Increase In Cytoplasmic Viscosity Research Articles

Membraneless Organelles and Viscosity Changes during Desiccation: A New Phase in Anhydrobiosis

AfrLEA6 is an intrinsically disordered protein found in the anhydrobiotic cysts of Artemia franciscana. AfrLEA6 shares homology with plant seed maturation proteins (SMPs), which have been linked to the longevity of orthodox seeds in the desiccated state. Therefore, it was hypothesized that ectopically expressing AfrLEA6 will increase the desiccation tolerance of a desiccation‐sensitive Drosophila melanogaster (Kc167) cell line. Surprisingly, ectopically expressed AfrLEA6 was found to undergo a liquid‐liquid phase separation (LLPS) in the cytoplasm of Kc167 cells. The overall architecture of the protein is composed of two SMP domains connected with a glycine‐ and proline‐rich spacer to a potential protein‐binding domain. It was determined that AfrLEA6 LLPS is dependent on the SMP domains, regulated by polysome assembly and disassembly, and may best be described as a specialized biphasic stress granule. It is speculated that AfrLEA6 stress granules protect desiccation‐sensitive proteins prior to glassy‐state formation during water loss. This hypothesis was strengthened by the observation that expression of AfrLEA6 in Kc167 cells increases their desiccation tolerance. Unexpectedly, AfrLEA6 also increases intracellular viscosity during desiccation compared to control cells, which reduced plasma membrane fusion between adjacent cells and may protect against internal sheer stresses. These data agree with recently reported cellular dielectrophoretic data, that AfrLEA6 expressed in Kc167 cells underwent a shift to become more entangled when cells were exposed to osmotic stress, thereby suggesting an increase intracellular viscosity. In A. franciscana, a rapid increase in cytoplasmic viscosity at relatively high‐water contents may be key to successfully entering the anhydrobiotic state, while trehalose glass formation at relatively low‐water contents may create a more stable intracellular environment for long‐term dormancy.Support or Funding InformationSupported by NSF IOS‐1659970.

GTx011, an Anti-Sickling Compound, Improves SS Blood Rheology By Reduction of HbS polymerization Via Allosteric Modulation of O2 Affinity

Sickle cell disease is caused by a mutation in the β-globin gene leading to production of hemoglobin S (HbS). HbS polymerizes under hypoxic conditions, leading to changes in cytoplasmic viscosity and formation of rigid, non-deformable sickled red blood cells (RBC). Loss of RBC deformability leads to abnormal blood rheology, diminished oxygen (O2) delivery and contributes to vaso-occlusion. GTx011 is a novel orally bioavailable small molecule that has been shown to inhibit HbS polymerization in vitro by maintaining oxygenated HbS under hypoxic conditions. We investigated the mechanism by which GTx011 modulates O2 affinity. Although it binds to the N-terminal α chain of Hb via a reversible Schiff base, 1D 1H NMR studies indicated that the compound allosterically influenced the intra-dimer interface of Hb (α122His and α103His), and the distal valine surrounding heme pockets for both the α and β chains, suggesting a unique solution phase structure. GTx011’s unique allosteric action upon the heme pocket allows it to improve O2 affinity without sterically blocking the release of O2. This modulation of O2 affinity delays the onset of HbS polymers and therefore we hypothesized will improve the abnormal blood rheology observed in SS blood.We report here the beneficial effect of GTx011 on SSRBC deformability using four independent whole cell systems: 1) the profile of SSRBC during passage through a gel filtration column, 2) the ability and corresponding pressure required for SSRBCs to pass through a polycarbonate filter under hypoxic conditions, 3) the tension required to aspirate SSRBCs into a micropipette under low O2 and 4) changes to blood viscosity during deoxygenation. Under deoxygenating conditions, GTx011 dose dependently enabled RBC deformability required for migration of SSRBCs through a gel filtration column. The pressure required to pass SSRBCs through a polycarbonate filter and the tension required to aspirate SSRBCs into a micropipette under hypoxic conditions were also reduced (Table 1). We have established a novel blood viscosity assay, which employs an enzymatic reaction (ascorbic acid/ascorbic oxidase) for deoxygenation of blood, followed by traditional measurements in cone plate viscometers. This protocol enabled us to reduce O2 levels without the technical limitations of using sodium dithionite, laser induced deoxygenation or passive deoxygenation via N2. Time dependent measures of viscosity, at a fixed shear rate, allowed us to quantify GTx011-induced delay in the onset of polymerization, and the increase in cytoplasmic viscosity. As in polymerization experiments, GTx011 dose dependently delayed the onset of hyperviscosity of SS blood under deoxygenating conditions. HbS modification ranging from 10-30%, was sufficient to achieve an improvement in SS blood hyperviscosity (Table 2)These data suggest that GTx011 is a promising agent that has the potential to improve SSRBC deformability and blood viscosity by inhibiting HbS polymerization, the pathophysiologic hallmark of sickle cell disease.Table 1AssayMigrationFilterabilityDeformabilityunit(% deformable cells)(mm Hg)(mDynes/cm2)[HbS]1 mM1 mM1 mM[GTx011](mM)00.5100.5100.51Readout1642893581621291013225Table 2AssayPolymerizationViscosity[HbS]50 μM1.5 mMGTx011 modified HbS(%)0103001030Delay time(min)131833212631 DisclosuresPatel:Global Blood Therapeutics: Employment, Equity Ownership. Cabrales:Global Blood Therapeutics: Consultancy. Dufu:Global Blood Therapeutics: Employment, Equity Ownership. Metcalf:Global Blood Therapeutics: Consultancy, Equity Ownership. Sinha:Global Blood Therapeutics: Employment, Equity Ownership.

