Mechanisms of endosmal permeability and release: a route for enhancing cytoplasmic delivery of endocytosed therapeutics.

Cells internalize macromolecules such as antibodies by
receptor-mediated endocytosis which results in the macromolecules being sequestered within
membrane-bound endosomes and lysosomes. Normally, endosomal leakage is minimal, and
cytoplasmic delivery is profoundly inefficient (Gilleron et al., 2013). However efficient release has
been observed following binding of adenovirus (Ad2) particles to cells (Meier et al., 2002). This
stimulates macropinocytosis whereby cells engulf large volumes of extracellular fluid by extending
protrusions from the cell surface which fuse together to form macropinosomes which are leaky in
Ad2-treated cells.
We have developed an assay for Ad2-mediated release of endosomal contents in HeLa cells. Cell
surface binding of Ad2 is sufficient to stimulate macropinocytosis of GFP with a nuclear import
sequence (NLS-GFP) and the resulting macropinosomes can burst, delivering NLS-GFP into the
cytoplasm. NLS-GFP can then translocate into the nucleus and be detected by
immunofluorescence.