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Title: Cellular Architecture Regulates Collective Calcium Signaling and Cell Contractility}
Article:
Abstract:
A key feature of multicellular systems is the ability of cells to function collectively in response to external stimuli. However, the mechanisms of intercellular cell signaling and their functional implications in diverse vascular structures are poorly understood. Using a combination of computational modeling and plasma lithography micropatterning, we investigate the roles of structural arrangement of endothelial cells in collective calcium signaling and cell contractility. Under histamine stimulation, endothelial cells in self-assembled and microengineered networks, but not individual cells and monolayers, exhibit calcium oscillations. Micropatterning, pharmacological inhibition, and computational modeling reveal that the calcium oscillation depends on the number of neighboring cells coupled via gap junctional intercellular communication, providing a mechanistic basis of the architecture-dependent calcium signaling. Furthermore, the calcium oscillation attenuates the histamine-induced cytoskeletal reorganization and cell contraction, resulting in differential cell responses in an architecture-dependent manner. Taken together, our results suggest that endothelial cells can sense and respond to chemical stimuli according to the vascular architecture via collective calcium signaling.
Introduction:
The calcium ion is a universal second messenger mediating a wide range of dynamic cell functions, such as exocytosis, contraction, transcription, and proliferation[1–3]. These biological processes that span time scales from microseconds to hours are regulated by diverse calcium signaling mechanisms. A hallmark of calcium signaling is the transient pulsing dynamics of cytosolic calcium concentration. The calcium dynamics are actively controlled by calcium release from intracellular stores and calcium influx from extracellular media, outflow by pumps and exchangers, and calcium binding proteins, which fine-tune the amplitude and duration of calcium pulses to regulate physiological functions. Calcium oscillation, for instance, can sensitize signal detection at low agonist concentration and its frequency can also encode additional information that selectively controls transcription factor activity[4–6]. The calcium dynamics are regulated by multiple intracellular calcium processing mechanisms, such as non-selective cation channels, voltage-dependent calcium channels, store-operated calcium channels, calcium-induced calcium release, and phospholipase C mediated inositol 1, 4, 5-trisphosphate activity[3]. Intercellular collective calcium signaling mechanisms can also modulate the calcium dynamics. In particular, gap junctional intercellular communication( GJIC) mediates coupling currents between neighboring cells triggered by an imbalance in the membrane potential. GJIC also allows calcium diffusion driven by a difference in the cytosolic calcium concentration[7, 8]. For instance, calcium waves can propagate among endothelial cells, and intercellular calcium signaling provides a robust mechanism for mechanosensing in endothelial networks[9, 10]. Three-dimensional coupling of β-cells can lead to robust synchronized calcium oscillations and insulin secretion at elevated glucose levels[11]. GJIC also regulates asymmetric neuronal fates[12] and controls collective chemosensing to generate synchronized and coordinated responses under adenosine triphosphate( ATP) stimulation[13, 14]. Physiologically, histamine is known to induce calcium signaling and cell contraction in endothelial cells. Interestingly, histamine-induced permeability is confined to venules rather than capillaries[15], suggesting an architecture dependence on the response to histamine. Nevertheless, the effects of structural arrangement, as seen in diverse vascular structures, on calcium signaling and their functional implications are poorly understood. In this study, we investigate architecture-dependent collective calcium signaling in human umbilical vein endothelial cells( HUVEC). Self-assembled capillary-like networks[16] and microengineered hexagonal cell networks[17] are created to evaluate the effects of cellular architecture on calcium signaling. Endothelial cell networks of arbitrary shapes and widths can be created by plasma lithography cell patterning[18]. The method has been demonstrated for investigating the effects of geometric confinements on various biological systems[19–22]. Histamine-induced calcium oscillations in the cell networks are compared with individual cells and monolayers to investigate the architecture dependence. Along with micropatterning and pharmacological inhibition, a computational model is developed to elucidate the effects of the number of neighboring cells on collective calcium signaling. The cytoskeletal reorganization and cell contraction induced by histamine are correlated with the calcium oscillation to study the function of collective calcium signaling in different cellular architectures.
