Biology subjects

Martin, J. G.

Publications and source records attributed to Martin, J. G..

10 recordsLinked to original sources

bioRxiv2026

Protein-based, live-cell PAINT microscopy with fluorogenic exchangeable HaloTag ligands

We report a generalizable method for single-molecule localization super-resolution microscopy in living cells. Live-cell PAINT (points accumulation for imaging in nanoscale topography) and single-molecule diffusivity mapping (SMdM) of intracellular protein targets are achieved by pairing exceptionally fluorogenic bis-trifluoromethyl rhodamine (BF) dyes with reversible HaloTag ligands and widely available HaloTag fusion proteins. This far-red small-molecule update to protein-based PAINT is readily incorporated into existing super-resolution microscopy workflows: wash-free staining and imaging are achieved for cell lines and primary neuron cultures for diverse protein targets. Pairing with photoconvertible fluorescent proteins further enables simultaneous two-color live-cell super-resolution microscopy and SMdM.

cell biology↗

bioRxiv2025

GPR17 structure and agonism with small molecules and oxysterols

GPR17 is a class A orphan GPCR that regulates myelination in the central nervous system. Inhibiting GPR17 is a potential therapeutic mechanism to induce remyelination. The limited understanding of the structural basis of GPR17 agonism and antagonism is a challenge for GPR17 drug discovery. We present novel cryo-EM structures of GPR17 with bound orthosteric antagonist or agonist/G protein heterotrimer with biophysical and cellular characterization to inform rational drug discovery. Both ligands bind towards a lateral edge of the orthosteric pocket, while an exceptionally long extracellular loop 2 occupies and limits access to this site. In contrast, pharmacological and photoaffinity studies indicate that endogenous oxysterol agonists of GPR17 bind allosterically. Characterization of oxysterol agonism versus a known orthosteric ligand in oligodendrocytes further supports an allosteric mechanism. Therefore, the design of more potent orthosteric ligands is limited by pocket size and accessibility. Targeting allosteric binding offers an alternative approach to GPR17 antagonism.

biochemistry↗

bioRxiv2025

CYFIP1 overexpression amplifies IL-6/STAT3 and IFN-γ/STAT1 signaling: potential implications for neuroinflammation and autism spectrum disorder

Autism spectrum disorder (ASD) encompasses a group of neurodevelopmental disorders characterised by impaired social interaction, delayed language development, and repetitive or restrictive behaviours. While both genetic and environmental factors contribute to ASD, the specific molecular mechanisms underlying these interactions remain unclear. In a recent preprint, we showed that cytokine signaling may be dysregulated in chromosome 15q-duplication syndrome (Dup(15q)), one of the most common syndromic forms of ASD. Specifically, Dup(15q) induced pluripotent stem cells (iPSC)-derived neurons exhibit an amplified Signal Transducer and Activator of Transcription-3 (STAT3) response to Interleukin-6 (IL-6), a proinflammatory cytokine often upregulated in ASD. To identify which gene in the 15q region may be responsible for modifying cytokine signalling responses in Dup(15q), we focus on the Cytoplasmic FMRP-Interacting Protein 1 (CYFIP1) gene, as increased CYFIP1 dosage in Dup(15q) is associated with increased severity of neurobehavioral symptoms in individuals with Dup(15q) and CYFIP1 dysregulation has been linked to altered expression of various genes involved in immunoregulatory signalling pathways. Here, we explore the eeects of CYFIP1 overexpression (CYFIP1-OE) on cytokine signaling, demonstrating that CYFIP1-OE in HEK293 cells modifies the expression and activity of several cytokine signaling-related transcription factors, including STAT3 and STAT1. Additionally, we use SH-SY5Y neuroblastoma cells to assess neuron-related phenotypes, showing that CYFIP1-OE alters IL-6-induced neurite outgrowth. These findings provide novel insights into how CYFIP1 may contribute to dysregulated cytokine responses in ASD, advancing our understanding of the molecular mechanisms that underlie neuroinflammatory processes in this disorder.

molecular biology↗

bioRxiv2025

Sleeping ORANGE: A CRISPR-Transposase Hybrid Approach to Boost Endogenous Protein Tagging Efficiency

