Selective autophagy is a fundamental cellular quality control pathway responsible for the targeted degradation of diverse cytosolic material, ranging from proteins to damaged organelles and invading pathogens. Despite its central importance in health and disease, the molecular and cellular principles governing how autophagosomes recognise, engulf, and degrade their cargo have remained poorly understood.

Using an integrated approach combining in situ cryo-electron tomography, live cell imaging, biophysical measurements, and multiscale simulations, we reveal the mechanistic principles of selective autophagy from the atomic to the cellular scale. Specifically, we show that the physical properties of the cargo are a critical determinant of autophagic fate. By determining the architecture of an autophagic cargo assembly by in situ cryo-ET at high resolution, we demonstrate that a single amino acid substitution is sufficient to shift the assembly material state from liquid-like to glass-like, impairing autophagic flux without disrupting local molecular organisation. Furthermore, we identify that receptor mobility at the cargo surface drives accumulation of autophagy initiation factors into dynamic hubs that nucleate phagophore biogenesis. Autophagosome formation subsequently progresses through well-characterised membrane intermediates guided by endoplasmic reticulum and vacuole contact sites. Features conserved even at the scale of intracellular bacterial pathogens, where the cargo surface itself templates phagophore formation through multiple parallel initiation events. At the pathological extreme, amyloid fibrils evade autophagic clearance by an incompatible material state.

Together, these findings establish cargo material state as a fundamental axis of autophagic regulation, with broad implications for understanding neurodegeneration, infection, and cellular homeostasis.