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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2026

Objective method to estimate the duration of the DNAreplication cycle without perturbation in Escherichia coli.

All cells, including unicellular organisms such as Escherichia coli, depend on the robustness of their cell cycle for proliferation. The cell cycle is central to the organisms highly resilient proliferation program and is thus a key focus in many areas of cell biology. However, studying the DNA replication cycle in bacteria remains challenging due to technical limitations. The durations of the replicative and post-replicative phases (known as the C and D periods, respectively) are inferred from the analysis of cellular DNA content distributions, obtained through microscopy or flow cytometry. This analysis typically requires pharmacological treatments to conduct replication run-off experiments, which help distinguish between cells that have or have not yet initiated DNA replication. Four decades ago, Skarstad, Steen and Boye showed that flow cytometry profiles measuring DNA amount per cell from exponentially growing cells could provide sufficient information to infer the durations of cell cycle periods at low growth rates. In this study, we propose an objective and automated approach that implements this idea to estimate cell cycle parameters for any growth conditions. Specifically, we validate a Nested Sampling method to estimate cell cycle parameters directly from flow cytometry data, eliminating the dependence on bacterial strain sensitivity to drugs. This tool, available as a Python package, allows for the accurate and minimally biased estimation of the C+D period under any biologically relevant conditions. Given its independence from pharmacological treatments, we anticipate broad adoption of this tool, especially as we show that most natural isolates of E. coli are not amenable to the state of the art replication run-off experiments.

microbiology↗

bioRxiv2024

Ionizable networks mediate pH-dependent allosteryin SH2 signaling proteins

Intracellular pH dynamics regulate normal cell biology, but most molecular drivers remain unknown. We developed a computational pipeline to identify pH-sensitive proteins and their mechanisms. We applied the pipeline to SHP2, a pH-sensitive signaling protein, with unknown mechanism. We found that SHP2 phosphatase activity is pH-sensitive in vitro and in cells, and mutation of identified His116 and Glu252 abolishes pH-sensitive function. We also discovered that Src is an unrecognized pH-dependent kinase and mutation of the identified ionizable network abolishes pH-sensitive activity. Importantly, we found that Src kinase activity was pH sensitive even in the presence of EGF and with a phosphomimetic (Src-Y527E) mutant required for auto-inhibitory SH2 domain binding. Thus, the identified pH-sensitive regulation of Src kinase activity functions in concert with established mechanisms of Src regulation by phosphorylation. Constant pH molecular dynamics simulations performed on both SHP2 and Src support allosteric regulation mediated by pH-dependent binding of inhibitory SH2 domains to the respective catalytic domains. Finally, we show that evolutionarily conserved putative pH-sensing networks were identified across SH2 domain-containing signaling proteins. Taken together, our computational, biophysical, and cellular analyses reveal a role for pHi dynamics in allosterically regulating activity of modular SH2 signaling proteins to control biology. One-sentence summaryThis paper investigates the role of pH in the allosteric regulation of signaling proteins with SH2 domains, specifically focusing on SHP2 and Src

biochemistry↗

bioRxiv2024

A proximity ligation screen identifies SNAT2 as a novel target of the MARCH1 E3 ubiquitin ligase

E3 ubiquitin ligases are part of various families of proteins and include hundreds of members, which play key roles in all aspects of cell biology. They generally regulate the half-life of other proteins but can also modulate their cellular localization and functions. The MARCH family of ubiquitin ligases is composed of 11 members and two closely related proteins, MARCH1 and MARCH8, share similar targets, while being active in different cell types. Although they appear to target principally immune cell components, such as MHC class II molecules and the co-stimulatory molecule CD86, the repertory of their targets remains to be fully documented. Here, to further define the MARCH1s interactome, we adapted a proximity-dependent biotin identification (BioID)-based screening approach in live HEK293 cells. We transfected a fusion protein consisting of mouse MARCH1 linked to YFP at its N-terminus and to the biotin ligase of Aquifex aeolicus at its C-terminus. Upon transient overexpression of this construct in the presence of exogenous biotin, we could recover biotinylated proteins that are presumably found within 10nm of MARCH1. To help in the identification of bona fide down-regulated specific targets, we compared MARCH1s interactome with the one obtained using a ubiquitination-deficient MARCH1 mutant (MARCH1W104A). CD98 and CD71, two previously described targets of MARCH1, were identified in this screen. Of 16 other biotinylated proteins identified by semi-quantitative mass spectrometry, 10 were tested directly by flow cytometry to monitor their expression in the presence or absence of transfected MARCH1. The protein levels of five of these endogenous targets, CD29, CD112, NKCC1, CD147 and SNAT2, confirmed their negative regulation by MARCH1 in this system. SNAT2 was particularly sensitive to the presence of MARCH1 and was found to be ubiquitinated on Western blots following immunoprecipitation. Thus, BioID2 is an effective mean of characterizing the interactome of MARCH1 and the identification of SNAT2 suggests a role of this ubiquitin ligase in cellular metabolism.

