ABSTRACT
The shape of cell is intimately connected to its function; however, we do not fully understand the underlying mechanism by which global shape regulates cell functions. Here, using a combination of theory, experiments and simulation, we investigated how global cell curvature can affect numerous subcellular activities and organization to control signal flow needed for phenotypic function. We find that global cell curvature regulates organelle location, inter-organelle distances and differential distribution of receptors in the plasma membrane. A combination of these factors leads to the modulation of signals transduced by the M3 muscarinic receptor/Gq/PLCβ pathway at the plasma membrane, amplifying Ca2+ dynamics in the cytoplasm and the nucleus as determined by increased activity of myosin light chain kinase in the cytoplasm and enhanced nuclear localization of the transcription factor NFAT. Taken together, our observations show a systems level phenomenon whereby global cell curvature affects subcellular organization and signaling to enable expression of phenotype.
INTRODUCTION
Cells utilize receptors on the plasma membrane to transduce a range of extracellular signals to regulate function in the cytoplasm and the nucleus1. Reaction kinetics of the biochemical interactions that comprise the signaling networks regulate the temporal dynamics of activation and inactivation of signaling components and effectors2. However, information flow within cells is not just temporally regulated, it is also spatially regulated by the shape of the cell3–5 and surface-to-volume ratio at the plasma membrane6. An additional layer of complexity is conferred by the spatial transfer of information by signaling reactions that occur within or at intracellular organelles. Recently, studies have shown that signaling at the endosomes plays an important role in prolonging cAMP dynamics through GPCRs7 and in EGFR dynamics8. An important compartmental regulation of organelle-based signaling is Ca2+ dynamics, since endoplasmic/sarcoplasmic reticulum is a regulatable Ca2+ store in cells. Ca2+ is a ubiquitous signaling molecule that controls many cellular functions including fertilization, proliferation, migration and cell death9-12. Ca2+ is able to participate in controlling this diverse array of functions due to the precise control of Ca2+ concentration across the cell. In vascular smooth muscle cells (VSMC), Ca2+ regulates both contractility and gene expression through IP3-mediated Ca2+ release by IP3R receptors located on the membrane of the sarcoplasmic reticulum (SR) and through Ca2+ influx at the plasma membrane13-15. Ca2+-calmodulin activates myosin light chain kinase (MLCK), which phosphorylates the light chain of myosin, initiating contraction16. Ca2+ also activates protein kinases and phosphatases that regulate transcription regulators that define the phenotypic status of VSMC17. Ca2+ activates calcineurin, which dephosphorylates the transcription factor nuclear factor of activated T-cells (NFAT) in the cytoplasm, resulting in its nuclear accumulation and expression of NFAT-regulated genes18. Ca2+ also activates calmodulin kinase II (CaMKII), that phosphorylates the transcription factor serum response factor (SRF)19 which, as a complex with myocardin, controls the expression of proteins necessary for the contractile function of VSMC20.
VSMC in the medial layer of blood vessels are not terminally differentiated and can undergo phenotypic transitions during injury and disease states21-23. VSMC shape and function are closely related; increasing elongation or aspect ratio (AR, defined as the ratio of the short axis to long axis) is correlated with differentiation and contractility 24, 25. How cell elongation is mechanistically linked to contractility is poorly understood. Based on the observations that cell shape and Ca2+ signaling closely regulate the contractile phenotype of differentiated VSMCs, we hypothesized that cell shape regulates organelle location, including the relative distances between plasma membrane, endoplasmic/sarcoplasmic reticulum (ER/SR) and the nucleus, to modulate cellular function. In other words, we sought to answer the question -- how does cell shape dependent organelle localization drive spatial control of information transfer? The answer to this question is critical for understanding how mechanochemical relationships control cell shape, signaling, and phenotype. We used micropatterned substrates to culture VSMCs in different shapes and developed theoretical and computational models to represent the spatio-temporal dynamics of Ca2+ transients mediated by Muscarinic Receptor 3 (M3R)/Phospholipase Cβ (PLCβ) pathway. Activation of M3R mediates contractility in VSMC by activating PLCβ resulting in phosphoinositide hydrolysis and IP3 mediated Ca2+ release from the SR26, 27. Our studies show an unexpected modulation of organelle location as a function of membrane curvature and that this change in organelle location results in signal amplification in the cytoplasm and nucleus.
