Identification of coilin interactors reveals coordinated control of Cajal body number and structure.

The cell nucleus contains distinct biomolecular condensates that form at specific genetic loci, organize chromosomes in 3D space, and regulate RNA processing. Among these, Cajal bodies (CBs) require key "scaffolding" proteins for their assembly, which is not fully understood. Here, we employ proximity biotinylation, mass spectrometry, and functional screening to comprehensively identify and test the functions of CB components.

A nuclear architecture screen in identifies Stonewall as a link between chromatin position at the nuclear periphery and germline stem cell fate.

The association of genomic loci to the nuclear periphery is proposed to facilitate cell type-specific gene repression and influence cell fate decisions. However, the interplay between gene position and expression remains incompletely understood, in part because the proteins that position genomic loci at the nuclear periphery remain unidentified. Here, we used an Oligopaint-based HiDRO screen targeting ∼1000 genes to discover novel regulators of nuclear architecture in cells.

A nuclear architecture screen in identifies Stonewall as a link between chromatin position at the nuclear periphery and germline stem cell fate.

The association of genomic loci to the nuclear periphery is proposed to facilitate cell-type specific gene repression and influence cell fate decisions. However, the interplay between gene position and expression remains incompletely understood, in part because the proteins that position genomic loci at the nuclear periphery remain unidentified. Here, we used an Oligopaint-based HiDRO screen targeting ~1000 genes to discover novel regulators of nuclear architecture in cells.

Super-resolution microscopy reveals focal organization of ER-associated Y-complexes in mitosis.

During mitotic entry of vertebrate cells, nuclear pore complexes (NPCs) are rapidly disintegrated. NPC disassembly is initiated by hyperphosphorylation of linker nucleoporins (Nups), which leads to the dissociation of FG repeat Nups and relaxation of the nuclear permeability barrier. However, less is known about disintegration of the huge nuclear and cytoplasmic rings, which are formed by annular assemblies of Y-complexes that are dissociated from NPCs as intact units.

Ribosome biogenesis factors-from names to functions.

The assembly of ribosomal subunits is a highly orchestrated process that involves a huge cohort of accessory factors. Most eukaryotic ribosome biogenesis factors were first identified by genetic screens and proteomic approaches of pre-ribosomal particles in Saccharomyces cerevisiae. Later, research on human ribosome synthesis not only demonstrated that the requirement for many of these factors is conserved in evolution, but also revealed the involvement of additional players, reflecting a more complex assembly pathway in mammalian cells.

Nuclear stabilization of p53 requires a functional nucleolar surveillance pathway.

The nucleolar surveillance pathway monitors nucleolar integrity and responds to nucleolar stress by mediating binding of ribosomal proteins to MDM2, resulting in p53 accumulation. Inappropriate pathway activation is implicated in the pathogenesis of ribosomopathies, while drugs selectively activating the pathway are in trials for cancer. Despite this, the molecular mechanism(s) regulating this process are poorly understood.

Genome-wide RNAi screen identifies novel players in human 60S subunit biogenesis including key enzymes of polyamine metabolism.

Ribosome assembly is an essential process that is linked to human congenital diseases and tumorigenesis. While great progress has been made in deciphering mechanisms governing ribosome biogenesis in eukaryotes, an inventory of factors that support ribosome synthesis in human cells is still missing, in particular regarding the maturation of the large 60S subunit. Here, we performed a genome-wide RNAi screen using an imaging-based, single cell assay to unravel the cellular machinery promoting 60S subunit assembly in human cells.

The nucleoporin Nup50 activates the Ran guanine nucleotide exchange factor RCC1 to promote NPC assembly at the end of mitosis.

During mitotic exit, thousands of nuclear pore complexes (NPCs) assemble concomitant with the nuclear envelope to build a transport-competent nucleus. Here, we show that Nup50 plays a crucial role in NPC assembly independent of its well-established function in nuclear transport. RNAi-mediated downregulation in cells or immunodepletion of Nup50 protein in Xenopus egg extracts interferes with NPC assembly.

Nuclear export of the pre-60S ribosomal subunit through single nuclear pores observed in real time.

Ribosomal biogenesis has been studied by biochemical, genetic and electron microscopic approaches, but live cell data on the in vivo kinetics are still missing. Here we analyse the export kinetics of the large ribosomal subunit (pre-60S particle) through single NPCs in human cells. We established a stable cell line co-expressing Halo-tagged eIF6 and GFP-fused NTF2 to simultaneously label pre-60S particles and NPCs, respectively.

Processing of the ribosomal ubiquitin-like fusion protein FUBI-eS30/FAU is required for 40S maturation and depends on USP36.

In humans and other holozoan organisms, the ribosomal protein eS30 is synthesized as a fusion protein with the ubiquitin-like protein FUBI. However, FUBI is not part of the mature 40S ribosomal subunit and cleaved off by an as-of-yet unidentified protease. How FUBI-eS30 processing is coordinated with 40S subunit maturation is unknown.

Mitotic disassembly and reassembly of nuclear pore complexes.

Nuclear pore complexes (NPCs) are huge protein assemblies within the nuclear envelope (NE) that serve as selective gates for macromolecular transport between nucleus and cytoplasm. When higher eukaryotic cells prepare for division, they rapidly disintegrate NPCs during NE breakdown such that nuclear and cytoplasmic components mix to enable the formation of a cytoplasmic mitotic spindle. At the end of mitosis, reassembly of NPCs is coordinated with the establishment of the NE around decondensing chromatin.

Embedded Microbubbles for Acoustic Manipulation of Single Cells and Microfluidic Applications.

