Biology subjects

Holt, L. J.

Publications and source records attributed to Holt, L. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Generative design of functional disordered regions in a developmental transcription factor

Rapid advances in protein structure design have not translated to intrinsically disordered regions (IDRs). This is largely due to our incomplete understanding of how IDR sequence encodes function. Here, we use stem cell differentiation as a model to map functional sequence features in the IDR of transcription factor RUNX2. We show that RUNX2 requires its C-terminal IDR to bind chromatin and regulate gene expression during osteogenesis. Surprisingly, linear IDR sequence is dispensable, and instead features of the conformational ensemble predict function. We develop an algorithm for rational IDR design based on biophysical principles, and use it to generate functional IDRs de novo. Together, these results illustrate the central role for TF IDRs in chromatin binding and extend generative protein design beyond the folded proteome.

synthetic biology↗

bioRxiv2026

ZATELLITE: a toolkit to visualize and manipulate human centromeres in live cells using synthetic zinc fingers

Changes in the number of chromosomes or their spatial organization within the nucleus have critical consequences for cell fate. Yet capturing the karyotype or three-dimensional architecture of chromatin in living cells remains limited by the difficulty in labeling endogenous loci non-invasively. The most widely used tools rely on dCas9, a bulky protein whose persistent DNA binding interferes with DNA and RNA metabolism, causes DNA damage, and is hard to multiplex. We developed ZATELLITE, an AI-enabled tool to target endogenous repetitive sequences with fluorescently-tagged synthetic zinc fingers. ZATELLITE probes have key advantages over dCas9: they are smaller, easier to multiplex and, critically, they do not cause DNA damage or chromosomal abnormalities, even after long-term labeling. We generated a collection of ZATELLITE probes to label the centromeres of nearly all human chromosomes, enabling the capture of genome organization and karyotype alterations in real time in living cells. Finally, we show that ZATELLITE can be used for simultaneous labeling and epigenetic editing of centromeres. Thus, ZATELLITE is a non-toxic, versatile tool for genome visualization and manipulation.

cell biology↗

bioRxiv2026

Nascent protein retention at polysomes reduces kinetic barriers to self-assembly

Living proteomes are necessarily far from equilibrium. It is paradoxical, then, that reducing the translation of new proteins -- which should promote equilibration -- instead prolongs life. We investigated the impact of translational flux to nucleation barriers that preserve the solubility of proteins destined to form amyloids or other assemblies. By manipulating translation initiation rates directly or indirectly, across yeast and human cells, and across a variety of supersaturable proteins, we find that accelerating translation initiation broadly accelerates nucleation irrespective of their global concentrations. We showed that this effect was confined to polysomes and was enhanced by N-terminal placement or other features that retained the nascent aggregating domain at polysomes. Finally, we show that intrinsically disordered regions with high tendencies to self-associate are specifically positioned to do so co-translationally, providing evidence that cotranslational nucleation has shaped proteome evolution.

cell biology↗

bioRxiv2025

Transcriptional consequences of herpes simplex virus 1 ICP4 inducible expression in uninfected cells