RESULTS
Reaction-Diffusion model predicts that the distance between PM and SR membrane affects signaling dynamics in the cytoplasmic volume
We tested the hypothesis that a change in membrane curvature and distance between two membranes affects signaling dynamics using a phenomenological reaction-diffusion model (Fig. 1a). A signaling molecule of interest, A, is produced at the PM with an on-rate kon (1/s) and binds to a receptor located at the SR membrane, with a rate koff (1/s) and is free to diffuse in the sandwiched cytoplasmic space and is degraded by a degrading enzyme with a rate kdeg (1/s). This model essentially captures the lifecycle of a second messenger such as IP3 that is produced at the PM through PIP2 hydrolysis by phospholipases, binds to inositol 3 phosphate receptor (IP3R) channel at the SR membrane and is degraded in the cytoplasm by 1,4,5-trisphosphate-5-phosphatase28. These events can be mathematically represented by the following system of reaction-diffusion equations. The dynamics of A in the cytoplasm are governed by diffusion and degradation and is given by,
Here, D is the diffusion coefficient of A (μm2/s), and CA is the concentration of A (μM). The boundary condition at the PM is a balance between the rate of production of A at the membrane and the diffusive flux from the membrane to the cellular interior and is given by,
Here, n is the normal vector to the surface and ∇ represents the gradient operator. At the SR membrane, similarly, we can write the boundary condition for the consumption of A as the balance of diffusive flux to the SR and consumption rate at the SR.
We solved these equations using finite element methods on three geometries (1) a rectangle (constant distance, zero curvature) (2) a circle, (constant distance, constant curvature) and (3) an elliptical sector, (varying distance, varying curvature) (Fig. 1b). In cases (1-2), the gradient of A is only along the radial direction (Fig. 1c). In triangular and trapezoidal geometries, varying PM-SR gradients result in two-dimensional gradients (Supplementary Fig. 1). However in case (3), the curvature of the membrane and the PM-SR distance will affect both the production and consumption of A at the PM and SR respectively. Hence, A varies both in the radial and angular directions, indicating that curvature and varying distances between the two membranes amplifies signaling gradients.
We next conducted dimensional analysis to identify when the dynamics of A would be diffusion-dominated or reaction-dominated. Non-dimensionalization of Eq. 1 results in a dimensionless group D/(kdegL2) the Damkohler number29. This dimensionless number is the ratio of diffusion time scale to reaction time scale. We found that the dynamics of A are diffusion dominated for physiological values of kdeg (Fig. 1d). Increasing kdeg many-fold or reducing the diffusion constant of A can result in the dynamics of A being reaction dominated for different PM-SR distances (Fig. 1d, shaded blue region). Both the increase in kdeg and the decrease in D, to obtain reaction-dominated regimes, are out of the range of physiological values, suggesting that the dynamics of A in the sandwiched space between the PM and SR are primarily diffusion dominated. From this simple phenomenological model, we predict that second messenger signaling networks such as IP3, where signaling occurs between PM and SR, will be impacted by global curvature and distance between the PM and the SR. This prediction raises the following questions: (1) does cell shape affect PM-SR distance? (2) Does changing PM-SR distance affect intracellular signaling dynamics? And (3) what is the impact of changing distance between organelles on signaling dynamics? We used VSMCs grown on micropatterned substrates to answer these questions.
Cell shape affects cytoskeleton organization and organelle location
We determined if changing cell shape affects organelle distribution and in particular PM-SR distance. In order to control the large scale cell shape, we used micropatterned substrates with the same surface area but increasing aspect ratio (AR, circles 1:1 to ellipses 1:8) (Fig. 2a). We investigated how cell shape affects cytoskeletal organization since the two are tightly interwoven30-32. Actin stress fibers increasingly oriented themselves along the long axis of the cell as the aspect ratio increased (Fig. 2b), indicating that the cells were responding to the mechanical forces and tension exerted by the substrate33. Microtubules became highly aligned and increasingly sparser in the cell tips compared to the cell body as the cell aspect ratio increased (Supplementary Fig. 2). Because cytoskeletal organization plays an important role in organelle distribution34, 35, we visualized the effect of cell shape on the bulk distribution and location of the mitochondria (Supplementary Fig. 3), endosomes (Supplementary Fig. 4) and Golgi membrane (Supplementary Fig. 5). These organelles increasingly localized to the cell center (perinuclear region) with increasing aspect ratio, similar to the characteristic central distribution of the endomembrane system in well-differentiated VSMC24. Because SR stores Ca2+, which controls both excitation-transcription coupling and contractility in VSMC36, we focused on SR distribution as a function of cell shape, using calnexin (Fig. 2c), protein disulfide isomerase (Fig. 2d), reticulon-4 (Fig. 2e) and bodipy glibenclamide (Supplementary Fig. 6) as SR markers. All four markers show that in circular cells, the SR was spread uniformly throughout the cell; increased aspect ratio induced the SR to localize in the perinuclear region and become significantly sparser, and mainly tubular, in the cell tips (Fig. 2c-e, Supplementary Fig. 6). Upon