Inheritance Of Cell Fate Determinants Research Articles

Temporally distinct roles of Aurora A in polarization of the C. elegans zygote.

During asymmetric cell division, cell polarity is coordinated with the cell cycle to allow proper inheritance of cell fate determinants and the generation of cellular diversity. In the Caenorhabditis elegans zygote, polarity is governed by evolutionarily conserved Partitioning-defective (PAR) proteins that segregate to opposing cortical domains to specify asymmetric cell fates. Timely establishment of PAR domains requires a cell cycle kinase, Aurora A (AIR-1 in C. elegans). Aurora A depletion by RNAi causes a spectrum of phenotypes including reversed polarity, excess posterior domains and no posterior domain. How depletion of a single kinase can cause seemingly opposite phenotypes remains obscure. Using an auxin-inducible degradation system and drug treatments, we found that AIR-1 regulates polarity differently at different times of the cell cycle. During meiosis I, AIR-1 acts to prevent later formation of bipolar domains, whereas in meiosis II, AIR-1 is necessary to recruit PAR-2 onto the membrane. Together, these data clarify the origin of multiple polarization phenotypes in RNAi experiments and reveal multiple roles of AIR-1 in coordinating PAR protein localization with cell cycle progression.

2019 – ASYMMETRIC CELL DIVISION MODULATES HUMAN HEMATOPOIETIC STEM CELL FATES

Effective manipulation of clinically relevant umbilical cord blood-derived human hematopoietic stem cell (hHSC) fate decisions remains an important goal for improved therapies. Based on observations in other model organisms, it was postulated that asymmetric cell division (ACD) regulates hHSC fate decisions of self-renewal and differentiation. In ACD, daughter cell fates are determined during cell division, e.g., by the asymmetric inheritance of cell fate determinants. Previous studies suggested the asymmetric inheritance of factors during progenitor divisions. However, due to the lack of tools for sufficient prospective hHSC enrichment, quantification of asymmetric inheritance and future daughter cell behaviors in the same experiment, hHSC ACD remained hypothetical. Here, we use continuous quantitative long-term single-cell imaging of highly purified hHSCs to identify factors and organelles that are asymmetrically inherited during hHSC divisions. Importantly, this asymmetric inheritance correlates with future asymmetric adhesion, motility, cell cycle progression, marker production, and metabolic activity in hHSC daughters. We further show, using novel real-time computational differentiation landscapes and in-silico hHSC purification, that this asymmetric inheritance also correlates with future expression of stem cell markers, and that culture conditions known to expand hHSCs increase symmetric cell divisions. We thus provide the first direct quantitative evidence that ACD exists in human hematopoietic stem and progenitor cells and modulates future daughter cells fates by the inheritance of factors during division. We provide insights into the underlying molecular mechanisms, and the exciting possibility to manipulate ACD and hHSC fate decision for improved therapy.

ASYMMETRIC CELL DIVISION CONTROLS HEMATOPOIETIC STEM CELL METABOLIC ACTIVATION AND DIFFERENTIATION

Hematopoietic stem cells (HSCs) can self-renew while differentiating to replenish the blood system lifelong. Asymmetric cell division (ACD) has been hypothesized as the mechanism balancing HSC self-renewal and differentiation. In this model, asymmetric daughter cell fates are determined by a mechanism linked to mitosis, e.g. the unequal inheritance of cell fate determinants into daughters. However, due to lacking technology, this mechanism could never been shown directly. Thus, it is also possible that HSCs do not divide asymmetrically, and differentiation is determined by post-mitotic mechanisms. Asymmetric inheritance of cell fate determinants has been reported in progenitor divisions of other tissues. However, low frequencies of HSCs and the technical challenges of continuous quantitative analysis of single suspension cells have prevented the study of HSC mitosis. Few studies therefore provide evidence for asymmetric inheritance of proteins during HSC division, and none was able to demonstrate that future daughter cell fates correlate with this asymmetric inheritance. Here, using continuous quantitative single cell imaging with clustering dimensionality reduction approaches of dynamic single cell behavior, we identify molecules and organelles that are asymmetrically inherited with the lysosomal machinery during HSC divisions. Importantly, we demonstrate that this asymmetric inheritance also predicts future metabolic activation, translation and the overall heterogeneity during differentiation of the HSC daughters. We further show that this mechanism is conserved from mouse to man. We provide the first direct quantitative evidence that HSCs utilize ACD, that future daughter cells fates are prospectively determined by asymmetric mitotic molecule inheritance, and provide insights into the underlying molecular mechanisms for improved therapeutic HSC manipulation.

