Biology subjects

Langer, J. D.

Publications and source records attributed to Langer, J. D..

At least 19 recordsLinked to original sources

bioRxiv2026

Mouse Neural Organoids Model the Mature In Vivo Synaptic Proteome

Neural organoids represent a promising approach for modelling neural circuits and neurological disease, yet their utility depends on whether they can recapitulate the molecular complexity of mature brain synapses. Here, we optimized a protocol to generate mature forebrain organoids from mouse embryonic stem cells and used time-resolved proteomics to define the emergence of synaptic proteins during maturation. Using fluorescence-activated synaptosome sorting coupled with mass spectrometry, we quantified the synapse-enriched proteome and found a high correlation with matched in vivo synaptic proteomes. These findings demonstrate that complex mature synaptic molecular architecture is present within an in vitro organoid environment.

neuroscience↗

bioRxiv2026

SUMO paralogues differentially affect phase separation and aggregation of intrinsically disordered proteins

SUMO, the small ubiquitin-like modifier, modulates interactions of folded proteins, but also affects the dynamics of biomolecular condensates. Moreover, SUMO can directly impinge on the biophysical properties of its targets, often increasing their solubility. TDP-43 is an RNA-binding protein containing an intrinsically disordered domain that participates in protein phase separation. Its aggregation, linked to neurodegenerative diseases, is counteracted by SUMOylation, but the underlying principles are poorly understood. We now reveal distinct mechanisms of how the two isoforms, SUMO1 and SUMO2, act on TDP-43. While SUMO1 inhibits phase separation by blocking TDP-43s self-assembly site, SUMO2 enhances phase separation via proximity-induced self-interaction involving its N-terminus and promotes a liquid-like state that prevents aggregation. The latter represents a general principle by which SUMO solubilises other targets. Our findings provide insight into how SUMOylation regulates transitions between soluble, liquid-like and aggregated states.

molecular biology↗

bioRxiv2026

Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

Ribosomes and thousands of mRNAs are localized near synapses to support local protein synthesis. Little is known, however, about how ribosomes are positioned and maintained in dendritic spines- the primary postsynaptic sites of excitatory neurotransmission. Here, using proximity labeling-mass spectrometry, we mapped the interactome of postsynaptic ribosomes, and discovered an unexpected interaction with AMPA receptor complex proteins. Co-immunoprecipitation and crosslinking mass spectrometry using rat cortical synaptosomes showed a direct, mRNA-independent interaction between postsynaptic proteins and intact ribosomes. Immunoprecipitation-ribosome profiling (IP-Ribo-seq) revealed not only the complete synaptic translatome but also that the AMPA receptor-associated subpopulation of synaptic ribosomes preferentially translates mRNAs encoding proteins related to post-synaptic density (PSD) scaffolding and cytoskeletal dynamics. Translation of one of the mRNA targets, Camk2a, was reduced in spines following ER sequestration of endogenous GluA1. Together, these results reveal a role for PSD proteins in positioning ribosomes near the postsynaptic membrane, providing a mechanism to couple synaptic activity with the local production of proteins needed for structural remodeling.

neuroscience↗

bioRxiv2025

Correlative MS Imaging for cellular identification and analysis of in situ cryo-ET

In situ cryo-electron tomography (cryo-ET) offers direct access to protein structures in their native state and environment. A central challenge for this technique is the identification and classification of imaged cells, as sample heterogeneity with respect to cell type and functional state can significantly impact the data. Currently, cells can be classified using subtomogram averaging or fluorescent labeling, which require sufficiently distinguishable macromolecules, genetic modifications, or the availability of suitable labels. In MALDI-MS Imaging (MSI) individual cells can be identified and classified reliably, making use of the distributions of phospholipid species, metabolites and small proteins with characteristic profiles for specific cell types. Here we present an integrated pipeline combining MSI and in situ cryo-ET for single-cell identification and classification. Our approach enables MSI data acquisition directly from EM grids and can be used on any eukaryotic single-cell cryo-ET sample without prior modification in less than 36 h. We apply this workflow to describe cell-to-cell heterogeneity in cultured HeLa cells, to distinguish between distantly related HeLa and Polytomella cells, and between primary cultured rat neurons and glial cells. Finally we present a data integration workflow combining cryo-ET, light microscopy and MSI aligned in the same coordinate system to facilitate efficient data visualization and interpretation. We anticipate that this approach will change the way that structural biologists can approach cellular heterogeneity in cryo-ET, as cells can be identified and classified reliably post acquisition.

