Prostate cancer cell migration induced by myopodin isoforms is associated with formation of morphologically and biochemically distinct actin networks

Abstract

Myopodin is an actin-binding protein that promotes cancer cell migration in response to serum stimulation and is associated with invasive tumor development. To determine whether enhanced migration reflects changes in actin cytoskeleton remodeling, fluorescence confocal microscopy was used to examine the composition and morphology of filamentous actin structures in mock-transduced cells vs. stably transduced PC3 cells expressing human myopodin isoforms, and the chemokinetic response of cells was quantified using transwell assays. The same approaches were used to analyze the effects of external migration stimuli, actin polymerization inhibitors or deletion of the isoform-specific amino- and/or carboxy termini on cell migration and actin bundle formation. Results indicate that the termini of the myopodin isoforms differentially alter the formation of morphologically distinct F-actin networks that also differ in their myosin and myopodin staining patterns. Furthermore, enhanced cell migration was reduced by >50% when actin bundle formation was impaired by myopodin-truncation, low concentrations of an actin polymerization inhibitor, or in the absence of an external migration stimulus. Human myopodin isoforms are therefore potent regulators of stress fiber formation, inducing the formation of biochemically and morphologically distinct F-actin networks in the cell body whose presence directly correlates with increased cell migration.

Synaptopodin-2 induces assembly of peripheral actin bundles and immature focal adhesions to promote lamellipodia formation and prostate cancer cell migration

“…Phalloidin staining of filamentous actin (F-actin) indicated the sheet-like protrusions generated in Synpo2-expressing cells contained actin-rich rims ~1 micrometer in diameter (Figure 2A ), a characteristic feature of lamellipodia [ 30 ], and Synpo2 predominantly colocalized with the induced SFs that formed in the cell body, as recently reported [ 17 ]. These actin-rich membrane protrusions and SFs were not evident in serum-stimulated, mock-transduced cells (Figure 2A ).…”

“…The majority of studies on Synpo2 in the context of invasive cancer cell migration use PC3 prostate cancer cells and ectopic expression of the Synpo2As isoform, a 698-residue isoform also referred to as ΔN-MYO1 [ 17 ]. Using this system, it was determined that conditioned medium from NIH 3T3 cells is a potent stimulator of PC3 cell motility, and ectopic expression of Synpo2 partially inhibits (~20-40%) this migratory response to variable extents [ 11 , 13 , 16 ].…”

“…This interesting family of actin regulators affects Rho signaling pathways, actin cytoskeleton dynamics and cell motility [ 43 ], although the relationships between these different processes are still poorly understood. Recent results revealed Synpo2 induces formation of ventral SFs in the cell body, the generation of which directly correlates with enhanced PC3 cell migration [ 17 ]. We now show that Synpo2-induced SF formation in the cell body is an incidental effect of retrograde F-actin flow from the cell periphery, and that SF formation reflects the role of Synpo2 as a newly identified effector of actin bundle and lamellipodia formation at the leading cell edge.…”

“…Previous studies provide some insights into how Synpo2 could affect cancer cell migration responses. We recently determined that all five isoforms of Synpo2 induce formation of, and co-localize with, morphologically and biochemically distinct ventral SFs in the cell body of PC3 cells following serum stimulation [ 17 ]. These results are consistent with our previous demonstration that Synpo2 activates RhoA [ 16 ], a key regulator of SF formation [ 18 ].…”

“…These results are consistent with our previous demonstration that Synpo2 activates RhoA [ 16 ], a key regulator of SF formation [ 18 ]. Inhibiting Synpo2-induced SF assembly also prevents Synpo2-enhanced prostate cancer cell migration in response to serum-stimulation [ 17 ], indicating a direct correlation between SF assembly and a Synpo2 pro-migratory phenotype. In addition, Synpo2 homologues from various species enhance actin nucleation, polymerization and bundling in vitro [ 19 , 20 ], and Synpo2 has been shown to interact with focal adhesions (FAs) and FA-associated proteins [ 12 , 13 , 21 ].…”