Defining signal thresholds in DNA microarrays: exemplary application for invasive cancer.

BACKGROUND: Genome-wide or application-targeted microarrays containing a subset of genes of interest have become widely used as a research tool with the prospect of diagnostic application. Intrinsic variability of microarray measurements poses a major problem in defining signal thresholds for absent/present or differentially expressed genes. Most strategies have used fold-change threshold values, but variability at low signal intensities may invalidate this approach and it does not provide information about false-positives and false negatives. RESULTS: We introduce a method to filter false-positives and false-negatives from DNA microarray experiments. This is achieved by evaluating a set of positive and negative controls by receiver operating characteristic (ROC) analysis. As an advantage of this approach, users may define thresholds on the basis of sensitivity and specificity considerations. The area under the ROC curve allows quality control of microarray hybridizations. This method has been applied to custom made microarrays developed for the analysis of invasive melanoma derived tumor cells. It demonstrated that ROC analysis yields a threshold with reduced missclassified genes in microarray experiments. CONCLUSIONS: Provided that a set of appropriate positive and negative controls is included on the microarray, ROC analysis obviates the inherent problem of arbitrarily selecting threshold levels in microarray experiments. The proposed method is applicable to both custom made and commercially available DNA microarrays and will help to improve the reliability of predictions from DNA microarray experiments.

Distribution and dynamics of gap junction channels revealed in living cells.

To study the structural composition and dynamics of gap junctions in living cells, we tagged their subunit proteins, termed connexins, with the autofluorescent tracer green fluorescent protein (GFP) and its cyan (CFP) and yellow (YFP) color variants. Tagged connexins assembled normally and channels were functional. High-resolution fluorescence images of gap junction plaques assembled from CFP and YFP tagged connexins revealed that the mode of channel distribution is strictly dependent on the connexin isoforms. Co-distribution as well as segregation into well-separated domains was observed. Based on accompanying studies we propose that channel distribution is regulated by intrinsic, connexin isoform specific signals. High-resolution time-lapse images revealed that gap junctions, contrary to previous expectations, are dynamic assemblies of channels. Channels within clusters and clusters themselves are mobile and constantly undergo structural rearrangements. Movements are complex and allow channels to move, comparable to other plasma membrane proteins not anchored to cytoskeletal elements. Comprehensive analysis, however, demonstrated that gap junction channel movements are not driven by diffusion described to propel plasma membrane protein movement. Instead, recent studies suggest that movements of gap junction channels are indirect and predominantly propelled by plasma membrane lipid flow that results from metabolic endo- and exocytosis.

Cell-free synthesis and assembly of connexins into functional gap junction membrane channels.

Several different gap junction channel subunit isotypes, known as connexins, were synthesized in a cell-free translation system supplemented with microsomal membranes to study the mechanisms involved in gap junction channel assembly. Previous results indicated that the connexins were synthesized as membrane proteins with their relevant transmembrane topology. An integrated biochemical and biophysical analysis indicated that the connexins assembled specifically with other connexin subunits. No interactions were detected between connexin subunits and other co-translated transmembrane proteins. The connexins that were integrated into microsomal vesicles assembled into homo- and hetero-oligomeric structures with hydrodynamic properties of a 9S particle, consistent with the properties reported for hexameric gap junction connexons derived from gap junctions in vivo. Further, cell-free assembled homo-oligomeric connexons composed of beta1 or beta2 connexin were reconstituted into synthetic lipid bilayers. Single channel conductances were recorded from these bilayers that were similar to those measured for these connexons produced in vivo. Thus, this is the first direct evidence that the synthesis and assembly of a gap junction connexon can take place in microsomal membranes. Finally, the cell-free system has been used to investigate the properties of alpha1, beta1 and beta2 connexin to assemble into hetero-oligomers. Evidence has been obtained for a selective interaction between individual connexin isotypes and that a signal determining the potential hetero-oligomeric combinations of connexin isotypes may be located in the N-terminal sequence of the connexins.

Single channel behavior of recombinant beta 2 gap junction connexons reconstituted into planar lipid bilayers.

