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Cell Volume Change Through Water Efflux Impacts Cell Stiffness And Cell volume change through water efflux impacts cell PNAS PLUS stiffness and stem cell fate Ming Guoa,1, Adrian F. Pegoraroa, Angelo Maoa,b, Enhua H. Zhouc,2, Praveen R. Aranyd,e, Yulong Hana,f, Dylan T. Burnetteg, Mikkel H. Jensena,h, Karen E. Kaszaa,i, Jeffrey R. Moorej, Frederick C. Mackintoshk,l,m, Jeffrey J. Fredbergc, David J. Mooneya,b, Jennifer Lippincott-Schwartzn,3, and David A. Weitza,o,3 aJohn A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138; bWyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA 02138; cHarvard T. H. Chan School of Public Health, Boston, MA 02115; dDepartment of Oral Biology, University at Buffalo, Buffalo, NY 14214; eDepartment of Biomedical Engineering, University at Buffalo, Buffalo, NY 14214; fBiomedical Engineering and Biomechanics Center, Xi’an Jiaotong University, Xi’an 710049, China; gDepartment of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232; hDepartment of Physics and Astronomy, California State University, Sacramento, CA 95819; iDepartment of Mechanical Engineering, Columbia University, New York, NY 10027; jDepartment of Biological Sciences, University of Massachusetts at Lowell, Lowell, MA 01854; kDepartment of Physics and Astronomy, VU University, 1081 HV Amsterdam, The Netherlands; lDepartment of Chemical and Biomolecular Engineering, Rice University, Houston, TX 77030; mCenter for Theoretical Biophysics, Rice University, Houston, TX 77030; nHoward Hughes Medical Institute, Janelia Research Campus, Ashburn, VA 20147; and oDepartment of Physics, Harvard University, Cambridge, MA 02138 Contributed by Jennifer Lippincott-Schwartz, August 26, 2017 (sent for review March 29, 2017; reviewed by Daniel A. Fletcher and Valerie M. Weaver) Cells alter their mechanical properties in response to their local similar manner to that observed for cells under external osmotic microenvironment; this plays a role in determining cell function and pressure: Both the cortical and cytoplasmic stiffness increase as can even influence stem cell fate. Here, we identify a robust and the volume decreases; the nuclear volume also decreases as cell unified relationship between cell stiffness and cell volume. As a cell volume decreases. Moreover, stem-cell differentiation is also spreads on a substrate, its volume decreases, while its stiffness strongly impacted by cell volume changes. These results suggest concomitantly increases. We find that both cortical and cytoplasmic that cells in different environments can change their volume cell stiffness scale with volume for numerous perturbations, in- through water efflux. This leads to changes in molecular crowding cluding varying substrate stiffness, cell spread area, and external and impacts the mechanics, physiology, and behavior of the cell, osmotic pressure. The reduction of cell volume is a result of water having far-reaching consequences on cell fate. CELL BIOLOGY efflux, which leads to a corresponding increase in intracellular molecular crowding. Furthermore, we find that changes in cell Results volume, and hence stiffness, alter stem-cell differentiation, regard- Bulk and Shear Moduli of Cells Increase as Cell Volume Decreases Under less of the method by which these are induced. These observations External Osmotic Compression. To directly probe the consequences reveal a surprising, previously unidentified relationship between of changes in cell volume, we control the external osmotic pres- cell stiffness and cell volume that strongly influences cell biology. sure through the addition of 300-Da polyethylene glycol (PEG 300) (8, 11). We measure the cell volume by fluorescently labeling PHYSICS cell volume | cell mechanics | molecular crowding | gene expression | stem cell fate Significance ell volume is a highly regulated property that affects myriad Cell volume is thought to be a well-controlled cellular charac- Cfunctions (1, 2). It changes over the course of the cell life teristic, increasing as a cell grows, while macromolecular den- cycle, increasing as the cell plasma membrane grows and the sity is maintained. We report that cell volume can also change amount of protein, DNA, and other intracellular material in- in response to external physical cues, leading to water influx/ creases (3). However, it can also change on a much more rapid efflux, which causes significant changes in subcellular macro- time scale, as, for example, on cell migration through confined molecular density. This is observed when cells spread out on a spaces (4, 5); in this case, the volume change is a result of water substrate: Cells reduce their volume and increase their molec- transport out of the cell. This causes