Dimitrios Vavylonis

Polymerization kinetics of ADP- and ADP-Pi-actin determined by fluorescence microscopy.

We used fluorescence microscopy to determine how polymerization of Mg-ADP-actin depends on the concentration of phosphate. From the dependence of the elongation rate on the actin concentration and direct observations of depolymerizing filaments, we measured the polymerization rate constants of ADP-actin and ADP-Pi-actin. Saturating phosphate reduces the critical concentration for polymerization of Mg-ADP-actin from 1.8 to 0.06 μM almost entirely by reducing the dissociation rate constants at both ends. Saturating phosphate increases the barbed end association rate constant of Mg-ADP-actin 15%, but this value is still threefold less than that of ATP-actin. Thus, ATP hydrolysis without phosphate dissociation must change the conformation of polymerized actin. Analysis of depolymerization experiments in the presence of phosphate suggests that phosphate dissociation near the terminal subunits is much faster than in the interior. Remarkably, 10 times more phosphate is required to slow the depolymerization of the pointed end than the barbed end, suggesting a weak affinity of phosphate near the pointed end. Our observations of single actin filaments provide clues about the origins of the difference in the critical concentration at the two ends of actin filaments in the presence of ATP.

Segmentation and tracking of cytoskeletal filaments using open active contours

We use open active contours to quantify cytoskeletal structures imaged by fluorescence microscopy in two and three dimensions. We developed an interactive software tool for segmentation, tracking, and visualization of individual fibers. Open active contours are parametric curves that deform to minimize the sum of an external energy derived from the image and an internal bending and stretching energy. The external energy generates (i) forces that attract the contour toward the central bright line of a filament in the image, and (ii) forces that stretch the active contour toward the ends of bright ridges. Images of simulated semiflexible polymers with known bending and torsional rigidity are analyzed to validate the method. We apply our methods to quantify the conformations and dynamics of actin in two examples: actin filaments imaged by TIRF microscopy in vitro, and actin cables in fission yeast imaged by spinning disk confocal microscopy.

Oscillatory Dynamics of Cdc42 GTPase in the Control of Polarized Growth

Pole to Pole How do fission yeast cells decide when to grow at a single end (or pole) of the cell or whether to grow in a multipolar manner? Das et al. (p. 239, published online 17 May) found that accumulation of the active form of the small guanine nucleotide–binding protein Cdc42 at the growing tip of the cell oscillated with a period of a few minutes. In cells growing at one pole, the oscillations were primarily present at that pole and during bipolar growth symmetrical anticorrelated oscillations were observed. Dynamic competition for Cdc42 between multiple growth zones could represent a flexible mechanism to modulate cell growth asymmetry. The regulation of a yeast cell-growth enzyme is dynamic rather than on-off. Cells promote polarized growth by activation of Rho-family protein Cdc42 at the cell membrane. We combined experiments and modeling to study bipolar growth initiation in fission yeast. Concentrations of a fluorescent marker for active Cdc42, Cdc42 protein, Cdc42-activator Scd1, and scaffold protein Scd2 exhibited anticorrelated fluctuations and oscillations with a 5-minute average period at polarized cell tips. These dynamics indicate competition for active Cdc42 or its regulators and the presence of positive and delayed negative feedbacks. Cdc42 oscillations and spatial distribution were sensitive to the amounts of Cdc42-activator Gef1 and to the activity of Cdc42-dependent kinase Pak1, a negative regulator. Feedbacks regulating Cdc42 oscillations and spatial self-organization appear to provide a flexible mechanism for fission yeast cells to explore polarization states and to control their morphology.

