Changes In Protein Localization Research Articles

A Cell-permeable, Activity-based Probe for Protein and Lipid Kinases

Protein and lipid kinases are two important classes of biomedically relevant enzymes. The expression and activity of many kinases are known to be dysregulated in a variety of diseases, and proteomic tools that can assess the presence and activity of these enzymes are likely to be useful for their evaluation. Because many of the mechanisms by which protein kinases can become unregulated involve post-translational modifications or changes in protein localization, they can only be detected by examining protein activity, sometimes within the context of the living cell. Wortmannin is a steroid-derived fungal metabolite that covalently inhibits both protein and lipid kinases. Here we describe the synthesis of three wortmannin derivatives, biotin-wortmannin, BODIPY-wortmannin, and tetramethylrhodamine-wortmannin. We demonstrate that these reagents exhibit reactivity similarly as wortmannin and react with members of the phosphatidylinositol 3-kinase and PI3-kinase related kinase families in cellular lysates. Moreover, in some cases these reagents can differentiate between the active and inactive forms of the enzyme, indicating that they are activity-based probes. The reagents also exhibit complementary properties. The biotin-wortmannin reagent is effective in the isolation of labeled proteins; all three can be used for protein labeling, and BODIPY-wortmannin is cell-permeable and can be used to label proteins within cells.

Localization of type IV collagen alpha chains in tooth germ development

The morphogenesis and morphological development of molar are regulated by the interaction between odontogenic epithelium and mesenchymal tissues. And Type IV collagen molecules, the major component of the dental basement membrane, are composed of three alpha chains. Using indirect immunofluorescence on cryosections, we characterized the changes in protein localization of alpha 1 to 6 chains of type IV collagen during molar development in mice molar and human tooth germ.ICR mouse embryos (detection of vaginal plug: day 0) and newborn mice were used. Human tooth germ was kindly provided by Dr. Yamamoto of Ohu University. Rat monoclonal antibodies recognizing type IV collagen alpha chains were raised against synthetic peptides of non-consensus amino acid sequences of the human and mice alpha (IV) chains. These monoclonal antibodies were kindly provided by Shigei medical research institute. Both mice and human tooth germ, we detected conversions in localization and composition of type IV collagen alpha chains working with tooth germ development. These results imply that there are time and site specificity, which may be involved in tooth morphogenesis and the tooth germ development, in type IV collagen alpha chains.

Near-Field Scanning Fluorescence Microscopy Study of Ion Channel Clusters in Cardiac Myocyte Membranes

Near-field scanning optical microscopy (NSOM) has been used to study the nanoscale distribution of voltage-gated L-type Ca 2+ ion channels, which play an important role in cardiac function. NSOM fluorescence imaging of immunostained cardiac myocytes (H9C2 cells) demonstrates that the ion channel is localized in small clusters with an average diameter of 100 nm. The clusters are randomly distributed throughout the cell membrane, with some larger fluorescent patches that high-resolution images show to consist of many small closely-spaced clusters. We have imaged unstained cells to assess the contribution of topography-induced artifacts and find that the topography-induced signal is <10% of the NSOM fluorescence intensity. We have also examined the dependence of the NSOM signal intensity on the tip-sample separation to assess the contributions from fluorophores that are significantly below the cell surface. This indicates that chromophores >∼200 nm below the probe will have negligible contributions to the observed signal. The ability to quantitatively measure small clusters of ion channels will facilitate future studies that examine changes in protein localization in stimulated cells and during cardiac development. Our work illustrates the potential of NSOM for studying membrane domains and protein localization/colocalization on a length scale which exceeds that available with optical microscopy.

