Biology subjects

Kutay, U.

Publications and source records attributed to Kutay, U..

13 recordsLinked to original sources

bioRxiv2026

RNF25 is activated as a response to amino acid starvation-induced ribosome collisions in competition with GCN2

Surveillance of mRNA translation relies on a suite of ribosome-associated quality control pathways. Recently, a novel pathway induced by trapping of translation factors in the ribosomal A-site has been described, involving ubiquitination of RPS27A/eS31 by the human E3 ubiquitin ligase RNF25. Here, we show that not only ribosome-stalling by low doses of translation inhibitors, but also amino acid starvation induces RPS27A/eS31 ubiquitination, identifying a natural trigger of RNF25 activation. Even under optimal growth conditions, RNF25 senses and resolves transient ribosome stalls. RPS27A/eS31 ubiquitination specifically depends on the ribosome collision sensor GCN1, a known cofactor of GCN2 involved in the integrated stress response. RNF25 and GCN2 both possess a GCN1-binding RWD domain, indicating a competitive relationship, with GCN2 acting as a negative regulator of RNF25 activation. Although both RNF25 and GCN2 respond to amino acid starvation, RPS27A/eS31 ubiquitination by RNF25 is not required for GCN2 activation, showing that both act in independent pathways. We propose that the RNF25 pathway acts as a first line of defence to resolve ribosome collisions, outcompeted by GCN2 binding to GCN1 under acute stress.

molecular biology↗

bioRxiv2025

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↗

bioRxiv2025

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↗

bioRxiv2025

A conserved mechanism of membrane fusion in nuclear pore complex assembly

The nuclear pore complex (NPC) forms a large channel that spans the double lipid bilayer of the nuclear envelope and is the central gateway for macromolecular transport between the nucleus and cytoplasm in eukaryotes. NPC biogenesis requires the coordinated assembly of over 500 proteins culminating in the fusion of the inner and outer nuclear membranes. The molecular mechanism of this membrane fusion step that occurs in all eukaryotes is unknown. Here, we elucidate the mechanism by which two paralogous transmembrane proteins, Brl1 and Brr6, mediate membrane fusion in S. cerevisiae. Both proteins form multimeric, ring-shaped complexes with membrane remodeling activity. Brl1 is enriched at NPC assembly sites via a nuclear export sequence and then interacts with Brr6 across the nuclear envelope lumen through conserved hydrophobic loops. Disrupting this interaction blocks fusion and halts NPC assembly. Molecular dynamics simulations suggest that the Brl1-Brr6 complex drives membrane fusion by forming a channel across bilayers that enables lipid exchange. Phylogenetic analyses reveal that Brl1/Brr6 homologues are broadly distributed across eukaryotes, and functional experiments in human cells and D. melanogaster establish CLCC1 as an NPC fusogen in metazoans. Together, our results uncover a novel, conserved mechanism for membrane fusion in eukaryotes.

cell biology↗

bioRxiv2025

Quantifying Intracellular Mechanosensitive Response upon Spatially Defined Mechano-Chemical Triggering

The mechanotransduction process relies on the interaction of mechanical and biochemical cues, transmitting cellular forces to intracellular organelles to activate biochemical pathways and elicit responses. This involves mechanoresponsive components like actin filaments, microtubules (MTs), and the lamin meshwork. Fluidic force microscopy (FluidFM), a force-controlled micropipette allows for the manipulation of intact cells mechanically and chemically, providing a novel approach to study mechanotransmission in cells in situ. FluidFM combined with fluorescence lifetime imaging microscopy (FLIM), enables high-resolution mapping of intracellular tension dynamics. Here, we used cells with varying nuclear lamina compositions to explore the laminas role in initiating mechanoresponse to external cues. We found that A-type and B-type lamins trigger nuclear mechanoresponse distinctly, with A-type lamins contributing to nuclear elasticity, whereas B-type lamins influence viscous response. Moreover, MTs underwent mechanical adaptation and assisted in releasing the tension in lamin A/C knockout (KO) cells, contrasting with healthy cells where MTs aid to preserve the tension locally rather than transferring it. This research provides insights into the dynamic mechanoresponse of cellular components and supports targeted therapies for mechanical stress-related diseases.

biophysics↗

bioRxiv2024

LBR and LAP2 mediate heterochromatin tethering to the nuclear periphery to preserve genome homeostasis

