UNASSIGNED: The diffusion of cell components such as proteins is crucial to the function of all living cells. The abundance of macromolecules in cells is likely to cause a state of macromolecular crowding, but its effects on the extent of diffusion remain poorly understood.
UNASSIGNED: Here we investigate the diffusion rate in three distinct locations in mesenchymal cell types, namely the open cytoplasm, the stress fibers in the open cytoplasm, and those below the nucleus using three kinds of biologically inert green fluorescent proteins (GFPs), namely a monomer, dimer, and trimer GFP. Fluorescence correlation spectroscopy (FCS) was used to determine the diffusion coefficients.
UNASSIGNED: We show that diffusion tends to be lowered on average in stress fibers and is significantly lower in those located below the nucleus. Our data suggest that the diffusive properties of GFPs, and potentially other molecules as well, are hindered by macromolecular crowding. However, although the size dependence on protein diffusion was also studied for monomer, dimer, and trimer GFPs, there was no significant difference in the diffusion rates among the GFPs of these sizes. These results could be attributed to the lack of significant change in protein size among the selected GFP multimers.
UNASSIGNED: The data presented here would provide a basis for better understanding of the complex protein diffusion in the nonuniform cytoplasm, shedding light on cellular responses to mechanical stress, their local mechanical properties, and reduced turnover in senescent cells.

Allogeneic serum and tissue-specific extracellular matrix have been shown to maintain permanently differentiated cell phenotype in culture. This is of particular importance for human tenocytes, a cell population that readily loses its function during ex vivo culture. With these in mind, herein we extracted human tenocytes using either foetal bovine serum or human serum, cultured them in the absence and presence of carrageenan and Ficoll®, the most widely used macromolecular crowding agents (to induce tissue-specific extracellular matrix deposition), and assessed cellular function, via metabolic activity, viability, proliferation and immunofluorescence for collagen related molecules, non-collagenous molecules and transmembrane molecules. At day 7, longest time point assessed, neither carrageenan nor Ficoll® significantly affected metabolic activity, viability and proliferation in either serum and human serum significantly increased metabolic activity and proliferation. At day 7, in the absence of macromolecular crowding, cells in human serum deposited significantly lower collagen type VI, biglycan, versican and tenomodulin than cells in foetal bovine serum. Interestingly, at day 7, in comparison to the no macromolecular crowding group, carrageenan in foetal bovine serum induced the highest effect, as judged by the highest number of significantly increased molecules (collagen type I, collagen type IV, collagen type V, collagen type VI, transforming growth factor β1, matrix metalloproteinase 14, lumican, versican, scleraxis and integrin α2β1). These data, although contradict previous observations where human serum outperformed foetal bovine serum, at the same time, support the use of foetal bovine serum in the development of cell-based medicines.

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.

The pre-integration steps of the HIV-1 viral cycle are some of the most valuable targets of recent therapeutic innovations. HIV-1 integrase (IN) displays multiple functions, thanks to its considerable conformational flexibility. Recently, such flexible proteins have been characterized by their ability to form biomolecular condensates as a result of Liquid-Liquid-Phase-Separation (LLPS), allowing them to evolve in a restricted microenvironment within cells called membrane-less organelles (MLO). The LLPS context constitutes a more physiological approach to study the integration of molecular mechanisms performed by intasomes (complexes containing viral DNA, IN, and its cellular cofactor LEDGF/p75). We investigated here if such complexes can form LLPS in vitro and if IN enzymatic activities were affected by this LLPS environment. We observed that the LLPS formed by IN-LEDGF/p75 functional complexes modulate the in vitro IN activities. While the 3\'-processing of viral DNA ends was drastically reduced inside LLPS, viral DNA strand transfer was strongly enhanced. These two catalytic IN activities appear thus tightly regulated by the environment encountered by intasomes.

