ROCK1 and LIMK2 interact in spread but not blebbing cancer cells

Abstract

Cancer cells migrating within a 3D microenvironment are able to adopt either a mesenchymal or amoeboid mode of migration. Amoeboid migration is characterised by membrane blebbing that is dependent on the Rho effectors, ROCK1/2. We identify LIMK2 as the preferred substrate for ROCK1 but find that LIMK2 did not induce membrane blebbing, suggesting that a LIMK2 pathway is not involved in amoeboid-mode migration. In support of this hypothesis, novel FRET data demonstrate a direct interaction between ROCK1 and LIMK2 in polarised but not blebbing cells. Our results point to a specific role for the ROCK1:LIMK2 pathway in mesenchymal-mode migration.

Figure 1. ROCK1 phosphorylates LIMK1 and LIMK2.

A) and B) CFP-LIMK1 or 2 and either CFP-ROCK1 or CFP alone were transiently transfected into MDA-MB231 cells and treated with 10 µM Y27632 for 24 hours. The resultant lysates were immunoblotted using anti-phospho-LIMK, -LIMK1, -LIMK2 and -ROCK1 antibodies. (nsb = non-specific binding of the anti-ROCK1 antibody to a protein/s at 220 kDa). C) Ratio of phospho/total LIMK1 CFP-LIMK1 and D) Ratio of phospho/total LIMK2. (* = P<0.05).

Figure 2. ROCK1 but not LIMK2 induces blebbing.

A) MDA-MB231 cells were transfected with GFP-ROCK1 or GFP-LIMK2, fixed and stained with Alexa Fluor 594-Phalloidin and Dapi. 150 cells over 3 independent experiments were scored for visible blebbing+/−s.e.m. P<0.05; ***P<0.001. B) Representative images from varying optical slices of a blebbing cell overexpressing GFP-ROCK1. (Bar = 20 µm).

Figure 3. ROCK1 and LIMK2 do not interact in blebbing cells.

MDA-MB231 cells were microinjected with GFP-ROCK1 and mRFP-LIMK2, fixed, imaged and analysed using FLIM microscopy and the TRI2 analysis programme. A) Images of the GFP lifetime and GFP and mRFP intensities across a typical blebbing cell was displayed for a cell expressing both the GFP-ROCK1 donor and the mRFP-LIMK2 acceptor and for comparison, only the GFP -ROCK1 donor. B) Histogram of the number of normalised pixel counts detected at each GFP lifetime. n = 9.

Figure 4. ROCK1 and LIMK2 interact in polarised cells.

MDA-MB231 cells were microinjected with GFP-ROCK1 and mRFP-LIMK2, fixed, imaged and analysed using FLIM microscopy and the TRI2 analysis programme. A) Images of the GFP lifetime and GFP and mRFP intensities across a typical elongated cell was displayed for a cell expressing both the GFP-ROCK1 donor and the mRFP-LIMK2 acceptor and for comparison, only the GFP -ROCK1 donor. B) Histogram of the number of normalised pixel counts detected at each GFP lifetime. C) A histogram of the average number of normalised pixel counts detected at each GFP lifetime in cells expressing both GFP-ROCK1 donor and mRFP-LIMK2 acceptor in cells of elongated or blebbing morphologies was constructed along with cells expressing only the GFP-ROCK1 donor. 18 cells over three independent experiments were imaged for each time point. D) A Histogram of the number of normalised pixel counts detected at each GFP lifetime for cells expressing both GFP-ROCK-1 donor and mRFP-LIMK-2 acceptor in MDA-MB231 cells pre-treated with Y27632. n = 9.