Daniel Z. Bar

Nuclear lamins: key regulators of nuclear structure and activities

•  The lamin molecule ‐  Domain organization of lamins ‐  Lamins are divided to type A and type B ‐  Post‐translational processing of lamin molecules ‐  Lamin molecules in evolution •  The supramolecular assembly of lamins ‐  From lamin monomer to lamin dimer ‐  From dimers to filaments ‐  The roles of the different domains in the assembly of lamins ‐  Laminopathic mutations affect lamin filament assembly ‐  Lamin assembly in vivo •  Lamin‐binding proteins ‐  Lamins, chromatin and epigenesis ‐  Lamin binding to DNA ‐  Lamin binding to chromatin ‐  Lamins affect chromatin organization and epigenesis ‐  Lamins are involved in many nuclear functions ‐  Lamins determine the shape and stiffness of the nucleus ‐  Lamins and DNA replication ‐  Lamins in transcription and splicing •  Lamins and aging ‐  Lamins and laminopathies ‐  Mutations in lamins and their associated proteins causing ‘laminopathies’ ‐  Animal models for laminopathies ‐  Molecular models for laminopathies •  Lamins and stem cells ‐  The Notch pathway ‐  The Wnt/β‐catenin pathway ‐  Other pathways •  Lamins and cancer ‐  Lamins as biomarkers for cancer ‐  Lamins and cancer regulating pathways ‐  Lamins and cancer related aneuploidy •  Lamin and viruses

Cell size and fat content of dietary-restricted Caenorhabditis elegans are regulated by ATX-2, an mTOR repressor

Significance Dietary restriction is a metabolic intervention that extends the lifespan and reduces animal size and fat content. We have used Caenorhabditis elegans to demonstrate that the homolog of human ATXN2, atx-2, is a major regulator of the animal response to dietary restriction. Down-regulation of atx-2 in dietary-restricted animals leads to increased animal size and fat levels, as well as accelerated development. Surprisingly, it does not affect the extended lifespan of dietary-restricted animals. These findings are relevant to mammals because Ataxin-2 knockout mice exhibit adult-onset obesity, owing to an unknown mechanism. atx-2 negatively regulates the mechanistic target of rapamycin pathway via its interaction with a GDP dissociation inhibitor β. Forced activation of this pathway may have therapeutic potential for obesity. Dietary restriction (DR) is a metabolic intervention that extends the lifespan of multiple species, including yeast, flies, nematodes, rodents, and, arguably, rhesus monkeys and humans. Hallmarks of lifelong DR are reductions in body size, fecundity, and fat accumulation, as well as slower development. We have identified atx-2, the Caenorhabditis elegans homolog of the human ATXN2L and ATXN2 genes, as the regulator of these multiple DR phenotypes. Down-regulation of atx-2 increases the body size, cell size, and fat content of dietary-restricted animals and speeds animal development, whereas overexpression of atx-2 is sufficient to reduce the body size and brood size of wild-type animals. atx-2 regulates the mechanistic target of rapamycin (mTOR) pathway, downstream of AMP-activated protein kinase (AMPK) and upstream of ribosomal protein S6 kinase and mTOR complex 1 (TORC1), by its direct association with Rab GDP dissociation inhibitor β, which likely regulates RHEB shuttling between GDP-bound and GTP-bound forms. Taken together, this work identifies a previously unknown mechanism regulating multiple aspects of DR, as well as unknown regulators of the mTOR pathway. They also extend our understanding of diet-dependent growth retardation, and offers a potential mechanism to treat obesity.

BAF-1 mobility is regulated by environmental stresses.

Barrier to autointegration factor (BAF) is an essential mobile protein that binds lamins, LEM-domain proteins, histones, and DNA. Under environmental stress, BAF becomes immobile. This phenomenon is not shared with other chromatin-binding proteins. The ability of BAF mutants to be immobilized by heat shock in gut cells correlated with normal or increased affinity for emerin.

Reversal of age-dependent nuclear morphology by inhibition of prenylation does not affect lifespan in Caenorhabditis elegans

Fibroblasts derived from Hutchinson-Gilford progeria syndrome (HGPS) patients and dermal cells derived from healthy old humans in culture display age-dependent progressive changes in nuclear architecture due to accumulation of farnesylated lamin A. Treating human HGPS cells or mice expressing farnesylated lamin A with farnesyl transferase inhibitors (FTIs) reverses nuclear phenotypes and extends lifespan. Aging adult Caenorhabditis elegans show changes in nuclear architecture resembling those seen in HGPS fibroblasts, as well as a decline in motility, phenotypes which are also inhibited by the FTI gliotoxin. However, it was not clear whether these effects were due to loss of farnesylation or to side effects of this drug. Here, we used a different FTI, manumycin, or downregulated polyprenyl synthetase with RNAi to test the roles of farnesylation in C. elegans aging. Our results show that the age-dependent changes in nuclear morphology depend on farnesylation. We also demonstrate that inhibition of farnesylation does not affect motility or lifespan, suggesting that the effects of blocking protein prenylation on nuclear morphology could be separated from their effects on motility and lifespan. These results provide further understanding of the role of lamin and farnesylation in the normal aging process and in HGPS.