biophysics↗

bioRxiv2025

SND3 is the membrane insertase within a fungal multipass translocon

During the biogenesis of most eukaryotic integral membrane proteins (IMPs), transmembrane domains are inserted into the endoplasmic reticulum membrane by a dedicated insertase or the SEC61 translocon. The SRP-independent (SND) pathway is the least understood route into the membrane, despite catering for a broad range of IMP types. Here, we show that Chaetomium thermophilum SND3 is a membrane insertase of novel fold. We further present a cryo-electron microscopy structure of a ribosome-associated SND3 translocon complex involved in co-translational IMP insertion. The structure reveals that the SND3 translocon additionally comprises the complete SEC61 translocon, CCDC47 and TRAP[a]. Here, the SEC61{beta} N-terminus works together with CCDC47 to prevent substrate access to the translocon. Instead, molecular dynamics simulations show that SND3 disrupts the lipid bilayer to promote IMP insertion via its membrane-embedded hydrophilic groove. Structural and sequence comparisons indicate that the SND3 translocon is a distinct multipass translocon for the insertion of multipass IMPs in fungi, euglenozoan parasites and other eukaryotic taxa.

biophysics↗

bioRxiv2025

Neuronal processes contain the essential components for the late steps of ribosome biogenesis

Neurons rely on spatial and temporal control of protein synthesis to respond rapidly and locally to external stimuli, a process facilitated by the dynamic localization and modification of ribosomes. While previous research has shown that neuronal activity can regulate ribosome localization and modify translation rates, little is known about ribosomal assembly within neuronal processes. Here, we investigated the potential for local ribosome maturation in rat neurons using proteomics, RNA sequencing, and imaging methods. We detected an abundance of ribosome biogenesis factors (RBFs) in distal neuronal compartments, particularly those associated with the late stages of ribosome assembly. Moreover, we detected cytosolic pre-rRNA species in dendrites, alongside the enzymes necessary for their processing, suggesting that local ribosome maturation can occur far from the nucleus. These findings challenge conventional models that confine ribosome biogenesis to nuclear and perinuclear regions and suggest that neurons may fine-tune local protein synthesis by regulating ribosome assembly near synaptic sites. This mechanism may enable rapid modulation of the translational capacity in response to physiological changes, regulating synaptic plasticity and local protein synthesis in neurons. Significance StatementNeurons require precise spatial and temporal regulation of protein synthesis to adapt rapidly to external stimuli, particularly at synapses. Our study challenges the view that new ribosomes can be made exclusively near the nucleus and reveals that ribosome biogenesis factors and pre-rRNA processing enzymes are present in distal neuronal compartments. These findings suggest that ribosome maturation can occur locally in dendrites, enabling rapid, spatially targeted modulation of translational capacity. This mechanism provides a different framework for understanding how neurons regulate synaptic plasticity and adapt to physiological changes. By demonstrating that ribosome assembly may extend beyond the nucleus, this work highlights a previously unrecognized layer of neuronal protein synthesis control, potentially transforming our understanding of how neurons orchestrate local responses to environmental cues.

cell biology↗

bioRxiv2025

The proteasome maturation factor POMP moonlights as a stress-induced transcriptional regulator

