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Myosin VI SI potentiates evoked exocytosis. (A) <t>RT-PCR</t> analysis of myosin VI isoform expression. β-Actin primers were used as a control. (B) Cartoons of GFP-tagged myosin VI constructs. The motor domain (purple) contains the actin- and ATP-binding sites, the reverse gear (761–814 aa, yellow) determines the unique directionality of myosin VI movement along actin, and the tail domain (orange) in the myosin VI SI (1,262 aa), but not the myosin VI NI (1,253 aa), contains a 9-aa insert. GFP-MyoVI-tail constructs lack the motor domain and are unable to interact with F-actin. (C) GFP-MyoVI protein expression in PC12 cells was evaluated by Western blotting using an anti–myosin VI antibody. Arrows indicate GFP-MyoVI-full proteins (175 kD) and GFP-MyoVI-tail proteins (85 kD). (D) NPY-hPLAP release was evaluated using a double-stimulation protocol in PC12 cells expressing GFP (control), GFP-MyoVI-SIfull, or GFP-MyoVI-NIfull. Released NPY-hPLAP is expressed as a percentage of total NPY-hPLAP ( n = 3). (E) NPY-hPLAP release was measured in wild-type (wt) and myosin VI stable knockdown (KD) PC12 cells overexpressing GFP (control), GFP-MyoVI-SIfull (+GFP-MyoVI-SIfull), or GFP-MyoVI-NIfull (+GFP-MyoVI-NIfull). Released NPY-hPLAP is expressed as a percentage of wild-type PC12 cells ( n = 3). Error bars are means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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Myosin VI SI potentiates evoked exocytosis. (A) <t>RT-PCR</t> analysis of myosin VI isoform expression. β-Actin primers were used as a control. (B) Cartoons of GFP-tagged myosin VI constructs. The motor domain (purple) contains the actin- and ATP-binding sites, the reverse gear (761–814 aa, yellow) determines the unique directionality of myosin VI movement along actin, and the tail domain (orange) in the myosin VI SI (1,262 aa), but not the myosin VI NI (1,253 aa), contains a 9-aa insert. GFP-MyoVI-tail constructs lack the motor domain and are unable to interact with F-actin. (C) GFP-MyoVI protein expression in PC12 cells was evaluated by Western blotting using an anti–myosin VI antibody. Arrows indicate GFP-MyoVI-full proteins (175 kD) and GFP-MyoVI-tail proteins (85 kD). (D) NPY-hPLAP release was evaluated using a double-stimulation protocol in PC12 cells expressing GFP (control), GFP-MyoVI-SIfull, or GFP-MyoVI-NIfull. Released NPY-hPLAP is expressed as a percentage of total NPY-hPLAP ( n = 3). (E) NPY-hPLAP release was measured in wild-type (wt) and myosin VI stable knockdown (KD) PC12 cells overexpressing GFP (control), GFP-MyoVI-SIfull (+GFP-MyoVI-SIfull), or GFP-MyoVI-NIfull (+GFP-MyoVI-NIfull). Released NPY-hPLAP is expressed as a percentage of wild-type PC12 cells ( n = 3). Error bars are means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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Myosin VI SI potentiates evoked exocytosis. (A) <t>RT-PCR</t> analysis of myosin VI isoform expression. β-Actin primers were used as a control. (B) Cartoons of GFP-tagged myosin VI constructs. The motor domain (purple) contains the actin- and ATP-binding sites, the reverse gear (761–814 aa, yellow) determines the unique directionality of myosin VI movement along actin, and the tail domain (orange) in the myosin VI SI (1,262 aa), but not the myosin VI NI (1,253 aa), contains a 9-aa insert. GFP-MyoVI-tail constructs lack the motor domain and are unable to interact with F-actin. (C) GFP-MyoVI protein expression in PC12 cells was evaluated by Western blotting using an anti–myosin VI antibody. Arrows indicate GFP-MyoVI-full proteins (175 kD) and GFP-MyoVI-tail proteins (85 kD). (D) NPY-hPLAP release was evaluated using a double-stimulation protocol in PC12 cells expressing GFP (control), GFP-MyoVI-SIfull, or GFP-MyoVI-NIfull. Released NPY-hPLAP is expressed as a percentage of total NPY-hPLAP ( n = 3). (E) NPY-hPLAP release was measured in wild-type (wt) and myosin VI stable knockdown (KD) PC12 cells overexpressing GFP (control), GFP-MyoVI-SIfull (+GFP-MyoVI-SIfull), or GFP-MyoVI-NIfull (+GFP-MyoVI-NIfull). Released NPY-hPLAP is expressed as a percentage of wild-type PC12 cells ( n = 3). Error bars are means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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m mulv reverse transcription system  (New England Biolabs)
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Myosin VI SI potentiates evoked exocytosis. (A) <t>RT-PCR</t> analysis of myosin VI isoform expression. β-Actin primers were used as a control. (B) Cartoons of GFP-tagged myosin VI constructs. The motor domain (purple) contains the actin- and ATP-binding sites, the reverse gear (761–814 aa, yellow) determines the unique directionality of myosin VI movement along actin, and the tail domain (orange) in the myosin VI SI (1,262 aa), but not the myosin VI NI (1,253 aa), contains a 9-aa insert. GFP-MyoVI-tail constructs lack the motor domain and are unable to interact with F-actin. (C) GFP-MyoVI protein expression in PC12 cells was evaluated by Western blotting using an anti–myosin VI antibody. Arrows indicate GFP-MyoVI-full proteins (175 kD) and GFP-MyoVI-tail proteins (85 kD). (D) NPY-hPLAP release was evaluated using a double-stimulation protocol in PC12 cells expressing GFP (control), GFP-MyoVI-SIfull, or GFP-MyoVI-NIfull. Released NPY-hPLAP is expressed as a percentage of total NPY-hPLAP ( n = 3). (E) NPY-hPLAP release was measured in wild-type (wt) and myosin VI stable knockdown (KD) PC12 cells overexpressing GFP (control), GFP-MyoVI-SIfull (+GFP-MyoVI-SIfull), or GFP-MyoVI-NIfull (+GFP-MyoVI-NIfull). Released NPY-hPLAP is expressed as a percentage of wild-type PC12 cells ( n = 3). Error bars are means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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Myosin VI SI potentiates evoked exocytosis. (A) <t>RT-PCR</t> analysis of myosin VI isoform expression. β-Actin primers were used as a control. (B) Cartoons of GFP-tagged myosin VI constructs. The motor domain (purple) contains the actin- and ATP-binding sites, the reverse gear (761–814 aa, yellow) determines the unique directionality of myosin VI movement along actin, and the tail domain (orange) in the myosin VI SI (1,262 aa), but not the myosin VI NI (1,253 aa), contains a 9-aa insert. GFP-MyoVI-tail constructs lack the motor domain and are unable to interact with F-actin. (C) GFP-MyoVI protein expression in PC12 cells was evaluated by Western blotting using an anti–myosin VI antibody. Arrows indicate GFP-MyoVI-full proteins (175 kD) and GFP-MyoVI-tail proteins (85 kD). (D) NPY-hPLAP release was evaluated using a double-stimulation protocol in PC12 cells expressing GFP (control), GFP-MyoVI-SIfull, or GFP-MyoVI-NIfull. Released NPY-hPLAP is expressed as a percentage of total NPY-hPLAP ( n = 3). (E) NPY-hPLAP release was measured in wild-type (wt) and myosin VI stable knockdown (KD) PC12 cells overexpressing GFP (control), GFP-MyoVI-SIfull (+GFP-MyoVI-SIfull), or GFP-MyoVI-NIfull (+GFP-MyoVI-NIfull). Released NPY-hPLAP is expressed as a percentage of wild-type PC12 cells ( n = 3). Error bars are means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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Image Search Results


