Two Functionally Distinct Sources of Actin Monomers Supply the Leading Edge of Lamellipodia

Abstract

Summary Lamellipodia, the sheet-like protrusions of motile cells, consist of networks of actin filaments (F-actin) regulated by the ordered assembly from and disassembly into actin monomers (G-actin). Traditionally, G-actin is thought to exist as a homogeneous pool. Here, we show that there are two functionally and molecularly distinct sources of G-actin that supply lamellipodial actin networks. G-actin originating from the cytosolic pool requires the monomer binding protein thymosin β4 (Tβ4) for optimal leading edge localization, is targeted to formins, and is responsible for creating an elevated G/F-actin ratio that promotes membrane protrusion. The second source of G-actin comes from recycled lamellipodia F-actin. Recycling occurs independently of Tβ4 and appears to regulate lamellipodia homeostasis. Tβ4-bound G-actin specifically localizes to the leading edge because it doesn’t interact with Arp2/3-mediated polymerization sites found throughout the lamellipodia. These findings demonstrate that actin networks can be constructed from multiple sources of monomers with discrete spatiotemporal functions.

Actin Turnover in Lamellipodial Fragments

“…S1 Biii). Measurements of the actin turnover rates in the lamellipodium support the nucleation-release model in some cell types [12,13,14,15], and the network treadmilling in other cell types [16] (see STAR Methods).…”

Coordinating Neuronal Actin–Microtubule Dynamics

“…Additionally, actinmonomer-binding or 'sequestering' proteins, including profilin and b-thymosin, restrict cellular G-actin availability to minimise spontaneous actin nucleation or polymerisation events [2,4]. However, both profilin and b-thymosin also positively support growth cone actin dynamics, by helping to localise G-actin to the dynamic, protruding edge [16,65]. Furthermore, profilin 'refreshes' the G-actin pool, mediating the conversion of ADP-actin to polymerisation-competent ATP-actin and also interacts with numerous other actin regulators, connecting these regulators with actin monomers [2,4,58].…”

“…F-actin disassembly is mediated by a collection of actin-binding proteins that can facilitate actinfilament depolymerisation or severing, such as those belonging to the Mical and actin-depolymerising factor (ADF)/cofilin families [78,79]. The actin monomers subsequently generated can then be translocated, either by diffusion or active transport, towards the cell edge and re-incorporated into polymerising filaments, thus completing the actin treadmill [16,65,76]. Polymerising F-actin filaments at the edge of the growth cone, which can be modelled as elastic Brownian ratchets, produce a protrusive force upon the cell membrane [80].…”

Competition and collaboration between different actin assembly pathways allows for homeostatic control of the actin cytoskeleton

“…Indeed, thymosin-b4-bound actin has been shown to contribute differentially to the lamellipodial actin network through formins, revealing an additional axis of competition for G-actin. 44 Furthermore, certain isoforms of the F-actin binding protein tropomyosin increase F-actin levels in adipose cells, indicating that the F-actin to G-actin ratio can potentially be modulated by F-actin binding proteins as well as actin assembly factors. 45 However, this effect may depend upon the ability of different tropomyosin isoforms to bind preferentially to F-actin generated by distinct formin isoforms.…”

“…In fact, this 'recycling' has recently been directly observed. 44 In addition to disassembly, F-actin bundling (via fascin, filamin, a-actinin, etc. ), myosin contractility and actin monomer sequestration (via thymosin-b4) can all be expected to affect G-actin levels and F-actin production.…”