Asymmetric Cell Division Research Articles

024 Asymmetric cell division for fate induction of chimeric antigen receptor (CAR) T cells

Early expansion and long-term persistence predict efficacy of genetically-engineered T cells. While this is thought to reflect induction of effector and memory T cell populations to provide both short-term clearance and long-lasting remission, the cellular mechanisms of fate induction after T cell activation through synthetic receptors are unknown. A better understanding of such processes could improve therapeutic outcome. Here we show that human T cells engineered to express chimeric antigen receptors (CARs) undergo asymmetric cell division (ACD) with distinct proximal and distal daughter cells that adopt effector and memory phenotypes, respectively.

Asymmetric Cell Division and Tumor Heterogeneity.

Asymmetric cell division (ACD) gives rise to two daughter cells with different fates after mitosis and is a fundamental process for generating cell diversity and for the maintenance of the stem cell population. The cancer stem cell (CSC) theory suggests that CSCs with dysregulated self-renewal and asymmetric cell division serve as a source of intra-tumoral heterogeneity. This heterogeneity complicates the diagnosis and treatment of cancer patients, because CSCs can give rise to aggressive clones that are metastatic and insensitive to multiple drugs, or to dormant tumor cells that are difficult to detect. Here, we review the regulatory mechanisms and biological significance of asymmetric division in tumor cells, with a focus on ACD-induced tumor heterogeneity in early tumorigenesis and cancer progression. We will also discuss how dissecting the relationship between ACD and cancer may help us find new approaches for combatting this heterogeneity.

Stem Cells

Applicability of stem cells in cell-based therapy and regenerative medicine provided hope for untreatable diseases; subsequently, stem cells bioengineering has become a promising scientific research topic. Different types of stem cells possess different characteristics; certain types are superior to others according to their advantages. Cell bias toward asymmetric division is the first step regarding tissue regeneration and homeostasis, and this event is stimulated by chemical and mechanical cues arising from the surrounding microenvironment. Inappropriate asymmetric cell division (ACD) consequently results in organ disrupt morphogenesis. Intracellular events including polar distribution of regulatory proteins and fate determinants are of significant importance to prepare cells toward asymmetric division. The assembly of polarity proteins on opposite sides of the cell would induce downstream signaling pathway, and this forms a fundamental mechanism to establish cell asymmetric division. This review addresses the current understanding regarding stem cells biology, niche-raised cues, and polarity of regularity proteins and fate determinants of ACD.

A niche for axial stem cells - A cellular perspective in amniotes

The head-tail axis in birds and mammals develops from a growth zone in the tail-end, which contains the node. This growth zone then forms the tailbud. Labelling experiments have shown that while many cells leave the node and tailbud to contribute to axial (notochord, floorplate) and paraxial (somite) structures, some cells remain resident in the node and tailbud. Could these cells be resident axial stem cells? If so, do the node and tailbud represent an instructive stem cell niche that specifies and maintains these stem cells? Serial transplantation and single cell labelling studies support the existence of self-renewing stem cells and heterotopic transplantations suggest that the node can instruct such self-renewing behaviour. However, only single cell manipulations can reveal whether self-renewing behaviour occurs at the level of a cell population (asymmetric or symmetric cell divisions) or at the level of single cells (asymmetric divisions only). We combine data on resident cells in the node and tailbud and review it in the context of axial development in chick and mouse, summarising our current understanding of axial stem cells and their niche and highlighting future directions of interest.

