Cell migration requires the coordination of an excitable signal transduction network involving Ras and PI3K pathways with cytoskeletal activity. We show that expressing activated Ras GTPase-family proteins in cells lacking PTEN or other mutations which increase cellular protrusiveness transforms cells into a persistently activated state. Leading- and trailing-edge markers were found exclusively at the cell perimeter and the cytosol, respectively, of the dramatically flattened cells. In addition, the lifetimes of dynamic actin puncta were increased where they overlapped with actin waves, suggesting a mechanism for the coupling between these two networks. All of these phenotypes could be reversed by inhibiting signal transduction. Strikingly, maintaining cells in this state of constant activation led to a form of cell death by catastrophic fragmentation. These findings provide insight into the feedback loops that control excitability of the signal transduction network, which drives migration.
Dictyostelium; cell migration; chemotaxis; signal transduction; waves.
Copyright © 2018 the Author(s). Published by PNAS.
Conflict of interest statement
The authors declare no conflict of interest.
Q62L expression of constitutively active RasC or Rap1 leads to dramatic cell spreading in pten cells. ( − A) Experimental scheme. Inhibitors used in this study are shown in red type. ( B) Untransfected control cells. ( C–F) Ras and Rap constructs were induced with doxycycline, and representative images were acquired at the indicated times for wild-type and constitutively active RasC expressed in ( C) AX2 cells and ( D) pten cells, and for wild-type and constitutively active Rap1 expressed in ( − E) AX2 cells and ( F) pten cells. ( − G) Isosurface renders of RasC/ pten cells (arrowhead) and RsC − Q62L/ pten cells (arrow). ( − H) Distribution of flatness coefficients of AX2 and pten cells expressing indicated constructs. ( − I and J) Inhibitions of the pancake phenotype by ( I) PP242 and ( J) LY294002.
Cytoskeletal changes in persistently activated cells. (
A– D) AX2 and pten cells expressing the indicated constructs and F-actin probe RFP-LimE − Δcoil. RasC Q62L expression changes the dynamics of F-actin waves, producing a persistent band of peripheral staining in pten cells. (Scale bars: − A– C, 5 μm; D, 4 μm.) ( E– H) Respective kymographs of cells in A–D. ( I) High-resolution images of actin probe LimE in parental pten ( − Left) and RasC Q62L/ pten ( − Right) cells. Top shows a view of the ventral surface; Bottom shows a side profile. (Scale bars: 5 μm.) ( J) Intermediate-wave F-actin wave pattern (GFP-LimE Δcoil) phenotypes induced by RasC Q62L expression in pten cells. (Scale bars: 5 μm.) Dox, doxycycline. −
Increased lifetime of F-actin puncta in persistently activated cells. (
A) Confocal section near the ventral surface of a RasC Q62L/ pten cell expressing F-actin probe-LimE − Δcoil. ( B) t-Stack of the cell in A showing lifetime of F-actin puncta. ( C) The changes in intensity over the lifetime of 10 different F-actin puncta from cells seen in Top are traced in the graphs shown Below. Blue traces on graphs correspond to boxed puncta in Top. ( D– F) Frequency distribution of puncta lifetimes. Wild-type and activated cells were treated with the indicated inhibitors for an hour before recording puncta lifetimes. Average lifetime of puncta in n = 100 different cells.
Signal transduction changes in persistently activated cells. Ras-GTP probe GFP-RBD expressed in (
A) pten cells with ( − B) associated kymograph, and in ( C) RasC Q62L /pten cells with ( − D) associated kymograph. ( E) Representative images of cells expressing TorC2 activity probe R1-Akt; arrows indicate patches of R1-Akt signal at the cell cortex. ( F) Portion of the cortex occupied by R1-Akt from experiments in E; n = 100 cells; error bars are SD. ( G– I) P-PKBR1 levels quantified from immunoblots ( G) show a marked increase in activated cells. PKB-substrate phosphorylation levels were similarly quantified in ( H) Rap1 G12V- and ( I) RasC Q62L-activated cells. n = threefold change normalized to empty vector-transformed cells.
Mutants with multiple protrusions can be converted to the pancake phenotype. (
A) Expression of RasC Q62L/KrsB − cells and RAM − mutants induces the spread, pancake phenotype. Representative images are shown of cells before (−) and 8 h after (+) the induction of RasC Q62L expression by doxycycline (Dox). CON and STEN markers expressed in RAM − pancake cells localize to a band at the periphery. ( B– E) Selected frames from movies of RAM13 − cells ( Left) before and after induction of RasC Q62L with doxycycline, expressing biosensors for F-actin probe LimE ( B and C) and PIP3 probe GFP-PH Crac ( D and E), with associated kymographs ( Right). (Kymograph vertical scale bars: 5 μm; horizontal scale bars: 30 s.)
Persistent activation of the STEN leads to cell death by sparagmosis. (
A) Persistent elevation of RasC Q62L. (Magnification: 40×.) ( B) Rap1 G12V signaling triggers fragmentation. (Magnification: 40×.) ( C) Doxycline added at time 0. Sparagmosis after 8 to 10 h, leading to the precipitous decline in survival in RasC Q62L and Rap1 G12V relative to control RasC and Rap1 expressing pten cells. ( − D) Sparagmosis in RAM − mutants expressing RasC Q62L and Rap1 G12V follows a similar time course to pten cells. − n = 3 experiments; error bars are SEM.
The STEN is required for pancake cell formation and sparagmosis. (
A) pkbR1 / − pten and ( − B) pkbA / − pten cells are unable to form pancake cells when RasC − Q62L expression is induced. Doxycycline was added at time 0. ( C) Quantification of data in A and B. ( D) Sparagmosis induction can blocked by rescuing pten cells with GFP-Pten or by dominant-negative Rap1. ( − E) TorC2 inhibitor PP242 and PI3K inhibitor LY294002, when added to cells for an hour before induction of RasC Q62L at time 0, effectively blocked sparagmosis induction, suggesting that PKB signaling and STEN activity are required for sparagmosis. For D and E, n = 3 experiments; error bars are SEM.
Model depicting the pathways leading to maximal activation of the STEN. Pairwise combination of constitutively active RasC or Rap1 activation with mutants which increase pseudopod formation leads to maximal activation of the STEN and the induction of sparagmosis, which leads to cell death. Maximal activation requires STEN activity and can be reversed or blocked by inhibiting it.
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Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.
Actin Cytoskeleton / physiology
Actin Cytoskeleton / ultrastructure
Dictyostelium / physiology*
Dictyostelium / ultrastructure
PTEN Phosphohydrolase / genetics
PTEN Phosphohydrolase / physiology
Protozoan Proteins / genetics
Protozoan Proteins / physiology*
Recombinant Proteins / metabolism
Signal Transduction / physiology*
rap1 GTP-Binding Proteins / genetics
rap1 GTP-Binding Proteins / physiology
rap1 GTP-Binding Proteins
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