Asymmetric Segregation Research Articles

Asymmetric Segregation of Aged Spindle Pole Bodies During Cell Division: Mechanisms and Relevance Beyond Budding Yeast?

Asymmetric cell division generates cell diversity and contributes to cellular aging and rejuvenation. Here, we review the molecular mechanisms enabling budding yeast to recognize spindle pole bodies (SPB, centrosome equivalent) based on their age, and guide their non-random mitotic segregation: SPB inheritance requires the distinction of old from new SPBs and is regulated by the SPB-inheritance network (SPIN) and the mitotic exit network (MEN). The SPIN marks the pre-existing SPB as old and the MEN recognizes these marks translating them into spindle orientation. We next revisit other molecules and structures that partition depending on their age rather than their abundance at mitosis as, for example, DNA, centrosomes, mitochondria, and histones in yeast and other systems. The recurrence of this differential behavior suggests a functional significance for numerous cell types, which we then discuss. We conclude that non-random segregation may facilitate asymmetric cell fate determination and thereby indirectly aging and rejuvenation. Also see the video abstract here: https://youtu.be/1sQ4rAomnWY.

Time-resolved transcriptomics in neural stem cells identifies a v-ATPase/Notch regulatory loop.

Drosophila melanogaster neural stem cells (neuroblasts [NBs]) divide asymmetrically by differentially segregating protein determinants into their daughter cells. Although the machinery for asymmetric protein segregation is well understood, the events that reprogram one of the two daughter cells toward terminal differentiation are less clear. In this study, we use time-resolved transcriptional profiling to identify the earliest transcriptional differences between the daughter cells on their way toward distinct fates. By screening for coregulated protein complexes, we identify vacuolar-type H+-ATPase (v-ATPase) among the first and most significantly down-regulated complexes in differentiating daughter cells. We show that v-ATPase is essential for NB growth and persistent activity of the Notch signaling pathway. Our data suggest that v-ATPase and Notch form a regulatory loop that acts in multiple stem cell lineages both during nervous system development and in the adult gut. We provide a unique resource for investigating neural stem cell biology and demonstrate that cell fate changes can be induced by transcriptional regulation of basic, cell-essential pathways.

1352 Basal layer divisions in epidermis over the human lifespan

While proliferation is decreased in aged tissues, no change in SC number was detected by our group or Bickenbachs. Lechler et al. examined types of SC divisions. Divisions perpendicular to the basement membrane were 8% in single-layered embryonic epidermis, 66% in multi-layer epidermis and 85% in adult mice. In humans there is a decrease in cells positive for the proliferation marker Ki67. Our aim was to quantify mitoses of basal keratinocytes in vivo, and examine the types of division, over the human lifespan. We quantified divisions in the basal layer in neonatal (0-7 days), adult (30-45 years) and aged (70-80 years) skin. Asymmetric segregation of polarity proteins is a defining characteristic of SCs. We visualized division orientation in the basal layer using gtubulin (spindle) and NUMA (polarity protein) in biopsies. Because of the limited divisions seen in vivo, we also quantified division type in keratinocytes from freshly obtained biopsies, in vitro, using Numb segregation to identify asymmetric SC divisions. The number of divisions/10cm of basal layer was significantly decreased with age [neonatal 7.6±2 (n=5) vs. adult 3.2±0.5 (n=4) vs. aged 1.5± 0.4 (n=8), p=0.001]. Both perpendicular (asymmetric) and parallel (symmetric) divisions were decreased with age. No significant change in the proportion of perpendicular (asymmetric) SC divisions was detected (neonatal 41.7±14.4% vs. adult 27.5±15.9% vs. aged 36.8±8%). Similar results were found in vitro [neonatal 29.1±7.4% (n=11) vs. adult 32.2% (n= 2) vs. aged 31.2±11.6% (n= 7)]. We conclude that epidermal basal cell divisions decrease over the human lifespan such that the number of divisions in 70+ year olds is 20% of that in neonates. However, the ratio of asymmetric/symmetric divisions remains unchanged. These findings could reflect a decrease in SC number, an increase in SC quiescence, and/or longer cell cycle duration in human epidermis.

