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Development from embryo to adult, organismal homeostasis and ageing are consecutive processes that rely on several functions of the nuclear envelope (NE). The NE compartmentalises the eukaryotic cells and provides physical stability to the genetic material in the nucleus. It provides spatiotemporal regulation of gene expression by controlling nuclear import and hence access of transcription factors to target genes as well as organisation of the genome into open and closed compartments. In addition, positioning of chromatin relative to the NE is important for DNA replication and repair and thereby also for genome stability. We discuss here the relevance of the NE in two classes of age-related human diseases. Firstly, we focus on the progeria syndromes Hutchinson–Gilford (HGPS) and Nestor–Guillermo (NGPS), which are caused by mutations in the LMNA and BANF1 genes, respectively. Both genes encode ubiquitously expressed components of the nuclear lamina that underlines the nuclear membranes. HGPS and NGPS patients manifest symptoms of accelerated ageing and cells from affected individuals show similar defects as cells from healthy old donors, including signs of increased DNA damage and epigenetic alternations. Secondly, we describe how several age-related neurodegenerative diseases, such as amyotrophic lateral sclerosis and Huntington’s disease, are related with defects in nucleocytoplasmic transport. A common feature of this class of diseases is the accumulation of nuclear pore proteins and other transport factors in inclusions. Importantly, genetic manipulations of the nucleocytoplasmic transport machinery can alleviate disease-related phenotypes in cell and animal models, paving the way for potential therapeutic interventions.

Control of gene expression in specific tissues and/or at certain stages of development allows the study and manipulation of gene function with high precision. Site-specific genome recombination by the flippase (FLP) and cyclization recombination (Cre) enzymes has proved particularly relevant. Joint efforts of many research groups have led to the creation of efficient FLP and Cre drivers to regulate gene expression in a variety of tissues in Caenorhabditis elegans. Here, we extend this toolkit by the addition of FLP lines that drive recombination specifically in distal tip cells, the somatic gonad, coelomocytes, and the epithelial P lineage. In some cases, recombination-mediated gene knockouts do not completely deplete protein levels due to persistence of long-lived proteins. To overcome this, we developed a spatiotemporally regulated degradation system for green fluorescent fusion proteins based on FLP-mediated recombination. Using 2 stable nuclear pore proteins, MEL-28/ELYS and NPP-2/NUP85 as examples, we report the benefit of combining tissue-specific gene knockout and protein degradation to achieve complete protein depletion. We also demonstrate that FLP-mediated recombination can be utilized to identify transcriptomes in a C. elegans tissue of interest. We have adapted RNA polymerase DamID for the FLP toolbox and by focusing on a well-characterized tissue, the hypodermis, we show that the vast majority of genes identified by RNA polymerase DamID are known to be expressed in this tissue. These tools allow combining FLP activity for simultaneous gene inactivation and transcriptomic profiling, thus enabling the inquiry of gene function in various complex biological processes.

Uncropped gel images of S2E and Uncropped gel images of S2F Fig.

(A) The 6 steps of ventral liver lobe formation correlate with fish standard length (SL). The numerical values that were used to generate the graph can be found in S1 Data. (B) Stage I: A small tissue extension at the tip of the left lobe is visible (n = 12 livers). (C) Stage II: a thin ventral lobe originates in the lower half of the left lobe (n = 6 livers). (D) Stage III: the thin ventral lobe shifts position towards the more anterior part of the left lobe (n = 4 livers). (E) Stage IV: the tip of the ventral lobe starts to expand (n = 7 livers). (F) Stage V: lateral-oriented expansion of the ventral lobe (n = 28 livers). (G) Stage VI: enlargement of all lobes in width (n = 6 livers). The blue areas in the schematics mark the region characteristic for the respective stage. (H) Schematic depicting the morphology of the liver in relation to the folding of the intestine in stages I-VI. A, anterior; P, posterior; R, right; L, left; RL, right lobe; LL, left lobe; VL, ventral lobe.

pbio.3002315.t001 - Lineage tracing identifies heterogeneous hepatoblast contribution to cell lineages and postembryonic organ growth dynamics

Schematics showing the current working models: (A) uni- and bipotent hepatoblast contributions to hepatocytes and BECs following heterogeneous lineage decisions. (B) Hepatoblasts contribute with heterogeneous proliferation behaviours to postembryonic liver growth. Cells from the embryonic left lobe contribute to the ventral lobe, including the formation of giant clusters (magenta). (C) The liver morphology changes dramatically simultaneous to the intestinal bending occurring during postembryonic growth (green). BEC, biliary epithelial cell; dpf, day postfertilization; SL, standard length.

