The magnified view: from ancient trinkets to single nuclear pore complexes.

A journey from the earliest known use of lenses and magnifying glasses in ancient times, through the development of microscopes and towards modern electron microscopy techniques. The evolving technology and improved microscopes enabled the discovery of intracellular organelles, the nucleus and nuclear pore complexes (NPCs). Current advances have led to composite three-dimensional models showing NPC structure in unprecedented detail but relying on the averaging of many images. A complementary approach is field emission scanning electron microscopy providing topographic surface images that are easily and intuitively interpreted by our brain. Recent advances in this technique have made it possible to expose nuclei from human cells and to focus on individual NPCs and their architectural features.

A single filament biomechanical study of the enteropathogenic Escherichia coli Type III secretion system reveals a high elastic aspect ratio.

Type III secretion systems (T3SSs) are syringe-like protein complexes used by some of the most harmful bacterial pathogens to infect host cells. While the T3SS filament, a long hollow conduit that bridges between bacteria and host cells, has been characterized structurally, very little is known about its physical properties. These filaments should endure shear and normal stresses imposed by the viscous mucosal flow during infection within the intestinal tract. We used atomic force microscopy (AFM) to probe the longitudinal and radial mechanical response of individual T3SS filaments by pulling on filaments extending directly from bacterial surfaces and later pressing into filaments that were detached from the bacteria. The measured longitudinal elastic moduli were higher by about two orders of magnitude than the radial elastic moduli. These proportions are commensurate with the role of the T3SS filament, which requires horizontal flexibility while maintaining its structural integrity to withstand intense stresses during infection.

The Effect of Outer Space and Other Environmental Cues on Bacterial Conjugation.

Bacterial conjugation is one of the most abundant horizontal gene transfer (HGT) mechanisms, playing a fundamental role in prokaryote evolution. A better understanding of bacterial conjugation and its cross talk with the environment is needed for a more complete understanding of HGT mechanisms and to fight the dissemination of malicious genes between bacteria. Here, we studied the effect of outer space, microgravity, and additional key environmental cues on transfer (tra) gene expression and conjugation efficiency, using the under studied broad-host range plasmid pN3, as a model. High resolution scanning electron microscopy revealed the morphology of the pN3 conjugative pili and mating pair formation during conjugation. Using a nanosatellite carrying a miniaturized lab, we studied pN3 conjugation in outer space, and used qRT-PCR, Western blotting and mating assays to determine the effect of ground physicochemical parameters on tra gene expression and conjugation. We showed for the first time that bacterial conjugation can occur in outer space and on the ground, under microgravity-simulated conditions. Furthermore, we demonstrated that microgravity, liquid media, elevated temperature, nutrient depletion, high osmolarity and low oxygen significantly reduce pN3 conjugation. Interestingly, under some of these conditions we observed an inverse correlation between tra gene transcription and conjugation frequency and found that induction of at least traK and traL can negatively affect pN3 conjugation frequency in a dose-dependent manner. Collectively, these results uncover pN3 regulation by various environmental cues and highlight the diversity of conjugation systems and the different ways in which they may be regulated in response to abiotic signals. IMPORTANCE Bacterial conjugation is a highly ubiquitous and promiscuous process, by which a donor bacterium transfers a large portion of genetic material to a recipient cell. This mechanism of horizontal gene transfer plays an important role in bacterial evolution and in the ability of bacteria to acquire resistance to antimicrobial drugs and disinfectants. Bacterial conjugation is a complex and energy-consuming process, that is tightly regulated and largely affected by various environmental signals sensed by the bacterial cell. Comprehensive knowledge about bacterial conjugation and the ways it is affected by environmental cues is required to better understand bacterial ecology and evolution and to find new effective ways to counteract the threating dissemination of antibiotic resistance genes between bacterial populations. Moreover, characterizing this process under stress or suboptimal growth conditions such as elevated temperatures, high salinity or in the outer space, may provide insights relevant to future habitat environmental conditions.

A Novel Homozygous Missense Variant in the LRRC32 Gene Is Associated With a New Syndrome of Cleft Palate, Progressive Vitreoretinopathy, Growth Retardation, and Developmental Delay.

