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

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.

Rab35 Targeting to the Plasma Membrane Is Dependent on the C-terminal Polybasic Cluster.

Rab35, a member of the Rab GTPase family, has been implicated in various cellular processes including cell motility and membrane trafficking. Although Rab35 is localized to the plasma membrane, Rab proteins that are identified to have high sequence homology with Rab35 exhibit distinct subcellular localization patterns. Comparing the amino acid sequences between Rab35 and its family members revealed a significant variation in an approximate 30-amino acid region of the C-terminus. This suggests that this region determines the subcellular localization of individual Rab proteins. To confirm this hypothesis, we constructed Rab35-Rab10 chimera proteins by exchanging their C-terminal domains with one another. Confocal microscopy of RAW264 cells expressing EGFP-fused Rab35-Rab10 chimeras has indicated that the C-terminal region of Rab35 is critical for its plasma membrane localization. Furthermore, we were able to determine that a basic amino acid cluster exists in the C-terminal region of Rab35 and that Rab35 localization shifts to the Golgi membrane when the number of basic amino acids in this region is reduced. Thus, it is likely that the approximate 30-amino acid C-terminal region containing basic clusters is responsible for Rab35 plasma membrane localization and that its preferential localization depends on the number of basic amino acids.