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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

BiFC-independent approaches demonstrate assembly and localization of the MIT complex at the tips of sticky fingers.(a) U2-OS cells stably expressing either 3xFlag-tagged integrin α5 or VC-tagged integrin α5 were plated on fibronectin for 2 h, fixed, permeabilized, stained with both Flag and Lpd antibodies and proximity ligation assay was performed to assess co-localization of both endogenous Lpd and integrin α5. Cells were counterstained with phalloidin and imaged by spinning disk confocal microscopy (SDCM). The proximity ligation signal is enriched at the cell edge and present at the tip of filopodia-like protrusions. U2-OS cells expressing α5-VC were used as negative control to show the specificity of the signal. Three representative cells for each condition are shown. Scale bar, 10 μm. (b) Cells described in a were stained with the indicated antibodies and imaged by SDCM. Polyclonal anti-EGFP antibody was used to detect VC-tagged integrin α5. Note the distribution of endogenous Lpd is similar in cells expressing either α5-Flag or α5-VC. Scale bar, 10 μm. (c) Tandem affinity purification approach showing assembly of a RIAM–integrin–talin complex. U2-OS cells expressing 3xFlag-RIAM, HA-talin and either αIIb-SBPβ3 or αIIb-SBPβ3(Y747A) were harvested and homogenized. Flag-RIAM was purified with anti-Flag resin and bound proteins were eluted with Flag peptides prior to a second purification with streptavidin resin to isolate αIIb-SBP integrin. Isolated proteins were analysed by Western blot with anti-HA, anti-β3 and anti-Flag antibodies. The talin-binding defective integrin β3(Y747A) mutant blocked the formation of the RIAM-αIIbβ3-talin complex.
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f6: BiFC-independent approaches demonstrate assembly and localization of the MIT complex at the tips of sticky fingers.(a) U2-OS cells stably expressing either 3xFlag-tagged integrin α5 or VC-tagged integrin α5 were plated on fibronectin for 2 h, fixed, permeabilized, stained with both Flag and Lpd antibodies and proximity ligation assay was performed to assess co-localization of both endogenous Lpd and integrin α5. Cells were counterstained with phalloidin and imaged by spinning disk confocal microscopy (SDCM). The proximity ligation signal is enriched at the cell edge and present at the tip of filopodia-like protrusions. U2-OS cells expressing α5-VC were used as negative control to show the specificity of the signal. Three representative cells for each condition are shown. Scale bar, 10 μm. (b) Cells described in a were stained with the indicated antibodies and imaged by SDCM. Polyclonal anti-EGFP antibody was used to detect VC-tagged integrin α5. Note the distribution of endogenous Lpd is similar in cells expressing either α5-Flag or α5-VC. Scale bar, 10 μm. (c) Tandem affinity purification approach showing assembly of a RIAM–integrin–talin complex. U2-OS cells expressing 3xFlag-RIAM, HA-talin and either αIIb-SBPβ3 or αIIb-SBPβ3(Y747A) were harvested and homogenized. Flag-RIAM was purified with anti-Flag resin and bound proteins were eluted with Flag peptides prior to a second purification with streptavidin resin to isolate αIIb-SBP integrin. Isolated proteins were analysed by Western blot with anti-HA, anti-β3 and anti-Flag antibodies. The talin-binding defective integrin β3(Y747A) mutant blocked the formation of the RIAM-αIIbβ3-talin complex.

Mentions: The foregoing studies established that BiFC could be used to observe the localization, dynamics and regulation of assembly of the MIT complex in living cells. The interaction of VN and VC domains, required to form BiFC, can increase the stability of the observed complex28. We performed a proximity ligation assay to visualize the localization of the MIT complex in the absence of BIFC. To perform this assay, we used a rabbit anti-Lpd antibody to stain endogenous Lpd in U2-OS cells and stained Flag-tagged recombinant integrin α5 with a murine anti-Flag antibody (Fig. 6b). We observed localization of the proximity ligation signal to the ends of actin filaments in protruding areas at the cell edge and at the tips of filopodia (Fig. 6a). Consistent with previous studies reporting that this method only detects a subset of protein complexes39, only a fraction of filopodia exhibited a signal at their tips. We also used tandem affinity purification to biochemically assess the formation of a RIAM/integrin/talin complex in the absence of BiFC. Sequential affinity chromatography under non-denaturing conditions isolated Flag-tagged RIAM followed by streptavidin affinity chromatography isolated integrin αIIb-SBPβ3 in a complex containing talin. The formation of this complex required the talin–integrin interaction because none of these components were identified when the tandem affinity purification was performed from cells bearing talin-binding defective αIIb-SBPβ3(Y747A) mutant (Fig. 6c). Finally, when we examined the effect of BiFC on cellular behaviour, we observed BiFC-positive cells exhibited similar numbers and lengths of finger-like protrusions to cells lacking BiFC (Supplementary Fig. 7). In summary, the presence of BiFC, which signals the formation of the MIT complex did not alter protrusion formation and, in the absence of BiFC, the MIT forms is localized to the ends of actin filaments in cell protrusions.