Proteostasis - the maintenance of proteins at proper concentrations, conformations, and subcellular locations - is essential for cellular function and is governed by tightly regulated protein synthesis and degradation pathways. The Proteasome Maturation Protein (POMP) is a key chaperone involved in assembling the proteasome, the primary complex responsible for protein degradation. Despite the conserved role of POMP, its loss produces contrasting proteostatic effects in yeast and mammalian cells, pointing to additional, unexplored functions. In this study, we investigated the possibility that POMP plays a moonlighting role in proteostasis regulation. We discovered that upon proteasome disruption, POMP rapidly accumulates in the nucleolus in a manner dependent on HSF1 and reactive oxygen species (ROS). Proteomic analysis of POMP interactors revealed RNA processing factors and transcriptomic profiling showed that nucleolar POMP orchestrates a protective transcriptional program. Our findings indicate POMP is a built-in sensor and effector within the proteasome assembly pathway, capable of buffering disturbances in proteasome function through a novel, non-canonical role. Notably, this mechanism is developmentally controlled and active in neurodegenerative disease contexts. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/650603v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@196cd2borg.highwire.dtl.DTLVardef@177d240org.highwire.dtl.DTLVardef@18c9bcborg.highwire.dtl.DTLVardef@483b94_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Integrating computational and experimental biophysics reveals novel insights into the RAD51-BRC4 interaction

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/642044v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@32e13org.highwire.dtl.DTLVardef@c60a6forg.highwire.dtl.DTLVardef@1d6fdceorg.highwire.dtl.DTLVardef@1b10541_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACTC_FLOATNO C_FIG The interaction between the RAD51 and BRCA2 proteins is central for homologous recombination, a crucial pathway ensuring high-fidelity DNA repair. Recruitment of RAD51 involves eight highly conserved regions on BRCA2, named BRC repeats. To date, only the interaction between the fourth BRC repeat (BRC4) and the RAD51 C-terminal domain has been structurally characterized, while the complex of full-length RAD51 with the peptide still remains elusive. Here, we report an integrative experimental and in silico approach to reconstruct the conformational ensemble in solution for full length RAD51 in complex with BRC4. We combined AlphaFold2, crosslinking mass spectrometry (XL-MS) and small angle x-ray scattering (SAXS) data with molecular dynamics simulations (MD). These data show that the full-length RAD51-BRC4 complex is a mixture of compact and elongated conformations. Detailed analysis of the reweighted ensemble, achieved through the maximum entropy principle, identifies key residues at the N-terminal-BRC4 interface mediating complex conformational dynamics. Our evidence provides robust atomic-level insights into the interaction of RAD51 and BRC4. These findings are crucial for understanding the molecular features underlying the recognition between RAD51 and BRCA2, which are essential for developing therapeutic intervention strategies in cancer treatment.

biophysics↗

bioRxiv2024

Cell type-Specific In Vivo Proteomes with a Multi-copy Mutant Methionyl t-RNA Synthetase Mouse Line

The functional diversity of cells is driven by the different proteins they express. While improvements in protein labeling techniques have allowed for the measurement of proteomes with increased sensitivity, measuring cell type-specific proteomes in vivo remains challenging. One of the most useful pipelines is bioorthogonal non-canonical amino acid tagging (BONCAT) with the MetRS* system, consisting of a transgenic mouse line expressing a mutant methionyl-tRNA synthetase (MetRS*) controlled by Cre recombinase expression. This system allows for cell type-specific labeling of proteins with a non-canonical amino acid (azidonorleucine, ANL), which can be subsequently conjugated to affinity or fluorescent tags using click chemistry. Click-modified proteins can then be visualized, purified and identified. The reduction in sample complexity allows for the detection of small changes in protein composition. Here we describe a multicopy MetRS* mouse line (3xMetRS* mouse line), which exhibits markedly enhanced ANL protein labeling, boosting the sensitivity and temporal resolution of the system and eliminating the need for working under methionine depletion conditions. Cell type-specific in vivo labeling is possible even in heterozygous animals, thus offering an enormous advantage for crossing the line into mutation and disease-specific backgrounds. Using the 3xMetRS* line we identified the in vivo proteome of a sparse cell population - the dopaminergic neurons of the olfactory bulb and furthermore determined newly synthesized proteins after short labeling durations following a single intraperitoneal ANL injection.

