Overlapping roles of JIP3 and JIP4 in promoting axonal transport of lysosomes in human iPSC-derived neurons.

The dependence of neurons on microtubule-based motors for the movement of lysosomes over long distances raises questions about adaptations that allow neurons to meet these demands. Recently, JIP3/MAPK8IP3, a neuronally enriched putative adaptor between lysosomes and motors, was identified as a critical regulator of axonal lysosome abundance. In this study, we establish a human induced pluripotent stem cell (iPSC)-derived neuron model for the investigation of axonal lysosome transport and maturation and show that loss of JIP3 results in the accumulation of axonal lysosomes and the Alzheimer's disease-related amyloid precursor protein (APP)-derived Aß42 peptide. We furthermore reveal an overlapping role of the homologous JIP4 gene in lysosome axonal transport. These results establish a cellular model for investigating the relationship between lysosome axonal transport and amyloidogenic APP processing and more broadly demonstrate the utility of human iPSC-derived neurons for the investigation of neuronal cell biology and pathology.

Pleiotropic requirements for human TDP-43 in the regulation of cell and organelle homeostasis.

TDP-43 is an RNA-binding protein that forms cytoplasmic aggregates in multiple neurodegenerative diseases. Although the loss of normal TDP-43 functions likely contributes to disease pathogenesis, the cell biological consequences of human TDP-43 depletion are not well understood. We, therefore, generated human TDP-43 knockout (KO) cells and subjected them to parallel cell biological and transcriptomic analyses. These efforts yielded three important discoveries. First, complete loss of TDP-43 resulted in widespread morphological defects related to multiple organelles, including Golgi, endosomes, lysosomes, mitochondria, and the nuclear envelope. Second, we identified a new role for TDP-43 in controlling mRNA splicing of Nup188 (nuclear pore protein). Third, analysis of multiple amyotrophic lateral sclerosis causing TDP-43 mutations revealed a broad ability to support splicing of TDP-43 target genes. However, as some TDP-43 disease-causing mutants failed to fully support the regulation of specific target transcripts, our results raise the possibility of mutation-specific loss-of-function contributions to disease pathology.

Lysosomes Dare Cells to be Different(iated).

In this issue of Cell Stem Cell, Villegas et al. (2019) used a genome-wide CRISPR screen to identify ESC pluripotency regulators, which generated insights into how signaling from lysosome enables cells to transcriptionally regulate their metabolism. Further investigation of human genetics identified a human developmental disease arising from a defect in this process.

C9orf72 binds SMCR8, localizes to lysosomes, and regulates mTORC1 signaling.

Hexanucleotide expansion in an intron of the C9orf72 gene causes amyotrophic lateral sclerosis and frontotemporal dementia. However, beyond bioinformatics predictions that suggested structural similarity to folliculin, the Birt-Hogg-Dubé syndrome tumor suppressor, little is known about the normal functions of the C9orf72 protein. To address this problem, we used genome-editing strategies to investigate C9orf72 interactions, subcellular localization, and knockout (KO) phenotypes. We found that C9orf72 robustly interacts with SMCR8 (a protein of previously unknown function). We also observed that C9orf72 localizes to lysosomes and that such localization is negatively regulated by amino acid availability. Analysis of C9orf72 KO, SMCR8 KO, and double-KO cell lines revealed phenotypes that are consistent with a function for C9orf72 at lysosomes. These include abnormally swollen lysosomes in the absence of C9orf72 and impaired responses of mTORC1 signaling to changes in amino acid availability (a lysosome-dependent process) after depletion of either C9orf72 or SMCR8. Collectively these results identify strong physical and functional interactions between C9orf72 and SMCR8 and support a lysosomal site of action for this protein complex.

Recruitment of folliculin to lysosomes supports the amino acid-dependent activation of Rag GTPases.

Birt-Hogg-Dubé syndrome, a human disease characterized by fibrofolliculomas (hair follicle tumors) as well as a strong predisposition toward the development of pneumothorax, pulmonary cysts, and renal carcinoma, arises from loss-of-function mutations in the folliculin (FLCN) gene. In this study, we show that FLCN regulates lysosome function by promoting the mTORC1-dependent phosphorylation and cytoplasmic sequestration of transcription factor EB (TFEB). Our results indicate that FLCN is specifically required for the amino acid-stimulated recruitment of mTORC1 to lysosomes by Rag GTPases. We further demonstrated that FLCN itself was selectively recruited to the surface of lysosomes after amino acid depletion and directly bound to RagA via its GTPase domain. FLCN-interacting protein 1 (FNIP1) promotes both the lysosome recruitment and Rag interactions of FLCN. These new findings define the lysosome as a site of action for FLCN and indicate a critical role for FLCN in the amino acid-dependent activation of mTOR via its direct interaction with the RagA/B GTPases.

