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Studies on the morphology and spreading of human endothelial cells define key inter- and intramolecular interactions for talin1.

Kopp PM, Bate N, Hansen TM, Brindle NP, Praekelt U, Debrand E, Coleman S, Mazzeo D, Goult BT, Gingras AR, Pritchard CA, Critchley DR, Monkley SJ - Eur. J. Cell Biol. (2010)

Bottom Line: The talin rod contains several actin-binding sites (ABS), and mutations in the C-terminal ABS that reduced actin-binding impaired talin1 function, whereas those that increased binding resulted in more stable FAs.The results show that both the N-terminal integrin and C-terminal actin-binding functions of talin are essential to cell spreading and FA assembly.Finally, mutations that relieve talin auto-inhibition resulted in the rapid and excessive production of FA, highlighting the importance of talin regulation within the cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Leicester, Leicester, UK.

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GFP-talin1 or talin2 rescues cell spreading and FA formation in talin1-depleted HUVEC. Cells were transfected with a talin1 siRNA or conRNA plus constructs encoding either GFP alone, mouse talin1-GFP or human talin2-GFP. Cells were replated on glass coverslips 72 h after transfection. (A) Epifluorescence images showing GFP localisation in cells 24 h after replating. (B, C) Time course of changes in cell morphology (B) and cell area (C). (D, E) FA number (D) and size (E) in different cell populations quantified using ImageJ. All results are expressed as mean ± s.e.m. Scale bars: 10 μm.
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fig3: GFP-talin1 or talin2 rescues cell spreading and FA formation in talin1-depleted HUVEC. Cells were transfected with a talin1 siRNA or conRNA plus constructs encoding either GFP alone, mouse talin1-GFP or human talin2-GFP. Cells were replated on glass coverslips 72 h after transfection. (A) Epifluorescence images showing GFP localisation in cells 24 h after replating. (B, C) Time course of changes in cell morphology (B) and cell area (C). (D, E) FA number (D) and size (E) in different cell populations quantified using ImageJ. All results are expressed as mean ± s.e.m. Scale bars: 10 μm.

Mentions: To confirm that the knockdown phenotype could be rescued by re-expressing talin1, HUVEC were transfected with the human talin1 siRNA together with a plasmid encoding GFP-tagged mouse talin1. After 72 h, cells were replated on glass coverslips, and analysed a further 24 h later. GFP-talin1 expression was confirmed by Western blotting (Fig. S3A). To ensure that phenotypic analysis was conducted on cells expressing similar levels of GFP-talin1, we only imaged those cells that could be visualised at exposure times between 400 and 600 ms on the epifluorescence microscope. Inspection of these images showed that GFP-mouse talin1 fully rescued the spreading defect in talin1 knockdown cells whereas GFP alone was unable to do so (Fig. 3A). Thus, the proportion of GFP-talin1 expressors that were spread (Fig. 3B and C) and the number of paxillin-containing FA (Fig. 3D) was about the same as in cells transfected with the conRNA and GFP. GFP-tagged talin1 localised to peripheral FA as well as to more central fibrillar adhesions (FB) (Fig. 3A arrow), although a cytoplasmic pool of what may be auto-inhibited talin was located around the nucleus. Analysis of the time course of cell spreading showed that talin1 knockdown cells were able to adhere and to spread at least initially (Fig. 3B and C), but this was not maintained, and after 1 h the cells had started to adopt an arborized morphology with multiple protrusions (Fig. 3B) which ultimately resulted in a reduced spread cell area (Fig. 3C). Cells rescued with wild-type GFP-talin1 showed a transient increase in arborized cells (5–7 h), but after 24 h the majority of cells (69%) were well spread. The talin1 knockdown phenotype could also be rescued by GFP-talin2 (Fig. 3A–C) which localised to FA and FB adhesions, although the size and number of paxillin-containing FA was slightly less than in cells expressing GFP-talin1 (Fig. 3D and E).


