Extrinsic Fluorescent Dyes as Tools for Protein Characterization

Noncovalent, extrinsic fluorescent dyes are applied in various fields of protein analysis, e.g. to characterize folding intermediates, measure surface hydrophobicity, and detect aggregation or fibrillation. The main underlying mechanisms, which explain the fluorescence properties of many extrinsic dyes, are solvent relaxation processes and (twisted) intramolecular charge transfer reactions, which are affected by the environment and by interactions of the dyes with proteins. In recent time, the use of extrinsic fluorescent dyes such as ANS, Bis-ANS, Nile Red, Thioflavin T and others has increased, because of their versatility, sensitivity and suitability for high-throughput screening. The intention of this review is to give an overview of available extrinsic dyes, explain their spectral properties, and show illustrative examples of their various applications in protein characterization

Genomic structure and alternative splicing of murine R2B receptor protein tyrosine phosphatases (PTPκ, μ, ρ and PCP-2)

BACKGROUND: Four genes designated as PTPRK (PTPκ), PTPRL/U (PCP-2), PTPRM (PTPμ) and PTPRT (PTPρ) code for a subfamily (type R2B) of receptor protein tyrosine phosphatases (RPTPs) uniquely characterized by the presence of an N-terminal MAM domain. These transmembrane molecules have been implicated in homophilic cell adhesion. In the human, the PTPRK gene is located on chromosome 6, PTPRL/U on 1, PTPRM on 18 and PTPRT on 20. In the mouse, the four genes ptprk, ptprl, ptprm and ptprt are located in syntenic regions of chromosomes 10, 4, 17 and 2, respectively. RESULTS: The genomic organization of murine R2B RPTP genes is described. The four genes varied greatly in size ranging from ~64 kb to ~1 Mb, primarily due to proportional differences in intron lengths. Although there were also minor variations in exon length, the number of exons and the phases of exon/intron junctions were highly conserved. In situ hybridization with digoxigenin-labeled cRNA probes was used to localize each of the four R2B transcripts to specific cell types within the murine central nervous system. Phylogenetic analysis of complete sequences indicated that PTPρ and PTPμ were most closely related, followed by PTPκ. The most distant family member was PCP-2. Alignment of RPTP polypeptide sequences predicted putative alternatively spliced exons. PCR experiments revealed that five of these exons were alternatively spliced, and that each of the four phosphatases incorporated them differently. The greatest variability in genomic organization and the majority of alternatively spliced exons were observed in the juxtamembrane domain, a region critical for the regulation of signal transduction. CONCLUSIONS: Comparison of the four R2B RPTP genes revealed virtually identical principles of genomic organization, despite great disparities in gene size due to variations in intron length. Although subtle differences in exon length were also observed, it is likely that functional differences among these genes arise from the specific combinations of exons generated by alternative splicing

Rgnef (p190RhoGEF) Knockout Inhibits RhoA Activity, Focal Adhesion Establishment, and Cell Motility Downstream of Integrins

Cell migration is a highly regulated process that involves the formation and turnover of cell-matrix contact sites termed focal adhesions. Rho-family GTPases are molecular switches that regulate actin and focal adhesion dynamics in cells. Guanine nucleotide exchange factors (GEFs) activate Rho-family GTPases. Rgnef (p190RhoGEF) is a ubiquitous 190 kDa GEF implicated in the control of colon carcinoma and fibroblast cell motility.Rgnef exon 24 floxed mice (Rgnef(flox)) were created and crossed with cytomegalovirus (CMV)-driven Cre recombinase transgenic mice to inactivate Rgnef expression in all tissues during early development. Heterozygous Rgnef(WT/flox) (Cre+) crosses yielded normal Mendelian ratios at embryonic day 13.5, but Rgnef(flox/flox) (Cre+) mice numbers at 3 weeks of age were significantly less than expected. Rgnef(flox/flox) (Cre+) (Rgnef-/-) embryos and primary mouse embryo fibroblasts (MEFs) were isolated and verified to lack Rgnef protein expression. When compared to wildtype (WT) littermate MEFs, loss of Rgnef significantly inhibited haptotaxis migration, wound closure motility, focal adhesion number, and RhoA GTPase activation after fibronectin-integrin stimulation. In WT MEFs, Rgnef activation occurs within 60 minutes upon fibronectin plating of cells associated with RhoA activation. Rgnef-/- MEF phenotypes were rescued by epitope-tagged Rgnef re-expression.Rgnef-/- MEF phenotypes were due to Rgnef loss and support an essential role for Rgnef in RhoA regulation downstream of integrins in control of cell migration

Impaired healing of cutaneous wounds and colonic anastomoses in mice lacking thrombin-activatable fibrinolysis inhibitor

Plasmin and other components of the plasminogen activation system play an important role in tissue repair by regulating extracellular matrix remodeling, including fibrin degradation. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a procarboxypeptidase that, after activation, can attenuate plasmin-mediated fibrin degradation by removing the C-terminal lysine residues from fibrin, which play a role in the binding and activation of plasminogen. To test the hypothesis that TAR is an important determinant in the control of tissue repair, we investigated the effect of TAR deficiency on the healing of cutaneous wounds and colonic anastomoses. Histological examination revealed inappropriate organization of skin wound closure in the TAR knockout mice, including an altered pattern of epithelial migration. The time required to completley heal the cutaneous wounds was slightly delayed in TAFI-deficient mice. Healing of colonic anastomoses was also impaired, as reflected by decreased strength of the tissue at the site of the suture, and by bleeding complications in three of 14 animals. Together, these abnormalities resulted in increased mortality in TAFI-deficient mice after colonic anastomoses. Although our study shows that tissue repair, including re-epithelialization and scar formation, occurs in TAFI-deficient mice, TAR appears to be important for appropriate organization of the healing proces