Rab5 activation on macropinosomes requires ALS2, and subsequent Rab5 inactivation through ALS2 detachment requires active Rab7.

Macropinocytosis is a nonspecific bulk uptake of extracellular fluid. During endosome maturation, the Rab5-to-Rab7 switch machinery executes the conversion from early to late endosomes. However, how the Rab switch works during macropinosome maturation remains unclear. Here, we elucidate the Rab switch machinery in macropinosome maturation using Förster resonance energy transfer imaging. Rab5 is activated and concurrently recruited to macropinosomes during ruffle closure. ALS2 depletion abolishes transient Rab5 activation on macropinosomes, while ALS2 is recruited to macropinosomes simultaneously with Rab5 activation. Thus, we conclude ALS2 activates Rab5 on macropinosomes. The absence of active Rab7 prolongs ALS2 presence and Rab5 activation on macropinosomes, indicating that active Rab7 is necessary for Rab5 inactivation through ALS2 dissociation and plays key roles in the Rab switch on macropinosomes.

Mon1-Ccz1 activates Rab7 only on late endosomes and dissociates from the lysosome in mammalian cells.

Rab GTPases act as molecular switches regulating various aspects of membrane trafficking. Among them, Rab5 and Rab7 play central roles in the endolysosomal network. Although many effectors downstream of Rab7 have been elucidated, our present understanding of the mechanism regulating Rab7 activity is extremely limited. It has only recently been accepted that the Mon1-Ccz1 complex is a Rab7 guanine nucleotide exchange factor, but it still remains unclear what the location where Mon1-Ccz1 works with Rab7 is. To address what kind of change or switch exists in the regulatory mechanism upstream of Rab7 during its transition from the late endosome to lysosome, we examined Rab7 activity in steady-state cells and during EGF-induced macropinocytosis using a newly developed FRET sensor. A combination of a Rab7 sensor and confocal FRET imaging techniques revealed that the activation of Rab7 on late endosomes depends on Mon1-Ccz1 and is implicated in late-endosome-lysosome fusion. In contrast, Rab7 activity on lysosomes was independent of Mon1-Ccz1 and active Rab7 played a role in perinuclear clustering of lysosomes.

Longest neurite-specific activation of Rap1B in hippocampal neurons contributes to polarity formation through RalA and Nore1A in addition to PI3-kinase.

In a developing nervous system, axon-dendrite formation is instructed by extrinsic cues, and the mechanism whereby a developing neuron interprets these cues using intracellular signaling is particularly important. Studies using dissociated hippocampal neurons have identified many signaling pathways underlying neuronal polarization. Among the components of these pathways, Rap1B is essential for axon specification in hippocampal cultures. However, spatiotemporal regulation of Rap1B activity in polarizing neurons and how it affects neuronal polarization remain unclear. Herein, we investigated spatiotemporal activity-change of Rap1B and its target molecules in hippocampal neurons. FRET imaging showed that specific activation of Rap1B was observed at the tip of a future axon. To dissect downstream signaling, we used three effector mutants of Rap1B. Expression of Rap1B-G12V/E37G and G12V/Y40C mutants resulted in supernumerary axons. The targets of Rap1B-G12V/E37G were RalA and Nore1A, whereas Rap1B-G12V/Y40C activated PI3-kinase. RalA was activated in the tip of stage 3 axons, and RalA-S28N expression reduced the fraction of neurons with supernumerary axons induced by Rap1B-G12V/E37G. Furthermore, Nore1A depletion reduced the number of cells without axons. These results indicate that specific activation of Rap1B contributes to neuronal polarization via interaction with RalA and Nore1A in addition to PI3-kinase.