Endothelial cells regulate alveolar morphogenesis by constructing basement membranes acting as a scaffold for myofibroblasts.

Alveologenesis is a spatially coordinated morphogenetic event, during which alveolar myofibroblasts surround the terminal sacs constructed by epithelial cells and endothelial cells (ECs), then contract to form secondary septa to generate alveoli in the lungs. Recent studies have demonstrated the important role of alveolar ECs in this morphogenetic event. However, the mechanisms underlying EC-mediated alveologenesis remain unknown.

Combinatorial selective ER-phagy remodels the ER during neurogenesis.

The endoplasmic reticulum (ER) employs a diverse proteome landscape to orchestrate many cellular functions, ranging from protein and lipid synthesis to calcium ion flux and inter-organelle communication. A case in point concerns the process of neurogenesis, where a refined tubular ER network is assembled via ER shaping proteins into the newly formed neuronal projections to create highly polarized dendrites and axons. Previous studies have suggested a role for autophagy in ER remodelling, as autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic boutons, and the membrane-embedded ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy.

TGF-β specifies T versus T17 cell fates in murine CD4 T cells through c-Maf.

T follicular helper (T) cells are essential for effective antibody responses, but deciphering the intrinsic wiring of mouse T cells has long been hampered by the lack of a reliable protocol for their generation in vitro. We report that transforming growth factor-β (TGF-β) induces robust expression of T hallmark molecules CXCR5 and Bcl6 in activated mouse CD4 T cells in vitro. TGF-β-induced mouse CXCR5 T cells are phenotypically, transcriptionally, and functionally similar to in vivo-generated T cells and provide critical help to B cells.

Purification of time-resolved insulin granules reveals proteomic and lipidomic changes during granule aging.

Endocrine cells employ regulated exocytosis of secretory granules to secrete hormones and neurotransmitters. Secretory granule exocytosis depends on spatiotemporal variables such as proximity to the plasma membrane and age, with newly generated granules being preferentially released. Despite recent advances, we lack a comprehensive view of the molecular composition of insulin granules and associated changes over their lifetime.

Cullin-RING ligases employ geometrically optimized catalytic partners for substrate targeting.

Cullin-RING ligases (CRLs) ubiquitylate specific substrates selected from other cellular proteins. Substrate discrimination and ubiquitin transferase activity were thought to be strictly separated. Substrates are recognized by substrate receptors, such as Fbox or BCbox proteins.

Identification of structural and regulatory cell-shape determinants in Haloferax volcanii.

Archaea play indispensable roles in global biogeochemical cycles, yet many crucial cellular processes, including cell-shape determination, are poorly understood. Haloferax volcanii, a model haloarchaeon, forms rods and disks, depending on growth conditions. Here, we used a combination of iterative proteomics, genetics, and live-cell imaging to identify mutants that only form rods or disks.

In situ structure of actin remodeling during glucose-stimulated insulin secretion using cryo-electron tomography.

Actin mediates insulin secretion in pancreatic β-cells through remodeling. Hampered by limited resolution, previous studies have offered an ambiguous depiction as depolymerization and repolymerization. We report the in situ structure of actin remodeling in INS-1E β-cells during glucose-stimulated insulin secretion at nanoscale resolution.

Visualizing chaperone-mediated multistep assembly of the human 20S proteasome.

Dedicated assembly factors orchestrate stepwise production of many molecular machines, including the 28-subunit proteasome core particle (CP) that mediates protein degradation. Here, we report cryo-EM reconstructions of seven recombinant human subcomplexes that visualize all five chaperones and the three active site propeptides across a wide swath of the assembly pathway. Comparison of these chaperone-bound intermediates and a matching mature CP reveals molecular mechanisms determining the order of successive subunit additions, and how proteasome subcomplexes and assembly factors structurally adapt upon progressive subunit incorporation to stabilize intermediates, facilitate the formation of subsequent intermediates, and ultimately rearrange to coordinate proteolytic activation with gated access to active sites.

Mechanism of millisecond Lys48-linked poly-ubiquitin chain formation by cullin-RING ligases.

E3 ubiquitin ligases, in collaboration with E2 ubiquitin-conjugating enzymes, modify proteins with poly-ubiquitin chains. Cullin-RING ligase (CRL) E3s use Cdc34/UBE2R-family E2s to build Lys48-linked poly-ubiquitin chains to control an enormous swath of eukaryotic biology. Yet the molecular mechanisms underlying this exceptional linkage specificity and millisecond kinetics of poly-ubiquitylation remain unclear.

Integrating cellular electron microscopy with multimodal data to explore biology across space and time.

