A 3D micro-printed single cell micro-niche with asymmetric niche signals - An in vitro model for asymmetric cell division study.

Intricate microenvironment signals orchestrate to affect cell behavior and fate during tissue morphogenesis. However, the underlying mechanisms on how specific local niche signals influence cell behavior and fate are not fully understood, owing to the lack of in vitro platform able to precisely, quantitatively, spatially, and independently manipulate individual niche signals. Here, microarrays of protein-based 3D single cell micro-niche (3D-SCµN), with precisely engineered biophysical and biochemical niche signals, are micro-printed by a multiphoton microfabrication and micropatterning technology. Mouse embryonic stem cell (mESC) is used as the model cell to study how local niche signals affect stem cell behavior and fate. By precisely engineering the internal microstructures of the 3D SCµNs, we demonstrate that the cell division direction can be controlled by the biophysical niche signals, in a cell shape-independent manner. After confining the cell division direction to a dominating axis, single mESCs are exposed to asymmetric biochemical niche signals, specifically, cell-cell adhesion molecule on one side and extracellular matrix on the other side. We demonstrate that, symmetry-breaking (asymmetric) niche signals successfully trigger cell polarity formation and bias the orientation of asymmetric cell division, the mitosis process resulting in two daughter cells with differential fates, in mESCs.

BIG coordinates auxin and SHORT ROOT to promote asymmetric stem cell divisions in Arabidopsis roots.

KEY MESSAGE: BIG regulates ground tissue formative divisions by bridging the auxin gradient with SHR abundance in Arabidopsis roots. The formative divisions of cortex/endodermis initials (CEIs) and CEI daughter cells (CEIDs) in Arabidopsis roots are coordinately controlled by the longitudinal auxin gradient and the radial SHORT ROOT (SHR) abundance. However, the mechanism underlying this coordination remains poorly understood. In this study, we demonstrate that BIG regulates ground tissue formative divisions by bridging the auxin gradient with SHR abundance. Mutations in BIG gene repressed cell cycle progression, delaying the formative divisions within the ground tissues and impairing the establishment of endodermal and cortical identities. In addition, we uncovered auxin's suppressive effect on BIG expression, triggering CYCLIND6;1 (CYCD6;1) activation in an SHR-dependent fashion. Moreover, the degradation of RETINOBLASTOMA-RELATED (RBR) is jointly regulated by BIG and CYCD6;1. The loss of BIG function led to RBR protein accumulation, detrimentally impacting the SHR/SCARECROW (SCR) protein complex and the CEI/CEID formative divisions. Collectively, these findings shed light on a fundamental mechanism wherein BIG intricately coordinates the interplay between SHR/SCR and auxin, steering ground tissue patterning within Arabidopsis root tissue.

Stochastic journeys of cell progenies through compartments and the role of self-renewal, symmetric and asymmetric division.

Division and differentiation events by which cell populations with specific functions are generated often take place as part of a developmental programme, which can be represented by a sequence of compartments. A compartment is the set of cells with common characteristics; sharing, for instance, a spatial location or a phenotype. Differentiation events are transitions from one compartment to the next. Cells may also die or divide. We consider three different types of division events: (i) where both daughter cells inherit the mother's phenotype (self-renewal), (ii) where only one of the daughters changes phenotype (asymmetric division), and (iii) where both daughters change phenotype (symmetric division). The self-renewal probability in each compartment determines whether the progeny of a single cell, moving through the sequence of compartments, is finite or grows without bound. We analyse the progeny stochastic dynamics with probability generating functions. In the case of self-renewal, by following one of the daughters after any division event, we may construct lifelines containing only one cell at any time. We analyse the number of divisions along such lines, and the compartment where lines terminate with a death event. Analysis and numerical simulations are applied to a five-compartment model of the gradual differentiation of hematopoietic stem cells and to a model of thymocyte development: from pre-double positive to single positive (SP) cells with a bifurcation to either SP4 or SP8 in the last compartment of the sequence.

Vacuolar H+-ATPase determines daughter cell fates through asymmetric segregation of the nucleosome remodeling and deacetylase complex.

