Biology subjects

Lanz, M.

Publications and source records attributed to Lanz, M..

8 recordsLinked to original sources

bioRxiv2026

Synthetic expansion of non-coding DNA reveals physiological constraints on genome size

Genome size varies widely across eukaryotes, largely because of differences in non-coding DNA, but the physiological consequences of this variation remain unclear. To directly test how non-coding DNA abundance influences cellular physiology, we engineered a scalable genome-expansion system in the budding yeast S. cerevisiae that increases genome size while leaving the endogenous genome unchanged. By sequentially fusing yeast artificial chromosomes (YACs) carrying predominantly non-coding human DNA, we generated strains with up to 12.8 Mb of additional DNA, approximately doubling the native genome. Genome expansion reduced growth rate and increased cell size in proportion to the amount of non-coding DNA. Spike-in-normalized RNA-seq and ChIP-seq revealed that the non-coding DNA is pervasively transcribed, with a proportional amount of RNA polymerase II being redistributed from the endogenous genome to the added non-coding sequences. This resulted in a global decrease in the endogenous mRNA concentration. However, ribosome profiling and proteomics experiments revealed that there is little translation of YAC-associated transcripts. Our mathematical model shows that cellular growth rate decreases because non-coding DNA acts as a sink for transcriptional resources to lower the concentration of endogenous mRNA. Thus, our work links genome expansion to proliferative capacity and offers a mechanistic explanation for why the fastest-growing cells, such as yeast and bacteria, carry so little non-coding DNA.

Synthetic Biology↗

bioRxiv2026

Chromatin association promotes UBR5-mediated degradation of Rb

The retinoblastoma protein Rb is a cell cycle inhibitor that plays a central role in regulating the G1/S cell cycle transition. Un-/hypo-phosphorylated Rb suppresses E2F transcription activity by binding to E2F/DP dimers and recruiting chromatin remodelers to prevent cells from entering S phase. For cells to progress through the G1/S transition, Rb is inactivated by two mechanisms: the "classic" pathway of Rb hyperphosphorylation by Cyclin-CDK complexes, and a recently identified "degradation" mechanism driven by the E3 ubiquitin ligase UBR5. These two pathways are interconnected, as only the un-/hypo-phosphorylated Rb can be degraded, and the hyper-phosphorylated Rb is stabilized to promote its reaccumulation in preparation for the next cell division cycle. However, the molecular basis for how Rb is stabilized upon phosphorylation remains unclear. In this study, we found that UBR5 preferentially targets chromatin-associated proteins for degradation. Since Rbs chromatin association is modulated by its phosphorylation, we hypothesized that phosphorylation may affect Rb stability by altering its chromatin association. To test this, we constructed a series of un-phosphorylatable Rb variants with graded reductions in chromatin association. Consistent with our hypothesis, we observed a strong correlation between an Rb variants chromatin association and its half-life. Fusing these Rb variants to histone H1 increased chromatin association to similar levels and equalized their protein half-lives. Taken together, these findings show how phosphorylation stabilizes Rb by promoting its dissociation from chromatin. This provides a striking example for how sub-organellar protein localization may be used to regulate stability.

molecular biology↗

bioRxiv2026

Identification and inhibition of the Cyclin D Rb-docking interface that drives cell division

The animal cell division cycle is initiated by the cyclin-dependent kinases CDK4 and CDK6 in complex with D-type cyclins. Cyclin D-CDK4/6 complex formation is promoted by the assembly factors p21 and p27, which bind both subunits. p27 binds the hydrophobic patch on cyclin D that is similar to the patch used by other cell cycle cyclins to dock their substrates. This raised the question as to how cyclin D could find its substrates if its hydrophobic patch were already occupied? Here, we show that D-type cyclins use their A2 helix to dock the retinoblastoma protein Rb, a key substrate regulating cell cycle progression. The specific interface of cyclin Ds A2 helix is unique among cyclins and its mutation slows proliferation. Taken together, our work identifies a cyclin D-substrate docking mechanism that can be targeted by novel cancer therapeutics.

biochemistry↗

bioRxiv2024

Intracellular diffusion in the cytoplasm increases with cell size in fission yeast