cell biology↗

bioRxiv2024

p53 isoforms have a high aggregation propensity, interact with chaperones and lack binding to p53 interaction partners

The p53 transcription factor family consists of the three members p53, p63 and p73. Both p63 and p73 exist in different isoforms that are well characterized. Isoforms have also been identified for p53 and it has been proposed that they are responsible for increased cancer metastasis. In contrast to the p63 and p73 isoforms, which do not contain truncations in folded domains, most of the p53 isoforms contain only parts of either the DNA binding domain or the oligomerization domain. To better understand the effect of p53 isoforms in cancer we provide here a comprehensive biochemical characterization. With the exception of the {Delta}40p53 isoform none of the other variants can bind to DNA with high affinity and none can upregulate transcription. Probing with antibodies, DARPins and other interaction partners confirmed that isoforms harboring deletions in the DNA binding domain cannot interact specifically with them, but instead are bound to chaperones and other factors known to interact with misfolded proteins. Expression of isoforms with deletions in the DNA binding domain results in upregulation of cellular chaperones. If the expression level surpasses a threshold, the chaperone system can no longer keep these isoforms soluble resulting in aggregation and co-aggregation with other factors.

biophysics↗

bioRxiv2024

Engineering CRBN for rapid identification of next generation binders

The majority of clinical degrader candidates utilize an immunomodulatory imide drug (IMiD)-based derivative, that directs their target substrate to the E3 ligase receptor Cereblon (CRBN), however, concomitant neo-substrate degradation by IMiDs often results in severe off-target effects. Further biophysical screening is needed to discover CRBN binders that might overcome these safety concerns, but the previously reported CRBN protein constructs suffer significant limitations that reduce their applicability as tools for biophysical assays making large-scale screening efforts a costly endeavor. This is due to the requirement of CRBN co-expression with DDB1 in insect cells to produce soluble protein that contains all the reported structural features necessary for proper compound interaction. Here, a near full-length human CRBN construct was designed that retains these required features, but for the first time allows the generation of highly homogenous and cost-efficient expression in E.coli, eliminating the need for DDB1 co-expression. We have extensively profiled the new construct against the existing CRBN constructs in binary and ternary degrader complexes, showing it to be the "best of both worlds" in terms of binding characteristics and ease of protein production. We then designed the Enamine CRBN focused IMiD library of 4480-compounds and demonstrated applicability of the new construct to high throughput screening by identifying novel CRBN binders with high potency and ligand efficiency. The new methods described here should serve as valuable tools for the development of the next generation IMiD-based degraders.

biophysics↗

bioRxiv2023

The proteomic landscape of synaptic diversity across brain regions and cell types

Brain function relies on communication via neuronal synapses. Neurons build and diversify synaptic contacts using different protein combinations that define the specificity, function and plasticity potential of synapses. More than a thousand proteins have been globally identified in both pre- and postsynaptic compartments, providing substantial potential for synaptic diversity. While there is ample evidence of diverse synaptic structures, states or functional properties, the diversity of the underlying individual synaptic proteomes remains largely unexplored. Here we used 7 different Cre-driver mouse lines crossed with a floxed mouse line in which the presynaptic terminals were fluorescently labeled (SypTOM) to identify the proteomes that underlie synaptic diversity. We combined microdissection of 5 different brain regions with fluorescent-activated synaptosome sorting to isolate and analyze using quantitative mass spectrometry 18 types of synapses and their underlying synaptic proteomes. We discovered ~1800 unique synapse type-enriched proteins and allocated thousands of proteins to different types of synapses. We identify commonly shared synaptic protein modules and highlight the hotspots for proteome specialization. A protein-protein correlation network classifies proteins into modules and their association with synaptic traits reveals synaptic protein communities that correlate with either neurotransmitter glutamate or GABA. Finally, we reveal specializations and commonalities of the striatal dopaminergic proteome and outline the proteome diversity of synapses formed by parvalbumin, somatostatin and vasoactive intestinal peptide-expressing cortical interneuron subtypes, highlighting proteome signatures that relate to their functional properties. This study opens the door for molecular systems-biology analysis of synapses and provides a framework to integrate proteomic information for synapse subtypes of interest with cellular or circuit-level experiments.