Real-time imaging of yeast cells reveals several distinct mechanisms of curing of the [URE3] prion

The [URE3] yeast prion is the self-propagating amyloid form of the Ure2 protein. [URE3] is cured by overexpression of several yeast proteins, including Ydj1, Btn2, Cur1, Hsp42, and human DnaJB6. To better understand [URE3] curing, we used real-time imaging with a yeast strain expressing a GFP-labeled full-length Ure2 construct to monitor the curing of [URE3] over time. [URE3] yeast cells exhibited numerous fluorescent foci, and expression of the GFP-labeled Ure2 affected neither mitotic stability of [URE3] nor the rate of [URE3] curing by the curing proteins. Using guanidine to cure [URE3] via Hsp104 inactivation, we found that the fluorescent foci are progressively lost as the cells divide until they are cured; the fraction of cells that retained the foci was equivalent to the [URE3] cell fraction measured by a plating assay, indicating that the foci were the prion seeds. During the curing of [URE3] by Btn2, Cur1, Hsp42, or Ydj1 overexpression, the foci formed aggregates, many of which were 0.5 μm or greater in size, and [URE3] was cured by asymmetric segregation of the aggregated seeds. In contrast, DnaJB6 overexpression first caused a loss of detectable foci in cells that were still [URE3] before there was complete dissolution of the seeds, and the cells were cured. We conclude that GFP labeling of full-length Ure2 enables differentiation among the different [URE3]-curing mechanisms, including inhibition of severing followed by seed dilution, seed clumping followed by asymmetric segregation between mother and daughter cells, and seed dissolution.

Centrosome Asymmetry in Fly Neural Stem Cells is Established in Early Mitosis Through Dynamic Mother-to-Daughter Centriole Relocalization of Cnb and Polo

Centrosomes, the main microtubule organizing centers (MTOCs) of metazoan cells, contain an older ‘mother’ and a younger ‘daughter’ centriole. Stem cells either inherit the mother or daughter centriole-containing centrosome, providing a possible mechanism for biased delivery of cell fate determinants. However, the molecular mechanisms regulating centrosome asymmetry and biased centrosome segregation are unclear. Using 3D-Structured Illumination Microscopy (3D-SIM) we show that in fly neural stem cells (neuroblasts) the mitotic kinase Polo and its substrate the centriolar protein Centrobin (Cnb) relocalize from the mother to the daughter centriole during mitosis. Cnb’s dynamic relocalization is regulated by Polo-mediated phosphorylation, whereas Polo requires both Wdr62 and Cnb. This switch in protein localization generates a centrosome that contains two molecularly distinct centrioles which separate at the end of mitosis. We propose that in fly neuroblasts the establishment of centriole asymmetry during mitosis is necessary for biased interphase MTOC activity, spindle orientation and asymmetric centrosome segregation.

The Fate Choice Between Effector and Memory T Cell Lineages: Asymmetry, Signal Integration, and Feedback to Create Bistability.

CD8+ T cells clear primary infections with intracellular pathogens and provide long-term immunity against reinfection. Two different types of CD8+ T cells are responsible for these functions: short-lived effector T cells and memory T cells. The cellular relationship between these two types of CD8+ T cells has been subject to much investigation. Both cell types can derive from a single naïve CD8+ T cell precursor. Their generation requires a fate choice early during a T cell response. As a result, two populations of T cells emerge. One of these consists of terminally differentiated short-lived effector T cells. The other contains cells able to develop into long-lived memory T cells. A foundation for development of these two populations may be laid during the first division of an activated naïve T cell precursor, as a consequence of asymmetric segregation of fate-determining factors into the daughter cells. Nonetheless, the binary choice between the two lineages is strongly influenced by signals, which ensure that the differentiation process is matched with the needs posed by the infection. Here, we will discuss the genetic and metabolic programs governing differentiation of these two lineages as well as the processes leading to their induction and consolidation to create bistability. These processes involve extensive lateral inhibition between the programs as well as positive feedback between the genetic programs and the signaling pathways responsible for their induction. These features will be highlighted by discussing the role of the Notch signaling pathway in guiding the decision between the two lineages.

Emerging roles for sphingolipids in cellular aging.

