Compositions and methods for monitoring transmembrane trafficking
View Patent ↗Provided herein are compositions and methods for monitoring the movement of analytes and/or cellular components across biological membranes (e.g., cell surface internalization). In particular, reporter constructs are provided, the transmembrane movement of which (e.g., by endocytosis) is monitored by methods described herein.
1. A method of detecting endocytosis in a living cell comprising:
(a) tethering a reporter element to a cell-surface analyte to create a fusion element, wherein a detectable signal from said reporter element is altered upon endocytosis of said fusion element; and
(b) detecting said detectable signal from said reporter element outside the living cell;
(c) allowing endocytosis of said fusion element;
(d) detecting an altered detectable signal from said reporter element within the living cell; and
(e) comparing said detectable signal to said altered detectable signal, thereby detecting endocytosis.
2. The method of detecting endocytosis of claim 1 , wherein said detectable signal and said altered detectable signal are emitted upon interaction of said reporter element with a reporter substrate.
3. The method of claim 2 , further comprising a step between steps (a) and (b) of contacting said reporter element with said reporter substrate.
4. The method of claim 1 , wherein said cell surface analyte comprises a cell surface receptor.
5. The method of claim 1 , wherein said cell-surface analyte is non-covalently associated with a component of the cell surface.
6. The method of claim 1 , wherein said reporter element and said cell-surface analyte are expressed as a fusion protein.
7. The method of claim 1 , wherein said reporter element comprises a luciferase enzyme.
8. The method of claim 7 , wherein the luciferase enzyme is a beetle or Oplophorus luciferase enzyme or a functionally equivalent variant or mutant thereof.
9. The method of claim 1 , wherein step (d) comprises detecting said second signal within an endosome.
10. The method of claim 9 , wherein said second signal is detectably altered within said endosome compared to said first signal outside the living cell.
11. The method of claim 1 , further comprising a step between steps (b) and (c) of triggering endocytosis.
12. The method of claim 3 , wherein said reporter substrate is permeable to the plasma membrane of said living cell.
13. The method of claim 3 , wherein said reporter substrate is completely or largely excluded from the intracellular space of said cell.
14. The method of claim 3 , wherein said reporter is an enzyme and is capable of substrate turnover.
15. The method of claim 3 , wherein said reporter substrate is a luciferin, luciferin derivative, coelenterazine, or a coelenterazine derivative.
16. The method of claim 1 , wherein said fusion element is incorporated into cell membrane such that said reporter element is localized in extracellular space.
17. The method of claim 1 , wherein endocytosis of said reporter element results in movement of said reporter element from an extracellular space into an endosome.
18. The method of detecting endocytosis of claim 6 , wherein said fusion protein is expressed endogenously within said living cell.
19. The method of detecting endocytosis of claim 6 , wherein said fusion protein is added exogenously to medium of said living cell.
20. The method of claim 1 , wherein said altered detectable signal is less than said detectable signal.