immunology↗

bioRxiv2024

Inter-kingdom signaling by the Legionella autoinducer LAI-1 involves the antimicrobial guanylate binding protein GBP

The causative agent of Legionnaires disease, Legionella pneumophila, is an amoebae-resistant environmental bacterium, which replicates intracellularly in a distinct compartment, the "Legionella-containing vacuole" (LCV). L. pneumophila employs the -hydroxyketone compound LAI-1 (Legionella autoinducer-1) for intra-species and inter-kingdom signaling. LAI-1 promotes intracellular replication and inhibits the migration of mammalian cells and Dictyostelium discoideum. In this study, we revealed that LAI-1 and "clickable" azido-LAI-1 derivatives inhibit the migration of D. discoideum and localize to LCVs. Azido-LAI-1 colocalizes with the LCV markers calnexin, P4C, and AmtA, but not with mitochondrial or lipid droplet markers. Intriguingly, LAI-1 dependent inhibition of D. discoideum migration involves the single guanylate-binding protein (GBP), a member of the GBP family of large GTPases, which in metazoan organisms promote cell autonomous immunity. D. discoideum lacking GBP ({Delta}gnbp) allows more efficient intracellular replication of L. pneumophila, without apparently compromising LCV remodeling or integrity, and GBP-GFP localizes to the ER at LCV-ER membrane contact sites (MCS). However, the peri-LCV localization of LAI-1 and GBP is not mutually dependent. Synthetic LAI-1 inhibits the expansion/remodeling of LCVs (but not vacuoles harboring avirulent L. pneumophila) in a GBP-dependent manner. Taken together, the work shows that LAI-1 localizes to LCVs, and LAI-1-dependent inter-kingdom signaling involves D. discoideum GBP, which localizes to LCV-ER MCS and acts as an antimicrobial factor by restricting the intracellular growth of L. pneumophila. Author SummarySmall molecule inter-kingdom signaling between pathogens and host cells represents a crucial but only partly understood aspect of microbial virulence. The amoeba-resistant opportunistic pathogen Legionella pneumophila employs the compound LAI-1 (Legionella autoinducer-1) for intra-species and inter-kingdom signaling. In metazoan cells, the conserved and wide-spread family of guanylate-binding protein (GBP) large GTPases usually comprises several distinct paralogues, which are implicated in pathogen detection, inflammation, cell death pathways, and cell autonomous immunity. In the social amoeba Dictyostelium discoideum, only a single GBP gene of unknown function is present. Using approaches from organic chemistry, genetics, cell biology and infection biology, we reveal that GBP is involved in the inhibition of D. discoideum migration and pathogen vacuole expansion/remodeling by LAI-1 as well as in intracellular growth of L. pneumophila. This study provides a novel link between small molecule inter-kingdom signaling and GBP-dependent cell autonomous immunity.

microbiology↗

bioRxiv2019

The hematopoietic landscape at single-cell resolution reveals unexpected stem cell features in naked mole-rats

