Biology subjects

Bunn, C.

Publications and source records attributed to Bunn, C..

4 recordsLinked to original sources

bioRxiv2025

AlveolEye: Rapid and precise lung morphometry guided by computer vision

Rigorous and reproducible evaluation of lung tissue under different conditions is necessary to interpret development, injury, and pharmacologic interventions. Common histological measurements in the distal lung include mean linear intercept (MLI) as a metric of alveolarization and airspace volume density (ASVD) as a metric of airspaces relative to tissue. Historically, these have been performed manually in a time-intensive process, with reproducible trends, but a high degree of variability between individuals. To improve the reproducibility and throughput of lung morphometry, we developed AlveolEye, an open source, semi-automated, computer vision-assisted tool that rapidly and reproducibly calculates MLI and ASVD from images of standard hematoxylin and eosin (H&E) stained tissue sections. AlveolEye-assisted MLI calculation closely aligns with manually-derived measurements for corresponding images, with preservation of trends in measurements between non-injured controls and neonatal mice subjected to two different injury models. Analyzing human tissue of varying ages suggests that the approach developed in AlveolEye is generalizable across species. Notably, AlveolEye markedly reduced the average variation across individual analyzers, with the greatest improvement in precision among individuals with the least experience in performing lung morphometry. The design of AlveolEye is intentionally semi-automated, preserving the investigators ability to assess and adjust parameters based on sample characteristics. AlveolEye facilitates efficient lung morphological measurements on larger sample sizes, allowing for greater statistical power for preclinical studies, and improves precision across individual observers, allowing for improved rigor in experimental design and execution.

developmental biology↗

bioRxiv2023

Mechanistic and evolutionary insights into isoform-specific 'supercharging' in DCLK family kinases

Catalytic signaling outputs of protein kinases are dynamically regulated by an array of structural mechanisms, including allosteric interactions mediated by intrinsically disordered segments flanking the conserved catalytic domain. The Doublecortin Like Kinases (DCLKs) are a family of microtubule-associated proteins characterized by a flexible C-terminal autoregulatory tail segment that varies in length across the various human DCLK isoforms. However, the mechanism whereby these isoform-specific variations contribute to unique modes of autoregulation is not well understood. Here, we employ a combination of statistical sequence analysis, molecular dynamics simulations and in vitro mutational analysis to define hallmarks of DCLK family evolutionary divergence, including analysis of splice variants within the DCLK1 sub-family, which arise through alternative codon usage and serve to supercharge the inhibitory potential of the DCLK1 C-tail. We identify co-conserved motifs that readily distinguish DCLKs from all other Calcium Calmodulin Kinases (CAMKs), and a Swiss-army assembly of distinct motifs that tether the C-terminal tail to conserved ATP and substrate-binding regions of the catalytic domain to generate a scaffold for auto-regulation through C-tail dynamics. Consistently, deletions and mutations that alter C-terminal tail length or interfere with co-conserved interactions within the catalytic domain alter intrinsic protein stability, nucleotide/inhibitor-binding, and catalytic activity, suggesting isoform-specific regulation of activity through alternative splicing. Our studies provide a detailed framework for investigating kinome-wide regulation of catalytic output through cis-regulatory events mediated by intrinsically disordered segments, opening new avenues for the design of mechanistically-divergent DCLK1 modulators, stabilizers or degraders.

evolutionary biology↗

bioRxiv2022

Epithelial Outgrowth Through Mesenchymal Rings Drives Alveologenesis

Determining how alveoli are formed and maintained is critical to understanding lung organogenesis and regeneration after injury. While technological barriers have heretofore limited real-time observation of alveologenesis, we have now used scanned oblique plane illumination microscopy of living lung slices to observe specific cellular behaviors at high resolution over several days. Contrary to the prevailing paradigm that alveoli form by airspace subdivision via ingrowing septa, we find that alveoli form by ballooning epithelial outgrowth supported by stable mesenchymal ring structures. Our systematic analysis allowed creation of a computational model of finely-timed cellular structural changes that drive alveologenesis under normal conditions or with perturbed intercellular Wnt signaling. This new paradigm and platform can be leveraged for mechanistic studies and screening for therapies to promote lung regeneration. One-Sentence SummaryLong-term live analysis of neonatal lungs supports a dynamic epithelial outgrowth model for alveologenesis.

developmental biology↗

bioRxiv2021

Evolution of functional diversity in the holozoan tyrosine kinome

The emergence of multicellularity is strongly correlated with the expansion of tyrosine kinases, a conserved family of signaling enzymes that regulates pathways essential for cell-to-cell communication. Although tyrosine kinases have been classified from several model organisms, a molecular-level understanding of tyrosine kinase evolution across all holozoans is currently lacking. Using a hierarchical sequence constraint-based classification of diverse holozoan tyrosine kinases, we construct a new phylogenetic tree that identifies two ancient clades of cytoplasmic and receptor tyrosine kinases separated by the presence of an extended insert segment in the kinase domain connecting the D and E helices. Present in nearly all receptor tyrosine kinases, this fast-evolving insertion imparts diverse functionalities such as post-translational modification sites and regulatory interactions. Eph and EGFR receptor tyrosine kinases are two exceptions which lack this insert, each forming an independent lineage characterized by unique functional features. We also identify common constraints shared across multiple tyrosine kinase families which warrant the designation of three new subgroups: Src Module (SrcM), Insulin Receptor Kinase-Like (IRKL), and Fibroblast, Platelet-derived, Vascular, and growth factor Receptors (FPVR). Subgroup-specific constraints reflect shared autoinhibitory interactions involved in kinase conformational regulation. Conservation analyses describe how diverse tyrosine kinase signaling functions arose through the addition of family-specific motifs upon subgroup-specific features and co-evolving protein domains. We propose the oldest tyrosine kinases, IRKL, SrcM, and Csk, originated from unicellular pre-metazoans and were co-opted for complex multicellular functions. The increased frequency of oncogenic variants in more recent tyrosine kinases suggests that lineage-specific functionalities are selectively altered in human cancers.

evolutionary biology↗