The Influence of Ions on the Assembly of Intermediate Filaments
5.4 Vimentin Filaments Assembled in the Presence of Hexammine-cobalt(III) Chloride
With the trivalent salt hexammine-cobalt(III) chloride the assembly of vimentin is investigated as well. Ion concentrations in a range from 0.01 mM to 0.5 mM are chosen for the SAXS experi-ments (Fig. 5.14).
The scattering profiles for vimentin assembled with hexammine-cobalt(III) chloride show that the curves for vimentin assembled with 0.2 mM and 0.5 mM have a similar behavior as the pre-cipitated samples assembled at high CaCl2 and MgCl2concentrations. For both hexammine-cobalt(III) chloride concentrations the precipitation is confirmed by visual inspection. An in-crease of the intensity at lowq-values can be observed with increasing ion concentration. The scattering curves for non-precipitated vimentin are steeper with increasing ion concentration.
The data is analyzed with the Guinier analysis and the derivatives are calculated. The results are summarized in Fig. 5.15 and Table 5.3, where precipitated vimentin filaments are either marked with a green circle in the Figure or a * in the Table. Data from precipitated vimentin is analyzed but will not be discussed, as the Guinier approximation is not valid anymore.
The radius of gyration of vimentin increases with increasing ion concentrations (Fig. 5.15a, Ta-ble 5.3). Values for the radius of gyration up to 10 nm are reached for non-precipitated vimentin.
TheI(0) values retrieved from Guinier analysis show an increase with increasing ion concentra-tions (Fig. 5.15b, Table 5.3).
Analyzing the mean steepness, the values are more negative with increasing ion concentration.
92 Chapter 5. The Influence of Ions on the Assembly of Intermediate Filaments
Figure 5.14:Scattering profiles of vimentin assembled with the trivalent ion hexammine-cobalt(III) chloride in the range of 0.01 mM to 0.5 mM. At a concentration of 0.2 mM the protein precipitated. In the scattering curves this can be seen by a change in the overall shape. For non aggregated vimentin filaments, the intensity at low q values increases with increasing ion concentration and the curves get steeper. In black the scattering curve for vimentin without any additional ions for assembly is shown.
Table 5.3:Results of the Guinier analysis of vimentin assembled withhexammine-cobalt(III) chlorideat different concentrations. The calculated Rcand I(0)values are listed. Precipitated vimentin is marked with *.
Ion concentration (mM) Rc(nm) I(0) (a.u.)
This indicates that the radius of the vimentin filament increases with increasing ion concentra-tion (Fig. 5.15c). Calculating the second derivative (curvature), an increase of the curvature is observed for vimentin assembled with increasing ion concentrations for non-precipitated vi-mentin filaments (Fig. 5.15d). This shows as well, that the radius of the filament increases with increasing ion concentration. Thus, the results gained from calculating the derivatives are in agreement with the Guinier analysis.
Applying the micelle model from Pedersen with the additional term to include tetramers to the data, yields information about the radiusRof the assembled filament and the radius of gyration
5.4. Vimentin Filaments Assembled in the Presence of Hexammine-cobalt(III) Chloride 93
Figure 5.15:Analysis of the scattering profiles of vimentin assembled with the trivalent ion hexammine-cobalt(III) chloride. (a) From Guinier analysis, the Rc values are calculated. With increasing ion concentration the radius increases. (b) The extracted I(0)values increase for increasing ion concentrations. (c) For increasing ion concentrations, values of the mean steepness decrease for concentrations up to 0.2 mM.
(d) The curvature increases up to a concentration of 0.1 mM hexammine-cobalt(III) chloride. Data from precipitated vimentin is marked with green circles.
of the tailsRg (Fig. 5.16).
An overall increase of the radius of the filaments can be observed with increasing ion concen-tration. Data from the radius of gyration analysis follows the same overall increasing trend, however vimentin filaments assembled at low ion concentrations (up to 0.05 mM hexammine-cobalt(III) chloride),Rg do not show a steady increase. For the amount of tetramers in the so-lution, first an increase from the first to the second ion concentration is observed, which is unexpected. After this increase in tetrameric contribution, the amount of tetramers in the sam-ple decreases with increasing ion concentration and reduces to zero at an ion concentration of
94 Chapter 5. The Influence of Ions on the Assembly of Intermediate Filaments
Figure 5.16:Analysis of vimentin assembled with hexammine-cobalt(III) chloride using the model. (a) Radius of the vimentin filament at different salt concentrations. The retrieved radius of the vimentin filaments in-crease with increasing ion concentration. (b) Calculated radii of gyration for the tails. An overall inin-crease of the radius of gyration of the tails is found with increasing ion concentration. Data from precipitated vimentin is marked with green circles.
0.08 mM (Fig. 5.17).
0 0.1concentra�on (mM)0.2 0.3 0.4 0.5
frac�on of tetramers
Figure 5.17:Fraction of tetramers in the measured solution for vimentin protein assembled with hexammine-cobalt(III) chloride. The amount of tetramers increases between ion concentrations of 0.01 to 0.02 mM.
After that, the amount of tetramers in the system decreases again until the filaments precipitate.
The amount of tetramers first increases and then decreases again. When comparing the values retrieved from Guinier analysis and the model-based analysis, slightly smaller but still similar values for the radius are found.
Again, fluorescence microscopy images are taken for vimentin assembled with 0.05 mM
5.5. Vimentin Filaments Assembled in the Presence of Spermine 95 hexammine-cobalt(III) chloride (Fig. 5.18). Assembling vimentin filaments with the trivalent ion under investigation, networks are formed like in the cases of divalent ions. Compared to networks formed with divalent ions however, the assembled vimentin networks found with hexammine-cobalt(III) chloride, are smaller in size.
25 µm
0.05 mM Hexammine-cobalt(III) chloride
Figure 5.18:Fluorescence microscopy image of vimentin assembled with 0.05 mM hexammine-cobalt(III) chloride.
As with the divalent ions, the filaments form networks, however the networks here seem smaller in size than when vimentin is assembled in the presence of the divalent ions.