Successful viral infections reflect the balanced outcome of a tightly regulated program of viral gene expression and manipulation of the host cell environment to favor production of new infectious particles. The productive (lytic) replication cycle of herpes simplex virus 1 (HSV-1) is dependent on the essential transcription factor ICP4 encoded by one of five immediate-early genes. At different steps in the HSV-1 temporal cascade, ICP4 either positively or negatively regulates transcription of immediate-early, early, and late HSV-1 genes, largely through sequence-specific binding to cis-acting regulatory elements. In contrast, the direct regulation of host transcription by ICP4 is less well understood. In this study we exogenously expressed doxycycline-inducible wild-type and mutant ICP4 proteins in uninfected primary human fibroblasts and performed RNA-Seq to identify ICP4-driven changes to the host transcriptome. Cross-referencing our findings to a published dataset of ICP4-dependent changes to the host transcriptome in HSV-1 infected cells provided validation for a subset of differentially-expressed genes regulated by ICP4. Furthermore, disrupting the ICP4 DNA-binding domain was sufficient to alter the cellular gene transcriptional program responsive to ICP4. This indicates that the DNA-binding domain of ICP4, which is required for site-specific DNA binding to the virus genome, may also regulate binding to the host genome. Together, these data provide a comprehensive transcriptomic analysis of how wild-type and mutant ICP4 expression impact cellular gene expression in uninfected cells. IMPORTANCEHerpes simplex virus 1 (HSV-1) is a widespread human pathogen responsible for a variety of mild to severe disease states. The viral lifecycle is driven by the essential viral transcription factor ICP4, which ensures the coordinated expression of most viral genes. Expression of ICP4-regulated genes and viral lifecycle progression impacts host gene expression; therefore, determining the specific impact of ICP4 on host gene expression has been challenging. Here, we provide a more direct analysis, examining changes in the transcriptome of primary fibroblasts when ICP4 is expressed alone. Differentially expressed genes were identified and cross-referenced to published data from infections with wild-type and ICP4-null viruses. We show that mutations in ICP4 that disrupt interactions with the host transcriptional machinery or interfere with site-specific DNA binding further modified this ICP4-driven remodeling of the host transcription. These data provide a comprehensive transcriptomic analysis of ICP4-driven dysregulation of host gene expression in uninfected cells.

microbiology↗

bioRxiv2025

Polarization Increases Nuclear Stiffness in Macrophages Despite Reduction in Lamin A/C Levels

Macrophages are innate immune cells contributing to tissue homeostasis and various pathologies. Signals from their environment can lead macrophages to adapt distinct functional phenotypes, a process called polarization. Because macrophages have been previously shown to degrade the nuclear envelope proteins lamin A/C upon pro-inflammatory polarization, and lamins are considered key determinants of nuclear deformability, we aimed to address the effect of pro-inflammatory stimulation on nuclear mechanics. We present the surprising finding that polarized bone marrow-derived macrophages have less deformable nuclei than unpolarized macrophages, despite their reduced lamin A/C levels. Furthermore, pro-inflammatory macrophages exhibited altered chromatin dynamics relative to unpolarized macrophages, including redistribution of trimethylated histone H3K9 (H3K9me3) from the nuclear periphery to the interior and increased chromatin compaction. Our findings suggest a model in which pro-inflammatory stimulation of macrophages induces chromatin changes that drive nuclear stiffening, and that in these cells, chromatin, rather than the nuclear lamina, is the major driver for resisting nuclear deformation. These findings may have functional relevance for the physiological function of polarized macrophages, as the mechanical properties of the nucleus can influence how these cells adapt and respond to their environments in the context of cell migration or inflammatory disease pathologies.

cell biology↗

bioRxiv2025

The Deformability of the Mammalian Cell Nucleus is Determined by the Identity of the Lamin Rod Domain

Lamins are nuclear intermediate filament proteins with diverse functions, ranging from organizing chromatin and regulating gene expression to providing structural support to the nucleus. Mammalian cells express two types of lamins, A-type and B-type, which, despite their similar structure and biochemical properties, exhibit distinct differences in expression, interaction partners, and function. One major difference is that A-type lamins have a significantly larger effect on the mechanical properties of the nucleus, which are crucial for protecting the nucleus from cytoskeletal forces, enabling cell migration through confined spaces, and contributing to cellular mechanotransduction. The molecular mechanism underlying this difference has remained unresolved. Here, we applied custom-developed biophysical and proteomic assays to lamin-deficient cell lines engineered to express specific full-length lamin proteins, lamin truncations, or chimeras combining domains from A- and B-type lamins, to systematically determine their contributions to nuclear mechanics. We found that although all expressed lamins contribute to the biophysical properties of the nuclear interior and confer some mechanical stability to the nuclear envelope, which is sufficient to protect the nuclear envelope from small cell-intrinsic forces and ensure proper positioning of nuclear pores, A-type lamins endow cells with a unique ability to resist large forces on the nucleus. Surprisingly, this effect was conferred through the A-type lamin rod domain, rather than the head or tail domains, which diverge more substantially between A- and B-type lamins and play important roles in lamin network formation. Collectively, our work provides an improved understanding of the distinct functions of different lamins in mammalian cells and may also explain why mutations in the A-type lamin rod domain cause more severe muscle defects in mouse models than other mutations.

