Asymmetric Distribution of DNA Between Daughter Cells with Final Symmetry Breaking During Aging of Human Fibroblasts

AbstractCytotoxic immune cells are endowed with a high degree of heterogeneity in their lytic function, but how this heterogeneity is generated is still an open question. We therefore investigated if human CD8+ T cells could segregate their lytic components during telophase, using imaging flow cytometry, confocal microscopy and live cell imaging. We show that CD107a+-intracellular vesicles, perforin and granzyme B unevenly segregate in a constant fraction of telophasic cells during each division round. Mathematical modeling posits that unequal lytic molecule inheritance by daughter cells results from the random distribution of lytic granules on the two sides of the cleavage furrow. Finally, we establish that the level of lytic compartment in individual CTL dictates CTL killing capacity. Together, our results show the stochastic asymmetric distribution of effector molecules in dividing CD8+ T cells. They propose uneven mitotic repartition of pre-packaged lytic components as a mechanism generating non-hereditary functional heterogeneity in CTL.

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CENP-C regulates centromere assembly, asymmetry and epigenetic age in Drosophila germline stem cells

10.1101/2020.11.02.364794 ◽

2020 ◽

Author(s):

Ben L Carty ◽

Anna A Dattoli ◽

Elaine M Dunleavy

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Germ Line ◽

Asymmetric Distribution ◽

Germline Stem Cells ◽

Daughter Cells ◽

Sister Chromatids ◽

Asymmetric Divisions ◽

Centromere Assembly

AbstractGermline stem cells divide asymmetrically to produce one new daughter stem cell and one daughter cell that will subsequently undergo meiosis and differentiate to generate the mature gamete. The silent sister hypothesis proposes that in asymmetric divisions, the selective inheritance of sister chromatids carrying specific epigenetic marks between stem and daughter cells impacts cell fate. To facilitate this selective inheritance, the hypothesis specifically proposes that the centromeric region of each sister chromatid is distinct. In Drosophila germ line stem cells (GSCs), it has recently been shown that the centromeric histone CENP-A (called CID in flies) - the epigenetic determinant of centromere identity - is asymmetrically distributed between sister chromatids. In these cells, CID deposition occurs in G2 phase such that sister chromatids destined to end up in the stem cell harbour more CENP-A, assemble more kinetochore proteins and capture more spindle microtubules. These results suggest a potential mechanism of ‘mitotic drive’ that might bias chromosome segregation. Here we report that the inner kinetochore protein CENP-C, is required for the assembly of CID in G2 phase in GSCs. Moreover, CENP-C is required to maintain a normal asymmetric distribution of CID between stem and daughter cells. In addition, we find that CID is lost from centromeres in aged GSCs and that a reduction in CENP-C accelerates this loss. Finally, we show that CENP-C depletion in GSCs disrupts the balance of stem and daughter cells in the ovary, shifting GSCs toward a self-renewal tendency. Ultimately, we provide evidence that centromere assembly and maintenance via CENP-C is required to sustain asymmetric divisions in female Drosophila GSCs.

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Active gelation breaks time-reversal-symmetry of mitotic chromosome mechanics

10.1101/296566 ◽

2018 ◽

Author(s):

Matthäus Mittasch ◽

Anatol W. Fritsch ◽

Michael Nestler ◽

Juan M. Iglesias-Artola ◽

Kaushikaram Subramanian ◽

...

Keyword(s):

Symmetry Breaking ◽

Time Reversal ◽

Mitotic Chromosome ◽

Metaphase Plate ◽

Time Reversal Symmetry ◽

Newtonian Mechanics ◽

Arrow Of Time ◽

Cell Nuclei ◽

Mitotic Chromosomes ◽

Daughter Cells

AbstractIn cell division, mitosis is the phase in which duplicated sets of chromosomes are mechanically aligned to form the metaphase plate before being segregated in two daughter cells. Irreversibility is a hallmark of this process, despite the fundamental laws of Newtonian mechanics being time symmetric.Here we show experimentally that mitotic chromosomes receive the arrow of time by time-reversal-symmetry breaking of the underlying mechanics in prometaphase. By optically inducing hydrodynamic flows within prophase nuclei, we find that duplicated chromatid pairs initially form a fluid suspension in the nucleoplasm: although showing little motion on their own, condensed chromosomes are free to move through the nucleus in a time-reversible manner. Actively probing chromosome mobility further in time, we find that this viscous suspension of chromatin transitions into a gel after nuclear breakdown. This gel state, in which chromosomes cannot be moved by flows, persists even when chromosomes start moving to form the metaphase plate. Complemented by minimal reconstitution experiments, our active intra-nuclear micro-rheology reveals time-reversal-symmetry breaking of chromosome mechanics to be caused by the transition from a purely fluid suspension into an active gel.Graphical abstractOne sentence summaryFlows induced in living cell nuclei reveal the rheological changes that bring chromosomes under mechanical control during mitosis.

