A novel mode of capping protein-regulation by twinfilin.

Adam B Johnston,Britney Johnson,Denise M Hilton,Patrick Mcconnell,Meghan T Harris,Avital Simone,Bruce L Goode,Gaya K Amarasinghe,John A Cooper

doi:10.7554/elife.41313

Adam B Johnston, Britney Johnson + Show 7 more

Open Access

https://doi.org/10.7554/elife.41313

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Journal: eLife	Publication Date: Oct 23, 2018
Citations: 41	License type: CC BY 4.0

Affiliation: Brandeis University, Washington University in St. Louis

Abstract

Cellular actin assembly is controlled at the barbed ends of actin filaments, where capping protein (CP) limits polymerization. Twinfilin is a conserved in vivo binding partner of CP, yet the significance of this interaction has remained a mystery. Here, we discover that the C-terminal tail of Twinfilin harbors a CP-interacting (CPI) motif, identifying it as a novel CPI-motif protein. Twinfilin and the CPI-motif protein CARMIL have overlapping binding sites on CP. Further, Twinfilin binds competitively with CARMIL to CP, protecting CP from barbed-end displacement by CARMIL. Twinfilin also accelerates dissociation of the CP inhibitor V-1, restoring CP to an active capping state. Knockdowns of Twinfilin and CP each cause similar defects in cell morphology, and elevated Twinfilin expression rescues defects caused by CARMIL hyperactivity. Together, these observations define Twinfilin as the first 'pro-capping' ligand of CP and lead us to propose important revisions to our understanding of the CP regulatory cycle.

Highlights

Of cellular actin structures with distinct architectural and dynamic properties requires the convergence and coordination of numerous actin assembly, stabilization, and disassembly mechanisms
An alignment of the three mouse and three human Twinfilin isoforms, along with the single Twinfilin genes expressed in S. cerevisiae and D. melanogaster (Figure 1A), revealed a region that includes two of the basic residues mutated in the yeast twf1-11 mutant
We discovered that Twinfilin binds to Capping Protein (CP) using an orphan CPI-like sequence in its C-terminal tail region, and through this interaction protects CP from inhibition and/or barbed end displacement by CARMIL and V-1

Summary

Introduction

Of cellular actin structures with distinct architectural and dynamic properties requires the convergence and coordination of numerous actin assembly, stabilization, and disassembly mechanisms. One enigmatic example is the interaction of Twinfilin with Capping Protein (CP) These two conserved proteins directly interact with high-affinity, and yet have seemingly opposite effects on the barbed ends of actin filaments. Cell Biology eLife digest Plant and animal cells are supported by skeleton-like structures that can grow and shrink beneath the cell membrane, pushing and pulling on the edges of the cell This scaffolding network – known as the cytoskeleton – contains long strands, or filaments, made from many identical copies of a protein called actin. Despite the high affinity of the Twinfilin-CP interaction (Kd ~10 nM for the yeast homologs [Poukkula et al, 2011]), studies have revealed no significant effects of Twinfilin on the barbed end capping activity of CP in vitro, and reciprocally, no obvious effect of CP on Twinfilin interactions with ADP-actin monomers (Falck et al, 2004). These and other data lead us to propose important revisions to current models for the CP regulatory cycle

Results

Discussion

Materials and methods