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Novel method for real-time monitoring of ATP release reveals multiple phases of autocrine purinergic signalling during immune cell activation.

ABSTRACT: The activation of immune cells must be tightly regulated to allow an effective immune defence while limiting collateral damage to host tissues. Cellular ATP release and autocrine stimulation of purinergic receptors are recognized as critical regulators of immune cell activation. However, the study of purinergic signalling has been hampered by the short half-life of the released ATP and its breakdown products as well as the lack of real-time imaging methods to study spatiotemporal dynamics of ATP release.To overcome these limitations, we optimized imaging methods that allow monitoring of ATP release with conventional microscopy using the recently developed small molecular ATP probes 1-2Zn(II) and 2-2Zn(II) for imaging of ATP in the extracellular space and release at the surface of living cells.1-2Zn(II) allowed imaging of <1 ?m ATP in the extracellular space, while 2-2Zn(II) provided unprecedented insights into the spatiotemporal dynamics of ATP release from neutrophils and T cells. Stimulation of these cells caused virtually instantaneous ATP release, which was followed by a second phase of ATP release that was localized to the immune synapse of T cells and the leading edge of polarized neutrophils. Imaging these ATP signalling processes along with mitochondrial probes provided evidence for a close spatial relationship between mitochondrial activation and localized ATP release in T cells and neutrophils.We believe that these novel live cell imaging methods can be used to define the roles of purinergic signalling in immune cell activation and in the regulation of other complex physiological processes.

SUBMITTER: Ledderose C

PROVIDER: S-EPMC4293224 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Novel method for real-time monitoring of ATP release reveals multiple phases of autocrine purinergic signalling during immune cell activation.

Ledderose C C Bao Y Y Zhang J J Junger W G WG

Acta physiologica (Oxford, England) 20141229 2

<h4>Aims</h4>The activation of immune cells must be tightly regulated to allow an effective immune defence while limiting collateral damage to host tissues. Cellular ATP release and autocrine stimulation of purinergic receptors are recognized as critical regulators of immune cell activation. However, the study of purinergic signalling has been hampered by the short half-life of the released ATP and its breakdown products as well as the lack of real-time imaging methods to study spatiotemporal dy ...[more]

PMID: 25482154

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Project description:Stimuli-responsive drug delivery vehicles have garnered immense interest in recent years due to unparalleled progress made in material science and nanomedicine. However, the development of stimuli-responsive devices with integrated real-time monitoring capabilities is still in its nascent stage because of the limitations of imaging modalities. In this paper, we describe the development of a polypeptide-wrapped mesoporous-silica-coated multicolor upconversion nanoparticle (UCNP@MSN) as an adenosine triphosphate (ATP)-responsive drug delivery system (DDS) for long-term tracking and real-time monitoring of drug release. Our UCNP@MSN with multiple emission peaks in UV-NIR wavelength range was functionalized with zinc-dipicolylamine analogue (TDPA-Zn(2+)) on its exterior surface and loaded with small-molecule drugs like chemotherapeutics in interior mesopores. The drugs remained entrapped within the UCNP-MSNs when the nanoparticles were wrapped with a compact branched polypeptide, poly(Asp-Lys)-b-Asp, because of multivalent interactions between Asp moieties present in the polypeptide and the TDPA-Zn(2+) complex present on the surface of UCNP-MSNs. This led to luminescence resonance energy transfer (LRET) from the UCNPs to the entrapped drugs, which typically have absorption in UV-visible range, ultimately resulting in quenching of UCNP emission in UV-visible range while retaining their strong NIR emission. Addition of ATP led to a competitive displacement of the surface bound polypeptide by ATP due to its higher affinity to TDPA-Zn(2+), which led to the release of the entrapped drugs and subsequent elimination of LRET. Monitoring of such ATP-triggered ratiometric changes in LRET allowed us to monitor the release of the entrapped drugs in real-time. Given these results, we envision that our proposed UCNP@MSN-polypeptide hybrid nanoparticle has great potential for stimuli-responsive drug delivery as well as for monitoring biochemical changes taking place in live cancer and stem cells.

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Dataset Information

Novel method for real-time monitoring of ATP release reveals multiple phases of autocrine purinergic signalling during immune cell activation.

Publications

Novel method for real-time monitoring of ATP release reveals multiple phases of autocrine purinergic signalling during immune cell activation.

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