Search bioRxiv⌕ Search

Biology subjects

uit de Bos, J.

Publications and source records attributed to uit de Bos, J..

6 recordsLinked to original sources

Dystonia-associated Torsins sustain CLCC1 function to promote membrane fusion of the nuclear envelope for NPC biogenesis

DYT1 early-onset dystonia is a severe, incurable disorder of the central nervous system caused by mutations in the gene encoding Torsin1A (Tor1A, DYT1). Torsins are ER-resident AAA+-ATPases implicated in lipid metabolism, nuclear pore complex (NPC) biogenesis, and lipoprotein secretion, yet their molecular function that underlies the disease pathology has remained incompletely understood. Here, we have utilized Drosophila melanogaster and human somatic cells as experimental models to shed light on their mode-of-action. Fly germ cells lacking dTorsin are arrested in development and display defects in the final steps of NPC biogenesis due to a failure in fusion of the inner and outer nuclear membranes. Using proximity labelling of Torsin1A in human cells, we identify the conserved membrane protein chloride channel CLIC-like protein 1 (CLCC1) as a novel Torsin binding partner. Absence of human CLCC1 or its Drosophila homolog dClcc1 phenocopied the membrane fusion defects at NPC assembly sites observed upon Torsin deletion. Furthermore, CLCC1 is enriched at arrested fusion sites, suggesting it to be a candidate for the elusive NE membrane fusogen. Importantly, CLCC1/dClcc1 overexpression is sufficient to rescue NPC biogenesis and developmental defects associated with Torsin-loss-of-function. Taken together, our data suggest that Torsin-regulated CLCC1 activity drives membrane fusion during NPC biogenesis and reveal that modulating CLCC1 expression is a promising therapeutic prospect for DYT1 dystonia.

cell biology↗

Persistent contacts between Climp63 and microtubules cause mitotic defects and nuclear fragmentation

The endoplasmic reticulum (ER) is the most elaborate endomembrane system in mammalian cells. To generate its characteristic shape, ER-resident proteins directly affect membrane topology, while interactions with the microtubule (MT) network determine ER positioning. The ER undergoes continuous remodeling with the most drastic rearrangements taking place during mitosis. A critical aspect is the reconfiguration of MT-ER interactions that accompanies spindle formation. One abundant ER-MT tether is constituted by the ER sheet-associated membrane protein Climp63, which binds MTs in a phosphorylation-sensitive manner. Here, we demonstrate that expression of a phosphodeficient Climp63 mutant in HeLa cells prevents dissolving of ER-MT contacts in mitosis, resulting in severe mitotic delays. Moreover, during mitotic exit, cells fail to properly enclose all chromosomes into a single nucleus, leading to excessive nuclear fragmentation. The emerging micronuclei assemble NE components with nuclear pore complexes, in contrast to what is known for micronuclei formed from lagging chromosomes. We further show that the N-terminal 28 amino acids are sufficient for the interaction between Climp63 and MTs with phosphorylation of serine S17 by CDK1 being critical for the mitotic release of the ER from MTs. Overall, our results demonstrate that aberrant Climp63 activity severely affects mitosis. One may speculate that mitotic aberrations may contribute to the poor prognosis associated with Climp63 overexpressing cancers.

cell biology↗

Torsin ATPases influence chromatin interaction of the Torsin regulator LAP1

The inner nuclear membrane is functionalized by diverse transmembrane proteins that associate with nuclear lamins and/or chromatin. When cells enter mitosis, membrane-chromatin contacts must be broken to allow for proper chromosome segregation; yet how this occurs remains ill-understood. Unexpectedly, we observed that an imbalance in the levels of the lamina-associated polypeptide 1 (LAP1), an activator of ER-resident Torsin AAA+-ATPases, causes a failure in membrane removal from mitotic chromatin, accompanied by chromosome segregation errors and changes in post-mitotic nuclear morphology. These defects are dependent on a hitherto unknown chromatin-binding region of LAP1 that we have delineated. LAP1-induced NE abnormalities are efficiently suppressed by expression of wild-type but not ATPase-deficient Torsins. Furthermore, a dominant-negative Torsin induces chromosome segregation defects in a LAP1-dependent manner. These results indicate that association of LAP1 with chromatin in the nucleus can be modulated by Torsins in the perinuclear space, shedding new light on the LAP1-Torsin interplay.

