Nucleation and Stability of Arp2/3-Generated Actin Filaments

Core Concepts

The author explores how VCA domains accelerate the nucleation of linear filaments by SPIN90-Arp2/3 while also destabilizing existing branches. The study reveals quantitative differences in the responses of linear and branched Arp2/3-generated filaments to regulatory proteins.

Abstract

The content delves into the activation mechanisms of the Arp2/3 complex by VCA and SPIN90, highlighting how different proteins impact the stability and nucleation of actin filaments. Key findings include the destabilization of branches by VCA domains, the stabilization effect of cortactin, and the impact of mechanical stress on debranching. The study sheds light on how regulatory factors influence the dynamics and turnover of actin filament networks.

Stats

Solid lines are exponential fits, yielding nucleation rates knuc=(1.06±0.03)×10−3 s-1 without VCA. Nucleation rates with VCA from N-WASP were (3.23±0.08)×10−3 s-1. Branch density was 0.95, 0.43, and 0.27 branches/µm with VCA domains from N-WASP, WASP, and WASH. GMF accelerated detachment with an apparent KD=25±11 nM. Cortactin stabilized SPIN90-Arp2/3 at pointed ends with 10 nM preventing filament dissociation over 1000 seconds.

Quotes

"VCA domains accelerate the nucleation of filaments by SPIN90-Arp2/3." "GMF accelerates detachment of filaments from SPIN90-Arp2/3." "Cortactin stabilizes SPIN90-Arp2/3 at filament pointed ends."

Key Insights Distilled From

Nucleation and stability of branched versus linear Arp2/3-generated actin filaments

by Cao,L., Ghas... at www.biorxiv.org 05-06-2022

https://www.biorxiv.org/content/10.1101/2022.05.06.490861v1

Deeper Inquiries

How do conformational differences between activated Arp2/3 complexes in branching versus linear filament generation affect their responses to regulatory proteins?

The conformational variances between activated Arp2/3 complexes in branching and linear filament generation play a crucial role in determining their responses to regulatory proteins. These differences impact how proteins like VCA, GMF, and cortactin interact with the Arp2/3 complex and influence its stability and turnover. For instance, the cryo-EM structures reveal that small changes in inter-subunit contacts within the Arp2/3 complex occur depending on whether it is activated for branching or linear filament formation. These structural distinctions likely lead to altered binding affinities and interactions with regulatory factors. In the context of this study, it was observed that VCA domains from different NPFs destabilize both branches and SPIN90-Arp2/3-nucleated filaments but exhibit quantitative variations in their effectiveness. This suggests that the specific conformations adopted by Arp2/3 complexes under different activation mechanisms determine how they respond to regulatory proteins. The ability of these complexes to bind G-actin, hydrolyze ATP, or withstand mechanical stress may be influenced by these conformational disparities.

What implications do the findings have for understanding actin network dynamics in cellular processes like endocytosis?

The findings presented shed light on how actin network dynamics are regulated during cellular processes such as endocytosis. Understanding how different activation mechanisms of the Arp2/3 complex (branching via NPFs like WASP/WAVE vs. linear nucleation via SPIN90) are controlled independently provides insights into the intricate regulation of actin cytoskeleton remodeling. In endocytosis, where dynamic actin networks play a critical role in membrane invagination and vesicle formation, the balance between branched and linear filaments is essential for proper function. The ability of regulatory proteins like cortactin, GMF, and VCA to modulate stability at branch junctions or pointed ends influences network architecture during processes like vesicle internalization. Moreover, considering factors like aging-induced debranching effects on branches but not on SPIN90-nucleated filaments highlights distinct behaviors based on activation mechanisms. This knowledge can inform researchers about potential targets for manipulating actin dynamics during endocytic events for therapeutic interventions or further mechanistic studies.

How might future research explore additional factors influencing stability and turnover in actin filament networks beyond those studied in this content?

Future research could delve deeper into exploring other factors that influence stability and turnover within actin filament networks beyond those investigated here: Post-translational modifications: Investigating how phosphorylation or acetylation of key components involved in regulating actin dynamics impact network stability. Actors at membrane interfaces: Exploring interactions between membrane-bound regulators (like BAR domain-containing proteins) with cytoskeletal elements during processes such as cell migration or organelle transport. Cross-talk with microtubules: Understanding how crosstalk between microtubule-associated proteins (MAPs) influences cytoskeletal organization through coordinated actions on both microtubules and actins. Role of molecular motors: Studying motor protein involvement (myosins/dyneins/kinesins) in mediating force generation within dynamic actomyosin assemblies impacting overall network integrity. By investigating these additional facets along with known regulators identified so far, researchers can gain a comprehensive understanding of the multifaceted control mechanisms governing actomyosin dynamics essential for various cellular functions across diverse biological contexts.