Diffusion in the cytoplasm can greatly impact cellular processes, yet regulation of macromolecular diffusion remains poorly understood. There is increasing evidence that cell size affects the density and macromolecular composition of the cytoplasm. Here, we studied whether cell size affects diffusion at the scale of macromolecules tens of microns in diameter. We analyzed the diffusive motions of intracellular genetically-encoded multimeric 40 nm nanoparticles (cytGEMs) in the cytoplasm of the fission yeast Schizosaccharomyces pombe. Using cell size mutants, we showed that cytGEMs diffusion coefficients decreased in smaller cells and increased in larger cells. This increase in diffusion in large cells may be due to a decrease in the DNA-to-Cytoplasm ratio, as diffusion was not affected in large multinucleate cytokinesis mutants. In investigating the underlying causes of altered cytGEMs diffusion, we found that the proteomes of large and small cells exhibited size-specific changes, including the sub-scaling of ribosomal proteins in large cells. Comparison with a similar dataset from human cells revealed that features of size-dependent proteome remodeling were conserved. These studies demonstrate that cell size is an important parameter in determining the biophysical properties and the composition of the cytoplasm.

cell biology↗

bioRxiv2023

Stringent Control Over Cytoplasmic and Membrane Densities Defines Regulation of Cell Geometry in Escherichia coli

Microbes exhibit precise control over their composition and geometry in order to adapt and grow in diverse environments. However, the mechanisms that orchestrate this simultaneous regulation, and how they are causally linked, remains poorly understood. In this work, we derive and experimentally test a biophysical model of cell size regulation in Escherichia coli which relates the cellular surface-to-volume ratio to the total macromolecular composition and partitioning of the proteome between cellular compartments. Central to this model is the observation that the macromolecular density of the cytoplasm and the protein density within the cell membranes are maintained at a constant ratio across growth conditions. Using quantitative mass spectrometry, single-cell microscopy, and biochemical assays, we show this model quantitatively predicts a non-linear relationship between the surface-to-volume ratio, proteome localization, and the total ribosome content of the cell. This model holds under perturbations of intracellular ppGpp concentrations-thereby changing the ribosomal content-demonstrating that cellular geometry is not strictly determined by the cellular growth rate. These findings provide a biophysical link between the coregulation of proteome organization and cellular geometry, offering a quantitative framework for understanding bacterial size regulation across conditions.

microbiology↗

bioRxiv2021

Trimethylamine N-Oxide (TMAO) links peripheral insulin resistance and cognitive deficiencies in a senescence accelerated mouse model

It has been established that ageing is the major risk factor for cognitive deficiency or neurodegenerative diseases such as Alzheimers disease (AD) and it is becoming increasingly evident that insulin resistance is another factor. Biological plausibility for a link between insulin resistance and dementia is relevant for understanding disease etiology, and to form bases for prevention efforts to decrease disease burden. The dysfunction of the insulin signaling system and glucose metabolism has been proposed to be responsible for brain aging. Normal insulin signaling in the brain is required to mediate growth, metabolic functions, and the survival of neurons and glia. Insulin receptors are densely expressed in the olfactory bulb, the cerebral cortex and the hippocampus and regulate neurotransmitter release and receptor recruitment. In normal elderly individuals, reduced glucose tolerance and decreased insulin levels in the aged brain are typically observed. Furthermore, insulin signaling is aberrantly activated in the AD brain, leading to non-responsive insulin receptor signaling. The senescence accelerated mouse (SAMP8) mouse was one of the accelerated senescence strains that spontaneously developed from breeding pairs of the AKR/J series. The SAMP8 mouse develops early learning and memory deficits (between 6 and 8 months) together with other characteristics similar to those seen in Alzheimers disease. The present project proposes the investigation of the missing link between aging, insulin resistance and dementia. Peripheral but not central insulin resistance was found in SAMP8 mice accompanied by cognitive deficiencies. Furthermore, a marked peripheral inflammatory state (i.e. significantly higher adipose tissue TNF- and IL6 levels) were observed in SAMP8 mice, followed by neuroinflammation that could be due to a higher cytokine leaking into the brain across a aging-disrupted BBB. Moreover, aging-induced gut dysbiosis produces higher TMAO that could also contribute to the peripheral and central inflammatory tone as well as to the cognitive deficiencies observed in SAMP8 mice. All those alterations were reversed by DMB, a treatment inhibits the transformation of choline, carnitine and crotonobetaine, decreaseing TMAO levels. The ever-increasing incidence of neurodegenerative diseases not only limits the life quality of the affected individuals and their families but also poses an enormous demand on the societies. Thus, it is instrumental to pursue novel promising approaches to prevent and treat it at the highest possible speed to rapidly translate them to clinical practice. From this point of view, data obtained from this project will be instrumental to validate the principle approach of microbial dysbiosis and increased TMAO secretion as a key link between aging, insulin resistance and dementia. Collectively, the proposed experiments ideally integrate the aim to promote a novel approach to improve the lives of those suffering from cognitive disturbances.