increased concentration of ular crowding due to an accompanying water efflux. Exploring intracellular material and molecular crowding, having numerous this phenomenon further, we removed water from mesen- important consequences (6, 7). Alternately, the volume of a cell chymal stem cells through osmotic pressure and found this was can be directly changed through application of an external osmotic sufficient to alter their differentiation pathway. Based on these pressure. This forces water out of the cell, which also decreases results, we suggest cells chart different differentiation and cell volume, increases the concentration of intracellular material, behavioral pathways by sensing/altering their cytoplasmic and intensifies molecular crowding. Application of an external volume and density through changes in water influx/efflux. osmotic pressure to reduce cell volume also has other pronounced Author contributions: M.G., J.L.-S., and D.A.W. designed research; M.G., A.F.P., A.M., manifestations: For example, it leads to a significant change in cell E.H.Z., P.R.A., Y.H., D.T.B., and K.E.K. performed research; M.G., A.M., E.H.Z., P.R.A., mechanics, resulting in an increase in stiffness (8); it also impacts D.T.B., M.H.J., K.E.K., J.R.M., and D.J.M. contributed new reagents/analytic tools; M.G., folding and transport of proteins (9), as well as condensation of A.F.P., Y.H., F.C.M., J.J.F., and D.A.W. analyzed data; and M.G., A.F.P., J.L.-S., and D.A.W. chromatin (10). These dramatic effects of osmotic-induced volume wrote the paper. change on cell behavior raise the question of whether cells ever Reviewers: D.A.F., University of California, Berkeley; and V.M.W., University of California, change their volume through water efflux under isotonic condi- San Francisco. tions, perhaps to modulate their mechanics and behavior through The authors declare no conflict of interest. 1 changes in molecular crowding. Present address: Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139. Here, we demonstrate that when cells are cultured under the 2Present address: Ophthalmology, Novartis Institutes of BioMedical Research, Cambridge, same isotonic conditions, but under stiffer extracellular environ- MA 02139. ments, they reduce their cell volume through water efflux out of 3To whom correspondence may be addressed. Email: [email protected]. the cell, and this has a large and significant impact on cell me- org or [email protected]. chanics and cell physiology. Specifically, as a cell spreads out on a This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. stiff substrate, its volume decreases, and the cell behaves in a 1073/pnas.1705179114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1705179114 PNAS Early Edition | 1of10 the cytoplasm and cell surface and use confocal microscopy to and is defined as B = −VdP/dV ∼−VΔP/ΔV. By varying the identify the boundaries of the cell in 3D (12, 13). The measured osmotic pressure and measuring the resultant volume of A7 cells volumes have been shown to be consistent with those measured cultured on a glass substrate, we measure B and find that it in- by atomic force microscopy (AFM) (8). We further validate our creases as cell volume decreases, as shown by the points at the confocal measurements by comparing them to those made by top in Fig. 1A. Using the expression for the volume dependence using high-resolution structured illumination microscopy (SIM) on of P, we can predict the behavior of the bulk modulus, B = 2 the same sample; the measured cell volumes are consistent for NkBTV/(V − Vmin) . This functional form provides an excellent both techniques, indicating a measurement uncertainty of <10% fit to the data, as shown by the dashed line through the points at (Fig. S1). Because cell volume naturally changes during the cell the top in Fig. 1A. The value of Vmin determined by the fit is cycle, we make all our observations after starving cells overnight; 2,053 ± 30 μm3; this agrees with the minimum volume measured however, measurements made under standard culture conditions when the cells are exposed to extreme osmotic compression to exhibit the same behavior, albeit with increased variability. remove all osmotically active water, ∼2,100 μm3. The fit also To explore the dependence of cell volume on external osmotic provides a value for N, and this gives a concentration of pressure, we culture A7 cells on glass substrates and add increasing ∼200 mM, which is consistent with the known salt concentration concentrations of PEG 300 to the medium; PEG 300 does not within a cell (15). The excellent fitting with a constant N suggests penetrate the cell membrane and thus increases the external os- that the total amounts of ions
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