Naked mole-rats are the longest-lived rodents endowed with resistance to cancer and age-related diseases, yet their stem cell characteristics remain enigmatic. We profiled the naked mole-rat hematopoietic system down to single-cell resolution, and identified several unique features likely contributing to longevity. In adult naked mole-rats red blood cells are formed in spleen and marrow, a neotenic feature beneficial for hypoxic environments and to prevent anemia. Platelet numbers are lower compared to short-lived mice, which may preclude age-related platelet increase and thrombosis. T cells mature in thymus and lymph nodes, providing a supply of T cells after age-related thymus involution. The pool of quiescent stem cells is higher than in mice, and HSCs overexpress an oxidative phosphorylation signature, revealing a new paradigm of stem cell metabolism to benefit longevity and oppose oncogenesis. Our work provides a platform to study immunology and stem cell biology in an animal model of healthy aging. HIGHLIGHTSO_LIFlow cytometry labelling panel to purify viable naked mole-rat HSPCs C_LIO_LIThe spleen as the major site of erythropoiesis in the naked mole-rat C_LIO_LINaked mole-rats show extrathymic T-cell development under homeostatic conditions C_LIO_LINaked mole-rat hematopoietic stem cells (HSCs) have high OXPHOS activity C_LI

immunology↗

bioRxiv2023

Selective Enhancer Dependencies in MYC-Intact and MYC-Rearranged Germinal Center B-cell Diffuse Large B-cell Lymphoma

High expression of MYC and its target genes define a subset of germinal center B-cell diffuse large B-cell lymphoma (GCB-DLBCL) associated with poor outcomes. Half of these high-grade cases show chromosomal rearrangements between the MYC locus and heterologous enhancer-bearing loci, while focal deletions of the adjacent non-coding gene PVT1 are enriched in MYC-intact cases. To identify genomic drivers of MYC activation, we used high-throughput CRISPR-interference (CRISPRi) profiling of candidate enhancers in the MYC locus and rearrangement partner loci in GCB-DLBCL cell lines and mantle cell lymphoma (MCL) comparators that lacked common rearrangements between MYC and immunoglobulin (Ig) loci. Rearrangements between MYC and non-Ig loci were associated with unique dependencies on specific enhancer subunits within those partner loci. Notably, fitness dependency on enhancer modules within the BCL6 super-enhancer (BCL6-SE) cluster regulated by a transcription factor complex of MEF2B, POU2F2, and POU2AF1 was higher in cell lines bearing a recurrent MYC::BCL6-SE rearrangement. In contrast, GCB-DLBCL cell lines without MYC rearrangement were highly dependent on a previously uncharacterized 3 enhancer within the MYC locus itself (GCBME-1), that is regulated in part by the same triad of factors. GCBME-1 is evolutionarily conserved and active in normal germinal center B cells in humans and mice, suggesting a key role in normal germinal center B cell biology. Finally, we show that the PVT1 promoter limits MYC activation by either native or heterologous enhancers and demonstrate that this limitation is bypassed by 3 rearrangements that remove PVT1 from its position in cis with the rearranged MYC gene. Key pointsO_LICRISPR-interference screens identify a conserved germinal center B cell MYC enhancer that is essential for GCB-DLBCL lacking MYC rearrangements. C_LIO_LIFunctional profiling of MYC partner loci reveals principles of MYC enhancer-hijacking activation by non-immunoglobulin rearrangements. C_LI

cancer biology↗

bioRxiv2025

Extracellular matrix remodeling supports Hydra vulgaris head regeneration and stem cell invasion

The small freshwater cnidarian Hydra vulgaris is a classic model for investigating the genetic regulation of whole-body regeneration, but the underlying cell biology is comparatively underexplored. Hydra has a simple body plan consisting of two epithelial monolayers separated by an extracellular matrix (ECM). This ECM contains conserved components such as collagen and laminin, making Hydra well suited for dissecting ECM function during regeneration. Following head amputation and wound closure, we observe a retraction of ECM proteins from the wound site, creating a region of low ECM protein accumulation that persists for several days during head regeneration. Several matrix metalloproteinase (MMP) genes are expressed during this process, and MMP inhibition reduces the size of the ECM gap and results in a regenerative outcome with gross morphological defects. We further find that interstitial stem cells (ISCs), which originate in the ectoderm, localize in the regenerating head endoderm near regions of reduced ECM. This suggests that the ECM gap facilitates stem cell invasion to populate the new head with neurons and gland cells. However, inhibition of collagen cross-linking reveals that collagen synthesis is also required for regeneration, indicating that Hydra must balance ECM degradation and synthesis to complete regeneration. Together, these findings highlight ECM remodeling as a critical and conserved feature of regeneration. Summary statementThis study uses Hydra vulgaris, a highly regenerative freshwater cnidarian, to study remodeling of extracellular matrix proteins and stem cell invasion during tissue regeneration.

