Structure-function relationships in the stem cell's mechanical world A: seeding protocols as a means to control shape and fate of live stem cells

Shape and fate are intrinsic manifestations of form and function at the cell scale. Here we hypothesize that seeding density and protocol affect the form and function of live embryonic murine mesenchymal stem cells (MSCs) and their nuclei. First, the imperative for study of live cells was demonstrated in studies showing changes in cell nucleus shape that were attributable to fixation per se. Hence, we compared live cell and nuclear volume and shape between groups of a model MSC line (C3H10T1/2) seeded at, or proliferated from 5,000 cells/cm2 to one of three target densities to achieve targeted development contexts. Cell volume was shown to be dependent on initial seeding density whereas nucleus shape was shown to depend on developmental context but not seeding density. Both smaller cell volumes and flatter nuclei were found to correlate with increased expression of markers for mesenchymal condensation as well as chondrogenic and osteogenic differentiation but a decreased expression of pre-condensation and adipogenic markers. Considering the data presented here, both seeding density and protocol significantly alter the morphology of mesenchymal stem cells even at very early stages of cell culture. Thus, these design parameters may play a critical role in the success of tissue engineering strategies seeking to recreate condensation events. However, a better understanding of how these changes in cell volume and nucleus shape relate to the differentiation of MSCs is important for prescribing precise seeding conditions necessary for the development of the desired tissue type. In a companion study (Part B, following), we address the effect of concomitant volume and shape changing stresses on spatiotemporal distribution of the cytoskeletal proteins actin and tubulin. Taken together, these studies bring us one step closer to our ultimate goal of elucidating the dynamics of nucleus and cell shape change as tissue templates grow (cell proliferation) and specialize (cell differentiation).     

Endocytosis and exocytosis protect cells against severe membrane tension variations

The ability of cells to regulate their shape and volume is critical for many cell functions. How endocytosis and exocytosis, as important ways of membrane trafficking, affect cellular volume regulation is still unclear. Here, we develop a theoretical framework to study the dynamics of cell volume, endocytosis, and exocytosis in response to osmotic shocks and mechanical loadings. This model can not only explain observed dynamics of endocytosis and exocytosis during osmotic shocks but also predict the dynamics of endocytosis and exocytosis during cell compressions. We find that a hypotonic shock stimulates exocytosis, while a hypertonic shock stimulates endocytosis; and exocytosis in turn allows cells to have a dramatic change in cell volume but a small change in membrane tension during hyposmotic swelling, protecting cells from rupture under high tension. In addition, we find that cell compressions with various loading speeds induce three distinct dynamic modes of endocytosis and exocytosis. Finally, we show that increasing endocytosis and exocytosis rates reduce the changes in cell volume and membrane tension under fast cell compression, whereas they enhance the changes in cell volume and membrane tension under slow cell compression. Together, our findings reveal critical roles of endocytosis and exocytosis in regulating cell volume and membrane tension.     

Cell Growth and Division: I. A Mathematical Model with Applications to Cell Volume Distributions in Mammalian Suspension Cultures

A mathematical model is formulated for the development of a population of cells in which the individual members may grow and divide or die. A given cell is characterized by its age and volume, and these parameters are assumed to determine the rate of volume growth and the probability per unit time of division or death. The initial value problem is formulated, and it is shown that if cell growth rate is proportional to cell volume, then the volume distribution will not converge to a time-invariant shape without an added dispersive mechanism. Mathematical simplications which are possible for the special case of populations in the exponential phase or in the steady state are considered in some detail. Experimental volume distributions of mammalian cells in exponentially growing suspension cultures are analyzed, and growth rates and division probabilities are deduced. It is concluded that the cell volume growth rate is approximately proportional to cell volume and that the division probability increases with volume above a critical threshold. The effects on volume distribution of division into daughter cells of unequal volumes are examined in computer models.     

Formation of cytoplasts from giant protoplasts in culture

Summary Protoplasts isolated from calli ofNicotiana plumbaginifolia can dramatically increase in volume without showing indications of cell wall synthesis. After reaching a critical size, the plasma-rich giant protoplasts show multiple formation of cytoplasts, which are released from the mother cells. The anucleate cytoplasts display the same increase in size as the nucleate protoplasts. Both cell types retain a spherical shape for several months, indicating that no major synthesis of cell wall occurred.     

