Hot-wiring clathrin-mediated endocytosis in human cells

Abstract

Clathrin-mediated endocytosis (CME) is the major route of receptor internalization at the plasma membrane. Analysis of constitutive CME is complicated by the fact that initiation of endocytic events is unpredictable. When and where a clathrin-coated pit will form and what cargo it will contain are difficult to foresee. Here we describe a series of genetically encoded reporters that allow the initiation of CME on demand. This is achieved by inducibly tethering a clathrin "hook" to a plasma membrane "anchor". Our design incorporates temporal and spatial control of initiation using chemical and optical tools, and the cargo is defined. Since this system bypasses multiple steps in vesicle creation, we term it "hot-wiring". In this paper, we use hot-wired endocytosis to define the functional interactions between clathrin and the β2 subunit of the AP2 complex. However, there are numerous applications for this new technology, which we hope will be broadly useful to the field.

FGFR3–TACC3 cancer gene fusions cause mitotic defects by removal of endogenous TACC3 from the mitotic spindle

“…Details of pTACC3 are given in the electronic supplementary material. CD8-TACC3-mCherry was constructed by amplifying TACC3 (649–838 aa) by PCR, digesting with Bam HI and Age I and cloning into CD8-mCherry, available from previous work [ 32 ]. Details of siRNA sequences to specifically knockdown FT3 fusions in RT112 or RT4 cells are given in the electronic supplementary material.…”