developmental biology↗

bioRxiv2020

Direct observation of genome surveillance by CRISPR-Cas in bacteria

CRISPR-Cas systems are prokaryotic adaptive immune systems that have been well characterized biochemically, but in vivo spatiotemporal regulation and cell biology remains largely unaddressed. Here, we used fluorescent fusion proteins to study the localization of the Type I-F CRISPR-Cas system native to Pseudomonas aeruginosa. When targeted to an integrated prophage, the crRNA-guided (Csy) complex and a majority of Cas3 molecules in the cell are recruited to a single focus. When lacking a target in the cell, however, the Csy complex is broadly nucleoid bound, while Cas3 is diffuse in the cytoplasm. Nucleoid association for the Csy proteins is crRNA-dependent, and inhibited by expression of anti-CRISPR AcrIF2, which blocks PAM binding. The Cas9 nuclease is also nucleoid localized, only when gRNA-bound, which is abolished by PAM mimic, AcrIIA4. Our findings reveal PAM-dependent nucleoid surveillance and spatiotemporal regulation in Type I CRISPR-Cas that separates the nuclease-helicase Cas3 from the crRNA-guided surveillance complex.

microbiology↗

bioRxiv2025

22q11 deletion selectively alters progenitor states and projection neuron identities in the developing cerebral cortex

The developmental origin of Layer 2/3 projection neuron (PN) pathology in the frontal association cortex due to heterozygous 22q11 gene deletion--the genetic foundation of elevated risk for schizophrenia and related psychiatric disorders in 22q11.2 Deletion Syndrome (22q11DS)--reflects cell state-dependent, temporally restricted vulnerability of transcriptionally diverse subsets of intermediate progenitors and neuroblasts. The molecular and cell biological consequences of these evanescent state-dependent changes include divergent proliferative capacity of the most highly proliferative progenitors, enhanced neurogenic gene expression, altered DNA methylation, and increased numbers of immediate neuroblast progeny at peak neurogenesis in fetal frontal cortex of the LgDel 22q11DS mouse model. These altered cell states prefigure a post-natal cohort of upper layer PNs generated at the peak of Layer 2/3 PN genesis--not before or after--whose frequencies are significantly diminished and molecular identities are substantially divergent in medial frontal association, but not primary somatosensory or visual cortices. Thus, frontal association cortices in 22q11DS and schizophrenia more broadly may be pathogenic targets due to vulnerabilities of expanded, highly proliferative, transcriptionally dynamic populations of intermediate progenitors that increase L2/3 PN frequency to enhance cortico-cortical connectivity.

neuroscience↗

bioRxiv2019

Lytic infection with murine gammaherpesvirus 68 activates host and viral RNA polymerase III-dependent promoters to enhance non-coding RNA expression

