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LKB1 kinase-dependent and -independent defects disrupt polarity and adhesion signaling to drive collagen remodeling during invasion.

Konen J, Wilkinson S, Lee B, Fu H, Zhou W, Jiang Y, Marcus AI - Mol. Biol. Cell (2016)

Bottom Line: The majority of LKB1 mutations are truncations that disrupt its kinase activity and remove its C-terminal domain (CTD).Instead, cell polarity is overseen by the kinase-independent function of its CTD and more specifically its farnesylation.This occurs through a mesenchymal-amoeboid morphological switch that signals through the Rho-GTPase RhoA.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory University, Atlanta, GA 30322 Graduate Program in Cancer Biology, Emory University, Atlanta, GA 30322.

No MeSH data available.


Related in: MedlinePlus

Constitutively active RhoA restores mesenchymal polarity in LKB1 farnesylation-mutant cells. (A) Schematic of double-stable cell lines expressing either empty GFP control or GFP-tagged, wild-type LKB1 or LKB1 C430S with constitutively active RhoA (Q63L) or cdc42 (Q61L). (B) Spheroids of H157 cells expressing either empty GFP control or GFP-tagged, wild-type LKB1 or LKB1 C430S and spheroids of these cells also expressing constitutively active RhoA or cdc42 were embedded in a collagen type I matrix. At 24 h postembedding, cells were fixed and stained with phalloidin. Amoeboid and mesenchymal morphologies (described in Figure 1) were quantified as a percentage back to the total number of cells invaded in each spheroid. Four spheroids. Scale, 20 μm. Arrows, mesenchymal cells; arrowheads, amoeboid cells. (C) The percentage of mesenchymal cells was quantified for each cell line at 24 h postembedding. (D) Each cell line was tracked over time. Cell tracks were plotted from a single point of origin. (E) Meandering index was calculated using the cell tracks from D. Meandering index is defined as the linear distance divided by the total path length; 30 cells. ****p ≤ 0.0001.
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Figure 4: Constitutively active RhoA restores mesenchymal polarity in LKB1 farnesylation-mutant cells. (A) Schematic of double-stable cell lines expressing either empty GFP control or GFP-tagged, wild-type LKB1 or LKB1 C430S with constitutively active RhoA (Q63L) or cdc42 (Q61L). (B) Spheroids of H157 cells expressing either empty GFP control or GFP-tagged, wild-type LKB1 or LKB1 C430S and spheroids of these cells also expressing constitutively active RhoA or cdc42 were embedded in a collagen type I matrix. At 24 h postembedding, cells were fixed and stained with phalloidin. Amoeboid and mesenchymal morphologies (described in Figure 1) were quantified as a percentage back to the total number of cells invaded in each spheroid. Four spheroids. Scale, 20 μm. Arrows, mesenchymal cells; arrowheads, amoeboid cells. (C) The percentage of mesenchymal cells was quantified for each cell line at 24 h postembedding. (D) Each cell line was tracked over time. Cell tracks were plotted from a single point of origin. (E) Meandering index was calculated using the cell tracks from D. Meandering index is defined as the linear distance divided by the total path length; 30 cells. ****p ≤ 0.0001.

Mentions: The present data show that LKB1 CTD, and specifically its farnesylation, promotes both mesenchymal cell polarity and RhoA activity. We next sought to determine whether LKB1 promotes mesenchymal polarization through RhoA signaling. We created double stable cells expressing either empty GFP or GFP-LKB1 wild type or a C430S farnesylation mutant and the constitutively active form of either RhoA (Q63L) or cdc42 (Q61L) (Figure 4A). Cells reexpressing wild-type LKB1 and either constitutively active RhoA or cdc42 maintain a mesenchymal polarization similar to cells reexpressing wild-type LKB1 alone (Figure 4, B and C). Similarly, rescuing cdc42 activity in farnesylation-mutant cells results in a predominantly amoeboid phenotype, similar to LKB1 farnesylation-mutant cells alone (Figure 4, B and C), suggesting that LKB1 does not signal to cdc42 to promote mesenchymal polarization. However, upon rescuing RhoA activity in LKB1 farnesylation-mutant cells, cells reacquire a mesenchymal polarization during 3D invasion (Figure 4, B and C). Of importance, this result is consistent with mesenchymal polarization in cells reexpressing wild-type LKB1. Restoring either RhoA or cdc42 activity in empty GFP control cells fails to restore this mesenchymal polarization (Figure 4, B and C). Given that our previous data highlight the role of LKB1 farnesylation in promoting mesenchymal polarity, these data suggest that this occurs through LKB1 signaling to RhoA, as rescuing RhoA activity in farnesylation-compromised cells restores mesenchymal polarity.


