Single-cell transcriptomics unveils xylem cell development and evolution.

BACKGROUND: Xylem, the most abundant tissue on Earth, is responsible for lateral growth in plants. Typical xylem has a radial system composed of ray parenchyma cells and an axial system of fusiform cells. In most angiosperms, fusiform cells comprise vessel elements for water transportation and libriform fibers for mechanical support, while both functions are performed by tracheids in other vascular plants such as gymnosperms. Little is known about the developmental programs and evolutionary relationships of these xylem cell types. RESULTS: Through both single-cell and laser capture microdissection transcriptomic profiling, we determine the developmental lineages of ray and fusiform cells in stem-differentiating xylem across four divergent woody angiosperms. Based on cross-species analyses of single-cell clusters and overlapping trajectories, we reveal highly conserved ray, yet variable fusiform, lineages across angiosperms. Core eudicots Populus trichocarpa and Eucalyptus grandis share nearly identical fusiform lineages, whereas the more basal angiosperm Liriodendron chinense has a fusiform lineage distinct from that in core eudicots. The tracheids in the basal eudicot Trochodendron aralioides, an evolutionarily reversed trait, exhibit strong transcriptomic similarity to vessel elements rather than libriform fibers. CONCLUSIONS: This evo-devo framework provides a comprehensive understanding of the formation of xylem cell lineages across multiple plant species spanning over a hundred million years of evolutionary history.

Single-particle tracking localization microscopy reveals nonaxonemal dynamics of intraflagellar transport proteins at the base of mammalian primary cilia.

Primary cilia play a vital role in cellular sensing and signaling. An essential component of ciliogenesis is intraflagellar transport (IFT), which is involved in IFT protein recruitment, axonemal engagement of IFT protein complexes, and so on. The mechanistic understanding of these processes at the ciliary base was largely missing, because it is challenging to observe the motion of IFT proteins in this crowded region using conventional microscopy. Here, we report short-trajectory tracking of IFT proteins at the base of mammalian primary cilia by optimizing single-particle tracking photoactivated localization microscopy for IFT88-mEOS4b in live human retinal pigment epithelial cells. Intriguingly, we found that mobile IFT proteins "switched gears" multiple times from the distal appendages (DAPs) to the ciliary compartment (CC), moving slowly in the DAPs, relatively fast in the proximal transition zone (TZ), slowly again in the distal TZ, and then much faster in the CC. They could travel through the space between the DAPs and the axoneme without following DAP structures. We further revealed that BBS2 and IFT88 were highly populated at the distal TZ, a potential assembly site. Together, our live-cell single-particle tracking revealed region-dependent slowdown of IFT proteins at the ciliary base, shedding light on staged control of ciliary homeostasis.

Super-Resolution Imaging Reveals TCTN2 Depletion-Induced IFT88 Lumen Leakage and Ciliary Weakening.

The primary cilium is an essential organelle mediating key signaling activities, such as sonic hedgehog signaling. The molecular composition of the ciliary compartment is distinct from that of the cytosol, with the transition zone (TZ) gated the ciliary base. The TZ is a packed and organized protein complex containing multiple ciliopathy-associated protein species. Tectonic 2 (TCTN2) is one of the TZ proteins in the vicinity of the ciliary membrane, and its mutation is associated with Meckel syndrome. Despite its importance in ciliopathies, the role of TCTN2 in ciliary structure and molecules remains unclear. Here, we created a CRISPR/Cas9 TCTN2 knockout human retinal pigment epithelial cell line and conducted quantitative analysis of geometric localization using both wide-field and super-resolution microscopy techniques. We found that TCTN2 depletion resulted in partial TZ damage, loss of ciliary membrane proteins, leakage of intraflagellar transport protein IFT88 toward the basal body lumen, and cilium shortening and curving. The basal body lumen occupancy of IFT88 was also observed in si-RPGRIP1L cells and cytochalasin-D-treated wild-type cells, suggesting varying lumen accessibility for intraflagellar transport proteins under different perturbed conditions. Our findings support two possible models for the lumen leakage of IFT88, i.e., a tip leakage model and a misregulation model. Together, our quantitative image analysis augmented by super-resolution microscopy facilitates the observation of structural destruction and molecular redistribution in TCTN2-/- cilia, shedding light on mechanistic understanding of TZ-protein-associated ciliopathies.

Super-resolution architecture of mammalian centriole distal appendages reveals distinct blade and matrix functional components.

Distal appendages (DAPs) are nanoscale, pinwheel-like structures protruding from the distal end of the centriole that mediate membrane docking during ciliogenesis, marking the cilia base around the ciliary gate. Here we determine a super-resolved multiplex of 16 centriole-distal-end components. Surprisingly, rather than pinwheels, intact DAPs exhibit a cone-shaped architecture with components filling the space between each pinwheel blade, a new structural element we term the distal appendage matrix (DAM). Specifically, CEP83, CEP89, SCLT1, and CEP164 form the backbone of pinwheel blades, with CEP83 confined at the root and CEP164 extending to the tip near the membrane-docking site. By contrast, FBF1 marks the distal end of the DAM near the ciliary membrane. Strikingly, unlike CEP164, which is essential for ciliogenesis, FBF1 is required for ciliary gating of transmembrane proteins, revealing DAPs as an essential component of the ciliary gate. Our findings redefine both the structure and function of DAPs.