THE ROLE OF THE POLARITY PROTEIN PAR3 IN HAEMATOPOIESIS AND LEUKAEMIA

Cell polarity is a critical feature of multiple aspects of cell biology e.g. cell division and differentiation. Disrupted polarity network is well established in epithelial cancers where the polarity regulators act as tumour-suppressors or oncogenes. In the haematopoietic system, polarity proteins regulate asymmetric cell division (ACD) of haematopoietic stem cells (HSCs). ACD is central to the maintenance of normal haematopoiesis, ensuring the retention of self-renewal and differentiation potentials of HSCs. Disrupted ACD is associated with leukaemia transformation as leukemic cells lose the ability to differentiate and instead gain the ability to self-renew. Considering this and the role of polarity proteins in ACD, understanding how they regulate HSCs ACD or contribute to leukaemia is of major importance. We examined the role of polarity protein PAR3 in acute myeloid leukaemia (AML). To determine the role of PAR3 loss in haematopoiesis, we utilised a Par3 conditional knockout mouse model. For the human model, Par3 was knocked out in AML cell lines using CRISPR/Cas9. We found that PAR3 is differentially expressed in haematopoietic progenitors and their more differentiated progeny. PAR3 loss in a pre-clinical model of murine AML led to a significant increase in the proportion and number of granulocyte/macrophage progenitors, which are the hyperproliferative cancer stem cells. This was associated with deregulation of MAPK and HIPPO signalling pathways in PAR3 KO animals. To study the cellular and molecular mechanisms of PAR3 in human AML, we have successfully knocked out the Par3 in several human AML cell lines. The role of PAR3 in ACD is being studied by examining the cell cycle status using live cell imaging, cell differentiation, polarised inheritance of cell fate determinants, and mitotic spindle positioning. In conclusion, PAR3 acts as a tumour suppressor in AML.

Asymmetric Inheritance of Cell Fate Determinants: Focus on RNA.

During the last decade, and mainly primed by major developments in high-throughput sequencing technologies, the catalogue of RNA molecules harbouring regulatory functions has increased at a steady pace. Current evidence indicates that hundreds of mammalian RNAs have regulatory roles at several levels, including transcription, translation/post-translation, chromatin structure, and nuclear architecture, thus suggesting that RNA molecules are indeed mighty controllers in the flow of biological information. Therefore, it is logical to suggest that there must exist a series of molecular systems that safeguard the faithful inheritance of RNA content throughout cell division and that those mechanisms must be tightly controlled to ensure the successful segregation of key molecules to the progeny. Interestingly, whilst a handful of integral components of mammalian cells seem to follow a general pattern of asymmetric inheritance throughout division, the fate of RNA molecules largely remains a mystery. Herein, we will discuss current concepts of asymmetric inheritance in a wide range of systems, including prions, proteins, and finally RNA molecules, to assess overall the biological impact of RNA inheritance in cellular plasticity and evolutionary fitness.

2021 - Asymmetric Cell Division Controls Hematopoietic Stem Cell Metabolic Activation and Differentiation

Hematopoietic stem cells (HSCs) maintain their numbers while also differentiating to replenish the entire blood system lifelong. Asymmetric cell division has been hypothesized as the underlying mechanism balancing HSC self-renewal and differentiation. In this model, future asymmetric daughter cell fates are prospectively determined by a mechanism linked to mitosis, e.g. the unequal inheritance of cell fate determinants to the two daughters. However, due to lacking technology, this mechanism could never been shown directly. It is therefore also possible that HSCs do not divide asymmetrically, and commitment to differentiation is determined by post-mitotic mechanisms. Asymmetric inheritance of cell fate determinants has been reported in in- and vertebrate progenitor divisions of other tissues. However, the low frequency of HSCs and the technical challenges associated with reliable continuous quantitative analysis of single suspension cells have prevented the study of events during HSC mitosis. Few studies therefore provide evidence for asymmetric inheritance of proteins during hematopoietic stem and progenitor cell (HSPC) division, and none was able to demonstrate that future daughter cell fates correlate with this asymmetric inheritance. Here, using novel continuous quantitative live single cell imaging, we identify several molecules which are asymmetrically inherited along with the lysosomal machinery during HSC divisions. Importantly, we demonstrate that this asymmetric inheritance also predicts future metabolic activation and differentiation of the HSC daughters. We provide the first direct quantitative evidence that HSCs utilize asymmetric cell division, that future daughter cells fates are prospectively determined by asymmetric mitotic molecule inheritance, and provides insights into the underlying molecular mechanisms for improved therapeutic HSC manipulation.