RNA polymerase III (pol III) transcribes multiple non-coding (nc) RNAs that are essential for cellular function. Pol III-dependent transcription is also engaged during certain viral infections, including the gammaherpesviruses ({gamma}HVs), where pol III-dependent viral ncRNAs promote pathogenesis. Additionally, several host ncRNAs are upregulated during {gamma}HV infection and play integral roles in pathogenesis by facilitating viral establishment and gene expression. Here, we sought to investigate how pol III promoters and transcripts are regulated during gammaherpesvirus infection using the murine gammaherpesvirus 68 ({gamma}HV68) system. To compare the transcription of host and viral pol III-dependent ncRNAs, we analyzed a series of pol III promoters for host and viral ncRNAs using a luciferase reporter optimized to measure pol III activity. We measured promoter activity from the reporter gene at the translation level via luciferase activity and at the transcription level via RT-qPCR. We further measured endogenous ncRNA expression at single cell-resolution by flow cytometry. These studies demonstrated that lytic infection with {gamma}HV68 increased the transcription from multiple host and viral pol III promoters, and further identified the ability of accessory sequences to influence both baseline and inducible promoter activity after infection. RNA flow cytometry revealed the induction of endogenous pol III-derived ncRNAs that tightly correlated with viral gene expression. These studies highlight how lytic gammaherpesvirus infection alters the transcriptional landscape of host cells to increase pol III-derived RNAs, a process that may further modify cellular function and enhance viral gene expression and pathogenesis. IMPORTANCEGammaherpesviruses are a prime example of how viruses can alter the host transcriptional landscape to establish infection. Despite major insights into how these viruses modify RNA polymerase II-dependent generation of messenger RNAs, how these viruses influence the activity of host RNA polymerase III remains much less clear. Small non-coding RNAs produced by RNA polymerase III are increasingly recognized to play critical regulatory roles in cell biology and virus infection. Studies of RNA polymerase III dependent transcription are complicated by multiple promoter types and diverse RNAs with variable stability and processing requirements. Here, we characterized a reporter system to directly study RNA polymerase III-dependent responses during gammaherpesvirus infection and utilized single-cell flow cytometry-based methods to reveal that gammaherpesvirus lytic replication broadly induces pol III activity to enhance host and viral non-coding RNA expression within the infected cell.

microbiology↗

bioRxiv2020

Hsf1 is SUMOylated in the activated trimeric state

The heat shock response (HSR) is a transcriptional program of organisms to counteract an imbalance in protein homeostasis. It is orchestrated in all eukaryotic cells by heat shock factor 1 (Hsf1). Despite very intensive research, the intricacies of the Hsf1 activation-attenuation cycle remain elusive at a molecular level. Posttranslational modifications belong to one of the key mechanisms proposed to adapt the Hsf1 activity to the needs of individual cells and phosphorylation of Hsf1 at multiple sites has attracted much attention. According to cell biological and proteomics data, Hsf1 is also modified by SUMO (small ubiquitin-like modifier) at several sites. How SUMOylation affects Hsf1 activity at a molecular level is still unclear. Here, we analyzed Hsf1 SUMOylation in vitro with purified components to address questions that could not be answered in cell culture models. In vitro Hsf1 is primarily conjugated at lysine 298 with a single SUMO, though we did detect low level SUMOylation at other sites. None of the tested E3 SUMO ligases increased SUMOylation efficacy as compared to the level in the presence of high concentrations of the E2 Ubc9. We provide evidence that Hsf1 trimerization and phosphorylation at serines 303 and 307 increases SUMOylation efficiency, suggesting that Hsf1 is SUMOylated in its activated state. Hsf1 can be SUMOylated when DNA-bound, and SUMOylation of Hsf1 does neither alter DNA binding affinity nor does it affect Hsc70 and DnaJB1-mediated monomerization of Hsf1 trimers and concomitant dislocation from DNA. We propose that SUMOylation acts at the transcription level of the HSR.

biochemistry↗

bioRxiv2026

NEK1 autophosphorylation is disrupted by amyotrophic lateral sclerosis-associated missense variants: activity biomarkers and structural insights

Rare variants in NEK1, encoding a serine/threonine kinase, are amongst the most consistently implicated genetic contributors to amyotrophic lateral sclerosis (ALS), reported in approximately 2-3% of cases. Yet, whilst recent studies have characterised the cell biological consequences of NEK1 loss-of-function, the biochemical effects of ALS-associated missense variants on kinase activity have not been directly investigated. This distinction is mechanistically important, because missense alleles encode mutant proteins rather than simply reducing protein dosage. Here, we provide the most comprehensive cell-based phosphoproteomic map of NEK1 phosphorylation to date, identifying ten recurrent phosphorylation sites across independent expression and acquisition conditions. We experimentally assign pSer14, pThr156 and pSer418 as NEK1 autophosphorylation sites using kinase-dead controls, targeted extracted ion chromatogram analysis, phosphosite mutagenesis and phosphospecific antibodies. Leveraging activation-loop pThr156 as a readout of NEK1 activity, we assessed nine ALS-associated missense variants spanning the major functional regions of the protein. Amongst catalytic-domain variants, R261C produced the most robust reduction in pThr156 autophosphorylation, R232C produced a smaller reduction, and R232H increased pThr156; the basic-region variant, A313T, also showed a smaller reduction. Structural modelling provides a mechanistic framework for understanding these variant-specific effects. Our study establishes the first activity-based framework for functional classification of NEK1 missense variants, and provides direct evidence that ALS-associated missense variants can alter NEK1 autophosphorylation through a mechanism distinct from simple haploinsufficiency. The phosphospecific antibodies, isogenic cell lines and curated phosphoproteomic datasets generated here provide a community resource for future studies of NEK1 regulation, variant interpretation and therapeutic target validation.