Asymmetric cell divisions (ACDs) generate two daughter cells with identical genetic information but distinct cell fates through epigenetic mechanisms. However, the process of partitioning different epigenetic information into daughter cells remains unclear. Here, we demonstrate that the nucleosome remodeling and deacetylase (NuRD) complex is asymmetrically segregated into the surviving daughter cell rather than the apoptotic one during ACDs in Caenorhabditis elegans. The absence of NuRD triggers apoptosis via the EGL-1-CED-9-CED-4-CED-3 pathway, while an ectopic gain of NuRD enables apoptotic daughter cells to survive. We identify the vacuolar H+-adenosine triphosphatase (V-ATPase) complex as a crucial regulator of NuRD's asymmetric segregation. V-ATPase interacts with NuRD and is asymmetrically segregated into the surviving daughter cell. Inhibition of V-ATPase disrupts cytosolic pH asymmetry and NuRD asymmetry. We suggest that asymmetric segregation of V-ATPase may cause distinct acidification levels in the two daughter cells, enabling asymmetric epigenetic inheritance that specifies their respective life-versus-death fates.

Signaling proteins in HSC fate determination are unequally segregated during asymmetric cell division.

Hematopoietic stem cells (HSCs) continuously replenish mature blood cells with limited lifespans. To maintain the HSC compartment while ensuring output of differentiated cells, HSCs undergo asymmetric cell division (ACD), generating two daughter cells with different fates: one will proliferate and give rise to the differentiated cells' progeny, and one will return to quiescence to maintain the HSC compartment. A balance between MEK/ERK and mTORC1 pathways is needed to ensure HSC homeostasis. Here, we show that activation of these pathways is spatially segregated in premitotic HSCs and unequally inherited during ACD. A combination of genetic and chemical perturbations shows that an ERK-dependent mechanism determines the balance between pathways affecting polarity, proliferation, and metabolism, and thus determines the frequency of asymmetrically dividing HSCs. Our data identify druggable targets that modulate HSC fate determination at the level of asymmetric division.

Differential growth regulates asymmetric size partitioning in Caulobacter crescentus.

Cell size regulation has been extensively studied in symmetrically dividing cells, but the mechanisms underlying the control of size asymmetry in asymmetrically dividing bacteria remain elusive. Here, we examine the control of asymmetric division in Caulobacter crescentus, a bacterium that produces daughter cells with distinct fates and morphologies upon division. Through comprehensive analysis of multi-generational growth and shape data, we uncover a tightly regulated cell size partitioning mechanism. We find that errors in division site positioning are promptly corrected early in the division cycle through differential growth. Our analysis reveals a negative feedback between the size of daughter cell compartments and their growth rates, wherein the larger compartment grows slower to achieve a homeostatic size partitioning ratio at division. To explain these observations, we propose a mechanistic model of differential growth, in which equal amounts of growth regulators are partitioned into daughter cell compartments of unequal sizes and maintained over time via size-independent synthesis.

NMY-2, TOE-2 and PIG-1 regulate Caenorhabditis elegans asymmetric cell divisions.

Asymmetric cell division is an important mechanism that generates cellular diversity during development. Not only do asymmetric cell divisions produce daughter cells of different fates, but many can also produce daughters of different sizes, which we refer to as Daughter Cell Size Asymmetry (DCSA). In Caenorhabditis elegans, apoptotic cells are frequently produced by asymmetric divisions that exhibit DCSA, where the smaller daughter dies. We focus here on the divisions of the Q.a and Q.p neuroblasts, which produce larger surviving cells and smaller apoptotic cells and divide with opposite polarity using both distinct and overlapping mechanisms. Several proteins regulate DCSA in these divisions. Previous studies showed that the PIG-1/MELK and TOE-2 proteins regulate DCSA in both the Q.a and Q.p divisions, and the non-muscle myosin NMY-2 regulates DCSA in the Q.a division but not the Q.p division. In this study, we examined endogenously tagged NMY-2, TOE-2, and PIG-1 reporters and characterized their distribution at the cortex during the Q.a and Q.p divisions. In both divisions, TOE-2 localized toward the side of the dividing cell that produced the smaller daughter, whereas PIG-1 localized toward the side that produced the larger daughter. As previously reported, NMY-2 localized to the side of Q.a that produced the smaller daughter and did not localize asymmetrically in Q.p. We used temperature-sensitive nmy-2 mutants to determine the role of nmy-2 in these divisions and were surprised to find that these mutants only displayed DCSA defects in the Q.p division. We generated double mutant combinations between the nmy-2 mutations and mutations in toe-2 and pig-1. Because previous studies indicate that DCSA defects result in the transformation of cells fated to die into their sister cells, the finding that the nmy-2 mutations did not significantly alter the Q.a and Q.p DCSA defects of toe-2 and pig-1 mutants but did alter the number of daughter cells produced by Q.a and Q.p suggests that nmy-2 plays a role in specifying the fates of the Q.a and Q.p that is independent of its role in DCSA.