The interrelationship of cell growth and division in haploid and diploid cells of Saccharomyces cerevisiae

The coordination of cell growth and division has been examined in isogenic haploid and diploid strains of Saccharomyces cerevisiae. The average cell volume of the haploid and diploid cells was unaffected by a range of environmental conditions and generation times. For most environments and generation times the mean cell volume of diploid cells was between 1.52 and 1.83 of the haploid cell volume. Both haploid and diploid cell volumes were reduced drastically when the cells were grown in the chemostat with glucose as the limiting substrate. In this environment diploid cells have the same mean cell volume as haploid cells. Diploid cells are more elongated than haploid cells, and the characteristic shape (eccentricity) of the cells is unaffected by all environmental conditions and generation times tested. Mother cell volume increased during the cell cycle, although the pattern of this increase was affected by the environmental conditions. Under most growth conditions detectable mother cell volume increase occurred only during the budding phase, whereas under conditions of carbon limitation detectable increase only occurred during the unbudded phase. A consequence of this result is that the mean cell volume of haploids at bud initiation is relatively constant in all environments, including carbon limitation. This suggests that there is a critical size for bud initiation for haploids which is constant and independent of environmental conditions. The results for diploids are more complex. Coordination of growth and division in haploid cells can be explained by a simple model initially developed for prokaryotes by Donachie. A modification of this model is proposed to account for the results with diploids.     

Joint modeling of cell and nuclear shape variation

Modeling cell shape variation is critical to our understanding of cell biology. Previous work has demonstrated the utility of nonrigid image registration methods for the construction of nonparametric nuclear shape models in which pairwise deformation distances are measured between all shapes and are embedded into a low-dimensional shape space. Using these methods, we explore the relationship between cell shape and nuclear shape. We find that these are frequently dependent on each other and use this as the motivation for the development of combined cell and nuclear shape space models, extending nonparametric cell representations to multiple-component three-dimensional cellular shapes and identifying modes of joint shape variation. We learn a first-order dynamics model to predict cell and nuclear shapes, given shapes at a previous time point. We use this to determine the effects of endogenous protein tags or drugs on the shape dynamics of cell lines and show that tagged C1QBP reduces the correlation between cell and nuclear shape. To reduce the computational cost of learning these models, we demonstrate the ability to reconstruct shape spaces using a fraction of computed pairwise distances. The open-source tools provide a powerful basis for future studies of the molecular basis of cell organization.     

Glutaraldehyde mediated echinocyte/discocyte transformation is Ca2+ dependent.

Echinocytes, which were produced from freshly banked blood by repeated washes in phosphate buffered saline, undergo a transformation to the discoid shape within less than 30 seconds of incubation in isotonic 0.05% glutaraldehyde pH 7.4. This echinocyte/discocyte transformation is not associated with a change of cell volume or critical hemolysis volume although a slight decrease of cellular deformability and a 4-8 fold increase of K+ efflux within 1 hour after glutaraldehyde incubation provide evidence of the fixative's attack on the cell membrane. Trypsination prior to the incubation in isotonic glutaraldehyde could not inhibit the shape change. Hypertonic glutaraldehyde solutions partially prevent the E/D transformation with regard to both the osmolarity of the medium and the permeability of the cell membrane. The glutaraldehyde stimulated transformation is entirely inhibited in the presence of a chelating agent the efficiency of which is overcome by addition of a more-than-equivalent amount of Ca2+. The mutual action of either agent is discussed, however, the mechanism of the phenomenon remains unclear.     

Hydrodynamics and cell volume oscillations in the pollen tube apical region are integral components of the biomechanics of Nicotiana tabacum pollen tube growth

Pollen tube growth is localized at the apex and displays oscillatory dynamics. It is thought that a balance between intracellular turgor pressure (hydrostatic pressure, reflected by the cell volume) and cell wall loosening is a critical factor driving pollen tube growth. We previously demonstrated that water flows freely into and out of the pollen tube apical region dependent on the extracellular osmotic potential, that cell volume changes reflect changes in the intracellular pressure, and that cell volume changes differentially induce, increases or decreases in specific phospholipid signals. This article shows that manipulation of the extracellular osmotic potential rapidly induces modulations in pollen tube growth rate frequencies, demonstrating that changes in the intracellular pressure are sufficient to reset the pollen tube growth oscillator. This indicates a direct link between intracellular hydrostatic pressure and pollen tube growth. Altering hydrodynamic flow through the pollen tube by replacing extracellular H₂O with ²H₂O adversely affects both cell volume and growth rate oscillations and induces aberrant morphologies. Normal growth and cell morphology are rescued by replacing ²H₂O with H₂O. Further studies revealed that the cell volume oscillates in the pollen tube apical region. These cell volume oscillations were not from changes in cell shape at the tip and were detectable up to 30 μm distal to the tip (the longest length measured). Cell volume in the apical region oscillates with the same frequency as growth rate oscillations but surprisingly the cycles are phase-shifted by 180°. Raman microscopy yields evidence that hydrodynamic flow out of the apex may be part of the biomechanics that drive cellular expansion. The combined results suggest that hydrodynamic loading/unloading in the apical region induces cell volume oscillations and has a role in driving cell elongation and pollen tube growth.     

