Multinucleated cells are important in many organisms, but the mechanisms governing the movements of nuclei sharing a common cytoplasm are not understood. In the hyphae of the plant pathogenic fungus Ashbya gossypii, nuclei move back and forth, occasionally bypassing each other, preventing the formation of nuclear clusters. This is essential for genetic stability. These movements depend on cytoplasmic microtubules emanating from the nuclei that are pulled by dynein motors anchored at the cortex. Using three-dimensional stochastic simulations with parameters constrained by the literature, we predict the cortical anchor density from the characteristics of nuclear movements. The model accounts for the complex nuclear movements seen in vivo, using a minimal set of experimentally determined ingredients. Of interest, these ingredients power the oscillations of the anaphase spindle in budding yeast, but in A. gossypii, this system is not restricted to a specific nuclear cycle stage, possibly as a result of adaptation to hyphal growth and multinuclearity.

The organisation of mammalian genomes into loops and topologically associating domains (TADs) contributes to chromatin structure, gene expression and recombination. TADs and many loops are formed by cohesin and positioned by CTCF. In proliferating cells, cohesin also mediates sister chromatid cohesion, which is essential for chromosome segregation. Current models of chromatin folding and cohesion are based on assumptions of how many cohesin and CTCF molecules organise the genome. Here we have measured absolute copy numbers and dynamics of cohesin, CTCF, NIPBL, WAPL and sororin by mass spectrometry, fluorescence-correlation spectroscopy and fluorescence recovery after photobleaching in HeLa cells. In G1-phase, there are ~250,000 nuclear cohesin complexes, of which ~ 160,000 are chromatin-bound. Comparison with chromatin immunoprecipitation-sequencing data implies that some genomic cohesin and CTCF enrichment sites are unoccupied in single cells at any one time. We discuss the implications of these findings for how cohesin can contribute to genome organisation and cohesion.

This protocol describes an innovative study to investigate the relationship between sleep, shift work and the immune response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2; coronavirus disease 2019 [COVID-19]) vaccination. As the COVID-19 pandemic is a global crisis with devastating health, social and economic impacts, there is a pressing need for effective vaccination programmes. Previous influenza and hepatitis vaccination studies suggest that lack of sleep can negatively alter immune responsiveness, while circadian misalignment most likely may also play an important role in the immune response to vaccination. Our present study will be the first to address this question in actual shift workers and in relation to COVID-19 vaccination. We hypothesise that the occurrence of recent night shifts and diminished sleep will negatively alter the immune response to vaccination in shift workers compared to dayworkers. We aim to recruit 50 shift workers and 50 dayworkers. Participants will receive an mRNA-based vaccination, through the Dutch vaccination programme. To assess immune responsiveness, blood will be drawn at baseline (before first vaccination), 10 days after first vaccination, the day prior to the second vaccination; and 28 days, 6 and 12 months after the second vaccination. Actigraphy and daily sleep e-diaries will be implemented for 7 days around each vaccination to assess sleep. The Pittsburgh Sleep Quality Index will be used to monitor sleep in the long term. Optimising the efficacy of the COVID-19 vaccines is of outmost importance and results of this study could provide insights to develop sleep and circadian-based interventions to enhance vaccination immunity, and thereby improve global health.

Eukaryotic genomes are partitioned into chromosomes that form compact and spatially well-separated mechanical bodies during mitosis. This enables chromosomes to move independently of each other for segregation of precisely one copy of the genome to each of the nascent daughter cells. Despite insights into the spatial organization of mitotic chromosomes and the discovery of proteins at the chromosome surface, the molecular and biophysical bases of mitotic chromosome structural individuality have remained unclear. Here we report that the proliferation marker protein Ki-67 (encoded by the MKI67 gene), a component of the mitotic chromosome periphery, prevents chromosomes from collapsing into a single chromatin mass after nuclear envelope disassembly, thus enabling independent chromosome motility and efficient interactions with the mitotic spindle. The chromosome separation function of human Ki-67 is not confined within a specific protein domain, but correlates with size and net charge of truncation mutants that apparently lack secondary structure. This suggests that Ki-67 forms a steric and electrostatic charge barrier, similar to surface-active agents (surfactants) that disperse particles or phase-separated liquid droplets in solvents. Fluorescence correlation spectroscopy showed a high surface density of Ki-67 and dual-colour labelling of both protein termini revealed an extended molecular conformation, indicating brush-like arrangements that are characteristic of polymeric surfactants. Our study thus elucidates a biomechanical role of the mitotic chromosome periphery in mammalian cells and suggests that natural proteins can function as surfactants in intracellular compartmentalization.

