Nuclear envelopathies comprise a heterogeneous group of diseases caused by mutations in genes enc... more 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.
Laryngeal and tracheal morbidity is a common complication of endotracheal tube (ETT)-based airway... more Laryngeal and tracheal morbidity is a common complication of endotracheal tube (ETT)-based airway management, and manifests as local irritation, inflammation, and edema. Systemic corticosteroids are commonly administered to manage these conditions; however, their efficacy is inadequate and limited by potential severe side effects. In the present study, a steroid delivery system for local therapy was developed to generate relatively high local drug concentrations and to improve drug efficacy. ETTs were coated with electrospun poly (lactic-coglycolic acid) (PLGA) nanofibers loaded with mometasone furoate (MF), creating a microscale thick layer. MF exhibited sustained release from coated ETTs over 14 days in vitro. An in vivo efficacy study in rats demonstrated the therapeutic benefit of MF-coated ETTs over bare ETTs, as measured by reduced laryngeal mucosal thickness and submucosal laryngeal edema. The fiber coating remained intact during tube intubation and extubation, demonstrating good adhesion to the tubes even after 24 h in aqueous solution at 37°C. These findings demonstrate the potential of drug-loaded ETTs to revolutionize the standard of care for endotracheal intubation.
The integrase (Int) proteins of coliphages HK022 and λ, are phosphorylated in one or more of thei... more The integrase (Int) proteins of coliphages HK022 and λ, are phosphorylated in one or more of their tyrosine residues. In Int of HK022 the phosphorylated residue(s) belong to its core-binding/catalytic domains. Wzc, a protein tyrosine kinase of Escherichia coli, is not required for Int phosphorylation in vivo, however, it can transphosphorylate the conserved Tyr 342 catalytic residue of Int in vitro. Int purified from cells that overexpress Wzc has a reduced activity in vitro. In vivo, the lysogenization of wild type HK022 as well as of λ is not affected by the overexpression of Wzc. However, the nin5 mutant of λ, which lacks a protein-tyrosine phosphatase gene, shows a significantly reduced lysogenization. It is suggested that phosphorylation of Int by Wzc down regulates the activity of Int.
We report biallelic missense and frameshift pathogenic variants in the gene encoding human nucleo... more 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.
Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membran... more 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.
Mitosis in higher eukaryotes is marked by the sequential assembly of two massive structures: the ... more 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.
Bjog: An International Journal Of Obstetrics And Gynaecology, Aug 24, 2019
Objectives: We wished to resolve the controversy regarding the presence of a microbiota in the pl... more Objectives: We wished to resolve the controversy regarding the presence of a microbiota in the placenta. Design: Classical and molecular microbiological study. Setting: All samples were collected during Cesarean section. Population: Twenty-eight human placentas and 6 murine placentas. Methods: All 28 human placentas were checked for 16S rRNA gene amplification products. Three locations from 4 selected human placentas and 3 "environmental controls" for each placenta were placed in 7 culture media. The 4 selected human placentas were further analyzed using Gram stain, immunohistochemistry for bacteria, electron microscopy, and TaqMan RT-qPCR. Six placentas from 3 SPF mice were cut into 4 pieces each, and further analyzed for 16S rRNA gene amplification. Main Outcome Measures: Microbiological and molecular evidence for bacteria. Results: None of the placental cultures used for the full analysis, or their environmental cultures, were positive for bacterial growth. All the other methods, including electron microscopy, 16S rRNA gene amplification and TaqMan RT-qPCR showed no evidence for bacteria. Immunohistochemistry showed negligible bacterial counts. None of the murine placentas showed evidence for 16S rRNA gene amplification.
Cleft lip and/or cleft palate are a common group of birth defects that further classify into synd... more 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 nonsense 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 β (TGF-β). 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-β 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.
Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of mic... more Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits. Video Link The video component of this article can be found at https://www.jove.com/video/55610/ 4,5. SPBs nucleate two classes of microtubules, spindle microtubules and astral microtubules. Spindle microtubules project into the nucleoplasm and are important for attaching to chromosomes via kinetochore microtubules and for stabilizing the spindle via overlapping interpolar microtubules 6. In contrast, astral microtubules project outwards into the cytosol and are relatively rare compared to the dense network of spindle microtubules. During mitosis, pre-anaphase cells have only 1-2 astral microtubules emanating from either SPB; these
Proceedings of the National Academy of Sciences of the United States of America, Feb 5, 2008
Activating transcription factor 2 (ATF2) regulates transcription in response to stress and growth... more Activating transcription factor 2 (ATF2) regulates transcription in response to stress and growth factor stimuli. Here, we use a mouse model in which ATF2 was selectively deleted in keratinocytes. Crossing the conditionally expressed ATF2 mutant with K14-Cre mice (K14.ATF2 f/f) resulted in selective expression of mutant ATF2 within the basal layer of the epidermis. When subjected to a two-stage skin carcinogenesis protocol [7,12-dimethylbenz[a]anthracene/phorbol 12-tetradecanoate 13-acetate (DMBA/TPA)], K14.ATF2 f/f mice showed significant increases in both the incidence and prevalence of papilloma development compared with the WT ATF2 mice. Consistent with these findings, keratinocytes of K14.ATF2 f/f mice exhibit greater anchorage-independent growth compared with ATF2 WT keratinocytes. Papillomas of K14.ATF2 f/f mice exhibit reduced expression of presenilin1, which is associated with enhanced -catenin and cyclin D1, and reduced Notch1 expression. Significantly, a reduction of nuclear ATF2 and increased -catenin expression were seen in samples of squamous and basal cell carcinoma, as opposed to normal skin. Our data reveal that loss of ATF2 transcriptional activity serves to promote skin tumor formation, thereby indicating a suppressor activity of ATF2 in skin tumor formation. -catenin ͉ keratinocyte ͉ presenilin ͉ cyclin D1 ͉ papilloma
Biochemical and Biophysical Research Communications, Mar 1, 2018
Intracellular membrane fusion depends on the presence of specific mediators, the vesicle (v-) and... more 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.
Aspergillus fumigatus is the most common mold species to cause disease in immunocompromised patie... more 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.
LAP1 is an inner nuclear membrane protein encoded by TOR1AIP1. A homozygous c.961C > T loss of... more 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.
High-resolution scanning electron microscopy provides three-dimensional surface images of nuclear... more 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.
Bacterial conjugation is a highly ubiquitous and promiscuous process, by which a donor bacterium ... more 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.
The nuclear pore complex (NPC) is an essential gateway between the cell nucleus and the cytoplasm... more The nuclear pore complex (NPC) is an essential gateway between the cell nucleus and the cytoplasm. The NPC is formed by multiple copies of ∼30 different proteins called nucleoporins, which can be divided into scaffold, membrane-anchored and barrier components. Thousands of phenylalanine-glycine (FG) repeats, found in barrier nucleoporins, interact to form the selective permeability barrier of the NPC channel. Shuttling nuclear transport receptors are able to interact with these FG repeats and mediate the passage of large macromolecular cargoes through the barrier. Combinations of shuttling receptors, their adaptors and localisation signals in cargo molecules define a wide array of nuclear import and export pathways. Recent research has pointed to some dynamic features in NPC components, as well as a number of nucleoporin-related human diseases which are characterised by highly cell-type-specific phenotypes. Key Concepts The nuclear pore complex (NPC) is a massive and elaborate structure formed by multiple copies of ∼30 different nucleoporins. The selective permeability barrier of the NPC is formed by hydrophobic interactions among FG repeats found in a large group of nucleoporins. Ions and small molecules are able to passively diffuse through the aqueous central channel of the NPC. Macromolecules transported through the NPC must contain specific targeting signals for nuclear import and nuclear export. Targeting signals are recognised by shuttling transport receptors which mediate the passage through the NPC channel by interacting with FG repeats. A small number of inherited diseases have been linked to mutations in human nucleoporins and are characterised by cell-type-specific phenotypes. Keywords: nuclear pore complex; nucleoporins; nuclear envelope; nuclear pore scaffold; nuclear transport; FG repeats; karyopherins
Delivery of DNA to the cell nucleus is an essential step in many types of viral infection, transf... more 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 β 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.
The mechanical response of individual T3SS filaments was measured by pulling on filaments extendi... more The mechanical response of individual T3SS filaments was measured by pulling on filaments extending out from bacterial surfaces and by pressing into detached filaments. The longitudinal and radial elastic moduli yielded an aspect ratio of ∼1 : 220.