Aging is a gradual loss of physiological functions as organisms' progress in age. Although aging in multicellular organisms is complex, some fundamental mechanisms and pathways may be shared from the single cellular yeast to human. Budding yeast Saccharomyces cerevisiae has been established model system for aging studies. A yeast cell divides asymmetrically to produce two cells that differ in size and age. The one that is smaller coming from bud is a newborn cell that with a full replicative potential head irrespective of the replicative age of its mother-the larger cell from which the bud grows out before division. The age asymmetry between daughter and mother is thought to be dependent on asymmetric segregation of certain factors such as protein aggregates, extrachromosomal DNA (ERCs) and dysfunctional organelles during successive cell divisions of the yeast replicative lifespan (RLS). It is also thought that certain plasma membrane proteins, in particular multidrug-resistant (MDR) proteins, asymmetrically partition between the mother and the bud based on the age of the polypeptides. Functional decline associated with the molecular aging of those proteins contributes to the fitness decline at advance age. In our recent study, we showed that sphingolipids facilitate the age-dependent segregation of MDRs between daughter and mother cell. In this review, we highlight and discuss the potential mechanisms by which sphingolipids regulate the aging process in yeast and cells of vertebrate animals including human.

Increased Numb protein expression predicts poor clinical outcomes in esophageal squamous cell carcinoma patients

ABSTRACTNumb is a protein whose asymmetric segregation during cell division determines cell fate and has numerous functions relevant to multiple fields of study, including developmental neurobiology and cancer biology. Little is known about the role of Numb in esophageal squamous cell carcinoma (ESCC), the predominant histological esophageal carcinoma in Asian populations. In this study, we focused on the expression and biologic functions of Numb in the context of ESCC. From analysis of tissue microarrays with 212 patients, it was found that Numb was significantly upregulated in ESCC tissues compared with corresponding non-cancerous tissues. Kaplan-Meier survival analysis suggests that higher expression of Numb was significantly associated with a high tumor recurrence (p = 0.015) and poor overall post-surgical survival (p = 0.016). Using multiple Cox regression, the expression of Numb was determined to be an independent predictor of poor prognosis. When siRNA was used to knockdown Numb in ESCC cell lines, there was a consistent increase in caspase-3 dependent apoptosis and inhibition of cellular proliferation, as well as downregulation of expression of the cancer stem cell markers Oct-4, SOX-2 and Nanog. In addition, downregulated Numb expression was not significantly associated with the migration of ESCC cells. These results indicate that Numb acts as an oncoprotein and has potential as a novel prognostic biomarker and therapeutic target in ESCC patients.

Mitochondria-Specific Monoclonal Antibodies in Eggs and Embryos of the Ascidian Halocynthia roretzi.

ABSTRACTAscidian embryos have become an important model for embryological studies, offering a simple example for mechanisms of cytoplasmic components segregation. It is a well-known example that the asymmetric segregation of mitochondria into muscle lineage cells occurs during ascidian embryogenesis. However, it is still unclear which signaling pathway is involved in this process. To obtain molecular markers for studying mechanisms involved in the asymmetric distribution of mitochondria, we have produced monoclonal antibodies, Mito-1, Mito-2 and Mito-3, that specifically recognize mitochondriarich cytoplasm in cells of the ascidian Halocynthia roretzi embryos. These antibodies stained cytoplasm like reticular structure in epidermis cells, except for nuclei, at the early tailbud stage. Similar pattern was observed in vital staining of mitochondria with DiOC2, a fluorescent probe of mitochondria. Immunostaining with these antibodies showed that mitochondria are evenly distributed in the animal hemisphere blastomeres at cleavage stages, whereas not in the vegetal hemisphere blastomeres. Mitochondria were transferred to the presumptive muscle and nerve cord lineage cells of the marginal zone in the vegetal hemisphere more than to the presumptive mesenchyme, notochord and endoderm lineage of the central zone. Therefore, it is suggested that these antibodies will be useful markers for studying mechanisms involved in the polarized distribution of mitochondria during ascidian embryogenesis.