Even though tissue-engineered medicines are under intense academic, clinical, and commercial investigation, only a handful of products have been commercialised, primarily due to the costs associated with their prolonged manufacturing. While macromolecular crowding has been shown to enhance and accelerate extracellular matrix deposition in eukaryotic cell culture, possibly offering a solution in this procrastinating tissue-engineered medicine development, there is still no widely accepted macromolecular crowding agent. With these in mind, we herein assessed the potential of gum Arabic, gum gellan, gum karaya, and gum xanthan as macromolecular crowding agents in WS1 skin fibroblast cultures (no macromolecular crowding and carrageenan were used as a control). Dynamic light scattering analysis revealed that all macromolecules had negative charge and were polydispersed. None of the macromolecules affected basic cellular function. At day 7 (the longest time point assessed), gel electrophoresis analysis revealed that all macromolecules significantly increased collagen type I deposition in comparison to the non-macromolecular crowding group. Also at day 7, immunofluorescence analysis revealed that carrageenan; the 50 µg/mL, 75 µg/mL, and 100 µg/mL gum gellan; and the 500 µg/mL and 1000 µg/mL gum xanthan significantly increased both collagen type I and collagen type III deposition and only carrageenan significantly increased collagen type V deposition, all in comparison to the non-macromolecular crowding group at the respective time point. This preliminary study demonstrates the potential of gums as macromolecular crowding agents, but more detailed biological studies are needed to fully exploit their potential in the development of tissue-engineered medicines.

BACKGROUND: The contractile phenotype of vascular smooth muscle cells (VSMCs) results in good diastolic and contractile capacities, and its altered function is the main pathophysiological basis for diseases such as hypertension. VSMCs exist as a synthetic phenotype in vitro, making it challenging to maintain a contractile phenotype for research. It is widely recognized that the common medium in vitro is significantly less crowded than in the in vivo environment. Additionally, VSMCs have a heightened sense for detecting changes in medium crowding. However, it is unclear whether macromolecular crowding (MMC) helps maintain the VSMCs contractile phenotype.
OBJECTIVE: This study aimed to explore the phenotypic, behavioral and gene expression changes of VSMCs after increasing the crowding degree by adding carrageenan (CR).
METHODS: The degree of medium crowding was examined by a dynamic light scattering assay; VSMCs survival and activity were examined by calcein/PI cell activity and toxicity and CCK-8 assays; VSMCs phenotypes and migration were examined by WB and wound healing assays; and gene expression was examined by transcriptomic analysis and RT-qPCR.
RESULTS: Notably, 225 μg/mL CR significantly increased the crowding degree of the medium and did not affect cell survival. Simultaneously, CR significantly promoted the contraction phenotypic marker expression in VSMCs, shortened cell length, decreased cell proliferation, and inhibited cell migration. CR significantly altered gene expression in VSMCs. Specifically, 856 genes were upregulated and 1207 genes were downregulated. These alterations primarily affect the cellular ion channel transport, microtubule movement, respiratory metabolism, amino acid transport, and extracellular matrix synthesis. The upregulated genes were primarily involved in the cytoskeleton and contraction processes of VSMCs, whereas the downregulated genes were mainly involved in extracellular matrix synthesis.
CONCLUSIONS: The in vitro study showed that VSMCs can maintain the contractile phenotype by sensing changes in the crowding of the culture environment, which can be maintained by adding CR.

Metabolites play crucial roles in cellular processes, yet their diffusion in the densely packed interiors of cells remains poorly understood, compounded by conflicting reports in existing studies. Here, we employ pulsed-gradient stimulated-echo NMR and Brownian/Stokesian dynamics simulations to elucidate the behavior of nano- and subnanometer-sized tracers in crowded environments. Using Ficoll as a crowder, we observe a linear decrease in tracer diffusivity with increasing occupied volume fraction, persisting─somewhat surprisingly─up to volume fractions of 30-40%. While simulations suggest a linear correlation between diffusivity slowdown and particle size, experimental findings hint at a more intricate relationship, possibly influenced by Ficoll\'s porosity. Simulations and numerical calculations of tracer diffusivity in the E. coli cytoplasm show a nonlinear yet monotonic diffusion slowdown with particle size. We discuss our results in the context of nanoviscosity and discrepancies with existing studies.

