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
Specificity of the  myosin VI tail antibody  (PGT). (a) Schematic diagram of the structure of myosin VI showing the different  domains which were expressed in E. coli as GST-fusion proteins and used to  raise antibodies. A BamHI  fragment encoding aa 308– 631 of the head domain was  used to raise the H ab; a HindIII fragment of the myosin VI  tail encoding aa 742–1,030  was used to raise the CCT ab,  a PstI fragment encoding the  COOH-terminal globular tail  was used to raise the PGT ab  and an ab was raised against  the whole tail (T ab) encoding aa 846–1,254. (b) Immunoblot of whole cell extracts  of NRK and A431 cells showing the specificity of the affinity purified PGT ab. (c) Immunoprecipitation of myosin  VI from [35S]methionine/ cysteine-labeled NRK and  A431 cells. Lanes 1 and 3  show immunoprecipitations  using the preimmune serum,  whereas for lanes 2 and 4, 10  μg of the affinity purified  PGT ab was used. (d) Immunoblot of immunoprecipitations from A431 cells of myosin VI probed with antibodies  to different myosins. Lane 1,  immunoprecipitation using the preimmune serum; lane 2, with  the tail ab (PGT) to myosin VI; lane 3, with the tail ab (T) to myosin VI. The immunoprecipitates were blotted onto nitrocellulose and probed with the polyclonal antibodies to myosin I  (myr2), myosin VI (MVI), myosin II (MII), and myosin V (MV).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132970&req=5

Figure 2: Specificity of the myosin VI tail antibody (PGT). (a) Schematic diagram of the structure of myosin VI showing the different domains which were expressed in E. coli as GST-fusion proteins and used to raise antibodies. A BamHI fragment encoding aa 308– 631 of the head domain was used to raise the H ab; a HindIII fragment of the myosin VI tail encoding aa 742–1,030 was used to raise the CCT ab, a PstI fragment encoding the COOH-terminal globular tail was used to raise the PGT ab and an ab was raised against the whole tail (T ab) encoding aa 846–1,254. (b) Immunoblot of whole cell extracts of NRK and A431 cells showing the specificity of the affinity purified PGT ab. (c) Immunoprecipitation of myosin VI from [35S]methionine/ cysteine-labeled NRK and A431 cells. Lanes 1 and 3 show immunoprecipitations using the preimmune serum, whereas for lanes 2 and 4, 10 μg of the affinity purified PGT ab was used. (d) Immunoblot of immunoprecipitations from A431 cells of myosin VI probed with antibodies to different myosins. Lane 1, immunoprecipitation using the preimmune serum; lane 2, with the tail ab (PGT) to myosin VI; lane 3, with the tail ab (T) to myosin VI. The immunoprecipitates were blotted onto nitrocellulose and probed with the polyclonal antibodies to myosin I (myr2), myosin VI (MVI), myosin II (MII), and myosin V (MV).

Mentions: The amino acid sequences of the myosins in each class are very conserved in different vertebrate species and it is usually the case that antibodies for a myosin from one species will cross-react with the same myosin in others. Antibodies were raised against different domains of chicken intestinal brush border myosin VI (shown schematically in Fig. 2 a) and affinity purified on GST- or His-tagged myosin VI fusion proteins. After affinity purification each of the antibodies recognized myosin VI on immunoblots of NRK and A431 fibroblasts. The predicted globular tail antibody (ab [PGT]) which was subsequently used for immunofluorescence reacted with a single band of the predicted molecular mass of 148 kD (Fig. 2 b). In NRK cells this antibody also recognized a band of lower molecular mass, which was probably a degradation product, since it varied between preparations. The tail ab (PGT) recognized myosin VI on immunoblots and also the native protein in whole cell extracts as shown by immunoprecipitation (Fig. 2 c). For specific, high yield immunoprecipitation of myosin VI, the whole tail ab (T) was found to be most effective. Immunoprecipitates of myosin VI from A431 cells with the tail antibodies (PGT and T) did not coprecipitate other myosins (Fig. 2 d).