cell biology↗

MCART1 is required for mitochondrial NAD transport

The nicotinamide adenine dinucleotide (NAD+/NADH) pair is a cofactor in redox reactions and is particularly critical in mitochondria as it connects substrate oxidation by the tricarboxylic acid (TCA) cycle to ATP generation by the electron transport chain (ETC) and oxidative phosphorylation. While a mitochondrial NAD+ transporter has been identified in yeast, how NAD enters mitochondria in higher eukaryotes is unknown. Here, we mine gene essentiality data from human cell lines to identify MCART1 (SLC25A51) as co-essential with ETC components. MCART1-null cells have large decreases in TCA cycle flux, mitochondrial respiration, ETC complex I activity, and mitochondrial levels of NAD+ and NADH. Isolated mitochondria from cells lacking or overexpressing MCART1 have greatly decreased or increased NAD uptake in vitro, respectively. Moreover, MCART1 and NDT1, a yeast mitochondrial NAD+ transporter, can functionally complement for each other. Thus, we propose that MCART1 is the long sought mitochondrial transporter for NAD in human cells.

cell biology↗

Natural genetic variation in C. elegans reveals genomic loci controlling metabolite levels

Metabolic homeostasis is sustained by complex biological networks responding to nutrient availability. Disruption of this equilibrium involving intricate interactions between genetic and environmental factors can lead to metabolic disorders, including obesity and type 2 diabetes. To identify the genetic factors controlling metabolism, we applied a quantitative genetic strategy using a Caenorhabditis elegans population consisting of 199 recombinant inbred lines (RILs) originally derived from crossing parental strains Bristol N2 and Hawaii CB4856. We focused on the genetic factors that control metabolite levels and measured fatty acid (FA) and amino acid (AA) composition in the 199 RILs using targeted metabolomics. For both FA and AA profiles, we observed large variation in metabolite levels with 32-82% heritability between the RILs. We performed metabolite-metabolite correlation analysis and detected strongly co-correlated metabolite clusters. To identify natural genetic variants responsible for the observed metabolite variations, we performed QTL mapping and detected 36 significant metabolite QTL (mQTL). We focused on the mQTL that displayed high significant linkage and heritability, including an mQTL for the FA C14:1 on chromosome I, and another mQTL for the FA C18:2 on chromosome IV. Using introgression lines (ILs) we were able to narrow down both mQTL to a 1.4 Mbp and a 3.6 Mbp region, respectively. Overall, this systems approach provides us with a powerful platform to study the genetic basis of C. elegans metabolism. It also allows us to investigate additional interventions, such as nutrients and stresses that maintain or disturb the regulatory network controlling metabolic homeostasis, and identify gene-by-environment interactions.

genomics↗

Condensin’s ATPase Machinery Drives and Dampens Mitotic Chromosome Condensation

Chromosome condensation by condensin is essential for faithful chromosome segregation. Metazoans have two complexes, named condensin I and II. Both are thought to act by creating looped structures in DNA, but how they do so is unknown. Condensins SMC subunits together form a composite ATPase with two pseudo-symmetric ATPase sites. We reveal that these sites have opposite functions in the condensation process. One site drives condensation, while the other site rather has a dampening function. Mutation of this dampener site hyperactivates both condensin I and II complexes. We find that hyperactive condensin I efficiently shortens chromosomes in the total absence of condensin II. The two complexes form loops with different lengths, and specifically condensin II is key to the decatenation of sister chromatids and the formation of a straight chromosomal axis.

molecular biology↗