Quantifying cell-matrix adhesion dynamics in living cells using interference reflection microscopy.

Focal adhesions and podosomes are integrin-mediated cell-substratum contacts that can be visualized using interference reflection microscopy (IRM). Here, we have developed automated image-processing procedures to quantify adhesion turnover from IRM images of live cells. Using time sequences of images, we produce adhesion maps that reveal the spatial changes of adhesions and contain additional information on the time sequence of these changes. Such maps were used to characterize focal adhesion dynamics in mouse embryo fibroblasts lacking one or both alleles of the vinculin gene. Loss of vinculin expression resulted in increased assembly, disassembly and/or in increased translocation of focal adhesions, suggesting that vinculin is important for stabilizing focal adhesions. This method is also useful for studying the rapid dynamics of podosomes as observed in primary mouse dendritic cells.

The dependence of DNase I activity on the conformation of oligodeoxynucleotides.

We have developed a sensitive continuous assay for nucleases using proton release. The assay has been applied to the determination of the kinetics of DNase I acting on short, defined deoxyoligonucleotides. The dependence of Kcat/K(m) on sequence and structure of short oligonucleotide substrates has been measured: increasing lengths of AnTn sequences decrease the rate of cleavage. G.A mismatches in which the bases pair using imino protons are cleaved quite effectively by DNase I. In contrast, tandem G.A mismatches which use amino pairing and have BII phosphodiesters, are refractory to DNase I. Also, the DNA strands of DNA.RNA hybrid duplexes are not cleaved by DNase I. These results show that the global conformation of a duplex and the details of its minor groove affect the cleavage efficiency by DNase I. The assay has also been used to measure the inhibition constant of the minor-groove-binding ligand propamidine. A value of 3 microM has been determined for binding to the sequence d(CGCGAATTCGCG)2, showing that dissociation constants can be determined even when there are no convenient optical signals for titrations.