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)

BiFC-independent approaches demonstrate assembly and localization of the MIT complex at the tips of sticky fingers.(a) U2-OS cells stably expressing either 3xFlag-tagged integrin α5 or VC-tagged integrin α5 were plated on fibronectin for 2 h, fixed, permeabilized, stained with both Flag and Lpd antibodies and proximity ligation assay was performed to assess co-localization of both endogenous Lpd and integrin α5. Cells were counterstained with phalloidin and imaged by spinning disk confocal microscopy (SDCM). The proximity ligation signal is enriched at the cell edge and present at the tip of filopodia-like protrusions. U2-OS cells expressing α5-VC were used as negative control to show the specificity of the signal. Three representative cells for each condition are shown. Scale bar, 10 μm. (b) Cells described in a were stained with the indicated antibodies and imaged by SDCM. Polyclonal anti-EGFP antibody was used to detect VC-tagged integrin α5. Note the distribution of endogenous Lpd is similar in cells expressing either α5-Flag or α5-VC. Scale bar, 10 μm. (c) Tandem affinity purification approach showing assembly of a RIAM–integrin–talin complex. U2-OS cells expressing 3xFlag-RIAM, HA-talin and either αIIb-SBPβ3 or αIIb-SBPβ3(Y747A) were harvested and homogenized. Flag-RIAM was purified with anti-Flag resin and bound proteins were eluted with Flag peptides prior to a second purification with streptavidin resin to isolate αIIb-SBP integrin. Isolated proteins were analysed by Western blot with anti-HA, anti-β3 and anti-Flag antibodies. The talin-binding defective integrin β3(Y747A) mutant blocked the formation of the RIAM-αIIbβ3-talin complex.
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f6: BiFC-independent approaches demonstrate assembly and localization of the MIT complex at the tips of sticky fingers.(a) U2-OS cells stably expressing either 3xFlag-tagged integrin α5 or VC-tagged integrin α5 were plated on fibronectin for 2 h, fixed, permeabilized, stained with both Flag and Lpd antibodies and proximity ligation assay was performed to assess co-localization of both endogenous Lpd and integrin α5. Cells were counterstained with phalloidin and imaged by spinning disk confocal microscopy (SDCM). The proximity ligation signal is enriched at the cell edge and present at the tip of filopodia-like protrusions. U2-OS cells expressing α5-VC were used as negative control to show the specificity of the signal. Three representative cells for each condition are shown. Scale bar, 10 μm. (b) Cells described in a were stained with the indicated antibodies and imaged by SDCM. Polyclonal anti-EGFP antibody was used to detect VC-tagged integrin α5. Note the distribution of endogenous Lpd is similar in cells expressing either α5-Flag or α5-VC. Scale bar, 10 μm. (c) Tandem affinity purification approach showing assembly of a RIAM–integrin–talin complex. U2-OS cells expressing 3xFlag-RIAM, HA-talin and either αIIb-SBPβ3 or αIIb-SBPβ3(Y747A) were harvested and homogenized. Flag-RIAM was purified with anti-Flag resin and bound proteins were eluted with Flag peptides prior to a second purification with streptavidin resin to isolate αIIb-SBP integrin. Isolated proteins were analysed by Western blot with anti-HA, anti-β3 and anti-Flag antibodies. The talin-binding defective integrin β3(Y747A) mutant blocked the formation of the RIAM-αIIbβ3-talin complex.
Mentions: The foregoing studies established that BiFC could be used to observe the localization, dynamics and regulation of assembly of the MIT complex in living cells. The interaction of VN and VC domains, required to form BiFC, can increase the stability of the observed complex28. We performed a proximity ligation assay to visualize the localization of the MIT complex in the absence of BIFC. To perform this assay, we used a rabbit anti-Lpd antibody to stain endogenous Lpd in U2-OS cells and stained Flag-tagged recombinant integrin α5 with a murine anti-Flag antibody (Fig. 6b). We observed localization of the proximity ligation signal to the ends of actin filaments in protruding areas at the cell edge and at the tips of filopodia (Fig. 6a). Consistent with previous studies reporting that this method only detects a subset of protein complexes39, only a fraction of filopodia exhibited a signal at their tips. We also used tandem affinity purification to biochemically assess the formation of a RIAM/integrin/talin complex in the absence of BiFC. Sequential affinity chromatography under non-denaturing conditions isolated Flag-tagged RIAM followed by streptavidin affinity chromatography isolated integrin αIIb-SBPβ3 in a complex containing talin. The formation of this complex required the talin–integrin interaction because none of these components were identified when the tandem affinity purification was performed from cells bearing talin-binding defective αIIb-SBPβ3(Y747A) mutant (Fig. 6c). Finally, when we examined the effect of BiFC on cellular behaviour, we observed BiFC-positive cells exhibited similar numbers and lengths of finger-like protrusions to cells lacking BiFC (Supplementary Fig. 7). In summary, the presence of BiFC, which signals the formation of the MIT complex did not alter protrusion formation and, in the absence of BiFC, the MIT forms is localized to the ends of actin filaments in cell protrusions.

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