Structural plasticity of mouse intestinal microvilli revealed by tissue-level cryo-ET

Cryo-electron tomography (cryo-ET) enables three-dimensional imaging of cellular architecture in a near-native state, but its use in intact mammalian tissues remains technically challenging. Here, using an optimized tissuelevel cryo-ET pipeline that integrates high-pressure freezing, cryo-CLEM, and an improved serial lift-out cryo-FIB workflow, we reconstructed brush-border regions of mouse small intestine from 43 tomographic tilt series and analyzed 490 individual microvilli. Microvilli formed a quasi-regular lattice (mean inter-microvillar spacing ≈ 61 nm) with diameters of 72–114 nm and clearly resolved actin core bundles. Each protrusion bore lateral, nanobristle-like projections that were morphologically heterogeneous and, in rare cases, bridged adjacent microvilli. Importantly, we identified 27 branched microvilli (~5.5%) including Y-shaped and multi-branched forms; the organization of actin bundles in these structures is consistent with a tip-to-base division mechanism for generating daughter protrusions. These observations reveal previously underappreciated structural plasticity in the mammalian brush border and support a fission-like pathway for microvillus renewal.