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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

The MIT complex directs protrusion.NIH-3T3 cells expressing the membrane marker mCherry-K-Ras-Caax and either control shRNA (CT) or RIAM shRNA (KD) were transiently transfected with constructs encoding BFP-RIAM(WT), talin-binding defective BFP-RIAM(4E) mutant or BFP alone. (a) Cell lysates were analysed by Western blot with anti-RIAM antibody. Tubulin was used as a loading control. (b) Cells were plated on fibrinogen for 2 h, fixed, stained with anti-paxillin antibody and imaged with TIRFM. Scale bar, 10 μm. (c) Cells were plated on fibrinogen for 2 h and imaged at 5 s interval for 3 min. An actively protruding part of the cell edge was computationally detected as described in Methods, and the changes in protrusion area over time were plotted. Each point depicts the mean ± s.e.m. of three independent experiments. A total of 3–5 cells for each condition have been analysed in each experiment. Two-way analysis of variance with Bonferroni's test were used to compare the populations: KD versus CT, P<0.001; KD+RIAM(WT) versus CT, P=NS; KD+RIAM(4E) versus CT, P<0.001. (d) The MIT complex is the molecular basis of ‘sticky fingers' at the leading edge. The N terminus of the MRL protein binds and recruits talin to the integrin to induce activation. The C terminus of MRL protein increases processive actin polymerization in part by recruiting ENA/VASP, thereby propelling the movement of the ‘sticky fingers'.
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f8: The MIT complex directs protrusion.NIH-3T3 cells expressing the membrane marker mCherry-K-Ras-Caax and either control shRNA (CT) or RIAM shRNA (KD) were transiently transfected with constructs encoding BFP-RIAM(WT), talin-binding defective BFP-RIAM(4E) mutant or BFP alone. (a) Cell lysates were analysed by Western blot with anti-RIAM antibody. Tubulin was used as a loading control. (b) Cells were plated on fibrinogen for 2 h, fixed, stained with anti-paxillin antibody and imaged with TIRFM. Scale bar, 10 μm. (c) Cells were plated on fibrinogen for 2 h and imaged at 5 s interval for 3 min. An actively protruding part of the cell edge was computationally detected as described in Methods, and the changes in protrusion area over time were plotted. Each point depicts the mean ± s.e.m. of three independent experiments. A total of 3–5 cells for each condition have been analysed in each experiment. Two-way analysis of variance with Bonferroni's test were used to compare the populations: KD versus CT, P<0.001; KD+RIAM(WT) versus CT, P=NS; KD+RIAM(4E) versus CT, P<0.001. (d) The MIT complex is the molecular basis of ‘sticky fingers' at the leading edge. The N terminus of the MRL protein binds and recruits talin to the integrin to induce activation. The C terminus of MRL protein increases processive actin polymerization in part by recruiting ENA/VASP, thereby propelling the movement of the ‘sticky fingers'.

Mentions: As noted above, MRL proteins drive cell protrusion and play a role in cell migration and we have shown here that they form an MIT complex at the tips of protrusive structures that help guide migration. Because MRL proteins may have multiple functions, we asked if their capacity to enter the MIT complex enabled them to support cell protrusion. To test this idea, we silenced RIAM in NIH-3T3 cells and rescued its expression with a mutant in the talin-binding RIAM(4E) N-terminal domain that preserves binding sites for Rap1 and proline-rich sequences that interact with VASP24 (Fig. 8a). Blocking the RIAM–talin interaction led to a dramatic reduction in RIAM recruitment to paxillin-staining adhesions (Fig. 8b). As previously reported23, silencing RIAM markedly inhibited lamellipodial protrusion; rescue with wild-type RIAM restored protrusion whereas the RIAM(4E) mutant had no effect (Fig. 8c; Supplementary Movie 12). Therefore, RIAM in the MIT complex supports cellular protrusion.


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)

The MIT complex directs protrusion.NIH-3T3 cells expressing the membrane marker mCherry-K-Ras-Caax and either control shRNA (CT) or RIAM shRNA (KD) were transiently transfected with constructs encoding BFP-RIAM(WT), talin-binding defective BFP-RIAM(4E) mutant or BFP alone. (a) Cell lysates were analysed by Western blot with anti-RIAM antibody. Tubulin was used as a loading control. (b) Cells were plated on fibrinogen for 2 h, fixed, stained with anti-paxillin antibody and imaged with TIRFM. Scale bar, 10 μm. (c) Cells were plated on fibrinogen for 2 h and imaged at 5 s interval for 3 min. An actively protruding part of the cell edge was computationally detected as described in Methods, and the changes in protrusion area over time were plotted. Each point depicts the mean ± s.e.m. of three independent experiments. A total of 3–5 cells for each condition have been analysed in each experiment. Two-way analysis of variance with Bonferroni's test were used to compare the populations: KD versus CT, P<0.001; KD+RIAM(WT) versus CT, P=NS; KD+RIAM(4E) versus CT, P<0.001. (d) The MIT complex is the molecular basis of ‘sticky fingers' at the leading edge. The N terminus of the MRL protein binds and recruits talin to the integrin to induce activation. The C terminus of MRL protein increases processive actin polymerization in part by recruiting ENA/VASP, thereby propelling the movement of the ‘sticky fingers'.
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Related In: Results  -  Collection

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f8: The MIT complex directs protrusion.NIH-3T3 cells expressing the membrane marker mCherry-K-Ras-Caax and either control shRNA (CT) or RIAM shRNA (KD) were transiently transfected with constructs encoding BFP-RIAM(WT), talin-binding defective BFP-RIAM(4E) mutant or BFP alone. (a) Cell lysates were analysed by Western blot with anti-RIAM antibody. Tubulin was used as a loading control. (b) Cells were plated on fibrinogen for 2 h, fixed, stained with anti-paxillin antibody and imaged with TIRFM. Scale bar, 10 μm. (c) Cells were plated on fibrinogen for 2 h and imaged at 5 s interval for 3 min. An actively protruding part of the cell edge was computationally detected as described in Methods, and the changes in protrusion area over time were plotted. Each point depicts the mean ± s.e.m. of three independent experiments. A total of 3–5 cells for each condition have been analysed in each experiment. Two-way analysis of variance with Bonferroni's test were used to compare the populations: KD versus CT, P<0.001; KD+RIAM(WT) versus CT, P=NS; KD+RIAM(4E) versus CT, P<0.001. (d) The MIT complex is the molecular basis of ‘sticky fingers' at the leading edge. The N terminus of the MRL protein binds and recruits talin to the integrin to induce activation. The C terminus of MRL protein increases processive actin polymerization in part by recruiting ENA/VASP, thereby propelling the movement of the ‘sticky fingers'.
Mentions: As noted above, MRL proteins drive cell protrusion and play a role in cell migration and we have shown here that they form an MIT complex at the tips of protrusive structures that help guide migration. Because MRL proteins may have multiple functions, we asked if their capacity to enter the MIT complex enabled them to support cell protrusion. To test this idea, we silenced RIAM in NIH-3T3 cells and rescued its expression with a mutant in the talin-binding RIAM(4E) N-terminal domain that preserves binding sites for Rap1 and proline-rich sequences that interact with VASP24 (Fig. 8a). Blocking the RIAM–talin interaction led to a dramatic reduction in RIAM recruitment to paxillin-staining adhesions (Fig. 8b). As previously reported23, silencing RIAM markedly inhibited lamellipodial protrusion; rescue with wild-type RIAM restored protrusion whereas the RIAM(4E) mutant had no effect (Fig. 8c; Supplementary Movie 12). Therefore, RIAM in the MIT complex supports cellular protrusion.

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