neuroscience↗

bioRxiv2023

VAP spatially stabilizes dendritic mitochondria to locally fuel synaptic plasticity

Synapses are pivotal sites of memory formation and undergo plasticity in response to external inputs. Consequently, synapses are hotspots of energy consumption and are susceptible to dysfunction when their energy supplies are perturbed. Mitochondria are stabilized near synapses via cytoskeletal tethering and serve as local energy supplies to fuel synaptic plasticity. However, the mechanisms that tether and stabilize neuronal mitochondria for long durations and determine the spatial dendritic segment supported during synaptic plasticity are unknown. We identified a list of novel mitochondrial-cytoskeletal interactors in neurons using APEX-based proximity labeling. We narrowed down the protein candidates that exclusively tether mitochondria to actin near postsynaptic spines using high-resolution Airyscan confocal imaging. We find that VAP, the vesicle-associated membrane protein-associated protein implicated in Amyotrophic Lateral Sclerosis and interacts with the endoplasmic reticulum, stabilizes mitochondria via actin near the spines. To test if the VAP-dependent stable mitochondrial compartments can locally support synaptic plasticity, we investigated individual spines stimulated by two-photon glutamate uncaging for spine plasticity induction and their adjacent spines. We find that, along with actin, VAP functions as a spatial stabilizer of mitochondrial compartments to sustain synaptic plasticity for up to ~60 min and as a spatial ruler that determines the ~30 m length of the dendritic segment supporting synaptic plasticity.

neuroscience↗

bioRxiv2022

An Abundance of Free Proteasomal Regulatory (19S) Particles Regulate Neuronal Synapses Independent of the Proteasome

The major protein-degradation machine, the proteasome, functions at brain synapses and regulates long-term information storage. Here we found that the two essential subcomplexes of the proteasome, the regulatory (19S) and catalytic (20S) particles that recognize and degrade substrates, are differentially distributed within individual rat cortical neurons. Using superresolution microscopy, we discovered a surprising abundance of free particles (19S) near synapses. The free neuronal 19S particles bind and deubiquitylate Lys63-ubiquitin, a non-proteasome targeting ub-linkage. Pull-down assays revealed a significant over-representation of synaptic molecules as Lys63 interactors. Inhibition of 19S deubiquitylase activity significantly altered excitatory synaptic transmission and reduced the synaptic availability of AMPA receptors at multiple trafficking points in a proteasome-independent manner. Together, these results reveal a moonlighting function of the regulatory proteasomal subcomplex near synapses. One-Sentence SummaryThe 19S subcomplex of the major protein-degradation machine, the proteasome, functions without its 20S partner to populate and regulate synapses.

neuroscience↗

bioRxiv2022

Isolation of a novel heterodimeric PSII complex via strep-tagged PsbO

The multi-subunit membrane protein complex Photosystem II (PSII) catalyzes the light-driven oxidation of water and with this the initial step of photosynthetic electron transport in plants, algae, and cyanobacteria. Its biogenesis is coordinated by a network of auxiliary proteins that facilitate the stepwise assembly of individual subunits and cofactors, forming various intermediate complexes until fully functional mature PSII is present at the end of the process. In the current study, we purified PSII complexes from a mutant line of the thermophilic cyanobacterium Thermosynechococcus vestitus BP-1 in which the extrinsic subunit PsbO, characteristic for active PSII, was fused with an N-terminal Twin-Strep-tag. Three distinct PSII complexes were separated by ion-exchange chromatography after the initial affinity purification. Two complexes differ in their oligomeric state (monomeric and dimeric) but share the typical subunit composition of mature PSII. They are characterized by the very high oxygen-evolving activity of approx. 6,000 {micro}mol O2{middle dot} (mg Chl{middle dot}h)-1. Analysis of the third (heterodimeric) PSII complex revealed lower oxygen-evolving activity of approx. 3,000 {micro}mol O2{middle dot} (mg Chl{middle dot}h)-1 and manganese content of 2.7 ({+/-} 0.2) per reaction center compared to 3.7 ({+/-} 0.2) of fully active PSII. Mass spectrometry and time-resolved fluorescence spectroscopy further indicated that PsbO is partially replaced by Psb27 in this PSII fraction, thus implying a role in the repair of the complex.

