Nucleation Promoting Factors (NPFs) (e.g. WASP, Scar/WAVE) modulate actin filament nucleation by bringing together actin monomers and pre-existing actin filaments, for example, during filopodial initiation where they recruit the Arp2/3 complex. NPFs compete with profilin for binding to free actin (which inhibits actin nucleotide exchange) ; these combined functions promote actin-filament assembly at the barbed end.
Mammalian NPFs are broadly grouped into 2 classes:Class I proteins are classified into five groups:
1) Wiskott-Aldrich Syndrome protein (WASP) and neuronal-enriched homologue of WASP (N-WASP)
2) WASP family Verprolin-homologous (WAVE) proteins (aka Suppressor of cAMP receptor [Scar])
3) WASP and Scar homologue (WASH)
4) WASP homologue associated with actin, membranes and microtubules (WHAMM)
5) junction-mediating regulatory protein (JMY)
Class II includes proteins such as cortactin
The conserved verpolin-cofilin-homology and acidic-rich (VCA) domain at the carboxy terminus of WASP and Scar family members binds directly to the Arp2/3 complex to increase its nucleation activity . WASp also associates with other signaling components (e.g. haemopoietic cell kinase) and formins to modulate actin polymerization for cell polarization and chemotaxis in neutrophils . WASp and formins also cooperate to control the balance between lamellipodial protrusion activity in epithelial cells . The WASP family of NPFs are normally auto-inhibited due to protein interactions which prevent the NPF from associating with actin and the Arp2/3 complex; they are activated by *Rho GTPases and PIP2  (reviewed in ). In contrast, the Scar/WAVE proteins have constitutive activity . NPF accessory proteins also modulate the activity of NPFs.
Examples of NPF accessory proteins include Verprolin (yeast), which modulates the activity of WASp with type I myosins, to promote actin assembly by Arp2/3 complex . WASp-interacting proteins (WIPs) will also regulate the WASp activity. For example WIP  not only inhibits N-WASP, but also promotes nucleation and activation of the [Arp2/3 complex through the coordinated binding of actin and another NPF, cortactin . SPIN90/WISH (SH3 protein interacting with Nck, 90 kDa/WASP-interacting SH3 protein) ) on the other hand increases actin assembly in dendritic filopodia/spines independently of N-WASP through its association with the neuron-specific scaffolding protein, PSD-95. Surprisingly, indirect evidence shows that WIPs are required for WASp function .
In another example, the WAVE complex will inhibit Scar/WAVE proteins from activating the Arp2/3 complex. The NPF activity of Scar/WAVE is restored during nucleation when Rac-GTP causes the dissociation of the WAVE complex from WAVE1 . Similarly, IRSp53 has been implicated in both lamellipodia and filopodia formation/protrusion by augmenting the Rac-GTP-induced activation of WAVE NPF activity .
Proteins of the Ena/VASP family contribute to cell movement, axon guidance, neural tube closure and shape change in vertebrate cells by modulating actin filament organization and dynamics; these effects are achieved in part by regulating the morphology and behavior of actin-based structures such as lamellipodia and filopodia (reviewed in ). Ena/VASP proteins also modulate actin dynamics at sites of cell-ECM and cell-cell interactions and they are concentrated to the proximal portion of phosphotyrosine-rich domains at the ends of F-actin stress fibers .
Ena/VASP proteins promote actin filament elongation by tethering actin filaments to sites of active actin assembly . Ena/VASP proteins recruit actin nucleation and initiation factors (e.g. Arp2/3 complex, formins) and promote F-actin assembly through profilin-binding (reviewed in ). The rate of Ena/VASP assisted actin filament elongation is determined by the recruitment of G-actin. This will occur via a G-actin binding site (GAB) that lies within the EVH2 domain and shares close sequence homology to WASP homology 2 motifs . Ena/VASP proteins are also thought to accumulate at the plasma membrane where they alter actin polymerization by antagonizing the barbed (+) end capping proteins, thereby enabling the incorporation of actin into longer filaments ; however, controversy over their exact mechanism still exists (reviewed in ). In addition, Ena/VASP may promote actin assembly by an unknown mechanism that is independent of initiation factors, however, this has not been demonstrated in intact cells .
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