Asymmetric cell division of hematopoietic stem cells: asymmetric inheritance of cell fate determinants and its role in cell fate decisions

Asymmetric cell division of hematopoietic stem cells: asymmetric inheritance of cell fate determinants and its role in cell fate decisions

Lis1 regulates asymmetric division in hematopoietic stem cells and in leukemia.

Cell fate can be controlled through asymmetric division and segregation of protein determinants. But the regulation of this process in the hematopoietic system is poorly understood. Here we show that the dynein binding protein Lis1 (Pafah1b1) is critically required for blood formation and hematopoietic stem cell function. Conditional deletion of Lis1 in the hematopoietic system led to a severe bloodless phenotype, depletion of the stem cell pool and embryonic lethality. Further, the loss of Lis1 accelerated cell differentiation, in part through defects in spindle positioning and inheritance of cell fate determinants. Finally, deletion of Lis1 blocked propagation of myeloid leukemia and led to a marked improvement in animal survival, suggesting that Lis1 is also required for oncogenic growth. These data identify a key role for Lis1 in hematopoietic stem cells, and mark the directed control of asymmetric division as a critical regulator of normal and malignant hematopoietic development.

Rho GTPase Activating Protein p190-B Regulates Hematopoietic Stem Cell Fate Decision

AbstractAbstract 575Hematopoietic stem cells (HSC) are undifferentiated cells that are capable of self-renewal and production of all mature blood and immune cells. The molecular mechanisms that determine HSC self renewal and cell fate decision still remain poorly understood. We have previously shown that p190-B GTPase Activating Protein (GAP), a negative regulator of Rho activity, is a critical regulator of HSC self renewal. In these studies, while p190-B-deficiency did not alter HSC cell cycle kinetics or survival either in vivo and in vitro, the loss of p190-B conferred enhanced serial transplantation capacity and also preserved the repopulating capacity of HSC/Ps during ex vivo cytokine-induced culture compared to WT HSC/Ps. Together, these data suggest that p190-B modulates HSC self renewal decisions during cell division (Xu et al, Blood 2009).In the current study, we tested the hypothesis that p190-B modulates the balance of asymmetric/symmetric self renewal divisions. To do so, single LSK cells isolated from fetal livers were cultured in the presence of cytokines known to promote self renewal. After 1–2 divisions, the media was replaced with cytokines that promote both proliferation and differentiation. The kinetics of the first 1–2 divisions of WT and p190-B–/– LSK was similar (n=450). However, after 10 days, the size of clones derived from p190-B−/− LSK cells were larger. Importantly, the frequency of clones exhibiting multipotent lineage differentiation was also higher from p190-B−/− LSK compared with WT (18 multipotent clones out of 43 (42%) vs 10 out of 39 (26%)). Therefore, p190-B may modulate the balance of asymmetric/symmetric HSC self renewal divisions. One fundamental mechanism of asymmetric cell division is the asymmetric inheritance of cell fate determinants, which is controlled by the asymmetric distribution of the cytoskeleton and polarity cues. Because Rho GTPases are key regulators of cytoskeleton polarity, we next examined cell shape and polarity (of WT and p190-B–/– LSK cells). WT cells exhibited an asymmetric shape with two distinct cell poles, including one pole enriched with microtubules. In contrast, p190-B–/– LSK appeared round with a homogenous distribution of microtubules. Using image flow spectral analysis (Imagestream), we analyzed the distribution of numb, a conserved cell fate determinant, which is unequally distributed during stem cell division in order to generate distinct daughter cells. Significantly more p190-B–/– LSK showed a symmetric numb distribution than WT LSK (41±5.8% vs 29±4.6%, n>1000 cells from 3 independent experiments, p<0.02). To determine the importance of this cell shape change, we examined the p38MAPK signaling pathway, since p190-B is known to regulate p38MAPK activity (Sordella et al, Dev Cell 2002) and p38MAPK has been implicated HSC self renewal decision (Ito et al, Nat Med 2006). Flow cytometry analysis revealed that p38MAPK activity increased in WT LSK-CD150+ cells during serial transplantation. p190-B-deficiency prevents the elevation of p38MAPK activity in HSC in secondary transplant recipients (mean fluorescence intensity in HSC isolated from non transplanted animals: 1182±439; from secondary recipients of WT HSC: 2218±680=; from secondary recipients of p190-B−/− HSC: 1540±711**, n=7, *p<0.01 compared to non transplanted mice; ** p<0.01 compared to WT and ns compared to non transplanted mice). Remarkably, inhibition of p38MAPK activity in WT LSK led to alterations in shape and polarity that was similar to p190-B–/– LSK. Furthermore, p38MAPK inhibition significantly increased LSK competitive repopulation activity after 7 days in culture, compared to vehicle treated LSK cells (chimera 53±1.7 vs 23±18%, n=4, p<0.02).Together, these data suggest that p190-B regulates HSC/P self renewal by modulating cell fate decision as the cell divide and that HSC/P shape integrity is critical for this process. This study implies that modulating cell polarity via Rho GTPase may provide rational to devise new therapeutic approaches to clinical HSC transplantation protocols. Disclosures:Filippi:American Heart Association: Research Funding.