Nuclear envelopathies comprise a heterogeneous group of diseases caused by mutations in genes enc... more 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.
Laryngeal and tracheal morbidity is a common complication of endotracheal tube (ETT)-based airway... more Laryngeal and tracheal morbidity is a common complication of endotracheal tube (ETT)-based airway management, and manifests as local irritation, inflammation, and edema. Systemic corticosteroids are commonly administered to manage these conditions; however, their efficacy is inadequate and limited by potential severe side effects. In the present study, a steroid delivery system for local therapy was developed to generate relatively high local drug concentrations and to improve drug efficacy. ETTs were coated with electrospun poly (lactic-coglycolic acid) (PLGA) nanofibers loaded with mometasone furoate (MF), creating a microscale thick layer. MF exhibited sustained release from coated ETTs over 14 days in vitro. An in vivo efficacy study in rats demonstrated the therapeutic benefit of MF-coated ETTs over bare ETTs, as measured by reduced laryngeal mucosal thickness and submucosal laryngeal edema. The fiber coating remained intact during tube intubation and extubation, demonstrating good adhesion to the tubes even after 24 h in aqueous solution at 37°C. These findings demonstrate the potential of drug-loaded ETTs to revolutionize the standard of care for endotracheal intubation.
The integrase (Int) proteins of coliphages HK022 and λ, are phosphorylated in one or more of thei... more The integrase (Int) proteins of coliphages HK022 and λ, are phosphorylated in one or more of their tyrosine residues. In Int of HK022 the phosphorylated residue(s) belong to its core-binding/catalytic domains. Wzc, a protein tyrosine kinase of Escherichia coli, is not required for Int phosphorylation in vivo, however, it can transphosphorylate the conserved Tyr 342 catalytic residue of Int in vitro. Int purified from cells that overexpress Wzc has a reduced activity in vitro. In vivo, the lysogenization of wild type HK022 as well as of λ is not affected by the overexpression of Wzc. However, the nin5 mutant of λ, which lacks a protein-tyrosine phosphatase gene, shows a significantly reduced lysogenization. It is suggested that phosphorylation of Int by Wzc down regulates the activity of Int.
We report biallelic missense and frameshift pathogenic variants in the gene encoding human nucleo... more 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.
Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membran... more 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.
Mitosis in higher eukaryotes is marked by the sequential assembly of two massive structures: the ... more 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.
Bjog: An International Journal Of Obstetrics And Gynaecology, Aug 24, 2019
Objectives: We wished to resolve the controversy regarding the presence of a microbiota in the pl... more Objectives: We wished to resolve the controversy regarding the presence of a microbiota in the placenta. Design: Classical and molecular microbiological study. Setting: All samples were collected during Cesarean section. Population: Twenty-eight human placentas and 6 murine placentas. Methods: All 28 human placentas were checked for 16S rRNA gene amplification products. Three locations from 4 selected human placentas and 3 "environmental controls" for each placenta were placed in 7 culture media. The 4 selected human placentas were further analyzed using Gram stain, immunohistochemistry for bacteria, electron microscopy, and TaqMan RT-qPCR. Six placentas from 3 SPF mice were cut into 4 pieces each, and further analyzed for 16S rRNA gene amplification. Main Outcome Measures: Microbiological and molecular evidence for bacteria. Results: None of the placental cultures used for the full analysis, or their environmental cultures, were positive for bacterial growth. All the other methods, including electron microscopy, 16S rRNA gene amplification and TaqMan RT-qPCR showed no evidence for bacteria. Immunohistochemistry showed negligible bacterial counts. None of the murine placentas showed evidence for 16S rRNA gene amplification.
Cleft lip and/or cleft palate are a common group of birth defects that further classify into synd... more 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 nonsense 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 β (TGF-β). 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-β 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.
Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of mic... more Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits. Video Link The video component of this article can be found at https://www.jove.com/video/55610/ 4,5. SPBs nucleate two classes of microtubules, spindle microtubules and astral microtubules. Spindle microtubules project into the nucleoplasm and are important for attaching to chromosomes via kinetochore microtubules and for stabilizing the spindle via overlapping interpolar microtubules 6. In contrast, astral microtubules project outwards into the cytosol and are relatively rare compared to the dense network of spindle microtubules. During mitosis, pre-anaphase cells have only 1-2 astral microtubules emanating from either SPB; these
Proceedings of the National Academy of Sciences of the United States of America, Feb 5, 2008
Activating transcription factor 2 (ATF2) regulates transcription in response to stress and growth... more Activating transcription factor 2 (ATF2) regulates transcription in response to stress and growth factor stimuli. Here, we use a mouse model in which ATF2 was selectively deleted in keratinocytes. Crossing the conditionally expressed ATF2 mutant with K14-Cre mice (K14.ATF2 f/f) resulted in selective expression of mutant ATF2 within the basal layer of the epidermis. When subjected to a two-stage skin carcinogenesis protocol [7,12-dimethylbenz[a]anthracene/phorbol 12-tetradecanoate 13-acetate (DMBA/TPA)], K14.ATF2 f/f mice showed significant increases in both the incidence and prevalence of papilloma development compared with the WT ATF2 mice. Consistent with these findings, keratinocytes of K14.ATF2 f/f mice exhibit greater anchorage-independent growth compared with ATF2 WT keratinocytes. Papillomas of K14.ATF2 f/f mice exhibit reduced expression of presenilin1, which is associated with enhanced -catenin and cyclin D1, and reduced Notch1 expression. Significantly, a reduction of nuclear ATF2 and increased -catenin expression were seen in samples of squamous and basal cell carcinoma, as opposed to normal skin. Our data reveal that loss of ATF2 transcriptional activity serves to promote skin tumor formation, thereby indicating a suppressor activity of ATF2 in skin tumor formation. -catenin ͉ keratinocyte ͉ presenilin ͉ cyclin D1 ͉ papilloma
Biochemical and Biophysical Research Communications, Mar 1, 2018
Intracellular membrane fusion depends on the presence of specific mediators, the vesicle (v-) and... more 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.
Aspergillus fumigatus is the most common mold species to cause disease in immunocompromised patie... more 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.
LAP1 is an inner nuclear membrane protein encoded by TOR1AIP1. A homozygous c.961C > T loss of... more 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.
High-resolution scanning electron microscopy provides three-dimensional surface images of nuclear... more 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.
Bacterial conjugation is a highly ubiquitous and promiscuous process, by which a donor bacterium ... more 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.
The nuclear pore complex (NPC) is an essential gateway between the cell nucleus and the cytoplasm... more The nuclear pore complex (NPC) is an essential gateway between the cell nucleus and the cytoplasm. The NPC is formed by multiple copies of ∼30 different proteins called nucleoporins, which can be divided into scaffold, membrane-anchored and barrier components. Thousands of phenylalanine-glycine (FG) repeats, found in barrier nucleoporins, interact to form the selective permeability barrier of the NPC channel. Shuttling nuclear transport receptors are able to interact with these FG repeats and mediate the passage of large macromolecular cargoes through the barrier. Combinations of shuttling receptors, their adaptors and localisation signals in cargo molecules define a wide array of nuclear import and export pathways. Recent research has pointed to some dynamic features in NPC components, as well as a number of nucleoporin-related human diseases which are characterised by highly cell-type-specific phenotypes. Key Concepts The nuclear pore complex (NPC) is a massive and elaborate structure formed by multiple copies of ∼30 different nucleoporins. The selective permeability barrier of the NPC is formed by hydrophobic interactions among FG repeats found in a large group of nucleoporins. Ions and small molecules are able to passively diffuse through the aqueous central channel of the NPC. Macromolecules transported through the NPC must contain specific targeting signals for nuclear import and nuclear export. Targeting signals are recognised by shuttling transport receptors which mediate the passage through the NPC channel by interacting with FG repeats. A small number of inherited diseases have been linked to mutations in human nucleoporins and are characterised by cell-type-specific phenotypes. Keywords: nuclear pore complex; nucleoporins; nuclear envelope; nuclear pore scaffold; nuclear transport; FG repeats; karyopherins
Delivery of DNA to the cell nucleus is an essential step in many types of viral infection, transf... more 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 β 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.
The mechanical response of individual T3SS filaments was measured by pulling on filaments extendi... more The mechanical response of individual T3SS filaments was measured by pulling on filaments extending out from bacterial surfaces and by pressing into detached filaments. The longitudinal and radial elastic moduli yielded an aspect ratio of ∼1 : 220.
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Papers by Boris Fichtman