Autosomal Trisomy and Triploidy Are Corrected During Female Meiosis in Caenorhabditis elegans

Trisomy and triploidy, defined as the presence of a third copy of one or all chromosomes, respectively, are deleterious in many species including humans. Previous studies have demonstrated that Caenorhabditis elegans with a third copy of the X chromosome are viable and fertile. However, the extra X chromosome was shown to preferentially segregate into the first polar body during oocyte meiosis to produce a higher frequency of euploid offspring than would be generated by random segregation. Here, we demonstrate that extra autosomes are preferentially eliminated by triploid C. elegans and trisomy IV C. elegans Live imaging of anaphase-lagging chromosomes and analysis of REC-8 staining of metaphase II spindles revealed that, in triploids, some univalent chromosomes do not lose cohesion and preferentially segregate intact into the first polar body during anaphase I, whereas other autosomes segregate chromatids equationally at anaphase I and eliminate some of the resulting single chromatids during anaphase II. We also demonstrate asymmetry in the anaphase spindle, which may contribute to the asymmetric segregation. This study reveals a pathway that allows aneuploid parents to produce euploid offspring at higher than random frequency.

Particle segregation in turbulent Couette–Poiseuille flow with vanishing wall shear

Inertial particles dispersed in wall-bounded flows in pipes and channels are known to accumulate close to the walls. The segregation ability depends greatly on the inertia-selection effects of the near-wall quasi-coherent turbulent structures, which are formed near both walls where shear stresses are high. Here, however, we investigated if and how particles segregate in the vicinity of walls in absence of mean shear. A tailor-made turbulent Couette–Poiseuille flow was designed such that the mean shear vanished at the moving wall, thereby resulting in an asymmetric flow with conventional near-wall turbulent structures only at one wall. In addition, Large-Scale Structures (LSSs) were observed in the flow, which greatly influenced the distribution of the inertial particles. Particles of five different inertia groups were embedded in the directly simulated turbulence field and examined. It was found that particles of high inertia segregated near the stationary wall where mean shear prevailed, but also near the moving wall where mean shear was absent. However, due to the qualitatively distinct near-wall flow structures, the inertia effects on the actual segregation were different at the two walls. Mechanisms causing the asymmetric wall-normal segregation were explored with the focus on the moving-wall region, where the quasi-coherent turbulent structures were absent, and the local fluid structures were dominated by imprints of the LSSs.

Polarity, Asymmetry and Aging:Are there Yayatis among Bacteria?

Bacteria have been shown to age. In an exponentially growing population some cells progressively slow down and stop dividing. This is thought to be due to asymmetric damage segregation in which old pole cells retain damaged components and the new pole cells receive newly synthesized components. Polarity implies functional asymmetry with a predefined direction with or without morphological difference. Cellular polarity and division asymmetry are common to yeast, bacteria and stem cells of multi-cell organisms. A number of processes in bacteria, including formation of endospores, flagella, stalks or buds show clear polar biases.

Unique sequence organization and small RNA expression of a "selfish" B chromosome.

B chromosomes are found in numerous plants and animals. These nonessential, supernumerary chromosomes are often composed primarily of noncoding DNA repeats similar to those found within transcriptionally "silenced" heterochromatin. In order to persist within their resident genomes, many B chromosomes exhibit exceptional cellular behaviors, including asymmetric segregation into gametes and induction of genome elimination during early development. An important goal in understanding these behaviors is to identify unique B chromosome sequences and characterize their transcriptional contributions. We investigated these properties by examining a paternally transmitted B chromosome known as paternal sex ratio (PSR), which is present in natural populations of the jewel wasp Nasonia vitripennis. To facilitate its own transmission, PSR severely biases the sex ratio by disrupting early chromatin remodeling processes. Through cytological mapping and other approaches, we identified multiple DNA repeats unique to PSR, as well as those found on the A chromosomes, suggesting that PSR arose through a merger of sequences from both within and outside the N. vitripennis genome. The majority of PSR-specific repeats are interspersed among each other across PSR's long arm, in contrast with the distinct "blocks" observed in other organisms' heterochromatin. Through transcriptional profiling, we identified a subset of repeat-associated, small RNAs expressed by PSR, most of which map to a single PSR-specific repeat. These RNAs are expressed at much higher levels than those arising from A chromosome-linked repeats, suggesting that in addition to its sequence organization, PSR's transcriptional properties differ substantially from the pericentromeric regions of the normal chromosomes.