Gliotoxin reverses age-dependent nuclear morphology phenotypes, ameliorates motility, but fails to affect lifespan of adult Caenorhabditis elegans

Specific mutations in human LMNA or loss of ZMPSTE26 activity cause abnormal processing of lamin A and early aging diseases, including Hutchinson Gilford progeria syndrome (HGPS). HGPS fibroblasts in culture undergo age-dependent progressive changes in nuclear architecture. Treating these cells with farnesyl transferase inhibitors (FTIs) reverse these nuclear phenotypes and also extend lifespan of mice HGPS models. Dermal cells derived from healthy old humans also accumulate the abnormally processed lamin A. However, the effect of FTIs on normal aging cells was not tested. Aging adult C. elegans cells show changes in nuclear architecture similar to HGPS fibroblasts and down regulating lamin expression in adult C. elegans reduces their lifespan. Here, we show that nuclei of adult C. elegans, in which lamin is down-regulated, have similar phenotypes to normal aging nuclei, but at an earlier age. We further show that treating adult C. elegans with the FTI gliotoxin reverses nuclear phenotypes and improves motility of aging worms. However, the average lifespan of the gliotoxin-treated animals was similar to that of untreated animals. These results suggest that lamins are involved in the process of normal aging in C. elegans.

A novel somatic mutation achieves partial rescue in a child with Hutchinson-Gilford progeria syndrome

Background Hutchinson-Gilford progeria syndrome (HGPS) is a fatal sporadic autosomal dominant premature ageing disease caused by single base mutations that optimise a cryptic splice site within exon 11 of the LMNA gene. The resultant disease-causing protein, progerin, acts as a dominant negative. Disease severity relies partly on progerin levels. Methods and results We report a novel form of somatic mosaicism, where a child possessed two cell populations with different HGPS disease-producing mutations of the same nucleotide—one producing severe HGPS and one mild HGPS. The proband possessed an intermediate phenotype. The mosaicism was initially discovered when Sanger sequencing showed a c.1968+2T>A mutation in blood DNA and a c.1968+2T>C in DNA from cultured fibroblasts. Deep sequencing of DNA from the probands blood revealed 4.7% c.1968+2T>C mutation, and 41.3% c.1968+2T>A mutation. Conclusions We hypothesise that the germline mutation was c.1968+2T>A, but a rescue event occurred during early development, where the somatic mutation from A to C at 1968+2 provided a selective advantage. This type of mosaicism where a partial phenotypic rescue event results from a second but milder disease-causing mutation in the same nucleotide has not been previously characterised for any disease.

Small GTPases in C. elegans metabolism

ABSTRACT The mechanistic target of rapamycin (mTOR) is an evolutionary conserved protein with a serine/threonine kinase activity that regulates cell growth, proliferation, motility, survival, protein synthesis, autophagy and transcription. It is embedded in 2 large protein complexes: mTORC1 and mTORC2. Regulation of specific mTOR pathway functions depends on multiple GTPases, that act either as regulators of mTOR protein complexes, coupling energy availability with mTORC1 activity, or as downstream effectors of both mTORC1 and mTORC2. In this commentary, we highlight the advantages of studying the mTOR pathway in C. elegans, including the subcellular localization of the signaling pathway components and the animal phenotypes following tissue specific protein over-expression or knockdown. One important regulator that is not limited to the mTOR pathway is RHEB. We discuss in vitro and in vivo data suggesting that RHEB can function as an inhibitor of mTOR when not bound to GTP. RHEB-1 itself is regulated by Rab GDP dissociation inhibitor β, which directly binds to ATX-2. We also highlight the roles of these proteins in dietary restriction-depended reduction in animal size and fat content.

Addendum: Biotinylation by antibody recognition—a method for proximity labeling

In the version of this article initially published, the introduction focused on methods for characterizing the nuclear envelope and did not include a comprehensive overview of proximity-based methods, some of which have similarly utilized antibody-conjugated peroxidase for proximity labeling by biotin deposition. The authors apologize for the omission of the following references: Kotani, N. et al., Proc. Natl Acad. Sci. USA 105, 7405–7409 (2008); Hashimoto, N. et al., Proteomics 12, 3154–3163 (2012); Li, X. W. et al., J. Biol. Chem. 289, 14434–14447 (2014); and Rees, J. S. et al., Curr. Protoc. Protein Sci. 80, 19.27.1–19.27.18 (2015).