Cleft lip and/or cleft palate are a common group of birth defects that further classify into syndromic and non-syndromic forms. The syndromic forms are usually accompanied by additional physical or cognitive abnormalities. Isolated cleft palate syndromes are less common; however, they are associated with a variety of congenital malformations and generally have an underlying genetic etiology. A single report in 2019 described a novel syndrome in three individuals, characterized by cleft palate, developmental delay and proliferative retinopathy due to a homozygous non-sense mutation in the LRRC32 gene encoding glycoprotein A repetitions predominant (GARP), a cell surface polypeptide crucial for the processing and maturation of transforming growth factor ß (TGF-ß). We describe a patient who presented with cleft palate, prenatal and postnatal severe growth retardation, global developmental delay, dysmorphic facial features and progressive vitreoretinopathy. Whole exome sequencing (WES) revealed a very rare homozygous missense variant in the LRRC32 gene, which resulted in substitution of a highly conserved isoleucine to threonine. Protein modeling suggested this variant may negatively affect GARP function on latent TGF-ß activation. In summary, our report further expands the clinical features of cleft palate, proliferative retinopathy and developmental delay syndrome and emphasizes the association of LRRC32 pathogenic variants with this new syndrome.

High-Resolution Imaging and Analysis of Individual Nuclear Pore Complexes.

Field emission scanning electron microscopy (FESEM) is a well-established technique for acquiring three-dimensional surface images of nuclear pore complexes (NPCs). We present an optimized protocol for the exposure of mammalian cell nuclei and direct surface imaging of nuclear envelopes by FESEM, allowing for a detailed morphological comparison of individual NPCs, without the need for averaging techniques. This provides a unique high resolution tool for studying the effects of cellular stress, specific genetic manipulations and inherited diseases on the ultrastructure of human NPCs.

The Cytopathic Effect of Different Toxin Concentrations From Different Clostridioides difficile Sequence Types Strains in Vero Cells.

Clostridioides difficile is one of the leading causes of healthcare-associated diarrhea, with severity ranging from mild, self-limiting disease, to life-threatening toxic megacolon. C. difficile infection (CDI) pathogenesis is mediated by the TcdA and TcdB toxins. This work aimed to draw correlations between toxin levels, bacterial strains, and disease severity in 63 CDI patients. C. difficile typing was performed by multi-locus sequence types (MLST). Toxin concentrations were measured using the TOX A/B test. In addition, cell cytotoxicity assay was performed following Vero cell exposure to stool extracts (24 h). The most prevalent sequence types (ST) were ST2, ST4, ST6, ST13, ST37, ST42, and ST104, with highest toxin levels produced by ST42 and ST104 (302.5 and 297.1 ng/ml, respectively). These strains had a stronger cytopathic effect (CPE) on Vero cells as compared to strains with lower toxin concentrations (p < 0.001), as manifested by lower cell counts and higher percentages of cell rounding and adhesion loss. Although no association was found between ST, toxin concentrations, and disease severity, a diverse in vitro effect of different STs on the viability and activity of Vero cells was observed. These findings suggest that disease severity is affected by both host immune responses and by bacterial characteristics.

Generation and characterization of iPSC lines from two nuclear envelopathy patients with a homozygous nonsense mutation in the TOR1AIP1 gene.

LAP1 is an inner nuclear membrane protein encoded by TOR1AIP1. A homozygous c.961C > T loss of function mutation in TOR1AIP1 that affects both isoforms of LAP1 was recently described. This mutation leads to the development of a severe multisystemic nuclear envelopathy syndrome. Here we describe the generation and characterization of two human induced pluripotent stem cell (hiPSC) lines derived from skin fibroblasts of two patients carrying the homozygous c.961C > T mutation. These novel lines can be used as a powerful tool to investigate the molecular mechanism by which LAP1 deficiency leads to the development of this severe hereditary disorder.

Expanding the phenotype of NUP85 mutations beyond nephrotic syndrome to primary autosomal recessive microcephaly and Seckel syndrome spectrum disorders.

Primary autosomal recessive microcephaly and Seckel syndrome spectrum disorders (MCPH-SCKS) include a heterogeneous group of autosomal recessive inherited diseases characterized by primary (congenital) microcephaly, the absence of visceral abnormalities, and a variable degree of cognitive impairment, short stature and facial dysmorphism. Recently, biallelic variants in the nuclear pore complex (NPC) component nucleoporin 85 gene (NUP85) were reported to cause steroid-resistant nephrotic syndrome (SRNS). Here, we report biallelic variants in NUP85 in two pedigrees with an MCPH-SCKS phenotype spectrum without SRNS, thereby expanding the phenotypic spectrum of NUP85-linked diseases. Structural analysis predicts the identified NUP85 variants cause conformational changes that could have an effect on NPC architecture or on its interaction with other NUPs. We show that mutant NUP85 is, however, associated with a reduced number of NPCs but unaltered nucleocytoplasmic compartmentalization, abnormal mitotic spindle morphology, and decreased cell viability and proliferation in one patient's cells. Our results also indicate the link of common cellular mechanisms involved in MCPH-SCKS spectrum disorders and NUP85-associated diseases. In addition to the previous studies, our results broaden the phenotypic spectrum of NUP85-linked human disease and propose a role for NUP85 in nervous system development.

