Biology subjects

Guyer, A.

Publications and source records attributed to Guyer, A..

4 recordsLinked to original sources

bioRxiv2024

Optogenetic control of Nodal signaling patterns

A crucial step in early embryogenesis is the establishment of spatial patterns of signaling activity. Tools to perturb morphogen signals with high resolution in space and time can help reveal how embryonic cells decode these signals to make appropriate fate decisions. Here, we present new optogenetic reagents and an experimental pipeline for creating designer Nodal signaling patterns in live zebrafish embryos. Nodal receptors were fused to the light-sensitive heterodimerizing pair Cry2/CIB1N, and the Type II receptor was sequestered to the cytosol. The improved optoNodal2 reagents eliminate dark activity and improve response kinetics, without sacrificing dynamic range. We adapted an ultra-widefield microscopy platform for parallel light patterning in up to 36 embryos and demonstrated precise spatial control over Nodal signaling activity and downstream gene expression. Patterned Nodal activation drove precisely controlled internalization of endodermal precursors. Further, we used patterned illumination to generate synthetic signaling patterns in Nodal signaling mutants, rescuing several characteristic developmental defects. This study establishes an experimental toolkit for systematic exploration of Nodal signaling patterns in live embryos.

developmental biology↗

bioRxiv2023

Non-homologous end joining shapes the genomic rearrangement landscape of chromothripsis from mitotic errors

Errors in mitosis can generate micronuclei that entrap mis-segregated chromosomes, which are susceptible to catastrophic fragmentation through a process termed chromothripsis. The reassembly of fragmented chromosomes by error-prone DNA double-strand break (DSB) repair generates a spectrum of simple and complex genomic rearrangements that are associated with human cancers and disorders. How specific DSB repair pathways recognize and process these lesions remains poorly understood. Here we used CRISPR/Cas9 to systematically inactivate distinct DSB processing or repair pathways and interrogated the rearrangement landscape of fragmented chromosomes from micronuclei. Deletion of canonical non-homologous end joining (NHEJ) components, including DNA-PKcs, LIG4, and XLF, substantially reduced the formation of complex rearrangements and shifted the rearrangement landscape toward simple alterations without the characteristic patterns of cancer-associated chromothripsis. Following reincorporation into the nucleus, fragmented chromosomes localize within micronuclei bodies (MN bodies) and undergo successful ligation by NHEJ within a single cell cycle. In the absence of NHEJ, chromosome fragments were rarely engaged by polymerase theta-mediated alternative end-joining or recombination-based mechanisms, resulting in delayed repair kinetics and persistent 53BP1-labeled MN bodies in the interphase nucleus. Prolonged DNA damage signaling from unrepaired fragments ultimately triggered cell cycle arrest. Thus, we provide evidence supporting NHEJ as the exclusive DSB repair pathway generating complex rearrangements following chromothripsis from mitotic errors.

cell biology↗

bioRxiv2022

Mitotic clustering of pulverized chromosomes from micronuclei

Complex genome rearrangements can be generated by the catastrophic shattering of mis-segregated chromosomes trapped within micronuclei through a process known as chromothripsis. Since each chromosome harbors a single centromere, how acentric fragments derived from shattered chromosomes are inherited between daughter cells during mitosis remains unknown. Here we tracked micronucleated chromosomes by live-cell imaging and show that acentric fragments cluster in close spatial proximity throughout mitosis for biased partitioning to a single daughter cell. Mechanistically, the CIP2A-TOPB1 complex prematurely associates with DNA lesions within ruptured micronuclei during interphase, which poises chromosome fragments for clustering upon mitotic entry. Inactivation of CIP2A or TOPBP1 caused pulverized chromosomes to untether and disperse throughout the mitotic cell, consequently resulting in the mis-accumulation of DNA fragments in the cytoplasm. The inheritance of shattered chromosomes by a single daughter cell suggests that micronucleation can drive complex rearrangements that lack the DNA copy number oscillations characteristic of canonical chromothripsis. Comprehensive analysis of pan-cancer whole-genome sequencing data revealed clusters of DNA copy number-neutral rearrangements - termed balanced chromothripsis - across diverse cancer types resulting in the acquisition of known driver events. Thus, distinct patterns of chromothripsis can be explained by the spatial mitotic clustering of pulverized chromosomes from micronuclei.

cell biology↗

bioRxiv2019

Selection of Candida albicans Trisomy during Oropharyngeal Infection Results in a Commensal-Like Phenotype

When the fungus Candida albicans proliferates in the oropharyngeal cavity during experimental oropharyngeal candidiasis (OPC), it undergoes large-scale genome changes at a much higher frequency than when it grows in vitro. Previously, we identified a specific whole chromosome amplification, trisomy of Chr 6 (Chr6x3), that was highly overrepresented among strains recovered from the tongues of mice with OPC. To determine the functional significance of this trisomy, we assessed the virulence of two Chr6 trisomic strains and a Chr5 trisomic strain in the mouse model of OPC. We also analyzed the expression of virulence-associated traits in vitro. All three trisomic strains exhibited characteristics of a commensal during OPC in mice. They achieved the same oral fungal burden as the diploid progenitor strain but caused significantly less weight loss and elicited a significantly lower inflammatory host response. In vitro, all three trisomic strains had reduced capacity to adhere to and invade oral epithelial cells and increased susceptibility to neutrophil killing. Whole genome sequencing of pre- and post-infection isolates found that the trisomies were usually maintained. Most post-infection isolates also contained de novo point mutations, but these were not conserved. While in vitro growth assays did not reveal phenotypes specific to de novo point mutations, they did reveal novel phenotypes specific to each lineage. These data reveal that during OPC, clones that are trisomic for Chr5 or Chr6 are selected and they facilitate a commensal-like phenotype.

genomics↗