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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 in NRK cells. (a and b) Indirect immunofluorescence localization of myosin VI using the affinity purified tail ab (PGT) at 10 μg/ml. Myosin VI is especially  enriched in the juxtanuclear area and in the dynamic leading  edge of the fibroblast. There is also a cytosolic pool. (c–e) Immunoelectron microscopic localization of myosin VI on frozen thin  sections with affinity purified tail ab (PGT) followed by protein  A–gold. The highest concentration of myosin VI is visible at  plasma membrane profiles with dynamic membrane protrusions.  Bars: (b) 20 μm; (e) 500 nm.
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Figure 3: Localization of myosin VI in NRK cells. (a and b) Indirect immunofluorescence localization of myosin VI using the affinity purified tail ab (PGT) at 10 μg/ml. Myosin VI is especially enriched in the juxtanuclear area and in the dynamic leading edge of the fibroblast. There is also a cytosolic pool. (c–e) Immunoelectron microscopic localization of myosin VI on frozen thin sections with affinity purified tail ab (PGT) followed by protein A–gold. The highest concentration of myosin VI is visible at plasma membrane profiles with dynamic membrane protrusions. Bars: (b) 20 μm; (e) 500 nm.

Mentions: Using the tail ab (PGT) on NRK cells, myosin VI was observed by indirect immunofluorescence to show a dual localization (Fig. 3, a and b) at the leading edge and in the juxtanuclear area. There was also a considerable amount of myosin VI present as a cytosolic pool presumably reflecting the fact that this myosin shuttles between actin/ membrane bound and soluble pools. Myosin VI was localized by immunoelectron microscopy on ultrathin frozen sections of NRK cells, using the tail ab (PGT) followed by protein A–gold (Fig. 3, c–e), which showed that it was enriched at the plasma membrane in regions with a highly dynamic appearance. For example, in Fig. 3 c the plasma membrane profile showing many protrusions at the top of the cell has a higher concentration of myosin VI than the smooth plasma membrane profile at the bottom of the cell. Myosin VI was found to be particularly enriched at the tips of filopodia, microvilli and also in ruffles (Fig. 3, d and e). Such membrane ruffles extend from the cell surface, as seen in Fig. 3, d and e, and may close up to form endocytic macropinosomes after touching another ruffle or falling back onto the cell surface. In cross section such newly formed macropinosomes in the NRK cells appeared heavily labeled with anti-myosin VI. Quantitation of the amount of myosin VI on unruffled plasma membrane compared with membrane protrusions revealed more than double the amount of myosin VI in areas with active membrane protrusions. We counted a total of 512 gold particles on 180 μm of unruffled plasma membrane i.e., 2.8 gold particles/μm of membrane and 884 gold particles on 120 μm of membrane protrusions i.e., 7.4 gold particles/μm of membrane. It was difficult to show specific labeling of myosin VI in the juxtanuclear area by EM, because of the high amount of myosin VI present as a cytosolic pool.


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 in NRK cells. (a and b) Indirect immunofluorescence localization of myosin VI using the affinity purified tail ab (PGT) at 10 μg/ml. Myosin VI is especially  enriched in the juxtanuclear area and in the dynamic leading  edge of the fibroblast. There is also a cytosolic pool. (c–e) Immunoelectron microscopic localization of myosin VI on frozen thin  sections with affinity purified tail ab (PGT) followed by protein  A–gold. The highest concentration of myosin VI is visible at  plasma membrane profiles with dynamic membrane protrusions.  Bars: (b) 20 μm; (e) 500 nm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132970&req=5

Figure 3: Localization of myosin VI in NRK cells. (a and b) Indirect immunofluorescence localization of myosin VI using the affinity purified tail ab (PGT) at 10 μg/ml. Myosin VI is especially enriched in the juxtanuclear area and in the dynamic leading edge of the fibroblast. There is also a cytosolic pool. (c–e) Immunoelectron microscopic localization of myosin VI on frozen thin sections with affinity purified tail ab (PGT) followed by protein A–gold. The highest concentration of myosin VI is visible at plasma membrane profiles with dynamic membrane protrusions. Bars: (b) 20 μm; (e) 500 nm.
Mentions: Using the tail ab (PGT) on NRK cells, myosin VI was observed by indirect immunofluorescence to show a dual localization (Fig. 3, a and b) at the leading edge and in the juxtanuclear area. There was also a considerable amount of myosin VI present as a cytosolic pool presumably reflecting the fact that this myosin shuttles between actin/ membrane bound and soluble pools. Myosin VI was localized by immunoelectron microscopy on ultrathin frozen sections of NRK cells, using the tail ab (PGT) followed by protein A–gold (Fig. 3, c–e), which showed that it was enriched at the plasma membrane in regions with a highly dynamic appearance. For example, in Fig. 3 c the plasma membrane profile showing many protrusions at the top of the cell has a higher concentration of myosin VI than the smooth plasma membrane profile at the bottom of the cell. Myosin VI was found to be particularly enriched at the tips of filopodia, microvilli and also in ruffles (Fig. 3, d and e). Such membrane ruffles extend from the cell surface, as seen in Fig. 3, d and e, and may close up to form endocytic macropinosomes after touching another ruffle or falling back onto the cell surface. In cross section such newly formed macropinosomes in the NRK cells appeared heavily labeled with anti-myosin VI. Quantitation of the amount of myosin VI on unruffled plasma membrane compared with membrane protrusions revealed more than double the amount of myosin VI in areas with active membrane protrusions. We counted a total of 512 gold particles on 180 μm of unruffled plasma membrane i.e., 2.8 gold particles/μm of membrane and 884 gold particles on 120 μm of membrane protrusions i.e., 7.4 gold particles/μm of membrane. It was difficult to show specific labeling of myosin VI in the juxtanuclear area by EM, because of the high amount of myosin VI present as a cytosolic pool.

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