neuroscience↗

bioRxiv2020

Impact of nanoscale hindrances on the relationship between lipid packing and diffusion in model membranes

Membrane models have allowed for precise study of the plasma membranes biophysical properties, helping to unravel both structural and dynamic motifs within cell biology. Free standing and supported bilayer systems are popular models to reconstitute the membrane related processes. Although it is well-known that each have their advantages and limitations, comprehensive comparison of their biophysical properties is still lacking. Here, we compare the diffusion and lipid packing in giant unilamellar vesicles, planar and spherical supported membranes and cell-derived giant plasma membrane vesicles. We apply florescence correlation spectroscopy, spectral imaging and super-resolution STED-FCS to study the diffusivity, lipid packing and nanoscale architecture of these membrane systems, respectively. Our data show that lipid packing and diffusivity is tightly correlated in free-standing bilayers. However, nanoscale interactions in the supported bilayers cause deviation from this correlation. This data is essential to develop accurate theoretical models of the plasma membrane and will serve as a guideline for suitable model selection in future studies to reconstitute biological processes.

biophysics↗

bioRxiv2024

Programmable soft DNA hydrogels stimulate cellular endocytic pathways and proliferation

Hydrogels are pivotal in tissue engineering, regenerative medicine, and drug delivery applications. DNA molecules stand out among various biomaterials due to their unparalleled precision, programmability, and customization. In this study, we introduce a palate of novel cellular scaffolding platforms made of pure DNA-based hydrogel systems while improving the shortcomings of the existing platforms. DNA strands can form complex supramolecular branched structures essential for designing novel functional materials by its precise sequence-based self-assembly. These unique geometric scaffolds offer a soft, cushiony platform, ideal for culturing cells to mimic the complex native in vivo environments better. Each hydrogel comprises repeating units of branched DNA supramolecular structures, each possessing a distinct number of branching arms. The epithelial cells grown over these hydrogels show dynamic changes at multiple levels, from morphology to protein expression patterns, enhanced membrane traffic, and proliferation. The DNA hydrogels explored here are mechanically weak and soft and thus appropriate for applications in cell biology. This research lays the groundwork for developing a DNA hydrogel system with a higher dynamic range of stiffness, which will open exciting avenues for tissue engineering and beyond. Graphical abstract illustrating diverse branched DNA supramolecular architectures forming DNA hydrogels of various geometric profiles, each put to use in the cell culture applications. O_FIG O_LINKSMALLFIG WIDTH=160 HEIGHT=200 SRC="FIGDIR/small/595930v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1a3d4cborg.highwire.dtl.DTLVardef@e771cforg.highwire.dtl.DTLVardef@e0d5b4org.highwire.dtl.DTLVardef@ee42d3_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Nutrient cycling is an important mechanism for homeostasis in plant cells

