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.

BackgroundTumor 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.MethodsFor 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.ResultsBesides 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.ConclusionWe 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.

Besides being expressed on professional antigen-presenting cells, HLA class II antigens are expressed on various tumors of non-lymphoid origin, including a subset of colorectal cancers (CRC). Information about the regulation of HLA class II antigen expression is important for a better understanding of their role in the interactions between tumor and immune cells. Whether lack of HLA class II antigen expression in tumors reflects the selective immune destruction of HLA class II antigen-expressing tumor cells is unknown. To address this question, we tested whether lack of HLA class II antigen expression in CRC was associated with immune cell infiltration. We selected microsatellite-unstable (MSI-H) CRC, because they show pronounced tumor antigen-specific immune responses and, in a subset of tumors, lack of HLA class II antigen expression due to mutations inactivating HLA class II-regulatory genes. We examined HLA class II antigen expression, mutations in regulatory genes, and CD4-positive T cell infiltration in 69 MSI-H CRC lesions. Mutations in RFX5, CIITA, and RFXAP were found in 13 (28.9%), 3 (6.7%), and 1 (2.2%) out of 45 HLA class II antigen-negative tumors. CD4-positive tumor-infiltrating lymphocyte counts were significantly higher in HLA class II antigen-negative tumors harboring mutations in HLA class II-regulatory genes (107.4 T cells per 0.25 mm(2)) compared to tumors without mutations (55.5 T cells per 0.25 mm(2), p = 0.008). Our results suggest that the outgrowth of tumor cells lacking HLA class II antigen expression due to mutations of regulatory genes is favored in an environment of dense CD4-positive T cell infiltration.