close inspection of Airy scan images of these SR markers, we qualitatively observe that in circular cells, the SR appeared to be equidistant from the plasma membrane along the periphery, i.e. there were no angular variations of PM-SR distance, while elliptical cells show a large angular variation in the PM-SR distance (Fig. 2d-e bottom panels and Supplementary Fig. 6, inset). We used transmission electron microscopy (TEM) to quantitatively confirm that the PM-SR distance is shape dependent. The cell periphery of circular cells and the cell body of elliptical cells showed long patches of smooth SR that were positioned close to the plasma membrane. PM-SR distance was indeed dependent on the shape of the cell: PM-SR distance in cell body of elliptical cells was significantly smaller compared to circular cells (Fig. 2g). In the tips of elliptical cells, the SR membrane formed fewer contacts with the plasma membrane, and showed significantly higher PM-SR distance (Fig. 2g). These results are consistent with the recent observation in neurons that PM-ER contacts are more extensive in the cell body compared to elongated projections such as dendrites and axons37. While other groups have reported that cell shape can affect organelle location38, 39, to our knowledge, this is the first quantitative characterization of PM-SR distances and the SR abundance along the juxtamembrane region with controlled cell shape variation. We then determined the relationship between global cell shape and distances between the nucleus and various organelles in VSMC. Nuclear aspect ratio increased with cellular aspect ratio (Fig. 2i) while nuclear size (area μm2) decreased with increasing cell aspect ratio (Fig. 2j). The nucleus became increasingly oriented along the major axis of the cell as the whole-cell aspect ratio increased, evidenced by polar graphs showing the orientation histograms of nuclei of cells in increasing cell AR (Fig. 2k). Circular shaped cells showed a random nuclear orientation whereas increasing the cellular aspect ratio progressively oriented the nucleus in the geometric center of the cell. PM-nuclear distance in the major axis of the cell increased with cell aspect ratio (Fig. 2m) while the PM-nuclear distance in the minor axis decreased with cell aspect ratio (Fig. 2n). These results indicate that in VSMC, cell elongation resulted in nuclear elongation, reduced nuclear size, and a decrease in the PM-nuclear distance in the minor axis of the cell (i.e. near the center of the cell). Thus, we show that cell shape affects not only organelle location, but also affects PM-SR distance, nuclear shape and PM-nuclear distances.
Computational models predict that IP3 spatio-temporal dynamics depend on nanometer scale changes in PM-SR distances
We next asked if changing PM-SR distance will impact the dynamics of IP3 in VSMCs. It is currently not possible to manipulate PM-SR distances with precise control in cells. Therefore, we developed a computational model for investigating how changing PM-SR distance would affect IP3 dynamics. This model is composed of a system of multi-compartmental partial differential equations, representing the reaction-diffusion of cytoplasmic species in the volume, coupled with boundary fluxes at the membranes. The reactions capture the biochemical interactions from the ligand binding and activation of M3R to IP3 production by PLC-mediated hydrolysis of PIP2 release from the SR (Fig. 3a, Supplementary Table 1). As before, we tested three different geometries – rectangles, circles, and ellipses. Each geometry consists of compartments representing the plasma membrane, the cytoplasm, the SR (modeled as thin rectangles) and the SR membrane (Fig. 3b). To test the effect of changing PM-SR distance on signaling dynamics independent of curvature, we computed the spatio-temporal profiles of IP3 in rectangular geometries. We found that varying the PM-SR distance played a critical role in determining the dynamics of IP3 (Fig. 3b, Supplementary Fig. 8). When the PM-SR distance is 50 nm, IP3 is concentrated between the PM and the SR, but as the PM-SR distance increases, the IP3 microdomain dissipates and no local increase in concentration is observed. Even in this highly idealized scenario, we find that there is a clear dependence of IP3 spatio-temporal dynamics on PM-SR distance. Furthermore, the SR acts as a diffusion barrier for IP3, preventing diffusion of IP3 away from the PM-SR region. The dynamics of IP3 in locations with and without SR (Fig. 3c) were also different, indicating that the diffusion distance set up by the PM-SR coupling plays an important role in governing the dynamics of second messengers such as IP3 and potentially Ca2+.
Next, we investigated the role of curvature coupling to PM-SR distances on IP3 and dynamics. We designed geometries that represented circular cells or elliptical cells with distance parameters based on experimental measurements from TEM analyses (Fig. 2g). We investigated the role of PM-SR distance in circular geometries. Again, we modeled the SR as thin rectangles and this time placed the SR at 98 nm away from the PM based on the experimental measurements. We found that the region between the PM and the SR acts as an IP3 microdomain since the SR not only consumes IP3 but also acts as a diffusion trap (Fig. 3e). However, we did not observe any angular variation along the periphery of the domain where the SR was present. We found that the angular variation for IP3 and Ca2+ dynamics was a result of the presence or absence of SR (Fig. 3c). There is a small radial variation at early times because the SR act as diffusion barrier but over longer times, these cytoplasmic gradients disappear (See different time points in Fig. 3c).