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Stochastic asymmetric repartition of lytic machinery in dividing CD8+ T cells generates heterogeneous killing behavior

eLife ◽

10.7554/elife.62691 ◽

2021 ◽

Vol 10 ◽

Author(s):

Fanny Lafouresse ◽

Romain Jugele ◽

Sabina Müller ◽

Marine Doineau ◽

Valérie Duplan-Eche ◽

...

Keyword(s):

T Cells ◽

Asymmetric Distribution ◽

Lytic Granules ◽

Daughter Cells ◽

Lytic Function ◽

Intracellular Vesicles ◽

Open Question ◽

High Degree ◽

Two Sides ◽

Lytic Compartment

Cytotoxic immune cells are endowed with a high degree of heterogeneity in their lytic function, but how this heterogeneity is generated is still an open question. We therefore investigated if human CD8+ T cells could segregate their lytic components during telophase, using imaging flow cytometry, confocal microscopy and live cell imaging. We show that CD107a+-intracellular vesicles, perforin and granzyme B unevenly segregate in a constant fraction of telophasic cells during each division round. Mathematical modeling posits that unequal lytic molecule inheritance by daughter cells results from the random distribution of lytic granules on the two sides of the cleavage furrow. Finally, we establish that the level of lytic compartment in individual CTL dictates CTL killing capacity. Together, our results show the stochastic asymmetric distribution of effector molecules in dividing CD8+ T cells. They propose uneven mitotic repartition of pre-packaged lytic components as a mechanism generating non-hereditary functional heterogeneity in CTL.

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CENP-C functions in centromere assembly, the maintenance of CENP-A asymmetry and epigenetic age in Drosophila germline stem cells

PLoS Genetics ◽

10.1371/journal.pgen.1009247 ◽

2021 ◽

Vol 17 (5) ◽

pp. e1009247

Author(s):

Ben L. Carty ◽

Anna A. Dattoli ◽

Elaine M. Dunleavy

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Germ Line ◽

Asymmetric Distribution ◽

Germline Stem Cells ◽

Daughter Cells ◽

Sister Chromatids ◽

Asymmetric Divisions ◽

Centromere Assembly

Germline stem cells divide asymmetrically to produce one new daughter stem cell and one daughter cell that will subsequently undergo meiosis and differentiate to generate the mature gamete. The silent sister hypothesis proposes that in asymmetric divisions, the selective inheritance of sister chromatids carrying specific epigenetic marks between stem and daughter cells impacts cell fate. To facilitate this selective inheritance, the hypothesis specifically proposes that the centromeric region of each sister chromatid is distinct. In Drosophila germ line stem cells (GSCs), it has recently been shown that the centromeric histone CENP-A (called CID in flies)—the epigenetic determinant of centromere identity—is asymmetrically distributed between sister chromatids. In these cells, CID deposition occurs in G2 phase such that sister chromatids destined to end up in the stem cell harbour more CENP-A, assemble more kinetochore proteins and capture more spindle microtubules. These results suggest a potential mechanism of ‘mitotic drive’ that might bias chromosome segregation. Here we report that the inner kinetochore protein CENP-C, is required for the assembly of CID in G2 phase in GSCs. Moreover, CENP-C is required to maintain a normal asymmetric distribution of CID between stem and daughter cells. In addition, we find that CID is lost from centromeres in aged GSCs and that a reduction in CENP-C accelerates this loss. Finally, we show that CENP-C depletion in GSCs disrupts the balance of stem and daughter cells in the ovary, shifting GSCs toward a self-renewal tendency. Ultimately, we provide evidence that centromere assembly and maintenance via CENP-C is required to sustain asymmetric divisions in female Drosophila GSCs.

