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 comprising:
a) expressing a fusion protein in a cell comprising a cell membrane, wherein said fusion protein comprises a cell-surface receptor protein and a reporter protein, and said expressing results in the co-expression of the cell surface receptor protein and the reporter protein as a single fusion protein in the cell, wherein the reporter protein is displayed extracellularly when the cell-surface receptor is incorporated into the cell membrane of the cell;
b) delivering a reporter substrate to the cell, wherein an association between the reporter protein and reporter substrate produces a detectable signal when the association occurs extracellularly and an altered signal when the association occurs intracellularly; and
c) monitoring said detectable signal and said altered signal, wherein a transition from said detectable signal to said altered signal indicates endocytosis of the fusion protein.
2. The method of detecting endocytosis of claim 1 , wherein monitoring of said detectable signal comprises:
i) inducing endocytosis of said fusion protein; and
ii) detecting said detectable signal and said altered signal after inducing endocytosis.
3. The method of detecting endocytosis of claim 1 , wherein monitoring of said detectable signal comprises:
i) detecting said signal prior to endocytosis;
ii) allowing endocytosis of said fusion protein;
iii) detecting said signal following endocytosis; and
iv) comparing said signal from step (i) to said signal from step (iii) to detect endocytosis.
4. The method of claim 1 , wherein said reporter protein comprises a luciferase enzyme.
5. The method of claim 4 , wherein the luciferase enzyme is a beetle or Oplophorus luciferase enzyme or a mutant or variant thereof.
6. The method of claim 1 , wherein endocytosis results in movement of said reporter protein from an extracellular space into an endosome.
7. The method of claim 6 , wherein said signal is detectably altered within said endosome compared to the extracellular space.
8. The method of claim 6 , wherein said signal is reduced within said endosome compared to the extracellular space.
9. The method of claim 1 , wherein said reporter substrate is a luciferin, luciferin derivative, coelenterazine or coelenterazine derivative.
10. The method of claim 3 , further comprising a step between steps (i) and (ii) of triggering endocytosis.