University of Illinois Urbana-Champaign

Mechanisms and regulation of ATP-dependent, steady state actin subunit exchange: Concurrent treadmilling & dynamic instability

Duttagupta, Madhura

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Description

Title
Mechanisms and regulation of ATP-dependent, steady state actin subunit exchange: Concurrent treadmilling & dynamic instability
Author(s)
Duttagupta, Madhura
Issue Date
2025-04-25
Director of Research (if dissertation) or Advisor (if thesis)
Brieher, William M
Doctoral Committee Chair(s)
Brieher, William M
Committee Member(s)
  • Sokac, Anna M
  • Freeman, Brian
  • Zhang, Kai
Department of Study
Cell & Developmental Biology
Discipline
Cell and Developmental Biology
Degree Granting Institution
University of Illinois Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
  • Dynamic Instability in actin
  • Barbed end fluctuations
  • BEF
Abstract
Precise control of structure and function in eukaryotic cells is possible due to the highly dynamic nature of actin networks and hinges on the irreversible ATPase activity of the actin protein itself. The canonical view of actin dynamics centers around ‘treadmilling’ which is a cycle of an actin monomer undergoing polymerization, ATP hydrolysis, phosphate release, and subsequent depolymerization. However, substantial experimental evidence and theoretical modelling suggests that the treadmilling model cannot explain the ensemble of cytoskeletal properties exhibited by cells. For chapter 1 of my thesis I decided to re-investigate steady state actin dynamics at a mechanistic level. I observed that steady state actin filaments undergo ATP-dependent, barbed end length fluctuations (BEFs). I discovered that BEFs have two prominent modes: a low amplitude-high frequency mode and a high amplitude-low frequency mode. Surprisingly pointed ends remained inert regardless of the type of BEF the barbed end was undergoing. While filaments treadmill, the dominant mode of filament turnover at steady state is a form of dynamic instability in which barbed ends interconvert between growing and shrinking phases with a stable filament core. In vivo actin dynamics occur at much faster timescales than in vitro. The slow rate of depolymerization becomes the limiting factor and actin binding proteins must regulate both polymerization and depolymerization reactions such that the rate of turnover can meet physiological demands. Cofilin and profilin are two essential proteins that accelerate steady state actin turnover rates, via complicated mechanisms. Under the treadmilling framework the consensus, however, is that Cofilin enhances depolymerization while profilin accelerates nucleotide exchange on actin monomers and funnels them to the barbed ends. For chapter 2, I decided to study how profilin and cofilin accelerate the new steady state dynamics established under the BEF framework. I found that profilin and cofilin, together, amplify low amplitude BEFs and activate pointed end depolymerization. These filaments appear to be treadmilling and simultaneously exhibiting BEFs. I named this treadmilling associated fluctuations (TAFs). TAFs lead to occasional filament stalling where there is no net growth because the tip interconverts between growth and shrinkage. However continuous pointed end depolymerization leads to whole filament elimination. Polymer mass and filament numbers remain steady because cofilin-mediated nucleation balances whole filament elimination. The overall effect is a version of dynamic instability in which newly nucleated filaments show net growth before converting to net shrinkage and eventual elimination. This chapter also provokes us to view profilin and cofilin with an alternative lens where cofilin is now promoting polymerization while profilin leans on the depolymerization mechanism. Therefore, for my final chapter I investigated the role of profilin in actin depolymerization. I found that under conditions of depolymerization, profilin enhances the barbed end off rate. When cofilin is added, profilin further enhances the barbed end depolymerization rate in a synergistic manner. Additionally, I discovered that profilin also accelerates cofilin mediated severing of actin without creating new heterotypic junctions by displacing bound cofilin from the filaments. My work in this thesis provides an alternative framework for cellular actin dynamics which can parsimoniously explain many observations that could not be accounted for by treadmilling. Further the BEF framework of actin dynamics opens up new possibilities of regulation at the barbed end by allowing a choice for nucleotide selectivity which was not possible under the treadmilling framework.
Graduation Semester
2025-05
Type of Resource
Thesis
Handle URL
https://hdl.handle.net/2142/129545
Copyright and License Information
Copyright 2025 Madhura Duttagupta

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