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Nanoscale Viscosity of Cytoplasm Is Conserved in HumanCell Lines

Karina Kwapiszewska†,*,Krzysztof Szczepański†,Tomasz Kalwarczyk†,Bernadeta Michalska‡,Paulina Patalas-Krawczyk‡,Jędrzej Szymański‡,Tomasz Andryszewski†,Michalina Iwan†,Jerzy Duszyński‡,Robert Hołyst†,*
†Instituteof Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, Warsaw, 01-224, Poland
‡NenckiInstitute of Experimental Biology, Pasteura 3, Warsaw, 02-093, Poland
*

Email:kkwapiszewska@ichf.edu.pl.

*

Email:rholyst@ichf.edu.pl.

Received 2020 Jun 5; Accepted 2020 Jul 31; Issue date 2020 Aug 20.

Copyright © 2020 American Chemical Society

This is an open access article published under a Creative Commons Attribution (CC-BY)License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.

PMCID: PMC7450658  PMID:32787203

Abstract

graphic file with name jz0c01748_0006.jpg

Metabolicreactions in living cells are limited by diffusion ofreagents in the cytoplasm. Any attempt to quantify the kinetics ofbiochemical reactions in the cytosol should be preceded by carefulmeasurements of the physical properties of the cellular interior.The cytoplasm is a complex, crowded fluid characterized by effectiveviscosity dependent on its structure at a nanoscopic length scale.In this work, we present and validate the model describing the cytoplasmicnanoviscosity, based on measurements in seven human cell lines, fornanoprobes ranging in diameters from 1 to 150 nm. Irrespective ofcell line origin (epithelial–mesenchymal, cancerous–noncancerous,male–female, young–adult), we obtained a similar dependenceof the viscosity on the size of the nanoprobes, with characteristiclength-scales of 20 ± 11 nm (hydrodynamic radii of major crowdersin the cytoplasm) and 4.6 ± 0.7 nm (radii of intercrowder gaps).Moreover, we revealed that the cytoplasm behaves as a liquid for lengthscales smaller than 100 nm and as a physical gel for larger lengthscales.

Metabolism at the cellular levelis considered as a network of reactions between biomolecules.1,2 These reactions maintain a balance where any prolonged disturbancecan lead to pathological changes, including cell death or systemicdiseases.3,4 From the physical point of view, a reactioncan occur when molecules of reagents approach each other. In an equilibrium-statesolution, Brownian motion (free diffusion) is a source of the movementof particles, and an increase of diffusion rate increases the probabilityof molecular encounters leading to biochemical reactions. The cytoplasmis a complex and crowded medium, where diffusion of biomolecules ishindered, and therefore diffusion can be treated as a factor limitingreaction rates in a cell.5,6 Decrease of diffusionrates would decrease rates of metabolic reactions and could lead tocell damage.7

According to the Stokes–Sutherland–Einsteinrelation,8,9 the diffusion coefficient depends inverselyon hydrodynamic drag,f = 6πηeffrp, whererp is the hydrodynamicradius of a probe and ηeff is an effective viscosityof the medium. Many reports show that viscosity of the cytoplasm isnot constant, but rather spatially heterogeneous.10−12 Additionally,according to our research, scale-dependent heterogeneity of cytoplasmicviscosity is even more pronounced.13−15 We found that objectsof different sizes can experience different viscosity: the viscosityincreases with the increasing size of the object.13 It is an outcome of the complex composition of cytoplasm—variouscomponents provide obstacles at different length-scales: the onlyobstacle of similar or smaller size can hinder the diffusion of aprobe (seeFigure1: I). Our previous, detailed works on polymer and colloidal solutionsresulted in a comprehensive model of length-scale dependent viscosity(LSDV), applicable for complex fluids13,16−19

graphic file with name jz0c01748_m001.jpg1

where η0 is the viscosityof a reference buffer, A is a pre-exponential factor of the orderof 1, ξ andRH are length scalescharacteristic for a given system, anda is an exponentof the order of unity.RH can be interpretedas a hydrodynamic radius of the main crowders, while ξ refersto an effective intercrowder gap, including a weak interactions factor.18,20 In such a fluid, small molecules (rp ≪ ξ) experience viscosity of the solvent, while bigtracers (rp ≫RH) experience viscosity measurable by macroscopic methods.To distinguish viscosity experienced by nanoobjects, we introducea term of nanoviscosity. We further presented applicability of thismodel to complex biological fluids, like cytosol of prokaryotic andeukaryotic cells,5,13 and we experimentally provedand applied this model for determination of oligomerization stateof proteins in living cells.15,21

Figure 1.