Islr regulates satellite cells asymmetric division through the SPARC/p-ERK1/2 signaling pathway.

Satellite cells (SCs) are adult muscle stem cells responsible for muscle regeneration after acute and chronic muscle injuries. The balance between stem cell self-renewal and differentiation determines the kinetics and efficiency of skeletal muscle regeneration. This study assessed the function of Islr in SC asymmetric division. The deletion of Islr reduced muscle regeneration in adult mice by decreasing the SC pool. Islr is pivotal for SC proliferation, and its deletion promoted the asymmetric division of SCs. A mechanistic search revealed that Islr bound to and degraded secreted protein acidic and rich in cysteine (SPARC), which activated p-ERK1/2 signaling required for asymmetric division. These findings demonstrate that Islr is a key regulator of SC division through the SPARC/p-ERK1/2 signaling pathway. These data provide a basis for treating myopathy.

Asymmetric T-cell division: insights from cutting-edge experimental techniques and implications for immunotherapy.

Asymmetric cell division is a fundamental process conserved throughout evolution, employed by both prokaryotic and eukaryotic organisms. Its significance lies in its ability to govern cell fate and facilitate the generation of diverse cell types. Therefore, attaining a detailed mechanistic understanding of asymmetric cell division becomes essential for unraveling the complexities of cell fate determination in both healthy and pathological conditions. However, the role of asymmetric division in T-cell biology has only recently been unveiled. Here, we provide an overview of the T-cell asymmetric division field with the particular emphasis on experimental methods and models with the aim to guide the researchers in the selection of appropriate in vitro/in vivo models to study asymmetric division in T cells. We present a comprehensive investigation into the mechanisms governing the asymmetric division in various T-cell subsets underscoring the importance of the asymmetry in fate-determining factor segregation and transcriptional and epigenetic regulation. Furthermore, the intricate interplay of T-cell receptor signaling and the asymmetric division geometry are explored, shedding light on the spatial organization and the impact on cellular fate.

Kin17 regulates proper cortical localization of Miranda in Drosophila neuroblasts by regulating Flfl expression.

During asymmetric division of Drosophila larval neuroblasts, the fate determinant Prospero (Pros) and its adaptor Miranda (Mira) are segregated to the basal cortex through atypical protein kinase C (aPKC) phosphorylation of Mira and displacement from the apical cortex, but Mira localization after aPKC phosphorylation is not well understood. We identify Kin17, a DNA replication and repair protein, as a regulator of Mira localization during asymmetric cell division. Loss of Kin17 leads to aberrant localization of Mira and Pros to the centrosome, cytoplasm, and nucleus. We provide evidence to show that the mislocalization of Mira and Pros is likely due to reduced expression of Falafel (Flfl), a component of protein phosphatase 4 (PP4), and defects in dephosphorylation of serine-96 of Mira. Our work reveals that Mira is likely dephosphorylated by PP4 at the centrosome to ensure proper basal localization of Mira after aPKC phosphorylation and that Kin17 regulates PP4 activity by regulating Flfl expression.

The contribution of asymmetric cell division to phenotypic heterogeneity in cancer.

Cells have evolved intricate mechanisms for dividing their contents in the most symmetric way during mitosis. However, a small proportion of cell divisions results in asymmetric segregation of cellular components, which leads to differences in the characteristics of daughter cells. Although the classical function of asymmetric cell division (ACD) in the regulation of pluripotency is the generation of one differentiated daughter cell and one self-renewing stem cell, recent evidence suggests that ACD plays a role in other physiological processes. In cancer, tumor heterogeneity can result from the asymmetric segregation of genetic material and other cellular components, resulting in cell-to-cell differences in fitness and response to therapy. Defining the contribution of ACD in generating differences in key features relevant to cancer biology is crucial to advancing our understanding of the causes of tumor heterogeneity and developing strategies to mitigate or counteract it. In this Review, we delve into the occurrence of asymmetric mitosis in cancer cells and consider how ACD contributes to the variability of several phenotypes. By synthesizing the current literature, we explore the molecular mechanisms underlying ACD, the implications of phenotypic heterogeneity in cancer, and the complex interplay between these two phenomena.

Imaging and Analysis of Drosophila Neural Stem Cell Asymmetric Division.

