Michael A. Model

Possible causes of apoptotic volume decrease: an attempt at quantitative review

Cell shrinkage and dehydration are essential characteristics of apoptosis, and loss of as much as half of the initial cell volume is not uncommon. This phenomenon is usually explained by efflux of K(+) and Cl(-). We reexamine this hypothesis on the basis of the available data for ion concentrations and the requirements for osmotic equilibrium and electroneutrality. In addition to ion loss, we discuss the possible impacts of several other processes: efflux of low-molecular-weight osmolytes, acidification of the cytosol, effects of water channels and pumps, heterogeneity of intracellular water, and dissociation of apoptotic bodies. We conclude that most mammalian cells are theoretically capable of reducing their volume by 15-20% through ion loss or a decrease in cytosolic pH, although, in reality, the contribution of these mechanisms to apoptotic shrinkage may be smaller. Transitions between osmotically active and inactive water pools might influence cell volume as well; these mechanisms are poorly understood but are amenable to experimental study. Dissociation of apoptotic bodies is a separate mechanism of volume reduction and should be monitored closely; this can be best achieved by measurement of intracellular water, rather than cell volume.

Optical determination of intracellular water in apoptotic cells.

•  Determination of intracellular water can be achieved by a combination of brightfield and transmission‐through‐dye imaging, all realized on a standard transmission microscope. •  The method permits sensitive detection of water loss in cells exposed to apoptotic agents. •  Cell water measurements are not affected by separation of apoptotic bodies and can be applied to samples where measurements of cell volume alone would be insufficient.

Imaging the Cell's Third Dimension

Introduction Shape and size are among the most basic and obvious characteristics of a cell (or of any physical object, for that matter). When a cell is observed through a microscope, one only sees its projection onto the image plane. Rather paradoxically, there are no easy techniques to visualize and measure cell’s third dimension—thickness. For example, confocal microscopy requires fluorescent labeling, multiple scanning with a high-power objective, possibly correction for the refractive index mismatch, and even then, generation of a complete three-dimensional profile is not very straightforward or precise. Other techniques for imaging the cell vertical profile, such as ion conductance [1], digital holographic [2], or atomic force microscopy [3], are rather complex and not available to most users. The related parameter of cell volume has also been elusive for traditional microscopy. Sometimes, the volumes of adherent cells are estimated from their diameters, even though it is evident that the extent of cell spreading cannot always correlate with the volume. Others detach cells prior to measurement and analyze them on a Coulter sizer; not only does this preclude continuous observation of the same cell, but the effect of detachment on cell volume has not been sufficiently characterized. Relative changes of the volume during rapid swelling or shrinkage can be observed by loading a fluorescent dye into the cytosol and measuring the fluorescence intensity with a confocal microscope [4]; to our knowledge, applicability of this method to slowly developing processes has not been investigated. These and some other techniques have been reviewed [5]; it seems that a method that would combine high precision, high speed, and low cost is still lacking. The question is important, though. Although the significance of cell thickness per se has been little explored, cell volume regulation has been a subject of intensive research. Cell volume is critical for understanding the cell response to anisosmotic environment [6], the development of apoptosis [7], and the cellular basis of a number of diseases, including ischemic injury [8] and brain edema [9]. Much evidence has accumulated that cell volume can have direct impact on cell behavior [10].

Dual Response of Human Leukemia U937 Cells to Hypertonic Shrinkage: Initial Regulatory Volume Increase (RVI) and Delayed Apoptotic Volume Decrease (AVD)

Background/Aims: Osmotic cell shrinkage is a powerful trigger of suicidal cell death or apoptosis, which is paralleled and enforced by apoptotic volume decrease (AVD). Cells counteract cell shrinkage by volume regulatory increase (RVI). The present study explored the response of human U937 cells to hypertonic solution thus elucidating the relationship between RVI and AVD. Methods: Cell water, concentration of monovalent ions and the appearance of apoptotic markers were followed for 0.5-4 h after the cells were transferred to a hypertonic medium. Intracellular water, K+, Na+, and Cl– content, ouabain-sensitive and -resistant Rb+ influxes were determined by measurement of the cell buoyant density in Percoll density gradient, flame emission analysis and 36Cl– assay, respectively. Fluorescent microscopy of live cells stained by acridine orange and ethidium bromide was used to verify apoptosis. Results: After 2-4 h incubation in hypertonic media the cell population was split into light (L) and heavy (H) fractions. According to microscopy and analysis of monovalent ions the majority of cells in the L population were healthy, while the H fractions were enriched with apoptotic cells. The density of L cells was decreasing with time, while the density of H cells was increasing, thus reflecting the opposite effects of RVI and AVD. At the same time, some of the cells were shifting from L to H fractions, indicating that apoptosis was gradually extending to cells that were previously displaying normal RVI. Conclusion: The findings suggest that apoptosis can develop in cells capable of RVI.

