In Vivo Formation of Vacuolated Multi-phase Compartments Lacking Membranes

• The N and C termini of TDP43 can drive the formation of giant liquid droplets in cells
• Droplets contain nucleoplasm-filled vacuoles despite lacking membranes
• ALS-causative point mutants in the C terminus alter phase properties
• Nearly identical TDP43 variants assemble into coexisting, yet largely immiscible phases

SummaryEukaryotic cells contain membrane-less organelles, including nucleoli and stress granules, that behave like liquid droplets. Such endogenous condensates often have internal substructure, but how this is established in the absence of membrane encapsulation remains unclear. We find that the N- and C-terminal domains of TDP43, a heterogeneous nuclear ribonucleoprotein implicated in neurodegenerative diseases, are capable of driving the formation of sub-structured liquid droplets in vivo. These droplets contain dynamic internal “bubbles” of nucleoplasm, reminiscent of membrane-based multi-vesicular endosomes. A conserved sequence embedded within the intrinsically disordered region (IDR) of TDP43 promotes the formation of these multi-phase assemblies. Disease-causing point mutations in the IDR can change the propensity to form bubbles, protein dynamics within the phase, or phase-environment exchange rates. Our results show that a single IDR-containing protein can nucleate the assembly of compartmentalized liquid droplets approximating the morphological complexity of membrane-bound organelles.

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