a and b). Colocalization with other Golgi markers (GM130 for the cis-Golgi, mannosidase II for the medial Golgi and galactosyltransferase for the trans-Golgi network [TGN]) was also observed (data not shown) consistent with myosin VI being present in the Golgi complex and the TGN.

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