LKB1 kinase-dependent and -independent defects disrupt polarity and adhesion signaling to drive collagen remodeling during invasion.

Konen J, Wilkinson S, Lee B, Fu H, Zhou W, Jiang Y, Marcus AI - Mol. Biol. Cell (2016)

Constitutively active RhoA restores mesenchymal polarity in LKB1 farnesylation-mutant cells. (A) Schematic of double-stable cell lines expressing either empty GFP control or GFP-tagged, wild-type LKB1 or LKB1 C430S with constitutively active RhoA (Q63L) or cdc42 (Q61L). (B) Spheroids of H157 cells expressing either empty GFP control or GFP-tagged, wild-type LKB1 or LKB1 C430S and spheroids of these cells also expressing constitutively active RhoA or cdc42 were embedded in a collagen type I matrix. At 24 h postembedding, cells were fixed and stained with phalloidin. Amoeboid and mesenchymal morphologies (described in Figure 1) were quantified as a percentage back to the total number of cells invaded in each spheroid. Four spheroids. Scale, 20 μm. Arrows, mesenchymal cells; arrowheads, amoeboid cells. (C) The percentage of mesenchymal cells was quantified for each cell line at 24 h postembedding. (D) Each cell line was tracked over time. Cell tracks were plotted from a single point of origin. (E) Meandering index was calculated using the cell tracks from D. Meandering index is defined as the linear distance divided by the total path length; 30 cells. ****p ≤ 0.0001.
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Related In: Results  -  Collection

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Figure 4: Constitutively active RhoA restores mesenchymal polarity in LKB1 farnesylation-mutant cells. (A) Schematic of double-stable cell lines expressing either empty GFP control or GFP-tagged, wild-type LKB1 or LKB1 C430S with constitutively active RhoA (Q63L) or cdc42 (Q61L). (B) Spheroids of H157 cells expressing either empty GFP control or GFP-tagged, wild-type LKB1 or LKB1 C430S and spheroids of these cells also expressing constitutively active RhoA or cdc42 were embedded in a collagen type I matrix. At 24 h postembedding, cells were fixed and stained with phalloidin. Amoeboid and mesenchymal morphologies (described in Figure 1) were quantified as a percentage back to the total number of cells invaded in each spheroid. Four spheroids. Scale, 20 μm. Arrows, mesenchymal cells; arrowheads, amoeboid cells. (C) The percentage of mesenchymal cells was quantified for each cell line at 24 h postembedding. (D) Each cell line was tracked over time. Cell tracks were plotted from a single point of origin. (E) Meandering index was calculated using the cell tracks from D. Meandering index is defined as the linear distance divided by the total path length; 30 cells. ****p ≤ 0.0001.
Mentions: The present data show that LKB1 CTD, and specifically its farnesylation, promotes both mesenchymal cell polarity and RhoA activity. We next sought to determine whether LKB1 promotes mesenchymal polarization through RhoA signaling. We created double stable cells expressing either empty GFP or GFP-LKB1 wild type or a C430S farnesylation mutant and the constitutively active form of either RhoA (Q63L) or cdc42 (Q61L) (Figure 4A). Cells reexpressing wild-type LKB1 and either constitutively active RhoA or cdc42 maintain a mesenchymal polarization similar to cells reexpressing wild-type LKB1 alone (Figure 4, B and C). Similarly, rescuing cdc42 activity in farnesylation-mutant cells results in a predominantly amoeboid phenotype, similar to LKB1 farnesylation-mutant cells alone (Figure 4, B and C), suggesting that LKB1 does not signal to cdc42 to promote mesenchymal polarization. However, upon rescuing RhoA activity in LKB1 farnesylation-mutant cells, cells reacquire a mesenchymal polarization during 3D invasion (Figure 4, B and C). Of importance, this result is consistent with mesenchymal polarization in cells reexpressing wild-type LKB1. Restoring either RhoA or cdc42 activity in empty GFP control cells fails to restore this mesenchymal polarization (Figure 4, B and C). Given that our previous data highlight the role of LKB1 farnesylation in promoting mesenchymal polarity, these data suggest that this occurs through LKB1 signaling to RhoA, as rescuing RhoA activity in farnesylation-compromised cells restores mesenchymal polarity.

Bottom Line: The majority of LKB1 mutations are truncations that disrupt its kinase activity and remove its C-terminal domain (CTD).Instead, cell polarity is overseen by the kinase-independent function of its CTD and more specifically its farnesylation.This occurs through a mesenchymal-amoeboid morphological switch that signals through the Rho-GTPase RhoA.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory University, Atlanta, GA 30322 Graduate Program in Cancer Biology, Emory University, Atlanta, GA 30322.

No MeSH data available.


Related in: MedlinePlus