In the hippocampal formation of Alzheimer's disease (AD) patients, both focal and diffuse deposits of Aβ peptides appear in a subregion- and layer-specific manner. Recently, pyroglutamate (pGlu or pE)-modified Aβ peptides were identified as a highly pathogenic and seeding Aβ peptide species. Since the pE modification is catalyzed by glutaminyl cyclase (QC) this enzyme emerged as a novel pharmacological target for AD therapy. Here, we reveal the role of QC in the formation of different types of hippocampal pE-Aβ aggregates. First, we demonstrate that both, focal and diffuse pE-Aβ deposits are present in defined layers of the AD hippocampus. While the focal type of pE-Aβ aggregates was found to be associated with the somata of QC-expressing interneurons, the diffuse type was not. To address this discrepancy, the hippocampus of amyloid precursor protein transgenic mice was analysed. Similar to observations made in AD, focal (i.e. core-containing) pE-Aβ deposits originating from QC-positive neurons and diffuse pE-Aβ deposits not associated with QC were detected in Tg2576 mouse hippocampus. The hippocampal layers harbouring diffuse pE-Aβ deposits receive multiple afferents from QC-rich neuronal populations of the entorhinal cortex and locus coeruleus. This might point towards a mechanism in which pE-Aβ and/or QC are being released from projection neurons at hippocampal synapses. Indeed, there are a number of reports demonstrating the reduction of diffuse, but not of focal, Aβ deposits in hippocampus after deafferentation experiments. Moreover, we demonstrate in neurons by live cell imaging and by enzymatic activity assays that QC is secreted in a constitutive and regulated manner. Thus, it is concluded that hippocampal pE-Aβ plaques may develop through at least two different mechanisms: intracellularly at sites of somatic QC activity as well as extracellularly through seeding at terminal fields of QC expressing projection neurons.

To understand how multiprotein complexes assemble and function on chromatin, we combined quantitative analysis of the mammalian nucleotide excision DNA repair (NER) machinery in living cells with computational modeling. We found that individual NER components exchange within tens of seconds between the bound state in repair complexes and the diffusive state in the nucleoplasm, whereas their net accumulation at repair sites evolves over several hours. Based on these in vivo data, we developed a predictive kinetic model for the assembly and function of repair complexes. DNA repair is orchestrated by the interplay of reversible protein-binding events and progressive enzymatic modifications of the chromatin substrate. We demonstrate that faithful recognition of DNA lesions is time consuming, whereas subsequently, repair complexes form rapidly through random and reversible assembly of NER proteins. Our kinetic analysis of the NER system reveals a fundamental conflict between specificity and efficiency of chromatin-associated protein machineries and shows how a trade off is negotiated through reversibility of protein binding.

Cohesin is a multisubunit protein complex that links sister chromatids from replication until segregation. The lack of obvious cohesin-targeting-specific sequences on DNA, as well as cohesin's molecular arrangement as a large ring, has led to the working hypothesis that cohesin acts as a direct topological linker. To preserve the identity of sister chromatids, such a linkage would need to stably persist throughout the entire S and G2 phases of the cell cycle. Unexpectedly, cohesin binds chromatin already in telophase, and a large fraction dissociates from chromosomes during prophase in a phosphorylation-dependent manner, whereas initiation of anaphase requires proteolytic cleavage of only a small fraction of cohesin. These observations raised the question of how and when cohesin interacts with chromatin during the cell cycle. Here, we report a cell-cycle dependence in the stability of cohesin binding to chromatin. Using photobleaching and quantitative live-cell imaging, we identified several cohesin pools with different chromatin binding stabilities. Although all chromatin bound cohesin dissociated after a mean residence time of less than 25 min before replication, about one-third of cohesin was bound much more stably after S phase and persisted until metaphase, consistent with long-lived links mediating cohesion between sister chromatids.

