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At least 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Chlamydomonas CHT7 is involved in repressing DNA replication and mitotic genes during synchronous growth

In the green alga Chlamydomonas reinhardtii, regulation of the cell cycle in response to external cues is critical for survival in a changing environment. The loss of the nuclear COMPROMISED HYDROLYSIS OF TRIACYLGLYCEROLS 7 (CHT7) protein affects the expression of many genes especially in response to nitrogen availability. Cells lacking CHT7 exhibit abnormal cell morphology following nitrogen deprivation and fail to resume normal cell division after N resupply. To investigate the function of CHT7 in the regulation of cell cycle-related pathways, cells were synchronized, and RNA-seq analysis was performed during various stages of the cell cycle. In the cht7 mutant following nitrogen deprivation, the cells were not dividing, but a subset of cell cycle genes involved in DNA replication and mitosis were found to be derepressed, suggesting that the CHT7 protein plays a role in cell cycle regulation that is opposite to that of the mitotic cyclin-dependent kinases. Furthermore, genes for cell wall synthesis and remodeling were found to be abnormally induced in nondividing cht7 cells; this misregulation may deplete cellular resources and thus contribute to cell death following nitrogen deprivation. Lastly, 43 minimally characterized kinases were found to be highly misregulated in cht7. Further analysis suggested that some of these CHT7-regulated kinases may be related to the MAP3K and Aurora-like kinases, while others are unique. Together, these results suggest a role of CHT7 in transcriptional regulation of the cell cycle and reveal several pathways and genes whose expression appears to be subject to a CHT7-mediated regulatory network.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2001

Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes

Senescence and genomic integrity are thought to be important barriers in the development of malignant lesions. Human fibroblasts undergo a limited number of cell divisions before entering an irreversible arrest, called senescence. Here we show that human mammary epithelial cells (HMECs) do not conform to this paradigm of senescence. In contrast to fibroblasts, HMECs exhibit an initial growth phase that is followed by a transient growth plateau (termed selection or M0; refs 3-5), from which proliferative cells emerge to undergo further population doublings (approximately 20-70), before entering a second growth plateau (previously termed senescence or M1; refs 4-6). We find that the first growth plateau exhibits characteristics of senescence but is not an insurmountable barrier to further growth. HMECs emerge from senescence, exhibit eroding telomeric sequences and ultimately enter telomere-based crisis to generate the types of chromosomal abnormalities seen in the earliest lesions of breast cancer. Growth past senescent barriers may be a pivotal event in the earliest steps of carcinogenesis, providing many genetic changes that predicate oncogenic evolution. The differences between epithelial cells and fibroblasts provide new insights into the mechanistic basis of neoplastic transformation.

NASA Discipline Radiation Health↗
DOE OSTI2025

Cyanobacterial circadian regulation enhances bioproduction under subjective nighttime through rewiring of carbon partitioning dynamics, redox balance orchestration, and cell cycle modulation

Abstract Background The industrial feasibility of photosynthetic bioproduction using cyanobacterial platforms remains challenging due to insufficient yields, particularly due to competition between product formation and cellular carbon demands across different temporal phases of growth. This study investigates how circadian clock regulation impacts carbon partitioning between storage, growth, and product synthesis in Synechococcus elongatus PCC 7942, and provides insights that suggest potential strategies for enhanced bioproduction. Results After entrainment to light-dark cycles, PCC 7942 cultures transitioned to constant light revealed distinct temporal patterns in sucrose production, exhibiting three-fold higher productivity during subjective night compared to subjective day despite moderate down-regulation of genes from the photosynthetic apparatus. This enhanced productivity coincided with reduced glycogen accumulation and halted cell division at subjective night time, suggesting temporal separation of competing processes. Transcriptome analysis revealed coordinated circadian clock-driven adjustment of the cell cycle and rewiring of energy and carbon metabolism, with over 300 genes showing differential expression across four time points. The subjective night was characterized by altered expression of cell division-related genes and reduced expression of genes involved in glycogen synthesis, while showing upregulation of glycogen degradation pathways, alternative electron flow components, the pentose phosphate pathway, and oxidative decarboxylation of pyruvate. These molecular changes created favorable conditions for product formation through enhanced availability of major sucrose precursors (glucose-1-phosphate and fructose-6-phosphate) and maintained redox balance through multiple mechanisms. Conclusions Our analysis of circadian regulatory rewiring of carbon metabolism and redox balancing suggests two potential approaches that could be developed for improving cyanobacterial bioproduction: leveraging natural circadian rhythms for optimizing cultivation conditions and timing of pathway induction, and engineering strains that mimic circadian-driven metabolic shifts through controlled carbon flux redistribution and redox rebalancing. While these strategies remain to be tested, they could theoretically improve the efficiency of photosynthetic bioproduction by enabling better temporal separation between cell growth, carbon storage accumulation, and product synthesis phases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Deciphering metabolic differentiation during Bacillus subtilis sporulation