cell biology↗

bioRxiv2025

Herpes simplex virus-1 fluidizes the nucleus enabling condensate formation

Molecular processes are profoundly influenced by the biophysical properties of the cell interior. However, the mechanisms that control these physical properties, and the processes they impact remain poorly understood, especially in the nucleus. We hypothesized that some viruses might change the biophysical properties of the nucleus to favor virus survival and replication and found that herpes simplex virus 1 (HSV-1) increases the mesoscale fluidity of the nucleus. The HSV-1 protein ICP4 caused fluidization and enabled growth of synthetic nuclear condensates. Conversely, conditions that decreased nuclear fluidity inhibited the growth of viral replication compartment condensates and reduced infectious virus production. Together, our data suggest that ICP4 increases nuclear fluidity to promote the formation of condensates that drive the progression of the HSV-1 life cycle. We speculate that a key function of ICP4 is to overcome the crowding and elastic confinement within cell nuclei that are a fundamental barrier to virus replication.

cell biology↗

bioRxiv2025

Giant KASH proteins and ribosomes synergistically establish cytoplasmic biophysical properties in vivo

Understanding how cells control their biophysical properties during development remains a fundamental challenge. While cytoplasmic macromolecular crowding affects multiple cellular processes in single cells, its regulation in living animals remains poorly understood. Using genetically encoded multimeric nanoparticles for in vivo rheology, we discovered that C. elegans tissues maintain distinct cytoplasmic biophysical properties that differ from those observed across diverse systems, including bacteria, yeast species, and cultured mammalian cells. We identified two conserved mechanisms controlling cytoplasmic macromolecular diffusion: ribosome concentration, a known regulator of cytoplasmic crowding, works in concert with a previously unknown function for the giant KASH protein ANC-1 scaffolding the endoplasmic reticulum. These findings reveal mechanisms by which tissues establish and maintain distinct cytoplasmic biophysical properties, with implications for understanding cellular organization across species. One-Sentence SummaryLiving tissues maintain unique intracellular biophysical properties under the control of cytoplasmic constraints and crowding.

biophysics↗

bioRxiv2024

The phase separation landscape of genome-wide genetic perturbations

Biomolecular organization is central to cell function. While phase separation is a key mechanism orchestrating this organization, we lack a comprehensive view of genes that can globally influence this process in vivo. To identify such genes, we combined functional genomics and synthetic biology. We developed a bioorthogonal system that can identify changes in the intracellular milieu that globally tune phase separation. We measured in vivo phase diagrams of a synthetic system across >25 million cells in 2,888 yeast knockouts, and identified 68 genes whose deletion alters the phase boundaries of the synthetic system, an unexpected result given the systems bioorthogonal design. Genes involved in TORC1 signaling and metabolism, particularly carbohydrate-, amino acid- and nucleotide synthesis were enriched. The mutants that changed phase separation also showed high pleiotropy, suggesting that phase separation interrelates with many aspects of biology. Highlights- A synthetic protein system reveals the genetic and environmental tunability of protein phase separation - Genetic knockouts affecting phase separation are highly pleiotropic - Carbohydrate, amino acid, and nucleotide metabolism contribute to modulating phase separation potential - Protein phase separation is a globally tunable property of the intracellular environment O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/620319v1_ufig1.gif" ALT="Figure 1"> View larger version (81K): org.highwire.dtl.DTLVardef@4b8403org.highwire.dtl.DTLVardef@1c80503org.highwire.dtl.DTLVardef@c10bf2org.highwire.dtl.DTLVardef@1f76c71_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

LINE-1 ribonucleoprotein complexes bind DNA to enable nuclear entry during mitosis