Local Delivery of Mometasone Furoate from an Eluting Endotracheal Tube Reduces Airway Morbidity Following Long-Term Animal Intubation.

BACKGROUND: upper airway complications are common sequelae of endotracheal tube (ETT) intubation, and systemic corticosteroids are considered a mainstay treatment for this problem. Drug-eluting ETT may present an attractive option for topical steroid delivery while avoiding systemic side effects and improving the therapeutic outcome. The objective of the present study is to evaluate the reduction of tube-related tracheal morbidity via a self-designed steroid-eluting ETT with controlled sustained release properties in an animal model. METHODS: steroid-eluting ETTs were coated by poly(lactic-co-glycolic acid) -electrospun nanofibers loaded with mometasone furoate (MF) as a model drug. Animals were randomly assigned into three equal groups: non-intubated, blank-ETT, and loaded-ETT. The intubation interval was 1 week. Specimens were analyzed by histology, specific fibrosis staining, and scanning electron microscopy (SEM). RESULTS: the blank-ETT group exhibited a significant increase in tracheal mucosal thickness compared to the loaded-ETT and control groups. Average tracheal mucosal thickness was 112 ± 34, 242 ± 49, and 113 ± 43 µm in the control, blank-ETT, and loaded-ETT groups, respectively. The blank-ETT group exhibited a significant increase in tracheal fibrosis compared to the loaded-ETT and control groups. Relative fibrosis values were 0.07 ± 0.05, 0.154 ± 0.1, and 0.0984 ± 0.084% for the control, blank-ETT, and loaded-ETT groups, respectively. While SEM imaging showed normal surface structures in the control group, intubated blank-ETT rats showed severe surface structural damage, whereas only mild damage was observed in the loaded-ETT group. CONCLUSIONS: local sustained release of MF via a self-designed drug-eluting ETT is a potential therapeutic approach which may significantly reduce tube-related upper airway morbidity.

Pathogenic Variants in NUP214 Cause "Plugged" Nuclear Pore Channels and Acute Febrile Encephalopathy.

We report biallelic missense and frameshift pathogenic variants in the gene encoding human nucleoporin NUP214 causing acute febrile encephalopathy. Clinical symptoms include neurodevelopmental regression, seizures, myoclonic jerks, progressive microcephaly, and cerebellar atrophy. NUP214 and NUP88 protein levels were reduced in primary skin fibroblasts derived from affected individuals, while the total number and density of nuclear pore complexes remained normal. Nuclear transport assays exhibited defects in the classical protein import and mRNA export pathways in affected cells. Direct surface imaging of fibroblast nuclei by scanning electron microscopy revealed a large increase in the presence of central particles (known as "plugs") in the nuclear pore channels of affected cells. This observation suggests that large transport cargoes may be delayed in passage through the nuclear pore channel, affecting its selective barrier function. Exposure of fibroblasts from affected individuals to heat shock resulted in a marked delay in their stress response, followed by a surge in apoptotic cell death. This suggests a mechanistic link between decreased cell survival in cell culture and severe fever-induced brain damage in affected individuals. Our study provides evidence by direct imaging at the single nuclear pore level of functional changes linked to a human disease.

Combined loss of LAP1B and LAP1C results in an early onset multisystemic nuclear envelopathy.

Nuclear envelopathies comprise a heterogeneous group of diseases caused by mutations in genes encoding nuclear envelope proteins. Mutations affecting lamina-associated polypeptide 1 (LAP1) result in two discrete phenotypes of muscular dystrophy and progressive dystonia with cerebellar atrophy. We report 7 patients presenting at birth with severe progressive neurological impairment, bilateral cataract, growth retardation and early lethality. All the patients are homozygous for a nonsense mutation in the TOR1AIP1 gene resulting in the loss of both protein isoforms LAP1B and LAP1C. Patient-derived fibroblasts exhibit changes in nuclear envelope morphology and large nuclear-spanning channels containing trapped cytoplasmic organelles. Decreased and inefficient cellular motility is also observed in these fibroblasts. Our study describes the complete absence of both major human LAP1 isoforms, underscoring their crucial role in early development and organogenesis. LAP1-associated defects may thus comprise a broad clinical spectrum depending on the availability of both isoforms in the nuclear envelope throughout life.

NEK3-mediated SNAP29 phosphorylation modulates its membrane association and SNARE fusion dependent processes.

