Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Monday Speaker Abstracts

RECONSTITUTING ESCRT-III MEDIATED MEMBRANE FISSION IN VITRO

Aurelien Roux 1 ; Javier Espadas 1 ; Anna Pfitzner 1 ; Cesar Bernat 1 ; Henry Zivkovitch 1 ; 1 University of Geneva, Biochemistry, Geneva, Switzerland

The ESCRT-III complex is the only fission machinery known to break lipid membranes from within the neck, in an orientation inverse to dynamin. It is also one of the most ancient membrane remodelling machinery, the only one present in old Archaea species. In the recent years, we have discovered essential features of the ESCRT-III that explain how it deforms the membrane. But the high dynamics of the fission reaction is difficult to reconstitute in vitro. By using Archeal proteins, I will present recent data towards the reconstitution of ESCRT-III mediated membrane fission.

AUTOPHAGY-LYSOSOMAL PATHWAYS IN NEURONS AND ASTROCYTES

Sandra Maday 1 ; David K Sidibe 1 ; Max H Stempel 1 ; Maeve L Coughlan 1 ; Christina Miranda 1 ; 1 Perelman School of Medicine at the University of Pennsylvania, Department of Neuroscience, Philadelphia, PA, USA

Neurons and astrocytes collaborate to construct trillions of synaptic connections in the brain. Most neurons and astrocytes are post-mitotic and long-lived, requiring robust quality control pathways to regulate the integrity of the proteome. Moreover, neurons and astrocytes have cell type-specific functions that place unique demands on the proteome. A key regulator of the proteome is autophagy, a lysosomal degradation pathway. During autophagy, cellular components are packaged into autophagosomes which fuse with lysosomes to enable cargo degradation. Loss of autophagy causes neurodevelopmental defects and neurodegeneration in mice and humans. How autophagy is regulated in neurons and astrocytes to facilitate cell-type specific functions and responses to cellular stress is largely unknown. Here, I present our work on defining pathways for autophagy in neurons and astrocytes. We developed a robust system to coculture primary neurons and astrocytes that recapitulates morphological, proteomic, and functional signatures of astrocytes in vivo. We find that the autophagy receptor p62 and its substrate engagement are differentially regulated in neurons and astrocytes. Metabolic stress increases p62-positive puncta in neurons and astrocytes, but only in neurons do these p62 puncta robustly associate with ubiquitin and require ubiquitination for their formation. These differences were also revealed at baseline. Moreover, deletion of the ubiquitin-associated (UBA) domain reduced p62 puncta only in neurons. In astrocytes, deletion of the UBA domain reduced p62 localization to aggresomes with proteasome-inhibition. Thus, our data suggest that neurons and astrocytes manage quality control differently and p62 has cell-type-specific functions that are elicited in distinct paradigms of cellular stress. Since ALS-linked mutations fall within distinct functional domains of p62, our study provides insights into cell-type-specific contributions to ALS. In fact, ALS-linked mutations in p62 that impair ubiquitin-binding disrupt baseline selective autophagy preferentially in neurons. Our work sets the stage for defining intercellular pathways between neurons and astrocytes to regulate the proteome.

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