BackgroundInvestigating the subcellular distribution of proteins is crucial for understanding complex cell behaviours and disease mechanisms, and fluorescence microscopy has become a key tool for visualising protein localisation. Endogenous protein tagging, where the sequence for a tag (typically a peptide or fluorescent protein) is integrated into the native genetic sequence encoding a protein of interest, enables proteins to be visualised without the need for antibodies against the target protein. ORANGE (Open Resource for the Application of Neuronal Genome Editing) is a CRISPR-Cas9-based endogenous protein tagging technique which relies on homology-independent targeted integration (HITI)-mediated gene editing. Utilising HITI as the DNA repair pathway of choice gives ORANGE the advantage of being more efficient than classical homology-directed repair (HDR)-based endogenous protein tagging techniques and additionally, means it can be used in post-mitotic cells. ResultsWe applied the ORANGE system to tag three proteins, CYFIP1, JAKMIP1, and STAT3, and confirmed that the expressed fusion proteins demonstrate expected subcellular localisations through fluorescence microscopy. Unexpectedly, the efficiency of ORANGE editing was less than 1% in HEK293 cells, despite high transfection efficiency. To improve the editing efficiency associated with ORANGE, we combined the ORANGE method with an established Sleeping Beauty transposase/CRISPR-Cas9 fusion technique, which has been shown to enhance HITI-mediated gene editing. Using this new method, which we term Sleeping ORANGE, we successfully tagged CYFIP1 with the fluorescent protein mNeonGreen. Importantly, quantitative analysis by fluorescence microscopy and flow cytometry demonstrated an increase in editing efficiency using Sleeping ORANGE, with an approximately 12.85-fold increase in the percentage of mNeonGreen-expressing cells at 72 hours post-transfection relative to populations of cells edited with the ORANGE method. ConclusionsWe have incorporated the DNA-binding domain of the Sleeping Beauty transposase to create a new system that improves the gene-editing efficiency of the ORANGE technique. With further developments to optimise CRISPR gRNA design and reduce off-target effects, the Sleeping ORANGE technique may form a valuable tool for researchers to better understand subcellular localisation and dynamics.

molecular biology↗

bioRxiv2025

Exploring the functions of JAKMIP1 in neuronal IL-6/STAT3 signaling and its relevance to chromosome 15q-duplication syndrome

Growing evidence supports neuroinflammation as a risk factor for neurodevelopmental and psychiatric disorders. Interleukin 6 (IL-6), a classical pro-inflammatory cytokine, has been associated with autism spectrum disorder (ASD)-related phenotypes. To better understand molecular factors that modify neuronal cytokine responses in ASD, we investigated potential roles for JAKMIP1, a gene linked to chromosome 15q-duplication syndrome (Dup15q; a form of syndromic ASD), in regulating IL-6/STAT3 signaling. We observe that JAKMIP1 deficiency impairs IL-6/STAT3 signaling and IL-6-induced neuritogenesis in SH-SY5Y cells; and discover that JAKMIP1 may regulate STAT3 expression via its C-terminus, which exhibits nucleoplasmic localization. Additionally, we find that IL-6/STAT3 signaling is altered in Dup15q hiPSCs-derived cortical neurons, which display heightened responsiveness to IL-6; though it is unclear whether and how JAKMIP1 contributes to this. Overall, our findings identify JAKMIP1 as a modulator of neuronal IL-6/STAT3 signaling and support that ASD-linked genetic variants can alter the inflammatory landscape of ASD.

neuroscience↗

bioRxiv2024

Multilevel selection on individual and group social behaviour in the wild

The degree to which phenotypes are shaped by multilevel selection - the theoretical framework proposing natural selection occurs at more than one level of biological organisation - is a classic debate in biology. Though social behaviours are a common theoretical example for multilevel selection, it is unknown if and how multilevel selection acts on sociality in the wild. We studied the relative strength of multilevel selection on both individual behaviour and group social structure, quantified with social networks and 19 years of data from a wild, free-living mammal, the yellow-bellied marmot (Marmota flaviventer). Contextual analysis revealed multilevel selection in specific fitness and life history contexts, with selection for group social structure being just as strong, if not stronger, than individual social behaviour. We also found antagonistic multilevel selection within and between levels, potentially explaining why increased sociality is not as beneficial or heritable in this system comparatively to other social taxa. Thus, the evolutionary dynamics of hierarchal or nested biological traits should be assessed at multiple levels simultaneously to tell a more accurate and comprehensive story. Overall, we provide empirical evidence suggesting that multilevel selection acts on social relationships and structures in the wild, and provide direct evidence for a classic, unanswered question in biology.