Myosin VI SI potentiates evoked exocytosis. (A) RT-PCR analysis of myosin VI isoform expression. β-Actin primers were used as a control. (B) Cartoons of GFP-tagged myosin VI constructs. The motor domain (purple) contains the actin- and ATP-binding sites, the reverse gear (761–814 aa, yellow) determines the unique directionality of myosin VI movement along actin, and the tail domain (orange) in the myosin VI SI (1,262 aa), but not the myosin VI NI (1,253 aa), contains a 9-aa insert. GFP-MyoVI-tail constructs lack the motor domain and are unable to interact with F-actin. (C) GFP-MyoVI protein expression in PC12 cells was evaluated by Western blotting using an anti–myosin VI antibody. Arrows indicate GFP-MyoVI-full proteins (175 kD) and GFP-MyoVI-tail proteins (85 kD). (D) NPY-hPLAP release was evaluated using a double-stimulation protocol in PC12 cells expressing GFP (control), GFP-MyoVI-SIfull, or GFP-MyoVI-NIfull. Released NPY-hPLAP is expressed as a percentage of total NPY-hPLAP ( n = 3). (E) NPY-hPLAP release was measured in wild-type (wt) and myosin VI stable knockdown (KD) PC12 cells overexpressing GFP (control), GFP-MyoVI-SIfull (+GFP-MyoVI-SIfull), or GFP-MyoVI-NIfull (+GFP-MyoVI-NIfull). Released NPY-hPLAP is expressed as a percentage of wild-type PC12 cells ( n = 3). Error bars are means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Journal: The Journal of Cell Biology

Article Title: Myosin VI small insert isoform maintains exocytosis by tethering secretory granules to the cortical actin

doi: 10.1083/jcb.201204092

Figure Lengend Snippet: Myosin VI SI potentiates evoked exocytosis. (A) RT-PCR analysis of myosin VI isoform expression. β-Actin primers were used as a control. (B) Cartoons of GFP-tagged myosin VI constructs. The motor domain (purple) contains the actin- and ATP-binding sites, the reverse gear (761–814 aa, yellow) determines the unique directionality of myosin VI movement along actin, and the tail domain (orange) in the myosin VI SI (1,262 aa), but not the myosin VI NI (1,253 aa), contains a 9-aa insert. GFP-MyoVI-tail constructs lack the motor domain and are unable to interact with F-actin. (C) GFP-MyoVI protein expression in PC12 cells was evaluated by Western blotting using an anti–myosin VI antibody. Arrows indicate GFP-MyoVI-full proteins (175 kD) and GFP-MyoVI-tail proteins (85 kD). (D) NPY-hPLAP release was evaluated using a double-stimulation protocol in PC12 cells expressing GFP (control), GFP-MyoVI-SIfull, or GFP-MyoVI-NIfull. Released NPY-hPLAP is expressed as a percentage of total NPY-hPLAP ( n = 3). (E) NPY-hPLAP release was measured in wild-type (wt) and myosin VI stable knockdown (KD) PC12 cells overexpressing GFP (control), GFP-MyoVI-SIfull (+GFP-MyoVI-SIfull), or GFP-MyoVI-NIfull (+GFP-MyoVI-NIfull). Released NPY-hPLAP is expressed as a percentage of wild-type PC12 cells ( n = 3). Error bars are means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Poly(A) + RNA was reverse transcribed, and PCR products were produced using the RT-PCR kit (ProtoScript M-MuLV Taq; New England Biolabs, Inc.).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Construct, Binding Assay, Western Blot