Monitoring Signaling Processes in Living Cells Using Biosensors

This collection of nine animations shows how different types of biosensors report changes in cellular processes through the production of a visually detectable signal. Biosensors can be created by attaching one or more fluorescent proteins (such as green fluorescent protein) to a target protein or peptide or by attaching a fluorescent dye that is sensitive to its environment to a protein or peptide. Conformational changes in proteins in response to ligand binding, changes in the concentration of cellular metabolites or signaling messengers, changes in protein localization, and changes in protein activity or covalent modification can all be detected with biosensors. These animations can be used separately or together to illustrate how molecular biology, chemistry, and microscopy have converged to allow cellular processes to be visualized in living cells. Several of the animations describe the production of a fluorescent resonance energy transfer (FRET) signal.

Monitoring signaling processes in living cells using biosensors.

This collection of nine animations shows how different types of biosensors report changes in cellular processes through the production of a visually detectable signal. Biosensors can be created by attaching one or more fluorescent proteins (such as green fluorescent protein) to a target protein or peptide or by attaching a fluorescent dye that is sensitive to its environment to a protein or peptide. Conformational changes in proteins in response to ligand binding, changes in the concentration of cellular metabolites or signaling messengers, changes in protein localization, and changes in protein activity or covalent modification can all be detected with biosensors. These animations can be used separately or together to illustrate how molecular biology, chemistry, and microscopy have converged to allow cellular processes to be visualized in living cells. Several of the animations describe the production of a fluorescent resonance energy transfer (FRET) signal.

Sequential changes in localization of repair-related proteins (heat shock protein 70, ubiquitin and vascular endothelial growth factor) in the different stages of myocardial infarction.

The myocardium expresses vascular endothelial growth factor (VEGF), heat shock protein 70 (HSP70), and ubiquitin immediately after the onset of cardiac ischaemia. This study demonstrated the sequential changes in localization of these proteins, in addition to fibronectin and troponin T (TnT), in human hearts with myocardial infarction (MI). Myocardial tissues from 40 autopsied MI cases were immunostained with the five antibodies against VEGF, HSP70, ubiquitin, fibronectin and TnT. Fibronectin was recognized only in the cardiomyocytes with infarction. Although TnT, HSP70, ubiquitin and VEGF were detected in the affected myocardium in the early stages, their expression in cardiomyocytes around infarcted foci were more intense. The cardiomyocytes with coagulative myocytolysis were positive for fibronectin, but negative or weakly positive for TnT, HSP70, ubiquitin and VEGF. In contrast, the cardiomyocytes with colliquative myocytolysis were strongly positive for TnT, HSP70, ubiquitin and VEGF, but negative for fibronectin. Immunostaining using antibodies to fibronectin, TnT, HSP70, ubiquitin and VEGF is useful for the discrimination between infarcted myocytes and ischaemia-damaged myocytes in the human heart with MI at autopsy.

Expression of Spicule Matrix Proteins in the Sea Urchin Embryo during Normal and Experimentally Altered Spiculogenesis

During its embryonic development, the sea urchin embryo forms an endoskeletal calcitic spicule. This instance of biomineralization is experimentally accessible and also offers the advantage of occurring within a developmental context. Here we investigate the time course of appearance and localization of two proteins among the four dozen that constitute the protein matrix of the skeletal spicule. SM50 and SM30 have been studied in some detail, and polyclonal antisera have been prepared against them (C. E. Killian and F. H. Wilt, 1996, J. Biol. Chem. 271, 9150–9159). Using these antibodies we describe here the localization and time course of accumulation of these two proteins in Strongylocentrotus purpuratus, both in the intact embryo and in micromere cultures. We also investigate the disposition of the matrix proteins, SM50, SM30, and PM27, in the three-dimensional spicule by studying changes in protein localization during experimental manipulation of isolated skeletal spicules. We conclude that SM50, PM27, and SM30 probably play different roles in biomineralization, based on their localization and patterns of expression. It is unlikely that these proteins are solely structural elements within the mineral. SM50 and PM27 may play a role in defining the extracellular space in which spicule deposition occurs, while SM30 may play a role in secretion of spicule components. Finally, we report on the effects of serum on expression of some primary mesenchyme-specific proteins in micromere cultures; withholding serum severely depresses accumulation of SM30 but has only modest effects on the accumulation of other proteins.