Figure 1

Principle of the researchon cytoplasmic nanoviscosity. (I) Assumptionsof the length-scale dependent viscosity (LSDV) model: (Ia) cytoplasmis a complex liquid containing components of various sizes. Thus,diffusion of the probes of different hydrodynamic radii (rp) is hindered by different cytoplasmic obstacles. Inthe result (Ib), effective viscosity (ηeff) probedby tracers of different sizes increase with the size of the tracer.(II) To examine ηeff, fluorescently labeled tracersare introduced to the cytoplasm—the mode of introduction isoptimized for a given probe. (III) Next, FCS measurements are performed:(IIIa) Confocal spot is positioned in the cytoplasmic area of thecell, and fluorescence fluctuations are registered, (IIIb) autocorrelationcurve (ACC) is calculated for the acquired data, and (IIIc) ACC isfitted with a proper diffusion model, and diffusion coefficient ofthe tracer is derived. (IV) Data collected for a set of tracers ina given cell line is used for quantitative description of the LSDVmodel: (IVa) ηeff experienced by the given probeis calculated from the diffusion coefficient, andrp (IVb) results are plotted and fitted witheq1; (IVc) LSDV profiles are comparedbetween different cell lines.

The LSDV model relies onRH and ξparameters, which reflect the length scales characterizing the structureof the fluid. For the simplest case of complex fluid—a singlepolymer in a continuous solvent—RH is defined as a hydrodynamic radius of polymer molecules, whileξ is mesh size or distance between intersections of polymerchains.19 In the case of the cytoplasm,there are different types of crowders (proteins, macromolecular complexes,organelles, or cytoskeleton), and thus only effectiveRH,eff and ξeff can be derived. Theseparameters seemed to be unique for every cell type and culture conditions.Cells of different types differ in terms of morphology, function,or gene expression. These differences can also have an impact on nanoviscosity-likenumbers, and types of metabolites and proteins would vary.

Inthis paper, we present a systematic, experimental study on nanoviscosityprofiles of seven different cell types. The principle of this workis shown inFigure1. Biologically inert tracers (dye molecules, fluorescent proteins,fluorescently labeled polymers, and nanoparticles) of sizerp were introduced into cells, and their diffusioncoefficients were measured by fluorescence correlation spectroscopy(FCS). Many works utilize FCS or its variants in cells;10,22−30 however, the systematic study on the nanoviscosity at differentlength scales—necessary for proper data analysis—isstill needed. Performance of FCS in living cells enabled reliableresults achievable in mild, physiologically relevant conditions.14,15,31 Tracers were chosen to coverall length scales essential for cell physiology (diameters from 1to 150 nm): metabolites, macromolecular complexes, proteins, nucleicacids, and vesicles. Cell lines were chosen to cover representativesof each group: cancerous or normal; epithelial or mesenchymal; maleor female donor. Effective viscosity was measured at different lengthscales in every cell line, and it was confirmed that effective viscosityof cytoplasm is length-scale dependent on the majority of human celllines.

Length-Scale Dependent Viscosity of Cytoplasm

The LSVDmodel predicts that tracers of different hydrodynamicradii would experience different effective viscosity of cytoplasm,as only those obstacles which are of similar or smaller size thanthe tracer would have an impact on ηeff (Figure1, panel I). To confirmthis prediction, tracers of defined hydrodynamic radii, ranging from0.65 to 81 nm, were introduced to cytoplasmic area of cells via microinjection(dextrans and nanoparticles), passive inflow (Calcein-AM), or biosynthesisupon transfection (proteins) (Figure1, panel II). We applied the core–shell typeof nanoparticles to avoid the impact of nanoparticle size on FCS measurements.32,33 Full information on tracers used in the experiments is presentedin Supporting Information 1 and 2 (SI 1, SI 2). Cells filled with tracers at final concentrations of 1–100nM in the cytoplasm were further examined under the confocal microscope.Focal volume was positioned in the cytoplasmic area of viable cells,and FCS data was acquired (Figure1, panel III). Each FCS experiment was preceded withcareful calibration (seeSI 1).14,34 Diffusion coefficients were derived for each type of probe (seeSI 3 for details), and results were averagedfor each of the cell lines considered in this study. Diffusion coefficientsobtained in the cytoplasm (D) were compared to diffusioncoefficients measured in water (D0) forthe same probes and temperature. Following the Stokes–Sutherland–Einsteinrelation, relative viscosity was calculated as follows: ηeff/η0 =D0/D. ηeff/η0 experiencedby the probe was plotted againstrp foreach of the cell lines (Figure1, panel IV).

