Membrane blebbing as a transient manifestation of lipid insertion during the cell cycle.
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Biomedical subjects
L G Tertoolen
Publications and source records attributed to L G Tertoolen.
Characterization of 42K+ and 86Rb+ transport and electrical membrane properties in exponentially growing neuroblastoma cells.
For measuring K+ efflux from exponentially growing neuroblastoma cells (clone Neuro-2A), two methods were used, a sampling method and a washing method. Both methods indicated that K+ efflux kinetics were as from a two-compartment system, but the two compartments could only be resolved completely using the washing method. A fast compartment, containing 143 +/- 16 nmol K+/10(6) cells, was found to be associated to the cell surface, and a slow compartment, containing 151 +/- 7 nmol K+/10(6) cells, was found to represent the intracellular K+. The rate constant of the slow compartment was 0.0164 +/-0.0005 min-1, and the K+ efflux rate was 2.46 +/- 0.14 nmol K+/10(6) cells per min. Using the appropriate conditions to measure K+ influx, the kinetics of influx were equal to the kinetics of efflux, indicating steady-state conditions. In addition a comparison was made between 42K+ and 86Rb+ as radioactive tracers for K+ flux. It was found that 86Rb+ was specifically bound on both the inside and the outside of the cells, and for this reason was not a suitable tracer for studying K+ flux kinetics in neuro-2A cells. A membrane potential of -42.9 +/- 1.3 mV and intracellular K+ activity of 108.1 +/- 3.0 mM were measured using conventional and ion-selective microelectrodes. A correlation was made between the K+ flux and electrophysiological data, using the equations of electrodiffusion theory. Thus, the permeabilities of K+ and Na+ were calculated as (3.9 +/- 0.4) . 10(-8) cm/s and (0.6 and 0.1) . 10(-8) cm/s respectively, together with K+ conductance of (2.8 +/- 0.3) . 10(-6) omega-1/cm2.
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Cation transport and growth regulation in neuroblastoma cells. Modulations of K+ transport and electrical membrane properties during the cell cycle.
Cation transport and membrane potential were studied during the cell cycle of neuroblastoma cells (clone Neuro-2A) to investigate the role of these parameters in growth regulation. The cells were synchronized by selective detachment of mitotic cells. The membrane potential and intracellular K+ activity were measured with conventional and K+-selective microelectrodes respectively. Both the membrane potential and K+ activity were high in mitosis, decreased to half maximal in G1 phase, and rose again during S phase. K+ efflux across the plasma membrane was studied with 42K+ as a radioactive tracer using a washing method for cells grown in monolayer and a continuous efflux method for mitotic cells in suspension. The intracellular K+ content and unidirectional K+ efflux rate obtained from these measurements showed modulations during the cell cycle similar to those of the membrane potential. Using equations of electrodiffusion theory the membrane permeabilities to K+ and Na+ were calculated. These permeabilities were high in mitosis, decreased rapidly in G1 phase and increased during S phase, followed by a transient decrease in G2 phase. A rapid increase was observed between G2 phase and the next mitosis. A similar pattern was obtained for the K+ conductance. K+ resistance changes during the cell cycle were similar to changes in the specific membrane resistance, measured by microelectrodes, except for the early cell cycle phases (mitosis and G1). These studies clearly demonstrate large modulations of the passive membrane permeability properties during the cell cycle. These modulations can be correlated with physicochemical membrane variations during the cell cycle, such as membrane fluidity and lateral mobility of lipids.
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Quantitative analysis of the numerical and lateral distribution of intramembrane particles in freeze-fractured biological membranes.
An improved method is presented for a quantitative analysis of freeze-fractured biological membranes. The analysis provides the numerical distribution of intramembrane particles (IMP) as a function of their diameter and a measure for the lateral IMP-distribution as a function of the IMP-diameter in terms that allow a statistical comparison between different replicas. In analyzing the lateral IMP-distribution a distinction between random, aggregative and dispersive distributions can be made for different IMP-diameter classes. Such an analysis makes the interpretation of the ultrastructural appearance of biological membranes possible in terms of the dynamic behavior of the represented membrane components. The method is demonstrated by comparing the structure of the plasma membrane of synchronized neuroblastoma cells after glutaraldehyde and formaldehyde fixation at pH 6.0.
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No junctional communication between epithelial cells in hydra.