Instructive role of aPKCζ subcellular localization in the assembly of adherens junctions in neural progenitors

In the neurogenic phase of CNS development, the proliferating progenitors are found medially within the neuroepithelium. The adherens junctions on the apical membrane of proliferating neural progenitors allow for cell-cell adhesion and medial stratification. In contrast, differentiating neuronal precursors delaminate and migrate laterally, establishing the laminar layers. Apical adherens junctions also establish the apical-basal polarity in neural progenitors, which in turn is postulated to lead to asymmetric inheritance of cell fate determinants during neurogenic divisions. The signaling pathways and cellular mechanisms that regulate the assembly and asymmetric localization of adherens junctions in neural progenitors remain elusive. Here we show that atypical PKCzeta/lambda (aPKCzeta/lambda) localizes at the apical membrane of proliferating neural stem cells, but not postmitotic neuronal precursors, in the developing chicken neural tube. This precise subcellular compartmentalization of the kinase activity provides an instructive signal for apical assembly of adherens junctions in a PI3K, Rac/Cdc42 signaling-dependent pathway. Apical aPKCzeta coordinates neural stem cell proliferation and the overall stratification of cell types within the neural tube.

Dare to be different: asymmetric cell division in Drosophila, C. elegans and vertebrates.

One widespread mechanism for the generation of diverse cell types is the unequal inheritance of cell fate determinants. Several such determinants have been identified in the fruitfly Drosophila melanogaster and the worm Caenorhabditis elegans and the molecular machinery responsible for their asymmetric segregation is beginning to be unraveled. To divide asymmetrically, cells establish an axis of polarity, orient the mitotic spindle along this axis and localize cell fate determinants to one side of the cell. During cytokinesis, determinants are then segregated into one of the two daughter cells where they direct cell fate. Here, we outline the steps and factors that are involved in this process in Drosophila and C. elegans and discuss their potential conservation in vertebrates.

Novel patterns of ectopic cell plate growth and lipid body distribution in the Arabidopsis gemini pollen1 mutant.

The nature of aberrant gametophytic cell divisions and altered pollen cell fate in the gemini pollen1 (gem1) mutant was investigated through ultrastructural analysis. The earliest noticeable defect in gem1 was the appearance of extended membrane profiles at the early bicellular stage. These were replaced by ectopic internal walls, which divided the cytoplasm into twin or multiple cell compartments. Complete or partial internal walls were callosic with highly complex profiles, indicating failed guidance or deregulated cell plate growth. Extended membrane profiles and delayed callose synthesis at division sites further suggested a novel pattern of cell plate assembly in gem1. Multiple cell compartments in gem1 adopted vegetative cell fate with regard to lipid body distribution. In the wild type, lipid bodies appear specifically in the vegetative cell, whereas in gem1, lipid bodies accumulated in all cytoplasmic compartments. Our results support the hypothesis that altered pollen cell fate in gem1 results from abnormal inheritance of cell fate determinants as a result of disturbed cytokinesis.