Abstract The bacteriumBacillus subtilisundergoes asymmetric cell division during sporulation, producing a mother cell and a smaller forespore connected by the SpoIIQ-SpoIIIA (or Q-A) channel. The two cells differentiate metabolically, and the forespore becomes dependent on the mother cell for essential building blocks. Here, we investigate the metabolic interactions between mother cell and forespore using genome-scale metabolic and expression models as well as experiments. Our results indicate that nucleotides are synthesized in the mother cell and transported in the form of nucleoside di- or tri-phosphates to the forespore via the Q-A channel. However, if the Q-A channel is inactivated later in sporulation, then glycolytic enzymes can form an ATP and NADH shuttle, providing the forespore with energy and reducing power. Our integrated in silico and in vivo approach sheds light into the intricate metabolic interactions underlying cell differentiation inB. subtilis, and provides a foundation for future studies of metabolic differentiation.

Science & Technology - Other Topics↗
DOE OSTI2021

Dual role of Fam208a during zygotic cleavage and early embryonic development

Maintenance of genome stability is essential for every living cell as genetic information is repeatedly challenged during DNA replication in each cell division event. Errors, defects, delays, and mistakes that arise during mitosis or meiosis lead to an activation of DNA repair processes and in case of their failure, programmed cell death, i.e. apoptosis, could be initiated. Fam208a is a protein whose importance in heterochromatin maintenance has been described recently. In this work, we describe the crucial role of Fam208a in sustaining genome stability during cellular division. }The targeted depletion of Fam208a in mice using CRISPR/Cas9 led to embryonic lethality before E12.5. We also used the siRNA approach to downregulate Fam208a in zygotes to avoid the influence of maternal RNA in the early stages of development. This early downregulation increased arresting of the embryonal development at the two-cell stage and the occurrence of multipolar spindles formation. To investigate this further, we used the yeast two-hybrid (Y2H) system and identified new putative interaction partners Gpsm2, Svil, and Itgb3bp. Their co-expression with Fam208a was assessed by RT-qPCR profiling and in situ hybridization [1] in multiple murine tissues. Based on these results we proposed that Fam208a functions within the HUSH complex by interaction with Mphosph8 as these proteins are not only able to physically interact but also co-localise. We are bringing new evidence that Fam208a is a multi-interacting protein affecting genome stability on the cell division level at the earliest stages of development and by interaction with methylation complex in adult tissues. }In addition to its epigenetic functions, Fam208a appears to have an important role in the zygotic division, possibly via interaction with newly identified putative partners Gpsm2, Svil, and Itgb3bp.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Identification and characterization of distinct cell cycle stages in cardiomyocytes using the FUCCI transgenic system

Understanding the regulatory mechanism by which cardiomyocyte proliferation transitions to endoreplication and cell cycle arrest during the neonatal period is crucial for identifying proproliferative factors and developing regenerative therapies. }We used a transgenic mouse model based on the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system to isolate and characterize cycling cardiomyocytes at different cell cycle stages at a single-cell resolution. Single-cell transcriptome analysis of cycling and noncycling cardiomyocytes was performed at postnatal days 0 (P0) and 7 (P7). }The FUCCI system proved to be efficient for the identification of cycling cardiomyocytes with the highest mitotic activity at birth, followed by a gradual decline in the number of cycling and mitotic cardiomyocytes during the neonatal period. Cardiomyocytes showed premature cell cycle exit at G1/S shortly after birth and delayed G1/S progression during endoreplication at P7. Single-cell RNA-seq confirmed previously described signaling pathways involved in cardiomyocyte proliferation (Erbb2 and Hippo/YAP), and maturation-related transcriptional changes during postnatal development, including the metabolic switch from glycolysis to fatty acid oxidation in cardiomyocytes. Importantly, we generated transcriptional profiles specific to cell division and endoreplication in cardiomyocytes at different developmental stages that may facilitate the identification of genes important for adult cardiomyocyte proliferation and heart regeneration. }In conclusion, the FUCCI mouse provides a valuable system to study cardiomyocyte cell cycle activity at single cell resolution that can help to decipher the switch from cardiomyocyte proliferation to endoreplication, and to revert this process to facilitate endogenous repair.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1997

