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Protein-based nanomachines drive every cellular process. An explosion of high-resolution structures of multiprotein complexes has improved our understanding of what these machines look like and how they work, but we still know relatively little about how they assemble in living cells. For example, it has only recently been appreciated that many complexes assemble co-translationally, with at least one subunit still undergoing active translation while already interacting with other subunits. One aspect that is particularly understudied is assembly order, the idea that there is a stepwise order to the subunit-subunit associations that underlies the efficient assembly of the quaternary structure. Here, we integrate a review of the methodological approaches commonly used to query assembly order within a discussion of studies of the 20S proteasome core particle, septin protein complexes, and the histone octamer. We highlight shared and distinct properties of these complexes that illustrate general themes applicable to most other multisubunit assemblies.

Thrombin is generated from prothrombin through sequential cleavage at two sites by the enzyme complex prothrombinase, composed of a serine protease, factor (f) Xa and a cofactor, fVa, on phospholipid membranes. In a recent paper published in Blood, Ruben et al. (Ruben et al. 2022 Blood 139, 3463-3473 (doi:10.1182/blood.2022015807)) reported a major breakthrough in the field: the cryogenic electron microscopy structures of human prothrombinase on nanodiscs at 5.5 Å resolution (7TPQ) and of a catalytically inert human prothrombinase with its substrate prothrombin in the absence of any membrane at 4.1 Å resolution (7TPP). As is the norm in structural biology, the original paper was reviewed without access to the coordinates and maps, and it was therefore not possible for referees to assess the validity of the structures or their interpretations. In this article, we provide a post hoc analysis of the quality of the reported coordinates and maps, and look closely at the claimed intermolecular contacts on which the supposed breakthrough depends. We demonstrate that the work is deeply flawed, with not a single claimed intermolecular contact supported by the map, and conclude that the two reported structures do not contain any useful information regarding the assembly or function of the prothrombinase complex.

Primordial germ cells (PGCs) are the founder cells that develop into mature gametes. PGCs emerge during weeks 2-3 of human embryo development. Pluripotency genes are reactivated during PGC specification, including Krüppel-like factor KLF4, but its precise role in PGC development is unclear. Here, we investigated the role of KLF4 in PGC development using our in vitro model for human PGC-like cells (hPGCLCs). We demonstrate that the depletion of KLF4 reduces the efficiency of hPGCLC specification, resulting in hPGCLCs with an aberrant transcriptome. Cut-and-run and transcriptomic analyses reveal that KLF4 represses somatic markers involved in neuronal and endodermal differentiation while promoting the expression of genes associated with PGC specification, such as PAX5, and epigenetic regulators, including DNMT3L and REST. KLF4 targets in hPGCLCs showed significant co-enrichment of motifs for SP and STAT factors, which are known to regulate cell cycle and migration genes. KLF4 contributes to human PGC development by activating genes involved in PGC specification and cell cycle regulation, while repressing somatic genes to maintain PGC identity.

The vertebrate visual cycle hinges on enzymatically converting all-trans-retinol (at-ROL) into 11-cis-retinal (11c-RAL), the chromophore that binds to opsins in photoreceptors, forming light-responsive pigments. When struck by a photon, these pigments activate the phototransduction pathway and initiate the process of vision. The enzymatic isomerization of at-ROL, crucial for restoring the visual pigments and preparing them to receive new light stimuli, relies on various enzymes found in both the photoreceptors and retinal pigment epithelium cells. To function effectively, retinoids must shuttle between these two cell types. Retinol-binding protein 3 (RBP3), located in the interphotoreceptor matrix, probably plays a pivotal role in this transport mechanism. Comprised of four retinoid-binding modules, RBP3 also binds fatty acids, potentially aiding retinal function by facilitating the loading and unloading of different retinoids at specific cell types thereby directing the cycle. In this study, we present a 3.67 Å cryoEM structure of porcine RBP3, along with molecular docking analysis and corroborative in-solution small-angle X-ray scattering data for titration of RBP3 with relevant ligands, that also give insights on RBP3 conformational adaptability.

