Methods to extract and analyze fluid from human pluripotent stem cell-derived choroid plexus organoids.

The choroid plexus (ChP) is a highly vascularized tissue lining the ventricular space of the brain. The ChP generates cerebrospinal fluid (CSF) and forms a protective barrier in the central nervous system (CNS). Recently, a three-dimensional human pluripotent stem cell (hPSC)-derived ChP organoid model has been developed.

BrainPhys neuronal medium optimized for imaging and optogenetics in vitro.

The capabilities of imaging technologies, fluorescent sensors, and optogenetics tools for cell biology are advancing. In parallel, cellular reprogramming and organoid engineering are expanding the use of human neuronal models in vitro. This creates an increasing need for tissue culture conditions better adapted to live-cell imaging.

Conserved and unique features of the fission yeast core Atg1 complex.

Although the human ULK complex mediates phagophore initiation similar to the budding yeast Saccharomyces cerevisiae Atg1 complex, this complex contains ATG101 but not Atg29 and Atg31. Here, we analyzed the fission yeast Schizosaccharomyces pombe Atg1 complex, which has a subunit composition that resembles the human ULK complex. Our pairwise coprecipitation experiments showed that while the interactions between Atg1, Atg13, and Atg17 are conserved, Atg101 does not bind Atg17.

Molecular interactions of the Saccharomyces cerevisiae Atg1 complex provide insights into assembly and regulatory mechanisms.

The Atg1 complex, which contains 5 major subunits: Atg1, Atg13, Atg17, Atg29, and Atg31, regulates the induction of autophagy and autophagosome formation. To gain a better understanding of the overall architecture and assembly mechanism of this essential autophagy regulatory complex, we have reconstituted a core assembly of the Saccharomyces cerevisiae Atg1 complex composed of full-length Atg17, Atg29, and Atg31, along with the C-terminal domains of Atg1 (Atg1[CTD]) and Atg13 (Atg13[CTD]). Using chemical-crosslinking coupled with mass spectrometry (CXMS) analysis we systematically mapped the intersubunit interaction interfaces within this complex.

Structural biology of the macroautophagy machinery.

Macroautophagy is a conserved degradative process mediated through formation of a unique double-membrane structure, the autophagosome. The discovery of autophagy-related (Atg) genes required for autophagosome formation has led to the characterization of approximately 20 genes mediating this process. Recent structural studies of the Atg proteins have provided the molecular basis for their function.

The role of Atg29 phosphorylation in PAS assembly.

Macroautophagy (hereafter autophagy) initiates at the phagophore assembly site (PAS), where most of the AuTophaGy-related (Atg) proteins are at least transiently localized. As the first protein complex targeted to the PAS, the Atg17-Atg31-Atg29 complex serves as the scaffold for other Atg proteins and plays a critical role for the organization of the PAS, and in autophagy initiation. We recently showed that this complex is constitutively formed and activated by the phosphorylation of Atg29 when autophagy is induced.

Structural characterization of the Saccharomyces cerevisiae autophagy regulatory complex Atg17-Atg31-Atg29.

Atg17, in complex with Atg29 and Atg31, constitutes a key module of the Atg1 kinase signaling complex and functions as an important organizer of the phagophore assembly site in the yeast Saccharomyces cerevisiae. We have determined the three-dimensional reconstruction of the full S. cerevisiae Atg17-Atg31-Atg29 complex by single-particle electron microscopy.

Atg29 phosphorylation regulates coordination of the Atg17-Atg31-Atg29 complex with the Atg11 scaffold during autophagy initiation.

Macroautophagy (hereafter autophagy) functions in the nonselective clearance of cytoplasm. This process participates in many aspects of cell physiology, and is conserved in all eukaryotes. Autophagy begins with the organization of the phagophore assembly site (PAS), where most of the AuTophaGy-related (Atg) proteins are at least transiently localized.