The oral mucosa represents a defensive barrier between the external environment and the rest of the body. Oral mucosal cells are constantly bathed in hypotonic saliva (normally one-third tonicity compared to plasma) and are repeatedly exposed to environmental stresses of tonicity, temperature, and pH by the drinks we imbibe (e.g., hypotonic: water, tea, and coffee; hypertonic: assorted fruit juices, and red wines). In the mouth, the broad-spectrum antiviral mediator MxA (a dynamin-family large GTPase) is constitutively expressed in healthy periodontal tissues and induced by Type III interferons (e.g., IFN-λ1/IL-29). Endogenously induced human MxA and exogenously expressed human GFP-MxA formed membraneless biomolecular condensates in the cytoplasm of oral carcinoma cells (OECM1 cell line). These condensates likely represent storage granules in equilibrium with antivirally active dispersed MxA. Remarkably, cytoplasmic MxA condensates were exquisitely sensitive sensors of hypotonicity-the condensates in oral epithelium disassembled within 1-2 min of exposure of cells to saliva-like one-third hypotonicity, and spontaneously reassembled in the next 4-7 min. Water, tea, and coffee enhanced this disassembly. Fluorescence changes in OECM1 cells preloaded with calcein-AM (a reporter of cytosolic \"macromolecular crowding\") confirmed that this process involved macromolecular uncrowding and subsequent recrowding secondary to changes in cell volume. However, hypertonicity had little effect on MxA condensates. The spontaneous reassembly of GFP-MxA condensates in oral epithelial cells, even under continuous saliva-like hypotonicity, was slowed by the protein-phosphatase-inhibitor cyclosporin A (CsA) and by the K-channel-blocker tetraethylammonium chloride (TEA); this is suggestive of the involvement of the volume-sensitive WNK kinase-protein phosphatase (PTP)-K-Cl cotransporter (KCC) pathway in the regulated volume decrease (RVD) during condensate reassembly in oral cells. The present study identifies a novel subcellular consequence of hypotonic stress in oral epithelial cells, in terms of the rapid and dynamic changes in the structure of one class of phase-separated biomolecular condensates in the cytoplasm-the antiviral MxA condensates. More generally, the data raise the possibility that hypotonicity-driven stresses likely affect other intracellular functions involving liquid-liquid phase separation (LLPS) in cells of the oral mucosa.

Cells remodel their cytoskeletal networks to adapt to their environment. Here, we analyze the mechanisms utilized by the cell to tailor its microtubule landscape in response to changes in osmolarity that alter macromolecular crowding. By integrating live-cell imaging, ex vivo enzymatic assays, and in vitro reconstitution, we probe the impact of cytoplasmic density on microtubule-associated proteins (MAPs) and tubulin posttranslational modifications (PTMs). We find that human epithelial cells respond to fluctuations in cytoplasmic density by modulating microtubule acetylation, detyrosination, or MAP7 association without differentially affecting polyglutamylation, tyrosination, or MAP4 association. These MAP-PTM combinations alter intracellular cargo transport, enabling the cell to respond to osmotic challenges. We further dissect the molecular mechanisms governing tubulin PTM specification and find that MAP7 promotes acetylation and inhibits detyrosination. Our data identify MAP7 in modulating the tubulin code, resulting in microtubule cytoskeleton remodeling and alteration of intracellular transport as an integrated mechanism of cellular adaptation.

Osmotic stress conditions occur at multiple stages of plant life. Changes in water availability caused by osmotic stress induce alterations in the mechanical properties of the plasma membrane, its interaction with the cell wall, and the concentration of macromolecules in the cytoplasm. We summarize the reported players involved in the sensing mechanisms of osmotic stress in plants. We discuss how changes in macromolecular crowding are perceived intracellularly by intrinsically disordered regions (IDRs) in proteins. Finally, we review methods for dynamically monitoring macromolecular crowding in living cells and discuss why their implementation is required for the discovery of new plant osmosensors. Elucidating the osmosensing mechanisms will be essential for designing strategies to improve plant productivity in the face of climate change.