skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Attention:

The NSF Public Access Repository (PAR) system and access will be unavailable from 9:30 PM ET on Friday, January 23 until 7:00 AM ET on Saturday, January 24 due to maintenance. We apologize for the inconvenience.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: Deep learning-driven imaging of cell division and cell growth across an entire eukaryotic life cycle
Abstract The life cycle of biomedical and agriculturally relevant eukaryotic microorganisms involves complex transitions between proliferative and non-proliferative states such as dormancy, mating, meiosis, and cell division. New drugs, pesticides, and vaccines can be created by targeting specific life cycle stages of parasites and pathogens. However, defining the structure of a microbial life cycle often relies on partial observations that are theoretically assembled in an ideal life cycle path. To create a more quantitative approach to studying complete eukaryotic life cycles, we generated a deep learning-driven imaging framework to track microorganisms across sexually reproducing generations. Our approach combines microfluidic culturing, life cycle stage-specific segmentation of microscopy images using convolutional neural networks, and a novel cell tracking algorithm, FIEST, based on enhancing the overlap of single cell masks in consecutive images through deep learning video frame interpolation. As proof of principle, we used this approach to quantitatively image and compare cell growth and cell cycle regulation across the sexual life cycle ofSaccharomyces cerevisiae. We developed a fluorescent reporter system based on a fluorescently labeled Whi5 protein, the yeast analog of mammalian Rb, and a new High-Cdk1 activity sensor, LiCHI, designed to report during DNA replication, mitosis, meiotic homologous recombination, meiosis I, and meiosis II. We found that cell growth preceded the exit from non-proliferative states such as mitotic G1, pre-meiotic G1, and the G0 spore state during germination. A decrease in the total cell concentration of Whi5 characterized the exit from non-proliferative states, which is consistent with a Whi5 dilution model. The nuclear accumulation of Whi5 was developmentally regulated, being at its highest during meiotic exit and spore formation. The temporal coordination of cell division and growth was not significantly different across three sexually reproducing generations. Our framework could be used to quantitatively characterize other single-cell eukaryotic life cycles that remain incompletely described. An off-the-shelf user interfaceYeastvisionprovides free access to our image processing and single-cell tracking algorithms.  more » « less
Award ID(s):
2235451
PAR ID:
10609126
Author(s) / Creator(s):
; ; ; ; ; ;
Publisher / Repository:
bioRxiv
Date Published:
Format(s):
Medium: X
Institution:
bioRxiv
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; ; (, Molecular Biology of the Cell)
    Kellogg, Douglas (Ed.)
    The life cycle of eukaryotic microorganisms involves complex transitions between states such as dormancy, mating, meiosis, and cell division, which are often studied independently from each other. Therefore, most microbial life cycles are theoretical reconstructions from partial observations of cellular states. Here we show that complete microbial life cycles can be directly and continuously studied by combining microfluidic culturing, life cycle stage-specific segmentation of micrographs, and a novel cell tracking algorithm, FIEST, based on deep learning video frame interpolation. As proof of principle, we quantitatively imaged and compared cell growth and the activity state of the cell division kinase, Cdk1, across the life cycle of Saccharomyces cerevisiae for up to three sexually reproducing generations. Our analysis of S. cerevisiae's life cycle provided the following new insights: 1) the accumulation of cell cycle regulators, such as Whi5, is tailored to each life cycle stage; 2) cell growth always preceded exit from nonproliferative states in our conditions; 3) the temporal coordination of meiotic events is the same across sexually reproducing populations when each generation is exposed to same conditions; 4) information such as cell size and morphology resets after each sexual reproduction cycle. Image processing and tracking algorithms are available as the Python package Yeastvision, which could be used study pathogens such as Candida glabrata, Cryptococcus neoformans, Colletotrichum acutatum, and other unicellular systems. 
    more » « less
  2. ; ; (, Proceedings of the National Academy of Sciences)
    Cells must couple cell-cycle progress to their growth rate to restrict the spread of cell sizes present throughout a population. Linear, rather than exponential, accumulation of Whi5, was proposed to provide this coordination by causing a higher Whi5 concentration in cells born at a smaller size. We tested this model using the inducible GAL1 promoter to make the Whi5 concentration independent of cell size. At an expression level that equalizes the mean cell size with that of wild-type cells, the size distributions of cells with galactose-induced Whi5 expression and wild-type cells are indistinguishable. Fluorescence microscopy confirms that the endogenous and GAL1 promoters produce different relationships between Whi5 concentration and cell volume without diminishing size control in the G1 phase. We also expressed Cln3 from the GAL1 promoter, finding that the spread in cell sizes for an asynchronous population is unaffected by this perturbation. Our findings indicate that size control in budding yeast does not fundamentally originate from the linear accumulation of Whi5, contradicting a previous claim and demonstrating the need for further models of cell-cycle regulation to explain how cell size controls passage through Start. 
    more » « less
  3. ; ; ; ; ; ; ; ; ; ; et al (, PLOS Biology)
    Keeney, Scott (Ed.)