We also conducted simulations for elliptical geometries with the SR at the variable distance from the PM as per experimental measurements such that the shortest PM-SR distance is 67 nm and the farthest PM-SR distance is more than 150 nm. This allowed us investigate how varying curvature and varying PM-SR distance affects IP3 dynamics. We found that the curvature of the plasma membrane, coupled with variable PM-SR distance gave rise to variable IP3 dynamics that was a function of PM-SR distance along the periphery (Fig. 3f). We found that the total amount of IP3 produced over 5 min was higher in elliptical cells than in circular cells (Fig. 3g). Thus, our simulations indicate that PM-SR distance affects the IP3 dynamics and curvature coupling serves to amplify this effect.
Cell shape affects receptor activation on the membrane and intracellular calcium dynamics
We tested the model predictions that distance between PM and SR can affect the dynamics of Ca2+ mediated by the M3R/IP3/Ca2+ pathway13, 40. We stained for M3R in circular and elliptical cells under three different conditions - unstimulated, stimulated with carbachol, and stimulated with carbachol in the presence of hypertonic sucrose, which inhibits receptor endocytosis41-43. In both the basal state and stimulated states, M3R was uniformly distributed on the plasma membrane of both circular and elliptical cells (Supplementary Fig. 9). Interestingly, in elliptical cells, when M3R was stimulated and endocytosis was inhibited, M3R accumulated in the cell body compared to the cell tips while there was no observable spatial asymmetry in the distribution of M3R in circular cells (Fig. 4a and Supplementary Fig. 9). We then investigated the effect of shape on cytoplasmic and nuclear Ca2+ dynamics upon stimulation of M3R in patterned VSMC (Fig. 4b-c). In the cytoplasmic region, circular and elliptical showed similar peak Ca2+ amplitudes. However, elliptical cells showed a slower rate of decrease in Ca2+ compared to circular cells, resulting in a slightly higher temporally integrated Ca2+ compared to circular cells, although the differences were not statistically significant (p=0.057, two-tailed t-test). Since small changes in second messenger signals can have large functional effects by being amplified by signaling networks44, we measured downstream myosin light chain kinase (MLCK) activity using a CaM-sensor FRET probe45, 46 (Fig. 4e-g). Elliptical cells showed a higher degree of MLCK FRET probe localization on actin filaments (Fig. 4e) and higher maximal activation compared to circular cells (Fig. 4f-g), indicating that the shape-induced increase in cytoplasmic Ca2+ signal propagates and is amplified downstream through MLCK activation, consistent with the previous finding that higher aspect ratio VSMC are more contractile47, 48. To our knowledge, this is the first demonstration of a direct relationship between cell shape, Ca2+ signaling and contractility. The differences in nuclear Ca2+ between circle and elliptical cells were distinct from cytoplasmic Ca2+ (Fig. 4h). In the nuclear region, the peak Ca2+ amplitudes of circle and elliptical cells were similar. However, there is a notable delay in the rate of nuclear Ca2+ increase to maximum in elliptical cells compared to circular cells, but the decay times in elliptical cells were slower as well, resulting in a significantly higher temporally integrated Ca2+ in elliptical cells compared to circular cells (Fig. 4i). These results indicate that cell shape affects nuclear Ca2+ transients mediated by M3R/PLCβ and elliptical cells is more prolonged, resulting in higher integrated Ca2+ compared to circular cells. Increase in nuclear Ca2+ in elliptical cells is likely to impact the nucleo-cytoplasmic transport of NFAT, which exhibits a Ca2+/calcineurin dependent translocation to the nucleus18, 49. We measured NFAT-GFP localization dynamics in live VSMCs in elliptical and circular micropatterns in response to Gαq activation through M3R stimulation (Fig. 4j-m). Elliptical cells exhibited greater NFAT-GFP nuclear localization compared to circular cells (Fig. 4k) and on average displayed higher maximal NFATnuc/cyto (Fig. 4l). We further validated the differences in NFAT translocation by immunofluorescence of NFAT1 (Fig. 4m, Supplementary Fig. 10a). At basal levels, NFATnuc/cyto were similar between circular and elliptical cells. However, elliptical cells displayed higher nuclear NFAT compared to circular cells at 30 minutes and 1 hour after stimulation, consistent with live-cell NFAT-GFP translocation results. We asked the question whether other Ca2+ dependent transcription factors are also impacted by shape. Ca2+ also triggers the nuclear localization of SRF through nuclear Ca2+/CaMKIV19 and Rho/ROCK/actin dynamics50, 51. Elliptical VSMC show increased nuclear SRF compared to circular cells at both basal and stimulated levels (Supplementary Fig. 10b). In contrast there was no difference in myocardin intensity or translocation dynamics between circle and elliptical cells (Supplementary Fig. 10c) suggesting that myocardin is constitutively active20, 52 Taken together, these results indicate that shape-induced modulation of Ca2+ signaling alters the activities of Ca2+ dependent transcription factors.