Mechanical signals in plant development: a new method for single cell studies

Cell division, which is critical to plant development and morphology, requires the orchestration of hundreds of intracellular processes. In the end, however, cells must make critical decisions, based on a discrete set of mechanical signals such as stress, strain, and shear, to divide in such a way that they will survive the mechanical loads generated by turgor pressure and cell enlargement within the growing tissues. Here we report on a method whereby tobacco protoplasts swirled into a 1.5% agarose entrapment medium will survive and divide. The application of a controlled mechanical load to agarose blocks containing protoplasts orients the primary division plane of the embedded cells. Photoelastic analysis of the agarose entrapment medium can identify the lines of principal stress within the agarose, confirming the hypothesis that cells divide either parallel or perpendicular to the principal stress tensors. The coincidence between the orientation of the new division wall and the orientation of the principal stress tensors suggests that the perception of mechanical stress is a characteristic of individual plant cells. The ability of a cell to determine a shear-free orientation for a new partition wall may be related to the applied load through the deformation of the matrix material. In an isotropic matrix a uniaxial load will produce a rotationally symmetric strain field, which will define a shear-free plane. Where high stress intensities combine with the loading geometry to produce multiaxial loads there will be no axis of rotational symmetry and hence no shear free plane. This suggests that two mechanisms may be orienting the division plane, one a mechanism that works in rotationally symmetrical fields, yielding divisions perpendicular to the compressive tensor, parallel to the long axis of the cell, and one in asymmetric fields, yielding divisions parallel to the short axis of the cell and the compressive tensor.

NASA Discipline Number 40-50↗
DOE OSTI2021

The oncogenic role of meiosis-specific Aurora kinase C in mitotic cells

Highlights: • AURKC is expressed in meiotic cells and some cancers. • AURKC expression increases cell migration and transformation of human mitotic cells. • Deletion of AURKC in U2OS cells decreases cell migration and soft agar growth. • Transcriptome analysis reveals that AURKC alters the extracellular matrix. Aberrant expression of meiosis-specific genes in cancer has recently emerged as a driver of some cancer formation. Aurora kinase C (AURKC) is a member of the Aurora kinase family of proteins known to regulate chromosome segregation during cell divisions. AURKC is normally expressed in meiotic cells; however, elevated levels of AURKC mRNA and protein are frequently measured in cancer cells. To understand the function of AURKC in cancer cells, expression was induced in noncancerous, human retina pigmented epithelial cells. While AURKC expression did not alter cell proliferation over 72 h, it did increase cell migration and anchorage independent growth in soft agar suggesting an oncogenic role in mitotically dividing cells. To evaluate AURKC as a potential therapeutic target, a frameshift mutation in the gene was introduced in U2OS osteosarcoma cells using CRISPR-Cas9 technology resulting in a premature stop codon. Cancer cells lacking AURKC displayed no change in cell proliferation over 72 h but did migrate less and formed fewer colonies in soft agar. Whole transcriptome sequencing analysis uncovered over 400 differentially expressed genes in U2OS cells with and without AURKC. GO analysis revealed alterations in proteinaceous extracellular matrix genes including COL1A1. These data indicate that therapeutics targeting AURKC could decrease cancer cell metastasis and disease progression. Because AURKC is transcriptionally silenced in normal mitotic cells, its disruption could specifically target cancer cells limiting the toxic side effects associated with current therapeutics.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Saccharomyces cerevisiae CellWall Remodeling in the Absence of Knr4 and Kre6 Revealed by Nano-FourierTransform Infrared Spectroscopy

The cell wall integrity (CWI) signaling pathway regulates yeast cell wall biosynthesis, cell division, and responses to external stress. The cell wall, comprised of a dense network of chitin, β-1,3- and β-1,6- glucans, and mannoproteins, is very thin, <100 nm. Alterations in cell wall composition may activate the CWI pathway. Saccharomyces cerevisiae, a model yeast, was used to study the role of individual wall components in altering the structure and biophysical properties of the yeast cell wall. Near-field Fourier transform infrared spectroscopy (nano-FT-IR) was used for the first direct, spectrochemical identification of cell wall composition in a background (wild-type) strain and two deletion mutants from the yeast knock-out collection: kre6Δ and knr4Δ. Killer toxin resistant 6 (Kre6) is an integral membrane protein required for biosynthesis of β-1,6-glucan, while Knr4 is a cell signaling protein involved in the control of cell wall biosynthesis, in particular, biosynthesis and deposition of chitin. Complementary spectral data were obtained with far-field (FF)-FT-IR, in transmission, and with attenuated total reflectance (ATR) spectromicroscopy with 3 –10 μm wavelength-dependent spatial resolution. The FF-FT-IR spectra of cells and spectra of isolated cell wall components showed that components of the cell body dominated transmission spectra and were still evident in ATR spectra. In contrast, the nano-FT-IR at ~25 nm spatial resolution could be used to characterize the yeast wall chemical structure. Our results show that the β-1,6-glucan content is decreased in kre6Δ, while all glucan content is decreased in the knr4Δ cell wall. The latter may be thinner than in wild type, since not only are mannan and chitin detectable by nano-FT-IR, but also lipid membranes and protein, indicative of cell interior.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1992

