Chemical synapses are the major sites of communication between neurons in the nervous system and mediate either excitatory or inhibitory signaling. At excitatory synapses, glutamate is the primary neurotransmitter and upon release from presynaptic vesicles, is detected by postsynaptic glutamate receptors, which include ionotropic AMPA and NMDA receptors. Here we have developed methods to identify glutamatergic synapses in brain tissue slices, label AMPA receptors with small gold nanoparticles (AuNPs), and prepare lamella for cryo-electron tomography studies. The targeted imaging of glutamatergic synapses in the lamella is facilitated by fluorescent pre- and postsynaptic signatures, and the subsequent tomograms allow for identification of key features of chemical synapses, including synaptic vesicles, the synaptic cleft and AuNP-labeled AMPA receptors. These methods pave the way for imaging natively derived brain regions at high resolution, using unstained, unfixed samples preserved under near-native conditions.

The scaffolding function of receptor interacting protein kinase 1 (RIPK1) confers intrinsic and extrinsic resistance to immune checkpoint blockades (ICBs) and has emerged as a promising target for improving cancer immunotherapies. To address the challenge posed by a poorly defined binding pocket within the intermediate domain, we harnessed proteolysis targeting chimera (PROTAC) technology to develop a first-in-class RIPK1 degrader, LD4172. LD4172 exhibited potent and selective RIPK1 degradation both in vitro and in vivo. Degradation of RIPK1 by LD4172 triggered immunogenic cell death (ICD) and enriched tumor-infiltrating lymphocytes and substantially sensitized the tumors to anti-PD1 therapy. This work reports the first RIPK1 degrader that serves as a chemical probe for investigating the scaffolding functions of RIPK1 and as a potential therapeutic agent to enhance tumor responses to immune checkpoint blockade therapy.

Reducing fibrous aggregates of protein tau is a possible strategy for halting progression of Alzheimer’s disease (AD). Previously we found that in vitro the D-peptide D-TLKIVWC disassembles tau fibrils from AD brains (AD-tau) into benign segments with no energy source present beyond ambient thermal agitation. This disassembly by a short peptide was unexpected, given that AD-tau is sufficiently stable to withstand disassembly in boiling SDS detergent. To consider D peptide-mediated disassembly as a potential therapeutic for AD, it is essential to understand the mechanism and energy source of the disassembly action. We find assembly of D-peptides into amyloid-like fibrils is essential for tau fibril disassembly. Cryo-EM and atomic force microscopy reveal that these D-peptide fibrils have a right-handed twist and embrace tau fibrils which have a left-handed twist. In binding to the AD-tau fibril, the oppositely twisted D-peptide fibril produces a strain, which is relieved by disassembly of both fibrils. This strain-relief mechanism appears to operate in other examples of amyloid fibril disassembly and provides a new direction for the development of first-in-class therapeutics for amyloid diseases.

Significant technical challenges exist when measuring synaptic connections between neurons in living brain tissue. The patch clamping technique, when used to probe for synaptic connections, is manually laborious and time-consuming. To improve its efficiency, we pursued another approach: instead of retracting all patch clamping electrodes after each recording attempt, we cleaned just one of them and reused it to obtain another recording while maintaining the others. With one new patch clamp recording attempt, many new connections can be probed. By placing one pipette in front of the others in this way, one can “walk” across the tissue, termed “patch-walking.” We performed 136 patch clamp attempts for two pipettes, achieving 71 successful whole cell recordings (52.2%). Of these, we probed 29 pairs (i.e., 58 bidirectional probed connections) averaging 91 μm intersomatic distance, finding 3 connections. Patch-walking yields 80-92% more probed connections, for experiments with 10-100 cells than the traditional synaptic connection searching method.

Starvation triggers bacterial spore formation, a committed differentiation program that transforms a vegetative cell into a dormant spore. Cells in a population enter sporulation non-uniformly to secure against the possibility that favorable growth conditions, which puts sporulation-committed cells at a disadvantage, may resume. This heterogeneous behavior is initiated by a passive mechanism: stochastic activation of a master transcriptional regulator. Here, we identify a cell-cell communication pathway that actively promotes phenotypic heterogeneity, wherein Bacillus subtilis cells that start sporulating early utilize a calcineurin-like phosphoesterase to release glycerol, which simultaneously acts as a signaling molecule and a nutrient to delay non-sporulating cells from entering sporulation. This produced a more diverse population that was better poised to exploit a sudden influx of nutrients compared to those generating heterogeneity via stochastic gene expression alone. Although conflict systems are prevalent among microbes, genetically encoded cooperative behavior in unicellular organisms can evidently also boost inclusive fitness.