Diffusion gradients of morphogens have been inferred as a basis for the control of morphogenesis in hydra, and morphogenetic substances have been found which, on the basis of their molecular weight (MW), should be able to pass gap junctions. There have been several reports of the presence of gap junctions between epithelial cells of hydra. However, until now, there has been no report published on whether these junctions enable the epithelial cells to exchange molecules of small molecular weight, as has been described in other organisms. Therefore we decided to investigate the communicative properties of the junctional membranes by electrophysiological methods and by intracellular-dye iontophoresis. We report here that no electrotonic coupling is detectable between epithelial cells of Hydra attenuata in: (1) intact animals, (2) head-regenerating animals, (3) cell re-aggregates, and (4) hydra that have become nervefree. Furthermore we show that epithelial cells are unable to exchange low-molecular weight fluorescent dyes.
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Intercellular communication patterns are involved in cell determination in early molluscan development.
The formation of specialized intercellular junctions, allowing the passage of low-molecular weight regulatory molecules, has been considered as a possible mechanism for regulating embryonic development. No direct evidence for this concept has been found in early development. In the mollusc Pattella vulgata it was demonstrated that cell positioning and specific cellular interactions are key factors in the control of early development. We have now investigated the pattern of intercellular communication during early development of this embryo by intracellular iontophoresis of the fluorescent dye Lucifer Yellow CH. We demonstrate that the formation of regional- and temporal-specific cell-to-cell coupling is correlated with the determination of the mesentoblast--the stem cell of the mesoderm--and the establishment of dorso-ventral polarity.
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The plasma-membrance IMP pattern as related to animal/vegetal polarity in the amphibian egg.
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Freeze-fracture electron microscopy of preexisting and nascent cell membrane in cleaving eggs of Xenopus laevis.
During cell division in the Xenopus egg (diameter 1.25 mm) new cell membrane is formed in the furrow region (rate of growth approx 4-10(4) mum2/min). Freeze-fracture electron microscopy has produced the following data. Preexisting plasma membrane faces show a reversed polarity with respect to particle distribution, i.e. more particles are attached to the E-face (density 1600-2200 particles/mum2) than to the P-face (300 particles/mum2). A frequency histogram of 2331 measured intramembranous particles does not show a continuous range of sizes. The following sizes were very obvious: 95 A (12%), 125 A (30%) and 180 A (6%). At the tips of surface protrusions both the E- and the P- face are particle-free. Nascent cell membrane fracture faces are more difficult to obtain. The particle density is low (E-face 300-500 particles/mum2). Lowering the ambient temperature to 5 degrees C for approx. 5 mins does not change the normal particle pattern, but it improves the output in nascent membrane fracture faces. The fact that in the Xenopus egg preexisting and nascent membrane regions are continuous but nevertheless maintain their highly different particle densities is noteworthy. The freeze-fracture data are discussed in relation to, among other things, the known values of the specific resistances of these membrane regions.
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Phorbol ester and diacylglycerol mimic growth factors in raising cytoplasmic pH.
There is now good evidence that cytoplasmic pH (pHi) may have an important role in the metabolic activation of quiescent cells. In particular, growth stimulation of mammalian fibroblasts leads to a rapid increase in pHi (refs 3-6), due to activation of a Na+/H+ exchanger in the plasma membrane, and this alkalinization is necessary for the initiation of DNA synthesis. However, the mechanism by which mitogens activate the Na+/H+ exchanger to raise pHi is not known, although an increase in cytoplasmic free Ca2+ ([Ca2+]i) has been postulated as the primary trigger. We now present data suggesting that the Na+/H+ exchanger is set in motion through protein kinase C, a phospholipid- and Ca2+-dependent enzyme normally activated by diacylglycerol produced from inositol phospholipids in response to external stimuli. Using newly developed pH microelectrodes and fluorimetric techniques, we show that a tumour promoting phorbol ester and synthetic diacylglycerol, both potent activators of kinase C (refs 12-15), mimic the action of mitogens in rapidly elevating pHi in different cell types. Furthermore, we demonstrate that, contrary to previous views, an early rise in [Ca2+]i is not essential for the activation of Na+/H+ exchange and the resultant increase in pHi. Finally, we suggest that an alkaline pHi shift, mediated by Na+/H+ exchange, may be a common signal in the action of those hormones which elicit the breakdown of inositol phospholipids.
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