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.