Biology subjects

Anguiano, M.

Publications and source records attributed to Anguiano, M..

5 recordsLinked to original sources

bioRxiv2026

Computational design of a versatile, zero-radius proximity labeling enzyme

The ability to map protein interactomes and organelle proteomes is foundational for achieving a molecular understanding of living cells. Proximity labeling (PL) provides a powerful strategy for this, but existing enzymes and photocatalysts are limited by their spatial resolution, reliance on biotin, and/or in vivo compatibility. Here we report FlexID, an engineered promiscuous ligase that catalyzes the rapid attachment of diverse small-molecule probes to proximal endogenous proteins. Critically, FlexID operates through a zero-radius, direct-contact mechanism, offering superior spatial precision compared to existing PL tools. We engineered FlexID by combining the strengths of sequence- and structure-trained computational models to enhance its catalytic activity and structural stability. Biophysical analysis revealed that specific conformational changes in FlexID improve its ability to recognize diverse target proteins while simultaneously preventing the premature release of the reactive intermediate. We demonstrate FlexID's versatility through in vivo proximity labeling, comprehensive organelle proteome mapping, and a high-throughput, fluorescence-based screen for molecular glues. Our work shows that computational methods can be harnessed to create mechanistically distinct PL enzymes and establishes FlexID as a flexible, high-resolution tool for mapping protein interactions and proteomes in living cells.

bioengineering↗

bioRxiv2025

Acute in vivo proximity labeling for membrane targeted proteomics in neuronal circuits

MOTIVATIONDespite the growing use of proximity labeling tools, there are limited techniques available to identify subcellularly localized proteins within targeted neuronal circuits on acute timescales in vivo. We engineered membrane targeted versions of the proximity labeling enzyme TurboID and validated their use for proteomic discovery in the mouse brain. We optimized an in vivo protocol to identify proteins in cell bodies and long range neuronal projections, and proteins differentially detected during acute drug labeling windows. SUMMARYA major goal within molecular systems neuroscience is to bridge the study of neuronal circuit function with changes in protein expression and localization in awake behaving animals. However, there are limited tools for capturing changes in subcellularly-defined proteomes within neuronal circuits during activity-gated timescales in vivo. Here, we engineered targeted versions of the proximity labeling enzyme TurboID, to tag proteins at the neuronal membrane during a user-delivered biotin injection. We optimized a labeling strategy that enables a one-to-two-hour labeling window and tagged proteins in medial prefrontal cortex (mPFC) cell bodies and corresponding axons in a downstream projection. We performed proteomics to identify proteins enriched in mPFC cell bodies and terminals, and upregulated in mPFC cell bodies following an acute cocaine injection. These advancements enable the detection of proteins at the subcellular level within short labeling windows, allowing identification of stimulus-specific proteomes in behaving mice.

neuroscience↗

bioRxiv2023

Rapid, biochemical tagging of cellular activity history in vivo

Intracellular calcium (Ca2+) is ubiquitous to cell signaling across all biology. While existing fluorescent sensors and reporters can detect activated cells with elevated Ca2+ levels, these approaches require implants to deliver light to deep tissue, precluding their noninvasive use in freely-behaving animals. Here we engineered an enzyme-catalyzed approach that rapidly and biochemically tags cells with elevated Ca2+ in vivo. Ca2+-activated Split-TurboID (CaST) labels activated cells within 10 minutes with an exogenously-delivered biotin molecule. The enzymatic signal increases with Ca2+ concentration and biotin labeling time, demonstrating that CaST is a time-gated integrator of total Ca2+ activity. Furthermore, the CaST read-out can be performed immediately after activity labeling, in contrast to transcriptional reporters that require hours to produce signal. These capabilities allowed us to apply CaST to tag prefrontal cortex neurons activated by psilocybin, and to correlate the CaST signal with psilocybin-induced head-twitch responses in untethered mice.

neuroscience↗

bioRxiv2022

The circadian clock conveys thermal and photoperiodic cues to modulate EYES ABSENT via the neuropeptide PDF to regulate seasonal physiology

Organisms adapt to seasonal changes in photoperiod and temperature to survive; however, the mechanisms by which these signals are integrated in the brain are poorly understood. We previously reported that EYES ABSENT (EYA) in Drosophila shows higher levels in cold temperature or short photoperiod, and genetic ablation of eya in the fly brain inhibits reproductive dormancy, suggesting that EYA promotes winter physiology. Nevertheless, the mechanisms by which EYA senses seasonal cues are unclear. Pigment-Dispersing Factor (PDF) is a neuropeptide important for photoentrainment and regulation of circadian output rhythms. Interestingly, PDF also regulates reproductive dormancy, suggesting that it may mediate the function of the circadian clock in modulating seasonal physiology. In this study, we investigated the role of PDF signaling in mediating the impact of EYA on seasonal biology. First, we subjected flies to different photoperiodic and temperature regimes and observed that PDF abundance is lower in cold and short days, compared to warm and long days. Interestingly, the response of PDF to seasonal cues is opposite of what was observed for EYA. We then determined the potential for PDF to convey seasonal cues and modulate EYA function in seasonality by assessing coexpression of EYA and PDF receptor. Our results indicated that PDF receptor (PDFR) is indeed coexpressed with EYA in the fly brain, including in the circadian clock neuronal network and neurons in the pars intercerebralis. We then manipulated PDF signaling in eya+ cells to show that PDF modulates seasonal adaptations in daily activity rhythm and ovary development via EYA-dependent and independent mechanisms. At the molecular level, manipulating PDF signaling impacted EYA protein abundance. Specifically, we showed that protein kinase A (PKA), an effector of PDF signaling, phosphorylates EYA and promotes its degradation. This explains the opposite responses of PDF and EYA abundance to changes in seasonal cues. In summary, our results support a model in which PDF signaling negatively modulates EYA levels to regulate seasonal physiology, linking the circadian clock to the modulation of seasonal adaptations.

neuroscience↗

bioRxiv2022

Novel cerebello-amygdala connections provide missing link between cerebellum and limbic system

The cerebellum is emerging as a powerful regulator of cognitive and affective processing and memory in both humans and animals and has been implicated in affective disorders. How the cerebellum supports affective function remains poorly understood. The short-latency (just a few ms) functional connections that were identified between the cerebellum and amygdala -a structure crucial for the processing of emotion and valence-more than 4 decades ago raise the exciting, yet untested, possibility that a cerebellum-amygdala pathway communicates information important for emotion. The major hurdle in rigorously testing this possibility is the lack of knowledge about the anatomy and functional connectivity of this pathway. Our initial anatomical tracing studies in mice excluded the existence of a direct monosynaptic pathway between cerebellum and amygdala. Using transneuronal tracing techniques, we have identified a novel disynaptic pathway that connects the cerebellar output nuclei to the basolateral amygdala. This pathway recruits the understudied intralaminar thalamus as a node. Using ex vivo optophysiology and super-resolution microscopy, we provide the first evidence for the functionality of the pathway, thus offering a missing mechanistic link between the cerebellum and amygdala. This discovery provides a connectivity blueprint between the cerebellum and a key structure of the limbic system. As such, it is the requisite first step toward obtaining new knowledge about cerebellar function in emotion, thus fundamentally advancing understanding of the neurobiology of emotion, which is perturbed in mental and autism spectrum disorders.

neuroscience↗