Biology subjects

Lemke, E. A.

Publications and source records attributed to Lemke, E. A..

13 recordsLinked to original sources

bioRxiv2026

A Phosphorylation-Induced Micellization switch in the low complexity domain of TDP-43

Abstract textPhase separation (PS) of the low-complexity domain (LCD) of TDP-43 is linked to pathogenic aggregates in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). Here, we show that extensive phosphorylation of the LCD C-terminus redirects its self-assembly. Coarse-grained Monte Carlo simulations predicted that 12 Ser phosphorylations partition the 148-residue LCD into a hydrophobic N-terminal and highly charged C-terminal block, favoring finite-sized micellization over macroscopic PS. In vitro, LCD phosphorylated by casein kinase 1 delta (CK1{delta}; mean of 12 phosphorylations by native mass spectrometry) and phosphomimetic 12D/12DD mutants formed spherical nanoparticles ({approx} 20-50 nm) above a low-micromolar critical micelle concentration, whereas the unphosphorylated LCD underwent reversible PS that matured into fibrils. Increasing ionic strength shifted the mutants toward anisotropic morphologies (worm-like 12D micelles and rigid 12DD nanocylinders). Turbidity assays and confocal imaging directly visualized the absence of PS in the phosphorylated form. Negative-stain and cryo-EM confirmed the spherical micellar architecture for the phosphorylated LCD and 12D/12DD mimics. Our data identify phosphorylation as a molecular switch tuning macrophase separation and fibril formation of TDP-43 LCD, providing a framework for an aggregation-protective role through microphase separation into size-limited micelles. Whether these assemblies are stable or kinetically trapped on pathological timescales remains unclear.

biophysics↗

bioRxiv2026

A Unifying Thermodynamic Model for Phase Separation and Aging of Biopolymers

Protein condensates that form via phase separation typically become more viscous over time and can harden in a process referred to as "molecular aging". Several mechanisms have been identified for this phenomenon. Of these, the ones involving enhanced {beta}-sheet or -strand interactions are of pathological relevance since they have been associated with neurodegeneration. Although there is much understanding of biopolymer phase behavior, an inclusive thermodynamic framework that unifies phase separation and {beta}-sheet-based aging is lacking. We present a time-dependent, multi-component extension of associating polymer theory that describes phase separation and aging of an intrinsically disordered protein (IDP) capable of associating through local, reversible folding. The model shows how the Second Law of Thermodynamics applies throughout, whether phase separation precedes and encourages aging or, vice versa, whether the increase in "stickiness" during aging drives phase separation. Our calculations show how the time-dependence of the average valency of associating sites determines the aging kinetics and the development of viscoelastic properties of a biocondensate. The agreement between our calculations and the change in dynamics of condensates of perfect repeat analogues of nucleoporin-98 not only validates the theory but also identifies these Nup98 variants as model systems for studying aging.

biophysics↗

bioRxiv2025

O-GlcNAcylation and an importin-β radial gradient keep the FG barrier liquid in live-cell nuclear pores

Summary ParagraphThe nuclear pore complex (NPC) regulates the molecular traffic between nucleus and cytoplasm.1,2 Its permeability barrier is formed by intrinsically disordered proteins (IDPs) known as FG-nucleoporins (FG-NUPs), whose physical state has long been debated.3-13 Deciphering how FG-NUPs behave inside living cells is crucial for understanding how the NPC achieves selective and rapid transport. Here, by combining site-specific labelling with picosecond time-resolved fluorescence anisotropy, we reveal that FG domains exhibit nanosecond-scale, liquid-like mobility in live cells, yet undergo a liquid-to-solid transition in vitro. Experiments and coarse-grained molecular dynamics simulations further show that importin-{beta}, a major nuclear transport receptor, and O-linked {beta}-N-acetylglucosamine (O-GlcNAc), a key post-translational modification of FG-NUPs together stabilise the dynamic FG network. Finely balanced FG-FG interactions modulated by O-GlcNAcylation, along with FG-importin-{beta} interactions, maintain liquidity and enrich importin-{beta} near the NPC periphery, while extended FG domains remain in the central channel to form the transport barrier. These findings reconcile conflicting models of FG-NUP organisation4,6,8,10,14,15 and explain the recent observations in high-resolution MINFLUX studies of importin-{beta} depletion from the pore centre in mammalian cells.16 Beyond resolving debates over FG-NUP behaviour, our study underscores the importance of studying IDPs in their cellular context, with broader implications for understanding IDP-related diseases, including viral infections, cancer, and neurodegenerative disorders.

