Phase separation in necrotic cells

Hollembeak

Kurokawa

2021

Cell Physiol Biochem

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., proteins) results in a shift of chemical equilibria towards the formation of macromolecular complexes and oligomers. This suggests a physiological mechanism of converting cell density changes into cellular responses. In this review, we start by providing a general overview of MC; then we examine the available experimental evidence that MC may act as a direct signaling factor in several types of cellular activities: mechano- and osmosensing, cell volume recovery in anisosmotic solutions, and apoptotic shrinkage. The latter phenomenon is analyzed in particular detail, as persistent shrinkage is known both to cause apoptosis and to occur during apoptosis resulting from other stimuli. We point to specific apoptotic reactions that involve formation of macromolecular complexes and, therefore, may provide a link between shrinkage and downstream responses.

Section: Mechano-and Osmosensingmentioning

confidence: 99%

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

Hollembeak

Kurokawa

2021

Cell Physiol Biochem

“…We have previously observed a similar situation in severely compromised cells at late stages of free-radical-induced damage when necrotic blebs gradually lose proteins [ 22 ]. That situation was apparently caused by a decrease in protein solubility and their extensive aggregation.…”

Section: Discussionmentioning

confidence: 57%

“…We next asked if these vacuoles are surrounded by a lipid membrane or represent a phase transition, as in necrotic blebs [ 22 ]. One way to approach this question would be to stain them with a fluorescent marker and test their mobility by fluorescence recovery after photobleaching, as was done in the aforementioned work.…”

Section: Resultsmentioning

confidence: 99%

Stability of Intracellular Protein Concentration under Extreme Osmotic Challenge

Hollembeak

2021

Cells

Self Cite

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.

“…Exclusion-based methods for cell volume measurement are some of the most direct and easy to implement. Our lab has been using TTD extensively for various research applications, often combining it with fluorescence or with quantitative phase imaging (Kasim et al, 2013;Gibbons et al, 2016;Rana et al, 2017;Beals et al, 2018;Model et al, 2018). FLEX in its modern implementation is also finding its uses Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Both methods require a shallow chamber, only slightly deeper than cell thickness, to enable comparison of the cell with the background. Both TTD and FLEX have been used for various biological applications (Kasim et al, 2013;Gabella et al, 2014;Gibbons et al, 2016;Monnier et al, 2016;Rana et al, 2017;Beals et al, 2018;Model et al, 2018;Roussel et al, 2018;Perez-Gonzalez et al, 2019). It appears, however, that the accuracy of FLEX has not been thoroughly tested, except for a semiquantitative comparison with atomic force microscopy (Bottier et al, 2011) and by correlation with diameters of round cells (Cadart et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Comparison of cell volume measurements by fluorescence and absorption exclusion microscopy

2020

Journal of Microscopy

Summary There are two light microscopic methods for cell volume measurement based on volume exclusion. One method, sometimes referred to as FLEX, utilises negative staining by an external fluorescent dye, and cell volume is found from attenuation of fluorescence under a wide‐field microscope. The other method (TTD) is based on exclusion of an external absorbing dye, resulting in an increased intensity of transmission image. In this work, we compared these two methods. TTD measurements were consistent, reproducible and identical to those obtained by confocal scanning. In our hands, FLEX based on either sodium fluorescein of fluorescent dextran, usually resulted in underestimation of cell volume, which were insignificant in shallow chambers but became more severe with increased chamber depth. We have not been able to exactly pinpoint the source of the problem; it may have been undetected accumulation of dye in the cells or, more likely, some unappreciated aspects of image formation under epi‐illumination. We also discuss applicability of both methods to in‐flow volume measurements. Lay Description Cell volume is a parameter important for many cell biological and physiological applications, and many different methods have been proposed for its measurement. Two light microscopic methods based on volume exclusion deserve special attention due to their speed and simplicity. In one of them (transmission‐through‐dye, or TTD), cells are placed in a shallow chamber, and a strongly absorbing external dye is added to the cell‐containing medium. The sample is imaged in transmission at a wavelength of maximum dye absorption. Because cells with intact membranes exclude the dye, they appear brighter on a transmission image, and their contrast quantitatively reflects cell thickness. By summation of thickness values over the cell area, cell volume can be obtained. The other method sometimes referred to as FLEX utilises exclusion of a fluorescent dye. Cells appear darker than the background under wide‐field fluorescence observation in accordance with their thicknesses, and cell volume is computed by thickness summation over the area, like in TTD. In this work, we compared the accuracy of TTD and FLEX for volume measurements. TTD and confocal scanning produced virtually identical results, which suggests that TTD data are accurate. On the other hand, cell volumes measured by FLEX were consistently smaller than by TTD. The discrepancy always increased with the depth of the chamber, although the exact relationship varied between experiments. By contrast, TTD results were insensitive to chamber depth. Thus, it appears that FLEX underestimates cell volume. The reason for that is not entirely clear. Accumulation of the fluorescent dye inside the cell could be a possibility, although we found no evidence for that. Most likely, the reason lies with some unappreciated aspects of wide‐field fluorescence image formation, which has not been well‐characterised for the type of negative staining used in FLEX. In our opinion, TTD is the method of c...