Asymmetric inheritance of the yeast chaperone Hsp26p and its functional consequences

The yeast Hsp26 protein, a conserved a-crystallin small heatshock chaperone, is assembled in to oligomeric complexes, microscopically visible as distinct cytoplasmic foci. We studied at single cell resolution the dynamics of Hsp26p foci assembly, the mode of their inheritance in to progeny cells and the physiological significance of Hsp26p function. We showed that Hsp26p foci are formed upon cells' entry in to stationary phase, but upon re-entry to proliferation they are asymmetrically retained in the mother cells and are absent from the newborn daughters. Despite the fact that Hsp26p assists re-solubilization of aggregation-prone proteins it does not extend chronological life span nor does it increase the tolerance of either mother or daughters against lethal stresses. Upon sequential HSP26 inductions, newly synthesized Hsp26p is readily incorporated in pre-existing foci, generating larger in size, but similar in appearance foci. At extreme heat-shock conditions, Hsp26p foci break apart into smaller granules dispersed in both mothers and growing buds, while recovery at normal temperature results in Hsp26p foci reassembly. These results suggested that such a complicated mechanism of dynamic Hsp26p assembly and disassembly, as well as asymmetric segregation may contribute to fine tuning regulation of protein aggregates' refolding, cell fitness and survival.

Does Ceramide Form Channels? The Ceramide-Induced Membrane Permeabilization Mechanism

Ceramide is a sphingolipid involved in several cellular processes, including apoptosis. It has been proposed that ceramide forms large and stable channels in the mitochondrial outer membrane that induce cell death through direct release of cytochrome c. However, this mechanism is still debated because the membrane permeabilizing activity of ceramide remains poorly understood. To determine whether the mechanism of ceramide-induced membrane leakage is consistent with the hypothesis of an apoptotic ceramide channel, we have used here assays of calcein release from liposomes. When assaying liposomes containing sphingomyelin and cholesterol, we observed an overall gradual phenomenon of contents release, together with some all-or-none leakage (at low ceramide concentrations or short times). The presence of channels in the bilayer should cause only an all-or-none leakage. When liposomes poor in sphingomyelin/cholesterol or mimicking the lipid composition of the mitochondrial outer membrane were tested, we did not detect any leakage. In consequence, the hypothesis of formation of large ceramide channels in the membrane is not consistent with our results. Instead we propose that the presence of ceramide in one of the membrane monolayers causes a surface area mismatch between both monolayers, which leads to vesicle collapse. The gradual phenomenon of calcein release would be due to a competition between two ceramide effects; namely, lateral segregation that facilitates permeabilization, and at longer times, trans-bilayer flip-flop that opposes asymmetric lateral segregation and causes a mismatch.

Comparison of tumor biology of two distinct cell sub-populations in lung cancer stem cells.

Characterization of the stem-like properties of cancer stem cells (CSCs) remain indirect and qualitative, especially the ability of CSCs to undergo asymmetric cell division for self renewal and differentiation, a unique property of cells of stem origin. It is partly due to the lack of stable cellular models of CSCs. In this study, we developed a new approach for CSC isolation and purification to derive a CSC-enriched cell line (LLC-SE). By conducting five consecutive rounds of single cell cloning using the LLC-SE cell line, we obtained two distinct sub-population of cells within the Lewis lung cancer CSCs that employed largely symmetric division for self-renewal (LLC-SD) or underwent asymmetric division for differentiation (LLC-ASD). LLC-SD and LLC-ASD cell lines could be stably passaged in culture and be distinguished by cell morphology, stem cell marker, spheroid formation and subcutaneous tumor initiation efficiency, as well as orthotopic lung tumor growth, progression and survival. The ability LLC-ASD cells to undergo asymmetric division was visualized and quantified by the asymmetric segregation of labeled BrdU and NUMB to one of the two daughter cells in anaphase cell division. The more stem-like LLC-SD cells exhibited higher capacity for tumorigenesis and progression and shorter survival. As few as 10 LLC-SD could initiate subcutaneous tumor growth when transplanted to the athymic mice. Collectively, these observations suggest that the SD-type of cells appear to be on the top of the hierarchical order of the CSCs. Furthermore, they have lead to generated cellular models of CSC self-renewal for future mechanistic investigations.

Mouse blastomeres acquire ability to divide asymmetrically before compaction.

