A phosphoinositide and RAB switch controls early macropinocytosis
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Evaluation Summary:
This study is of interest to the broader audience of cell biologists, as it aims to dissect the hierarchy of protein recruitment and lipid conversion events that may provide a potential mechanism for the formation, maturation and fate of macropinosomes. The conclusions are based on the observation that pharmacological inhibition of the lipid kinase VPS34, which generates the signalling lipid PI3P on endosomes, prevents accumulation of Rab5 on macropinosomes, blocking their maturation and causing them to re-fuse with the plasma membrane.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Macropinocytosis is a non-selective endocytic process by which cells take up large amounts of extracellular fluids into giant vesicles known as macropinosomes. This mechanism is used by immune cells to sample the surroundings for antigens and can be exploited by cancer cells for nutrient uptake. What determines the fate of macropinosomes after they have been internalized is largely unknown. Here we investigate the role of the phosphatidylinositol 3-kinase VPS34/PIK3C3 and its product phosphatidylinositol 3-phosphate (PtdIns3P) in macropinosome fate determination. Inhibition of VPS34 led to a decrease in macropinosome survival and fluid phase uptake as well as preventing recruitment of early endosomal factors, including the small GTPase RAB5 and its effectors, to the forming macropinosomes. Instead, forming macropinosomes under VPS34 inhibition accumulated regulators of endocytic recycling, including RAB8A, RAB10, RAB11A, and PtdIns4P, which led to fusion of macropinosomes with the plasma membrane.
Whereas RAB5 was critical for macropinosome formation, macropinosome fusion with the plasma membrane depended on RAB8A. Thus, macropinosome maturation is regulated by a PtdIns3P-controlled switch that balances macropinosome fate between the default, endolysosomal maturation and an alternative, secretory route.
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Evaluation Summary:
This study is of interest to the broader audience of cell biologists, as it aims to dissect the hierarchy of protein recruitment and lipid conversion events that may provide a potential mechanism for the formation, maturation and fate of macropinosomes. The conclusions are based on the observation that pharmacological inhibition of the lipid kinase VPS34, which generates the signalling lipid PI3P on endosomes, prevents accumulation of Rab5 on macropinosomes, blocking their maturation and causing them to re-fuse with the plasma membrane.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
Spangenberg and co-workers show that macropinocytosis in tissue culture cells is inhibited by SAR405, which targets the PI3kinase, Vps34. This kinase produces PI3P on endosomes, which is important for their trafficking. Inhibited cells still form early macropinosomes, but these do not progress through the endocytic system and instead the authors argue that they fuse back to the plasma membrane and release their contents, thus making fluid uptake very inefficient.
The basic observation suggesting a requirement for PI3P in macropinosome trafficking is interesting, though not completely surprising. Since macropinocytosis is a cyclic process in which membrane is taken in, trafficked through the endosomal system and returned to the cell surface it is expected that blocking one step will eventually block the whole …
Reviewer #1 (Public Review):
Spangenberg and co-workers show that macropinocytosis in tissue culture cells is inhibited by SAR405, which targets the PI3kinase, Vps34. This kinase produces PI3P on endosomes, which is important for their trafficking. Inhibited cells still form early macropinosomes, but these do not progress through the endocytic system and instead the authors argue that they fuse back to the plasma membrane and release their contents, thus making fluid uptake very inefficient.
The basic observation suggesting a requirement for PI3P in macropinosome trafficking is interesting, though not completely surprising. Since macropinocytosis is a cyclic process in which membrane is taken in, trafficked through the endosomal system and returned to the cell surface it is expected that blocking one step will eventually block the whole process, possibly through an indirect mechanism. Thus inhibitor experiments such as this are difficult to understand mechanistically (though the fact that SAR405 works quickly is encouraging).
Inhibited macropinosomes may fail to retain fluid efficiently for at least three different reasons: they do not seal properly and so drain back to the surface; or they lose fluid in small vesicles which traffic back to the surface; or they fuse back to the surface as the authors suggest. The author's conclusion is not well-supported by their data in my opinion. For instance, in several cases newly formed macropinosomes appear to shrink over a period of 20 seconds (Fig 1E), which is inconsistent with a mechanism based on fusion back to the plasma membrane.
One problem is that the authors cannot visualise the inhibited macropinosomes very well because they lack their Phafin2 reporter. However, macropinosomes can be followed using FITC dextran either supplied continuously, or after a short pulse, and observation by oblique plane microscopy might allow the authors to follow the shrinking macropinosomes and so distinguish between these possibilities.
A second general problem is whether the inhibition is indirect. Apart from the possibility of backing up the endocytic system already mentioned, it is likely that inhibiting Vps34 also perturbs other phosphoinositides, of which PIP3 is essential for macropinoscytosis, and PI3,4P2 is likely important in early trafficking. It is therefore very surprising that neither of these was imaged.
Some of the data appears to be over-interpreted. For instance, it is claimed that Rab5 is not recruited in the presence of SAR405, yet the quantitation in Fig 3B shows comparable or higher levels than control for the first 100 sec and substantial levels throughout. Similarly Rabankyrin5.
Fig 2D suggests that less than half of FITC dextran uptake is due to macropinocytosis (assuming EIPA and SAR405 block it completely). What other routes are used for this residual uptake, and why are they not sensitive to SAR405, as CME would be expected to be?
PI3P undoubtedly has an important role in macropinosome trafficking, but teasing it out will be difficult and complex. The author's conclusions may be correct, but they are short of convincing as they stand.