The transcription factor TFEB links mTORC1 signaling to transcriptional control of lysosome homeostasis.

Lysosomes are the major cellular site for clearance of defective organelles and digestion of internalized material. Demand on lysosomal capacity can vary greatly, and lysosomal function must be adjusted to maintain cellular homeostasis. Here, we identified an interaction between the lysosome-localized mechanistic target of rapamycin complex 1 (mTORC1) and the transcription factor TFEB (transcription factor EB), which promotes lysosome biogenesis. When lysosomal activity was adequate, mTOR-dependent phosphorylation of TFEB on Ser(211) triggered the binding of 14-3-3 proteins to TFEB, resulting in retention of the transcription factor in the cytoplasm. Inhibition of lysosomal function reduced the mTOR-dependent phosphorylation of TFEB, resulting in diminished interactions between TFEB and 14-3-3 proteins and the translocation of TFEB into the nucleus, where it could stimulate genes involved in lysosomal biogenesis. These results identify TFEB as a target of mTOR and suggest a mechanism for matching the transcriptional regulation of genes encoding proteins of autophagosomes and lysosomes to cellular need. The closely related transcription factors MITF (microphthalmia transcription factor) and TFE3 (transcription factor E3) also localized to lysosomes and accumulated in the nucleus when lysosome function was inhibited, thus broadening the range of physiological contexts under which this regulatory mechanism may prove important.

Emerging roles for p120-catenin in cell adhesion and cancer.

Although originally identified as a Src substrate, p120-catenin (p120) is now known to regulate cell-cell adhesion through its interaction with the cytoplasmic tail of classical and type II cadherins. New evidence indicates that p120 regulates cadherin turnover at the cell surface, thereby controlling the amount of cadherin available for cell-cell adhesion. This function is necessary but not sufficient to promote strong adhesion, which is further controlled by signals acting on the amino-terminal p120 regulatory domain. p120 also modulates the activities of RhoA, Rac, and Cdc42, suggesting that along with other Src substrates, p120 regulates actin dynamics. Thus, p120 is a master regulator of cadherin abundance and activity, and likely participates in regulating the balance between adhesive and motile cellular phenotypes. This review summarizes recent progress in understanding mechanisms of p120 action, and discusses new implications with respect to roles for p120 in disease and cancer.

Regulation of p120-catenin nucleocytoplasmic shuttling activity.

P120-catenin is the prototypic member of a subfamily of Armadillo repeat domain (Arm domain) proteins involved in cell-cell adhesion. Interestingly, all members of the p120 subfamily have also been observed in the nucleus, suggesting that they have additional roles that have yet to be determined. Here, we have developed a novel model system for studying the nucleocytoplasmic shuttling capabilities of p120. We show that simultaneous deletion of both of the conventional nuclear localization sequences (NLSs) in p120 had little effect on its nuclear localization. Instead, the Armadillo repeat domain was essential, and deletion of Arm repeat 3 or Arm repeat 5 eliminated nuclear entry despite the presence of both NLSs. In addition, deletion of Arm repeat 8 resulted in constitutive nuclear localization of p120-3A in both E-cadherin-positive and -negative cell lines. Thus, the core shuttling functions are dependent on the Arm domain. We have also identified two regions within the N-terminus of p120 that modulate nuclear shuttling dynamics of p120. In cadherin-deficient cells, normal epithelial morphology could be restored by both WT-E-cadherin and p120 uncoupled E-cadherin mutants, but only WT-E-cadherin strongly reduced nuclear localization of p120. Moreover, structural changes in p120 that reduced its affinity for E-cadherin increased p120 nuclear localization. Thus, reduced shuttling in the presence of E-cadherin is principally due to sequestration, a condition that is probably dynamic under normal circumstances but completely lost in metastatic cells that have downregulated E-cadherin. Notably, Arm repeats 3 and 5 are necessary for both E-cadherin binding and nuclear translocation, indicating that these repeats have dual roles. Surprisingly, in the absence of E-cadherin there was significant colocalization of cytoplasmic p120 with elements of the tubulin cytoskeleton, particularly in perinuclear locations. Depolymerizing microtubules with nocodazole increased nuclear p120, whereas stabilizing tubulin with taxol reduced nuclear p120 and strongly increased p120 association with microtubules. Thus, p120 has intrinsic nucleocytoplasmic shuttling activity that is modulated, in part, by extrinsic factors such as cadherin binding and interactions with the microtubule network.