animal behavior and cognition↗

bioRxiv2021

Evasion of Toll-like Receptor Recognition by Escherichia coli is mediated via Population Level Regulation of Flagellin Production

Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract infections. Analysis of the innate immune response in immortalised urothelial cells suggests that the bacterial flagellar subunit, flagellin, is key in inducing host defences. A panel of 40 clinical uro-associated Escherichia coli isolates recovered from either asymptomatic bacteruria (ASB), cystitis or pyelonephritis patients, were characterised for motility and their ability to induce an innate response in urothelial cells stably transfected with a NF{kappa}B luciferase reporter. Twenty-four isolates (60%) were identified as motile with strains recovered from cystitis patients exhibiting a bipolar motility distribution pattern (P < 0.005) and associated with a 2-5 fold increase in NF{kappa}B signalling. Although two isolates were associated with swarm sizes of >7 cm and NF{kappa}B activities of >30 fold (P = 0.029), data overall suggested bacterial motility and the NF{kappa}B signalling response were not directly correlated. To explore whether the signalling response reflected antigenic variation flagellin was purified from 11 different isolates and the urothelial cell challenges repeated. Purified flagellin filaments generated comparable (30.4{+/-}1.8 to 46.1{+/-}2.5 fold, P = NS) NF{kappa}B signalling responses, irrespective of either the source of the isolate or H-serotype. These data argued against any variability between isolates being related to flagellin itself. To determine the roles, if any, of flagellar abundance in inducing these responses flagellar hook numbers of a range of cystitis and ABU isolates were quantified using a plasmid encoded flagellar hook gene flgEA240C. Foci data suggested isolates were averaging between 1 and 2 flagella per cell, while only 10 to 60% each isolates population exhibited foci. These data suggested selective pressures exist in the urinary tract that allow uro-associated E. coli strains to maintain motility exploiting population heterogeneity to prevent host TLR5 recognition.

microbiology↗

bioRxiv2019

Deubiquitination of phosphoribosyl-ubiquitin conjugates by PDE domain-containing Legionella effectors

Posttranslational protein modification by ubiquitin (Ub) is a central eukaryotic mechanism that regulates a plethora of physiological processes. Recent studies unveiled an unconventional type of ubiquitination mediated by the SidE family of Legionella pneumophila effectors, such as SdeA, that catalyzes the conjugation of Ub to a serine residue of target proteins via a phosphoribosyl linker (hence named PR-ubiquitination). Comparable to the deubiquitinases (DUBs) in the canonical ubiquitination pathway, here we show that two Legionella effectors, named DupA (deubiquitinase for PR-ubiquitination) and DupB, reverse PR-ubiquitination by specific removal of phosphoribosyl-Ub (PR-Ub) from substrates. Both DupA and DupB are fully capable of rescuing the Golgi fragmentation phenotype caused by exogenous expression of SdeA in mammalian cells. We further show that deletion of these two genes results in significant accumulation of PR-ubiquitinated species in host cells infected with Legionella. In addition, we have identified a list of specific PR-ubiquitinated host targets and show that DupA and DupB play a role in modulating the association of PR-ubiquitinated host targets with Legionella containing vacuoles (LCV). Together, our data establish a complete PR-ubiquitination and deubiquitination cycle and demonstrate the intricate control that Legionella has over this unusual Ub-dependent posttranslational modification.\n\nStatement of significanceUbiquitination is a vital posttranslational modification in eukaryotes. A variety of microbial pathogens exploit this pathway during their infection. Legionella pneumophila, the causative bacterial pathogen of Legionnaires disease, has been show to hijack host ubiquitination pathway via a large number of effectors. Recent studies revealed a family of effectors catalyzing a novel type of Ub-dependent posttranslational modification, namely PR-ubiquitination. Here we report two new players, DupA and DupB, involved in this unconventional pathway. We found that DupA and DupB function as PR-Ub specific DUBs and play a role in regulating the PR-ubiquitination levels of host targets. Our results not only provide an expanding view of the PR-ubiquitination pathway, but may also facilitate the future identification of PR-ubiquitination pathways in eukaryotes.

biochemistry↗