Intracellular membrane fusion depends on the presence of specific mediators, the vesicle (v-) and the target (t-) SNAREs (Soluble N-ethylmaleimide-sensitive factor, NSF, attachment protein SNAP receptors), whose interaction brings apposing membranes to close proximity and initiates their fusion. SNAP29 (synaptosomal-associated protein 29), a t-SNARE protein, is involved in multiple fusion events during intracellular transport and affects structure of organelles such as the Golgi apparatus and focal adhesions. Mutations in SNAP29 gene result in CEDNIK (Cerebral dysgenesis, neuropathy, ichthyosis and palmoplantar keratoderma) syndrome. In the present study, we show that NEK3 (NIMA-never in mitosis gene A-related kinase 3)-mediated serine 105 (S105) phosphorylation of SNAP29 directs its membrane association, without which cells present defective focal adhesion formation, impaired Golgi structure and attenuated cellular recycling. In contrast to a phosphorylation-defective serine 105 to alanine (S105A) mutant, wildtype SNAP29, partially rescued the abnormal morphology of a CEDNIK patient derived fibroblasts. Our results highlight the importance of NEK3-mediated S105 phosphorylation of SNAP29 for its membrane localization and for membrane fusion dependent processes.

Phenotypic and Proteomic Analysis of the Aspergillus fumigatus ΔPrtT, ΔXprG and ΔXprG/ΔPrtT Protease-Deficient Mutants.

Aspergillus fumigatus is the most common mold species to cause disease in immunocompromised patients. Infection usually begins when its spores (conidia) are inhaled into the airways, where they germinate, forming hyphae that penetrate and destroy the lungs and disseminate to other organs, leading to high mortality. The ability of hyphae to penetrate the pulmonary epithelium is a key step in the infectious process. A. fumigatus produces extracellular proteases that are thought to enhance penetration by degrading host structural barriers. This study explores the role of the A. fumigatus transcription factor XprG in controlling secreted proteolytic activity and fungal virulence. We deleted xprG, alone and in combination with prtT, a transcription factor previously shown to regulate extracellular proteolysis. xprG deletion resulted in abnormal conidiogenesis and formation of lighter colored, more fragile conidia and a moderate reduction in the ability of culture filtrates (CFs) to degrade substrate proteins. Deletion of both xprG and prtT resulted in an additive reduction, generating a mutant strain producing CF with almost no ability to degrade substrate proteins. Detailed proteomic analysis identified numerous secreted proteases regulated by XprG and PrtT, alone and in combination. Interestingly, proteomics also identified reduced levels of secreted cell wall modifying enzymes (glucanases, chitinases) and allergens following deletion of these genes, suggesting they target additional cellular processes. Surprisingly, despite the major alteration in the secretome of the xprG/prtT null mutant, including two to fivefold reductions in the level of 24 proteases, 18 glucanases, 6 chitinases, and 19 allergens, it retained wild-type virulence in murine systemic and pulmonary models of infection. This study highlights the extreme adaptability of A. fumigatus during infection based on extensive gene redundancy.

Mechanisms of Bacterial (Serratia marcescens) Attachment to, Migration along, and Killing of Fungal Hyphae.

We have found a remarkable capacity for the ubiquitous Gram-negative rod bacterium Serratia marcescens to migrate along and kill the mycelia of zygomycete molds. This migration was restricted to zygomycete molds and several basidiomycete species. No migration was seen on any molds of the phylum Ascomycota. S. marcescens migration did not require fungal viability or surrounding growth medium, as bacteria migrated along aerial hyphae as well.S. marcescens did not exhibit growth tropism toward zygomycete mycelium. Bacterial migration along hyphae proceeded only when the hyphae grew into the bacterial colony. S. marcescens cells initially migrated along the hyphae, forming attached microcolonies that grew and coalesced to generate a biofilm that covered and killed the mycelium. Flagellum-defective strains of S. marcescens were able to migrate along zygomycete hyphae, although they were significantly slower than the wild-type strain and were delayed in fungal killing. Bacterial attachment to the mycelium does not necessitate type 1 fimbrial adhesion, since mutants defective in this adhesin migrated equally well as or faster than the wild-type strain. Killing does not depend on the secretion of S. marcescens chitinases, as mutants in which all three chitinase genes were deleted retained wild-type killing abilities. A better understanding of the mechanisms by which S. marcescens binds to, spreads on, and kills fungal hyphae might serve as an excellent model system for such interactions in general; fungal killing could be employed in agricultural fungal biocontrol.

Imaging metazoan nuclear pore complexes by field emission scanning electron microscopy.

