Label-Free Quantification of Protein Density in Living Cells.

Intracellular water content, W, and protein concentration, P, are essential characteristics of living cells. Healthy cells maintain them within a narrow range, but often become dehydrated under severe stress; moreover, persistent loss of water (an increase in P) can lead to apoptotic death. It is very likely that protein concentration affects cellular metabolism and signaling through macromolecular crowding (MC) effects, to which P is directly related, but much remains unknown in this area.

Delayed vacuolation in mammalian cells caused by hypotonicity and ion loss.

Prolonged exposure of mammalian cells to hypotonic environments stimulates the development of sometimes large and numerous vacuoles of unknown origin. Here, we investigate the nature and formation of these vacuoles, which we term LateVacs. Vacuolation starts after osmotic cell swelling has subsided and continues for many hours thereafter.

Stability of Intracellular Protein Concentration under Extreme Osmotic Challenge.

Cell volume (CV) regulation is typically studied in short-term experiments to avoid complications resulting from cell growth and division. By combining quantitative phase imaging (by transport-of-intensity equation) with CV measurements (by the exclusion of an external absorbing dye), we were able to monitor the intracellular protein concentration (PC) in HeLa and 3T3 cells for up to 48 h. Long-term PC remained stable in solutions with osmolarities ranging from one-third to almost twice the normal.

Studying cell volume beyond cell volume.

The first part of the paper describes two simple microscopic techniques that we use in our laboratory. One measures cell volumes in adherent cultures and the other measures cell dry mass; both measurements are done on the same instrument (a standard bright-field transmission microscope with only one or two narrow-band color filters added) and on the same cells. The reason for combining cell volume with dry mass is that the ratio of the two-dry mass concentration (MC)-is an important and insufficiently utilized biological parameter.

Macromolecular Crowding: a Hidden Link Between Cell Volume and Everything Else.

High density of intracellular macromolecules creates a special condition known as macromolecular crowding (MC). One well-established consequence of MC is that only a slight change in the concentration of macromolecules (e.g.

A Reverse-Osmosis Model of Apoptotic Shrinkage.

The standard theory of apoptotic volume decrease (AVD) posits activation of potassium and/or chloride channels, causing an efflux of ions and osmotic loss of water. However, in view of the multitude of possible channels that are known to support apoptosis, a model based on specific signaling to a channel presents certain problems. We propose another mechanism of apoptotic dehydration based on cytoskeletal compression.

Evidence for macromolecular crowding as a direct apoptotic stimulus.

Potassium loss and persistent shrinkage have both been implicated in apoptosis but their relationship and respective roles remain controversial. We approached this problem by clamping intracellular sodium and potassium in HeLa or MDCK cells using a combination of ionophores. Although ionophore treatment caused significant cell swelling, the initial volume could be restored and further reduced by application of sucrose.

Cell Volume Measurements by Optical Transmission Microscopy.

Cell volume is an important parameter in studying cell adaptation to anisosmotic stress, activation of monovalent ion channels, and cell death. This article describes a method for measurement of the volumes 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.

Calibration and characterization of intracellular Asante Potassium Green probes, APG-2 and APG-4.

The response of fluorescent ion probes to ions is affected by intracellular environment. To properly calibrate them, intracellular and extracellular concentrations of the measured ion must be made equal. In the first, computational, part of this work, we show, using the example of potassium, that the two requirements for ion equilibration are complete dissipation of membrane potential and high membrane permeability for both potassium and sodium.

Intracellular Macromolecules in Cell Volume Control and Methods of Their Quantification.

The traditional theories of cell volume regulation focus on monovalent ions and small organic osmolytes. The main subject of this review is macromolecular content of the cell and its role in cell volume. We start by reviewing general information about cellular macromolecules and present some quantitative relationships.

Staurosporine-induced apoptotic water loss is cell- and attachment-specific.

Apoptotic volume decrease (AVD) is a characteristic cell shrinkage observed during apoptosis. There are at least two known processes that may result in the AVD: exit of intracellular water and splitting of cells into smaller fragments. Although AVD has traditionally been attributed to water loss, direct evidence for that is often lacking.

Intermolecular G-Quadruplex Induces Hyaluronic Acid-DNA Superpolymers Causing Cancer Cell Swelling, Blebbing, and Death.

Over the past decade, nanomedicine has gained considerable attraction through its relevance, for example, in "smart" delivery, thus creating platforms for novel treatments. Here, we report a natural polymer-DNA conjugate that undergoes self-assembly in a K-dependent fashion to form a G-quadruplex (GQ) and generate superpolymeric structures. We derivatized a thiolated conjugate of the naturally occurring glycosaminoglycan polymer hyaluronic acid (HASH) with short G-rich DNA (HASH-DNA) that can form an intermolecular noncanonical GQ structure.

Bright-Field Microscopy of Transparent Objects: A Ray Tracing Approach.

The formation of a bright-field microscopic image of a transparent phase object is described in terms of elementary geometrical optics. Our approach is based on the premise that the image replicates the intensity distribution (real or virtual) at the front focal plane of the objective. The task is therefore reduced to finding the change in intensity at the focal plane caused by the object.

Methods for cell volume measurement.

Volume is an essential characteristic of a cell, and this review describes the main methods of its measurement that have been used in the past several decades. The discussed methods include various implementations of light scattering, estimates based on one or two cell dimensions, surface scanning, fluorescence confocal and transmission slice-by-slice imaging, intracellular volume markers, displacement of extracellular solution, quantitative phase imaging, radioactive methods, and some others. Suitability of these methods to some typical samples and applications is discussed.

Calibrated brightfield-based imaging for measuring intracellular protein concentration.

Intracellular protein concentration is an essential cell characteristic, which manifests itself through the refractive index. The latter can be measured from two or more mutually defocused brightfield images analyzed using the TIE (transport-of-intensity equation). In practice, however, TIE does not always achieve quantitatively accurate results on biological cells.

Unidirectional Flux Balance of Monovalent Ions in Cells with Na/Na and Li/Na Exchange: Experimental and Computational Studies on Lymphoid U937 Cells.

Monovalent ion traffic across the cell membrane occurs via various pathways. Evaluation of individual fluxes in whole cell is hampered by their strong interdependence. This difficulty can be overcome by computational analysis of the whole cell flux balance.

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.

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.

Computation of pump-leak flux balance in animal cells.

Background/aims: Many vital processes in animal cells depend on monovalent ion transport across the plasma membrane via specific pathways. Their operation is described by a set of nonlinear and transcendental equations that cannot be solved analytically. Previous computations had been optimized for certain cell types and included parameters whose experimental determination can be challenging.

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.

Optical determination of intracellular water in apoptotic cells.

Intracellular water plays a critical role in apoptotic and necrotic cell death. We describe a method for quantifying cell water by application of two previously described variants of transmission microscopy. By taking two axially displaced brightfield images, the phase shift of the transmitted wave was computed using the transport-of-intensity equation.