A decrease in bulk water and mannitol and accumulation of trehalose and trehalose-based oligosaccharides define a two-stage maturation process towards extreme stress resistance in ascospores of Neosartorya fischeri (Aspergillus fischeri).

Fungal propagules survive stresses better than vegetative cells. Neosartorya fischeri, an Aspergillus teleomorph, forms ascospores that survive high temperatures or drying followed by heat. Not much is known about maturation and development of extreme stress resistance in fungal cells. This study provides a novel two-step model for the acquisition of extreme stress resistance and entry into dormancy. Ascospores of 11- and 15-day-old cultures exhibited heat resistance, physiological activity, accumulation of compatible solutes and a steep increase in cytoplasmic viscosity. Electron spin resonance spectroscopy indicated that this stage is associated with the removal of bulk water and an increase of chemical stability. Older ascospores from 15- to 50-day-old cultures showed no changes in compatible solute content and cytoplasmic viscosity, but did exhibit a further increase of heat resistance and redox stability with age. This stage was also characterized by changes in the composition of the mixture of compatible solutes. Mannitol levels decreased and the relative quantities of trehalose and trehalose-based oligosaccharides increased. Dormant ascospores of N. fischeri survive in low-water habitats. After activation of the germination process, the stress resistance decreases, compatible solutes are degraded and the cellular viscosity drops. After 5 h, the hydrated cells enter the vegetative stage and redox stability has decreased notably.

Ballistic intracellular nanorheology reveals ROCK-hard cytoplasmic stiffening response to fluid flow

Cells in vivo are constantly subjected to mechanical shear stresses that play important regulatory roles in various physiological and pathological processes. Cytoskeletal reorganizations that occur in response to shear flow have been studied extensively, but whether the cytoplasm of an adherent cell adapts its mechanical properties to respond to shear is largely unknown. Here we develop a new method where fluorescent nanoparticles are ballistically injected into the cells to probe, with high resolution, possible local viscoelastic changes in the cytoplasm of individual cells subjected to fluid flow. This new assay, ballistic intracellular nanorheology (BIN), reveals that shear flow induces a dramatic sustained 25-fold increase in cytoplasmic viscosity in serum-starved Swiss 3T3 fibroblasts. By contrast, cells stimulated with the actin contractile agonist LPA show highly transient stiffening of much lower amplitude, despite the formation of similar cytoskeletal structures. Shear-induced cytoplasmic stiffening is attenuated by inhibiting actomyosin interactions and is entirely eliminated by specific Rho-kinase (ROCK) inhibition. Together, these results show that biochemical and biophysical stimuli may elicit the formation of qualitatively similar cytoskeleton structures (i.e. stress fibers and focal adhesions), but induces quantitatively different micromechanical responses. Our results suggest that when an adherent cell is subjected to shear stresses, its first order of action is to prevent detachment from its substratum by greatly stiffening its cytoplasm through enhanced actin assembly and Rho-kinase mediated contractility.