Herpetomonas samuelpessoai: Role of subpellicular microtubules in shape transitions of trypanosomatids

There is a close association between changes in cell volume and shape transition of Herpetomonas samuelpessoai. A rearrangement of the spatial organization of subpellicular microtubules provides the structural basis for the process of shape transition. A model is presented which accounts for the relationship between microtubule arrangement, changes in cell volume, and transition from elongate (promastigote) to the more spherical (para- and opisthomastigote) forms. Its central feature consists of an asymmetrical departure from the regularly helicoidal distribution of the microtubules upon induction of transition. While some microtubules become more linear, others assume a compensatory overspiralized course, allowing for a modification of volume with slight or no change of the cell surface area.     

Cell volume and plasma membrane osmotic water permeability in epithelial cell layers measured by interferometry.

The development of strategies to measure plasma membrane osmotic water permeability (Pf) in epithelial cells has been motivated by the identification of a family of molecular water channels. A general approach utilizing interferometry to measure cell shape and volume was developed and applied to measure Pf in cell layers. The method is based on the cell volume dependence of optical path length (OPL) for a light beam passing through the cell. The small changes in OPL were measured by interferometry. A mathematical model was developed to relate the interference signal to cell volume changes for cells of arbitrary shape and size. To validate the model, a Mach-Zehnder interference microscope was used to image OPL in an Madin Darby Canine Kidney (MDCK) cell layer and to reconstruct the three-dimensional cell shape (OPL resolution < lambda/25). As predicted by the model, a doubling of cell volume resulted in a change in OPL that was proportional to the difference in refractive indices between water and the extracellular medium. The time course of relative cell volume in response to an osmotic gradient was computed from serial interference images. To measure cell volume without microscopy and image analysis, a Mach-Zehnder interferometer was constructed in which one of two interfering laser beams passed through a flow chamber containing the cell layer. The interference signal in response to an osmotic gradient was analyzed to quantify the time course of relative cell volume. The calculated MDCK cell plasma membrane Pf of 6.1 x 10(-4) cm/s at 24 degrees C agreed with that obtained by interference microscopy and by a total internal reflection fluorescence method. Interferometry was also applied to measure the apical plasma membrane water permeability of intact toad urinary bladder; Pf increased fivefold after forskolin stimulation to 0.04 cm/s at 23 degrees C. These results establish and validate the application of interferometry to quantify cell volume and osmotic water permeability in cell layers.     

Stomatal mechanics: volume changes during opening

Abstract. The determination of guard cell lumen volume in relation to its geometric characteristic dimensions is presented. Stomatal movements can be divided into two stages: Spannungsphase and motorphase, with a transition point between them. During the Spannungsphase movement, the lumen volume increases as a result of the change in its geometric shape. At transition, the lumen volume is approximated by a portion of a circular tube with a rounded cross-section. The volume increase during the motorphase comes from three different sources: expansion by wall stretching, increasing inner cross-section of a guard cell by wall thinning, and polar expansion. The relationship between the aperture and the lumen volume is also established. The results set forth in this geometric model are essential to studies of the pressure interaction between a guard cell and its surrounding epidermal cells.     

Electric sizing of platelets stimulated by adenosine diphosphate (ADP)

The platelet-rich plasma stimulated by a low dose of adenosine diphosphate (ADP) was examined by an electric counter, oscilloscopy, an electron microscopy and packed cell volume analysis. The size distribution curve obtained by the electric counter shifted to the left, and the cell volume was decreased. On the oscillograph, those small cells were seen as pulse signals with a regular shape. However, microscopic observation did not find as many microplatelets or fragments. Also, packed cell volume analysis showed no difference between ADP-stimulated and unstimulated samples. On the electronmicrograph, a slight aggregation was seen, with many of the ADP-stimulated platelets exhibiting spiny forms. The shape factors influences strongly the electric sizing, thus, the deformation of platelets may be the reason for the disagreement between the results by electric observation and those by other methods. Our results indicated that the small platelets observed on the size distribution curve do not always reflect the actual small cells.     

