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At least 73 records · Page 4

Centrosome and microtubule instability in aging Drosophila cells

Several cytoskeletal changes are associated with aging which includes alterations in muscle structure leading to muscular atrophy, and weakening of the microtubule network which affects cellular secretion and maintenance of cell shape. Weakening of the microtubule network during meiosis in aging oocytes can result in aneuploidy or trisomic zygotes with increasing maternal age. Imbalances of cytoskeletal organization can lead to disease such as Alzheimer's, muscular disorders, and cancer. Because many cytoskeletal diseases are related to age we investigated the effects of aging on microtubule organization in cell cultures of the Drosophila cell model system (Schneider S-1 and Kc23 cell lines). This cell model is increasingly being used as an alternative system to mammalian cell cultures. Drosophila cells are amenable to genetic manipulations and can be used to identify and manipulate genes which are involved in the aging processes. Immunofluorescence, scanning, and transmission electron microscopy were employed for the analysis of microtubule organizing centers (centrosomes) and microtubules at various times after subculturing cells in fresh medium. Our results reveal that centrosomes and the microtubule network becomes significantly affected in aging cells after 5 days of subculture. At 5-14 days of subculture, 1% abnormal out of 3% mitoses were noted which were clearly distinguishable from freshly subcultured control cells in which 3% of cells undergo normal mitosis with bipolar configurations. Microtubules are also affected in the midbody during cell division. The midbody in aging cells becomes up to 10 times longer when compared with midbodies in freshly subcultured cells. During interphase, microtubules are often disrupted and disorganized, which may indicate improper function related to transport of cell organelles along microtubules. These results are likely to help explain some cytoskeletal disorders and diseases related to aging.

Non-NASA Center↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗

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)↗

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.↗

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.↗

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↗

PCM1 coordinates centrosome asymmetry with polarized endosome dynamics to regulate daughter cell fate

Vertebrate radial glia progenitors (RGPs) balance self-renewal and differentiation through asymmetric cell division (ACD), which involves unequal centrosome inheritance. How centrosome asymmetry directs cell fate remains poorly understood. Here, we identify Pericentriolar material 1 (Pcm1) as a key player in this process. In zebrafish embryonic RGPs, Pcm1 is asymmetrically associated with Cep83, a mother centrosome marker. Using in vivo time-lapse imaging and nanoscale-resolution expansion microscopy, we detect Pcm1 on Notch ligand-containing endosomes, where it interacts–either directly or indirectly–with Par-3 and dynein. Loss of pcm1 disrupts endosome dynamics, increasing neuronal differentiation at the expense of RGP self-renewal. Mechanistically, Pcm1 facilitates the transition from Rab5b to Rab11a and promotes the assembly of Par-3 and dynein macromolecular complexes on recycling endosomes. Furthermore, we find conserved PARD3-PCM1-CEP83-RAB11 associations in human cortical brain organoids. Our findings uncover that Pcm1 links centrosome asymmetry to polarized endosome trafficking, thereby regulating RGP fate decisions.

Cell fate and cell lineage↗

Unique trajectory of gene family evolution from genomic analysis of nearly all known species in an ancient yeast lineage

Gene gains and losses are a major driver of genome evolution; their precise characterization can provide insights into the origin and diversification of major lineages. Here, we examined gene family evolution of 1154 genomes from nearly all known species in the medically and technologically important yeast subphylum Saccharomycotina. We found that yeast gene family evolution differs from that of plants, animals, and filamentous ascomycetes, and is characterized by smaller overall gene numbers yet larger gene family sizes for a given gene number. Faster-evolving lineages (FELs) in yeasts experienced significantly higher rates of gene losses—commensurate with a narrowing of metabolic niche breadth—but higher speciation rates than their slower-evolving sister lineages (SELs). Gene families most often lost are those involved in mRNA splicing, carbohydrate metabolism, and cell division and are likely associated with intron loss, metabolic breadth, and non-canonical cell cycle processes. Our results highlight the significant role of gene family contractions in the evolution of yeast metabolism, genome function, and speciation, and suggest that gene family evolutionary trajectories have differed markedly across major eukaryotic lineages.