The beta 2 gap junction protein (Cx26) was expressed in an insect cell line by infection with a baculovirus vector containing the rat beta 2 cDNA. Isolated beta 2 gap junction connexons were reconstituted into planar lipid bilayers. Single channel activity was observed with a unitary conductance of 35-45 pS in 200 mM KCl. Channels with conductance values of 60 pS and 90-110 pS also coexisted with the lower conducting channel suggesting that there are channels with different conductance properties within a population of connexons. Channel activity was observed at voltages of up to 150 mV. Furthermore, the characterization of these channel properties from the beta 2 connexons that were generated by this heterologous expression system has provided the basis for identifying an endogenous beta 2 connexon channel in material reconstituted from native rat liver gap junctions.

Artificial transmembrane ion channels from self-assembling peptide nanotubes.

Naturally occurring membrane channels and pores are formed from a large family of diverse proteins, peptides and organic secondary metabolites whose vital biological functions include control of ion flow, signal transduction, molecular transport and production of cellular toxins. But despite the availability of a large amount of biochemical information about these molecules, the design and synthesis of artificial systems that can mimic the biological function of natural compounds remains a formidable task. Here we present a simple strategy for the design of artificial membrane ion channels based on a self-assembled cylindrical beta-sheet peptide architecture. Our systems--essentially stacks of peptide rings--display good channel-mediated ion-transport activity with rates exceeding 10(7) ions s-1, rivalling the performance of many naturally occurring counterparts. Such molecular assemblies should find use in the design of novel cytotoxic agents, membrane transport vehicles and drug-delivery systems.

Single channel behavior of matrix porin of Escherichia coli.

Porins are trimeric proteins in the outer membranes of Gram-negative bacteria. Several of them, among them matrix porin of Escherichia coli, form symmetrically voltage-gated ion channels in planar bilayers. Trimers exhibit negative resistance at potentials larger than +/- 90mV. Here we show that, after two pores within a trimer close irreversibly, the remaining third pore shows channel properties distinct from those observed in the trimer. This residual pore exhibits an asymmetric current-voltage dependence with a pronounced polarity-dependent shift toward low potentials and rates of channel-closing and opening that are one to two orders of magnitude faster than those observed for single channels in a reversibly voltage-dependent trimer. Rectification of single channels thus resembles that of a voltage-gated channel type observed in outer membrane patches of E.coli spheroblasts, hinting at the relevance of the phenomenon in vivo.

Plasticity of Escherichia coli porin channels. Dependence of their conductance on strain and lipid environment.

The conductance properties of three members of the porin family which form channels across the outer membrane of Gram-negative bacteria were compared. With their endogenous lipopolysaccharide (LPS) bound, the closely related porins F and C from Escherichia coli reveal significantly different conductance steps and closing potentials, with values of 0.82 nS (nanosiemens) and 89 mV for F-type channels, and 0.49 nS and 158 mV for C-type pores (1 M NaCl), respectively. On the basis of their closing potentials, the two channel types can be distinguished unequivocally. If reconstituted in asolectin and extraneous LPS, porin C forms F-type in addition to C-type channels. Substitution of asolectin by mitochondrial lipids yields the native C-type pores only. Both channel types can be induced to assume the mutually other channel configuration by variation of ionic strength. A multiplicity of channel subtypes is observed by variation of the pH of the medium. The three channels within a trimer are, however, consistently of the same type. Since structural studies have revealed a single channel per monomer, the several conductance steps observed are likely to reflect distinct configurations of the same channel. Best channel recoveries were observed if endogenous LPS remained associated to porin during purification. Significant yields could nevertheless be obtained also if LPS was removed from porin and replaced with various precursors or chemically synthesized analogues. As function requires the presence of glycolipids, yet crystallization is perturbed by heterodisperse endogenous LPS, the smallest monodisperse analogues yielding good channel recovery were determined. The minimal synthetic moiety is a monoglucosaminetetraacyl compound. The characteristics of porin B from E. coli BE are shown to be indistinguishable from those of porin F. The conductance properties of this porin, refolded from random coil configuration, are indistinguishable from those exhibited by native protein. The formation of channels is thus encoded by the sequence of the mature polypeptide alone.