biophysics↗

bioRxiv2025

Differential conformational expansion of Nup98-HOXA9 oncoprotein in micro- and macrophases

Transcription factors (TFs) play a central role in gene regulation by binding to specific DNA sequences and orchestrating the transcriptional machinery. A majority of eukaryotic TFs have a block copolymer architecture, with at least one block being a folded DNA interaction domain, and another block being highly enriched in intrinsic disorder. Multivalent interactions in the intrinsically disordered region (IDR) can contribute to phase separation into macroscopic condensates. In this study, we focus on Nup98-HOXA9 (NHA9), a chimeric transcription factor implicated in leukemogenesis, in which two FG-repeat-rich IDRs derived from Nup98 get fused to the C-terminal part of transcription factor HOXA9. By integrating experiments and simulations, we examined the structural dynamics of NHA9s FG domain across assembly states. We found that the FG domain has different conformational compactness in the monomeric state, oligomeric, and densely packed condensate state. Notably, the oligomeric state exhibits micelle-like organisation, with the DNA-binding domain exposed at the periphery. While their architecture is non-random, their sizes depend on NHA9 concentration, consistent with non-core-shell spherical micelles. Molecular dynamics simulations support the expansion behaviour of NHA9s FG domain as oligomeric assemblies grow in size and reveal micelle-like structural features in oligomeric assemblies. These findings offer molecular insight into the phase behaviour of NHA9 and highlight the dynamic conformational transitions of IDRs during condensate formation, with implications for understanding transcriptional regulation in cancer.

biophysics↗

bioRxiv2024

Direct RNA sequencing (RNA004) allows for improved transcriptome assessment and near real-time tracking of methylation for medical applications

Direct RNA sequencing (DRS) is a nanopore-based technique for analyzing RNA in its native form, promising breakthroughs in diagnostics and biomarker development. Coupled to RNA002 sequencing chemistry, its clinical implementation has been challenging due to low throughput, low accuracy, and lack of large-scale RNA-modification models. In this study, we evaluate the improvements achieved by pairing the latest RNA004 chemistry with novel modified-base-calling models for pseudouridine and N6-methyladenosine using diverse RNA samples from cell lines, synthetic oligos, and human blood. Finally, we present the first clinical application of DRS by confirming the loss of RNA methylation in a patient carrying truncating mutations in the methyltransferase METTL5. Conclusively, the combined use of RNA004 chemistry with the base-calling models significantly improved the throughput, accuracy, and site-specific detection of modifications. From this perspective, we offer an outlook on the potential suitability of DRS for use in routine diagnostics, the first comprehensive benchmark of human peripheral blood and quality assessments of RNA therapeutics.

bioinformatics↗

bioRxiv2024

Chaperone quality control in liquid-phase separated organelles

Molecular chaperones, central to the cellular proteostasis network, play an essential role in preventing the formation and proliferation of harmful aggregates associated with neurodegenerative diseases. Notably, for many intrinsically disordered proteins (IDPs), which are prone to form such damaging deposits, the formation of nano-clusters and phase separation into organelles prior to aggregation have been observed. The impact of molecular chaperones on such assemblies, remains unclear. In our study, we concentrated on the family of small heat shock proteins (sHsps), which are typically dynamic and form large oligomeric structures. While sHsps are mainly structured/folded proteins, they can undergo transient multivalent interactions, like many IDPs. Thus, sHsps might be a suitable regulator for vital and ubiquitous formation of membrane-less organelles in eukaryotic cells rich in IDPs and to inhibit aberrant aggregation. Here we show, using microfluidic diffusional sizing, that the formation of nano-clusters of FUS, associated with neurodegenerative diseases can be inhibited by the presence of sHsps. Furthermore, we identify that, depending on their assembly state, sHsps are capable of targeting specifically the interface between the dense droplet phase and the dilute phase not only of FUS but also of TDP-43, likely because the interface is the primary starting point for fibril formation or protein aggregation in general. Our findings emphasise the impact of molecular chaperones on maintaining the homeostasis of IDPs in the dilute and condensed phase. This could help to understand how chaperone dysregulation can influence aberrant protein association.

