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. In this study, we quantified intracellular water in staurosporine-treated cells using a previously described optical microscopic technique that combines volume measurements with quantitative phase analysis. Water loss was observed in detached HeLa and in adherent MDCK but not in adherent HeLa cells. At the same time, adherent HeLa and adherent MDCK cells exhibited visually similar apoptotic morphology, including fragmentation and activation of caspase-3. Morphological changes and caspase activation were prevented by chloride channel blockers DIDS and NPPB in both adherent and suspended HeLa cells, while potassium channel blocker TEA was ineffective. We conclude that staurosporine-induced dehydration is not a universal cell response but depends on the cell type and substrate attachment and can only be judged by direct water measurements. The effects of potassium or chloride channel blockers do not always correlate with the AVD.

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. Therefore, we have developed a calibration procedure that involves successive imaging of cells in solutions containing different amounts of added protein. This allows one to directly relate the output of TIE (T) to intracellular protein concentration C (g/L). The resultant relationship has a simple form: C ≈ 1.0(T/V), where V is the cell volume (µm3 ) and 1.0 is an empirical coefficient. We used calibrated TIE imaging to characterize the regulatory volume increase (RVI) in adherent HeLa cells placed in a hyperosmotic solution. We found that while no RVI occurs over the first 30-60 min, the protein concentration fully recovers after 20 h. Because interpretation of such long experiments may depend on whether protein concentration varies significantly throughout the cell cycle, we measured this parameter in three cell lines: HeLa, MDCK and DU145. Our data indicate that protein concentration remains relatively stable in these cells. © 2017 International Society for Advancement of Cytometry.

Phase separation in necrotic cells.

Necrotic cells are known to develop characteristic membrane blebs. We measured protein concentration within necrotic blebs and found that it can be reduced by as much as twenty-fold compared to the main cell body (CB). These results raise two questions: 1. Why do proteins vacate the bleb? 2. How can osmotic equilibrium be maintained between the bleb and CB? Our photobleaching and ultracentrifugation experiments indicate extensive protein aggregation. We hypothesize that protein aggregation within the CB shifts the chemical equilibrium and draws proteins out of the bleb; at the same time, aggregation reduces the effective molar concentration of protein in the CB, so that osmotic equilibrium between high-protein CB and low-protein necrotic blebs becomes possible.

Spike-protein proteolytic antibodies in COVID-19 convalescent plasma contribute to SARS-CoV-2 neutralization.

Understanding the mechanisms of antibody-mediated neutralization of SARS-CoV-2 is critical in combating the COVID-19 pandemic. Based on previous reports of antibody catalysis, we investigated the proteolysis of spike (S) by antibodies in COVID-19 convalescent plasma (CCP) and its contribution to viral neutralization. Quenched fluorescent peptides were designed based on S epitopes to sensitively detect antibody-mediated proteolysis. We observed epitope cleavage by CCP from different donors which persisted when plasma was heat-treated or when IgG was isolated from plasma. Further, purified CCP antibodies proteolyzed recombinant S domains, as well as authentic viral S. Cleavage of S variants suggests CCP antibody-mediated proteolysis is a durable phenomenon despite antigenic drift. We differentiated viral neutralization occurring via direct interference with receptor binding from that occurring by antibody-mediated proteolysis, demonstrating that antibody catalysis enhanced neutralization. These results suggest that antibody-catalyzed damage of S is an immunologically relevant function of neutralizing antibodies against SARS-CoV-2.

Melanization of Candida auris Is Associated with Alteration of Extracellular pH.

Candida auris is a recently emerged global fungal pathogen, which causes life-threatening infections, often in healthcare settings. C. auris infections are worrisome because the fungus is often resistant to multiple antifungal drug classes. Furthermore, C. auris forms durable and difficult to remove biofilms. Due to the relatively recent, resilient, and resistant nature of C. auris, we investigated whether it produces the common fungal virulence factor melanin. Melanin is a black-brown pigment typically produced following enzymatic oxidation of aromatic precursors, which promotes fungal virulence through oxidative stress resistance, mammalian immune response evasion, and antifungal peptide and pharmaceutical inactivation. We found that certain strains of C. auris oxidized L-DOPA and catecholamines into melanin. Melanization occurred extracellularly in a process mediated by alkalinization of the extracellular environment, resulting in granule-like structures that adhere to the fungus' external surface. C. auris had relatively high cell surface hydrophobicity, but there was no correlation between hydrophobicity and melanization. Melanin protected the fungus from oxidative damage, but we did not observe a protective role during infection of macrophages or Galleria mellonella larvae. In summary, C. auris alkalinizes the extracellular medium, which promotes the non-enzymatic oxidation of L-DOPA to melanin that attaches to its surface, thus illustrating a novel mechanism for fungal melanization.