Shape and Volume Changes in Frog Erythrocytes Following Ultraviolet Radiation

Frog erythrocytes in Ringer's solution were exposed to ultraviolet radiation and then followed in camera lucida drawings for changes in shape and dimension. Cell thickness was found to increase while cell width remained constant throughout the period prior to hemolysis. The cell shortened and bulged at the ends during the middle third of the prolytic period while a region around the cell center remained constricted. When this constricted region gave way, the cell became spherical and hemolyzed. Cell volume as calculated from the cell's dimensions increased linearly with time throughout the prolytic period to hemolysis then dropped rapidly to a constant value somewhat higher than the original cell volume. These changes in shape and volume are consistent with a colloid osmotic type of hemolysis but with other factors acting to limit the rate of swelling and the forms assumed during the swelling process. The relationship between the time of hemolysis and the cell surface area exposed to the ultraviolet is discussed as it applies to the site of ultraviolet damage.     

CORRELATED EVOLUTION OF GENOME SIZE AND CELL VOLUME IN DIATOMS (BACILLARIOPHYCEAE)1

A correlation between genome size and cell volume has been observed across diverse assemblages of eukaryotes. We examined this relationship in diatoms (Bacillariophyceae), a phylum in which cell volume is of critical ecological and biogeochemical importance. In addition to testing whether there is a predictive relationship across extant species, we tested whether evolutionary divergences in genome size were correlated with evolutionary divergences in cell size (using independent contrasts). We estimated total DNA content for 16 diatom species using a flow cytometer and estimated cell volumes using critical dimensions with scaling equations. Our independent contrast analyses indicated a significant correlated evolution between genome size and cell volume. We then explored the evolutionary and ecological implications of this evolutionary relationship. Diatom cell volume is an important component of the global carbon cycle; therefore, understanding the mechanisms that drive diatom genome evolution has both evolutionary and ecological importance.     

THE IMPORTANCE OF DIATOM CELL SIZE IN COMMUNITY ANALYSIS1

The large variation in size and shape in diatoms is shown by morphometric measurements of 515 benthic and pelagic diatom species from the Baltic Sea area. The largest mean cell dimension (mostly the apical axis) varied between 4.2 and 653 μm, cell surface area between 55 and 344,000 μm², and cell volume between 21 and 14.2 × 10⁶μm³. The shape‐related index, length to width ratio, was between 1.0 and 63.3 and the shape‐ and size‐related index, surface area to volume ratio, was between 0.02 and 3.13. Diatom community analysis by multivariate statistics is usually based on counts of a fixed number of diatom valves with species scores irrespective of cell size. This procedure underestimates the large species for two reasons. First, the importance of a species with higher cell volume is usually larger in a community. Second, larger species usually have lower abundances and their occurrence in the diatom counts is stochastic. This article shows that co‐occurring small and large diatom species can respond very differently to environmental constraints. Large epiphytic diatoms responded most to macroalgal host species and small epiphytic diatoms most to environmental conditions at the sampling site. Large epilithic diatoms responded strongly to salinity, whereas small epilithic diatoms did so less clearly. The conclusion is that different scale‐dependent responses are possible within one data set. The results from the test data also show that important ecological information from diatom data can be missed when the large species are neglected or underestimated.     

