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.

Keywords: Rab10; endocytosis; live-cell imaging; macrophages; macropinocytosis; tubules.

Figure 1

Rab10 dynamics in live RAW264 cells under the optogenetic control of PA-Rac1 activity. (A) The blue boxed area of a cell expressing ECFP-PA-Rac1 was irradiated with a blue laser to photoactivate PA-Rac1. Phase-contrast (left) and mCherry-Rab10 (right) images were acquired at the indicated times after PA-Rac1 activation (ON) and deactivation (OFF). Scale bar =10 μm. (B) Enlarged images of the white boxed area in (A) Following local PA-Rac1 activation, Rab10-positive premacropinosomes were formed in the area. After PA-Rac1 OFF, a few Rab10-positive tubules extended from a premacropinosome (arrowheads). Scale bar = 2 μm. (C) Representative phase-contrast and EGFP-Rab10 fluorescence images of RAW264 cells during PA-Rac1 ON-OFF cycles. (D) Confocal time-lapse microscopy of EGFP-Rab10 in RAW264 cells during PA-Rac1 ON-OFF cycles. Selected frames from the time-lapse movie are presented. Elapsed times are shown in the frame. Scale bar = 10 μm. The corresponding movie is available in the Supplementary Material Movie 2 .

Figure 2

Short-lived Rab10-positive premacropinosomal structures exist in RAW264 cells under near-physiological conditions. Confocal live-cell imaging of RAW 264 cells expressing EGFP-Rab10 stimulated with 10 nM phorbol myristate acetate (PMA). Elapsed times after the addition of PMA are shown in the frame. Arrows indicate a Rab10-positive premacropinosome which disappears within a few minutes. Asterisks indicate a Rab10-negative macropinosomal structure that remains as a phase-bright macropinosome. Scale bar = 10 μm. The corresponding movie is available in the Supplementary Material Movie 3 .

Figure 3

Rab10-positive tubules originate from unclosed macropinocytic cups (premacropinosomes). Rab10-positive macropinosome-like structures were induced by PA-Rac1 ON-OFF cycling. After confirming the formation of Rab10-positive tubules elongated from macropinosome-like structures, the FM4-64 membrane-impermeable dye was added to the medium to label the cell surface plasma membrane during image recording. (A) Images acquired just before the addition of FM4-64 (time 0 sec). Rab10-positive tubules elongated from Rab10-positive macropinosome-like structures were confirmed (arrows). (B) The EGFP-Rab10 and FM4-64 images were acquired at 20 sec after the FM4-64 addition. Although phase-bright Rab10-negative macropinosomes (blue asterisks) were unlabeled with the FM4-64, Rab10-positive macropinocytic structures with extending tubules (arrows) were labeled with the FM4-64. Scale bar = 10 μm.

Figure 4

The retrograde movement of Rab10-positive tubules is dependent on microtubules. (A) Epifluorescence microscopy of mCherry-Rab10 and EGFP-tubulin in live RAW 264 cells during PA-Rac1 ON-OFF cycling. Arrow indicates a Rab10-positive tubule extended from a peripheral premacropinosome. Scale bar=10 μm. (B) Higher-magnification view of extending Rab10-positive tubules (red arrows) along microtubules (green arrows). Scale bar = 2 μm. (C) The extension of EGFP-Rab10 tubules was inhibited in nocodazole-treated RAW264 cells, while many Rab10-positive premacropinosomes (arrows) were formed. Scale bar = 10 μm. The corresponding movies are available in the ( Supplementary Material Movies 4 – 6 ).

Figure 5

Localizations of EHBP1 and EHD1 on Rab10-positive premacropinosomes and tubules. (A) EHBP1 colocalized with Rab10 in premacropinosomes and tubules. Scale bar = 10 μm. (B) EHD1 predominantly localized on Rab10-positive tubules extended from premacropinosomes. Scale bar = 5 μm. Corresponding movies are available in the ( Supplementary Material Movies 7 , 8 ).

Figure 6

Rab5, an early endocytic marker, is not recruited to Rab10-positive premacropinosomes and tubules. (A) Live-cell microscopy of RAW264 cells expressing EGFP-Rab5a and mCherry-Rab10. Scale bar = 10 μm. (B) Enlarged micrographs of the boxed area in (A). Selective frames at two-time points as indicated. Rab5a-positive macropinosomes are Rab10-negative (asterisk). Rab10-positive compartments disappear within a few minutes without becoming Rab5a-positive, suggesting that Rab10-positive compartments are distinct from those of the classical macropinocytic pathway. Scale bar = 5 μm. The corresponding movie is available in the ( Supplementary Material Movie 9 ).

Figure 7

Rab10-positive premacropinosomes and tubules are distinct from conventional degradative endocytic or recycling pathways. Rab10-positive premacropinosomes and tubules are not labeled with the late endosome/lysosome markers, Rab7- (A) or LAMP1 (B). The recycling endosomal markers, Rab4 (C), Rab11 (D), and sorting nexin 1 (SNX1) (E) are faintly observed in Rab10-positive premacropinosomes and tubules, although they are only transiently recruited to the Rab10-negative conventional macropinosomes. Only Rab8a colocalized with Rab10 in the premacropinosomes and extending tubules (F). All scale bars = 10 μm. The corresponding movies are available in the ( Supplementary Material Movies 10 – 14 ).

Figure 8

Premacropinosome formation and the recruitment of Rab10 are not PI3K-dependent. The PI(3,4,5)P₃/PI(3,4)P₂ production by class I PI3K was monitored through the expression of mCitrine-Akt-PH domain (Akt-PH, green) in RAW264 cells during PA-Rac1 ON-OFF cycling (A) Although only small amounts of Akt-PH were observed on Rab10-positive premacropinosomes (arrows), Rab10-negative premacropinosomes were strongly positive for Akt-PH (arrowheads) at the timing of macropinosome closure. Elapsed time is shown in the frame. (B) In the presence of 10 μM LY294002, a PI3K inhibitor, the formation of Rab10-positive premacropinosomes was frequently seen (open arrows), whereas Akt-PH recruitment to the membrane was completely abolished. All scale bars = 10 μm. The corresponding movies are available in the ( Supplementary Material Movie 15 ).