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42835-AC7
Liquid Crystalline Formation of Filamentous Actin Assembly
This PRF grant supported our study of the isotropic-nematic (I-N) liquid crystalline transition of F-actin. Over the two-year grant period, we have determined the nematic order parameter of F-actin. We have observed the first order I-N transition with samples prepared from short actin filaments, and characterized in detail the growth of co-existing domains coupled with the actin polymerization kinetics. For samples containing long F-actin, we revealed the origin of the continuous nature of the I-N transition, by measuring the single filament motion in the range of concentrations traversing the I-N transition. The results of these measurements help test predictions of recent theories developed to address physical properties of a large class of semiflexible polymers, which form entangled networks at extremely low volume fractions. Understanding such systems may prove helpful in certain industrial and biomedical applications. Below, we describe briefly each of the main findings made over the past two years.
I. The nematic order parameter of F-actin
F-actin forms a nematic liquid crystalline phase at a few mg/ml in protein concentration. We found that the alignment of actin filaments approaches a plateau value between 0.7 and 0.75 using a combination of techniques including fluorescence and polarization microscopy, single filament tracing, and small angle X-ray scattering. This value is significantly below the theoretical limit of 1.0 for complete alignment. The significant extent of misalignment remaining in the nematic phase implies interesting rheological properties, which we indeed have observed by tracing the motions of single filaments. By comparing the birefringence measurements with the orientational order parameter determined by X-ray and fluorescence imaging of probe filaments, we have determined the specific birefringence of completely aligned F-actin. This result allows for convenient determination of the nematic order parameter of F-actin in future studies solely by the birefringence measurements.
II. I-N coexistence in short F-actin samples
By preparing actin samples of over 10 mg/ml concentrations and short average length of between 1 and 2 micrometers, we recently succeeded in confirming the 1st order nature of I-N transition as expected in a rodlike system. The observed features include spontaneous occurrence of density fluctuation, domain formation and co-existence of isotropic and nematic droplets. We also noted slow approach towards equilibrium and metastability, which is suggestive why the first order features were not clearly observed in all the previous studies by several groups of researchers. In addition, we have characterized the kinetic properties of the phase separation, featuring both nucleation and growth and spinodal decomposition.
III. Anomalous filament motion coupled with the I-N transition
By recording the motion of fluorescently labeled filaments as tracers, we have measured the restricted diffusion of F-actin in their entangled network. Such motions appear nearly one dimensional and along the filament axis, as expected based on the tube model for polymer dynamics. Remarkably, we found that the diffusion constant monotonically decreases with increasing concentration, even as F-actin undergoes the I-N transition. Different properties are found when labeled fd viruses or microtubules are introduced into the F-actin samples, suggesting that the counter-intuitive finding may be due to weak interaction between actin filament as they are parallelly aligned and collide with each other driven by constant Brownian motion. Such a weak interaction is of electrostatic nature, since we found the diffusion coefficient to decrease progressively as Mg2+ concentration was increased. Our model to explain such findings is also aided by a picture of what we refer as “entangled nematic”. Specifically, the remaining extent of entanglement in a nematic alignment of order parameter 0.7 or so continues to pose a severe constraint on the motion of their constituent filaments. Consequently, the tube model and the drag to longitudinal diffusion of F-actin is nearly unaffected by the nematic order.
Our study of the I-N transition of F-actin provided us with exciting findings and led to new physical models, which help us gain insight in F-actin as a typical filamentous system formed by self-assembly. The knowledge acquired through this particular study may also shed light on similar issues such as slow dynamics and meta-stability in other polymeric systems. Future studies may apply the findings of F-actin to other polymer physics issues including phase transition, rheology, glass transition, etc.