Acoustically excited microstructures have demonstrated significant potential for small-scale biomedical applications by overcoming major microfluidic limitations. Recently, the application of oscillating microbubbles has demonstrated their superiority over acoustically excited solid structures due to their enhanced acoustic streaming at low input power. However, their limited temporal stability hinders their direct applicability for industrial or clinical purposes.

The Diverse Cellular Functions of Inner Nuclear Membrane Proteins.

The nuclear compartment is delimited by a specialized expanded sheet of the endoplasmic reticulum (ER) known as the nuclear envelope (NE). Compared to the outer nuclear membrane and the contiguous peripheral ER, the inner nuclear membrane (INM) houses a unique set of transmembrane proteins that serve a staggering range of functions. Many of these functions reflect the exceptional position of INM proteins at the membrane-chromatin interface.

Torsin ATPases influence chromatin interaction of the Torsin regulator LAP1.

The inner nuclear membrane is functionalized by diverse transmembrane proteins that associate with nuclear lamins and/or chromatin. When cells enter mitosis, membrane-chromatin contacts must be broken to allow for proper chromosome segregation; yet how this occurs remains ill-understood. Unexpectedly, we observed that an imbalance in the levels of the lamina-associated polypeptide 1 (LAP1), an activator of ER-resident Torsin AAA+-ATPases, causes a failure in membrane removal from mitotic chromatin, accompanied by chromosome segregation errors and changes in post-mitotic nuclear morphology.

Structural basis for the final steps of human 40S ribosome maturation.

Eukaryotic ribosomes consist of a small 40S and a large 60S subunit that are assembled in a highly coordinated manner. More than 200 factors ensure correct modification, processing and folding of ribosomal RNA and the timely incorporation of ribosomal proteins. Small subunit maturation ends in the cytosol, when the final rRNA precursor, 18S-E, is cleaved at site 3 by the endonuclease NOB1.

USP16 counteracts mono-ubiquitination of RPS27a and promotes maturation of the 40S ribosomal subunit.

Establishment of translational competence represents a decisive cytoplasmic step in the biogenesis of 40S ribosomal subunits. This involves final 18S rRNA processing and release of residual biogenesis factors, including the protein kinase RIOK1. To identify novel proteins promoting the final maturation of human 40S subunits, we characterized pre-ribosomal subunits trapped on RIOK1 by mass spectrometry, and identified the deubiquitinase USP16 among the captured factors.

A Global Screen for Assembly State Changes of the Mitotic Proteome by SEC-SWATH-MS.

Living systems integrate biochemical reactions that determine the functional state of each cell. Reactions are primarily mediated by proteins. In proteomic studies, these have been treated as independent entities, disregarding their higher-level organization into complexes that affects their activity and/or function and is thus of great interest for biological research.

Molecular mechanism of the RNA helicase DHX37 and its activation by UTP14A in ribosome biogenesis.

Eukaryotic ribosome biogenesis is a highly orchestrated process involving numerous assembly factors including ATP-dependent RNA helicases. The DEAH helicase DHX37 (Dhr1 in yeast) is activated by the ribosome biogenesis factor UTP14A to facilitate maturation of the small ribosomal subunit. We report the crystal structure of DHX37 in complex with single-stranded RNA, revealing a canonical DEAH ATPase/helicase architecture complemented by a structurally unique carboxy-terminal domain (CTD).

Influenza virus uses transportin 1 for vRNP debundling during cell entry.

Influenza A virus is a pathogen of great medical impact. To develop novel antiviral strategies, it is essential to understand the molecular aspects of virus-host cell interactions in detail. During entry, the viral ribonucleoproteins (vRNPs) that carry the RNA genome must be released from the incoming particle before they can enter the nucleus for replication.

Dissociation of membrane-chromatin contacts is required for proper chromosome segregation in mitosis.

The nuclear envelope (NE) aids in organizing the interphase genome by tethering chromatin to the nuclear periphery. During mitotic entry, NE-chromatin contacts are broken. Here, we report on the consequences of impaired NE removal from chromatin for cell division of human cells.

Observing and tracking single small ribosomal subunits in vivo.

Ribosomes are formed of a small and a large subunit (SSU/LSU), both consisting of rRNA and a plethora of accessory proteins. While biochemical and genetic studies identified most of the involved proteins and deciphered the ribosomal synthesis steps, our knowledge of the molecular dynamics of the different ribosomal subunits and also of the kinetics of their intracellular trafficking is still limited. Adopting a labelling strategy initially used to study mRNA export we were able to fluorescently stain the SSU in vivo.

Mitotic Disassembly of Nuclear Pore Complexes Involves CDK1- and PLK1-Mediated Phosphorylation of Key Interconnecting Nucleoporins.

During interphase, the nuclear envelope (NE) serves as a selective barrier between cytosol and nucleoplasm. When vertebrate cells enter mitosis, the NE is dismantled in the process of nuclear envelope breakdown (NEBD). Disassembly of nuclear pore complexes (NPCs) is a key aspect of NEBD, required for NE permeabilization and formation of a cytoplasmic mitotic spindle.

Efficient protein targeting to the inner nuclear membrane requires Atlastin-dependent maintenance of ER topology.

Newly synthesized membrane proteins are targeted to the inner nuclear membrane (INM) by diffusion within the membrane system of the endoplasmic reticulum (ER), translocation through nuclear pore complexes (NPCs) and retention on nuclear partners. Using a visual in vitro assay we previously showed that efficient protein targeting to the INM depends on nucleotide hydrolysis. We now reveal that INM targeting is GTP-dependent.