Sea urchins, integral to marine ecosystems and valued as a delicacy in Asia and Europe, contain physiologically important long-chain (>C₂₀) polyunsaturated fatty acids (PUFA) in their gonads, including arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) and unusual non-methylene-interrupted fatty acids (NMI-FA) such as 20:2^Δ5,11. Although these fatty acids may partially be derived from their diet, such as macroalgae, the present study on Hemicentrotus pulcherrimus has uncovered multiple genes encoding enzymes involved in long-chain PUFA biosynthesis. Specifically, 3 fatty acid desaturases (FadsA, FadsC1 and FadsC2) and 13 elongation of very-long-chain fatty acids proteins (Elovl-like, Elovl1/7-like, Elovl2/5-like, Elovl4-like, Elovl8-like and Elovl6-like A-H) were identified in their genome and transcriptomes. Functional analysis showed that FadsA and FadsC2 function as a Δ5 desaturase and a Δ8 desaturase, respectively, enabling the conversion of 18:2n-6 and 18:3n-3 into ARA and EPA, respectively, along with Elovl, particularly Elovl6-like C. Elovl6-like C demonstrates elongase activity towards both C₁₈ PUFA and monounsaturated fatty acids. Consequently, FadsA and Elovl6-like C enable the synthesis of several NMI-FA, including 20:2^Δ5,11 and 20:3^Δ5,11,14, from C₁₈ precursors. This indicates that H. pulcherrimus can endogenously synthesize a wide variety of C₂₀ PUFA and NMI-FA, highlighting active biosynthesis pathways within sea urchins.

Epithelia are multicellular sheets that form barriers defining the internal and external environments. The constant stresses acting at this interface require that epithelial sheets are mechanically robust and provide a selective barrier to the hostile exterior. These properties are mediated by cellular junctions which are physically linked with heavily crosslinked cytoskeletal networks. Such hardwiring is facilitated by plakins, a family of giant modular proteins which serve as 'molecular bridges' between different cytoskeletal filaments and multiprotein adhesion complexes. Dysfunction of cytoskeletal crosslinking compromises epithelial biomechanics and structural integrity. Subsequent loss of barrier function leads to disturbed tissue homeostasis and pathological consequences such as skin blistering or intestinal inflammation. In this article, we highlight the importance of the cytolinker protein plectin for the functional organization of epithelial cytoskeletal networks. In particular, we focus on the ability of plectin to act as an integrator of the epithelial cytoarchitecture that defines the biomechanics of the whole tissue. Finally, we also discuss the role of cytoskeletal crosslinking in emerging aspects of epithelial mechanobiology that are critical for the maintenance of epithelial homeostasis.

Maintenance and breeding of experimental organisms are fundamental to life sciences, but both initial and running costs, and hands-on zootechnical demands can be challenging for many laboratories. Here, we first aimed to further develop a simple protocol for reliable inland culture of tunicate model species of the Ciona genus. We cultured both Ciona robusta and Ciona intestinalis in controlled experimental conditions, with a focus on dietary variables, and quantified growth and maturation parameters. From statistical analysis of these standardized datasets, we gained insights into the post-embryonic developmental physiology of Ciona and inferred an improved diet and culturing conditions for sexual maturation. We showed that body length is a critical determinant of both somatic and sexual maturation, which suggests the existence of systemic control mechanisms of resource allocation towards somatic growth or maturation and supports applying size selection as a predictor of reproductive fitness in our inland culture to keep the healthiest animals at low density in the system. In the end, we successfully established a new protocol, including size selection, to promote both sperm and egg production. Our protocol using small tanks will empower researchers to initiate inland Ciona cultures with low costs and reduced space constraints.

The established consensus sequence for human 5' splice sites masks the presence of two major splice site classes defined by preferential base-pairing potentials with either U5 snRNA loop 1 or the U6 snRNA ACAGA box. The two 5' splice site classes are separable in genome sequences, sensitized by specific genotypes and associated with splicing complexity. The two classes reflect the commitment to 5' splice site usage occurring primarily during 5' splice site transfer to U6 snRNA. Separating the human 5' splice site consensus into its two major constituents can help us understand fundamental features of eukaryote genome architecture and splicing mechanisms and inform treatment design for diseases caused by genetic variation affecting splicing.

The enterobacterial common antigen (ECA) is conserved in Gram-negative bacteria of the Enterobacterales order although its function is debated. ECA biogenesis depends on the Wzx/Wzy-dependent strategy whereby the newly synthesized lipid-linked repeat units, lipid III, are transferred across the inner membrane by the lipid III flippase WzxE. WzxE is part of the Wzx family and required in many glycan assembly systems, but an understanding of its molecular mechanism is hindered due to a lack of structural evidence. Here, we present the first X-ray structures of WzxE from Escherichia coli in complex with nanobodies. Both inward- and outward-facing conformations highlight two pairs of arginine residues that move in a reciprocal fashion, enabling flipping. One of the arginine pairs coordinated to a glutamate residue is essential for activity along with the C-terminal arginine rich tail located close to the entrance of the lumen. This work helps understand the translocation mechanism of the Wzx flippase family.