An integrated model of shape and organelle location provides insight into curvature coupling of Ca2+ dynamics in VSMCs
Our previous models were focused on localized PM-SR interactions in highly idealized geometries (Fig. 3). However, ultrastructural analyses have shown that the ER is a highly dynamic, tubular network that occupies roughly 10% of the cytosolic volume and extends from the nucleus to the cell periphery53. We developed an integrated whole cell, multi-compartmental partial differential equation model in Virtual Cell to derive relationships between experimental observations and models at the whole cell level (Fig. 5a-l). Upon stimulation of M3R, the concentration gradients of IP3 and cytoplasmic Ca2+ were highest in regions where PM-SR distance was lowest (cell body), and lowest in regions where PM-SR were farthest from each other (cell tips) (Fig. 5a and Fig. 5c), establishing an IP3 and Ca2+ gradient from the cell body to the tips which progressively became steeper with aspect ratio. More importantly, increasing the AR increased global IP3 and Ca2+ levels in both cytoplasmic and nuclear compartments (Fig. 5b, Fig. 5d-h). Hence, elliptical cells are predicted to exhibit increased global cytoplasmic and nuclear Ca2+ compared to circular cells upon stimulation of M3R due to decreased PM-SR and PM-nuclear distance in the short axis of the cell. This model failed to capture the delay in the rate of nuclear Ca2+ increase to maximum in elliptical cells (Fig. 4h). This suggest that the model specification was incomplete and additional details may be needed to make the model more realistic54. Since cell shape determines nuclear shape, we hypothesized that as the cell elongates, invaginations in the nuclear membrane may decrease. As nuclear invaginations have been shown to be related to nuclear calcium dynamics55-57, we introduced a calcium permeability term in the model, by introducing a second order reaction dependent on the nuclear permeability, NPC, using the same circle and elliptical geometries (Supplementary Fig. 12). NPC represents the net nuclear permeability which may be due to an increase in nuclear membrane surface area and/or increase in nuclear pores; if NPC=0, the nuclear membrane is completely impermeable to cytoplasmic Ca2+; if NPC=1, the nuclear membrane is completely permeable to the cytoplasmic Ca2+ and mirrors the cytoplasmic Ca2+ transient. When nuclear permeability in circular cells was three times higher compared to elliptical cells, there was closer agreement between the model and experimental temporal behavior in both cytoplasmic and nuclear Ca2+ (Fig. 5i-l), suggesting that nuclear membrane of elliptical cells are less permeable to cytoplasmic Ca2+, compared to circular cells. The net effect of decreased nuclear permeability led to higher integrated nuclear Ca2+ signals in cells, which increases its availability for slower downstream processes in the nucleus that are driven by Ca2+ 25, 26. Thus the whole-cell model, coupled with organelle location based on curvature, captures key findings of Ca2+ dynamics in VSMC of different shapes.
DISCUSSION
One of the key features of signal transduction is the spatial organization of information propagation. Here, we bring together several seemingly independent effects due to change in global cell shape to provide an integrated view of how curvature, affects organelle location as well as distribution of receptors in the plane of the membrane to modulate signal transduction and thus affect cellular function (Fig. 6). Shape and biochemical signaling are coupled together in a feedback loop to maintain phenotype: cell shape integrates external mechanical and chemical signals on the plasma membrane3, 4 while intracellular signaling cascades containing chemical information in the form of reaction kinetics, in turn regulate cell shape58, 59. We propose that shape-dependent endomembrane and nuclear organization serves as the critical link that connect these two components in the feedback loop. This intricate, non-linear coupling of geometric and chemical information can potentially lead to signal amplification to control phenotype.
