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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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Myosin VI concentrates in perinuclear recycling endosomes in LNCaP cellsTo analyse the relative distribution of myosin VI between the recycling endosome and the TGN, cryosections of LNCaP cells were double labelled with antibodies against endogenous myosin VI (10 nm gold) and TGN46 (15 nm gold) or with antibodies against myosin VI (10 nm gold) and TfR (15 nm gold). Myosin VI and TGN46 (a) or myosin VI and TfR (b) colocalise on tubular vesicular membranes near a stack of membranes, the Golgi complex. Bar: 300 nm. (B) The relative amounts of myosin VI associated with either the recycling endosome or the TGN compartment were quantified on pictures of the Golgi area at 64K magnification. In both experiments 100 myosin molecules were assessed for colocalisation with either TGN46 or TfR on the same length of membrane (about 45 μm each). To measure the length of the membrane the number of intersections with the Photoshop 1cm grid lines were counted (Rabouille et al JCB 1995). Whereas 68 out of 100 myosin VI molecules can be found on a membrane compartment also containing the TfR, only 38 out of 100 myosin VI molecules share a membrane with TGN46.
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Figure 3: Myosin VI concentrates in perinuclear recycling endosomes in LNCaP cellsTo analyse the relative distribution of myosin VI between the recycling endosome and the TGN, cryosections of LNCaP cells were double labelled with antibodies against endogenous myosin VI (10 nm gold) and TGN46 (15 nm gold) or with antibodies against myosin VI (10 nm gold) and TfR (15 nm gold). Myosin VI and TGN46 (a) or myosin VI and TfR (b) colocalise on tubular vesicular membranes near a stack of membranes, the Golgi complex. Bar: 300 nm. (B) The relative amounts of myosin VI associated with either the recycling endosome or the TGN compartment were quantified on pictures of the Golgi area at 64K magnification. In both experiments 100 myosin molecules were assessed for colocalisation with either TGN46 or TfR on the same length of membrane (about 45 μm each). To measure the length of the membrane the number of intersections with the Photoshop 1cm grid lines were counted (Rabouille et al JCB 1995). Whereas 68 out of 100 myosin VI molecules can be found on a membrane compartment also containing the TfR, only 38 out of 100 myosin VI molecules share a membrane with TGN46.

Mentions: The precise localisation of myosin VI in the perinuclear region was investigated at the ultrastructural level. Cryosections of LNCaP cells double labelled with antibodies to myosin VI and TfR or TGN46 demonstrate that myosin VI is associated with vesicular and tubular membranes at the Golgi complex, showing partial colocalisation with TGN 46 (a) and TfR (b) (figure 3 A). A morphometric analysis was performed to quantify the colocalisation of myosin VI with TfR or with TGN46 and determine whether myosin VI is recruited to the perinuclear endocytic recycling compartment (TfR) or to the trans-Golgi network (TGN46). In each double labelling experiment 100 myosin VI associated gold particles present on a vesicle or tubule were analysed for colocalisation with TfR or TGN46 on the same membrane structure. The same length of membrane of about 45 μm was scored in each experiment. Figure 3 B shows that 68 out of 100 myosin VI molecules are on a compartment that also contains TfR, whereas only 38 molecules out of 100 myosin VI molecules share a membrane with TGN46, indicating that most of the myosin VI in the perinuclear region is present in the TfR-positive endocytic recycling compartment and less is associated with the trans-Golgi network.


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)

Myosin VI concentrates in perinuclear recycling endosomes in LNCaP cellsTo analyse the relative distribution of myosin VI between the recycling endosome and the TGN, cryosections of LNCaP cells were double labelled with antibodies against endogenous myosin VI (10 nm gold) and TGN46 (15 nm gold) or with antibodies against myosin VI (10 nm gold) and TfR (15 nm gold). Myosin VI and TGN46 (a) or myosin VI and TfR (b) colocalise on tubular vesicular membranes near a stack of membranes, the Golgi complex. Bar: 300 nm. (B) The relative amounts of myosin VI associated with either the recycling endosome or the TGN compartment were quantified on pictures of the Golgi area at 64K magnification. In both experiments 100 myosin molecules were assessed for colocalisation with either TGN46 or TfR on the same length of membrane (about 45 μm each). To measure the length of the membrane the number of intersections with the Photoshop 1cm grid lines were counted (Rabouille et al JCB 1995). Whereas 68 out of 100 myosin VI molecules can be found on a membrane compartment also containing the TfR, only 38 out of 100 myosin VI molecules share a membrane with TGN46.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Myosin VI concentrates in perinuclear recycling endosomes in LNCaP cellsTo analyse the relative distribution of myosin VI between the recycling endosome and the TGN, cryosections of LNCaP cells were double labelled with antibodies against endogenous myosin VI (10 nm gold) and TGN46 (15 nm gold) or with antibodies against myosin VI (10 nm gold) and TfR (15 nm gold). Myosin VI and TGN46 (a) or myosin VI and TfR (b) colocalise on tubular vesicular membranes near a stack of membranes, the Golgi complex. Bar: 300 nm. (B) The relative amounts of myosin VI associated with either the recycling endosome or the TGN compartment were quantified on pictures of the Golgi area at 64K magnification. In both experiments 100 myosin molecules were assessed for colocalisation with either TGN46 or TfR on the same length of membrane (about 45 μm each). To measure the length of the membrane the number of intersections with the Photoshop 1cm grid lines were counted (Rabouille et al JCB 1995). Whereas 68 out of 100 myosin VI molecules can be found on a membrane compartment also containing the TfR, only 38 out of 100 myosin VI molecules share a membrane with TGN46.
Mentions: The precise localisation of myosin VI in the perinuclear region was investigated at the ultrastructural level. Cryosections of LNCaP cells double labelled with antibodies to myosin VI and TfR or TGN46 demonstrate that myosin VI is associated with vesicular and tubular membranes at the Golgi complex, showing partial colocalisation with TGN 46 (a) and TfR (b) (figure 3 A). A morphometric analysis was performed to quantify the colocalisation of myosin VI with TfR or with TGN46 and determine whether myosin VI is recruited to the perinuclear endocytic recycling compartment (TfR) or to the trans-Golgi network (TGN46). In each double labelling experiment 100 myosin VI associated gold particles present on a vesicle or tubule were analysed for colocalisation with TfR or TGN46 on the same membrane structure. The same length of membrane of about 45 μm was scored in each experiment. Figure 3 B shows that 68 out of 100 myosin VI molecules are on a compartment that also contains TfR, whereas only 38 molecules out of 100 myosin VI molecules share a membrane with TGN46, indicating that most of the myosin VI in the perinuclear region is present in the TfR-positive endocytic recycling compartment and less is associated with the trans-Golgi network.

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