Arp2/3 and Mena/VASP Require Profilin 1 for Actin Network Assembly at the Leading Edge

Abstract

Cells have many types of actin structures, which must assemble from a common monomer pool. Yet, it remains poorly understood how monomers are distributed to and shared between different filament networks. Simplified model systems suggest that monomers are limited and heterogeneous, which alters actin network assembly through biased polymerization and internetwork competition. However, less is known about how monomers influence complex actin structures, where different networks competing for monomers overlap and are functionally interdependent. One example is the leading edge of migrating cells, which contains filament networks generated by multiple assembly factors. The leading edge dynamically switches between the formation of different actin structures, such as lamellipodia or filopodia, by altering the balance of these assembly factors' activities. Here, we sought to determine how the monomer-binding protein profilin 1 (PFN1) controls the assembly and organization of actin in mammalian cells. Actin polymerization in PFN1 knockout cells was severely disrupted, particularly at the leading edge, where both Arp2/3 and Mena/VASP-based filament assembly was inhibited. Further studies showed that in the absence of PFN1, Arp2/3 no longer localizes to the leading edge and Mena/VASP is non-functional. Additionally, we discovered that discrete stages of internetwork competition and collaboration between Arp2/3 and Mena/VASP networks exist at different PFN1 concentrations. Low levels of PFN1 caused filopodia to form exclusively at the leading edge, while higher concentrations inhibited filopodia and favored lamellipodia and pre-filopodia bundles. These results demonstrate that dramatic changes to actin architecture can be made simply by modifying PFN1 availability.

The actin regulator profilin 1 is functionally associated with the mammalian centrosome

“…Profilin-actin serves as source of actin for microfilament polymerization and as such co-operates with actin nucleation and elongation promoting proteins (26,27,28,29,33,39,40). A possible scenario is therefore that the loss of profilin results in less of actin polymerization in the centrosomal region in analogy with what was recently reported for the cell edge (41); as a consequence, less of steric hindrance (8,42) would reduce a possible space constrain for enhanced de novo microtubule nucleation. Alternatively, because actin is a structural component of functional γTuRCs (22,23,24), centrosomal profilin may tune the availability of actin for γTuRC.…”

Delivering defined amounts of purified protein with high precision into living cells

“…Using the protein delivery assessment methods described in steps 6–8, we have shown that the amount of delivered protein is linearly proportional to the bath concentration in the electroporation reaction for FITC-labeled dextran (dextran-FITC) ( Figure 1 A), Alexa 647-labeled thymosin β4 (Tβ4-647) ( Figure 1 B), Tβ4-647 that has been co-electroporated with dextran-FITC ( Figure 1 C), and profilin 1 ( Figure 1 D) ( Skruber et al., 2020 ). Further, we have shown that proteins are functional after delivery by electroporation.…”

“…For imaging, coverslips need to be cleaned and coated with appropriate reagents to help the cells adhere to them. Below we detail coating the coverslips with laminin as in ( Skruber et al., 2020 ); however, the specific reagents (ex. fibronectin, poly-d-lysine) and incubation times/temperatures will vary depending on the cell type used.…”

“…This protocol will be useful for any researcher who is interested in protein concentration-dependent cellular phenotypes. For complete details on the use and execution of this protocol, please refer to Skruber et al. (2020) .…”

“…For complete details on the use and execution of this protocol, please refer to Skruber et al. (2020) .…”