Photorhabdus CipA and CipB spontaneously demix into distinct, isolable cytosolic crystals through orthogonal self-assembly in mammalian cells.

Authors

Haruki Hasegawa, Songyu Wang, Emma M Pelegri-O'Day

Published in

Experimental cell research. Volume 463. Issue 2. Pages 115209. Oct 22, 2026. Epub Oct 22, 2026.

Abstract

Crystalline inclusion proteins CipA and CipB from Photorhabdus luminescens serve as versatile scaffolds for clustering genetically fused heterologous proteins into light-refractile inclusion bodies in bacteria. Although various Cip-fusion proteins have been tested in microbial systems, the phase separation behaviors and biochemical attributes of Cip and Cip-fusion proteins in eukaryotic cellular environments remain poorly understood. This study demonstrates that CipA and CipB undergo crystallization in the cytosol of human embryonic kidney cells. Shortly after transfection, CipA and CipB become the most abundant proteins in mammalian cells and produce prominent cytosolic crystals often exceeding 10 μm in side length. Co-expression of CipA and CipB drives spontaneous demixing into two distinct crystal populations, and their orthogonality is maintained even when an unrelated third protein crystallizes in the same cytosol, permitting three crystal types to coexist simultaneously. Intracellular crystals are readily isolable from cells, and once purified, these crystals are stable in vitro at physiological pH. CipA and CipB crystals, however, exhibit distinct pH-dependent solubilization behaviors, differing in both dissolution kinetics and oligomeric profiles. When fused to GFP, both CipA and CipB enable Cip-GFP fusions to phase separate into condensate-like cytosolic protein droplets. While Cip-mCherry fusions do not drive spontaneous self-assembly, Cip-mCherry can still be efficiently recruited into Cip-GFP-generated condensate droplet only when their Cip-scaffold identity is matched. Although fused cargo types influence the phase separation threshold of individual Cip-fusion proteins, the findings support Cip as robust orthogonal self-assembly platforms for producing programmable cytosolic protein assemblies in mammalian cells.

PMID:
42913145
Bibliographic data and abstract were imported from PubMed on 24 Oct 2026.

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