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. 2017 Jul 15;130(14):2277–2291. doi: 10.1242/jcs.192781

Fig. 8.

Fig. 8.

A FAK-Rab5-Rac1 pathway mediates cell spreading and the response to LIPUS. (A) Representative images of B16 cells coexpressing the indicated constructs pretreated in suspension with either DMSO or FAKi for 1 h before being plated onto fibronectin and fixed after 45 min. Scale bar: 20 µm. (B) Measurements of mean cell area and mean cell circularity show that FAKi treatment reduces cell spreading, similar to expression of either Rac1N17 (DN) or Rab5N34 (DN). Data are mean±s.e.m. from three independent experiments; n=46–78. ***P<0.001 against the DMSO control; +++P<0.001 against WT Rac1 and WT Rab5-expressing DMSO control. (C) Representative images of B16 cells expressing Rac1L61 (CA) with either WT Rab5 or Rab5N34. Scale bar: 20 µm. (D) Quantification of the cell area shows that CA Rac1 expression rescues the defects in cell spreading associated with FAKi treatment. Coexpression of Rab5N34 with Rac1L61 reduces cell spreading to a similar extent to FAKi treatment. Data are mean±s.e.m. from three independent experiments; n=50–72. +++P<0.001 against CA Rac1 and WT Rab5-expressing DMSO control. (E) Mean cell area of B16 cells coexpressing the indicated Rac1 and Rab5 constructs, treated in suspension with either DMSO or dynasore. Note that dynasore treatment has a similar effect on cell area as coexpression of Rab5N34. Data are mean±s.e.m. from three independent experiments; n=21–40. ***P<0.001 against DMSO control. (F) B16 cells treated with either EHT1864 (10 µM) or dynasore (80 µM) were stimulated for 10 min with LIPUS to assess pFAK-Y397 levels. Western blots show that inhibition of either Rac1 or dynamin blocks the LIPUS-induced increase in pFAK levels (blots are representative of at least three independent experiments; α-tubulin was used as a loading control). (G) Model of how cells sense and respond to LIPUS: LIPUS stimulation is ‘sensed’ by vinculin at integrin-mediated cell-ECM adhesions, requiring the link between vinculin and the actin cytoskeleton. FAK signalling leads to Rab5-dependent activation of Rac1. Once Rac1 is active, a feedback mechanism promotes further FAK phosphorylation, thereby propagating Rac1 activity. Active Rac1 localizes at Rab5-positive vesicles to facilitate trafficking, promoting rearrangements of the actin cytoskeleton and increased cell motility.