Limits...
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.

Show MeSH

Related in: MedlinePlus

Binding of myosin VI to  actin filaments. Myosin VI was immunoprecipitated from A431 cells  under native conditions (a) and incubated with 10 μM of F-actin in  the presence (lane 2) or absence  (lane 3) of 2.5 mM MgATP. The  immunobeads containing bound  myosin VI were briefly centrifuged  and washed with or without  MgATP to remove nonbound actin.  The washed beads were run on  SDS-PAGE and stained with Coomassie blue. Lane 1 shows actin  alone and lane 4 an immunoprecipitation with preimmune serum incubated with actin. In b the immunoprecipitated myosin VI was  incubated with (lane 2) or without  (lane 1) recombinant PAK before  assessing binding to actin filaments  under the same conditions as described in a.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132970&req=5

Figure 8: Binding of myosin VI to actin filaments. Myosin VI was immunoprecipitated from A431 cells under native conditions (a) and incubated with 10 μM of F-actin in the presence (lane 2) or absence (lane 3) of 2.5 mM MgATP. The immunobeads containing bound myosin VI were briefly centrifuged and washed with or without MgATP to remove nonbound actin. The washed beads were run on SDS-PAGE and stained with Coomassie blue. Lane 1 shows actin alone and lane 4 an immunoprecipitation with preimmune serum incubated with actin. In b the immunoprecipitated myosin VI was incubated with (lane 2) or without (lane 1) recombinant PAK before assessing binding to actin filaments under the same conditions as described in a.

Mentions: Myosin VI from A431 cells, immunocaptured on a bead support, bound to F-actin in the absence of MgATP (rigor binding) and binding was significantly decreased (8–10-fold lower) in the presence of MgATP (Fig. 8 a). When the myosin VI was first phosphorylated in vitro by PAK there was no significant effect on its ability to bind actin in the presence of MgATP (Fig. 8 b). These results are in agreement with previous observations in Acanthamoeba myosin I where phosphorylation in the head domain leads to an increase in the actin-activated myosin I MgATPase activity (Maruta and Korn, 1977).


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)

Binding of myosin VI to  actin filaments. Myosin VI was immunoprecipitated from A431 cells  under native conditions (a) and incubated with 10 μM of F-actin in  the presence (lane 2) or absence  (lane 3) of 2.5 mM MgATP. The  immunobeads containing bound  myosin VI were briefly centrifuged  and washed with or without  MgATP to remove nonbound actin.  The washed beads were run on  SDS-PAGE and stained with Coomassie blue. Lane 1 shows actin  alone and lane 4 an immunoprecipitation with preimmune serum incubated with actin. In b the immunoprecipitated myosin VI was  incubated with (lane 2) or without  (lane 1) recombinant PAK before  assessing binding to actin filaments  under the same conditions as described in a.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132970&req=5

Figure 8: Binding of myosin VI to actin filaments. Myosin VI was immunoprecipitated from A431 cells under native conditions (a) and incubated with 10 μM of F-actin in the presence (lane 2) or absence (lane 3) of 2.5 mM MgATP. The immunobeads containing bound myosin VI were briefly centrifuged and washed with or without MgATP to remove nonbound actin. The washed beads were run on SDS-PAGE and stained with Coomassie blue. Lane 1 shows actin alone and lane 4 an immunoprecipitation with preimmune serum incubated with actin. In b the immunoprecipitated myosin VI was incubated with (lane 2) or without (lane 1) recombinant PAK before assessing binding to actin filaments under the same conditions as described in a.
Mentions: Myosin VI from A431 cells, immunocaptured on a bead support, bound to F-actin in the absence of MgATP (rigor binding) and binding was significantly decreased (8–10-fold lower) in the presence of MgATP (Fig. 8 a). When the myosin VI was first phosphorylated in vitro by PAK there was no significant effect on its ability to bind actin in the presence of MgATP (Fig. 8 b). These results are in agreement with previous observations in Acanthamoeba myosin I where phosphorylation in the head domain leads to an increase in the actin-activated myosin I MgATPase activity (Maruta and Korn, 1977).

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
Related in: MedlinePlus