biophysics↗

bioRxiv2023

Protein-specific crowding accelerates aging in phase-separated droplets

Crowding agents, such as polyethylene glycol (PEG, are often used to mimic the cellular cytoplasm in protein assembly studies. Despite the perception that crowding agents have an inert nature, recent work has shown they are not bystanders while proteins interact. Here, we explore the diverse effects of PEG on the phase separation and maturation of proteins. We use two proteins, the FG domain of Nup98 and bovine serum albumin (BSA), which represent an intrinsically disordered protein and a protein with well-established secondary structure, respectively. PEG expedites the maturation of Nup98, enhancing denser protein packing and fortifying hydrophobic interactions which hasten beta-sheet formation and subsequent droplet gelation. In contrast for BSA, PEG appears to enhance droplet stability and limits available solvent for the protein solubilization, without inducing significant changes to the secondary structure, pointing towards a significantly different behavior of the crowding agent. Interestingly, we detect almost no presence of PEG in Nup droplets whereas PEG is detectable within BSA droplets. Our findings demonstrate a nuanced interplay between crowding agents and proteins. PEG can accelerate protein maturation in LLPS systems but its partitioning and effect on protein structure in droplets is protein specific. This suggests that crowding phenomena are specific to each protein-crowding agent pair.

biophysics↗

bioRxiv2023

An intact amber-free HIV-1 system for in-virus protein bioorthogonal click labeling that delineates envelope conformational dynamics

The HIV-1 envelope (Env) glycoprotein is conformationally dynamic and mediates membrane fusion required for cell entry. Single-molecule fluorescence resonance energy transfer (smFRET) of Env using peptide tags has provided mechanistic insights into the dynamics of Env conformations. Nevertheless, using peptide tags risks potential effects on structural integrity. Here, we aim to establish minimally invasive smFRET systems of Env on the virus by combining genetic code expansion and bioorthogonal click chemistry. Amber stop-codon suppression allows site-specifically incorporating noncanonical/unnatural amino acids (ncAAs) at introduced amber sites into proteins. However, ncAA incorporation into Env (or other HIV-1 proteins) in the virus context has been challenging due to low copies of Env on virions and incomplete amber suppression in mammalian cells. Here, we developed an intact amber-free virus system that overcomes impediments from preexisting ambers in HIV-1. Using this system, we successfully incorporated dual ncAAs at amber-introduced sites into Env on intact virions. Dual-ncAA incorporated Env retained similar neutralization sensitivities to neutralizing antibodies as wildtype. smFRET of click-labeled Env on intact amber-free virions recapitulated conformational profiles of Env. The amber-free HIV-1 infectious system also permits in-virus protein bioorthogonal labeling, compatible with various advanced microscopic studies of virus entry, trafficking, and egress in living cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/530526v1_ufig1.gif" ALT="Figure 1000"> View larger version (30K): org.highwire.dtl.DTLVardef@10e7728org.highwire.dtl.DTLVardef@b1078aorg.highwire.dtl.DTLVardef@65f874org.highwire.dtl.DTLVardef@9ab0e8_HPS_FORMAT_FIGEXP M_FIG C_FIG Amber-free HIV-1 infectious systems actualized minimal invasive Env tagging for smFRET, versatile for in-virus bioorthogonal click labeling in advanced microscopic studies of virus-host interactions.

microbiology↗

bioRxiv2022

Deciphering the conformations and dynamics of FG-nucleoporins in situ

The [~]120 MDa nuclear pore complex (NPC) acts as a gatekeeper for the molecular traffic between the nucleus and the cytosol. Small cargo readily passes through the transport channel, yet large cargo requires specialized nuclear transport receptors. While the scaffold structure that anchors the NPC in the double-layered nuclear envelope has been resolved to remarkable details, the spatial organization of intrinsically disordered nucleoporins (NUPs) within the central channel remains enigmatic. These so-called FG-NUPs account for about one-third of the total mass of the NPC and form the actual transport barrier. Here we combined site-specific fluorescent labeling in non-fixed cells and fluorescent lifetime imaging microscopy (FLIM) to directly decipher the conformations of an essential constituent of the permeability barrier, NUP98, inside the functioning NPCs using Fluorescence resonance energy transfer (FRET). With detailed measurements of the distance distribution of eighteen NUP98 segments combined with coarse-grained modeling, we mapped the uncharted biochemical environment inside the nanosized transport channel. We found that good-solvent conditions for a polymer dominate the inside of the nanosized NPC, expand the FG-domain in situ and facilitate nuclear transport, in sharp contrast to the collapsed NUP98 FG-chain in aqueous solution. The combination of fluorescence microscopy, high-resolution electron tomography, and molecular simulation opens a window into the so-far unresolved organization of the FG-NUPs at the center of NPC function, allowing us to reconcile scientific models of nuclear transport.

