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A RIAM/lamellipodin-talin-integrin complex forms the tip of sticky fingers that guide cell migration.

Lagarrigue F, Vikas Anekal P, Lee HS, Bachir AI, Ablack JN, Horwitz AF, Ginsberg MH - Nat Commun (2015)

Bottom Line: The leading edge of migrating cells contains rapidly translocating activated integrins associated with growing actin filaments that form 'sticky fingers' to sense extracellular matrix and guide cell migration.Here we utilized indirect bimolecular fluorescence complementation to visualize a molecular complex containing a Mig-10/RIAM/lamellipodin (MRL) protein (Rap1-GTP-interacting adaptor molecule (RIAM) or lamellipodin), talin and activated integrins in living cells.These data reveal the molecular basis of the formation of 'sticky fingers' at the leading edge of migrating cells and show that an MIT complex drives these protrusions.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California San Diego, La Jolla, California 92093, USA.

ABSTRACT
The leading edge of migrating cells contains rapidly translocating activated integrins associated with growing actin filaments that form 'sticky fingers' to sense extracellular matrix and guide cell migration. Here we utilized indirect bimolecular fluorescence complementation to visualize a molecular complex containing a Mig-10/RIAM/lamellipodin (MRL) protein (Rap1-GTP-interacting adaptor molecule (RIAM) or lamellipodin), talin and activated integrins in living cells. This complex localizes at the tips of growing actin filaments in lamellipodial and filopodial protrusions, thus corresponding to the tips of the 'sticky fingers.' Formation of the complex requires talin to form a bridge between the MRL protein and the integrins. Moreover, disruption of the MRL protein-integrin-talin (MIT) complex markedly impairs cell protrusion. These data reveal the molecular basis of the formation of 'sticky fingers' at the leading edge of migrating cells and show that an MIT complex drives these protrusions.

No MeSH data available.


Related in: MedlinePlus

Formation of the MIT complex depends on talin interaction with the integrin.(a) Predicted effects of blocking talin's interactions on BiFC. Mutations of integrin β3(L746A) and β3(Y747A) interfere with talin binding. (b) U2-OS cells expressing VN-RIAM were infected with lentiviral particles encoding αIIb-VCβ3, αIIb-VCβ3(L746A) or αIIb-VCβ3(Y747A). Cells were harvested, fixed, stained with anti-αIIbβ3 (D57) antibody and BiFC fluorescence was measured by flow cytometry. The mean of BiFC fluorescence intensity of the D57-positive population was plotted. Data are expressed as percentage of cells expressing αIIb-VCβ3(WT). One-way analysis of variance with Bonferroni's test were used to compare the populations: *** P<0.001. The error bars display s.e.m. for four separate experiments. (c) U2-OS cells expressing VN-RIAM were transduced as described in b were plated on fibronectin for 2 h. The cells were fixed, stained with anti-αIIbβ3 (D57) antibody and imaged for BiFC by spinning disc confocal microscopy. Scale bar, 10 μm.
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f3: Formation of the MIT complex depends on talin interaction with the integrin.(a) Predicted effects of blocking talin's interactions on BiFC. Mutations of integrin β3(L746A) and β3(Y747A) interfere with talin binding. (b) U2-OS cells expressing VN-RIAM were infected with lentiviral particles encoding αIIb-VCβ3, αIIb-VCβ3(L746A) or αIIb-VCβ3(Y747A). Cells were harvested, fixed, stained with anti-αIIbβ3 (D57) antibody and BiFC fluorescence was measured by flow cytometry. The mean of BiFC fluorescence intensity of the D57-positive population was plotted. Data are expressed as percentage of cells expressing αIIb-VCβ3(WT). One-way analysis of variance with Bonferroni's test were used to compare the populations: *** P<0.001. The error bars display s.e.m. for four separate experiments. (c) U2-OS cells expressing VN-RIAM were transduced as described in b were plated on fibronectin for 2 h. The cells were fixed, stained with anti-αIIbβ3 (D57) antibody and imaged for BiFC by spinning disc confocal microscopy. Scale bar, 10 μm.

Mentions: To directly test whether BiFC required talin–integrin interaction, we introduced the β3(Y747A) mutation that inhibits the binding of talin, filamin and other cytoplasmic proteins or the β3(L746A) mutation that selectively reduces integrin interactions only with talin11 (Fig. 3a). In cells expressing either αIIb-VCβ3(L746A) or αIIb-VCβ3(Y747A) there was a dramatic reduction in the BiFC signal as assayed by quantitative flow cytometry (Fig. 3b) and by spinning disk confocal microscopy (Fig. 3c). Thus, we conclude that BiFC reports the presence of an MIT complex.


