Our **data** thus far clearly point to PIKfyve playing a role in the process of tau seeding in neurons. In order to explore this, we decided to further characterize K18 PFFs uptake and internalization following PIKfyve inhibition. Using K18 PFFs labeled with the pH-insensitive AF488, the number of K18 or FL Tau PFF-positive vesicles in primary hippocampal neurons was to our surprise not altered at 100 nM or 1 μM YM-201636 treatment (Fig. 5, A and B). These results seemed at first to be counterintuitive given our previous **data** using pHrodo labeled K18 PFFs that suggested reduced internalization. To address this conundrum, we tested if pharmacological inhibition of PIKfyve would reduce K18-pHrodo signal as we observed using the knockdown approach. Similar to PIKfyve knock-down, treatment of primary hippocampal neurons with YM-201636 displayed a dose-dependent reduction in K18-pHrodo signal (Fig. 5, C and D). Since the pHrodo signal is not only dependent on neuronal internalization of a labeled substrate but also on the transport into low-pH compartments, we evaluated intracellular acidification using LysoTracker. Interestingly, YM-201636 treatment did not show a decrease, but rather an increase in LysoTracker intensity at concentrations up to 1 μM before reducing due to toxicity (Fig. 5F). These **data** show that the reduction in K18-pHrodo signal is not due to impaired acidification but instead suggest that K18 PFFs are retained in nonacidic vesicles in the endosomal pathway. In support of this notion, PIKfyve inhibition has been linked to an intracellular vesicular phenotype consisting in enlarged endosomes, caused by the reduced maturation of MVB into lysosomes as well as lysosomal recycling (52,53). Indeed, when we treated primary hippocampal neurons with 1 μM YM-201636, we noticed a decrease in the number of LAMP1-positive organelles but an increase in their size, supported by 3D imaging and in line with previous reports (54, 55) (Figs. 5E and S8). Similar findings can be observed in HEK293T cells following 30 min treatment with 1 μM YM-201636, pointing to a cell-typeindependent phenotype (Fig. S9). These **data** suggest that the reduction in K18 PFFs internalization previously seen with Rac1 inhibition is carried out by a mechanism that is PIKfyveindependent, while PIKfyve could be important for the fate of the seed once internalized.