Induced pluripotent stem cells (iPSCs) hold promise to revolutionize studies of intracellular transport in live human neurons and to shed new light on the role of dysfunctional transport in neurodegenerative disorders. Here, we describe an approach for live imaging of axonal and dendritic transport in iPSC‐derived cortical neurons. We use transfection and transient expression of genetically‐encoded fluorescent markers to characterize the motility of Rab‐positive vesicles, including early, late and recycling endosomes, as well as autophagosomes and mitochondria in iPSC‐derived neurons. Comparing transport parameters of these organelles with data from primary rat hippocampal neurons, we uncover remarkable similarities. In addition, we generated lysosomal‐associated membrane protein 1 (LAMP1)‐enhanced green fluorescent protein (EGFP) knock‐in iPSCs and show that knock‐in neurons can be used to study the transport of endogenously labeled vesicles, as a parallel approach to the transient overexpression of fluorescently labeled organelle markers.

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Cells harbor numerous mesoscale membraneless compartments that house specific biochemical processes and perform distinct cellular functions. These protein and RNA-rich bodies are thought to form through multivalent interactions among proteins and nucleic acids resulting in demixing via liquid-liquid phase separation (LLPS). Proteins harboring intrinsically disordered regions (IDRs) predominate in membraneless organelles. However, it is not known whether IDR sequence alone can dictate the formation of distinct condensed phases. We identified a pair of IDRs capable of forming spatially distinct condensates when expressed in cells. When reconstituted in vitro, these model proteins do not co-partition, suggesting condensation specificity is encoded directly in the polypeptide sequences. Through computational modeling and mutagenesis, we identified the amino acids and chain properties governing homotypic and heterotypic interactions that direct selective condensation. These results form the basis of physicochemical principles that may direct subcellular organization of IDRs into specific condensates and reveal an IDR code that can guide construction of orthogonal membraneless compartments.

Lysosomes are membrane-bound organelles that regulate cellular proteostasis. Loss of lysosomal integrity initiates cell death pathways. Thus, cells must rely on quality control mechanisms for protection, including the selective isolation and degradation of damaged lysosomes by lysophagy. Here, we report that the selective autophagy receptor SQSTM1/p62 is an essential lysophagy receptor recruited to damaged lysosomes in both HeLa cells and neurons. p62 oligomers form liquid-like condensates that are critical in lysophagy. These condensates are regulated by the small heat shock protein HSP27, which binds p62 to prevent p62 aggregation and facilitate autophagosome formation. Mutations in p62 are implicated in Amyotrophic Lateral Sclerosis (ALS), and expression of ALS-associated mutations in p62 impair lysophagy, suggesting that deficits in this pathway may contribute to the cellular pathogenesis of ALS. Thus, p62 oligomers cooperate with HSP27 to promote lysophagy by forming a platform for autophagosome biogenesis at damaged lysosomes.

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