A RIAM/lamellipodin-talin-integrin complex forms the tip of sticky fingers that guide cell migration.

Lagarrigue F, Vikas Anekal P, Lee HS, Bachir AI, Ablack JN, Horwitz AF, Ginsberg MH - Nat Commun (2015)

Formation of the MIT complex depends on talin interaction with the integrin.(a) Predicted effects of blocking talin's interactions on BiFC. Mutations of integrin β3(L746A) and β3(Y747A) interfere with talin binding. (b) U2-OS cells expressing VN-RIAM were infected with lentiviral particles encoding αIIb-VCβ3, αIIb-VCβ3(L746A) or αIIb-VCβ3(Y747A). Cells were harvested, fixed, stained with anti-αIIbβ3 (D57) antibody and BiFC fluorescence was measured by flow cytometry. The mean of BiFC fluorescence intensity of the D57-positive population was plotted. Data are expressed as percentage of cells expressing αIIb-VCβ3(WT). One-way analysis of variance with Bonferroni's test were used to compare the populations: *** P<0.001. The error bars display s.e.m. for four separate experiments. (c) U2-OS cells expressing VN-RIAM were transduced as described in b were plated on fibronectin for 2 h. The cells were fixed, stained with anti-αIIbβ3 (D57) antibody and imaged for BiFC by spinning disc confocal microscopy. Scale bar, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4589889&req=5

f3: Formation of the MIT complex depends on talin interaction with the integrin.(a) Predicted effects of blocking talin's interactions on BiFC. Mutations of integrin β3(L746A) and β3(Y747A) interfere with talin binding. (b) U2-OS cells expressing VN-RIAM were infected with lentiviral particles encoding αIIb-VCβ3, αIIb-VCβ3(L746A) or αIIb-VCβ3(Y747A). Cells were harvested, fixed, stained with anti-αIIbβ3 (D57) antibody and BiFC fluorescence was measured by flow cytometry. The mean of BiFC fluorescence intensity of the D57-positive population was plotted. Data are expressed as percentage of cells expressing αIIb-VCβ3(WT). One-way analysis of variance with Bonferroni's test were used to compare the populations: *** P<0.001. The error bars display s.e.m. for four separate experiments. (c) U2-OS cells expressing VN-RIAM were transduced as described in b were plated on fibronectin for 2 h. The cells were fixed, stained with anti-αIIbβ3 (D57) antibody and imaged for BiFC by spinning disc confocal microscopy. Scale bar, 10 μm.
Mentions: To directly test whether BiFC required talin–integrin interaction, we introduced the β3(Y747A) mutation that inhibits the binding of talin, filamin and other cytoplasmic proteins or the β3(L746A) mutation that selectively reduces integrin interactions only with talin11 (Fig. 3a). In cells expressing either αIIb-VCβ3(L746A) or αIIb-VCβ3(Y747A) there was a dramatic reduction in the BiFC signal as assayed by quantitative flow cytometry (Fig. 3b) and by spinning disk confocal microscopy (Fig. 3c). Thus, we conclude that BiFC reports the presence of an MIT complex.

Bottom Line: The leading edge of migrating cells contains rapidly translocating activated integrins associated with growing actin filaments that form 'sticky fingers' to sense extracellular matrix and guide cell migration.Here we utilized indirect bimolecular fluorescence complementation to visualize a molecular complex containing a Mig-10/RIAM/lamellipodin (MRL) protein (Rap1-GTP-interacting adaptor molecule (RIAM) or lamellipodin), talin and activated integrins in living cells.These data reveal the molecular basis of the formation of 'sticky fingers' at the leading edge of migrating cells and show that an MIT complex drives these protrusions.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California San Diego, La Jolla, California 92093, USA.

ABSTRACT
The leading edge of migrating cells contains rapidly translocating activated integrins associated with growing actin filaments that form 'sticky fingers' to sense extracellular matrix and guide cell migration. Here we utilized indirect bimolecular fluorescence complementation to visualize a molecular complex containing a Mig-10/RIAM/lamellipodin (MRL) protein (Rap1-GTP-interacting adaptor molecule (RIAM) or lamellipodin), talin and activated integrins in living cells. This complex localizes at the tips of growing actin filaments in lamellipodial and filopodial protrusions, thus corresponding to the tips of the 'sticky fingers.' Formation of the complex requires talin to form a bridge between the MRL protein and the integrins. Moreover, disruption of the MRL protein-integrin-talin (MIT) complex markedly impairs cell protrusion. These data reveal the molecular basis of the formation of 'sticky fingers' at the leading edge of migrating cells and show that an MIT complex drives these protrusions.

No MeSH data available.


Related in: MedlinePlus