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)

Specificity of the  myosin VI tail antibody  (PGT). (a) Schematic diagram of the structure of myosin VI showing the different  domains which were expressed in E. coli as GST-fusion proteins and used to  raise antibodies. A BamHI  fragment encoding aa 308– 631 of the head domain was  used to raise the H ab; a HindIII fragment of the myosin VI  tail encoding aa 742–1,030  was used to raise the CCT ab,  a PstI fragment encoding the  COOH-terminal globular tail  was used to raise the PGT ab  and an ab was raised against  the whole tail (T ab) encoding aa 846–1,254. (b) Immunoblot of whole cell extracts  of NRK and A431 cells showing the specificity of the affinity purified PGT ab. (c) Immunoprecipitation of myosin  VI from [35S]methionine/ cysteine-labeled NRK and  A431 cells. Lanes 1 and 3  show immunoprecipitations  using the preimmune serum,  whereas for lanes 2 and 4, 10  μg of the affinity purified  PGT ab was used. (d) Immunoblot of immunoprecipitations from A431 cells of myosin VI probed with antibodies  to different myosins. Lane 1,  immunoprecipitation using the preimmune serum; lane 2, with  the tail ab (PGT) to myosin VI; lane 3, with the tail ab (T) to myosin VI. The immunoprecipitates were blotted onto nitrocellulose and probed with the polyclonal antibodies to myosin I  (myr2), myosin VI (MVI), myosin II (MII), and myosin V (MV).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Specificity of the myosin VI tail antibody (PGT). (a) Schematic diagram of the structure of myosin VI showing the different domains which were expressed in E. coli as GST-fusion proteins and used to raise antibodies. A BamHI fragment encoding aa 308– 631 of the head domain was used to raise the H ab; a HindIII fragment of the myosin VI tail encoding aa 742–1,030 was used to raise the CCT ab, a PstI fragment encoding the COOH-terminal globular tail was used to raise the PGT ab and an ab was raised against the whole tail (T ab) encoding aa 846–1,254. (b) Immunoblot of whole cell extracts of NRK and A431 cells showing the specificity of the affinity purified PGT ab. (c) Immunoprecipitation of myosin VI from [35S]methionine/ cysteine-labeled NRK and A431 cells. Lanes 1 and 3 show immunoprecipitations using the preimmune serum, whereas for lanes 2 and 4, 10 μg of the affinity purified PGT ab was used. (d) Immunoblot of immunoprecipitations from A431 cells of myosin VI probed with antibodies to different myosins. Lane 1, immunoprecipitation using the preimmune serum; lane 2, with the tail ab (PGT) to myosin VI; lane 3, with the tail ab (T) to myosin VI. The immunoprecipitates were blotted onto nitrocellulose and probed with the polyclonal antibodies to myosin I (myr2), myosin VI (MVI), myosin II (MII), and myosin V (MV).
Mentions: The amino acid sequences of the myosins in each class are very conserved in different vertebrate species and it is usually the case that antibodies for a myosin from one species will cross-react with the same myosin in others. Antibodies were raised against different domains of chicken intestinal brush border myosin VI (shown schematically in Fig. 2 a) and affinity purified on GST- or His-tagged myosin VI fusion proteins. After affinity purification each of the antibodies recognized myosin VI on immunoblots of NRK and A431 fibroblasts. The predicted globular tail antibody (ab [PGT]) which was subsequently used for immunofluorescence reacted with a single band of the predicted molecular mass of 148 kD (Fig. 2 b). In NRK cells this antibody also recognized a band of lower molecular mass, which was probably a degradation product, since it varied between preparations. The tail ab (PGT) recognized myosin VI on immunoblots and also the native protein in whole cell extracts as shown by immunoprecipitation (Fig. 2 c). For specific, high yield immunoprecipitation of myosin VI, the whole tail ab (T) was found to be most effective. Immunoprecipitates of myosin VI from A431 cells with the tail antibodies (PGT and T) did not coprecipitate other myosins (Fig. 2 d).

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