plant biology↗

bioRxiv2021

Co-translational assembly counteracts promiscuous interactions

During the co-translational assembly of protein complexes, a fully synthesized subunit engages with the nascent chain of a newly synthesized interaction partner. Such events are thought to contribute to productive assembly, but their exact physiological relevance remains underexplored. Here, we examined structural motifs contained in nucleoporins for their potential to facilitate co-translational assembly. We experimentally tested candidate structural motifs and identified several previously unknown co-translational interactions. We demonstrate by selective ribosome profiling that domain invasion motifs of beta-propellers, coiled-coils, and short linear motifs act as co-translational assembly domains. Such motifs are often contained in proteins that are members of multiple complexes (moonlighters) and engage with closely related paralogs. Surprisingly, moonlighters and paralogs assembled co-translationally in only one but not all of the relevant assembly pathways. Our results highlight the regulatory complexity of assembly pathways.

molecular biology↗

bioRxiv2021

Dynamic bi-directional phosphorylation events associated with the reciprocal regulation of synapses during homeostatic up- and down-scaling.

Homeostatic synaptic scaling allows for bi-directional adjustment of the strength of synaptic connections in response to changes in their input. Protein phosphorylation modulates many neuronal and synaptic processes, but it has not been studied on a global, proteome-wide scale during synaptic scaling. To examine this, we used LC-MS/MS analyses to measure changes in the phosphoproteome in response to up- or down-scaling in cultured cortical neurons over minutes to 24 hours. Out of 45,000 phosphorylation events measured, ~3,300 (associated with 1,280 phospho-proteins) were regulated by homeostatic scaling. The activity-sensitive phosphoproteins were predominantly located at synapses and involved in cytoskeletal reorganization. We identified many early transient phosphorylation events which could serve as sensors for the activity offset as well as late and/or persistent phosphoregulation that could represent effector mechanisms driving the homeostatic response. Much of the persistent phosphorylation was reciprocally regulated by up- or down-scaling, suggesting that the mechanisms underlying these two poles of synaptic regulation make use of a common signaling axis.

neuroscience↗

bioRxiv2021

Neuronal ribosomes dynamically exchange ribosomal proteins in a context-dependent manner.

Owing to their morphological complexity and dense network connections, neurons modify their proteomes locally, using mRNAs and ribosomes present in the neuropil (tissue enriched for dendrites and axons). Although ribosome biogenesis largely takes place in the nucleus and perinuclear region, neuronal ribosomal protein (RP) mRNAs have been frequently detected remotely, in dendrites and axons. Here, using imaging and ribosome profiling, we directly detected the RP mRNAs and their translation in the neuropil. Combining brief metabolic labeling with mass spectrometry, we found that a group of RPs quickly associated with translating ribosomes in the cytoplasm and that this incorporation is independent of canonical ribosome biogenesis. Moreover, the incorporation probability of some RPs was regulated by location (neurites vs. cell bodies) and changes in the cellular environment (in response to oxidative stress). Our results suggest new mechanisms for the local activation, repair and/or specialization of the translational machinery within neuronal processes, potentially allowing remote neuronal synapses a rapid solution to the relatively slow and energy-demanding requirement of nuclear ribosome biogenesis.

molecular biology↗