Long Interspersed Nuclear Element-1 (LINE-1) is an autonomous retrotransposon that makes up a significant portion of the human genome, contributing to genetic diversity and genome evolution. LINE-1 encodes two proteins, ORF1p and ORF2p, both essential for successful retrotransposition. ORF2p has endonuclease and reverse transcription activity, while ORF1p binds RNA. Many copies of ORF1p assemble onto the LINE-1 RNA to form a ribonucleoprotein complex (RNP). However, the functional role of RNPs in the LINE-1 life cycle is unclear. Using reconstitution assays on DNA curtains, we show that L1 RNPs gain DNA binding activity only when RNA is super-saturated with ORF1p. In cells, L1 RNPs bind to chromosomes during mitosis. Mutational analysis reveals that DNA binding is crucial for nuclear entry and LINE-1 retrotransposition activity. Thus, a key function of ORF1p is to form an RNP that gains access to the genome through DNA binding upon nuclear envelope breakdown.

biophysics↗

bioRxiv2024

Development and Characterization of 50 nanometer diameter Genetically Encoded Multimeric Nanoparticles

The mechanisms that regulate the physical properties of the cell interior remain poorly understood, especially at the mesoscale. Many crucial macromolecules and molecular assemblies such as ribosomes, RNA polymerase, and biomolecular condensates span the mesoscale size range, and changes in mesoscale physical properties have been suggested to be crucial for both normal physiology and disease. Therefore, we need better tools to study the cellular environment at this scale. Physical properties of the cell can be inferred through analysis of the motion of tracer nanoparticles, an approach called nanorheology. This requires the introduction of nanoparticles to cells, which can be labor intensive and slow. Genetically Encoded Multimeric nanoparticles (GEMs) were recently developed to address this limitation. GEMs consist of scaffold proteins fused to fluorescent tags that self-assemble into bright and stable nanoparticles of defined geometry. However, extremely sensitive microscopes were required to track previous (40nm-GEMs). Here, we describe the development and characterization of 50 nm diameter GEMs (50nm-GEMs) that are brighter and probe a slightly longer length scale. 50nm-GEMs will make high-throughput nanorheology accessible to a broader range of researchers and reveal new insights into the biophysical properties of cells.

biophysics↗

bioRxiv2024

Macromolecular interactions and geometrical confinement determine the 3D diffusion of ribosome-sized particles in live Escherichia coli cells

The crowded bacterial cytoplasm is comprised of biomolecules that span several orders of magnitude in size and electrical charge. This complexity has been proposed as the source of the rich spatial organization and apparent anomalous diffusion of intracellular components, although this has not been tested directly. Here, we use biplane microscopy to track the 3D motion of self-assembled bacterial Genetically Encoded Multimeric nanoparticles (bGEMs) with tunable size (20 to 50 nm) and charge (-2160 to +1800 e) in live Escherichia coli cells. To probe intermolecular details at spatial and temporal resolutions beyond experimental limits, we also developed a colloidal whole-cell model that explicitly represents the size and charge of cytoplasmic macromolecules and the porous structure of the bacterial nucleoid. Combining these techniques, we show that bGEMs spatially segregate by size, with small 20-nm particles enriched inside the nucleoid, and larger and/or positively charged particles excluded from this region. Localization is driven by entropic and electrostatic forces arising from cytoplasmic polydispersity, nucleoid structure, geometrical confinement, and interactions with other biomolecules including ribosomes and DNA. We observe that at the timescales of traditional single molecule tracking experiments, motion appears sub-diffusive for all particle sizes and charges. However, using computer simulations with higher temporal resolution, we find that the apparent anomalous exponents are governed by the region of the cell in which bGEMs are located. Molecular motion does not display anomalous diffusion on short time scales and the apparent sub-diffusion arises from geometrical confinement within the nucleoid and by the cell boundary.

biophysics↗

bioRxiv2024

Grb7, Grb10 and Grb14, encoding the growth factor receptor-bound 7 family of signalling adaptor proteins have overlapping functions in the regulation of fetal growth and post-natal glucose metabolism