The results obtained for six cell lines(HeLa, HepG2, MCF-7, A549,HSAEC, and U2-Os) are compiled inFigure2. Error bars represent standard deviationsreflecting the intercellular variability of the results. Possibleintracellular variability was neglected, as discussed inSI 4. For each of the cell lines listed above,the effective viscosity of the cytoplasm is increasing with the sizeof the probing tracer. Although absolute values of ηeff slightly differ in particular cell lines, the trend is common inall cells of this group. The results were fitted with the LSDV model(eq1), with followingparameters:RH = 20 ± 11 nm, ξ= 4.6 ± 0.7 nm,a = 0.57 ± 0.14. A wasfixed to 1.3 following our previous results.13 The values of the parameters of the LSDV model provide informationregarding the rheological structure of the cytosol.19 Exponenta < 1 is characteristic forentangled polymer solutions.18,19RH is attributed to the size of major crowders in the complexliquid.RH = 20 nm suggests that majorcrowders are of diameters ∼40 nm, which correspond to largecytoplasmic structures, such as vesicles, mRNA molecules, or ribosomes.35−37 The length-scale ξ is defined as an average radius of a meshcreated by major crowders.19 ξ ≈4.6 nm corresponds to the size of proteins. Thus, diffusion of proteinsis affected by big crowders in the cytoplasm, while smaller metabolitesexperience viscosity of the solvent.

Figure 2.

Figure 2

Nanoviscosity measured in six differentcell lines. Experimentalresults are presented as scatter. Each point represents the averagevalue obtained from at least 10 cells from two independent inoculations.Error bars correspond to standard deviations. Dashed line representsLSDV model (eq1) fittedto experimental data with the following parameters:A = 1.3 (fixed),RH = 20 ± 11 nm,ξ = 4.6 ± 0.7 nm,a = 0.57 ± 0.14.Shading represents the error of the model calculated using the totaldifferential method.

Our results, presentedinFigure2, were comparedto measurements reported by other groups.10,13,30,38−40 The results of the comparison are shown in SI5 (Figure SI5). In general, our results are in goodagreement with scattered data reported by other groups, with mismatchesthat could be attributed to different methods of measurements.

Gel-likeStructure of Cytoplasm

Diffusion coefficients of the probesof hydrodynamic radii smallerthan 50 nm could have been measured in the cytoplasm using FCS. Largerprobes, however, were more challenging: only a few autocorrelationcurves were interpretable, and it was much too little for proper dataanalysis. We decided to support the FCS technique with its variant—RasterImage Correlation Spectroscopy (RICS).41

Fluorescent nanoparticles of diameters exceeding 100 nm wereintroducedvia microinjection to the cytoplasm of HeLa cells and fibroblasts,and RICS analysis was performed. It turned out that no diffusion-dependentcorrelation could have been detected using RICS. Frame-by-frame analysisof the pictures revealed that long time and range translational diffusioncould not be detected for nanoparticles ofrp > 50 nm (seeSupplementary Movie). On the contrary, nanoparticles are trapped and oscillating insingle spots. It seems like large cytoplasmic structures—likecytoskeleton or endoplasmic reticulum—create a gel-like structureof the mesh size ∼100 nm. The size of the mesh differs in differentcells or regions, as nanoparticles ofrp = 68 nm exhibited free diffusion (proper FCS autocorrelation curves)in several cases in HeLa cells. On the other hand, the majority ofimage series of nanoparticles ofrp =68 or 81 nm revealed particle trapping. Our observation of a gel-likestructure filled with a liquid phase is in good correlation with previousatomic force microscopy measurements.38

