Biology subjects

Kolar, K.

Publications and source records attributed to Kolar, K..

4 recordsLinked to original sources

bioRxiv2023

Massive contractions of Myotonic Dystrophy Type 2-associated CCTG tetranucleotide repeats occur via double strand break repair with distinct requirements for helicases

Myotonic Dystrophy Type 2 (DM2) is a genetic disease caused by expanded CCTG DNA repeats in the first intron of CNBP. The number of CCTG repeats in DM2 patients ranges from 75-11,000, yet little is known about the molecular mechanisms responsible for repeat expansions or contractions. We developed an experimental system in Saccharomyces cerevisiae that enables selection of large-scale contractions of (CCTG)100 within the intron of a reporter gene and subsequent genetic analysis. Contractions exceeded 80 repeat units, causing the final repetitive tract to be well below the threshold for disease. We found that Rad51 and Rad52 are required for these massive contractions, indicating a mechanism that involves homologous recombination. Srs2 helicase was shown previously to stabilize CTG, CAG, and CGG repeats. Loss of Srs2 did not significantly affect CCTG contraction rates in unperturbed conditions. In contrast, loss of the RecQ helicase Sgs1 resulted in a 6-fold decrease in contraction rate with specific evidence that helicase activity is required for large-scale contractions. Using a genetic assay to evaluate chromosome arm loss, we determined that CCTG and reverse complementary CAGG repeats elevate the rate of chromosomal fragility compared to a low-repeat control. Overall, our results demonstrate that the genetic control of CCTG repeat contractions is notably distinct among disease-causing microsatellite repeat sequences.

genetics↗

bioRxiv2023

Polymodal sensory perception of mechanical and chemical cues drives robust settlement and metamorphosis of a marine pre-vertebrate zooplanktonic larva.

The Earths oceans brim with an incredible diversity of microscopic planktonic animals, many of which correspond to the transient larval stage in the life cycles of benthic marine organisms. The mechanisms by which marine larvae use their nervous system to sense and process diverse environmental cues (physical and chemical) in the water column and the benthos to settle and metamorphose is a major problem across the fields of neuroscience, development, evolution and ecology, yet they remain largely unclear. Here, we employ Ca2+ imaging and behavioral assays using the larval form of the protochordate Ciona intestinalis to characterise the mechanical and chemical stimuli these larvae respond to during settlement and metamorphosis. We also identify the polymodal sensory cells that detect these stimuli. Whole brain Ca2+ imaging further revealed that the presentation or removal of ethological chemosensory stimuli engages the activities of different neuronal sub-populations resulting in brain state changes, which may underlie behavioral action selections and metamorphosis. Finally, chemogenetic interrogation coupled to behavioral analysis reveals that peptidergic sensory neurons including polymodal cells capable of chemotactile perception and chemosensory/neurosecretory cells of proto-placodal ectoderm origin play a pivotal role in regulating stimulus induced settlement and metamorphosis. This work suggests that marine zooplanktonic larvae utilise their streamlined nervous systems to perform multimodal integration of ethologically physical and chemical cues to explore the oceanic water column and benthos.

neuroscience↗

bioRxiv2020

Mapping calcium dynamics in a developing tubular structure.

Calcium is a ubiquitous and versatile second messenger that plays a central role in the development and function of a wide range of cell types, tissues and organs. Despite significant recent progress in the understanding of calcium (Ca2+) signalling in organs such as the developing and adult brain, we have relatively little knowledge of the contribution of Ca2+ to the development of tubes, structures widely present in multicellular organisms. Here we image Ca2+ dynamics in the developing notochord of Ciona intestinalis. We show that notochord cells exhibit distinct Ca2+ dynamics during specific morphogenetic events such as cell intercalation, cell elongation and tubulogenesis. We used an optogenetically controlled Ca2+ actuator to show that sequestration of Ca2+ results in defective notochord cell intercalation, and pharmacological inhibition to reveal that stretch-activated ion channels (SACs), inositol triphosphate receptor (IP3R) signalling, Store Operated Calcium Entry (SOCE), Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and gap junctions are required for regulating notochord Ca2+ activity during tubulogenesis. Cytoskeletal rearrangements drive the cell shape changes that accompany tubulogenesis. In line with this, we show that Ca2+ signalling modulates reorganization of the cytoskeletal network across the morphogenetic events leading up to and during tubulogenesis of the notochord. We additionally demonstrate that perturbation of the actin cytoskeleton drastically remodels Ca2+ dynamics, suggesting a feedback mechanism between actin dynamics and Ca2+ signalling during notochord development. This work provides a framework to quantitatively define how Ca2+ signalling regulates tubulogenesis using the notochord as model organ, a defining structure of all chordates.

developmental biology↗

bioRxiv2019

Mesmerize: A highly versatile platform for calcium imaging analysis and creation of self-contained FAIR datasets.

Calcium imaging is an increasingly valuable technique for understanding neural circuits, neuroethology, and cellular mechanisms. The analysis of calcium imaging data presents challenges in image processing, data organization, analysis, and accessibility. Tools have been created to address these problems independently, however a comprehensive user-friendly package does not exist. Here we present "Mesmerize", an efficient, expandable and user-friendly analysis platform, which uses a Findable, Accessible, Interoperable and Reproducible (FAIR) system to encapsulate the entire analysis process, from raw data to interactive visualizations for publication. Mesmerize provides a user-friendly graphical interface to state-of-the-art analysis methods for signal extraction & downstream analysis. We demonstrate the broad scientific scope of Mesmerizes applications by analyzing neuronal datasets from mouse and a volumetric zebrafish dataset. We also applied contemporary time-series analysis techniques to analyze a novel dataset comprising neuronal, epidermal, and migratory mesenchymal cells of the protochordate Ciona intestinalis.

neuroscience↗