Cell division is a conserved process among eukaryotes. It is designed to segregate chromosomes into future daughter cells and involves a complex rearrangement of the cytoskeleton, including microtubules and actin filaments. An additional level of complexity is present in asymmetric dividing stem cells because cytoskeleton elements are also regulated by polarity cues. The neural stem cell system of the fruit fly represents a simple model to dissect the mechanisms that control cytoskeleton reorganization during asymmetric division. In this chapter, we propose to describe protocols that allow accurate analysis of microtubule reorganization during cell division in this model.

The people behind the papers - Thomas Mullan and Richard Poole.

Asymmetric cell divisions can produce daughter cells of different sizes, but it is unclear whether unequal cell cleavage is important for cell fate decisions. A new paper in Development explores the role of unequal cleavages in Caenorhabditis elegans embryos. To learn more about the story behind the paper, we caught up with first author Thomas Mullan and corresponding author Richard Poole, Associate Professor of Developmental Biology at University College London, UK.

A model about regulation on three division modes of stem cell.

We construct a multi-stage cell lineage model for cell division, apoptosis and movement. Cells are assumed to secrete and respond to negative feedback molecules which act as a control on the stem cell divisions (including self-renewal, asymmetrical cell division (ACD) and differentiation). The densities of cells and molecules are described by coupled reaction-diffusion partial differential equations, and the plane wavefront propagation speeds can be obtained analytically and verified numerically. It is found that with ACD the population and propagation of stem cells can be promoted but the negative regulation on self-renewal and differentiation will work slowly. Regulatory inhibition on differentiation will inversely increase stem cells but not affect the population and wave propagation of the cell lineage. While negative regulation on self-renewal and ACD will decrease the population of stem cells and slow down the propagation, and even drive stem cells to extinction. Moreover we find that inhibition on self-renewal has a strength advantage while inhibition on ACD has a range advantage to kill stem cells. Possible relations to model cancer development and therapy are also discussed.

Epigenetic regulation of asymmetric cell division by the LIBR-BRD4 axis.

Asymmetric cell division (ACD) is a mechanism used by stem cells to maintain the number of progeny. However, the epigenetic mechanisms regulating ACD remain elusive. Here we show that BRD4, a BET domain protein that binds to acetylated histone, is segregated in daughter cells together with H3K56Ac and regulates ACD. ITGB1 is regulated by BRD4 to regulate ACD. A long noncoding RNA (lncRNA), LIBR (LncRNA Inhibiting BRD4), decreases the percentage of stem cells going through ACD through interacting with the BRD4 mRNAs. LIBR inhibits the translation of BRD4 through recruiting a translation repressor, RCK, and inhibiting the binding of BRD4 mRNAs to polysomes. These results identify the epigenetic regulatory modules (BRD4, lncRNA LIBR) that regulate ACD. The regulation of ACD by BRD4 suggests the therapeutic limitation of using BRD4 inhibitors to treat cancer due to the ability of these inhibitors to promote symmetric cell division that may lead to tumor progression and treatment resistance.

Eclosion muscles secrete ecdysteroids to initiate asymmetric intestinal stem cell division in Drosophila.

During organ development, tissue stem cells first expand via symmetric divisions and then switch to asymmetric divisions to minimize the time to obtain a mature tissue. In the Drosophila midgut, intestinal stem cells switch their divisions from symmetric to asymmetric at midpupal development to produce enteroendocrine cells. However, the signals that initiate this switch are unknown. Here, we identify the signal as ecdysteroids. In the presence of ecdysone, EcR and Usp promote the expression of E93 to suppress Br expression, resulting in asymmetric divisions. Surprisingly, the primary source of pupal ecdysone is not from the prothoracic gland but from dorsal internal oblique muscles (DIOMs), a group of transient skeletal muscles that are required for eclosion. Genetic analysis shows that DIOMs secrete ecdysteroids during mTOR-mediated muscle remodeling. Our findings identify sequential endocrine and mechanical roles for skeletal muscle, which ensure the timely asymmetric divisions of intestinal stem cells.

Comprehensive and quantitative analysis of intracellular structure polarization at the apical-basal axis in elongating Arabidopsis zygotes.