In most eukaryotic cells, euchromatin is localized in the nuclear interior, whereas heterochromatin is enriched at the nuclear envelope (NE). This conventional chromatin organization is established by heterochromatin tethering to the NE, however its importance for cellular homeostasis is largely unexplored. Peripheral heterochromatin localization relies on redundant NE-tethering systems. One tether is constituted by the lamin B receptor (LBR) in mammals, but the enigmatic nature of the other tethers has hampered functional analyses. Here we demonstrate that the downregulation of abundant, ubiquitous NE proteins can induce the global detachment of heterochromatin from the NE. Among these factors, we identify LBR and LAP2 as major players in bulk heterochromatin attachment to the NE in pluripotent and differentiated mammalian cells. Their loss leads to repositioning of heterochromatin to the nuclear interior, changes in chromatin accessibility, deregulation of gene expression including activation of antiviral innate immunity, and defects in cell fate determination.

cell biology↗

bioRxiv2023

A nuclear architecture screen in Drosophila identifies Stonewall as a link between chromatin position at the nuclear periphery and germline stem cell fate

The association of genomic loci to the nuclear periphery is proposed to facilitate cell-type specific gene repression and influence cell fate decisions. However, the interplay between gene position and expression remains incompletely understood, in part because the proteins that position genomic loci at the nuclear periphery remain unidentified. Here, we used an Oligopaint-based HiDRO screen targeting [~]1000 genes to discover novel regulators of nuclear architecture in Drosophila cells. We identified the heterochromatin-associated protein, Stonewall (Stwl), as a factor promoting perinuclear chromatin positioning. In female germline stem cells (GSCs), Stwl binds and positions chromatin loci, including GSC differentiation genes, at the nuclear periphery. Strikingly, Stwl-dependent perinuclear positioning is associated with transcriptional repression, highlighting a likely mechanism for Stwls known role in GSC maintenance and ovary homeostasis. Thus, our study identifies perinuclear anchors in Drosophila and demonstrates the importance of gene repression at the nuclear periphery for cell fate.

cell biology↗

bioRxiv2021

Processing of the Ribosomal Ubiquitin-Like Fusion Protein FUBI-eS30/FAU is Required for 40S Maturation and Depends on USP36

In humans and other holozoan organisms, the ribosomal protein eS30 is synthesized as a fusion protein with the ubiquitin-like protein FUBI. However, FUBI is not part of the mature 40S ribosomal subunit and cleaved off by an as-of-yet unidentified protease. How FUBI-eS30 processing is coordinated with 40S subunit maturation is unknown. To study the mechanism and importance of FUBI-eS30 processing, we expressed non-cleavable mutants in human cells, which affected late steps of cytoplasmic 40S maturation, including the maturation of 18S rRNA and recycling of late-acting ribosome biogenesis factors. Differential affinity purification of wild-type and non-cleavable FUBI-eS30 mutants identified the deubiquitinase USP36 as a candidate FUBI-eS30 processing enzyme. Depletion of USP36 by RNAi or CRISPRi indeed impaired FUBI-eS30 processing and moreover, purified USP36 cut FUBI-eS30 in vitro. Together, these data demonstrate the functional importance of FUBI-eS30 cleavage and identify USP36 as a novel protease involved in this process.

cell biology↗

bioRxiv2021

The nucleoporin Nup50 activates the Ran guanyl-nucleotide exchange factor RCC1 to promote mitotic NPC assembly

During mitotic exit, thousands of nuclear pore complexes (NPCs) assemble concomitant with the nuclear envelope to build a transport-competent nucleus. We show here that Nup50 plays a crucial role in NPC assembly that is independent of its well-established function in nuclear transport. RNAi-mediated downregulation in cells or immunodepletion of the protein in Xenopus egg extracts interferes with NPC assembly. We define a conserved central region of 46 residues in Nup50 that is crucial for Nup153 and MEL28/ELYS binding, and NPC interaction. Surprisingly, neither NPC interaction nor binding of Nup50 to importin , {beta}, the GTPase Ran or chromatin is crucial for its function in the assembly process. Instead, we discovered that an N-terminal fragment of Nup50 can stimulate the Ran guanine exchange factor RCC1 and NPC assembly, indicating that Nup50 acts via the Ran system in mitotic NPC reformation. In support of this conclusion, Nup50 mutants defective in RCC1 binding and stimulation cannot replace the wild type protein in in vitro NPC assembly assays.

