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Overexpression of myosin VI in prostate cancer cells enhances PSA and VEGF secretion, but has no effect on endocytosis.

Puri C, Chibalina MV, Arden SD, Kruppa AJ, Kendrick-Jones J, Buss F - Oncogene (2009)

Bottom Line: Intracellular targeting seems to involve two myosin VI-interacting proteins, GIPC and LMTK2, both of which can be co-immunoprecipitated with myosin VI from LNCaP cells.The absence of Disabled-2 (Dab2), a tumour suppressor and myosin VI-binding partner, inhibits recruitment of myosin VI to endocytic structures at the plasma membrane in LNCaP cells, but interestingly has no effect on endocytosis.Small interfering RNA-mediated downregulation of myosin VI expression results in a significant reduction in prostate-specific antigen (PSA) and vascular endothelial growth factor (VEGF) secretion in LNCaP cells.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building, Cambridge, UK.

ABSTRACT
Tissue expression microarrays, employed to determine the players and mechanisms leading to prostate cancer development, have consistently shown that myosin VI, a unique actin-based motor, is upregulated in medium-grade human prostate cancers. Thus, to understand the role of myosin VI in prostate cancer development, we have characterized its intracellular localization and function in the prostate cancer cell line LNCaP. Using light and electron microscopy, we identified myosin VI on Rab5-positive early endosomes, as well as on recycling endosomes and the trans-Golgi network. Intracellular targeting seems to involve two myosin VI-interacting proteins, GIPC and LMTK2, both of which can be co-immunoprecipitated with myosin VI from LNCaP cells. The absence of Disabled-2 (Dab2), a tumour suppressor and myosin VI-binding partner, inhibits recruitment of myosin VI to endocytic structures at the plasma membrane in LNCaP cells, but interestingly has no effect on endocytosis. Small interfering RNA-mediated downregulation of myosin VI expression results in a significant reduction in prostate-specific antigen (PSA) and vascular endothelial growth factor (VEGF) secretion in LNCaP cells. Our results suggest that in prostate cancer cells, myosin VI regulates protein secretion, but the overexpression of myosin VI has no major impact on clathrin-mediated endocytosis.

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Intracellular localisation of myosin VI in LNCaP cellsThe endogenous myosin VI was detected with a polyclonal antibody to myosin VI (a, d, g, j, and m) and double labelled with a monoclonal antibody to AP2 (b), Rab5 (e), TfR (h and k) and TGN46 (n) in immunofluorescence. The merged images are shown in (c, f, i, l and o). Myosin VI is present in peripheral Rab5 and TfR positive endosomes and overlapps with the localisation of TfR and TGN46 in the perinuclear region. No myosin VI is colocalising with AP2 in clathrin-coated structures at the plasma membrane.
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Figure 2: Intracellular localisation of myosin VI in LNCaP cellsThe endogenous myosin VI was detected with a polyclonal antibody to myosin VI (a, d, g, j, and m) and double labelled with a monoclonal antibody to AP2 (b), Rab5 (e), TfR (h and k) and TGN46 (n) in immunofluorescence. The merged images are shown in (c, f, i, l and o). Myosin VI is present in peripheral Rab5 and TfR positive endosomes and overlapps with the localisation of TfR and TGN46 in the perinuclear region. No myosin VI is colocalising with AP2 in clathrin-coated structures at the plasma membrane.

Mentions: In LNCaP cells over-expression of endogeneous myosin VI causes it to accumulate in the cytosol and since not bound to cargo or target membranes, it could be removed by pre-permeabilising the cells with saponin prior to fixation (Morriswood et al., 2007). Under these conditions myosin VI was observed in a vesicular staining pattern throughout the cell, with high concentrations in cell extensions and in the perinuclear area (figure 2). To characterise the intracellular distribution of myosin VI we performed double labelling experiments with antibodies to marker proteins for the different subcellular compartments. Although LNCaP cells express myosin VI isoforms containing the LI-abc and bc inserts, no endogenous myosin VI was detected in AP-2 positive clathrin-coated structures at the plasma membrane (figure 2, a-c) due to the lack of Dab2 (see figure 4). The myosin VI in cell extensions in the cell periphery colocalises with Rab5 (figure 2, d-f) and the transferrin receptor (TfR) (figure 2, g-l), indicating that in LNCaP cells myosin VI is recruited to early endosomes and is also concentrated in the perinuclear region, where it colocalises with TGN46, a marker of the trans Golgi network (figure 2, m-o), and with TfR, which is present in the endocytic recycling compartment (figure 2, j-l). A similar juxtanuclear concentration of myosin VI was recently observed in surgical prostate cancer tissues (Wei et al., 2008). In the non-malignant prostate cell line PNT1A myosin VI colocalises with TfR and Rab5-positive early endocytic compartment in the cell periphery, however, less myosin VI is present in the perinuclear region at/around the Golgi complex. PTN1A cells do not express the myosin VI LI isoform and therefore although Dab2 is present, very little colocalisation with AP2 in clathrin-coated structures at the plasma membrane was detected (supplementary figure 1).


