Biology subjects

Kedziora, K.

Publications and source records attributed to Kedziora, K..

3 recordsLinked to original sources

bioRxiv2024

Cited4a limits cardiomyocyte dedifferentiation and proliferation during zebrafish heart regeneration

Cardiac regeneration involves the interplay of complex interactions between many different cell types, including cardiomyocytes. The exact mechanism that enables cardiomyocytes to undergo dedifferentiation and proliferation to replace lost cells has been intensely studied. Here we report a single nuclear RNA sequencing profile of the injured zebrafish heart and identify distinct cardiomyocyte populations in the injured heart. These cardiomyocyte populations have diverse functions, including stress response, myofibril assembly, proliferation and contraction. The contracting cardiomyocyte population also involves the activation of maturation pathways as an early response to injury. This intriguing finding suggests that constant maintenance of a distinctive terminally differentiated cardiomyocyte population is important for cardiac function during regeneration. To test this hypothesis, we determined that cited4a, a p300/CBP transcriptional coactivator, is induced after injury in the mature cardiomyocyte population. Moreover, loss-of-cited4a mutants presented increased dedifferentiation, proliferation and accelerated heart regeneration. Thus, suppressing cardiomyocyte maturation pathway activity in injured hearts could be an approach to promote heart regeneration.

developmental biology↗

bioRxiv2023

An in vitro platform for quantifying cell cycle phase lengths in primary human intestinal stem cells

Background and AimsThe intestinal epithelium exhibits dynamic control of cell cycle phase lengths, yet no experimental platform exists for directly analyzing cell cycle phases in living human intestinal stem cells (ISCs). Here, we develop primary human ISC lines with two different reporter constructs to provide fluorescent readouts to analyze cell cycle phases in cycling ISCs. Methods3D printing was used to construct a collagen press for making chamber slides that support primary human ISC growth and maintenance within the working distance of a confocal microscope objective. The PIP-FUCCI fluorescent cell cycle reporter and a variant with H2A-mScarlet that allows for automated tracking of cell cycle phases (PIP-H2A) were used in human ISCs along with live imaging and EdU pulsing. An analysis pipeline combining free-to-use programs and publicly available code was compiled to analyze live imaging results. ResultsChamber slides with soft collagen pressed to a thickness of 0.3 mm concurrently support ISC cycling and confocal imaging. PIP-FUCCI ISCs were found to be optimal for snapshot analysis wherein all nuclei are assigned to a cell cycle phase from a single image. PIP-H2A ISCs were better suited for live imaging since constant nuclear signal allowed for more automated analysis. CellPose2 and TrackMate were used together to track cycling cells. ConclusionsWe present two complete platforms for analyzing cell cycle phases in living primary human ISCs. The PIP-FUCCI construct allows for cell cycle phase assignment from one image of living cells, the PIP-H2A construct allows for semi-automated direct quantification of cell cycle phase lengths in human ISCs using our computational pipeline. These platforms hold great promise for future studies on how pharmaceutical agents affect the intestinal epithelium, how cell cycle is regulated in human ISCs, and more.

cell biology↗

bioRxiv2018

Cdt1 variants reveal unanticipated aspects of interactions with Cyclin/CDK and MCM important for normal genome replication.

The earliest step in DNA replication is origin licensing which is the DNA loading of MCM helicase complexes. The Cdt1 protein is essential for MCM loading during G1 phase of the cell cycle, yet the mechanism of Cdt1 function is still incompletely understood. We examined a collection of rare Cdt1 variants that cause a form of primordial dwarfism (Meier-Gorlin syndrome) plus one hypomorphic Drosophila allele to shed light on Cdt1 function. Three hypomorphic variants load MCM less efficiently than WT Cdt1, and their lower activity correlates with impaired MCM binding. A structural homology model of the human Cdt1-MCM complex position the altered Cdt1 residues at two distinct interfaces rather than the previously described single MCM interaction domain. Surprisingly, one dwarfism allele (Cdt1-A66T) is more active than WT Cdt1. This hypermorphic variant binds both Cyclin A and SCFSkp2 poorly relative to WT Cdt1. Detailed quantitative live cell imaging analysis demonstrated no change in stability of this variant however. Instead, we propose that Cyclin A/CDK inhibits Cdt1 licensing function independently of the creation of the SCFSkp2 phosphodegron. Together, these findings identify key Cdt1 interactions required for both efficient origin licensing and tight Cdt1 regulation to ensure normal cell proliferation and genome stability.

molecular biology↗