Cytomechanical Properties of Papaver Pollen Tubes Are Altered after Self-Incompatibility Challenge

Self-incompatibility (SI) in Papaver rhoeas triggers a ligand-mediated signal transduction cascade, resulting in the inhibition of incompatible pollen tube growth. Using a cytomechanical approach we have demonstrated that dramatic changes to the mechanical properties of incompatible pollen tubes are stimulated by SI induction. Microindentation revealed that SI resulted in a reduction of cellular stiffness and an increase in cytoplasmic viscosity. Whereas the former cellular response is likely to be the result of a drop in cellular turgor, we hypothesize that the latter is caused by as yet unidentified cross-linking events. F-actin rearrangements, a characteristic phenomenon for SI challenge in Papaver, displayed a spatiotemporal gradient along the pollen tube; this suggests that signal propagation occurs in a basipetal direction. However, unexpectedly, local application of SI inducing S-protein did not reveal any evidence for localized signal perception in the apical or subapical regions of the pollen tube. To our knowledge this represents the first mechanospatial approach to study signal propagation and cellular responses in a well-characterized plant cell system. Our data provide the first evidence for mechanical changes induced in the cytoplasm of a plant cell stimulated by a defined ligand.

Fluorogenic compound hydrolysis as a measure of toxicity-induced cytoplasmic viscosity and pH changes

If a fluorogenic compound, such as fluorescein diacetate, is added to a water solution containing living cells it becomes hydrolyzed by intracellular esterases into a fluorochrome whose fluorescence can be used to monitor the cytoplasmic pH and the cytoplasmic viscosity of the cells. In this paper we have used this technique to measure the effects of different concentrations of Co 2+ and Cd 2+ ions on the cytoplasmic pH and the cytoplasmic viscosity of a single cell culture. Our results indicate that the observed decrease in the efficiency of the intracellular hydrolyzation of fluorogenic substances in the presence of different concentrations of heavy metals could be caused by both a decrease in the cytoplasmic pH and an increase in the cytoplasmic viscosity. A decrease in cytoplasmic pH would decrease the effectiveness of the intracellular enzymes, whereas an increase in cytoplasmic viscosity would decrease diffusion which would also reduce the effectiveness of the reaction. The dependence of the reciprocal of the cytoplasmic viscosity on the concentration of these metals correlates well with published results on their toxicity.

Carbohydrate induced modulation of cell membrane I. Interaction of sialic acid with peripheral blood lymphocytes: A spin label study

Sialic acid is now known to serve as ligand for lymphocyte lectins known as selectins. Although its role as a ligand for adhesion molecules has been studied extensively, no studies have been performed to determine the physiological changes in lymphocytes after binding of sialic acid to lymphocyte lectin. We report for the first time that interaction of lymphocytes with sialic acid severely restricts the rotational mobility of the cell surface proteins as well as membrane lipids, as studied by EPR spectroscopy using spin probes. Binding of mucin totally immobilizes the lymphocyte membrane. Surprisingly the binding of sialic acid or mucin also immobilized the aqueous probe TEMPO, indicating an appreciable increase in cytoplasmic viscosity.

Inositol-less death in yeast results in a simultaneous increase in intracellular viscosity

Inositol auxotrophs of yeast developing on isositol-deficient medium continue protein synthesis for 4-6 h, lose viability rapidly after 6 h, and show an increase in cytoplasmic viscosity as measured by spin label rotational motion. Cycloheximide prevents the rapid loss of cell viability, stops protein synthesis, and simultaneously prevents an increase in cytoplasmic viscosity. From these observations, we infer that intracellular translational diffusion is upset as a consequence of inositol starvation. Cell death may be caused by a modified intracellular diffusion environment.

Heavy water inhibition of cell division: an approach to mechanism.

ABS>At concentrations higher than about 75%, deuterium oxide blocks cell division in the eggs of the sea urchin Arbacia punctulata and in those of the annelid Chaetopterus pergamentaceus. The block is reversible, although cells that have been kept is a deuterium-rich medium for long periods relative to the time for one mitotic cycle may show multipolar and irregular divisions after removal to normal water, and may subsequently cytolyze. The antimitotic action is manifested at all stages of the mitotic cycle and, to a significant extent, during cytokinesis. An important feature of the mitotic block is that it becomes effective almost immediately after the cells are placed in D2O and is quickly reversed when the cells are washed free of D2O. Whereas D2O blocks mitosis at all stages it does not destroy the achromatic apparatus, nor does it cause wholesale regression of cleavage furrows. Instead, it exerts a powerful stabilizing effect upon these structures. This appears to be related to a sharp and reversible increase in cytoplasmic viscosity evoked by immersion of nondividing cells in D2O. Examination of a number of alternative explanations of the effect leaves as the most useful working hypothesis a rigidification of the mitotic apparatus, possibly throughmore » a small increase in the strength of individual hydrogen bonds which cross-link macromolecules or small particles. For large, multiply cross-linked structures, such changes for individual bonds would produce very large cooperative effects in the direction required by the data.« less