Overexpression of myosin VI in prostate cancer cells enhances PSA and VEGF secretion, but has no effect on endocytosis.

Puri C, Chibalina MV, Arden SD, Kruppa AJ, Kendrick-Jones J, Buss F - Oncogene (2009)

Intracellular localisation of myosin VI in LNCaP cellsThe endogenous myosin VI was detected with a polyclonal antibody to myosin VI (a, d, g, j, and m) and double labelled with a monoclonal antibody to AP2 (b), Rab5 (e), TfR (h and k) and TGN46 (n) in immunofluorescence. The merged images are shown in (c, f, i, l and o). Myosin VI is present in peripheral Rab5 and TfR positive endosomes and overlapps with the localisation of TfR and TGN46 in the perinuclear region. No myosin VI is colocalising with AP2 in clathrin-coated structures at the plasma membrane.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2871299&req=5

Figure 2: Intracellular localisation of myosin VI in LNCaP cellsThe endogenous myosin VI was detected with a polyclonal antibody to myosin VI (a, d, g, j, and m) and double labelled with a monoclonal antibody to AP2 (b), Rab5 (e), TfR (h and k) and TGN46 (n) in immunofluorescence. The merged images are shown in (c, f, i, l and o). Myosin VI is present in peripheral Rab5 and TfR positive endosomes and overlapps with the localisation of TfR and TGN46 in the perinuclear region. No myosin VI is colocalising with AP2 in clathrin-coated structures at the plasma membrane.
Mentions: In LNCaP cells over-expression of endogeneous myosin VI causes it to accumulate in the cytosol and since not bound to cargo or target membranes, it could be removed by pre-permeabilising the cells with saponin prior to fixation (Morriswood et al., 2007). Under these conditions myosin VI was observed in a vesicular staining pattern throughout the cell, with high concentrations in cell extensions and in the perinuclear area (figure 2). To characterise the intracellular distribution of myosin VI we performed double labelling experiments with antibodies to marker proteins for the different subcellular compartments. Although LNCaP cells express myosin VI isoforms containing the LI-abc and bc inserts, no endogenous myosin VI was detected in AP-2 positive clathrin-coated structures at the plasma membrane (figure 2, a-c) due to the lack of Dab2 (see figure 4). The myosin VI in cell extensions in the cell periphery colocalises with Rab5 (figure 2, d-f) and the transferrin receptor (TfR) (figure 2, g-l), indicating that in LNCaP cells myosin VI is recruited to early endosomes and is also concentrated in the perinuclear region, where it colocalises with TGN46, a marker of the trans Golgi network (figure 2, m-o), and with TfR, which is present in the endocytic recycling compartment (figure 2, j-l). A similar juxtanuclear concentration of myosin VI was recently observed in surgical prostate cancer tissues (Wei et al., 2008). In the non-malignant prostate cell line PNT1A myosin VI colocalises with TfR and Rab5-positive early endocytic compartment in the cell periphery, however, less myosin VI is present in the perinuclear region at/around the Golgi complex. PTN1A cells do not express the myosin VI LI isoform and therefore although Dab2 is present, very little colocalisation with AP2 in clathrin-coated structures at the plasma membrane was detected (supplementary figure 1).

Bottom Line: Intracellular targeting seems to involve two myosin VI-interacting proteins, GIPC and LMTK2, both of which can be co-immunoprecipitated with myosin VI from LNCaP cells.The absence of Disabled-2 (Dab2), a tumour suppressor and myosin VI-binding partner, inhibits recruitment of myosin VI to endocytic structures at the plasma membrane in LNCaP cells, but interestingly has no effect on endocytosis.Small interfering RNA-mediated downregulation of myosin VI expression results in a significant reduction in prostate-specific antigen (PSA) and vascular endothelial growth factor (VEGF) secretion in LNCaP cells.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building, Cambridge, UK.

ABSTRACT
Tissue expression microarrays, employed to determine the players and mechanisms leading to prostate cancer development, have consistently shown that myosin VI, a unique actin-based motor, is upregulated in medium-grade human prostate cancers. Thus, to understand the role of myosin VI in prostate cancer development, we have characterized its intracellular localization and function in the prostate cancer cell line LNCaP. Using light and electron microscopy, we identified myosin VI on Rab5-positive early endosomes, as well as on recycling endosomes and the trans-Golgi network. Intracellular targeting seems to involve two myosin VI-interacting proteins, GIPC and LMTK2, both of which can be co-immunoprecipitated with myosin VI from LNCaP cells. The absence of Disabled-2 (Dab2), a tumour suppressor and myosin VI-binding partner, inhibits recruitment of myosin VI to endocytic structures at the plasma membrane in LNCaP cells, but interestingly has no effect on endocytosis. Small interfering RNA-mediated downregulation of myosin VI expression results in a significant reduction in prostate-specific antigen (PSA) and vascular endothelial growth factor (VEGF) secretion in LNCaP cells. Our results suggest that in prostate cancer cells, myosin VI regulates protein secretion, but the overexpression of myosin VI has no major impact on clathrin-mediated endocytosis.

Show MeSH
Related in: MedlinePlus