Time-lapse analysis of gravitropism in Ceratodon protonemata

The tip cell of the protonema of the moss Ceratodon purpureus (Hedw.) Brid. is negatively gravitropic when grown in the dark on supplemented agar. Gravitropism, plastid distribution, and plastid movement were studied in living cells using time-lapse video microscopy and infrared light. A wrong-way (downward) curvature preceded upward curvature and was detected as early as 2 minutes after reorientation. Upward curvature began 30-45 minutes after reorientation to the horizontal. Cell division temporarily reversed upward curvature, but did not inhibit wrong-way curvature. Since significant amyloplast sedimentation always occurred before the start of upward curvature, it is possible that these amyloplasts function as statoliths for upward curvature. However, no significant amyloplast sedimentation occurred before wrong-way curvature. Thus, this early phase of gravitropism cannot require plastid sedimentation for gravity sensing. Most plastids moved within and between zones, and plastid zonation was highly dynamic. Plastids moved toward the apex and toward the base of the cell at rates much slower than cytoplasmic streaming. Despite the dynamic nature of plastid movement and zonation, during upward curvature the distance between sedimented plastids and the apex stayed constant. Time-lapse analysis has revealed intriguing events not readily seen previously using destructive sampling.

NASA Discipline Number 40-50↗
NASA NTRS1986

Polyamine metabolism and osmotic stress. II. Improvement of oat protoplasts by an inhibitor of arginine decarboxylase

We have attempted to improve the viability of cereal mesophyll protoplasts by pretreatment of leaves with DL-alpha-difluoromethylarginine (DFMA), a specific 'suicide' inhibitor of the enzyme (arginine decarboxylase) responsible for their osmotically induced putrescine accumulation. Leaf pretreatment with DFMA before a 6 hour osmotic shock caused a 45% decrease of putrescine and a 2-fold increase of spermine titer. After 136 hours of osmotic stress, putrescine titer in DFMA-pretreated leaves increased by only 50%, but spermidine and spermine titers increased dramatically by 3.2- and 6-fold, respectively. These increases in higher polyamines could account for the reduced chlorophyll loss and enhanced ability of pretreated leaves to incorporate tritiated thymidine, uridine, and leucine into macromolecules. Pretreatment with DFMA significantly improved the overall viability of the protoplasts isolated from these leaves. The results support the view that the osmotically induced rise in putrescine and blockage of its conversion to higher polyamines may contribute to the lack of sustained cell division in cereal mesophyll protoplasts, although other undefined factors must also play a major role.

Non-NASA Center↗
NASA NTRS2000

Cloning and characterization of ftsZ and pyrF from the archaeon Thermoplasma acidophilum

To characterize cytoskeletal components of archaea, the ftsZ gene from Thermoplasma acidophilum was cloned and sequenced. In T. acidophilum ftsZ, which is involved in cell division, was found to be in an operon with the pyrF gene, which encodes orotidine-5'-monophosphate decarboxylase (ODC), an essential enzyme in pyrimidine biosynthesis. Both ftsZ and pyrF from T. acidophilum were expressed in Escherichia coli and formed functional proteins. FtsZ expression in wild-type E. coli resulted in the filamentous phenotype characteristic of ftsZ mutants. T. acidophilum pyrF expression in an E. coli mutant lacking pyrF complemented the mutation and rescued the strain. Sequence alignments of ODCs from archaea, bacteria, and eukarya reveal five conserved regions, two of which have homology to 3-hexulose-6-phosphate synthase (HPS), suggesting a common substrate recognition and binding motif. Copyright 2000 Academic Press.