Discussion:
In this study, the significance of cellular architecture in GJIC-mediated collective calcium signaling is identified. Unlike monolayers and individual cells that exhibit prolonged cytosolic calcium response, cells in capillary-like networks and hexagonal cell networks display short calcium pulses and oscillations after histamine treatment. Histamine-mediated calcium oscillations occur only when a small number of( ~2–6) cells are physically connected in self-assembled and microengineered networks. Systematic investigation using linear networks and computational modeling suggests that the calcium oscillation occurrence rate negatively correlates with the number of neighboring cells. Patterning cells in linear networks with low cell density and with different widths supports that the calcium oscillation is not due to the elongated morphology of the cells or physical confinement by the patterns. The dependence on the number of coupled cells leads to the architecture-dependent calcium signaling, since most cells in the networks connect to 2–5 cells while cells in monolayers typically connect to over 8 cells. Additionally, pharmacological inhibition, computational modeling and micropatterning revealed that the calcium oscillation is mediated by GJIC. Inhibition of GJIC with a gap junction blocker suppresses the calcium oscillation. With GJIC inhibition, the calcium dynamics of cells in capillary-like networks and hexagonal cell networks resemble the response of individual cells. These results collectively suggest that cellular architecture regulates collective calcium signaling via GJIC. A computational model is developed to explore the mechanisms of architecture-dependent calcium oscillations. The computational model captures the essence of calcium dynamics, including the transient pulses, the uniform amplitude, the concentration dependence, the irregular pattern, the occurrence rate, and the architecture dependence. The model provides insights into the mechanisms that drive the architecture-dependent calcium oscillation. In particular, the calcium fluxes are highly sensitive to the cytosolic calcium concentration. The coupling current due to electrical coupling perturbs the cytosolic calcium concentration, which generates the oscillatory activity in coupled cells[8]. The electrical coupling provides a potential mechanism on how calcium oscillations are generated in coupled cells, but not in individual cells. Incorporating electrical coupling in the model successfully creates the random distribution of calcium pulses and the irregular pattern observed in the experiment. The oscillation cannot be explained by intercellular calcium diffusion alone, as the oscillations are irregular and unsynchronized between neighboring cells. In contrast, the biochemical coupling through intercellular diffusion of calcium suppresses the oscillation by stabilizing the cytosolic calcium concentration in the cells. As supported by the computational model and linear networks with different widths, the stabilizing capability increases with the number of cells coupled and abolishes calcium oscillations when a large number of cells are connected( e. g., in monolayers). Since the stabilizing capability depends on the number of neighboring cells, the calcium oscillation occurs only when a small number of cells are coupled, providing a basis of the architecture-dependent calcium signaling( S7 and S8 Figs). Our computational model is based on a set of assumptions and simplifications, which will likely limit the predictive power of the model. Several aspects of the model should be considered. First, the contribution of voltage gated calcium channels to agonist-induced calcium entry in endothelial cells is inconclusive[33–35]. Endothelial cells are often considered to be electrically unexcitable and the effect of membrane potential on agonist-induced calcium signaling in endothelial cells remains controversial[36, 37]. Second, the permeability of vertebrate gap junction can be modulated by cytosolic ionic composition and connexin phosphorylation. The increase in cytosolic calcium concentration was shown to inhibit electrical coupling and dye transfer among cells[38]. Phosphorylation of connexin by protein kinases also resulted in internalization and degradation of gap junctions[39]. Our model may only represent the cell behaviors under specific conditions and do not rule out the possibility of other calcium signaling mechanisms. In general, calcium dynamics can be influenced by a large number of factors and mechanisms. Computational models of calcium dynamics and signaling in vascular regulation have been reviewed previously[40]. The relationships between the cytosolic calcium, membrane potential, coupling current and voltage gated channels can be context-dependent( e. g., agonist, concentration and duration) and each factor may influence the calcium dynamics through multiple feedback loops direct or indirectly. As an example, the inhibitory effects of verapamil( an L-type voltage gated calcium channels inhibitor) on histamine-induced calcium signaling in human umbilical vein endothelial cells were observed only at submaximal histamine concentrations, suggesting that voltage gated calcium channels might play a role at a specific range of agonist concentration in HUVECs[41]. Non-selective cation channels were also reported to be responsible for histamine-activated Ca2+ influx[42]. For histamine-activated calcium signaling in endothelial cells, it has been demonstrated that the membrane potential plays a key role for fine-tuning the calcium influx[43]. In particular, the membrane potential can effectively regulate calcium influx which is responsible for the plateau phase of agonist-mediated calcium dynamics. In addition, activation of the calcium-activated potassium channel triggers membrane hyperpolarization and, thus, provides a driving force for cation influx( Na+, K+ and Ca2+)[44]. In the future, additional studies should be performed to determine the contribution of voltage-gated calcium channels, membrane potential and gap junction mediated electrical coupling with the regulation of cytosolic calcium concentration and connexin phosphorylation. Our results underscore the possibility that cellular architecture modulates histamine-mediated cytoskeletal reorganization and cell contractility via GJIC-mediated collective calcium signaling. Unlike monolayers and individual cells that exhibit cytoskeletal reorganization, histamine has minimal effects on the actin cytoskeleton in capillary-like networks and hexagonal cell networks. To study the roles of calcium oscillation on cytoskeletal reorganization, histamine pulses that impose calcium oscillations are applied to individual cells and monolayers. The histamine pulses have a weak effect on the actin cytoskeleton compared to continuous histamine treatment. On the other hand, suppressing calcium oscillations in self-assembled networks by GJIC inhibition induces cytoskeletal reorganization and cell contraction. Therefore, the effects of histamine can be modulated by the calcium dynamics and are architecture-dependent. Physiologically, the architecture dependence may represent one of the ways of endothelial cells to respond to the stimuli differentially in various vascular structures. The architecture-dependent calcium oscillations might contribute to the specificity of histamine-induced permeability in venules, but not in capillaries[15]. Our results, however, do not rule out other potential mechanisms in generating the specificity such as the differences in calcium signaling mechanisms between venules and capillaries. Further investigation is required to test this hypothesis and study the architecture dependence in other models( S9 Fig). As a universal second messenger, collective calcium signaling may serve as a broad mechanism in regulating various cell functions. Other factors may also be considered in the computational and experimental models to decipher the regulation and functional implication of collective calcium signaling in diverse cellular architectures.
Cells can sense and respond to their microenvironments collectively to yield high-order complex multicellular behaviors . Despite its fundamental importance in cell physiology , the emerging mechanisms that drive collective cell signaling and their functional implications in diverse cellular structures , e . g . , as seen in various vascular architectures , remain poorly understood . These collective cell behaviors are often counterintuitive and cannot be understood by extrapolating the behaviors of individual cells . Using a combination of microscale cell patterning and computational modeling , we elucidate the architecture-dependent calcium signaling and contractility in endothelial cells via gap junction mediated calcium oscillation in an architecture-dependent manner . As calcium is one of the most important second messengers , collective calcium signaling may serve as a broad mechanism in regulating various cell functions . More importantly , our finding highlights a relatively unexplored mechanism of cellular regulation via cellular architectures .