The nuclear envelope, which protects and organizes the genome, is dismantled during mitosis. In the Caenorhabditis elegans zygote, nuclear envelope breakdown (NEBD) of the parental pronuclei is spatially and temporally regulated during mitosis to promote the unification of the maternal and paternal genomes. Nuclear pore complex (NPC) disassembly is a decisive step of NEBD, essential for nuclear permeabilization. By combining live imaging, biochemistry, and phosphoproteomics, we show that NPC disassembly is a stepwise process that involves Polo-like kinase 1 (PLK-1)–dependent and –independent steps. PLK-1 targets multiple NPC subcomplexes, including the cytoplasmic filaments, central channel, and inner ring. PLK-1 is recruited to and phosphorylates intrinsically disordered regions (IDRs) of several multivalent linker nucleoporins. Notably, although the phosphosites are not conserved between human and C. elegans nucleoporins, they are located in IDRs in both species. Our results suggest that targeting IDRs of multivalent linker nucleoporins is an evolutionarily conserved driver of NPC disassembly during mitosis.

Nucleoporins (Nups) assemble nuclear pores that form the permeability barrier between nucleoplasm and cytoplasm. Nucleoporins also localize in cytoplasmic foci proposed to function as pore pre‐assembly intermediates. Here, we characterize the composition and incidence of cytoplasmic Nup foci in an intact animal, C. elegans. We find that, in young non‐stressed animals, Nup foci only appear in developing sperm, oocytes and embryos, tissues that express high levels of nucleoporins. The foci are condensates of highly cohesive FG repeat‐containing nucleoporins (FG‐Nups), which are maintained near their solubility limit in the cytoplasm by posttranslational modifications and chaperone activity. Only a minor fraction of FG‐Nup molecules concentrate in Nup foci, which dissolve during M phase and are dispensable for nuclear pore assembly. Nucleoporin condensation is enhanced by stress and advancing age, and overexpression of a single FG‐Nup in post‐mitotic neurons is sufficient to induce ectopic condensation and organismal paralysis. We speculate that Nup foci are non‐essential and potentially toxic condensates whose assembly is actively suppressed in healthy cells.

Heterochromatin is characterized by an enrichment of repetitive elements and low gene density and is often maintained in a repressed state across cell division and differentiation. The silencing is mainly regulated by repressive histone marks such as H3K9 and H3K27 methylated forms and the heterochromatin protein 1 (HP1) family. Here, we analyzed in a tissue-specific manner the binding profile of the two HP1 homologs in Caenorhabditis elegans, HPL-1 and HPL-2, at the L4 developmental stage. We identified the genome-wide binding profile of intestinal and hypodermal HPL-2 and intestinal HPL-1 and compared them with heterochromatin marks and other features. HPL-2 associated preferentially to the distal arms of autosomes and correlated positively with the methylated forms of H3K9 and H3K27. HPL-1 was also enriched in regions containing H3K9me3 and H3K27me3 but exhibited a more even distribution between autosome arms and centers. HPL-2 showed a differential tissue-specific enrichment for repetitive elements conversely with HPL-1, which exhibited a poor association. Finally, we found a significant intersection of genomic regions bound by the BLMP-1/PRDM1 transcription factor and intestinal HPL-1, suggesting a corepressive role during cell differentiation. Our study uncovers both shared and singular properties of conserved HP1 proteins, providing information about genomic binding preferences in relation to their role as heterochromatic markers.

Nuclear pore complexes (NPCs) are composed of multiple copies of 30-35 nucleoporins (Nups; NPPs in C. elegans) embedded in the nuclear envelope (NE) and act as regulators of RNA and protein transport between the cytoplasm and nucleoplasm. In addition to their function in nucleocytoplasmic transport, the contact of Nups with chromatin at NPCs and in the nuclear interior has allowed the emergency of non-transport functions. These non-canonical functions of Nups are relevant to human pathologies and developmental phenotypes that arise as a result of Nup mutations. Moreover, there are Nups that gain a new localization upon certain stress conditions, such as heat stress. These ¿mobile Nups¿ have been detected both in human and fission yeast cells. The mechanisms underlying the mobility of Nups, and the phenotypes and pathologies associated with defects in these new roles of mobile Nups are just emerging in the field. The general hypothesis of this project is that specific nucleoporins have acquired new functions during evolution, acting therefore as moonlighting proteins. We recently discovered a novel heat stress-induced structure in fission yeast, termed nucleolar rings (NuRs), which accumulate of a specific subset of Nups (PMID: 33176152). To uncover moonlighting functions of nucleoporins conserved across species we apply acute heat stress to a variety of C. elegans strains with deletions and/or fluorescent tags in endogenous Nup (npp) loci. We report that certain Nups detach rapidly from NPCs upon exposure to heat stress whereas other Nups remain stable. Unexpectedly, the behaviour of individual Nups seems not to correlate with their position within the NPC. Benefitting from the many genome tagging and mutagenesis efforts in the community, we expect to perform a systematic analysis of most NPC components and thereby obtain a detailed view of Nups in stress resistance.

