Michael A. Model

Impermeability of the ascending limb of the Henle loop for water is traditionally regarded as essential for countercurrent multiplication in the kidney. Similar claims have been made about permeability properties of the collecting duct and some other epithelia. It is not clear, however, how a structure based on phospholipid bilayers can be water-impermeant if phospholipid bilayers themselves have measurable permeability. The presence of two membranes separated by the cytoplasm may only account for a several-fold reduction in permeability compared to a single bilayer. By analyzing published data, we conclude that these tubules do have a finite water permeability, especially the collecting duct. Although the results on isolated ascending limbs vary among authors, osmotic shock experiments clearly indicate that both the collecting duct and the ascending Henle loop are sufficiently water-permeable to observe volume regulation effects. We conclude that these epithelia by themselves do not display unusual resistance to water flow; it can be estimated that 20-50% of the fluid entering the tubules can be reabsorbed into a strongly hypertonic medulla. It is possible, however, that unstirred layers in the intact kidney may contribute to the apparent low permeability of the tubules.

To study macromolecular crowding (MC) in living cells, one needs a method to measure it. Several existing approaches to quantify MC address slightly different aspects of crowding. If we define MC through protein concentration, it can be measured by quantitative phase imaging coupled with volume determination; both can be realized on a standard bright-field microscope. Osmotic cell theory can help identify the essential factors that control MC. Nevertheless, there are still many gaps in our understanding of MC regulation and, in particular, of the interrelationship between MC and cell stress or damage. Experiments show that MC is subject to homeostatic control and returns to its resting values following various disturbances. Severe cell damage causes an accumulation of water and a decrease in MC; however, based on limited data, water accumulation is restricted to one area of the cell (necrotic bleb), while the rest of the cell remains at normal density. Similar heterogeneous water distribution is observed in vacuolated mammalian cells. Intermediate degrees of stress tend to produce dehydration and an increase in MC. Apoptotic shrinkage is one common example of stress-induced dehydration, but the effect may be more general. A hypothesis on its mechanism is proposed.

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. Obviously, in order to study the biological roles and regulation of MC in living cells, one needs a method to measure it. Simple and accurate measurements of P in adherent cells can be based on its relationship to refractive index. The latter can be derived from two or more (depending on the algorithm) mutually defocused brightfield images processed by the transport‐of‐intensity equation (TIE) that must be complemented by a determination of volume. Here, we describe the experimental considerations for both TIE imaging and for a particular method of cell volume measurement, transmission‐through‐dye (TTD). We also introduce an ImageJ plugin for solving TIE. TIE and TTD are fully compatible with each other as well as with fluorescence. A similar approach can be applied to subcellular organelles; however, in this case, the volume must be determined differently.© 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Sample preparation for TIE with or without TTDBasic Protocol 2: Acquisition of TIE and TTD imagesBasic Protocol 3: Calibration of TIEBasic Protocol 4: Measurement of the absorption coefficient of the medium used for TTDBasic Protocol 5: Image processing using FijiSupport Protocol 1: Installation and use of TIE pluginSupport Protocol 2: Automation of the double TTD/TIE processing using a Fiji macro

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. Most of the vacuoles are positive for the lysosomal marker LAMP-1 but not for the autophagosomal marker LC3. Vacuoles do not appear to have acidic pH, as they exclude LysoTracker and acridine orange; inhibiting the V-ATPase with bafilomycin A1 has no effect on their formation. No LateVacs were formed in cells with a knockout of the essential LRRC8A subunit of the volume-regulated anion channel (VRAC). Since the main feature of cells recovered from hypotonic swelling should be reduced chloride concentration, we tested if chloride depletion can act as a signal for vacuolation. Indeed, four different low-chloride buffers resulted in the development of similar vacuoles. Moreover, vacuolation was suppressed in WNK1/WNK3 double knockouts or by the inhibition of WNK kinase, which is activated by low chloride; in hypotonic media, the WNK inhibitor had a similar effect. However, exposing cells to a low-sodium, high-potassium medium also resulted in vacuoles, which were insensitive to WNK. We conclude that vacuole development can be triggered either by the loss of chloride or by the loss of sodium.

High density of intracellular proteins creates a very crowded environment, where the volume available to protein molecules becomes limited. This gives rise to macromolecular crowding (MC) effects, which include slower diffusion, formation of protein complexes and oligomers, and driving proteins into more compact shapes [ 1]. Recent years have seen a surge of interest in MC. Due to high sensitivity of the MC effects to small perturbations in protein concentration, crowding is believed to directly control many biological processes [ 2].

Protein concentration (PC) is an essential characteristic of cells and organelles; it determines the extent of macromolecular crowding effects and serves as a sensitive indicator of cellular health. A simple and direct way to quantify PC is provided by brightfield‐based transport‐of‐intensity equation (TIE) imaging combined with volume measurements. However, since TIE is based on geometric optics, its applicability to micrometer‐sized particles is not clear. Here, we show that TIE can be used on particles with sizes comparable to the wavelength. At the same time, we introduce a new ImageJ plugin that allows TIE image processing without resorting to advanced mathematical programs. To convert TIE data to PC, knowledge of particle volumes is essential. The volumes of bacteria or other isolated particles can be measured by displacement of an external absorbing dye (“transmission‐through‐dye” or TTD microscopy), and for spherical intracellular particles, volumes can be estimated from their diameters. We illustrate the use of TIE on Escherichia coli, mammalian nucleoli, and nucleolar fibrillar centers. The method is easy to use and achieves high spatial resolution.

Purpose Microscopic observation of live parasites in the stool is an important diagnostic tool in human and veterinary medicine. Because of the presence of large amounts of contaminating organic matter, microscopic analysis must be preceded by time-consuming pre-purification steps. Transmission-through-dye (TTD) optical microscopy obviates this problem. The purpose of this study is to illustrate the use of TTD for the analysis of stool samples. Methods TTD imaging is based on the exclusion of a strongly absorbing dye by living cells. A food colorant Acid Blue 9 (AB9) is added to a fecal suspension, and the sample is observed under transmitted illumination through a 630 nm bandpass filter. AB9 strongly absorbs red light, and it comes out of the sample significantly attenuated. However, if a viable cell of any origin is present in the sample, it excludes the dye and reduces the depth of the light-absorbing layer. Results Live cells or eggs appear bright red on a dark background, while most of the organic contaminants remain dark. The method has been demonstrated on schistosomes, hookworms, giardia, yeast, and other organisms. Conclusion TTD dramatically increases the visibility of live parasites and permits their direct observation in a fecal suspension. TTD can be enabled on any microscope by simply adding a red filter.

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. When cells were subjected to extreme hypoosmolarity (one-quarter of normal), their PC did not decrease as one might expect, but increased; a similar dehydration response was observed at high concentrations of ionophore gramicidin. Highly dilute media, or even moderately dilute in the presence of cytochalasin, caused segregation of water into large protein-free vacuoles, while the surrounding cytoplasm remained at normal density. These results suggest that: (1) dehydration is a standard cellular response to severe stress; (2) the cytoplasm resists prolonged dilution. In an attempt to investigate the mechanism behind the homeostasis of PC, we tested the inhibitors of the protein kinase complex mTOR and the volume-regulated anion channels (VRAC). The initial results did not fully elucidate whether these elements are directly involved in PC maintenance.