RESUMO
Phosphoinositides, phospholipids that are key cell-signal mediators, are present at very low levels in cellular membranes and within nuclei. Phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), a phosphoinositide barely present in resting cell membranes, is produced when cells receive either growth, proliferation, or movement signals. Aberrant PIP3 levels are associated with the formation of cancers. PIP3 pools are also present in the nucleus, specifically in the nucleolus. However, questions related to the organization and function of this lipid in such membraneless intranuclear structures remain unanswered. Therefore, chemical sensors for tracking cellular PIP3 are invaluable not only for timing signal initiation in membranes but also for identifying the organization and function of membraneless nuclear PIP3 pools. Because PIP3 is present in the inner leaflet of cell membranes and in the nucleus, cell-permeable, rapid-response fluorescent sensors would be ideal. We have designed two peptide-based, water-soluble, cell-permeable, ratiometric PIP3 sensors named as MFR-K17H and DAN-NG-H12G. MFR-K17H rapidly entered into the cell cytoplasm, distinctly reporting rapid (<1 min) time scales of growth factor-stimulated PIP3 generation and depletion within cell membranes in living cells. Importantly, MFR-K17H lighted up inherently high levels of PIP3 in triple-negative breast cancer cell membranes, implying future applications in the detection of enhanced PIP3 levels in cancerous cells. On the other hand, DAN-NG-H12G targeted intranuclear PIP3 pools, revealing that within membraneless structures, PIP3 resided in a hydrophobic environment. Together, both probes form a unique orthogonally targeted combination of cell-permeable, ratiometric probes that, unlike previous cell-impermeable protein-based sensors, are easy to apply and provide an unprecedented handle into PIP3-mediated cellular processes.
RESUMO
Central roles of Mn2+ ions in immunity, brain function, and photosynthesis necessitate probes for tracking this essential metal ion in living systems. However, developing a cell-permeable, fluorescent sensor for selective imaging of Mn2+ ions in the aqueous cellular milieu has remained a challenge. This is because Mn2+ is a weak binder to ligand-scaffolds and Mn2+ ions quench fluorescent dyes leading to turn-off sensors that are not applicable for in vivo imaging. Sensors with a unique combination of Mn2+ selectivity, µM sensitivity, and response in aqueous media are necessary for not only visualizing labile cellular Mn2+ ions live, but also for measuring Mn2+ concentrations in living cells. No sensor has achieved this combination thus far. Here we report a novel, completely water-soluble, reversible, fluorescent turn-on, Mn2+ selective sensor, M4, with a K d of 1.4 µM for Mn2+ ions. M4 entered cells within 15 min of direct incubation and was applied to image Mn2+ ions in living mammalian cells in both confocal fluorescence intensity and lifetime-based set-ups. The probe was able to visualize Mn2+ dynamics in live cells revealing differential Mn2+ localization and uptake dynamics under pathophysiological versus physiological conditions. In a key experiment, we generated an in-cell Mn2+ response curve for the sensor which allowed the measurement of the endogenous labile Mn2+ concentration in HeLa cells as 1.14 ± 0.15 µM. Thus, our computationally designed, selective, sensitive, and cell-permeable sensor with a 620 nM limit of detection for Mn2+ in water provides the first estimate of endogenous labile Mn2+ levels in mammalian cells.