The mouse preimplantation embryo generates the precursors of trophectoderm (TE) and inner cell mass (ICM) during the 8- to 16-cell stage transition, when the apico-basal polarized blastomeres undergo divisions that give rise to cells with different fate. Asymmetric segregation of polar domain at 8–16 cell division generate two cell types, polar cells which adopt an outer position and develop in TE and apolar cells which are allocated to inner position as the precursors of ICM. It is still not know when the blastomeres of 8-cell stage start to be determined to undergo asymmetric division. Here, we analyze the frequency of symmetric and asymmetric divisions of blastomeres isolated from 8-cell stage embryo before and after compaction. Using p-Ezrin as the polarity marker we found that size of blastomeres in 2/16 pairs cannot be used as a criterion for distinguishing symmetric and asymmetric divisions. Our results showed that at early 8-cell stage, before any visible signs of cortical polarity, a subset of blastomeres had been already predestined to divide asymmetrically. We also showed that almost all of 8-cell stage blastomeres isolated from compacted embryo divide asymmetrically, whereas in intact embryos, the frequency of asymmetric divisions is significantly lower. Therefore we conclude that in intact embryo the frequency of symmetric and asymmetric division is regulated by cell-cell interactions.

Characterization of the martensite phase formed during hydrogen ion irradiation in austenitic stainless steel

Microstructural changes in austenitic stainless steel caused by hydrogen ion irradiation were investigated using transmission electron microscopy (TEM). It has been confirmed that the irradiation induced the formation of martensite along the grain boundary; the martensite phase exhibited a crystal orientation relationship with the adjacent austenite phase. The results of this study also indicate that the concentration of Cr in the martensite phase is lower compared to that in the austenite matrix. The TEM results showed the development of asymmetric radiation-induced segregation (RIS) near the grain boundary, which leads to local changes in the chemical composition such as reduction of Cr near the grain boundary. The asymmetric RIS serves as a prerequisite for the formation of the martensite under hydrogen irradiation.

Antibody Uptake Assay in the Embryonic Zebrafish Forebrain to Study Notch Signaling Dynamics in Neural Progenitor Cells In Vivo.

Stem cells can generate cell fate heterogeneity through asymmetric cell division (ACD). ACD derives from the asymmetric segregation of fate-determining molecules and/or organelles in the dividing cell. Radial glia in the embryonic zebrafish forebrain are an excellent model for studying the molecular mechanisms regulating ACD of stem cells in vertebrates, especially for live imaging concerning in vivo molecular and cellular dynamics. Due to the current difficulty in expressing fluorescent reporter-tagged proteins at physiological levels in zebrafish for live imaging, we have developed an antibody uptake assay to label proteins in live embryonic zebrafish forebrain with high specificity. DeltaD is a transmembrane ligand in Notch signaling pathway in the context of ACD of radial glia in zebrafish. By using this assay, we have successfully observed the in vivo dynamics of DeltaD for studying ACD of radial glia in the embryonic zebrafish forebrain.

The Mitochondrial Metabolic Checkpoint and Reversing Stem Cell Aging

Cell cycle checkpoints are surveillance mechanisms in eukaryotic cells that monitor the condition of the cell, repair cellular damages, and allow the cell to progress through the various phases of the cell cycle when conditions become favorable. Recent advances in hematopoietic stem cell (HSC) biology highlight a mitochondrial metabolic checkpoint that is essential for HSCs to return to the quiescent state. As quiescent HSCs enter the cell cycle, mitochondrial biogenesis is induced, which is associated with increased mitochondrial protein folding stress and mitochondrial oxidative stress. Mitochondrial unfolded protein response and mitochondrial oxidative stress response are activated to alleviate stresses and allow HSCs to exit the cell cycle and return to quiescence. Other mitochondrial maintenance mechanisms include mitophagy and asymmetric segregation of aged mitochondria. Because loss of HSC quiescence results in the depletion of the HSC pool and compromised tissue regeneration, deciphering the molecular mechanisms that regulate the mitochondrial metabolic checkpoint in HSCs will increase our understanding of hematopoiesis and how it becomes dysregulated under pathological conditions and during aging. More broadly, this knowledge is instrumental for understanding the maintenance of cells that convert between quiescence and proliferation to support their physiological functions. DisclosuresNo relevant conflicts of interest to declare.