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Systematic analysis of asymmetric partitioning of yeast proteome between mother and daughter cells reveals “aging factors” and mechanism of lifespan asymmetry

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1506054112 ◽

2015 ◽

Vol 112 (38) ◽

pp. 11977-11982 ◽

Cited By ~ 30

Author(s):

Jing Yang ◽

Mark A. McCormick ◽

Jiashun Zheng ◽

Zhengwei Xie ◽

Mitsuhiro Tsuchiya ◽

...

Keyword(s):

Cell Division ◽

Asymmetric Cell Division ◽

Asymmetric Distribution ◽

Systematic Analysis ◽

Daughter Cells ◽

Yeast Proteome ◽

Mother And Daughter ◽

Asymmetric Partitioning ◽

Mother Cells ◽

Aging Factors

Budding yeast divides asymmetrically, giving rise to a mother cell that progressively ages and a daughter cell with full lifespan. It is generally assumed that mother cells retain damaged, lifespan limiting materials (“aging factors”) through asymmetric division. However, the identity of these aging factors and the mechanisms through which they limit lifespan remain poorly understood. Using a flow cytometry-based, high-throughput approach, we quantified the asymmetric partitioning of the yeast proteome between mother and daughter cells during cell division, discovering 74 mother-enriched and 60 daughter-enriched proteins. While daughter-enriched proteins are biased toward those needed for bud construction and genome maintenance, mother-enriched proteins are biased towards those localized in the plasma membrane and vacuole. Deletion of 23 of the 74 mother-enriched proteins leads to lifespan extension, a fraction that is about six times that of the genes picked randomly from the genome. Among these lifespan-extending genes, three are involved in endosomal sorting/endosome to vacuole transport, and three are nitrogen source transporters. Tracking the dynamic expression of specific mother-enriched proteins revealed that their concentration steadily increases in the mother cells as they age, but is kept relatively low in the daughter cells via asymmetric distribution. Our results suggest that some mother-enriched proteins may increase to a concentration that becomes deleterious and lifespan-limiting in aged cells, possibly by upsetting homeostasis or leading to aberrant signaling. Our study provides a comprehensive resource for analyzing asymmetric cell division and aging in yeast, which should also be valuable for understanding similar phenomena in other organisms.

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PolyQ-dependent RNA–protein assemblies control symmetry breaking

The Journal of Cell Biology ◽

10.1083/jcb.201407105 ◽

2015 ◽

Vol 208 (5) ◽

pp. 533-544 ◽

Cited By ~ 53

Author(s):

ChangHwan Lee ◽

Patricia Occhipinti ◽

Amy S. Gladfelter

Keyword(s):

Symmetry Breaking ◽

Cell Polarity ◽

Protein Interaction ◽

Dendritic Growth ◽

Physiological Function ◽

Low Complexity ◽

Asymmetric Distribution ◽

Ashbya Gossypii ◽

Polarized Growth ◽

Protein Assemblies

Dendritic growth in fungi and neurons requires that multiple axes of polarity are established and maintained within the same cytoplasm. We have discovered that transcripts encoding key polarity factors including a formin, Bni1, and a polarisome scaffold, Spa2, are nonrandomly clustered in the cytosol to initiate and maintain sites of polarized growth in the fungus Ashbya gossypii. This asymmetric distribution requires the mRNAs to interact with a polyQ-containing protein, Whi3, and a Pumilio protein with a low-complexity sequence, Puf2. Cells lacking Whi3 or Puf2 had severe defects in establishing new sites of polarity and failed to localize Bni1 protein. Interaction of mRNAs with Whi3 and Puf2 promotes enrichment of transcripts at established sites of polarized growth and clustering of polarity transcripts throughout the cell body. Thus, aggregation-prone proteins make functional assemblies to position polarity transcripts, and nonrandom positioning of transcripts is required for symmetry-breaking events. This reveals a physiological function for polyQ-driven assemblies in regulating cell polarity.