We used VSMC as a model system to unravel the complex relationship between global cell curvature, signaling, and endomembrane organization. During atherogenesis, disruption of local microenvironment in the medial layer of blood vessels causes VSMC to lose its native spindle shape, and subsequently lose contractile function24, 60. It was not clear how loss of shape can to a decrease in contractile function. This study provides a mechanistic explanation for the functional role of shape in VSMC contractile function. Cell shape governs membrane curvature, which enables the emergence of systems level properties; cell elongation simultaneously concentrates plasma membrane receptors in the flatter regions of the membrane and reduces the distance between the PM-SR and SR-nucleus in the same region, hence effectively forming a diffusion-restricted domain where receptors, SR and the nucleus become closer to each other, establishing high effective IP3 and Ca2+ concentration in the cell center. Given the slow diffusion coefficient of IP3 (≤10 μm2/s) which limits the range of action over which it can exert its signal61 and the dimensions of typical spindle-shaped VSMC (long axis ≥150 μm and short axis length of ≤ 10μm), control of endomembrane organization by cell shape is a physical, non-genomic mechanism by which IP3/Ca2+ signals can be locally amplified in order to achieve high concentration of Ca2+ globally. These can further regulate contractility and Ca2+-dependent gene expression in the nucleus. Although the effect of cell shape on individual readouts of signaling may appear small, the collective and progressive amplification of signal transduction through coupled reaction kinetics and spatial organelle organization is sufficient to result in different regimes of phenotypic function.
While we have extensively explored the role of cell shape in signaling here and previously3, 4, features that we have not considered here, such as the forces exerted by the extracellular microenvironment on the cell, and vice versa, also play a critical role in transmitting geometric information62, 63, trafficking64, and signal transduction65. Furthermore, the interaction between signaling and cytoskeletal remodeling can lead to changes in cell shape and local curvature66, 67. Our observations of increasing anisotropy and robustness in expression of actin myofibrils, along with increased nuclear SRF localization with aspect ratio indicate that cytoskeletal signaling also contributes towards the contractile phenotype of VSMC. Cytoskeletal and Ca2+ signaling may act in concert in maintaining the differentiated phenotype of spindle-shaped VSMC. We were able to uncover unique aspects of signal flow in cells based on geometry and chemical reaction cascades alone and coupling the role of cytoskeletal interactions is a focus of future studies. We conclude that being at the right place at the right time is critical for information to flow from one compartment to another and for short term signals like Ca2+ to have long lasting effects such as contractility and transcription factor activity. Like real estate, it seems that cells have the same motto – location, location, location!
METHODS
Cell culture
A10 cells, which are VSMC from thoracic/medial layer of rat aortas, were obtained from American Type Culture Collection (CRL-1476). A10 cells were maintained in Dulbecco’s modified eagle’s medium (DMEM, Gibco), supplemented with 10% Fetal Bovine Serum, 1% penicillin/streptomycin, at 37 °C and 5% CO2. Cells were transfected using Neon Transfection System (Life Technologies) according to manufacturer's instructions. Briefly, 5 ×105 cells were electroporated with 1 μg DNA in suspension buffer, with the following electroporation settings: 1400 V, 2 pulses, 20 ms pulses each. Cells were then suspended in DMEM supplemented with 10% FBS and then allowed to adhere on micropatterned surfaces. Forty-eight hours post-transfection, cells were imaged using Hanks Balanced Salt Solution (HBSS) supplemented with CaCl2, MgCl2 and 10 mM HEPES.
Micropatterning
Patterned surfaces were fabricated by conventional photolithography using SU8 photoresist68. Briefly, cover glass slides were cleaned by sonication in isopropanol and deionized water for 15 minutes and baked at 110 °C overnight and photolithography was subsequently performed using standard vacuum hard-contact mode. Before plating of cells onto micropatterns, microfabricated surfaces were washed with 50 μg/mL gentamicin and then incubated with 0.5% Pluronic for at least 3 hours. Micropatterns were then washed with PBS and were seeded with cells.
