Macropinocytosis is a prevalent and essential pathway in macrophages where it 
contributes to anti-microbial responses and innate immune cell functions. Cell 
surface ruffles give rise to phagosomes and to macropinosomes as 
multi-functional compartments that contribute to environmental sampling, 
pathogen entry, plasma membrane turnover and receptor signalling. Rapid, high 
resolution, lattice light sheet imaging demonstrates the dynamic nature of 
macrophage ruffling. Pathogen-mediated activation of surface and endosomal 
Toll-like receptors (TLRs) in macrophages upregulates macropinocytosis. Here, 
using multiple forms of imaging and microscopy, we track membrane-associated, 
fluorescently-tagged Rab8a expressed in live macrophages, using a variety of 
cell markers to demonstrate Rab8a localization and its enrichment on early 
macropinosomes. Production of a novel biosensor and its use for quantitative 
FRET analysis in live cells, pinpoints macropinosomes as the site for 
TLR-induced activation of Rab8a. We have previously shown that TLR signalling, 
cytokine outputs and macrophage programming are regulated by the GTPase Rab8a 
with PI3 Kγ as its effector. Finally, we highlight another effector, the 
phosphatase OCRL, which is located on macropinosomes and interacts with Rab8a, 
suggesting that Rab8a may operate on multiple levels to modulate 
phosphoinositides in macropinosomes. These findings extend our understanding of 
macropinosomes as regulatory compartments for innate immune function in 
macrophages. This article is part of the Theo Murphy meeting issue 
'Macropinocytosis'.