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Excitable dynamics of Ras triggers self-organized PIP3 signaling for spontaneous cell migration

10.1101/356105 ◽

2018 ◽

Cited By ~ 3

Author(s):

Seiya Fukushima ◽

Satomi Matsuoka ◽

Masahiro Ueda

Keyword(s):

Symmetry Breaking ◽

Cell Movement ◽

Direct Interaction ◽

Feedback Regulation ◽

Asymmetric Distribution ◽

Spatial Cues ◽

Downstream Signaling ◽

Self Organized ◽

Ras Activation ◽

Tightly Coupled

Spontaneous cell movement is underpinned by an asymmetric distribution of signaling molecules including small G proteins and phosphoinositides on the cell membrane. A fundamental question is the molecular mechanism for the spontaneous symmetry breaking. Here we report that GTP bound Ras (Ras-GTP) breaks the symmetry due to excitability even in the absence of extracellular spatial cues and cytoskeletal polarity as well as downstream signaling activities. A stochastic excitation of local and transient Ras activation induced PIP3 accumulation via direct interaction with PI3K, causing tightly coupled traveling waves propagating along the membrane. Comprehensive phase analysis of the waves of Ras-GTP and PIP3 metabolism-related molecules revealed the network structure of the excitable system including positive feedback regulation of Ras-GTP by PIP3. A mathematical model reconstituted a series of the observed symmetry breaking phenomena, illustrating an essential involvement of excitability in the cellular decision-making process.

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Mother-daughter asymmetry of pH underlies aging and rejuvenation in yeast

eLife ◽

10.7554/elife.03504 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 75

Author(s):

Kiersten A Henderson ◽

Adam L Hughes ◽

Daniel E Gottschling

Keyword(s):

Plasma Membrane ◽

Mother Cell ◽

Daughter Cell ◽

Asymmetric Distribution ◽

Replicative Aging ◽

Cytosolic Ph ◽

Daughter Cells ◽

Proton Atpase ◽

Mother And Daughter ◽

Mother Cells

Replicative aging in yeast is asymmetric–mother cells age but their daughter cells are rejuvenated. Here we identify an asymmetry in pH between mother and daughter cells that underlies aging and rejuvenation. Cytosolic pH increases in aging mother cells, but is more acidic in daughter cells. This is due to the asymmetric distribution of the major regulator of cytosolic pH, the plasma membrane proton ATPase (Pma1). Pma1 accumulates in aging mother cells, but is largely absent from nascent daughter cells. We previously found that acidity of the vacuole declines in aging mother cells and limits lifespan, but that daughter cell vacuoles re-acidify. We find that Pma1 activity antagonizes mother cell vacuole acidity by reducing cytosolic protons. However, the inherent asymmetry of Pma1 increases cytosolic proton availability in daughter cells and facilitates vacuole re-acidification and rejuvenation.

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In Vivo Pre-Instructed HSCs Robustly Execute Asymmetric Cell Divisions In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms21218225 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8225

Author(s):

Mukul Girotra ◽

Vincent Trachsel ◽

Aline Roch ◽

Matthias P. Lutolf

Keyword(s):

Single Cell ◽

Cell Fate ◽

Asymmetric Distribution ◽

Hematopoietic Stem ◽

Asymmetric Cell Divisions ◽

Native Bone ◽

Daughter Cells ◽

Cell Divisions

Hematopoietic stem cells (HSCs) are responsible for life-long production of all mature blood cells. Under homeostasis, HSCs in their native bone marrow niches are believed to undergo asymmetric cell divisions (ACDs), with one daughter cell maintaining HSC identity and the other committing to differentiate into various mature blood cell types. Due to the lack of key niche signals, in vitro HSCs differentiate rapidly, making it challenging to capture and study ACD. To overcome this bottleneck, in this study, we used interferon alpha (IFNα) treatment to ”pre-instruct” HSC fate directly in their native niche, and then systematically studied the fate of dividing HSCs in vitro at the single cell level via time-lapse analysis, as well as multigene and protein expression analysis. Triggering HSCs’ exit from dormancy via IFNα was found to significantly increase the frequency of asynchronous divisions in paired daughter cells (PDCs). Using single-cell gene expression analyses, we identified 12 asymmetrically expressed genes in PDCs. Subsequent immunocytochemistry analysis showed that at least three of the candidates, i.e., Glut1, JAM3 and HK2, were asymmetrically distributed in PDCs. Functional validation of these observations by colony formation assays highlighted the implication of asymmetric distribution of these markers as hallmarks of HSCs, for example, to reliably discriminate committed and self-renewing daughter cells in dividing HSCs. Our data provided evidence for the importance of in vivo instructions in guiding HSC fate, especially ACD, and shed light on putative molecular players involved in this process. Understanding the mechanisms of cell fate decision making should enable the development of improved HSC expansion protocols for therapeutic applications.

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