Actin polymerization kinetics, cap structure, and fluctuations

Polymerization of actin proteins into dynamic structures is essential to eukaryotic cell life, motivating many in vitro experiments measuring polymerization kinetics of individual filaments. Here, we model these kinetics, accounting for all relevant steps revealed by experiment: polymerization, depolymerization, random ATP hydrolysis, and release of phosphate (P(i)). We relate filament growth rates to the dynamics of ATP-actin and ADP-P(i)-actin caps that develop at filament ends. At the critical concentration of the barbed end, c(crit), we find a short ATP cap and a long fluctuation-stabilized ADP-P(i) cap. We show that growth rates and the critical concentration at the barbed end are intimately related to cap structure and dynamics. Fluctuations in filament lengths are described by the length diffusion coefficient, D. Recently Fujiwara et al. [Fujiwara, I., Takahashi, S., Takaduma, H., Funatsu, T. & Ishiwata, S. (2002) Nat. Cell Biol. 4, 666-673] and Kuhn and Pollard [Kuhn, J. & Pollard, T. D. (2005) Biophys. J. 88, 1387-1402] observed large length fluctuations slightly above c(crit), provoking speculation that growth may proceed by oligomeric rather than monomeric on-off events. For the single-monomer growth process, we find that D exhibits a pronounced peak below c(crit), due to filaments alternating between capped and uncapped states, a mild version of the dynamic instability of microtubules. Fluctuations just above c(crit) are enhanced but much smaller than those reported experimentally. Future measurements of D as a function of concentration can help identify the origin of the observed fluctuations.

Non-equilibrium in adsorbed polymer layers

High molecular weight polymer solutions have a powerful tendency to deposit adsorbed layers when exposed to even mildly attractive surfaces. The equilibrium properties of these dense interfacial layers have been extensively studied theoretically. A large body of experimental evidence, however, indicates that non-equilibrium effects are dominant whenever monomer–surface sticking energies are somewhat larger than kT, a common case. Polymer relaxation kinetics within the layer are then severely retarded, leading to non-equilibrium layers whose structure and dynamics depend on adsorption kinetics and layer ageing. Here we review experimental and theoretical work exploring these non-equilibrium effects, with emphasis on recent developments. The discussion addresses the structure and dynamics in non-equilibrium polymer layers adsorbed from dilute polymer solutions and from polymer melts and more concentrated solutions. Two distinct classes of behaviour arise, depending on whether physisorption or chemisorption is involved. A given adsorbed chain belonging to the layer has a certain fraction of its monomers bound to the surface, f, and the remainder belonging to loops making bulk excursions. A natural classification scheme for layers adsorbed from solution is the distribution of single-chain f values, P(f), which may hold the key to quantifying the degree of irreversibility in adsorbed polymer layers. Here we calculate P(f) for equilibrium layers; we find its form is very different to the theoretical P(f) for non-equilibrium layers which are predicted to have infinitely many statistical classes of chain. Experimental measurements of P(f) are compared to these theoretical predictions.

α-Actinin and fimbrin cooperate with myosin II to organize actomyosin bundles during contractile-ring assembly

During cytokinesis in Schizosaccharomyces pombe, the transient connections between nodes allow them to condense into the contractile ring. We find that α-actinin and fimbrin, two actin cross-linking proteins, are critical for node condensation as they stabilize transient linear actomyosin structures and thus modulate the morphology of the actomyosin network.

Irreversible adsorption from dilute polymer solutions

Abstract.We study irreversible polymer adsorption from dilute solutions theoretically. Universal features of the resultant non-equilibrium layers are predicted. Two broad cases are considered, distinguished by the magnitude of the local monomer-surface sticking rate Q: chemisorption (very small Q) and physisorption (large Q). Early stages of layer formation entail single-chain adsorption. While single-chain physisorption times

Spontaneous Cdc42 Polarization Independent of GDI-Mediated Extraction and Actin-Based Trafficking

\tau_{\rm ads}

Irreversibility and polymer adsorption

are typically micro- to milli-seconds, for chemisorbing chains of N units we find experimentally accessible times

Automated actin filament segmentation, tracking and tip elongation measurements based on open active contour models

\tau_{\rm ads} = Q^{-1} N^{3/5}