Biology subjects

Tiroumalechetty, A.

Publications and source records attributed to Tiroumalechetty, A..

3 recordsLinked to original sources

bioRxiv2026

Patient CACNA1A variant uncouples calcium channel conductance from synaptic output

Channelopathies are a class of neurodevelopmental disorders with often devastating consequences, and effective therapies depend on understanding how patient variants alter channel function. These effects are typically assessed by biophysical characterization in heterologous expression systems. Mutations in CACNA1A, which encodes the P/Q-type calcium channel CaV2.1, underlie a spectrum of neurological disorders in which symptoms are generally classified as loss-of-function (LoF) or gain-of-function (GoF). However, some patients present with overlapping phenotypes that defy this binary framework. Here we describe a CACNA1A variant for which heterologous assays fail to capture a key in vivo functional effect. We characterize a de novo variant of a highly conserved residue, D1634N, identified in a patient with a mixed clinical presentation that includes both LoF- and GoF-associated symptoms. Biophysical characterization in HEK293T cells supports a classic and severe LoF effect, including reduced current density and a right-shifted current-voltage relationship. In contrast, in vivo analysis of the corresponding endogenous variant in the C. elegans homolog reveals a paradoxical increase in spontaneous synaptic vesicle release, despite reduced channel expression. Molecular dynamics simulations predict that the mutation prolongs dwell time in a partially open state, potentially increasing calcium influx at rest. This model is supported by biophysical recordings of the human channel showing increased current at hyperpolarized potentials and by rescue of the C. elegans phenotype through genetic elevation of resting membrane potential. Together, these findings reconcile the patients clinical presentation by describing a complex, mixed-function variant, highlight the importance of cellular context in variant interpretation and therapeutic development, and establish C. elegans as a powerful in vivo platform for evaluating the functional consequences of pathogenic ion channel variants.

neuroscience↗

bioRxiv2025

WormSNAP: A software for fast, accurate, and unbiased detection of fluorescent puncta in C. elegans

The detection and characterization of fluorescent puncta are critical tasks in image analysis pipelines for fluorescence imaging. Existing methods for quantitative characterization of such puncta often suffer from biases and limitations, compromising the reliability and reproducibility of results. Moreover, the widespread adoption of many available analysis scripts is often hampered by over-optimization for specific samples, requiring extensive coding knowledge to repurpose for other datasets. We present WormSNAP (Worm SyNapse Analysis Program), a freely available, stand-alone, no-code approach to automated unbiased detection and characterization of 2D fluorescent puncta, originally developed to characterize images of the synapses residing in C. elegans nerve cords but suitable for broader 2D fluorescence image analysis. WormSNAP incorporates a local means thresholding algorithm and a user-friendly Graphical User Interface (GUI) for efficient and accurate analysis of large datasets, with user control of thresholding and restriction parameters and visualization options for further refinement. WormSNAP also calculates three types of correlation metrics for 2-channel images, enabling users to select the ideal metric for their dataset. WormSNAP provides robust and accurate fluorescent puncta detection in a variety of conditions, accelerating the image analysis workflow from data acquisition to figure generation.

neuroscience↗

bioRxiv2022

In vivo proximity ligation reveals endogenous candidate interactors of Neurexin's intracellular domain

Neurexins are highly-spliced transmembrane cell adhesion molecules that bind an array of partners via their extracellular domains. However, much less is known about the signaling pathways downstream of neurexins largely-invariant intracellular domain. C. elegans contains a single neurexin gene that we have previously shown is required for presynaptic assembly and stabilization. To gain insight into the signaling pathways mediating neurexins presynaptic functions, we employed a proximity ligation method, endogenously tagging neurexins intracellular domain with the promiscuous biotin ligase TurboID, allowing us to isolate adjacent biotinylated proteins by streptavidin pull-down and mass spectrometry. We compared our experimental strain to a control strain in which neurexin, endogenously tagged with TurboID, was dispersed from presynaptic active zones by the deletion of its C-terminal PDZ-binding motif. Using this approach we identified both known and novel intracellular interactors of neurexin, including active zone scaffolds, actin-binding proteins (including almost every member of the Arp2/3 complex), signaling molecules, and mediators of RNA trafficking, protein synthesis and degradation, among others. Characterization of mutants for candidate neurexin interactors revealed that they recapitulate aspects of the nrx-1 mutant phenotype, suggesting they may be involved in neurexin signaling. Finally, to investigate a possible role for neurexin in local actin assembly, we endogenously tagged its intracellular domain with actin depolymerizing and sequestering peptides (DeActs), and found that this led to defects in active zone assembly.

neuroscience↗