Nestor-Guillermo Progeria Syndrome (NGPS) is a premature ageing illness that affects a variety of tissues, leading to growth retardation, and severe skeletal defects. The syndrome is caused by a single amino acid substitution (A12T) in BAF1 (Barrier to Autointegration Factor 1), a highly conserved chromatin binding protein implicated in nuclear envelope (NE) breakdown, assembly and repair as well as chromatin compaction. We have modified the baf-1 locus in Caenorhabditis elegans to mimic the human NGPS mutation (baf-1(G12T)) to elucidate why a mutation in an essential protein expressed throughout development triggers the appearance of symptoms in children ~2 years after birth. We report that NE levels of lamin/LMN-1 and emerin/EMR-1 are reduced in baf-1(G12T) mutants, whereas errors in chromosome segregation are increased. The baf-1(G12T) mutation reduces fertility and lifespan and accelerates age-dependent nuclear morphology deterioration. Moreover, we found that baf-1(G12T) mutants are hypersensitive to NE perturbations, particularly to modifications affecting lamin/LMN-1. CRISPR-mediated gene knockout in NGPS fibroblasts unveiled a set of genes whose depletion alleviates the nuclear associated defects. When orthologs were silenced by RNAi in C. elegans, lis-1(PAFAH1B1/LIS1), vps-16(VPS16), smu-1(SMU1) and rps-1(RPS3A) reduced the embryonic lethality of sensitized baf-1(G12T) mutants. LIS1 is necessary for the correct differentiation and function of osteoclasts, regulating microtubule network and lysosomal dynamics. This offers a working model to explain the severe skeletal defects of NGPS patients. In support of these observations, we uncover that depletion of dlc-1(DYNLL2), vps-11(VPS11) and LINC (Linker of nucleoskeleton to cytoskeleton) complex subunits sun-1(SUN1) and zyg-12(HOOK1/2) also decreased the proportion of dead eggs of sensitized baf-1(G12T) worms. These results represent a first and encouraging list of candidate genes to be further explored for the

BAF-1 (Barrier to Autointegration Factor) is a highly conserved chromatin binding protein implicated in nuclear envelope (NE) breakdown, assembly and repair as well as chromatin compaction. It acts as a homodimer and is found in the nucleoplasm and enriched at the NE, where its localisation is interdependent on lamins and LEM-domain proteins (LAP2, emerin, and MAN1). Strikingly, a single amino acid substitution in human BAF (A12T) causes Nestor-Guillermo Progeria Syndrome (NGPS). This premature ageing illness affects a variety of tissues, leading to growth retardation, severe skeletal defects and scoliosis. We have modified the baf-1 locus in Caenorhabditis elegans to mimic the human NGPS mutation (baf- 1(G12T)) to elucidate why a mutation in an essential protein expressed throughout development triggers the appearance of symptoms ~2 years after birth. We report that lifespan and NE levels of lamin/LMN-1 and emerin/EMR-1 are reduced in baf-1(G12T) mutants, whereas errors in chromosome segregation are increased. Interestingly, the baf-1(G12T) mutation makes animals temperature sensitive in terms of brood size and fertilisation capacity. Moreover, we found that baf-1(G12T) mutants are hypersensitive to NE perturbations, particularly to modifications affecting lamin/LMN-1. To explore if the NGPS mutation affects BAF-1¿s association with chromatin, we determined the binding profiles for wild type and mutant BAF-1 through tissue-specific DamID. Globally, the profiles for the two proteins are very similar, but we also identified discrete genomic regions with altered association to BAF-1. We are currently correlating these observations with tissue-specific changes in gene expression.

Nuclear pore complexes (NPCs) are essential to communication of macromolecules between the cell nucleus and the surrounding cytoplasm. RNA synthesized in the nucleus is exported through NPCs to function in the cytoplasm, whereas transcription factors and other proteins are selectively and actively imported. In addition, many NPC constituents, known as nuclear pore proteins (nucleoporins or nups), also play critical roles in other processes, such as genome organization, gene expression, and kinetochore function. Thanks to its genetic amenability and transparent body, the nematode Caenorhabditis elegans is an attractive model to study NPC dynamics. We provide here an overview of available genome engineered strains and FLP/Frt-based tools to study tissue-specific functions of individual nucleoporins. We also present protocols for live imaging of fluorescently tagged nucleoporins in intact tissues of embryos, larvae, and adult and for analysis of interactions between nucleoporins and chromatin by DamID.