C. L. Bashford

Scanning ion conductance microscopy of living cells.

Currently there is a great interest in using scanning probe microscopy to study living cells. However, in most cases the contact the probe makes with the soft surface of the cell deforms or damages it. Here we report a scanning ion conductance microscope specially developed for imaging living cells. A key feature of the instrument is its scanning algorithm, which maintains the working distance between the probe and the sample such that they do not make direct physical contact with each other. Numerical simulation of the probe/sample interaction, which closely matches the experimental observations, provides the optimum working distance. The microscope scans highly convoluted surface structures without damaging them and reveals the true topography of cell surfaces. The images resemble those produced by scanning electron microscopy, with the significant difference that the cells remain viable and active. The instrument can monitor small-scale dynamics of cell surfaces as well as whole-cell movement.

Specialized scanning ion‐conductance microscope for imaging of living cells

A specialized scanning ion conductance microscope (SICM) for imaging living cells has been developed from a conventional patch‐clamp apparatus, which uses a glass micropipette as the sensitive probe. In contrast with other types of scanning probe microscope, the SICM probe has significant advantages for imaging living cells: it is most suitable for imaging samples immersed in water solutions; and since the probe senses ion current and does not need physical contact with the sample during the scan, any preliminary preparation of cells (fixation or adherence to a substrate) is unnecessary. We have successfully imaged murine melanocytes in growth medium. The microscope images the highly convoluted surface structures without damaging or deforming them, and reveals the true, three‐dimensional relief of the cells. This instrument has considerable ability to operate, potentially simultaneously, in applications as diverse as real‐time microscopy, electrophysiology, micromanipulation and drug delivery.

Rapid Switching of Ion Current in Narrow Pores: Implications for Biological Ion Channels

Ions flowing through purely synthetic filters made of polyethylene terephthalate which have been etched to produce narrow pores show: (i) rapid transitions between a high-conducting and a low-conducting state; (ii) selectivity of ion flow; and (iii) inhibition by divalent cations and protons. These features resemble those displayed by many biological ion channels. We interpret our results in terms of the special properties of ion conductance at an interface that may be observed whenever the contribution of bulk conductance is minimal.

Low conductance states of a single ion channel are not ‘closed’

We have used a polymer-exclusion method to estimate the sizes of the high and low-conductance states of Staphylococcus aureus α-toxin channels across planar lipid bilayers. Despite a >10-fold difference in conductance between high and low-conductance states, the size differs by <2-fold. We conclude that factors other than the dimensions have a strong influence on the conductance of α-toxin channels. We also show that the high conductance state is destabilized by the presence of high molecular weight polymers outside the channel, compatible with the removal of channel water as the high conductance state “shrinks” to the low conductance state.

Pore-forming toxins: Experiments with S. aureus α-toxin, C. perfringens θ-toxin and E. coli haemolysin in lipid bilayers, liposomes and intact cells

Abstract Three quite different bacterial toxins (S. aureus α-toxin, C. perfringens θ-toxin and E. coli haemolysin) induce the leakage of phosphorylated metabolites from Lettre cells and of calcein from liposomes; in each case leakage is inhibited by Zn2+ > Ca2+ > Mg2+. Inhibition is not due to displacement of toxin from the membrane, since divalent cations inhibit leakage through pre-formed pores. Electrical conductivity across phospholipid bilayers is induced by each of the three toxins; in each case the probability of channels being in the open state is reduced by divalent cations. Although the pores induced in phospholipid bilayers and liposomes vary greatly in size (θ-toxin ⪢ haemolysin > α-toxin), in Lettre cells the lesions appear more uniform, suggestive of a limiting effect in cells.

A novel explanation for fluctuations of ion current through narrow pores.

Fluctuation of ion current, between a high conductance and a low conductance state, through biological ion channels and pores is assumed to arise from conformational changes between an “open” and a “closed” configuration. Here we offer an additional mechanism that arises from changes in ionization of fixed charges within, or at the mouth of, a channel or pore. Our hypothesis, which is based on measurements of ion selectivity alongside ion current, applies to pores through some synthetic membranes and through channels—such as those created by certain toxins—that remain (at least partially) open in the low conductance state. It may also explain the phenomena of “open channel noise” and “substate behavior” that characterize several endogenous ion channels and should be considered when modeling the behavior of such channels.—Korchev, Y. E., Bashford, C. L., Alder, G. M., Apel, P. T., Edmonds, D. T., Lev, A. A., Nandi, K., Zima, A. V., Pasternak, C. A. A novel explanation for fluctuations of ion current through narrow pores. FASEB J. 11, 600–608 (1997)

Influence of pH on the uptake of 5-fluorouracil into isolated tumour cells.