Self-labeling protein tags such as HaloTag are powerful tools that can label fusion proteins with synthetic fluorophores for use in fluorescence microscopy. Here we introduce HaloTag variants with either increased or decreased brightness and fluorescence lifetime compared with HaloTag7 when labeled with rhodamines. Combining these HaloTag variants enabled live-cell fluorescence lifetime multiplexing of three cellular targets in one spectral channel using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. Additionally, the brightest HaloTag variant showed up to 40% higher brightness in live-cell imaging applications.

Fluorescence lifetime multiplexing requires fluorescent probes with distinct fluorescence lifetimes but similar spectral properties. Even though synthetic probes for many cellular targets are available for multicolor live-cell fluorescence microscopy, few of them have been characterized for their use in fluorescence lifetime multiplexing. Here, we demonstrate that, from a panel of 18 synthetic probes, eight pairwise combinations are suitable for fluorescence lifetime multiplexing in living mammalian cell lines. Moreover, combining multiple pairs in different spectral channels enables us to image four and with the help of self-labeling protein tags up to eight different biological targets, effectively doubling the number of observable targets. The combination of synthetic probes with fluorescence lifetime multiplexing is thus a powerful approach for live-cell imaging.

Faithful chromosome segregation, during meiosis, is of critical importance to prevent aneuploidy in the resulting embryo. In mammalian oocytes, the segregation of homologous chromosomes takes place with the spindle located at the cell's periphery. The spindle is often assembled close to the centre of the cell, which necessitates the actin network for spindle transport to the cell cortex. In this study, we investigate how the segregation of chromosomes is coordinated with the positioning of the metaphase I spindle. We develop different assays to perturb the spindle's position and to delay its relocation to the cell periphery. We find that anaphase is delayed until the spindle is positioned in close proximity with the oocyte cortex. We further show that the metaphase arrest is dependent on a functional actin network, in addition to the spindle assembly checkpoint. Our work provides the first evidence for the existence of a functional spindle position checkpoint.

Although splicing is essential for the expression of most eukaryotic genes, inactivation of splicing factors causes specific defects in mitosis. The molecular cause of this defect is unknown. Here, we show that the spliceosome subunits SNW1 and PRPF8 are essential for sister chromatid cohesion in human cells. A transcriptome-wide analysis revealed that SNW1 or PRPF8 depletion affects the splicing of specific introns in a subset of pre-mRNAs, including pre-mRNAs encoding the cohesion protein sororin and the APC/C subunit APC2. SNW1 depletion causes cohesion defects predominantly by reducing sororin levels, which causes destabilisation of cohesin on DNA. SNW1 depletion also reduces APC/C activity and contributes to cohesion defects indirectly by delaying mitosis and causing "cohesion fatigue". Simultaneous expression of sororin and APC2 from intron-less cDNAs restores cohesion in SNW1-depleted cells. These results indicate that the spliceosome is required for mitosis because it enables expression of genes essential for cohesion. Our transcriptome-wide identification of retained introns in SNW1- and PRPF8-depleted cells may help to understand the aetiology of diseases associated with splicing defects, such as retinosa pigmentosum and cancer.

Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B. We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells.

Huntingtin (HTT) fragments with extended polyglutamine tracts self-assemble into amyloid-like fibrillar aggregates. Elucidating the fibril formation mechanism is critical for understanding Huntington's disease pathology and for developing novel therapeutic strategies. Here, we performed systematic experimental and theoretical studies to examine the self-assembly of an aggregation-prone N-terminal HTT exon-1 fragment with 49 glutamines (Ex1Q49). Using high-resolution imaging techniques such as electron microscopy and atomic force microscopy, we show that Ex1Q49 fragments in cell-free assays spontaneously convert into large, highly complex bundles of amyloid fibrils with multiple ends and fibril branching points. Furthermore, we present experimental evidence that two nucleation mechanisms control spontaneous Ex1Q49 fibrillogenesis: (1) a relatively slow primary fibril-independent nucleation process, which involves the spontaneous formation of aggregation-competent fibrillary structures, and (2) a fast secondary fibril-dependent nucleation process, which involves nucleated branching and promotes the rapid assembly of highly complex fibril bundles with multiple ends. The proposed aggregation mechanism is supported by studies with the small molecule O4, which perturbs early events in the aggregation cascade and delays Ex1Q49 fibril assembly, comprehensive mathematical and computational modeling studies, and seeding experiments with small, preformed fibrillar Ex1Q49 aggregates that promote the assembly of amyloid fibrils. Together, our results suggest that nucleated branching in vitro plays a critical role in the formation of complex fibrillar HTT exon-1 aggregates with multiple ends.

