Rab10-Positive Tubular Structures Represent a Novel Endocytic Pathway That Diverges From Canonical Macropinocytosis in RAW264 Macrophages

Abstract

Using the optogenetic photo-manipulation of photoactivatable (PA)-Rac1, remarkable cell surface ruffling and the formation of a macropinocytic cup (premacropinosome) could be induced in the region of RAW264 macrophages irradiated with blue light due to the activation of PA-Rac1. However, the completion of macropinosome formation did not occur until Rac1 was deactivated by the removal of the light stimulus. Following PA-Rac1 deactivation, some premacropinosomes closed into intracellular macropinosomes, whereas many others transformed into long Rab10-positive tubules without forming typical macropinosomes. These Rab10-positive tubules moved centripetally towards the perinuclear Golgi region along microtubules. Surprisingly, these Rab10-positive tubules did not contain any endosome/lysosome compartment markers, such as Rab5, Rab7, or LAMP1, suggesting that the Rab10-positive tubules were not part of the degradation pathway for lysosomes. These Rab10-positive tubules were distinct from recycling endosomal compartments, which are labeled with Rab4, Rab11, or SNX1. These findings suggested that these Rab10-positive tubules may be a part of non-degradative endocytic pathway that has never been known. The formation of Rab10-positive tubules from premacropinosomes was also observed in control and phorbol myristate acetate (PMA)-stimulated macrophages, although their frequencies were low. Interestingly, the formation of Rab10-positive premacropinosomes and tubules was not inhibited by phosphoinositide 3-kinase (PI3K) inhibitors, while the classical macropinosome formation requires PI3K activity. Thus, this study provides evidence to support the existence of Rab10-positive tubules as a novel endocytic pathway that diverges from canonical macropinocytosis.

Morphological Evidence for Novel Roles of Microtubules in Macrophage Phagocytosis

“…Since we have previously compared and examined fixation protocols for clear visualization of microtubules in another experimental system [ 27 , 28 ], we could improve the fixation and staining protocols. In some previous studies from other groups, although the authors did not mention the microtubule networks, there are clear photographs showing the microtubule network in macrophages, similar to our results [ 29 , 30 ]. Analyses of macrophages using innovative imaging technologies are becoming increasingly important for a precise understanding of immunology.…”

The small GTPase RAB10 regulates endosomal recycling of the LDL receptor and transferrin receptor in hepatocytes

“…RAB10 has also been implicated in diverse areas of membrane trafficking in different cell types, including formation of noncanonical macropinosome tubules in macrophages ( 49 ), vesicle transportation from early endosome to recycling endosome in C. elegans ( 31 ), and Golgi to plasma membrane transport in macrophages ( 32 ). Consistent with this wide range of functions, RAB10 has been localized to multiple subcellular compartments in different cell types including the endoplasmic reticulum, trans Golgi network, early endosomes, recycling endosomes, phagosomes, and primary cilia ( 25 , 31 , 32 , 50 , 51 , 52 ).…”

Salmonella exploits membrane reservoirs for invasion of host cells

“…Prior studies have identified a wide spectrum of RAB10 activities in multiple intracellular compartments, including endosomes, the Golgi/TGN, the endoplasmic reticulum, GLUT4-containing exocytic vesicles, neuronal axons, and primary cilia 5 , 6 , 37 . Plus, a recent study from Kawai et al showed that in macrophages RAB10 + tubular structures can represent a novel endocytic pathway that diverges from canonical micropinocytosis under certain conditions (e.g., PI3-kinase inhibition) 38 . Here, our findings offer fresh perspectives on the multifaceted functions of RAB10 and membrane reservoirs in the context of bacterial invasion.…”