To investigate the possible dependence of 5-fluorouracil (5FU) uptake in tumours on the intra- (pHi) and extracellular (pHe) pH, a pH gradient (deltapH) was imposed across the plasma membrane of ascites tumour cells in vitro, similar to that known to occur in some solid tumours in vivo, by incubation in media of PHe 5-8. A > or = 2:1 (intracellular/extracellular) accumulation of radiolabelled 5FU occurred after 5 min incubation of the cells with 0.5 mM 5FU at pHe of 5.0, 5.5 or 6.0. 5FU metabolism is slow under these conditions, and 5FU uptake was not affected by longer incubations up to 20 min, nor by the absence of a sodium gradient. pHi was estimated from the distribution of the weak acid, 5.5-dimethyl-2,4-oxazolidione ([14C]DMO) across the cell membrane. There was significant correlation between the intracellular/extracellular 5FU ratio and pHe (from pHe 6-8), deltapH and pHi (P < 0.02). Similar results were obtained with HT29 cells. Incubation with a drug that made plasma membranes permeable to H+ significantly decreased 5FU uptake in Lettre cells. The co-transport of 5FU may occur on a proton symport using the proton motive force of the deltapH.

Heat shock proteins induce pores in membranes

Human heat shock protein (hsp) 70 and bacterial protein groEL promote leakage of calcein from liposomes induced by human serum albumin signal peptide, byS. aureus α toxin or by diphtheria toxin. Hsp 70 and groEL, as well as two mycobacterial homologues hsp 71 and hsp 65, induce ion conducting pores across planar lipid bilayers at low or neutral pH. It is concluded that hsp induce pores in membranes and that this may contribute to their action within cells.

Understanding the Tumor Metabolic Phenotype in the Genomic Era

Now, at the beginning of a new century, 80 years after Warburgs Nobel prize winning discoveries, we are beginning to make sense of the underlying causes of the well known metabolic phenotype of tumor cells. Building on decades of research to understand the interrelationships between respiration and glycolysis in cancer, the tumor metabolic phenotype can now begin to be understood in a genomic context. With the discovery of hypoxia inducible factor-1 (HIF-1), which is widely overexpressed across a broad range of cancers, modern molecular tools have allowed us to put together the pattern of events that might explain the metabolic differences between tumor and normal cells. HIF-1 controls cellular and systemic responses to oxygen availability and coordinates up-regulation of genes involved in many pathways concerned with tumour growth and metabolism including angiogenesis, glucose and energy metabolism, cellular proliferation, differentiation and viability, apoptosis, pH regulation and matrix metabolism. These findings begin to explain how glucose uptake and glycolysis could be up-regulated in cancer cells (through binding to a core DNA recognition sequence) in a co-ordinated and constitutive fashion that may also allow us to elucidate new targets for tumor therapy.

Energy-linked activities of the chromaffin granule membrane

The storage vesicles of the adrenal medulla (the chromaffin granules) contain unusually high levels of catecholamines (0.5 M) and nucleotides (0.125 M ATP) as well as substantial amounts of acidic protein [ 11. The membrane of the chromafiin granule contains several activities that may be important in maintaining these high metabolite levels. These include an electron transport chain [2] and, in particular, an ATPase activity that has been reported to be associated with the active transport of catecholamines [3]. Earlier work [4 ] with the storage vesicles extracted from bovine splenic nerve has shown that uncouplers and inhibitors of mitochondrial oxidative phosphorylation can affect both the uptake and the release of noradrenaline from these vesicles. In this report we shall indicate that the ATPase activity of the chromaffm granule membrane is reflected by changes in the fluorescence of the probe ANS and that such changes are abolished by uncoupling agents with a concomitant increase in ATPase activity.