Studies on the morphology and spreading of human endothelial cells define key inter- and intramolecular interactions for talin1.

Kopp PM, Bate N, Hansen TM, Brindle NP, Praekelt U, Debrand E, Coleman S, Mazzeo D, Goult BT, Gingras AR, Pritchard CA, Critchley DR, Monkley SJ - Eur. J. Cell Biol. (2010)

GFP-talin1 or talin2 rescues cell spreading and FA formation in talin1-depleted HUVEC. Cells were transfected with a talin1 siRNA or conRNA plus constructs encoding either GFP alone, mouse talin1-GFP or human talin2-GFP. Cells were replated on glass coverslips 72 h after transfection. (A) Epifluorescence images showing GFP localisation in cells 24 h after replating. (B, C) Time course of changes in cell morphology (B) and cell area (C). (D, E) FA number (D) and size (E) in different cell populations quantified using ImageJ. All results are expressed as mean ± s.e.m. Scale bars: 10 μm.
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fig3: GFP-talin1 or talin2 rescues cell spreading and FA formation in talin1-depleted HUVEC. Cells were transfected with a talin1 siRNA or conRNA plus constructs encoding either GFP alone, mouse talin1-GFP or human talin2-GFP. Cells were replated on glass coverslips 72 h after transfection. (A) Epifluorescence images showing GFP localisation in cells 24 h after replating. (B, C) Time course of changes in cell morphology (B) and cell area (C). (D, E) FA number (D) and size (E) in different cell populations quantified using ImageJ. All results are expressed as mean ± s.e.m. Scale bars: 10 μm.
Mentions: To confirm that the knockdown phenotype could be rescued by re-expressing talin1, HUVEC were transfected with the human talin1 siRNA together with a plasmid encoding GFP-tagged mouse talin1. After 72 h, cells were replated on glass coverslips, and analysed a further 24 h later. GFP-talin1 expression was confirmed by Western blotting (Fig. S3A). To ensure that phenotypic analysis was conducted on cells expressing similar levels of GFP-talin1, we only imaged those cells that could be visualised at exposure times between 400 and 600 ms on the epifluorescence microscope. Inspection of these images showed that GFP-mouse talin1 fully rescued the spreading defect in talin1 knockdown cells whereas GFP alone was unable to do so (Fig. 3A). Thus, the proportion of GFP-talin1 expressors that were spread (Fig. 3B and C) and the number of paxillin-containing FA (Fig. 3D) was about the same as in cells transfected with the conRNA and GFP. GFP-tagged talin1 localised to peripheral FA as well as to more central fibrillar adhesions (FB) (Fig. 3A arrow), although a cytoplasmic pool of what may be auto-inhibited talin was located around the nucleus. Analysis of the time course of cell spreading showed that talin1 knockdown cells were able to adhere and to spread at least initially (Fig. 3B and C), but this was not maintained, and after 1 h the cells had started to adopt an arborized morphology with multiple protrusions (Fig. 3B) which ultimately resulted in a reduced spread cell area (Fig. 3C). Cells rescued with wild-type GFP-talin1 showed a transient increase in arborized cells (5–7 h), but after 24 h the majority of cells (69%) were well spread. The talin1 knockdown phenotype could also be rescued by GFP-talin2 (Fig. 3A–C) which localised to FA and FB adhesions, although the size and number of paxillin-containing FA was slightly less than in cells expressing GFP-talin1 (Fig. 3D and E).

Bottom Line: The talin rod contains several actin-binding sites (ABS), and mutations in the C-terminal ABS that reduced actin-binding impaired talin1 function, whereas those that increased binding resulted in more stable FAs.The results show that both the N-terminal integrin and C-terminal actin-binding functions of talin are essential to cell spreading and FA assembly.Finally, mutations that relieve talin auto-inhibition resulted in the rapid and excessive production of FA, highlighting the importance of talin regulation within the cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Leicester, Leicester, UK.

Show MeSH
Related in: MedlinePlus