Biology spans a continuum of length and time scales. Individual experimental methods only glimpse discrete pieces of this spectrum but can be combined to construct a more holistic view. In this Review, we detail the latest advancements in volume electron microscopy (vEM) and cryo-electron tomography (cryo-ET), which together can visualize biological complexity across scales from the organization of cells in large tissues to the molecular details inside native cellular environments.

PNMA2 forms immunogenic non-enveloped virus-like capsids associated with paraneoplastic neurological syndrome.

The paraneoplastic Ma antigen (PNMA) proteins are associated with cancer-induced paraneoplastic syndromes that present with an autoimmune response and neurological symptoms. Why PNMA proteins are associated with this severe autoimmune disease is unclear. PNMA genes are predominantly expressed in the central nervous system and are ectopically expressed in some tumors.

The Calmodulin-interacting peptide Pcp4a regulates feeding state-dependent behavioral choice in zebrafish.

Animals constantly need to judge the valence of an object in their environment: is it potential food or a threat? The brain makes fundamental decisions on the appropriate behavioral strategy by integrating external information from sensory organs and internal signals related to physiological needs. For example, a hungry animal may take more risks than a satiated one when deciding to approach or avoid an object. Using a proteomic profiling approach, we identified the Calmodulin-interacting peptide Pcp4a as a key regulator of foraging-related decisions.

Comparing divisome organization between vegetative and sporulating at the nanoscale using DNA-PAINT.

Spore-forming bacteria have two distinct division modes: sporulation and vegetative division. The placement of the foundational division machinery component (Z-ring) within the division plane is contingent on the division mode. However, investigating if and how division is performed differently between sporulating and vegetative cells remains challenging, particularly at the nanoscale.

Molecular dynamics simulations of HIV-1 matrix-membrane interactions at different stages of viral maturation.

Although the structural rearrangement of the membrane-bound matrix (MA) protein trimers upon HIV-1 maturation has been reported, the consequences of MA maturation on the MA-lipid interactions are not well understood. Long-timescale molecular dynamics simulations of the MA multimeric assemblies of immature and mature virus particles with our realistic asymmetric membrane model have explored MA-lipid interactions and lateral organization of lipids around MA complexes. The number of stable MA-phosphatidylserine and MA-phosphatidylinositol 4,5-bisphosphate (PIP2) interactions at the trimeric interface of the mature MA complex is observed to be greater compared to that of the immature MA complex.

Doa10/MARCH6 architecture interconnects E3 ligase activity with lipid-binding transmembrane channel to regulate SQLE.

Transmembrane E3 ligases play crucial roles in homeostasis. Much protein and organelle quality control, and metabolic regulation, are determined by ER-resident MARCH6 E3 ligases, including Doa10 in yeast. Here, we present Doa10/MARCH6 structural analysis by cryo-EM and AlphaFold predictions, and a structure-based mutagenesis campaign.

Selective gene expression maintains human tRNA anticodon pools during differentiation.

Transfer RNAs are essential for translating genetic information into proteins. The human genome contains hundreds of predicted tRNA genes, many in multiple copies. How their expression is regulated to control tRNA repertoires is unknown.

STOPGAP, an open-source package for template matching, subtomogram alignment, and classification.

Cryo-electron tomography (cryo-ET) enables molecular-resolution 3D imaging of complex biological specimens such as viral particles, cellular sections, and in some cases, whole cells. This enables the structural characterization of molecules in their near-native environments, without the need for purification or separation, thereby preserving biological information such as conformational states and spatial relationships between different molecular species. Subtomogram averaging is an image processing workflow that allows users to leverage cryo-ET data to identify and localize target molecules, determine high-resolution structures of repeating molecular species, and classifying different conformational states.

Comparative Quantitative Proteomic Analysis of Melanoma Subtypes, Nevus-Associated Melanoma, and Corresponding Nevi.

A substantial part of cutaneous malignant melanomas develops from benign nevi. However, the precise molecular events driving the transformation from benign to malignant melanoma are not well-understood. We used laser microdissection and mass spectrometry to analyze the proteomes of melanoma subtypes, including superficial spreading melanomas (n = 17), nodular melanomas (n = 17), and acral melanomas (n = 15).

Full Mass Range ΦSDM Orbitrap Mass Spectrometry for DIA Proteome Analysis.

Optimizing data-independent acquisition methods for proteomics applications often requires balancing spectral resolution and acquisition speed. Here, we describe a real-time full mass range implementation of the phase-constrained spectrum deconvolution method (ΦSDM) for Orbitrap mass spectrometry that increases mass resolving power without increasing scan time. Comparing its performance to the standard enhanced Fourier transformation signal processing revealed that the increased resolving power of ΦSDM is beneficial in areas of high peptide density and comes with a greater ability to resolve low-abundance signals.