Naomi Feinstein

Transmission electron microscope studies of the nuclear envelope in Caenorhabditis elegans embryos

Nuclear membranes and nuclear pore complexes (NPCs) are conserved in both animals and plants. However, the lamina composition and the dimensions of NPCs vary between plants, yeast, and vertebrates. In this study, we established a protocol that preserves the structure of Caenorhabditis elegans embryonic cells for high-resolution studies with thin-section transmission electron microscopy (TEM). We show that the NPCs are bigger in C. elegans embryos than in yeast, with dimensions similar to those in higher eukaryotes. We also localized the C. elegans nuclear envelope proteins Ce-lamin and Ce-emerin by pre-embedding gold labeling immunoelectron microscopy. Both proteins are present at or near the inner nuclear membrane. A fraction of Ce-lamin, but not Ce-emerin, is present in the nuclear interior. Removing the nuclear membranes leaves both Ce-lamin and Ce-emerin associated with the chromatin. Eliminating the single lamin protein caused cell death as visualized by characteristic changes in nuclear architecture including condensation of chromatin, clustering of NPCs, membrane blebbing, and the presence of vesicles inside the nucleus. Taken together, these results show evolutionarily conserved protein localization, interactions, and functions of the C. elegans nuclear envelope.

Barrier to autointegration factor blocks premature cell fusion and maintains adult muscle integrity in C. elegans

Barrier to autointegration factor (BAF) binds double-stranded DNA, selected histones, transcription regulators, lamins, and LAP2–emerin–MAN1 (LEM) domain proteins. During early Caenorhabditis elegans embryogenesis, BAF-1 is required to organize chromatin, capture segregated chromosomes within the nascent nuclear envelope, and assemble lamin and LEM domain proteins in reforming nuclei. In this study, we used C. elegans with a homozygous deletion of the baf-1 gene, which survives embryogenesis and larval stages, to report that BAF-1 regulates maturation and survival of the germline, cell migration, vulva formation, and the timing of seam cell fusion. In the seam cells, BAF-1 represses the expression of the EFF-1 fusogen protein, but fusion still occurs in C. elegans lacking both baf-1 and eff-1. This suggests the existence of an eff-1–independent mechanism for cell fusion. BAF-1 is also required to maintain the integrity of specific body wall muscles in adult animals, directly implicating BAF in the mechanism of human muscular dystrophies (laminopathies) caused by mutations in the BAF-binding proteins emerin and lamin A.

Distinct Regions Specify the Targeting of Otefin to the Nucleoplasmic Side of the Nuclear Envelope

Otefin is a 45-kDa nuclear envelope protein with no apparent homology to other known proteins. It includes a large hydrophilic domain, a single carboxyl-terminal hydrophobic sequence of 17 amino acids, and a high content of serine and threonine residues. Cytological labeling located otefin on the nucleoplasmic side of the nuclear envelope. Chemical extraction of nuclei from Drosophila embryos revealed that otefin is a peripheral protein whose association with the nuclear envelope is stronger than that of lamin. Deletion mutants of otefin were expressed in order to identify regions that direct otefin to the nuclear envelope. These experiments revealed that the hydrophobic sequence at the carboxyl terminus is essential for correct targeting to the nuclear envelope, whereas additional regions in the hydrophilic domain of otefin are required for its efficient targeting and stabilization in the nuclear envelope.

A laminopathic mutation disrupting lamin filament assembly causes disease-like phenotypes in Caenorhabditis elegans

Mutations in human LMNA cause Emery-Dreifuss muscular dystrophy; however, a mechanistic link between the effect of mutations on lamin filament assembly and disease phenotype has not been established. Here we show that changes in lamin filament structure translate into disease phenotypes in Caenorhabditis elegans by altering the character of the nuclear lamina.

Bundle-forming pilus retraction enhances enteropathogenic Escherichia coli infectivity.

Enteropathogenic Escherichia coli (EPEC) and other pathogenic bacteria use dynamic type IV pili to adhere to the host. Here we show that the capacity of the EPEC type IV pili to retract is required for the breakdown of the host epithelial tight-junction barrier, efficient actin-pedestal formation, and translocation of effectors via the type III secretion system.

Ce-emerin and LEM-2: essential roles in Caenorhabditis elegans development, muscle function, and mitosis.

ETOC: Caenorhabditis elegans lacking both Ce-emerin and LEM-2 show that these proteins are essential for development of specific lineages, mitosis in somatic cells, and smooth muscle activity. Reduced life span and smooth muscle activity of LEM-2–null worms predicts human LEM2 gene links to diseases more severe than Emery-Dreifuss muscular dystrophy.

