In situ cryo electron tomography of cryo focused ion beam milled samples emerged in recent years as a powerful technique for structural studies of macromolecular complexes in their native cellular environment. The lamella-shaped samples, however, have a limited area and are created with a necessary pretilt. This severely limits the possibilities for recording tomographic tilt series in a high-throughput manner. Here, we utilise a geometrical sample model and optical image shift to record tens of tilt series in parallel, thereby saving time and gaining sample areas conventionally used for tracking of specimen movement. The parallel cryo electron tomography (PACE-tomo) method achieves a throughput faster than 5 min per tilt series and allows the collection of sample areas that were previously unreachable, thus maximising the amount of data from each lamella. Performance testing with ribosomes in vitro and in situ on state-of-the-art and general-purpose microscopes demonstrated the high-throughput and high-quality of PACE-tomo.

The mother centriole in a cell has two appendages, the distal appendage (DA) and subdistal appendage (SDA), which have roles in generating cilia and organizing the cellular microtubular network, respectively. In the knockout (KO) cells of Odf2, the component of the DA and SDA, both appendages simultaneously disappear. However, the molecular mechanisms by which the DA and SDA form independently but close to each other downstream of Odf2 are unknown. Here, using super‐resolution structured illumination microscopy (SR‐SIM), we found that the signal for GFP‐tagged Odf2 overlapped considerably with that of immunofluorescently labeled Cep128. We further found that Cep128 knockdown (KD) caused the dissociation of other SDA components from the centriole, including centriolin, Ndel1, ninein and Cep170, whereas Odf2 was still associated with the centriole. In contrast, the DA components remained associated with the centriole in Cep128 KD cells. Consistent with this observation, we identified Cep128 as an Odf2‐interacting protein by immunoprecipitation. Taken with the finding that Cep128 deletion decreased the stability of centriolar microtubules, our results indicate that Cep128 associates with Odf2 in the hierarchical assembly of SDA components to elicit the microtubule‐organizing function.

Apical constriction is critical for epithelial morphogenesis, including neural tube formation. Vertebrate apical constriction is induced by di‐phosphorylated myosin light chain (ppMLC)‐driven contraction of actomyosin‐based circumferential rings (CRs), also known as perijunctional actomyosin rings, around apical junctional complexes (AJCs), mainly consisting of tight junctions (TJs) and adherens junctions (AJs). Here, we revealed a ppMLC‐triggered system at TJ‐associated CRs for vertebrate apical constriction involving microtubules, LUZP1, and myosin phosphatase. We first identified LUZP1 via unbiased screening of microtubule‐associated proteins in the AJC‐enriched fraction. In cultured epithelial cells, LUZP1 was found localized at TJ‐, but not at AJ‐, associated CRs, and LUZP1 knockout resulted in apical constriction defects with a significant reduction in ppMLC levels within CRs. A series of assays revealed that ppMLC promotes the recruitment of LUZP1 to TJ‐associated CRs, where LUZP1 spatiotemporally inhibits myosin phosphatase in a microtubule‐facilitated manner. Our results uncovered a hitherto unknown microtubule‐LUZP1 association at TJ‐associated CRs that inhibits myosin phosphatase, contributing significantly to the understanding of vertebrate apical constriction.

g Claudin protein family members, of which there are at least 27 in humans and mice, polymerize to form tight junctions (TJs) between epithelial cells, in a tissue-and developmental stage-specific manner. Claudins have a paracellular barrier function. In addition, certain claudins function as paracellular channels for small ions and/or solutes by forming selective pores at the TJs, although the specific claudins involved and their functional mechanisms are still in question. Here we show for the first time that claudin-21, which is more highly expressed in the embryonic than the postnatal stages, acts as a paracellular channel for small cations, such as Na ؉ , similar to the typical channel-type claudins claudin-2 and -15. Claudin-21 also allows the paracellular passage of larger solutes. Our findings suggest that claudin-21-based TJs allow the passage of small and larger solutes by both paracellular channel-based and some additional mechanisms. Epithelial cell sheets form and cover every compartment of the body. Homeostasis is specifically maintained for organ and tissue function in each compartment. To create sheets with a barrier function, epithelial cells adhere to each other side by side through well-organized cell-cell adhesion systems (1), such as tight junctions (TJs), which maintain distinct environments by blocking the free exchange of molecules across the cell sheet (2, 3). Accumulating evidence indicates that the molecular strands that form the TJ barriers are composed primarily of polymerized claudins, which are adhesion molecules containing four transmembrane helices (2, 4). The claudins both create the paracellular barrier and determine which ions and/or molecules can selectively cross it (5-7).The claudins comprise a large gene family with at least 27 members, in humans or mice, that have distinct expression patterns and properties (2,3,8). Epithelial cells generally express multiple types of claudins; the particular claudin combination expressed appears to determine the specific properties of each TJbased paracellular permselective barrier (9-22). Two categories of claudins have been proposed, namely, the paracellular barrier type and the paracellular channel type, based on the transepithelial electrical resistance (TER) or on the cation and/or anion permeability of many epithelial cell lines (5, 7). Among some claudins characterized as channel-type claudins, such as claudin-2, -7, -10, -15, -16, and -17, claudin-2 and -15 have been studied extensively with respect to specific ions and water (9-11, 14, 17, 20, 21, 23, 24).The structure of claudin-15 was recently analyzed; this analysis revealed that two extracellular segments form a unique ␤ sheet domain fixed to a transmembrane four-helix bundle by a W-LW claudin consensus motif (25,26). The high-resolution structure of claudin-15 suggests that a ␤-barrel pore created by eight claudin molecules in the region between two cells can form a paracellular channel, which is regulated by charged residues on the claudins' extracellular domains.Although ...

Liquid-liquid phase separation (LLPS) is involved in various dynamic biological phenomena. In epithelial cells, dynamic regulation of junctional actin filaments tethered to the apical junctional complex (AJC) is critical for maintaining internal homeostasis against external perturbations; however, the role of LLPS in this process remains unknown. Here, after identifying a multifunctional actin nucleator, cordon bleu (Cobl), as an AJC-enriched microtubule-associated protein, we conducted comprehensive in vitro and in vivo analyses. We found that apical microtubules promoted LLPS of Cobl at the AJC, and Cobl actin assembly activity increased upon LLPS. Thus, microtubules spatiotemporally regulated junctional actin assembly for epithelial morphogenesis and paracellular barriers. Collectively, these findings established that LLPS of the actin nucleator Cobl mediated dynamic microtubule-actin cross-talk in junctions, which fine-tuned the epithelial barrier.