Comparative Genomics↗

RBE of Energetic Iron Ions for the Induction of Early and Late Chromosome Aberrations in Different Cell Types

Numerous published studies have reported the Relative Biological Effectiveness (RBE) values for chromosome aberrations induced by charged particles of different LET. The RBE for chromosome aberrations in human lymphocytes exposed ex vivo has been suggested to show a similar relationship as the quality factor for cancer induction. Therefore, increased chromosome aberrations in the astronauts' white blood cells post long-duration missions are used to determine the biological doses from exposures to space radiation. However, the RBE value is known to be very different for different types of cancer. Previously, we reported that, even though the RBE for initial chromosome damages was high in human lymphocytes exposed to Fe ions, the RBE was significantly reduced after multiple cell divisions post irradiation. To test the hypothesis that RBE values for chromosome aberrations are cell type dependent, and different between early and late damages, we exposed human lymphocytes ex vivo, and human mammary epithelial cells in vitro to various charged particles. Chromosome aberrations were quantified using the samples collected at first mitosis post irradiation for initial damages, and the samples collected after multiple generations for the remaining or late arising aberrations. Results of the study suggested that the effectiveness of high-LET charged particles for late chromosome aberrations may be cell type dependent, even though the RBE values are similar for early damages.

Zhang, Ye↗

Dependence of Early and Late Chromosomal Aberrations on Radiation Quality and Cell Types

Exposure to radiation induces different types of DNA damage, increases mutation and chromosome aberration rates, and increases cellular transformation in vitro and in vivo. The susceptibility of cells to radiation depends on genetic background and growth condition of cells, as well as types of radiation. Mammalian cells of different tissue types and with different genetic background are known to have different survival rate and different mutation rate after cytogenetic insults. Genomic instability, induced by various genetic, metabolic, and environmental factors including radiation, is the driving force of tumorigenesis. Accurate measurements of the relative biological effectiveness (RBE) is important for estimating radiation-related risks. To further understand genomic instability induced by charged particles and their RBE, we exposed human lymphocytes ex vivo, human fibroblast AG1522, human mammary epithelial cells (CH184B5F5/M10), and bone marrow cells isolated from CBA/CaH(CBA) and C57BL/6 (C57) mice to high energy protons and Fe ions. Normal human fibroblasts AG1522 have apparently normal DNA damage response and repair mechanisms, while mammary epithelial cells (M10) are deficient in the repair of DNA DSBs. Mouse strain CBA is radio-sensitive while C57 is radio-resistant. Metaphase chromosomes at different cell divisions after radiation exposure were collected and chromosome aberrations were analyzed as RBE for different cell lines exposed to different radiations at various time points up to one month post irradiation.

Lu, Tao↗

Elevated levels of N-lauroylethanolamine, an endogenous constituent of desiccated seeds, disrupt normal root development in Arabidopsis thaliana seedlings

N-Acylethanolamines (NAEs) are prevalent in desiccated seeds of various plant species, and their levels decline substantially during seed imbibition and germination. Here, seeds of Arabidopsis thaliana (L.) Heynh. were germinated in, and seedlings maintained on, micromolar concentrations of N-lauroylethanolamine (NAE 12:0). NAE 12:0 inhibited root elongation, increased radial swelling of root tips, and reduced root hair numbers in a highly selective and concentration-dependent manner. These effects were reversible when seedlings were transferred to NAE-free medium. Older seedlings (14 days old) acclimated to exogenous NAE by increased formation of lateral roots, and generally, these lateral roots did not exhibit the severe symptoms observed in primary roots. Cells of NAE-treated primary roots were swollen and irregular in shape, and in many cases showed evidence, at the light- and electron-microscope levels, of improper cell wall formation. Microtubule arrangement was disrupted in severely distorted cells close to the root tip, and endoplasmic reticulum (ER)-localized green fluorescent protein (mGFP5-ER) was more abundant, aggregated and distributed differently in NAE-treated root cells, suggesting disruption of proper cell division, endomembrane organization and vesicle trafficking. These results suggest that NAE 12:0 likely influences normal cell expansion in roots by interfering with intracellular membrane trafficking to and/or from the cell surface. The rapid metabolism of NAEs during seed imbibition/germination may be a mechanism to remove this endogenous class of lipid mediators to allow for synchronized membrane reorganization associated with cell expansion.

Non-NASA Center↗