Immunofluorescence of cells in micropatterns
Cells were seeded onto the micropatterned coverslips and were allowed to adhere and comply with the patterns for at least 24 hours. After assay treatments, cells were fixed with 4% paraformaldehyde (Electron Microscopy) for 15 minutes at room temperature, washed with PBS, permeabilized with 0.2% saponin for 30 minutes and blocked with 4% normal goat serum doped with 0.05% saponin for 1 hour. Cells were then incubated overnight with primary antibodies (sources and catalog numbers shown in table below) that had been diluted in blocking solution at 4 °C. Cells were washed with PBS and samples were incubated with secondary antibodies (Alexa 488, Alexa 568 and/or Alexa 647) for 1 hour at room temperature. For organelle staining, cells were counter-stained with Actin Green and DAPI counterstains in addition to the secondary antibodies. Cells were then imaged on a Zeiss LSM 880 microscope equipped with 60x objective. Same acquisition settings were applied across different conditions that were compared (laser power, gain settings, magnification, zoom, pixel size and slice thickness). For quantitative immunofluorescence of M3R, a Z-stack of 30-40 slices using a slice thickness of 0.5 μm were obtained for each cell. Z-stack datasets were then pared down to 21-22 slices encompassing the entire height of the cell (mean cell height ~ 10 μm). Alignment, registration and cropping were performed to ensure each image had the same x-y dimensions (circular cells = 253 × 246 pixels, elliptical cells (AR 1:10) = 106 × 512 pixels). Per condition, images of cells obtained from the same z-plane (3.0 μm from the confocal slice corresponding to the bottom region of the plasma membrane), were averaged to obtain the averaged distribution of M3R in different regions of the cell. For immunofluorescence of transcription factors, multi-channel images consisting of DAPI, Actin Green (Invitrogen), and primary antibodies for NFAT, SRF or myocardin were aligned and stitched using the ZEN 2014 software. Image analysis and quantification was performed using ImageJ scripts. Briefly, nuclei were segmented in the DAPI channel. Corresponding cytosol and whole cell objects were outlined utilizing the contrast enhanced phalloidin channel to define cell boundaries. Nuclear-to-cytoplasmic transcription factor ratio was defined as the ratio of the mean transcription factor intensity colocalizing with the nuclear object divided by the mean intensity of the corresponding cytosol object. All measurements were exported directly to csv files and were subsequently analyzed using MATLAB to generate plots.
Airy-scan Imaging of Live Cells
VSMC conforming in micropattern were simultaneously labeled with 1 μM CellMask Plasma Membrane tracker (Life Technologies), 1 μM CellMask ER marker (BODIPY TR Glibenclamide), in HBSS buffer supplemented with 1% Pyruvate, 1% HEPES and 1 mM Trolox, for 5 minutes at room temperature. Images were acquired using Zeiss LSM 880 using Airy-scan imaging equipped with 63× 1.4 Plan-Apochromat Oil objective lens at 30 °C. Z-stacks using with an interval of 0.15 μm were collected for the entire cell height which approximated 10-12 μm. Z-stack analyses and other post-acquisition processing were performed on ZEN software (Carl Zeiss).
Calcium Measurements
VSMC were seeded on micropattern coverslips. Calcium measurements in micropatterns were performed as previously described with modifications69. Briefly, cells in micropatterns were serum-starved for 12 hours and loaded with 5 μM of calcium green (dissolved in DMSO) for 30 minutes at room temperature, with Hanks Balanced Salt solution, (HBSS) supplemented with CaCl2, MgCl2 and 10 mM HEPES. Calcium Green was imaged using Zeiss 510 equipped with 40x Apochromat objective at acquisition frame rate of 4 fps (250 ms acquisition time), and Calcium Green was excited using Argon ion laser 488 at low transmittivity (1%) to prevent photobleaching. Image stacks acquired were then imported into Fiji/ImageJ. Background subtraction were performed on the time stacks by using a rolling ball radius of 50 pixels. Cytoplasm and nuclear regions of interest (ROI) were chosen by performing a maximum intensity projection of the time-stack and specifying a 5 μm radius circle within the the nuclear and cytoplasmic regions. To convert intensity values to Ca2+ concentration, modified Grynkiewicz equation was used, defined as: [Calcium] Where Fmin is the average fluorescence intensity of the ROI after addition of 100 μM BAPTA AM, Fmax is the average fluorescence intensity of the ROI after addition of 0.100 μM A23187. Integrated Ca2+ were obtained using the trapz() function in MATLAB.
FRET imaging
MLCK-FRET plasmid is a kind gift from Dr. James T. Stull (University of Texas Southwestern Medical Center). The MLCK-FRET plasmid is a calmodulin-binding based sensor where calmodulin binding sequence is flanked with eCFP and eYFP and exhibits decreased FRET upon binding with calmodulin46, 18. Cells expressing MLCK-FRET were imaged using Zeiss LSM 880 (Carl Zeiss, Jena, Germany), at 37 °C incubator, fitted with Plan-Apochromat 20x, equipped with 458 nm and 514 nm Argon ion laser lines for excitation of eCFP and eYFP respectively. Incident excitation light was split using an MBS 458 nm/514 nm beam splitter and collected on a 32-spectral array GaAsp detector. The fluorescence emission was collected from 463-520 nm (ECFP), 544-620 nm (FRET channel and eYFP channel). Intensity based ratiometric FRET were obtained using custom-written scripts in ImageJ and MATLAB. Since MLCK-FRET is a single-chain construct, decrease in FRET, and increase in MLCK binding to calmodulin, was expressed as the ratio of emission intensity at 520 nm/emission intensity at 510 nm normalized at the basal levels.
