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ROCK1 and LIMK2 interact in spread but not blebbing cancer cells.

Shea KF, Wells CM, Garner AP, Jones GE - PLoS ONE (2008)

Bottom Line: 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.Our results point to a specific role for the ROCK1:LIMK2 pathway in mesenchymal-mode migration.

View Article: PubMed Central - PubMed

Affiliation: Randall Division of Cell & Molecular Biophysics, King's College London, London, United Kingdom.

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.

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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.
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pone-0003398-g004: 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.

Mentions: Having established that ROCK1 and LIMK2 are not interacting in blebbing cells we analysed the localisation and interaction of ROCK1 and LIMK2 in spread cells. In spread cells the majority of LIMK2 and ROCK expression is localised in cytoplasm, but expression of both proteins can be detected in the nucleus (Fig. 4). In MDA-MB231 cells with a spread/polarised phenotype we detected a decreased GFP lifetime when ROCK1 and LIMK2 were co-expressed, showing that ROCK1 and LIMK2 interact in spread cells (Fig. 4). The GFP lifetime decrease is seen across the cell cytoplasm in a punctate distribution. In comparison, two polarised cells microinjected with only GFP-ROCK1 do not display any decrease in GFP lifetime (Fig. 4). There is no significant drop in GFP lifetime below control levels when cells expressing ROCK1 and LIMK2 are pre-incubated with the ROCK inhibitor Y27632 (Fig 4). Interestingly, in many cells there is a lack of any detectable interaction between ROCK1 and LIMK2 at the cell periphery (highlighted by arrowheads in Fig. 4).


ROCK1 and LIMK2 interact in spread but not blebbing cancer cells.

Shea KF, Wells CM, Garner AP, Jones GE - PLoS ONE (2008)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2561063&req=5

pone-0003398-g004: 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.
Mentions: Having established that ROCK1 and LIMK2 are not interacting in blebbing cells we analysed the localisation and interaction of ROCK1 and LIMK2 in spread cells. In spread cells the majority of LIMK2 and ROCK expression is localised in cytoplasm, but expression of both proteins can be detected in the nucleus (Fig. 4). In MDA-MB231 cells with a spread/polarised phenotype we detected a decreased GFP lifetime when ROCK1 and LIMK2 were co-expressed, showing that ROCK1 and LIMK2 interact in spread cells (Fig. 4). The GFP lifetime decrease is seen across the cell cytoplasm in a punctate distribution. In comparison, two polarised cells microinjected with only GFP-ROCK1 do not display any decrease in GFP lifetime (Fig. 4). There is no significant drop in GFP lifetime below control levels when cells expressing ROCK1 and LIMK2 are pre-incubated with the ROCK inhibitor Y27632 (Fig 4). Interestingly, in many cells there is a lack of any detectable interaction between ROCK1 and LIMK2 at the cell periphery (highlighted by arrowheads in Fig. 4).

Bottom Line: 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.Our results point to a specific role for the ROCK1:LIMK2 pathway in mesenchymal-mode migration.

View Article: PubMed Central - PubMed

Affiliation: Randall Division of Cell & Molecular Biophysics, King's College London, London, United Kingdom.

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.

Show MeSH
Related in: MedlinePlus