Homeostasis in living cells refers to the steady state of internal, physical, and chemical conditions. It is maintained by self-regulation of the dynamic cellular system. In order to gain insight into homeostatic mechanisms that keep cytosolic nutrient concentrations in plant cells within a homeostatic range, I performed computational cell biology experiments. Systems of membrane transporters were modelled mathematically followed by the simulation of their dynamics. The detailed analyses of what-if scenarios demonstrate that a single transporter type for a nutrient, irrespective whether it is a channel or a co-transporter, is not sufficient to set a desired cytosolic concentration. A cell cannot flexibly react on different external conditions. At least two different transporter types for the same nutrient are required, which are energized differently. The gain of flexibility in adjusting the nutrient concentration was accompanied by the establishment of energy-consuming nutrient cycles at the membrane suggesting that these sometimes called futile cycles are not as futile as they appear. This understanding may help in future to design new strategies for increasing nutrient use efficiency of crop plants taking into account the complex interplay of transporter networks at the cellular level. One sentence summaryFirst principles of membrane transport explain why the maintenance of a constant cytosolic nutrient concentration is often accompanied by the futile cycling of the nutrient across the membrane.

plant biology↗

bioRxiv2023

Lipid droplets as substrates for protein phase separation

Membrane-associated protein phase separation plays critical roles in cell biology, driving essential cellular phenomena from immune signaling to membrane traffic. Importantly, by restricting diffusion to a two-dimensional surface, lipid bilayers can nucleate phase separation at far lower concentrations compared to those required for phase separation in solution. How might other intracellular lipid substrates, such as lipid droplets, contribute to nucleation of phase separation? Distinct from bilayer membranes, lipid droplets consist of a phospholipid monolayer surrounding a core of neutral lipids, and they are energy storage organelles that protect cells from lipotoxicity and oxidative stress. Here, we show that intrinsically disordered proteins can undergo phase separation on the surface of synthetic and cell-derived lipid droplets. Specifically, we find that model disordered domains, FUS LC and LAF1-RGG, separate into protein-rich and protein-depleted phases on the surfaces of lipid droplets. Owing to the hydrophobic nature of interactions between FUS LC proteins, increasing ionic strength drives an increase in its phase separation on droplet surfaces. The opposite is true for LAF1-RGG, owing to the electrostatic nature of its interprotein interactions. In both cases, protein-rich phases on the surfaces of synthetic and cell-derived lipid droplets demonstrate molecular mobility indicative of a liquid-like state. Our results show that lipid droplets can nucleate protein condensates, suggesting that protein phase separation could be key in organizing biological processes involving lipid droplets.

biophysics↗

bioRxiv2024

Rapid and reversible dissolution of biomolecular condensates using light-controlled recruitment of a solubility tag

Biomolecular condensates are broadly implicated in both normal cellular regulation and disease. Consequently, several chemical biology and optogenetic approaches have been developed to induce phase separation of a protein of interest. However, few tools are available to perform the converse function--dissolving a condensate of interest on demand. Such a tool would aid in testing whether the condensate plays specific functional roles, a major question in cell biology and drug development. Here we report an optogenetic approach to selectively dissolve a condensate of interest in a reversible and spatially controlled manner. We show that light-gated recruitment of maltose-binding protein (MBP), a commonly used solubilizing domain in protein purification, results in rapid and controlled dissolution of condensates formed from proteins of interest. Our optogenetic MBP-based dissolution strategy (OptoMBP) is rapid, reversible, and can be spatially controlled with subcellular precision. We also provide a proof-of-principle application of OptoMBP, showing that disrupting condensation of the oncogenic fusion protein FUS-CHOP results in reversion of FUS-CHOP driven transcriptional changes. We envision that the OptoMBP system could be broadly useful for disrupting constitutive protein condensates to probe their biological functions.

bioengineering↗

bioRxiv2023

Spontanously breaking of symmetry in overlapping cell instance segmentation using diffusion models

Instance segmentation is the task of assigning unique identifiers to individual objects in images. Solving this task requires breaking the inherent symmetry that semantically similar objects must result in distinct outputs. Deep learning algorithms bypass this break-of-symmetry by training specialized predictors or by utilizing intermediate label representations. However, many of these approaches break down when faced with overlapping labels that can appear, e.g., in biological cell layers. Here, we discuss the reason for this failure and offer a novel approach for instance segmentation based on diffusion models that breaks this symmetry spontaneously. Our method outputs pixel-level instance segmentations matching the performance of models such as cellpose on the cellpose fluorescent cell dataset while also permitting overlapping labels.

bioinformatics↗