Cytoskeleton and Cell Motility

• V.6.iv Subproject 4 - Experiments on the Actin Cytoskeleton
• V.6.iv.a Introduction
• V.6.iv.b Background and Significance
• V.6.iv.c Aims
• V.6.iv.d Biochemical Assembly of F-actin: Thermodynamics
  • V.6.iv.d.1 Introduction
  • V.6.iv.d.2 Preliminary Results
  • V.6.iv.d.3 Methodology
  • V.6.iv.d.4 Significance
• V.6.iv.e Construction of a Composite Actin Network to Mimic Cytoskeletal Mechanics
  • V.6.iv.e.1 Introduction
  • V.6.iv.e.2 Preliminary Results
  • V.6.iv.e.3 Hypothesis
  • V.6.iv.e.4 Methodology
  • V.6.iv.e.5 Model
  • V.6.iv.e.6 Significance

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We will first attempt to confirm this surprising preliminary result using such as sedimentation, light scattering and fluorescence measurements, as well as differential scanning calorimetry (DSC). If we confirm our finding, we will propose a new model to explain actin polymerization by explicitly incorporating the role of hydration, or water activity. Given the unique structure of water and its physical properties such as strong hydrogen bonding and high polarizability, the hydrophobicity of a protein solution may change sharply within a relatively narrow temperature range, thus causing a non-monotonic change of association constant in protein assembly. We will further examine the influence of temperature on actin polymerization in the presence of regulatory proteins β-thymosin and cofilin/ADF. We will also explore if different actin isoforms have evolved to have different sensitivity to temperature.

The temperature dependence of actin assembly has both thermodynamic and cell biological consequences. The significant deviation from the van't Hoff law suggests the anomalous role of hydration in actin assembly. Successful analysis of actin assembly may help explain similar protein assemblies such as formation of microtubules, intermediate filaments, and more specialized polymers, such as sickle cell hemoglobin polymers triggered by hypoxia, bundles of major sperm protein (MSP), and protein aggregates such as those of beta peptide and tau, which lead to various forms of Alzheimer disease. Understanding the thermodynamics of this rich variety of protein assemblies represents a valuable area of study in its own right.

The cell biological consequences have become increasingly evident with the advent of modern molecular genetics. Genetic manipulation to up or down regulate expression levels of certain proteins via raising or lowering temperature is now routine, but we do not understand the relation between temperature and activity. A better understanding of such temperature manipulations may well depend on determining the temperature dependence of some key protein assembly like that of the cytoskeletal polymers.