The prediction of pathological changes on single cell behaviour is a challenging task for deep learning models. Indeed, in self-supervised learning methods, no prior labels are used for the training and all of the information for event predictions are extracted from the data themselves. We present here a novel self-supervised learning model for the detection of anomalies in a given cell population, StArDusTS. Cells are monitored over time, and analysed to extract time-series of dry mass values. We assessed its performances on different cell lines, showing a precision of 96% in the automatic detection of anomalies. Additionally, anomaly detection was also associated with cell measurement errors inherent to the acquisition or analysis pipelines, leading to an improvement of the upstream methods for feature extraction. Our results pave the way to novel architectures for the continuous monitoring of cell cultures in applied research or bioproduction applications, and for the prediction of pathological cellular changes.

The discovery that experimental delivery of dsRNA can induce gene silencing at target genes revolutionized genetics research, by both uncovering essential biological processes and creating new tools for developmental geneticists. However, the efficacy of exogenous RNA interference (RNAi) varies dramatically within the Caenorhabditis elegans natural population, raising questions about our understanding of RNAi in the lab relative to its activity and significance in nature. Here, we investigate why some wild strains fail to mount a robust RNAi response to germline targets. We observe diversity in mechanism: in some strains, the response is stochastic, either on or off among individuals, while in others, the response is consistent but delayed. Increased activity of the Argonaute PPW-1, which is required for germline RNAi in the laboratory strain N2, rescues the response in some strains but dampens it further in others. Among wild strains, genes known to mediate RNAi exhibited very high expression variation relative to other genes in the genome as well as allelic divergence and strain-specific instances of pseudogenization at the sequence level. Our results demonstrate functional diversification in the small RNA pathways in C. elegans and suggest that RNAi processes are evolving rapidly and dynamically in nature.

The efficient cytosolic delivery of proteins is critical for advancing novel therapeutic strategies. Current delivery methods are severely limited by endosomal entrapment, and detection methods lack sophistication in tracking the fate of delivered protein cargo. HaloTag, a commonly used protein in chemical biology and a challenging delivery target, is an exceptional model system for understanding and exploiting cellular delivery. Here, we employed a combinatorial strategy to direct HaloTag to the cytosol. We established the use of Virginia Orange, a pH-sensitive fluorophore, and Janelia Fluor 585, a similar but pH-agnostic fluorophore, in a fluorogenic assay to ascertain protein localization within human cells. Using this assay, we investigated HaloTag delivery upon modification with cell-penetrating peptides, carboxyl group esterification, and cotreatment with an endosomolytic agent. We found efficacious cytosolic entry with two distinct delivery methods. This study expands the toolkit for detecting the cytosolic access of proteins and highlights that multiple intracellular delivery strategies can be used synergistically to effect cytosolic access. Moreover, HaloTag is poised to serve as a platform for the delivery of varied cargo into human cells.

Focal adhesions (FAs) connect inner workings of cell to the extracellular matrix to control cell adhesion, migration and mechanosensing. Previous studies demonstrated that FAs contain three vertical layers, which connect extracellular matrix to the cytoskeleton. By using super-resolution iPALM microscopy, we identify two additional nanoscale layers within FAs, specified by actin filaments bound to tropomyosin isoforms Tpm1.6 and Tpm3.2. The Tpm1.6-actin filaments, beneath the previously identified α-actinin cross-linked actin filaments, appear critical for adhesion maturation and controlled cell motility, whereas the adjacent Tpm3.2-actin filament layer beneath seems to facilitate adhesion disassembly. Mechanistically, Tpm3.2 stabilizes ACF-7/MACF1 and KANK-family proteins at adhesions, and hence targets microtubule plus-ends to FAs to catalyse their disassembly. Tpm3.2 depletion leads to disorganized microtubule network, abnormally stable FAs, and defects in tail retraction during migration. Thus, FAs are composed of distinct actin filament layers, and each may have specific roles in coupling adhesions to the cytoskeleton, or in controlling adhesion dynamics.

The accelerating pace of technological advancements necessitates specialised expertise and cutting-edge instruments to maintain competitive research in life sciences. Core facilities - collaborative laboratories equipped with state-of-the-art tools and staffed by expert personnel - are vital resources that support diverse scientific endeavours. However, their adoption in lower-income communities has been comparatively stagnant due to both financial and cultural challenges. This paper explores the perils of not supporting core facilities on national research enterprises, underscoring the need for balanced investments in discovery science and crucial infrastructure support. We explore the implications from the perspectives of funders, university leaders and lab heads. We advocate for a paradigm shift to recognise these facilities as essential components of national research efforts. Core facilities are positioned not as optional but as strategic investments that can catalyse breakthroughs, particularly in environments with limited resources.