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Reviewer #2 (Public Review):
There are many interesting aspects of this work and it is one of the first studies to address how macropinosomes initiate their maturation. As much of the machinery is shared with other endocytic pathways it is possible (although not demonstrated) that the observed mechanisms indicate general aspects of trafficking.
Their conclusions are largely based on the observation that pharmacological inhibition of the lipid kinase VPS34, which generates the signalling lipid PI3P on endosomes, prevents accumulation of Rab5 on macropinosomes, blocking their maturation and causing them to re-fuse with the plasma membrane. This affect is clear, and well characterised, however several important mechanistic questions remain.
A major limitation is that they do not directly show whether the macropinosomes that apparently …
Reviewer #2 (Public Review):
There are many interesting aspects of this work and it is one of the first studies to address how macropinosomes initiate their maturation. As much of the machinery is shared with other endocytic pathways it is possible (although not demonstrated) that the observed mechanisms indicate general aspects of trafficking.
Their conclusions are largely based on the observation that pharmacological inhibition of the lipid kinase VPS34, which generates the signalling lipid PI3P on endosomes, prevents accumulation of Rab5 on macropinosomes, blocking their maturation and causing them to re-fuse with the plasma membrane. This affect is clear, and well characterised, however several important mechanistic questions remain.
A major limitation is that they do not directly show whether the macropinosomes that apparently re-fuse with the surface have actually completed closure in the first place. It remains possible that the macropinocytic cups only partially close, and Rab5/VPS34 activity is required for a final scission.
It is also unclear whether VPS34 is upstream or downstream of Rab5 in these studies. Previous work clearly shows that Rab5 is able to recruit and activate VPS34 to produce PI3P. The model proposed by the authors suggests that VPS34 activity is actually upstream of Rab5, causing its recruitment. Some of the data support this, but as VPS34, Rab5 and all their effectors are also important for other endocytic trafficking it is likely that this will cause indirect effects, such as retaining of Rab5 on other compartments to prevent its recruitment.
They try to address this by artificially targeting Rab5 constitutively to the membrane (Figure 6A-C) but these experiments lack some important controls, and it remains to be explained how an initial pool of VPS34 activity would be regulated. The dynamics of VPS34 localisation are also never directly addressed so further experiments are required to support this aspect of their model.
Nonetheless, they show that upon (apparent) closure, macropinosomes by default accumulate Rabs8 and 10 - normally associated with recycling endosomes. This is interesting because it implies they will naturally re-fuse with the plasma membrane unless the cell specifically drives them down a maturation pathway. In fact they show that, in their cells, only 60% of macropinosomes formed ever normally make it to maturation (Figure 2A). This is interesting, and as speculated by the authors, is possibly a safely mechanism to rid the cell of "accidental" vesicles. How this is regulated and mechanistically occurs is however not explored.
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Reviewer #3 (Public Review):
In this study, Spangenberg et al investigate the role of Vps34, a lipid kinase which catalyzes the formation of Phosphatidylinositol-3-phosphate PI(3)P from Phasphatidylinostol) during the early stages of micropinocytosis. A small molecule inhibitor of Vps34 blocks the switch from early PI(4)P-positive macropinosomes to PI(3)P-positive macropinosomes: Macropinosomes retain PI(4)P and Rab8 and fail to recruit Rab5 and its effectors. Unable to undergo endosome-like maturation, these impaired macropinosomes appear to regurgitate to the plasma membrane, potentially as a mechanism to maintain membrane homeostasis. These observations suggest the presence of a novel Rab switch from secretory Rabs such as Rab8 to endosomal Rabs such as Rab5 on newly forming macropinosomes.
The study presents an interesting and novel …
Reviewer #3 (Public Review):
In this study, Spangenberg et al investigate the role of Vps34, a lipid kinase which catalyzes the formation of Phosphatidylinositol-3-phosphate PI(3)P from Phasphatidylinostol) during the early stages of micropinocytosis. A small molecule inhibitor of Vps34 blocks the switch from early PI(4)P-positive macropinosomes to PI(3)P-positive macropinosomes: Macropinosomes retain PI(4)P and Rab8 and fail to recruit Rab5 and its effectors. Unable to undergo endosome-like maturation, these impaired macropinosomes appear to regurgitate to the plasma membrane, potentially as a mechanism to maintain membrane homeostasis. These observations suggest the presence of a novel Rab switch from secretory Rabs such as Rab8 to endosomal Rabs such as Rab5 on newly forming macropinosomes.
The study presents an interesting and novel mechanism in addition to the Rab5-to-Rab7-conversion in the endosomal degradative pathway. While it is yet unclear from the data presented whether the switch from Rab8 to Rab5 is blocked due to the lack of PI(3)P, or the persistence of PI(4)P, the functional importance for the interconversion of these lipids is evident. Regrettably, the data are not always well presented and/or explained, and a few simple experiments would aid to shed more light on the hierarchical control of the studied Rab and lipid switch. As such, the suggestion that PI(3)P directly recruits Rab5 is well beyond the available data and potentially misleading. It should be postulated more cautiously, or investigated further to provide supporting experimental evidence. For example, establishing the temporal correlation between PI(3)P generation and Rab5 recruitment, and demonstrating preferential binding of Rab5 to PI(3)P over other phosphatidylinositol species would be necessary to support this claim. The notion that Vps34-mediatied PI(3)P synthesis is important to recruit Rab5 as it competes with PI(4)P formation seems simpler and more plausible, as a PI(4)P -dependent Rab5 inhibitor and or Rab8 stabilizing factor could not be excluded, and a direct interaction between PI(3)P and Rab5 has not established. Overall, if these points were addressed, the study would provide an interesting glimpse into the regulation of a Rab switch to determine organelle fate.
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