Deconstructing the Late Phase of Vimentin Assembly by Total Internal Reflection Fluorescence Microscopy (TIRFM)

Quantitative imaging of intermediate filaments (IF) during the advanced phase of the assembly process is technically difficult, since the structures are several µm long and therefore they exceed the field of view of many electron (EM) or atomic force microscopy (AFM) techniques. Thereby quantitative studies become extremely laborious and time-consuming. To overcome these difficulties, we prepared fluorescently labeled vimentin for visualization by total internal reflection fluorescence microscopy (TIRFM). In order to investigate if the labeling influences the assembly properties of the protein, we first determined the association state of unlabeled vimentin mixed with increasing amounts of labeled vimentin under low ionic conditions by analytical ultracentrifugation. We found that bona fide tetrameric complexes were formed even when half of the vimentin was labeled. Moreover, we demonstrate by quantitative atomic force microscopy and electron microscopy that the morphology and the assembly properties of filaments were not affected when the fraction of labeled vimentin was below 10%. Using fast frame rates we observed the rapid deposition of fluorescently labeled IFs on glass supports by TIRFM in real time. By tracing their contours, we have calculated the persistence length of long immobilized vimentin IFs to 1 µm, a value that is identical to those determined for shorter unlabeled vimentin. These results indicate that the structural properties of the filaments were not affected significantly by the dye. Furthermore, in order to analyze the late elongation phase, we mixed long filaments containing either Alexa 488- or Alexa 647-labeled vimentin. The ‘patchy’ structure of the filaments obtained unambiguously showed the elongation of long IFs through direct end-to-end annealing of individual filaments

In Vitro Assembly Kinetics of Cytoplasmic Intermediate Filaments: A Correlative Monte Carlo Simulation Study.

Intermediate filament (IF) elongation proceeds via full-width "mini-filaments", referred to as "unit-length" filaments (ULFs), which instantaneously form by lateral association of extended coiled-coil complexes after assembly is initiated. In a comparatively much slower process, ULFs longitudinally interact end-to-end with other ULFs to form short filaments, which further anneal with ULFs and with each other to increasingly longer filaments. This assembly concept was derived from time-lapse electron and atomic force microscopy data. We previously have quantitatively verified this concept through the generation of time-dependent filament length-profiles and an analytical model that describes assembly kinetics well for about the first ten minutes. In this time frame, filaments are shorter than one persistence length, i.e. ~1 μm, and thus filaments were treated as stiff rods associating via their ends. However, when filaments grow several μm in length over hours, their flexibility becomes a significant factor for the kinetics of the longitudinal annealing process. Incorporating now additional filament length distributions that we have recorded after extended assembly times by total internal reflection fluorescence microscopy (TIRFM), we developed a Monte Carlo simulation procedure that accurately describes the underlying assembly kinetics for large time scales

MC simulations of filaments with a <i>l</i>_p of 1000 nm.

<p>MC simulations of filaments with a <i>l</i>_p of 1000 nm.</p

Observed length distributions can be well described by a simple assembly model.

<p>Length distribution profiles of vimentin measured at distinct time points are shown (grey bars): A, 10 min; B, 2 h and C, 4 h (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157451#pone.0157451.t002" target="_blank">Table 2</a>). To reduce the noise the size of the bins was set to 5 (B,C). The obtained data can be well described by our MC simulations. 10 simulations were performed at protein concentration of 17.5 g/l (for details see bottom <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157451#pone.0157451.t002" target="_blank">Table 2</a>) and mean values at the given <<i>l</i>_n> were calculated (black).</p

Assembly kinetics of vimentin.

<p>Assembly kinetics of vimentin.</p

Length distributions of vimentin intermediate filaments normalized to the persistence length.

<p>Filament length distributions were measured at three assembly times. The proportion of filaments lengths observed of the indicated lengths is shown at the specific time points.</p

MC simulated filament assembly.

<p>At a mean filament length of 5 ULFs length distribution profiles simulated at various concentrations were compared (A). The mean filament growth, <<i>l</i>_n>-1, over time is shown in (B). The volume, the protein concentration, the initial number of ULFs and the number of sweeps to reach the mean length are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157451#pone.0157451.t002" target="_blank">Table 2</a>. The black line, the grey bars and their standard errors were derived from 10 simulations. Varying the concentration results in comparable length distributions, but the time, or number of sweeps, to reach a given mean length is different.</p

Observed length distributions of cytoplasmic intermediate filaments compared with MC simulations.

<p>Mean lengths and assembly conditions are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157451#pone.0157451.t003" target="_blank">Table 3</a> (bold). 10 simulations were performed at protein concentration of 17.5 g/l (grey bars, for details see bottom <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157451#pone.0157451.t001" target="_blank">Table 1</a>). 4 simulations were performed at similar conditions, but the reaction solid angle and the bond angle was set to 25° resulting in a reduction of the persistence length to 1/3 (black line). In both cases the profile at a mean length of 7.9 ULF is shown. Interestingly the shape of the histogram is the same, but the number of sweeps to reach that distribution is decreased by a factor of 4 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157451#pone.0157451.t003" target="_blank">Table 3</a>).</p

<i>In Vitro</i> Assembly Kinetics of Cytoplasmic Intermediate Filaments: A Correlative Monte Carlo Simulation Study - Fig 2

<p><b>Comparison of IFs obtained by EM (A) AFM (B) and TIRFM (C).</b> Assembly was stopped after 10 min for EM and AFM and after 4 h for TIRFM.</p