Bacterial Proteins/chemistry/genetics/metabolism↗
DOE OSTI2025

Engineering quorum-sensing circuits in Synechococcus elongatus PCC 7942 towards self-inducible systems

Despite significant potential for cyanobacteria as sustainable bioproduction chases, there are limited examples of scaled cyanobacterial bioproduction. In part, this is because most cyanobacterial species are poorly adapted to bioreactor cultivation conditions and lack features that facilitate biomass growth and harvesting at scale. We explored quorum sensing (QS) pathways derived from heterotrophic microbes as a method for autoinduction of gene expression circuits coordinated to population density in cyanobacteria. Here, we integrated genetic modules designed to produce and detect the diffusible QS signal, acyl-homoserine lactones (AHLs), in the cyanobacterial model, Synechococcus elongatus PCC 7942 (S. elongatus). We demonstrate that S. elongatus heterologously produces sufficient AHL signals to activate gene expression in a dose-dependent and population density-responsive manner. A hybrid combination of AHL synthesis enzyme from Vibrio fischeri (Lux system) with the transcription factor receiver from Pseudomonas aeruginosa (Las system) provides an ideal activation ratio and mitigates toxicity observed with some AHL systems. As a proof of concept, we coupled the QS pathway to the expression of a cell division inhibitory gene, cdv3, facilitating late-phase cell elongation, cell sedimentation, and improved biomass recovery. Our findings provide a foundation for the development of auto-induction systems leverageable to improve cyanobacterial biotechnology applications.

Acyl homoserine lactones (AHL)↗
DOE OSTI2021

Single-cell RNA sequencing of batch Chlamydomonas cultures reveals heterogeneity in their diurnal cycle phase

The photosynthetic unicellular alga Chlamydomonas (Chlamydomonas reinhardtii) is a versatile reference for algal biology because of its ease of culture in the laboratory. Genomic and systems biology approaches have previously described transcriptome responses to environmental changes using bulk data, thus representing the average behavior from pools of cells. Here, we apply single-cell RNA sequencing (scRNA-seq) to probe the heterogeneity of Chlamydomonas cell populations under three environments and in two genotypes differing by the presence of a cell wall. First, we determined that RNA can be extracted from single algal cells with or without a cell wall, offering the possibility to sample natural algal communities. Second, scRNA-seq successfully separated single cells into nonoverlapping cell clusters according to their growth conditions. Cells exposed to iron or nitrogen deficiency were easily distinguished despite a shared tendency to arrest photosynthesis and cell division to economize resources. Notably, these groups of cells not only recapitulated known patterns observed with bulk RNA-seq but also revealed their inherent heterogeneity. A substantial source of variation between cells originated from their endogenous diurnal phase, although cultures were grown in constant light. We exploited this result to show that circadian iron responses may be conserved from algae to land plants. We document experimentally that bulk RNA-seq data represent an average of typically hidden heterogeneity in the population.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2002

Triangle Geometry Processing for Surface Modeling and Cartesian Grid Generation

Cartesian mesh generation is accomplished for component based geometries, by intersecting components subject to mesh generation to extract wetted surfaces with a geometry engine using adaptive precision arithmetic in a system which automatically breaks ties with respect to geometric degeneracies. During volume mesh generation, intersected surface triangulations are received to enable mesh generation with cell division of an initially coarse grid. The hexagonal cells are resolved, preserving the ability to directionally divide cells which are locally well aligned.

Aftosmis, Michael J.↗
NASA NTRS1993

A scientific role for Space Station Freedom: Research at the cellular level

The scientific importance of Space Station Freedom is discussed in light of the valuable information that can be gained in cellular and developmental biology with regard to the microgravity environment on the cellular cytoskeleton, cellular responses to extracellular signal molecules, morphology, events associated with cell division, and cellular physiology. Examples of studies in basic cell biology, as well as their potential importance to concerns for future enabling strategies, are presented.

Johnson, Terry C.↗
DOE OSTI2025

Quantitative multiplexed analysis of gene and protein expression patterns in Yarrowia lipolytica

In this report, we present coordinated observations of protein and mRNA transcript counts at the single-cell level in the oleaginous yeast model Yarrowia lipolytica. The transcription factor Xbp1p regulates entry into a quiescent state, representing a shift of resources to sequestration of nutrients rather than cell division. We observed the responses of wild-type and Δxbp1 cells to protein (by fluorescence) and transcript quantification and localization at both single-cell and population-averaged levels. Data were collected via single-molecule fluorescence in situ hybridization (smFISH) and qPCR under nitrogen depletion, a condition that drives lipid accumulation. These techniques reveal a complex and heterogeneous population of Xbp1p dynamics and downstream regulation. Our findings highlight the need for single-cell resolution analyses to describe cellular dynamics and regulatory processes.

Yarrowia lipolytica↗