biophysics↗

bioRxiv2021

Role of Solvent Compatibility in the Phase Behavior of Binary Solutions of Weakly Associating Multivalent Polymers

Condensate formation of biopolymer solutions, prominently those of various intrinsically disordered proteins (IDPs), is determined by "sticky" interactions between associating residues, multivalently present along the polymer backbone. Using a ternary mean field "stickers-and-spacers" model, we demonstrate that if sticker association is of the order of a few times the thermal energy, a delicate balance between specific binding and non-specific polymer-solvent interactions gives rise to a particularly rich ternary phase behavior under physiological circumstances. For a generic system represented by a solution comprising multi-associative scaffold and client polymers, the difference in solvent compatibility between the polymers modulates the nature of isothermal liquid-liquid phase separation (LLPS) between associative and segregative. The calculations reveal regimes of dualistic phase behavior, where both types of LLPS occur within the same phase diagram, either associated with the presence of multiple miscibility gaps, or a flip in the slope of the tie-lines belonging to a single coexistence region.

biophysics↗

bioRxiv2020

Bioorthogonal red and far-red fluorogenic probes for wash-free live-cell and super-resolution microscopy

Small-molecule fluorophores enable the observation of biomolecules in their native context with fluorescence microscopy. Specific labelling via bioorthogonal tetrazine chemistry confers minimal label size and rapid labelling kinetics. At the same time, fluorogenic tetrazine-dye conjugates exhibit efficient quenching of dyes prior to target binding. However, live-cell compatible long-wavelength fluorophores with strong fluorogenicity have been difficult to realize. Here, we report close proximity tetrazine-dye conjugates with minimal distance between tetrazine and fluorophore. Two synthetic routes give access to a series of cell permeable and impermeable dyes including highly fluorogenic far-red emitting derivatives with electron exchange as dominant excited state quenching mechanism. We demonstrate their potential for live-cell imaging in combination with unnatural amino acids, wash-free multi-colour and super-resolution STED and SOFI imaging. These dyes pave the way for advanced fluorescence imaging of biomolecules with minimal label size.

cell biology↗

bioRxiv2019

Molecular determinants of large cargo transport into the nucleus

Transport of molecules between the nucleus and the cytoplasm is tightly regulated by the nuclear pore complex (NPC). Even very large cargoes such as many pathogens, mRNAs and pre-ribosomal subunits can pass the NPC intact. Compared to small import complexes, for such large cargoes >15 nm there is very little quantitative understanding of the mechanism for efficient transport, the role of multivalent binding to nuclear transport receptors via nuclear localisation sequences (NLSs) and effects of size differences. Here, we assayed nuclear import kinetics in cells for a total of 30 large cargo models based on four capsid-like particles in the size range of 17-36 nm, with tuneable numbers of up to 240 NLSs. We show that the requirements for transport scale non-linearly with size and obey a minimal cut off of functional import requiring more than 10 NLS in the lowest case. Together, our results reveal the key molecular determinants on large cargo import kinetics in cells.

biophysics↗

bioRxiv2017

Architecture of TAF11/TAF13/TBP complex suggests novel regulatory state in General Transcription Factor TFIID function

General transcription factor TFIID is a key component of RNA polymerase II transcription initiation. Human TFIID is a megadalton-sized complex comprising TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). TBP binds to core promoter DNA, recognizing the TATA-box. We identified a ternary complex formed by TBP and the histone fold (HF) domain-containing TFIID subunits TAF11 and TAF13. We demonstrate that TAF11/TAF13 competes for TBP binding with TATA-box DNA, and also with the N-terminal domain of TAF1 previously implicated in TATA-box mimicry. In an integrative approach combining crystal coordinates, biochemical analyses and data from cross-linking mass-spectrometry (CLMS), we determine the architecture of the TAF11/TAF13/TBP complex, revealing TAF11/TAF13 interaction with the DNA binding surface of TBP. We identify a highly conserved C-terminal TBP-binding domain (CTID) in TAF13 which is essential for supporting cell growth. Our results thus have implications for cellular TFIID assembly and suggest a novel regulatory state for TFIID function.

biochemistry↗