NFAT imaging
HA-NFAT1(4-460)-GFP was a gift from Anjana Rao (Addgene plasmid # 11107). Patterned cells expressing NFAT-GFP was imaged using Zeiss 880, using Argon ion laser 488 nm, as described above and 1.4 NA objective, with an acquisition rate of 1 frame every 10 seconds. Time series image stacks were analyzed using ImageJ. Regions of interest of identical size were drawn in the cytoplasmic and nuclear regions of interest and the ratios of these intensities were computed over time.
Electron Microscopy
Micropatterned coverslips containing fixed A10 cells were embedded in Embed 812 resin (Electron Microscopy Sciences (EMS), Hatfield, PA) using the following protocol. Cells were rinsed in 200mM phosphate buffer (PB), osmicated with 1% osmium tetroxide/PB, washed with distilled water (dH20), and en bloc stained with aqueous 2% uranyl acetate, washed with dH2O and dehydrated via increasing ethanol (ETOH) series /distilled water (25%, 60%, 75%, 95% and 100% ETOH). Cells were further dehydrated using propylene oxide (PO), and embedded using ascending PO:EPON resin concentrations (2:1, 1:1, 1:2, pure). Prior to solidification, the coverslips were placed on 1”X 3” microscope slides, and multiple open ended embedding capsule with a 1 × 1 cm face (EMS) were placed on the coverslips covering the areas of interest. The resin was then polymerized in a vacuum oven at of 65 °C for 8–12 hours. After the first layer was solidified, the capsule was topped off with more resin and put back in the oven for another 8–12 hours. Capsules containing micropatterned cells were removed from coverslips using previously described methods70. Briefly, to separate the block from the coverslip, a hot plate was heated to 60×C and the microscope slide was placed directly on a pre-heated hot plate for exactly 3 minutes and 30 seconds. The slide was removed from the hot plate and the capsules carefully dislodged free from the coverslips. Once separated, the block face retains the cells within the micropatterns. The block was coarsely trimmed with a double-edged razorblade, and a Diatome cryotrim 45× mesa-trimming knife (EMS) was used to finely trim the block. Using as large a block face as possible, 70 nm ultrathin sections were cut from the block surface using an ultra-thin diamond knife (EMS), and a Leica EM UC7 ultramicrotome (Buffalo Grove, IL). All sections coming off the block face were collected. Sections were collected using a Perfect Loop (EMS) and transferred to a 2 × 1 mm formvar-carbon coated reinforced slot grid (EMS). The sample was dried on the grid and transferred to Hiraoka Staining Mats (EMS) for heavy metal staining. Grids were stained with 3% uranyl acetate in water for 40 minutes, washed and stained with Reynold’s lead citrate for 3 minutes, washed and allowed to dry. Electron microscopy images were taken using a Hitachi 7000 Electron Microscope (Hitachi High Technologies America, Inc.) equipped with an AMT Advantage CCD camera. Cells were viewed at low-magnification to identify areas of interest (cell tip versus cell body) before high magnification imaging. Images were transferred to Adobe Photoshop CS3 (version 10), and adjusted for brightness and contrast. Measurement of plasma membrane to ER distances from electron microscopy images were performed blindly. Briefly, sample information from images were removed and images were saved with a randomized filename. Image contrast was further enhanced using ImageJ using contrast-limited adaptive histogram equalization (CLAHE). Only images with discernible smooth ER closely apposed to the plasma membrane were analyzed and distances were measured at optimal xy orientations at 50 nm intervals using ImageJ. Data was graphed using Excel and MATLAB.
Statistics
Results are presented as mean±standard error of the mean from at least three independent experiments. Normality was determined using Shapiro-Wilk test using a p-value ≥ 0.05. If distribution is normal, a two-tailed Student’s t-test was performed. For datasets with non-normal distribution, two-tailed Mann-Whitney test was used. P < 0.05 was considered statistically significant.
Model Development
We formulated a phenomenological model to study the role of PM-SR distances. The complete derivation and mathematical solution is given in the supplementary material. Complete details of the simulations using finite-element methods in COMSOL and using finite-volume methods in Virtual Cell are given in the Supplementary Material.
ACKNOWLEDGEMENTS
We thank Dr. Eric Sobie, Dr. Marc Birtwistle and Dr. Evren Azeloglu for discussion and reagents. This work was supported NIH Grants GM072853 and the Systems Biology Center grant P50GM071558. RC was supported by a NIH postdoctoral fellowship F32GM116415 and research in PR’s laboratory is supported by ARO W911NF-16-1-0411, AFOSR FA9550-15-1-0124, and NSF PHY-1505017.
Footnotes
↵* Joint senior authors
One Sentence Summary: Changes in the global curvature of the plasma membrane alter the distances between the plasma membrane and sarcoplasmic reticulum, and the nucleus, and modulate the strength of Ca2+ signaling in vascular smooth muscle cells.
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