https://orcid.org/0000-0002-7828-8118
In 2010, Dr. Li Yu and colleagues identified the important role of autophagic lysosome reformation (ALR) in maintaining lysosome homeostasis in cells after prolonged starvation. Since then, Dr. Yu and his research group have contributed significantly to elucidating the molecular mechanisms that underlie ALR. Here, we summarize the progress of research on ALR and specifically highlight the roles of kinesin 1, clathrin and phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P2) in regulating this process.
A careful morphological examination of the autolysosomes in normal rat kidney cells in a time course from 0 h to 12 h starvation revealed the presence of tubular extensions after 8 h Citation[1]. These structures were then pinched off to form new lysosomal compartments, thus replenishing the number of lysosomes in the cells. This process is termed autophagic lysosome reformation. Moreover, Yu et al. demonstrated that ALR is dependent on MTOR (mechanistic target of rapamycin [serine/threonine kinase]) reactivation during prolonged starvation; once the cells are starved, MTOR is inhibited and autophagy is induced. If starvation persists, the recycled amino acids resulting from the autophagy influx reactivate MTOR, which in turn induces ALR.
Later studies from Yu and colleagues further investigated the molecular mechanisms regulating ALR. They first did a systematic screen to identify potential regulators Citation[2]. Initially, the elongated autolysosome tubular structures were purified. They then combined proteomic analysis, large-scale RNA-mediated interference screening and functional studies to look for candidate components that control autophagic lysosome reformation. Through this approach, they determined that both clathrin and PtdIns(4,5)P2 are required for ALR [2]. Knocking down of the clathrin heavy chain gene (Cltc) leads to enlarged autolysosomes without elongated tubular structures. In addition, they found that a pool of clathrin translocates to autolysosomes after starvation, suggesting clathrin is likely to directly regulate ALR. Furthermore, PtdIns(4,5)P2 is also observed on autolysosomes after starvation. Silencing of either the Pip5k1a or Pip5k1b isoforms of PtdIns4P 5-kinase, inhibits ALR; however, the 2 corresponding enzymes play differential roles in regulating ALR. In Pip5k1b knockdown cells, autolysosomes are enlarged and barely display tubular structures, implicating a role for PIP5K1B in the initiation of ALR. In contrast, with the silencing of Pip5k1a, elongated tubular structures from autolysosomes were stabilized and did not scission from the lysosome. This discrepancy can be explained by the observation that PIP5K1A localizes to the tips and buds of the tubules. This localization may promote the local synthesis of PtdIns(4,5)P2 and facilitate the budding and/or fission of the tubules. Moreover, they found that adaptor protein 2 that bridges clathrin with PtdIns(4,5)P2 is also required for ALR. These observations imply a model in which synthesis of PtdIns(4,5)P2 on autolysosomes recruits clathrin and promotes ALR during prolonged starvation.
The downstream factors that interact with PtdIns(4,5)P2 and clathrin with regard to ALR regulation were unknown until recently. Dr. Yu's research group reported that the kinesin 1 motor protein KIF5B is required for autolysosome tubulation Citation[3]. In kif5b knockout cells, autolysosome tubules are almost not observed after prolonged starvation. Expression of wild-type (WT) KIF5B, but not KIF5BT92N, a mutant lacking mobility, suppresses the defects. Next, they developed an in vitro reconstitution system to test whether KIF5B can drive autolysosome tubulation. Autolysosomes and KIF5B (WT or T92N mutant) were purified separately. They confirmed that both WT and mutant KIF5B are able to associate with the purified autolysosomes. However, with ATP, only KIF5BWT, but not KIF5BT92N, drives tubule formation from autolysosomes, suggesting KIF5B is able to directly mediate autolysosome tubulation, and this is dependent on its mobility.