Emerin and LEM2: essential roles in C. elegans development, muscle function and mitosis

ETOC: Caenorhabditis elegans lacking both Ce-emerin and LEM-2 show that these proteins are essential for development of specific lineages, mitosis in somatic cells, and smooth muscle activity. Reduced life span and smooth muscle activity of LEM-2–null worms predicts human LEM2 gene links to diseases more severe than Emery-Dreifuss muscular dystrophy.

Structural and physiological phenotypes of disease-linked lamin mutations in C. elegans.

The nuclear lamina is a major structural element of the nucleus and is predominately composed of the intermediate filament lamin proteins. Missense mutations in the human lamins A/C cause a family of laminopathic diseases, with no known mechanistic link between the position of the mutation and the resulting disease phenotypes. The Caenorhabditis elegans lamin (Ce-lamin) is structurally and functionally homologous to human lamins, and recent advances have allowed detailed structural analysis of Ce-lamin filaments both in vitro and in vivo. Here, we studied the effect of laminopathic mutations on Ce-lamin filament assembly in vitro and the corresponding physiological phenotypes in animals. We focused on three disease-linked mutations, Q159K, T164P, and L535P, which have previously been shown to affect lamin structure and nuclear localization. Mutations prevented the proper assembly of Ce-lamin into filament and/or paracrystalline arrays. Disease-like phenotypes were observed in strains expressing low levels of these mutant lamins, including decreased fertility and motility coincident with muscle lesions. In addition, the Q159K- and T164P-expressing strains showed a reduced lifespan. Thus, different disease-linked mutations in Ce-lamin exhibit major effects in vivo and in vitro. Using C. elegans as a model system, a comprehensive analysis of the effects of specific lamin mutations from the level of in vitro filament assembly to the physiology of the organism will help uncover the mechanistic differences between these different lamin mutations.

Polyglucosan neurotoxicity caused by glycogen branching enzyme deficiency can be reversed by inhibition of glycogen synthase

Uncontrolled elongation of glycogen chains, not adequately balanced by their branching, leads to the formation of an insoluble, presumably neurotoxic, form of glycogen called polyglucosan. To test the suspected pathogenicity of polyglucosans in neurological glycogenoses, we have modeled the typical glycogenosis Adult Polyglucosan Body Disease (APBD) by suppressing glycogen branching enzyme 1 (GBE1, EC 2.4.1.18) expression using lentiviruses harboring short hairpin RNA (shRNA). GBE1 suppression in embryonic cortical neurons led to polyglucosan accumulation and associated apoptosis, which were reversible by rapamycin or starvation treatments. Further analysis revealed that rapamycin and starvation led to phosphorylation and inactivation of glycogen synthase (GS, EC 2.4.1.11), dephosphorylated and activated in the GBE1‐suppressed neurons. These protective effects of rapamycin and starvation were reversed by overexpression of phosphorylation site mutant GS only if its glycogen binding site was intact. While rapamycin and starvation induce autophagy, autophagic maturation was not required for their corrective effects, which prevailed even if autophagic flux was inhibited by vinblastine. Furthermore, polyglucosans were not observed in any compartment along the autophagic pathway. Our data suggest that glycogen branching enzyme repression in glycogenoses can cause pathogenic polyglucosan buildup, which might be corrected by GS inhibition.

Expression and localization of muscarinic receptors in P19-derived neurons

The muscarinic acetylcholine receptors are important in a variety of physiological processes such as induction of secretion from various glands and regulation of pacemaker activity, muscle tone, and neurotransmission. To date, the muscarinic receptor family includes five members (designated m1–m5), of which m1–m4 are abundant in brain and in peripheral tissues, and m5 is found exclusively in brain, and even there at very low levels. The expression of m1–m5 receptor subtypes was studied in neurons derived from the murine embryonal carcinoma cell line P19. These cells serve as a model system for differentiation and maturation of neurons resembling CNS neurons. Our results show that P19 neurons express mainly the m2, m3, and m5 subtypes. Low levels of m1 receptors are also detected and m4 subtype is practically absent. Furthermore, muscarinic receptors in P19 neurons are functional in activating second messenger signaling pathways. The localization of m2 receptors is predominantly presynaptic, whereas the m5 subtype is mainly postsynaptic. Consequently, P19 cells provide a model system for the study of pre- and postsynaptic muscarinic acetylcholine-receptor subtypes in a proper neuronal context. This is particularly valid for the rare m5 receptors.