Cell-Shape Regulation of Smooth Muscle Cell Proliferation

Vascular smooth muscle cells (SMCs) play an important role in vascular remodeling. Heterogeneity and phenotypic changes in SMCs are usually accompanied by a morphological difference, i.e., elongated/spindle-like versus spread-out or epithelioid/rhomboid cell shapes. However, it is not known whether the cell shape directly regulates SMC proliferation, and what the underlying mechanisms are. In this study, microgrooves and micropatterned matrix islands were used to engineer the cell shape and investigate the associated biophysical and biological mechanisms. Compared to spread-out SMCs on nonpatterned surfaces, SMCs on micropatterned surfaces demonstrated elongated morphology, significantly lower cell and nucleus shape indexes, less spreading, a lower proliferation rate, and a similar response (but to a lesser extent) to platelet-derived growth factor, transforming growth factor-β, and mechanical stretching. DNA microarray profiling revealed a lower expression of neuron-derived orphan receptor-1 (NOR-1) in elongated SMCs. Knocking down NOR-1 suppressed DNA synthesis in SMCs, suggesting that NOR-1 is a mediator of cell elongation effects. Regulation of DNA synthesis in SMCs by the cell shape alone and a decrease in DNA synthesis in the case of small cell spreading area were achieved by micropatterning SMCs on matrix islands of different shapes and spreading areas. Changes in the cell shape also affected the nucleus shape, whereas variations in the cell spreading area modulated the nucleus volume, indicating a possible link between nucleus morphology (both shape and volume) and DNA synthesis. The findings of this investigation provide insight into cell shape effects on cell structure and proliferation, and have direct implications for vascular pathophysiology.     

Stereological Analysis of Cotyledon Cell Development in Phaseolus: II. THE DEVELOPING COTYLEDON

Quantitative changes in organelle volume, number, and area duringcotyledon cell development in Phaseolus vulgaris are describedduring the early period of reserve protein synthesis. RoughER undergoes a 400-fold increase in area per cell, and alsochanges in shape; changes in mitochondrial volume do not parallelthe changes which occur in respiration rate; nuclear volumeincreases, and relationships between cell and nuclear volume,DNA content, and cell weight are discussed; plastid divisionappears to occur in steps.     

Abnormalities of elastic and transporting properties of red blood cells under development of apoptosis

The functional properties of erythrocytes under development of apoptotic process in these cells were investigated by the low angle light scattering technique. Apoptosis induced by ionomycin was associated with an initial decrease of cell volume and caused formation of echinocytes. After that the cells restored their volume forming rounded erythrocytes with rugged membrane capable to agglomerate with each other. At the late stages of apoptosis, small fragmented cells can be revealed. Preapoptotic red blood cells (at all stages of apoptosis) manifested an enormous tolerance to hypotonic loading, whereas control cells hemolyzed just after reaching a critical volume (∼150 fl). Acidic hemolysis cannot differentiate between control and preapoptotic erythrocytes, the cells being hemolyzed not reaching the critical volume. Placing the control erythrocytes to a medium with ammonia ions instead of sodium ions caused an initial increase of cell volume above the critical point, and then it was also followed by hemolysis. Under ammonia loading, an initial rate of the cell volume growth and a ratio of the hemolyzed cells were significantly reduced in preapoptotic cells.     

Effect of the level of ATP and of the state of spectrin on osmotic properties of bovine erythrocytes

The effect of the intracellular level of ATP and of the state of spectrin on the critical cell volume of bovine erythrocyte was studied. The state of spectrin was changed by thermal denaturation, which for the bovine red cell took place at similar temperature as for the human erythrocyte. The increase of the ATP level and the spectrin denaturation increased the critical cell volume, while metabolic starvation decreased it. The changes of the ATP level did not influence the critical volume after the denaturation of spectrin. The results suggest that the ATP-dependent effect on the critical cell volume was caused by an alteration of the membrane extensibility due to the change of the membrane skeleton-lipid bilayer interaction(s).     

Morphology of yeast cell wall as affected by genetic manipulation of beta(1 --> 6) glycosidic linkage

The morphology of yeast cells as it is affected by the glycosidic linkages of constituent glucan was studied. Four different strains of Saccharomyces cerevisiae were studied. A cell wall matrix particle representing the intact original morphology and composed entirely of beta-glucan was prepared. Using prepared cell wall glucan particles, the morphology and cell wall matrix structure were examined. Genetic modification of the cell wall structure during growth results in the alteration of the shape and hydrodnamic volume of the intact cell wall particles. The shape and hydrodynamic volume of the cell wall particles can also be modified by in vitro chemical and enzymatic treatment. The shape factor and hydrodynamic volume of the whole glucan cell wall matrix particles were evaluated quantitatively using a rheological analysis. An increased degree of beta(1 --> 6) cross-linking in the cell wall matrix induces a nearly 2-fold increase in the shape factor and a 10-fold increase in the compression modulus of the glucan particles. The disruption of beta(1 --> 6) glycosidic cross-linking causes the particles to swell by up to 18% of their original volume. This was used as a strategy to isolate a yeast mutant with a high beta(1 --> 6) glycosidic content in the cell wall glucan.