Measurement of wheat germ agglutinin binding with a fluorescence microscope

Signal intensity in fluorescence microscopy is often measured relative to arbitrary standards. We propose a calibration method based on a solution of the same fluorophore, whose binding to cells needs to be quantified. The method utilizes the low sensitivity of intensity to the object distance in wide‐field imaging of uniform materials. Liquid layers of slowly varying depth were prepared by immersing a spherical lens into a drop of a fluorophore placed on a slide. Flatfield‐corrected images of the contact and surrounding areas showed linear dependence of the gray level on the depth of fluorescent liquid. This allowed conversion of the measured intensity into the number of molecules per unit area. The method was applied to different cell types stained by WGA‐Alexa 488 and WGA‐TRITC. Consistent results were obtained by comparing microscopy with flow cytometry, comparing imaging through different objectives and comparing different WGA conjugates. Reproducibility of calibration was within 97% when low magnification was used. Fluorescence of free and bound WGA was found to be different, however, and therefore precise measurement of the number of cell‐bound molecules was problematic in this particular system. We conclude that the method achieves reliable measurement of cellular staining in the units of soluble fluorophore. For probes whose fluorescent properties are unaffected by binding, quantification of staining in true molecular units should be possible.

CrossTalk opposing view: the triggering and progression of the cell death machinery can occur without cell volume perturbations

Cell death is accompanied by the dissipation of ion gradients across the plasma membrane which, in turn, may cause cell volume perturbations by altering the content of intracellular osmolytes and osmotically obliged water. It is well documented that cells maintain their volume with an accuracy of 2–3% and volume perturbations beyond this range affect diverse cellular functions including membrane electrical potential, oxygen burst, metabolism, proliferation and gene expression (Lang et al. 1993; Hoffmann et al. 2009). Considering this, the contrasting volume behaviour in cells undergoing two morphologically distinct modes of death, apoptosis and necrosis, was classified by Okada and co-workers as necrotic volume

Thickness profiling of formaldehyde-fixed cells by transmission-through-dye microscopy

Conventional light microscopy techniques are poorly suited for imaging the vertical cell dimension. This can be accomplished using transmission-through-dye (TTD) imaging, in which cell thickness is directly converted into image intensity in the presence of extracellular dye with strong absorption. We have previously described applications of TTD to living cells using the dye Acid Blue 9 (AB9) to generate contrast. In this work, we investigated the possibility of extending TTD to chemically fixed cells. This would depend on preservation of cell impermeability to the dye; by using a method based on fluorescence quenching, we found that formaldehyde-fixed cells remain impermeable to AB9. Fixation enables imaging of cell surfaces in the presence of high concentrations of AB9, bringing the vertical resolution to several nanometers per pixel; that is at least an order of magnitude better than resolution achievable with live cells. TTD images collected with high-NA objectives are often contaminated by Becke lines resulting from intracellular organelles, and we show how to distinguish them from features on the cell surface. Quantification of cell thickness and volume on fixed cells is also possible during the early stages of fixation; this can be useful, for example, for measuring volume kinetics following rapid introduction of a stimulus.

Volume measurements and fluorescent staining indicate an increase in permeability for organic cation transporter substrates during apoptosis

Extensive membrane blebbing is one of the earliest observable changes in HeLa cells stimulated with apoptosis inducers. Blebbing caused by actinomycin D or camptothecin, but not by anti-Fas antibody, is accompanied by an almost 10% volume increase as measured by transmission-through-dye microscopy. When the experiment is carried out in DMEM medium, the swelling appears to result from activation of amiloride-sensitive channels. Low-sodium choline-, but not N-methyl(-)D-glucamine-based, medium, also supports swelling during the blebbing phase of apoptosis; this indicates that the membrane becomes permeable to choline as well. Because choline can enter the cells through organic cation transporters (OCT), we tested three fluorescent dyes (2-[4-(dimethylamino)styryl]-1-methylpyridinium iodide, rhodamine 123 and ethidium bromide) that have been reported to utilize OCT for cell entry. Intact HeLa cells are poorly permeable for these fluorophores, and initially they accumulate on the plasma membranes. Blebbing results in an enhanced penetration of these dyes into the cell interior, as was demonstrated both by direct observation and by FRET. The increased membrane permeability is specific for OCT substrates; the other tested cationic dyes apparently cross the membrane by other routes and exhibit a markedly different behavior. Our results reveal a previously unknown feature of apoptosis and the utility of cationic dyes for studying membrane transport.

Cell Volume Measurements by Optical Transmission Microscopy

Cell volume is an important parameter in cell adaptation to anisosmotic stress, in the development of apoptosis and necrosis, and in the pathogenesis of several diseases. This unit describes a method for measuring the volume of adherent cells using a standard light microscope. A coverslip with attached cells is placed in a shallow chamber in a medium containing a strongly absorbing and cell‐impermeant dye, Acid Blue 9. When such a sample is imaged in transmitted light at a wavelength of maximum dye absorption (630 nm), the resulting contrast quantitatively reflects cell thickness. Once the thickness is known at every point, the volume can be computed as well. Technical details, interpretation of data, and possible artifacts are discussed. Measurements in absolute units require knowledge of the absorption coefficient, and a similar procedure for the measurement of absorption coefficient is described.

Measurement of bacterial volume by transmission-through-dye imaging

Transmission-through-dye (TTD) microscopy makes possible direct measurement of bacterial volume, irrespective of cell shape. The technique can be realized on any brightfield microscope and is applicable to bacteria of all shapes. TTD imaging requires that intact bacteria be immobilized on a flat transparent surface, such as a glass coverslip.