Spontaneous phosphoinositide 3-kinase signaling dynamics drive spreading and random migration of fibroblasts

Abstract

During directed cell migration (chemotaxis), cytoskeletal dynamics are stimulated and spatially biased by phosphoinositide 3-kinase (PI3K) and other signal transduction pathways. Live-cell imaging using total internal reflection fluorescence (TIRF) microscopy revealed that, in the absence of soluble cues, 3Ј-phosphoinositides are enriched in a localized and dynamic fashion during active spreading and random migration of mouse fibroblasts on adhesive surfaces. Surprisingly, we found that PI3K activation is uncoupled from classical integrin-mediated pathways and feedback from the actin cytoskeleton. Inhibiting PI3K significantly impairs cell motility, both in the context of normal spreading and when microtubules are dissociated, which induces a dynamic protrusion phenotype as seen by TIRF in our cells. Accordingly, during random migration, 3Ј-phosphoinositides are frequently localized to regions of membrane protrusion and correlate quantitatively with the direction and persistence of cell movement. These results underscore the importance of localized PI3K signaling not only in chemotaxis but also in basal motility/migration of fibroblasts. Journal of Cell Science 314 is largely PI3K dependent. This is seen most dramatically when microtubules are depolymerized using nocodazole, which induces rapid protrusion-retraction events as seen by TIRF. In the context of random fibroblast migration, formation of branched lamellipodia and turning events were found to coincide with localized enrichment of 3Ј-phosphoinositides. Results PI3K lipid products accumulate in a dynamic fashion during fibroblast spreadingTo assess the activation of PI3K signaling and its possible relation to adhesion-based motility, we established stable expression of the 3Ј-phosphoinositide-specific Akt pleckstrin-homology domain, fused with enhanced green fluorescent protein (EGFP-AktPH) in NIH3T3 fibroblasts and monitored these cells by TIRF microscopy as they attached and spread on glass coated with fibronectin or poly-D-lysine (Fig. 1). Glass coated with bovine serum albumin, which was also present in our imaging buffer, does not promote adhesion of these cells (results not shown). After allowing the cells to spread for 30-50 minutes, a saturating dose of PDGF was added to evaluate the maximal activation of PI3K in each cell, followed by a large dose of PI3K inhibitor to evaluate the fluorescence intensity associated with EGFP-AktPH in the cytosol; the latter is used to normalize the PI3K-dependent response (Schneider and Haugh, 2004).Cells spreading on fibronectin consistently showed dynamic enrichment of 3Ј-phosphoinositides, with transient bursts of signaling that were often localized in actively protruding regions at the cell periphery and other times showed a more global pattern ( Fig. 1A; supplementary material Movie 1). These patterns of PI3K signaling are distinct from the more stable, ring-like patterns seen in response to PDGF stimulation, which we have previously explained in quantitative detail (Schneider and Haugh, 2004). Analysis of a...

Quantification and visualization of phosphoinositides by quantum dot-labeled specific binding-domain probes

“…The PI3K-driven signaling pathway induces rapid reorganization of the actin cytoskeleton, which gives rise to not only lamellipodia but also peripheral and dorsal membrane ruffl es ( 22,23 ). These transient and dynamic changes in the actin cytoskeleton are initiated by the production of consistent with previous reports ( 24,25 ) ( Fig.…”

PI3K therapy reprograms mitochondrial trafficking to fuel tumor cell invasion

“…In addition to oxidative phosphorylation, Akt/mTOR signaling was identified here as a key regulator of mitochondrial trafficking and tumor cell invasion. This is consistent with a pivotal role of PI3K in directional cell movements (34), supporting chemotaxis at the leading edge of migration (35) and Rac1 activation (36). A third signaling requirement of this pathway involved FAK activity (18), which has also been implicated in cytoskeletal dynamics (37).…”

Gradients Of Rac1 Nanoclusters Support Spatial Patterns of Rac1 Signaling

“…Polarized migrating cells exhibit opposite gradients of PIP3/PIP2 with an enrichment of PIP3 at the front and PIP2 in the body (Petrie et al, 2009;Haugh et al, 2000;Weiger et al, 2009). Because PIP3 and PIP2 may organize in segregated nanoclusters (Wang and Richards, 2012), we believe that the front of the cell presents a larger number of PIP3 nanoclusters and the body a larger number of PIP2 ones.…”