<h3>Title</h3>
<h3>Cell-Permeable, Ratiometric, Fluorescent Sensors for Imaging Phosphoinositides</h3>
<p><strong><span style="text-decoration: underline;">Abstract</span> :</strong></p>
<p style="text-align: justify;">Phosphoinositides, a class of essential phospholipids, are key mediators of cell signalling and regulatory processes. Dynamic changes in concentrations and distributions of phosphoinositides act as initiating flags for cell signalling events.<sup>1</sup> Impaired phosphoinositide metabolism has been linked to multiple disorders including type-2-diabetes, bipolar disorder, cancer. Hence, tracking phosphoinositide dynamics can provide mechanistic insights into fundamental cellular processes, aid in disease diagnosis, and guide therapeutic routes. Fluorescence microscopy can provide with an appropriate imaging set-up to track phosphoinositides in living cells with high spatiotemporal resolution. Since, phosphoinositides are non-fluorescent, fluorescent sensors selective toward phosphoinositide are required. However, tracking phosphoinositides in living cells is extremely challenging. This is because, phosphoinositides are present in significantly lower concentrations (&lt;1-5 mol%) when compared to other cellular phospholipids. Further, reversible phosphorylation on the inositol headgroup of phosphatidylinositol leads to the presence of seven distinct phosphoinositides in the cell-membrane. Each member of the phosphoinositide family plays a distinct role in the cell. Phosphoinositides are also mostly present in the inner-leaflet of the cell membrane. Therefore in order to track the live cell dynamics of a specific phosphoinositide and elucidate its role in cellular processes we require selective, sensitive, reversible, and cell permeable probes.<sup>1</sup> Towards this goal, we have developed a peptide-based reversible detection scheme for selective tracking of phosphoinositides and have applied the strategy for sensing phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2)<sup>2</sup> and phosphatidylinositol-(3,4,5)-trisphosphate (PI(3,4,5)P3). We have harnessed insights from MD simulations on phosphoinositide binding peptides with phospholipid bilayers to fine-tune the selectivity and the sensitivity of the sensors. By leveraging the flexibility of our sensing scheme, we have altered binding scaffolds as well as dyes to make a sensor library for different phosphoinositides. We have successfully developed two orthogonally emitting sensors with high selectivity toward PI(4,5)P2 and PI(3,4,5)P3, respectively. Simultaneous imaging of PI(4,5)P2 and PI(3,4,5)P3 can shed light on the cellular processes like cell proliferation and migration under physiological and pathophysiological conditions where these phosphoinositides are key regulators. Both the sensors have been tested for in vitro selectivity against other biologically relevant phospholipids and are cell-permeable. Current studies are focused on tracking the phosphoinositide dynamics in living cells using these novel sensors. I will present the details of work done in last one year and on-going research on the applications of the novel sensors in live cell imaging.</p>
<p style="text-align: justify;"> [1] R. Kundu, A. Chandra, A. Datta, <em>Isr. J. Chem.,</em> <strong>2021<em>, </em></strong><em>61</em>, 199.</p>
<p>[2] S. Mondal, A. Rakshit, S. Pal, A. Datta. <em>ACS Chem. Biol.,</em> <strong>2016</strong>, <em>11</em>, 1834–1843.</p>