    In most sexually reproducing organisms crossing over between chromosome homologs during meiosis is essential to produce haploid gametes. Most crossovers that form in meiosis in budding yeast result from the biased resolution of double Holliday junction (dHJ) intermediates. This dHJ resolution step involves the actions of Rad2/XPG family nuclease Exo1 and the Mlh1-Mlh3 mismatch repair endonuclease. Here, we provide genetic evidence in baker’s yeast that Exo1 promotes meiotic crossing over by protecting DNA nicks from ligation. We found that structural elements in Exo1 that interact with DNA, such as those required for the bending of DNA during nick/flap recognition, are critical for its role in crossing over. Consistent with these observations, meiotic expression of the Rad2/XPG family member Rad27 partially rescued the crossover defect inexo1null mutants, and meiotic overexpression of Cdc9 ligase reduced the crossover levels ofexo1DNA-binding mutants to levels that approached theexo1null. In addition, our work identified a role for Exo1 in crossover interference. Together, these studies provide experimental evidence for Exo1-protected nicks being critical for the formation of meiotic crossovers and their distribution. 
    more » « less
  4. ; ; ; ; ; ; ; ; ; ; et al (, PLOS Biology)
    Martin, Sophie G. (Ed.)
    Commitment to cell division at the end of G1 phase, termed Start in the budding yeast Saccharomyces cerevisiae , is strongly influenced by nutrient availability. To identify new dominant activators of Start that might operate under different nutrient conditions, we screened a genome-wide ORF overexpression library for genes that bypass a Start arrest caused by absence of the G1 cyclin Cln3 and the transcriptional activator Bck2. We recovered a hypothetical gene YLR053c , renamed NRS1 for Nitrogen-Responsive Start regulator 1, which encodes a poorly characterized 108 amino acid microprotein. Endogenous Nrs1 was nuclear-localized, restricted to poor nitrogen conditions, induced upon TORC1 inhibition, and cell cycle-regulated with a peak at Start. NRS1 interacted genetically with SWI4 and SWI6 , which encode subunits of the main G1/S transcription factor complex SBF. Correspondingly, Nrs1 physically interacted with Swi4 and Swi6 and was localized to G1/S promoter DNA. Nrs1 exhibited inherent transactivation activity, and fusion of Nrs1 to the SBF inhibitor Whi5 was sufficient to suppress other Start defects. Nrs1 appears to be a recently evolved microprotein that rewires the G1/S transcriptional machinery under poor nitrogen conditions. 
    more » « less
  5. ; ; ; (, BMC Genomics)
    Abstract BackgroundMeiosis is a specialized cell division that underpins sexual reproduction in most eukaryotes. During meiosis, interhomolog meiotic recombination facilitates accurate chromosome segregation and generates genetic diversity by shuffling parental alleles in the gametes. The frequency of meiotic recombination inArabidopsishas a U-shaped curve in response to environmental temperature, and is dependent on the Type I, crossover (CO) interference-sensitive pathway. The mechanisms that modulate recombination frequency in response to temperature are not yet known. ResultsIn this study, we compare the transcriptomes of thermally-stressed meiotic-stage anthers frommsh4andmus81mutants that mediate the Type I and Type II meiotic recombination pathways, respectively. We show that heat stress reduces the number of expressed genes regardless of genotype. In addition,msh4mutants have a distinct gene expression pattern compared tomus81and wild type controls. Interestingly,ASY1,which encodes a HORMA domain protein that is a component of meiotic chromosome axes, is up-regulated in wild type andmus81but not inmsh4. In addition,SDSthe meiosis-specific cyclin-like gene,DMC1the meiosis-specific recombinase,SYN1/REC8the meiosis-specific cohesion complex component, andSWI1which functions in meiotic sister chromatid cohesion are up-regulated in all three genotypes. We also characterize 51 novel, previously unannotated transcripts, and show that their promoter regions are associated with A-rich meiotic recombination hotspot motifs. ConclusionsOur transcriptomic analysis ofmsh4andmus81mutants enhances our understanding of how the Type I and Type II meiotic CO pathway respond to environmental temperature stress and might provide a strategy to manipulate recombination levels in plants. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Text Encoded Conversion
  • XML
  • Save / Share this Record
  • Send to Email