Nanoviscosity in Different Cells

There is striking complianceof the nanoviscosity profiles obtainedfor different cell lines (Figure3a–d). It seems that the LSDV model is universalregardless of the original tissue, type of the cell, gender, or ageof the donor. Although values of nanoviscosity for given length scalescan slightly differ between different cells, the overall trend issimilar—the nanoviscosity is length-scale dependent. The majorityof batteries used in the study exhibit cytoplasmic viscosity of approximately2 viscosities of water for probes ofrp < 1 nm, while for probes ofrp >20 nm the nanoviscosity reaches the value of approximately 10 viscositiesof water. We assumed four factors that could impact nanometabolismvia nanoviscosity of the cytoplasm: tissue type (epithelial or mesenchymal),disease (cancerous or noncancerous), gender of the donor (male orfemale), and age of the donor (young or adult); seeSI 6. No differences could have been spotted between the cellgroups in any of the four categories. Additionally, for our furtherwork, we profiled nanoviscosity of other cell lines (primary mammaryepithelium and triple-negative breast cancer cells; data not shown),and their nanoviscosity is comparable with the values presented inFigure2. Stability of thecytoplasmic nanoviscosity is particularly surprising for the caseof cancer and healthy cells, which are reported to differ in termsof microscopic rheological parameters.42,43

Figure 3.

Figure 3

Comparisonof nanoviscosity in different cell types. Graphs representaverage relative nanoviscosity measured in the cytoplasm of differentcells and plotted against hydrodynamic radii of the tracers probingthe viscosity (data consistent withFigure2) (a–d) Cell lines used in the studywere divided into groups (seeSI 5), accordingto (a) tissue origin, (b) disease, (c) gender of donor, or (d) ageof donor. No deviations of the viscosity could have been observedbetween these groups. (e) Fibroblasts were the only cell line in whichnanoviscosity was found to differ from the major trend for small probes(rp < 10 nm).

The presented results show that nanoviscosity is somehow conservedin human cells, apart from the viscosity of the cytoplasmic matrixof small molecules (rp < 1 nm); theLSDV profiles—depending on the abundance of organelles andmacromolecules—are also the same. This stability is a surprisingresult, in terms of widely reported variability in cell sizes,44 as well as protein expression levels.45 In our previous work,31 we presented that nanoviscosity sensed by EGFP (rp = 2.3 nm) is also constant (with a slight, 30% increaseduring S phase) during the whole cell cycle of HeLa cells. These results,together with those presented in the present work, provide a pictureof stable nanoviscosity in human cells. Future questions arise fromthese observations: whether nanoviscosity has a biological impactand is conserved on a level optimal for cell homeostasis.

Fibroblasts ExhibitDifferent Nanoviscosity than Other Cells

Primary skin fibroblastsare the only cells for which nanoviscosityprofile is not length-scale dependent in the range of length scalesof 1 nm <rp < 20 nm. Thus, thenanoviscosity profile of fibroblasts deviates from the results forall other cell lines (Figure3: e). It is a surprising result, as other mesenchymal (Figure3: a) or noncancerous(Figure3: b) cellsexhibited “usual” LSDV profiles. On the other hand,cytoplasmic viscosities were similar in fibroblasts and other cellsfor the probes larger than 20 nm. The nanoviscosity for smaller probesin the cytoplasm of fibroblasts was independent of the passage number(seeSI 7).

To investigate a potentialsource of differences in nanoviscosity,we imaged large cytoplasmic obstacles (cytoskeleton: actin and tubulin,and endoplasmic reticulum, ER) in fibroblasts, HeLa, A549, and U2-Oscells (Figure4). Fibroblastswere imaged as cells of interest, according to their extraordinarynanoviscosity. HeLa and A549 were chosen as control cancer epithelialcells, while U2-Os were selected as control cancer mesenchymal cells.In the first experiment (Figure4: a), actin and tubulin were stained using ligandsspecific for these proteins (phalloidin-based and paclitaxel-based,respectively). At least ten cells were imaged for every cell type.No distinct differences in cytoskeleton abundances were observed.The second experiment (Figure4: b) included the immunostaining of ER. Again, at least tencells were imaged for every cell type. In this variant, it was observedthat the ER is much more abundant in fibroblasts than other cells.The abundance of the stained ER was quantified (seeSI 8), and results are presented inFigure5. We decided to take into account the totalsize of the ER, rather than the signal intensity, which may vary fromcell to cell according to different protein expression levels.46 A significant difference in ER abundance wasobserved between fibroblasts and other cells: ER covered an averageof 67% of the cytoplasmic area in fibroblasts, while in A549, HeLa,and U2-Os, it was 37%, 43%, and 38%, respectively. As a complement,the cytosol (liquid phase of cytoplasm) of the fibroblasts was compressedinto 33% of the cytoplasmic volume, while in other cells, it is anaverage of 61%.