High resolution three-dimensional surface images of nuclear pore complexes (NPCs) can be obtained by field emission scanning electron microscopy. We present a short retrospective view starting from the early roots of microscopy, through the discovery of the cell nucleus and the development of some modern techniques for sample preparation and imaging. Detailed protocols are presented for assembling anchored nuclei in a Xenopus cell-free reconstitution system and for the exposure of the nuclear surface in mammalian cell nuclei. Immunogold labeling of metazoan NPCs and a promising new technique for delicate coating with iridium are also discussed.

High-resolution scanning electron microscopy for the imaging of nuclear pore complexes and Ran-mediated transport.

High-resolution scanning electron microscopy provides three-dimensional surface images of nuclear pore complexes (NPCs) embedded in the nuclear envelope. Here, we describe a method for exposing the nuclear surface in mammalian tissue culture cells for imaging by scanning electron microscopy. Hypotonic treatment is followed by low-speed centrifugation onto polylysine-coated silicon chips, without the use of detergents. This helps to preserve NPCs close to their native morphology, embedded in undamaged nuclear membranes. This method is particularly advantageous for combining high-resolution imaging of NPCs with mammalian genetic systems.

Stress and aging at the nuclear gateway.

The nuclear pore complex (NPC) is a massive molecular machine embedded in the nuclear envelope and controlling traffic into and out of the cell nucleus. Here, we describe some of the outstanding research questions concerning the NPC, its assembly and functions. We also discuss recent findings that link the NPC and its immediate surroundings to the process of cellular aging. Scaffold and barrier nucleoporins are two major types of protein building blocks that make up the NPC. Surprisingly, these two groups of nucleoporins differ dramatically in their turnover rates. Recent work identifies some of the scaffold nucleoporins as the most extremely long-lived proteins in rat brain. Some of the consequences of these findings and new open questions arising from them are discussed. We also consider the evidence for a perturbed permeability barrier in nuclei from old cells and the alteration of nuclear transport pathways under stress conditions. Finally, we describe the connection between premature aging syndromes and the nuclear lamina, a filamentous protein network which underlies the nuclear envelope.

Inner/Outer nuclear membrane fusion in nuclear pore assembly: biochemical demonstration and molecular analysis.

Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.

Transportin mediates nuclear entry of DNA in vertebrate systems.

Delivery of DNA to the cell nucleus is an essential step in many types of viral infection, transfection, gene transfer by the plant pathogen Agrobacterium tumefaciens and in strategies for gene therapy. Thus, the mechanism by which DNA crosses the nuclear pore complex (NPC) is of great interest. Using nuclei reconstituted in vitro in Xenopus egg extracts, we previously studied DNA passage through the nuclear pores using a single-molecule approach based on optical tweezers. Fluorescently labeled DNA molecules were also seen to accumulate within nuclei. Here we find that this import of DNA relies on a soluble protein receptor of the importin family. To identify this receptor, we used different pathway-specific cargoes in competition studies as well as pathway-specific dominant negative inhibitors derived from the nucleoporin Nup153. We found that inhibition of the receptor transportin suppresses DNA import. In contrast, inhibition of importin beta has little effect on the nuclear accumulation of DNA. The dependence on transportin was fully confirmed in assays using permeabilized HeLa cells and a mammalian cell extract. We conclude that the nuclear import of DNA observed in these different vertebrate systems is largely mediated by the receptor transportin. We further report that histones, a known cargo of transportin, can act as an adaptor for the binding of transportin to DNA.

Transportin regulates major mitotic assembly events: from spindle to nuclear pore assembly.

Mitosis in higher eukaryotes is marked by the sequential assembly of two massive structures: the mitotic spindle and the nucleus. Nuclear assembly itself requires the precise formation of both nuclear membranes and nuclear pore complexes. Previously, importin alpha/beta and RanGTP were shown to act as dueling regulators to ensure that these assembly processes occur only in the vicinity of the mitotic chromosomes. We now find that the distantly related karyopherin, transportin, negatively regulates nuclear envelope fusion and nuclear pore assembly in Xenopus egg extracts. We show that transportin-and importin beta-initiate their regulation as early as the first known step of nuclear pore assembly: recruitment of the critical pore-targeting nucleoporin ELYS/MEL-28 to chromatin. Indeed, each karyopherin can interact directly with ELYS. We further define the nucleoporin subunit targets for transportin and importin beta and find them to be largely the same: ELYS, the Nup107/160 complex, Nup53, and the FG nucleoporins. Equally importantly, we find that transportin negatively regulates mitotic spindle assembly. These negative regulatory events are counteracted by RanGTP. We conclude that the interplay of the two negative regulators, transportin and importin beta, along with the positive regulator RanGTP, allows precise choreography of multiple cell cycle assembly events.