Mammalian genomes are spatially organized into compartments, topologically associating domains (TADs), and loops to facilitate gene regulation and other chromosomal functions. How compartments, TADs, and loops are generated is unknown. It has been proposed that cohesin forms TADs and loops by extruding chromatin loops until it encounters CTCF, but direct evidence for this hypothesis is missing. Here, we show that cohesin suppresses compartments but is required for TADs and loops, that CTCF defines their boundaries, and that the cohesin unloading factor WAPL and its PDS5 binding partners control the length of loops. In the absence of WAPL and PDS5 proteins, cohesin forms extended loops, presumably by passing CTCF sites, accumulates in axial chromosomal positions (vermicelli), and condenses chromosomes. Unexpectedly, PDS5 proteins are also required for boundary function. These results show that cohesin has an essential genome-wide function in mediating long-range chromatin interactions and support the hypothesis that cohesin creates these by loop extrusion, until it is delayed by CTCF in a manner dependent on PDS5 proteins, or until it is released from DNA by WAPL.

The nuclear pore complex (NPC) mediates nucleocytoplasmic transport through the nuclear envelope. How the NPC assembles into this double membrane boundary has remained enigmatic. Here, we captured temporally staged assembly intermediates by correlating live cell imaging with high-resolution electron tomography and super-resolution microscopy. Intermediates were dome-shaped evaginations of the inner nuclear membrane (INM), that grew in diameter and depth until they fused with the flat outer nuclear membrane. Live and super-resolved fluorescence microscopy revealed the molecular maturation of the intermediates, which initially contained the nuclear and cytoplasmic ring component Nup107, and only later the cytoplasmic filament component Nup358. EM particle averaging showed that the evagination base was surrounded by an 8-fold rotationally symmetric ring structure from the beginning and that a growing mushroom-shaped density was continuously associated with the deforming membrane. Quantitative structural analysis revealed that interphase NPC assembly proceeds by an asymmetric inside-out extrusion of the INM.

Fluorescent biosensors enable the study of cell physiology with spatiotemporal resolution; yet, most biosensors suffer from relatively low dynamic ranges. Here, we introduce a family of designed Förster resonance energy transfer (FRET) pairs with near-quantitative FRET efficiencies based on the reversible interaction of fluorescent proteins with a fluorescently labeled HaloTag. These FRET pairs enabled the straightforward design of biosensors for calcium, ATP and NAD+ with unprecedented dynamic ranges. The color of each of these biosensors can be readily tuned by changing either the fluorescent protein or the synthetic fluorophore, which enables simultaneous monitoring of free NAD+ in different subcellular compartments following genotoxic stress. Minimal modifications of these biosensors furthermore allow their readout to be switched to fluorescence intensity, fluorescence lifetime or bioluminescence. These FRET pairs thus establish a new concept for the development of highly sensitive and tunable biosensors.

Mammalian oocytes are filled with poorly understood structures called cytoplasmic lattices. First discovered in the 1960s and speculated to correspond to mammalian yolk, ribosomal arrays, or intermediate filaments, their function has remained enigmatic to date. Here, we show that cytoplasmic lattices are sites where oocytes store essential proteins for early embryonic development. Using super-resolution light microscopy and cryoelectron tomography, we show that cytoplasmic lattices are composed of filaments with a high surface area, which contain PADI6 and subcortical maternal complex proteins. The lattices associate with many proteins critical for embryonic development, including proteins that control epigenetic reprogramming of the preimplantation embryo. Loss of cytoplasmic lattices by knocking out PADI6 or the subcortical maternal complex prevents the accumulation of these proteins and results in early embryonic arrest. Our work suggests that cytoplasmic lattices enrich maternally provided proteins to prevent their premature degradation and cellular activity, thereby enabling early mammalian development.

Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate (pGlu) from glutamine precursors at the N-terminus of a number of peptide hormones, neuropeptides and chemokines. This post-translational modification stabilizes these peptides, protects them from proteolytical degradation or is important for their biological activity. However, QC is also involved in a pathogenic pGlu modification of peptides accumulating in protein aggregation disorders such as Alzheimer's disease and familial Danish and familial British dementia. Its isoenzyme (isoQC) was shown to contribute to aspects of inflammation by pGlu-modifying and thereby stabilizing the monocyte chemoattractant protein CCL2. For the generation of respective animal models and for pharmacological treatment studies the characterization of the mouse strain and brain region-specific expression of QC and isoQC is indispensible. In order to address this issue, we used enzymatic activity assays and specific antibodies to detect both QC variants by immunohistochemistry in nine different mouse strains. Comparing different brain regions, the highest enzymatic QC/isoQC activity was detected in ventral brain, followed by cortex and hippocampus. Immunohistochemical stainings revealed that QC/isoQC activity in cortex mostly arises from isoQC expression. For most brain regions, the highest QC/isoQC activity was detected in C3H and FVB mice, whereas low QC/isoQC activity was present in CD1, SJL and C57 mice. Quantification of QC- and isoQC-immunoreactive cells by unbiased stereology revealed a higher abundance of isoQC- than of QC-immunoreactive neurons in Edinger-Westphal nucleus and in substantia nigra. In the locus coeruleus, however, there were comparable densities of QC- and of isoQC-immunoreactive neurons. These observations are of considerable importance with regard to the selection of appropriate mouse strains for the study of QC/isoQC relevance in mouse models of neurodegeneration and neuroinflammation and for the testing of therapeutical interventions in these models.

Cohesin is a protein complex that is required to hold sister chromatids together. Cleavage of the Scc1 subunit of cohesin by the protease separase releases the complex from chromosomes and thereby enables the separation of sister chromatids in anaphase. In vertebrate cells, the bulk of cohesin dissociates from chromosome arms already during prophase and prometaphase without cleavage of Scc1. Polo-like kinase 1 (Plk1) and Aurora-B are required for this dissociation process, and Plk1 can phosphorylate the cohesin subunits Scc1 and SA2 in vitro, consistent with the possibility that cohesin phosphorylation by Plk1 triggers the dissociation of cohesin from chromosome arms. However, this hypothesis has not been tested yet, and in budding yeast it has been found that phosphorylation of Scc1 by the Polo-like kinase Cdc5 enhances the cleavability of cohesin, but does not lead to separase-independent dissociation of cohesin from chromosomes. To address the functional significance of cohesin phosphorylation in human cells, we have searched for phosphorylation sites on all four subunits of cohesin by mass spectrometry. We have identified numerous mitosis-specific sites on Scc1 and SA2, mutated them, and expressed nonphosphorylatable forms of both proteins stably at physiological levels in human cells. The analysis of these cells lines, in conjunction with biochemical experiments in vitro, indicate that Scc1 phosphorylation is dispensable for cohesin dissociation from chromosomes in early mitosis but enhances the cleavability of Scc1 by separase. In contrast, our data reveal that phosphorylation of SA2 is essential for cohesin dissociation during prophase and prometaphase, but is not required for cohesin cleavage by separase. The similarity of the phenotype obtained after expression of nonphosphorylatable SA2 in human cells to that seen after the depletion of Plk1 suggests that SA2 is the critical target of Plk1 in the cohesin dissociation pathway.

A long-standing question in quantitative biology is the relationship between mRNA and protein levels of the same gene. Here, we measured mRNA and protein abundance, the phenotypic state, and the population context in thousands of single human cells for 23 genes by combining a unique collection of cell lines with fluorescently tagged endogenous genomic loci and quantitative immunofluorescence with branched DNA single-molecule fluorescence in situ hybridization and computer vision. mRNA and protein abundance displayed a mean single-cell correlation of 0.732 at steady state. Single-cell outliers of linear correlations are in a specific phenotypic state or population context. This is particularly relevant for interpreting mRNA-protein relationships during acute gene induction and turnover, revealing a specific adaptation of gene expression at multiple steps in single cells. Together, we show that single-cell protein abundance can be predicted by multivariate information that integrates mRNA level with the phenotypic state and microenvironment of a particular cell.