Figure 4.

Figure 4

Confocal images of subcellular structures of four celllines: A549,HeLa, U2-Os, and Fibroblasts. (a) Staining of cytoskeletal proteins(actin and tubulin) showed no particular differences between celltypes. (b) Immunostaining of endoplasmic reticulum (ER) revealed ahigh abundance of ER in fibroblasts comparing to three other celllines. Scale bars correspond to 10 μm.

Figure 5.

Figure 5

Quantificationof ER abundance in different cell types. (a) Exampleconfocal images of ER in different cells. (b) Average abundance ofER (white pixels) and cytosol (black pixels) in cells of various types.

From the diffusion point of view, the endoplasmicreticulum isa set of membrane walls crossing the medium. Its presence is includedin the ηeff measured in our FCS experiments. Thefocal volume has a cross section of diameter ∼400 nm, whichcan consist of ER cisterna or other membrane obstacles (such as mitochondria,lysosomes, etc.). With the higher ER or organelle abundance, the numberof membrane walls increases. There is a known phenomenon of near-walldiffusion hindrance,47 causing an increaseof effective viscosity. Also, our previous studies on lamellar phasesrevealed an increase of continuous phase viscosity, comparing to thesame solvent with no lamella.48 These observationsare consistent with our measurements in fibroblasts—more abundantER can possibly cause matrix viscosity increase. This effect is lesspronounced for bigger length scales—for tracers ofrp > 20 nm, cytoplasmic viscosities of fibroblastsreach values similar to every other cell line examined in this study.

To conclude, we performed a systematic study on cytoplasmic nanostructurein seven different cell types. Cell lines used in this study representeddifferent origins (epithelial or mesenchymal, cancer or healthy, maleor female, young or adult). We probed cytoplasmic nanoviscosity atlength scales in the range of 1–150 nm, revealing length-scaledependent viscosity profiles present in the majority of cells. Weprovided the model equation describing nanoviscosity, and derivedlength scales characteristic for the cytoplasm. It was shown thatmRNA, ribosomes, and vesicles are major cytoplasmic crowders. It wasalso demonstrated that nanoparticles of diameters bigger than 100nm are unable to diffuse freely through the cytoplasm, suggestinga critical length scale crossover to gel-like structure in the cytoplasm.

The cytoplasmic nanoviscosity is conserved in the majority of humancell lines. The only cells differing from the major trend are fibroblasts.The potential source of this discrepancy can be the abundance of intracellularmembrane structures, which we identified at the example of the endoplasmicreticulum. Though, the length-scale dependent viscosity model seemsto be universal for human cells, regardless of age, disease, or typeof tissue. Moreover, in our previous work,31 we presented the stability of cytoplasmic viscosity for the wholecell cycle. All these results indicate that nanoviscosity can playa vital role in cellular homeostasis maintenance, and some unknownmechanism keeps it stable in single cells and between cell types.These observations open a new field of questions about the role andregulation of the physical properties of cells.

Acknowledgments

The authors would like to acknowledge Dr. KrzysztofSozański for his help with RICS experiments. This work wassupported by the Maestro grant UMO-2016/22/A/ST4/00017 from the NationalScience Centre, Poland.

Supporting Information Available

The Supporting Information isavailable free of charge athttps://pubs.acs.org/doi/10.1021/acs.jpclett.0c01748.

  • Materials and methods;Characteristics of fluorescenttracers; Autocorrelation curve fitting; Variability of nanoviscosity:intracellular vs intercellular; Comparison of cytoplasmic viscosityreported in different studies; Types of cells used in experiments;Effect of passage number on the viscosity of fibroblasts; Quantificationof ER abundance (PDF)

  • Supplementary movie: Raster Image Correlation Spectroscopy(MOV)

The authorsdeclare no competing financial interest.

Supplementary Material

jz0c01748_si_001.pdf (635.3KB, pdf)

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