As discussed above, PtdIns(4,5)P2 regulates ALR. The authors found that with ATP and KIF5B, the purified autolysosomes from Pip5k1b knockdown cells cannot be tubulated, suggesting that PtdIns(4,5)P2 is very likely required for KIF5B's function in ALR. Indeed, using lipid overlay and liposome sedimentation assays, Du et al. found that KIF5B interacts with PtdIns(4,5)P2. In addition, the cargo domain (amino acids 814–963) of KIF5B mediates the interaction. Finally, with the in vitro tubulation system, they found that KIF5B can drive tubulation of liposomes containing a PtdIns(4,5)P2 concentration higher than 10%. Thus, PtdIns(4,5)P2 on autolysosomes recruits KIF5B to drive ALR.
The physiological level of PtdIns(4,5)P2 in cellular membranes is relatively low; it is suggested that 5% may be the upper limit of local concentrations. Because KIF5B was not able to drive tubulation of liposomes containing 5% PtdIns(4,5)P2, some other factor may be involved in facilitating the ability of KIF5B to drive autolysosome tubulation under physiological conditions. As mentioned above, clathrin is also required for ALR and localizes at autolysosomes after starvation. Does clathrin play a role in KIF5B-mediated tubulation? The authors tested this hypothesis using the in vitro reconstitution system. When the purified autolysosomes were treated with trypsin, which “strips off” clathrin, the tubulation driven by KIF5B is impaired. But if the system is complemented with rat brain cytosol containing clathrin, tubulation of autolysosomes is rescued. Moreover, KIF5B is able to drive tubulation of liposomes containing 5% PtdIns(4,5)P2 when rat brain cytosol is present. These lines of evidence imply that clathrin is indispensible for the autolysosome tubulation driven by KIF5B.
It was also previously observed that clathrin and PtdIns(4,5)P2 are located on autolysosomes in the form of small buds/microdomains Citation[2]. Is it possible that clathrin promotes the formation of PtdIns(4,5)P2-enriched-microdomains, so that the local concentration of PtdIns(4,5)P2 is sufficient to recruit KIF5B to drive tubulation? To answer this question, the authors took advantage of giant unilamellar vesicles (GUVs) containing fluorescently labeled PtdIns(4,5)P2, which is evenly distributed on the GUVs. In the presence of rat brain cytosol, however, PtdIns(4,5)P2 forms microdomains on the GUVs. If the rat brain cytosol is pre-incubated with anti-clathrin antibody so that clathrin is removed, the PtdIns(4,5)P2 microdomains are not formed. In addition, when KIF5B is added, it colocalizes with the PtdIns(4,5)P2 microdomains on the GUVs. Moreover, in either Cltc or Pip5k1b knockdown cells, the colocalization of KIF5B with autolysosomes is diminished. These results collectively support the idea that clathrin promotes the formation of PtdIns(4,5)P2 microdomains on autolysosomes, which in turn facilitate the recruitment of KIF5B to drive its tubulation.
To sum up, Dr. Yu and his research group made significant contributions to unravel the molecular mechanisms of ALR. The understanding of ALR provides new insights into the maintenance of lysosome homeostasis, which is of great physiological importance. In addition, the observation that clathrin enriches PtdIns(4,5)P2 at specific microdomains on the lysosome membrane to allow the recruitment of its downstream effectors also provides novel clues to the studies of other cellular processes that involve clathrin.
Acknowledgements
This work was supported by NIH grant GM053396 to DJK.
References
- Yu L, McPhee CK, Zheng L, Mardones GA, Rong Y, Peng J, Mi N, Zhao Y, Liu Z, Wan F, et al. Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature. 2010;465:942–6. doi:10.1038/nature09076. PMID:20526321.
- Rong Y, Liu M, Ma L, Du W, Zhang H, Tian Y, Cao Z, Li Y, Ren H, Zhang C, et al. Clathrin and phosphatidylinositol-4,5-bisphosphate regulate autophagic lysosome reformation. Nat Cell Biol. 2012;14:924–34. doi:10.1038/ncb2557. PMID:22885770.
- Du W, Su QP, Chen Y, Zhu Y, Jiang D, Rong Y, Zhang S, Zhang Y, Ren H, Zhang C, et al. Kinesin 1 Drives Autolysosome Tubulation. Dev Cell. 2016;37:326–36. doi:10.1016/j.devcel.2016.04.014. PMID:27219061.