cell biology↗

bioRxiv2021

Nuclear export of the pre-60S ribosomal subunit through single nuclear pores observed in real time

Ribosomal subunit biogenesis within mammalian cells initiates in the nucleolus with the assembly of a 90S precursor particle, which is subsequently split into the pre-40S and pre-60S subunits. During further processing steps, pre-ribosomal subunits are loaded with export receptors, which enables their passage through the pore complexes (NPCs) into the cytoplasm. Here export factors are released and both subunits can form a mature ribosome. Ribosomal biogenesis has been studied in great detail by biochemical, genetic and electron microscopic approaches, however, until now live cell data on the in vivo kinetics are still missing. We analysed export kinetics of the large ribosomal subunit ("pre-60S particle") through single NPCs in living human cells. To assess the in vivo dynamics of this process, we established a stable cell line co-expressing Halo-tagged eIF6 and GFP-fused NTF2 to simultaneously label ribosomal 60S subunits (eIF6) and NPCs (NTF2). By combining single molecule tracking and super resolution confocal microscopy in a highly customized microscopic setup, we visualized the dynamics of single pre-60S particles during the interaction with and export through single NPCs. In this way we obtained unprecedented insights into this key cellular process. Our results revealed that for export events, maximum particle accumulation is found in the centre of the pore, while unsuccessful export terminates within the nuclear basket. The export process takes place with a single rate limiting step and an export dwell time of ~24 milliseconds. Only about 1/3 of attempted export events were successful. Given the molecular mass of the pre-60S particles our results show that the mass flux through a single NPC can reach up to ~125 MDa{middle dot}s-1 in vivo.

cell biology↗

bioRxiv2021

Nuclear stabilisation of p53 requires a functional nucleolar surveillance pathway

The nucleolar surveillance pathway (NSP) monitors nucleolar fidelity and responds to nucleolar stresses (i.e., inactivation of ribosome biogenesis) by mediating the inhibitory binding of ribosomal proteins (RPs) to mouse double minute 2 homolog (MDM2), a nuclear-localised E3 ubiquitin ligase, which results in p53 accumulation. Inappropriate activation of the NSP has been implicated in the pathogenesis of collection of human diseases termed "ribosomopathies", while drugs that selectively activate the NSP are now in trials for cancer. Despite the clinical significance, the precise molecular mechanism(s) regulating the NSP remain poorly understood. Using genome-wide loss of function screens, we demonstrate the ribosome biogenesis (RiBi) axis as the most potent class of genes whose disruption stabilises p53. Furthermore, we identified a novel suite of genes critical for the NSP, including a novel mammalian protein implicated in 5S ribonucleoprotein particle (5S-RNP) biogenesis, HEATR3. By selectively disabling the NSP, we unexpectedly demonstrate that a functional NSP is required for the ability of all nuclear acting stresses tested, including DNA damage, to robustly induce p53 accumulation. Together, our data demonstrates that the NSP has evolved as the dominant central integrator of stresses that regulate nuclear p53 abundance, thus ensuring RiBi is hardwired to cellular proliferative capacity.

cell biology↗

bioRxiv2020

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↗

bioRxiv2020

The final step of 40S ribosomal subunit maturation is controlled by a dual key lock

Preventing premature interaction of preribosomes with the translation apparatus is essential to translation accuracy. Hence, the final maturation step releasing functional 40S ribosomal subunits, namely processing of the 18S ribosomal RNA 3 end, is safeguarded by protein DIM2, which both interacts with the endoribonuclease NOB1 and masks the rRNA cleavage site. To elucidate the control mechanism that unlocks NOB1 activity, we performed cryo-EM analysis of late human pre-40S particles purified using a catalytically-inactive form of ATPase RIO1. These structures, together with in vivo and in vitro functional analyses, support a model in which ATP-loaded RIO1 cooperates with ribosomal protein RPS26/eS26 to displace DIM2 from the 18S rRNA 3 end, thereby triggering final cleavage by NOB1; release of ADP then leads to RIO1 dissociation from the 40S subunit. This dual key lock mechanism requiring RIO1 and RPS26 guarantees the precise timing of pre-40S particle conversion into translation-competent ribosomal subunits.

molecular biology↗