evolutionary biology↗

bioRxiv2020

Archaeogenomic Distinctiveness of the Isthmo-Colombian Area

The recently enriched genomic history of Indigenous groups in the Americas is still meagre concerning continental Central America. Here, we report ten pre-Hispanic (plus two early colonial) genomes and 84 genome-wide profiles from seven groups presently living in Panama. Our analyses reveal that pre-Hispanic demographic changes and isolation events contributed to create the extensive genetic structure currently seen in the area, which is also characterized by a distinctive Isthmo-Colombian Indigenous component. This component drives these populations on a specific variability axis and derives from the local admixture of different ancestries of northern North American origin(s). Two of these ancestries were differentially associated to Pleistocene Indigenous groups that also moved into South America leaving heterogenous footprints. An additional Pleistocene ancestry was brought by UPopI, a still unsampled population that remained restricted to the Isthmian area, expanded locally during the early Holocene, and left genomic traces up to the present.

genomics↗

bioRxiv2020

Microsaccades during high speed continuous visual search

Here, we provide an analysis of the microsaccades that occurred during continuous visual search and targeting of small faces that we pasted either into cluttered background photos or into a simple gray background. Subjects continuously used their eyes to target singular 3-degree upright or inverted faces in changing scenes. As soon as the participants gaze reached the target face, a new face was displayed in a different and random location. Regardless of the experimental context (e.g. background scene, no background scene), or target eccentricity (from 4 to 20 degrees of visual angle), we found that the microsaccade rate dropped to near zero levels within only 12 milliseconds after trial onset. There were almost never any microsaccades before the saccade to the face. One subject completed 118 consecutive trials without a single microsaccade. However, in about 20% of the trials, there was a single corrective microsaccade that occurred almost immediately after the preceding saccades offset. These corrective microsaccades were task oriented because their facial landmark targeting distributions matched those of saccades within both the upright and inverted face conditions. Our findings show that a single feedforward pass through the visual hierarchy for each stimulus is likely all that is needed to effectuate prolonged continuous visual search. In addition, we provide evidence that microsaccades can serve perceptual functions like correcting saccades or effectuating task-oriented goals during continuous visual search.

neuroscience↗

bioRxiv2020

Evidence for face selectivity in early vision

The commonly accepted "simple-to-complex" model of visual processing in the brain posits that visual tasks on complex objects such as faces are based on representations in high-level visual areas. Yet, recent experimental data showing the visual systems ability to localize faces in natural images within 100ms (Crouzet et al., 2010) challenge the prevalent hierarchical description of the visual system, and instead suggest the hypothesis of face-selectivity in early visual areas. In the present study, we tested this hypothesis with human participants in two eye tracking experiments, an fMRI experiment and an EEG experiment. We found converging evidence for neural representations selective for upright faces in V1/V2, with latencies starting around 40 ms post-stimulus onset. Our findings suggest a revision of the standard "simple-to-complex" model of hierarchical visual processing. Significance statementVisual processing in the brain is classically described as a series of stages with increasingly complex object representations: early visual areas encode simple visual features (such as oriented bars), and high-level visual areas encode representations for complex objects (such as faces). In the present study, we provide behavioral, fMRI, and EEG evidence for representations of complex objects - namely faces - in early visual areas. Our results challenge the standard "simple-to-complex" model of visual processing, suggesting that it needs to be revised to include neural representations for faces at the lowest levels of the visual hierarchy. Such early object representations would permit the rapid and precise localization of complex objects, as has previously been reported for the object class of faces.

neuroscience↗

bioRxiv2019

Stimulus Onset Hub: An open-source, low latency, and opto-isolated trigger box for neuroscientific research replicability and beyond.

There is currently a replication crisis in many fields of neuroscience and psychology, with some estimates claiming up to 64% of research in psychological science is not reproducible. Three common culprits which have been suspected to cause the failure to replicate such studies are small sample sizes, \"hypothesizing after the results are known,\" and \"p-hacking.\" Here, we introduce accurate stimulus timing as an additional possibility. Accurate stimulus onset timing is critical to almost all psychophysical research. Auditory, visual, or manual response time stimulus onsets are typically sent through wires to various machines that record data such as: eye gaze positions, electroencephalography, stereo electroencephalography, and electrocorticography. These stimulus onsets are collated and analyzed according to experimental condition. If there is variability in the temporal accuracy of the delivery of these onsets to external systems, the quality of the resulting data and scientific analyses will degrade. Here, we describe an approximately $200 Arduino based system and associated open-source codebase which achieved a 5.34 microsecond delay from the inputs to the outputs while electrically opto-isolating the connected external systems. Using an oscilloscope, the device is configurable for different environmental conditions particular to each laboratory (e.g. light sensor type, screen type, speaker type, stimulus type, temperature, etc). This low-cost open-source project delivered electrically isolated stimulus onset Transistor-Transistor Logic triggers with a median precision of 5.34 microseconds and was successfully tested with 7 different external systems that record eye and neurological data.

neuroscience↗