Global Hopf bifurcation in the ZIP regulatory system

Regulation of zinc uptake in roots of Arabidopsis thaliana has recently been modeled by a system of ordinary differential equations based on the uptake of zinc, expression of a transporter protein and the interaction between an activator and inhibitor. For certain parameter choices the steady state of this model becomes unstable upon variation in the external zinc concentration. Numerical results show periodic orbits emerging between two critical values of the external zinc concentration. Here we show the existence of a global Hopf bifurcation with a continuous family of stable periodic orbits between two Hopf bifurcation points. The stability of the orbits in a neighborhood of the bifurcation points is analyzed by deriving the normal form, while the stability of the orbits in the global continuation is shown by calculation of the Floquet multipliers. From a biological point of view, stable periodic orbits lead to potentially toxic zinc peaks in plant cells. Buffering is believed to be an efficient way to deal with strong transient variations in zinc supply. We extend the model by a buffer reaction and analyze the stability of the steady state in dependence of the properties of this reaction. We find that a large enough equilibrium constant of the buffering reaction stabilizes the steady state and prevents the development of oscillations. Hence, our results suggest that buffering has a key role in the dynamics of zinc homeostasis in plant cells.Comment: 22 pages, 5 figures, uses svjour3.cl

Zinc-Regulated DNA Binding of the Yeast Zap1 Zinc-Responsive Activator

The Zap1 transcription factor of Saccharomyces cerevisiae plays a central role in zinc homeostasis by controlling the expression of genes involved in zinc metabolism. Zap1 is active in zinc-limited cells and repressed in replete cells. At the transcriptional level, Zap1 controls its own expression via positive autoregulation. In addition, Zap1's two activation domains are regulated independently of each other by zinc binding directly to those regions and repressing activation function. In this report, we show that Zap1 DNA binding is also inhibited by zinc. DMS footprinting showed that Zap1 target gene promoter occupancy is regulated with or without transcriptional autoregulation. These results were confirmed using chromatin immunoprecipitation. Zinc regulation of DNA binding activity mapped to the DNA binding domain indicating other parts of Zap1 are unnecessary for this control. Overexpression of Zap1 overrode DNA binding regulation and resulted in constitutive promoter occupancy. Under these conditions of constitutive binding, both the zinc dose response of Zap1 activity and cellular zinc accumulation were altered suggesting the importance of DNA binding control to zinc homeostasis. Thus, our results indicated that zinc regulates Zap1 activity post-translationally via three independent mechanisms, all of which contribute to the overall zinc responsiveness of Zap1

The zinc transporter ZIP12 regulates the pulmonary vascular response to chronic hypoxia

The typical response of the adult mammalian pulmonary circulation to a low oxygen environment is vasoconstriction and structural remodelling of pulmonary arterioles, leading to chronic elevation of pulmonary artery pressure (pulmonary hypertension) and right ventricular hypertrophy. Some mammals, however, exhibit genetic resistance to hypoxia-induced pulmonary hypertension1, 2, 3. We used a congenic breeding program and comparative genomics to exploit this variation in the rat and identified the gene Slc39a12 as a major regulator of hypoxia-induced pulmonary vascular remodelling. Slc39a12 encodes the zinc transporter ZIP12. Here we report that ZIP12 expression is increased in many cell types, including endothelial, smooth muscle and interstitial cells, in the remodelled pulmonary arterioles of rats, cows and humans susceptible to hypoxia-induced pulmonary hypertension. We show that ZIP12 expression in pulmonary vascular smooth muscle cells is hypoxia dependent and that targeted inhibition of ZIP12 inhibits the rise in intracellular labile zinc in hypoxia-exposed pulmonary vascular smooth muscle cells and their proliferation in culture. We demonstrate that genetic disruption of ZIP12 expression attenuates the development of pulmonary hypertension in rats housed in a hypoxic atmosphere. This new and unexpected insight into the fundamental role of a zinc transporter in mammalian pulmonary vascular homeostasis suggests a new drug target for the pharmacological management of pulmonary hypertension

MagFRET : the first genetically encoded fluorescent Mg2+ sensor

Magnesium has important structural, catalytic and signaling roles in cells, yet few tools exist to image this metal ion in real time and at subcellular resolution. Here we report the first genetically encoded sensor for Mg2+, MagFRET-1. This sensor is based on the high-affinity Mg2+ binding domain of human centrin 3 (HsCen3), which undergoes a transition from a molten-globular apo form to a compactly-folded Mg2+-bound state. Fusion of Cerulean and Citrine fluorescent domains to the ends of HsCen3, yielded MagFRET-1, which combines a physiologically relevant Mg2+ affinity (Kd = 148 µM) with a 50% increase in emission ratio upon Mg2+ binding due to a change in FRET efficiency between Cerulean and Citrine. Mutations in the metal binding sites yielded MagFRET variants whose Mg2+ affinities were attenuated 2- to 100-fold relative to MagFRET-1, thus covering a broad range of Mg2+ concentrations. In situ experiments in HEK293 cells showed that MagFRET-1 can be targeted to the cytosol and the nucleus. Clear responses to changes in extracellular Mg2+ concentration were observed for MagFRET-1-expressing HEK293 cells when they were permeabilized with digitonin, whereas similar changes were not observed for intact cells. Although MagFRET-1 is also sensitive to Ca2+, this affinity is sufficiently attenuated (Kd of 10 µM) to make the sensor insensitive to known Ca2+ stimuli in HEK293 cells. While the potential and limitations of the MagFRET sensors for intracellular Mg2+ imaging need to be further established, we expect that these genetically encoded and ratiometric fluorescent Mg2+ sensors could prove very useful in understanding intracellular Mg2+ homeostasis and signaling

Genetically encoded fluorescent probes for Intracellular Zn2+ imaging

In this chapter we provide an overview of the various genetically encoded fluorescent Zn2+ sensors that have been developed over the past 5 to 10 years. We focus on sensors based on Förster resonance energy transfer (FRET), as these have so far proven to be the most useful for detecting Zn2+ in biological samples. Our goal is to provide a balanced discussion of the pros and cons of the various sensors and their application in intracellular imaging. Following the description of the various sensors, several recent applications of these sensors are discussed. We end the chapter by identifying remaining challenges in this field and discussing future perspectives

Genetically encoded fluorescent probes for Intracellular Zn2+ imaging

In this chapter we provide an overview of the various genetically encoded fluorescent Zn2+ sensors that have been developed over the past 5 to 10 years. We focus on sensors based on Förster resonance energy transfer (FRET), as these have so far proven to be the most useful for detecting Zn2+ in biological samples. Our goal is to provide a balanced discussion of the pros and cons of the various sensors and their application in intracellular imaging. Following the description of the various sensors, several recent applications of these sensors are discussed. We end the chapter by identifying remaining challenges in this field and discussing future perspectives