BackgroundThe growth factor receptor bound 7 (Grb7) family of signalling adaptor proteins comprises Grb7, Grb10 and Grb14. Each can interact with the insulin receptor and other receptor tyrosine kinases, where Grb10 and Grb14 inhibit insulin receptor activity. In cell culture studies they mediate functions including cell survival, proliferation, and migration. Mouse knockout (KO) studies have revealed physiological roles for Grb10 and Grb14 in glucose-regulated energy homeostasis. Both Grb10 KO and Grb14 KO mice exhibit increased insulin signalling in peripheral tissues, with increased glucose and insulin sensitivity and a modestly increased ability to clear a glucose load. In addition, Grb10 strongly inhibits fetal growth such that at birth Grb10 KO mice are 30% larger by weight than wild type littermates. ResultsHere, we generate a Grb7 KO mouse model. We show that during fetal development the expression patterns of Grb7 and Grb14 each overlap with that of Grb10. Despite this, Grb7 and Grb14 did not have a major role in influencing fetal growth, either alone or in combination with Grb10. At birth, in most respects both Grb7 KO and Grb14 KO single mutants were indistinguishable from wild type, while Grb7:Grb10 double knockout (DKO) were near identical to Grb10 KO single mutant and Grb10:Grb14 DKO mutants slightly smaller. In the developing kidney Grb7 had a subtle positive influence on growth. An initial characterisation of Grb7 KO adult mice revealed sexually dimorphic effects on energy homeostasis, with females having significantly smaller white adipose tissue (WAT) depots and an enhanced ability to clear glucose from the circulation, compared to wild type littermates. Males had elevated fasted glucose levels with a trend towards smaller WAT depots, without improved glucose clearance. ConclusionsGrb7 and Grb14 do not have significant roles as inhibitors of fetal growth, unlike Grb10, and instead Grb7 may promote growth of the developing kidney. In adulthood, Grb7 contributes subtly to glucose mediated energy homeostasis, raising the possibility of redundancy between all three adaptors in physiological regulation of insulin signalling and glucose handling.

developmental biology↗

bioRxiv2023

Mesoscale molecular assembly is favored by the active, crowded cytoplasm

The mesoscale organization of molecules into membraneless biomolecular condensates is emerging as a key mechanism of rapid spatiotemporal control in cells1. Principles of biomolecular condensation have been revealed through in vitro reconstitution2. However, intracellular environments are much more complex than test-tube environments: They are viscoelastic, highly crowded at the mesoscale, and are far from thermodynamic equilibrium due to the constant action of energy-consuming processes3. We developed synDrops, a synthetic phase separation system, to study how the cellular environment affects condensate formation. Three key features enable physical analysis: synDrops are inducible, bioorthogonal, and have well-defined geometry. This design allows kinetic analysis of synDrop assembly and facilitates computational simulation of the process. We compared experiments and simulations to determine that macromolecular crowding promotes condensate nucleation but inhibits droplet growth through coalescence. ATP-dependent cellular activities help overcome the frustration of growth. In particular, actomyosin dynamics potentiate droplet growth by reducing confinement and elasticity in the mammalian cytoplasm, thereby enabling synDrop coarsening. Our results demonstrate that mesoscale molecular assembly is favored by the combined effects of crowding and active matter in the cytoplasm. These results move toward a better predictive understanding of condensate formation in vivo.

