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

Analyzing the regulation of metabolic pathways in human breast cancer

<p>Abstract</p> <p>Background</p> <p>Tumor therapy mainly attacks the metabolism to interfere the tumor's anabolism and signaling of proliferative second messengers. However, the metabolic demands of different cancers are very heterogeneous and depend on their origin of tissue, age, gender and other clinical parameters. We investigated tumor specific regulation in the metabolism of breast cancer.</p> <p>Methods</p> <p>For this, we mapped gene expression data from microarrays onto the corresponding enzymes and their metabolic reaction network. We used Haar Wavelet transforms on optimally arranged grid representations of metabolic pathways as a pattern recognition method to detect orchestrated regulation of neighboring enzymes in the network. Significant combined expression patterns were used to select metabolic pathways showing shifted regulation of the aggressive tumors.</p> <p>Results</p> <p>Besides up-regulation for energy production and nucleotide anabolism, we found an interesting cellular switch in the interplay of biosynthesis of steroids and bile acids. The biosynthesis of steroids was up-regulated for estrogen synthesis which is needed for proliferative signaling in breast cancer. In turn, the decomposition of steroid precursors was blocked by down-regulation of the bile acid pathway.</p> <p>Conclusion</p> <p>We applied an intelligent pattern recognition method for analyzing the regulation of metabolism and elucidated substantial regulation of human breast cancer at the interplay of cholesterol biosynthesis and bile acid metabolism pointing to specific breast cancer treatment.</p

Width measurements of (A) unlabeled vimentin and (B) vimentin with 10% of Alexa 488 labeled subunits.

<p>Grey bars: filaments at 10 s of assembly; black bars: filaments at 1 h of assembly (339 to 1656 data points were collected per graph). Filaments are smooth and homogenous as shown by the EM images (bar = 200 nm).</p

Absorption spectra are used to accurately determine the labeling efficiency of Alexa 488 labeled vimentin.

<p>Peaks are evaluated at A_280nm for vimentin and at A_495nm for the Alexa 488 concentration. To separate the influence of Alexa 488 within the vimentin peak, spectra of pure dye (light grey) are subtracted from the labeled protein spectra (dark grey) to obtain normalized vimentin spectra (black).</p

Plot of the mean-square end-to-end distance as a function of the contour length.

<p>Nearly identical shapes of the curves indicate that filaments assembled for 2 hours (black, n = 168, 2–4 µm length) and 4 hours (grey, n = 91, 4–15 µm length) have comparable mechanical properties. The data were derived from 10% labeled vimentin equilibrated on glass and visualized with TIRFM.</p

Vimentin can be efficiently fluorescently labeled and purified.

<p>(A) Labeling of vimentin at 0.25 g/l (grey) and 1.0 g/l (black) with different Alexa 488 concentrations (2 h incubation at RT). Relative labeling efficiency was determined by applying the samples to a reducing SDS-PAGE and quantifying the intensity of the vimentin band. (B) Reducing SDS-PAGE of relevant fractions indicating the separation of labeled protein from free dye by size exclusion chromatography. (B 1) Vimentin before chromatographic separation; (B 2) Pool of major vimentin fractions; (B 3) Fraction between protein and dye; (B 4) Fraction of free dye. (C) Native PAGE (C 1) shows that free dye band of the major vimentin fraction (B 2) is induced by sample preparation for reducing SDS-PAGE; (C 2) free dye. Protein per lane: (B 2) 1.7 µg and (C 1) 2 µg.</p

Longitudinal assembly of IFs by end-to-end fusion of filaments.

<p>(A) Assembly starter units labeled with two different dyes, assembled separately for 30 min, mixed and further assembled for 2 days. (B) Striped filaments were subsequently observed with TIRFM (bar 3 µm).</p

Length distribution of labeled and unlabeled vimentin after defined time points visualized by AFM.

<p>After 10 min of assembly vimentin with (A) 0% and (B) 10% of Alexa 488 labeled subunits show comparable assembly kinetics (C); grey: unlabeled (n = 1534); red: 10% label (n = 1645). After an assembly of 1 day, vimentin with (D) 0% and (E) 10% of label show long filaments, in contrast to (F) 50% of label where assembly is clearly affected (Image sizes 10×10 µm).</p