A comprehensive and quantitative evaluation of multiple intracellular structures or proteins is a promising approach to provide a deeper understanding of and new insights into cellular polarity. In this study, we developed an image analysis pipeline to obtain intensity profiles of fluorescent probes along the apical-basal axis in elongating Arabidopsis thaliana zygotes based on two-photon live-cell imaging data. This technique showed the intracellular distribution of actin filaments, mitochondria, microtubules, and vacuolar membranes along the apical-basal axis in elongating zygotes from the onset of cell elongation to just before asymmetric cell division. Hierarchical cluster analysis of the quantitative data on intracellular distribution revealed that the zygote may be compartmentalized into two parts, with a boundary located 43.6% from the cell tip, immediately after fertilization. To explore the biological significance of this compartmentalization, we examined the positions of the asymmetric cell divisions from the dataset used in this distribution analysis. We found that the cell division plane was reproducibly inserted 20.5% from the cell tip. This position corresponded well with the midpoint of the compartmentalized apical region, suggesting a potential relationship between the zygote compartmentalization, which begins with cell elongation, and the position of the asymmetric cell division.

Morphogenesis in Trypanosoma cruzi epimastigotes proceeds via a highly asymmetric cell division.

Trypanosoma cruzi is a protist parasite that is the causative agent of Chagas disease, a neglected tropical disease endemic to the Americas. T. cruzi cells are highly polarized and undergo morphological changes as they cycle within their insect and mammalian hosts. Work on related trypanosomatids has described cell division mechanisms in several life-cycle stages and identified a set of essential morphogenic proteins that serve as markers for key events during trypanosomatid division. Here, we use Cas9-based tagging of morphogenic genes, live-cell imaging, and expansion microscopy to study the cell division mechanism of the insect-resident epimastigote form of T. cruzi, which represents an understudied trypanosomatid morphotype. We find that T. cruzi epimastigote cell division is highly asymmetric, producing one daughter cell that is significantly smaller than the other. Daughter cell division rates differ by 4.9 h, which may be a consequence of this size disparity. Many of the morphogenic proteins identified in T. brucei have altered localization patterns in T. cruzi epimastigotes, which may reflect fundamental differences in the cell division mechanism of this life cycle stage, which widens and shortens the cell body to accommodate the duplicated organelles and cleavage furrow rather than elongating the cell body along the long axis of the cell, as is the case in life-cycle stages that have been studied in T. brucei. This work provides a foundation for further investigations of T. cruzi cell division and shows that subtle differences in trypanosomatid cell morphology can alter how these parasites divide.

Selective retention of dysfunctional mitochondria during asymmetric cell division in yeast.

Decline of mitochondrial function is a hallmark of cellular aging. To counteract this process, some cells inherit mitochondria asymmetrically to rejuvenate daughter cells. The molecular mechanisms that control this process are poorly understood. Here, we made use of matrix-targeted D-amino acid oxidase (Su9-DAO) to selectively trigger oxidative damage in yeast mitochondria. We observed that dysfunctional mitochondria become fusion-incompetent and immotile. Lack of bud-directed movements is caused by defective recruitment of the myosin motor, Myo2. Intriguingly, intact mitochondria that are present in the same cell continue to move into the bud, establishing that quality control occurs directly at the level of the organelle in the mother. The selection of healthy organelles for inheritance no longer works in the absence of the mitochondrial Myo2 adapter protein Mmr1. Together, our data suggest a mechanism in which the combination of blocked fusion and loss of motor protein ensures that damaged mitochondria are retained in the mother cell to ensure rejuvenation of the bud.

Apical PAR protein caps orient the mitotic spindle in C. elegans early embryos.

During embryonic development, oriented cell divisions are important for patterned tissue growth and cell fate specification. Cell division orientation is controlled in part by asymmetrically localized polarity proteins, which establish functional domains of the cell membrane and interact with microtubule regulators to position the mitotic spindle. For example, in the 8-cell mouse embryo, apical polarity proteins form caps on the outside, contact-free surface of the embryo that position the mitotic spindle to execute asymmetric cell division. A similar radial or "inside-outside" polarity is established at an early stage in many other animal embryos, but in most cases, it remains unclear how inside-outside polarity is established and how it influences downstream cell behaviors. Here, we explore inside-outside polarity in C. elegans somatic blastomeres using spatiotemporally controlled protein degradation and live embryo imaging. We show that PAR polarity proteins, which form apical caps at the center of the contact-free membrane, localize dynamically during the cell cycle and contribute to spindle orientation and proper cell positioning. Surprisingly, isolated single blastomeres lacking cell contacts are able to break symmetry and form PAR-3/atypical protein kinase C (aPKC) caps. Polarity caps form independently of actomyosin flows and microtubules and can regulate spindle orientation in cooperation with the key polarity kinase aPKC. Together, our results reveal a role for apical polarity caps in regulating spindle orientation in symmetrically dividing cells and provide novel insights into how these structures are formed.