biophysics↗

bioRxiv2023

KaryoTap Enables Aneuploidy Detection in Thousands of Single Human Cells

Aneuploidy--the presence of chromosome gains and losses--is highly prevalent in human tumors, yet the contribution of chromosome missegregation rates and selection to shape these patterns remains poorly understood. To address this challenge at scale, we developed KaryoTap, a cost-effective single-cell DNA sequencing method combining custom targeted panels for the Tapestri platform with a Bayesian gaussian mixture model to detect chromosome- and chromosome arm-scale aneuploidy as well as integrated gRNAs and barcodes. KaryoTap achieves an average accuracy of >85% for arm events and >90% for chromosome events at <$1 per cell, allowing scalable analysis of tens of thousands of cells per experiment. Through KaryoTap-based analysis of 11,555 cells, we performed in vitro evolution screens on immortalized human cells from mammary gland, pancreas and melanocytes. By comparing aneuploidy frequencies immediately after reversine-induced missegregation versus after extended proliferation, we quantified positive and negative selection for specific aneuploidies. Most aneuploidies--both gains and losses--are under negative selection, yet some chromosomal gains are under positive selection (such as 8q and 7q). We found that proliferative selective pressures can explain tissue-specific patterns of chromosomal gains observed in human cancers. Critically, when positively selected events were excluded from analysis, correlations between in vitro and cancer gain frequencies strongly decreased or disappeared, whereas excluding negatively selected events largely preserved these correlations. These findings demonstrate that proliferative selection shapes the landscape of chromosomal gains in cancer, with a more prominent role for positive selection.

genomics↗

bioRxiv2023

GEMspa: a Napari plugin for analysis of single particle tracking data

The cellular environment is crowded with macromolecules and far from thermodynamic equilibrium. This active, crowded environment influences biochemical reactions and the formation of cellular structures such as membraneless organelles. These physical properties can change during normal physiology and in disease states such as neurodegenerative diseases and cancer, impacting cell behavior and function. Therefore, it is crucial to develop methods to characterize these properties. Microrheology is the inference of physical properties from the motion of tracer particles embedded within a material. This technique requires single particle tracking (SPT) and analysis of these tracks through the framework of soft-condensed matter physics. Analysis of SPT data can be challenging due to the lack of comprehensive user-friendly software tools. To address this, we introduce GEMspa, a software package implemented as a plugin for the open source image analysis platform, napari. GEMspa provides a GUI for a commonly used localization/tracking algorithm (via Trackpy), and a suite of methods to extract basic parameters describing particle motion. This platform aims to streamline the workflow of data analysis steps and allow researchers to visualize and optimize parameters for high-quality results, thereby making microrheology accessible to non-expert scientists.

biophysics↗

bioRxiv2023

mRNA condensation fluidizes the cytoplasm

The intracellular environment is packed with macromolecules of mesoscale size, and this crowded milieu significantly influences cell physiology. When exposed to stress, mRNAs released after translational arrest condense with RNA binding proteins, resulting in the formation of membraneless RNA protein (RNP) condensates known as processing bodies (P-bodies) and stress granules (SGs). However, the impact of the assembly of these condensates on the biophysical properties of the crowded cytoplasmic environment remains unclear. Here, we find that upon exposure to stress, polysome collapse and condensation of mRNAs increases mesoscale particle diffusivity in the cytoplasm. Increased mesoscale diffusivity is required for the efficient formation of Q-bodies, membraneless organelles that coordinate degradation of misfolded peptides that accumulate during stress. Additionally, we demonstrate that polysome collapse and stress granule formation has a similar effect in mammalian cells, fluidizing the cytoplasm at the mesoscale. We find that synthetic, light-induced RNA condensation is sufficient to fluidize the cytoplasm, demonstrating a causal effect of RNA condensation. Together, our work reveals a new functional role for stress-induced translation inhibition and formation of RNP condensates in modulating the physical properties of the cytoplasm to effectively respond to stressful conditions.

cell biology↗

bioRxiv2023

Increased mesoscale diffusivity in response to acute glucose starvation

Macromolecular crowding is an important parameter that impacts multiple biological processes. Passive microrheology using single particle tracking is a powerful means of studying macromolecular crowding. Here we monitored the diffusivity of self-assembling fluorescent nanoparticles (NS) in response to acute glucose starvation. mRNP diffusivity was reduced upon glucose starvation as previously reported. In contrast, we observed increased diffusivity of NS particles. Our results suggest that, upon glucose starvation, mRNP granule diffusivity may be reduced due to changes in physical interactions, while global crowding in the cytoplasm may be reduced.

biophysics↗