A combined in silico and in vivo approach to the structure-function annotation of SPD-2 provides mechanistic insight into its functional diversity

ABSTRACT Centrosomes are organelles that function as hubs of microtubule nucleation and organization, with key roles in organelle positioning, asymmetric cell division, ciliogenesis, and signaling. Aberrant centrosome number, structure or function is linked to neurodegenerative diseases, developmental abnormalities, ciliopathies, and tumor development. A major regulator of centrosome biogenesis and function in C. elegans is the conserved Spindle-defective protein 2 (SPD-2), a homolog of the human CEP-192 protein. CeSPD-2 is required for centrosome maturation, centriole duplication, spindle assembly and possibly cell polarity establishment. Despite its importance, the specific molecular mechanism of CeSPD-2 regulation and function is poorly understood. Here, we combined computational analysis with cell biology approaches to uncover possible structure–function relationships of CeSPD-2 that may shed mechanistic light on its function. Domain prediction analysis corroborated and refined previously identified coiled-coils and ASH (Aspm-SPD-2 Hydin) domains and identified new domains: a GEF domain, an Ig-like domain, and a PDZ-like domain. In addition to these predicted structural features, CeSPD-2 is also predicted to be intrinsically disordered. Surface electrostatic maps identified a large basic region unique to the ASH domain of CeSPD-2. This basic region overlaps with most of the residues predicted to be involved in protein–protein interactions. In vivo, ASH::GFP localized to centrosomes and centrosome-associated microtubules. Our analysis groups ASH domains, PapD, Usher chaperone domains, and Major Sperm Protein (MSP) domains into a single superfold within the larger Immunoglobulin superfamily. This study lays the groundwork for designing rational hypothesis-based experiments to uncover the mechanisms of CeSPD-2 function in vivo. Abbreviations:AIR, Aurora kinase; ASH, Aspm-SPD-2 Hydin; ASP, Abnormal Spindle Protein; ASPM, Abnormal Spindle-like Microcephaly-associated Protein; CC, coiled-coil; CDK, Cyclin-dependent Kinase; Ce, Caenorhabditis elegans; CEP, Centrosomal Protein; CPAP, centrosomal P4.1-associated protein; D, Drosophila; GAP, GTPase activating protein; GEF, GTPase guanine nucleotide exchange factor; Hs, Homo sapiens/Human; Ig, Immunoglobulin; MAP, Microtubule associated Protein; MSP, Major Sperm Protein; MDP, Major Sperm Domain-Containing Protein; OCRL-1, Golgi endocytic trafficking protein Inositol polyphosphate 5-phosphatase; PAR, abnormal embryonic PARtitioning of the cytosol; PCM, Pericentriolar material; PCMD, pericentriolar matrix deficient; PDZ, PSD95/Dlg-1/zo-1; PLK, Polo like kinase; RMSD, Root Mean Square Deviation; SAS, Spindle assembly abnormal proteins; SPD, Spindle-defective protein; TRAPP, TRAnsport Protein Particle; Xe, Xenopus; ZYG, zygote defective protein.

On Solutions to a Class of Functional Differential Equations with Time-Dependent Coefficients

In this paper, we study initial boundary value problems that involve functional (nonlocal) partial differential equations with variable coefficients. These problems arise in cell growth models with symmetric and asymmetric modes of division. We determine the general solution to the symmetric cell division problem for a certain class of coefficients and establish the convergence of solutions to a large time asymptotic solution. The existence of a steady size distribution (SSD) solution for an asymmetric cell division problem is established and is shown to be the large time-attracting solution for a certain class of coefficients. The rate of convergence of solutions towards the SSD solution is affected by the choice of coefficients and remains unaffected by the asymmetry in cell division. The uniqueness of solutions to the initial boundary value problem is also established.

The neurogenic fate of the hindbrain boundaries relies on Notch3-dependent asymmetric cell divisions.

Elucidating the cellular and molecular mechanisms that regulate the balance between progenitor cell proliferation and neuronal differentiation in the construction of the embryonic brain demands the combination of cell lineage and functional approaches. Here, we generate the comprehensive lineage of hindbrain boundary cells by using a CRISPR-based knockin zebrafish transgenic line that specifically labels the boundaries. We unveil that boundary cells asynchronously engage in neurogenesis undergoing a functional transition from neuroepithelial progenitors to radial glia cells, coinciding with the onset of Notch3 signaling that triggers their asymmetrical cell division. Upon notch3 loss of function, boundary cells lose radial glia properties and symmetrically divide undergoing neuronal differentiation. Finally, we show that the fate of boundary cells is to become neurons, the subtype of which relies on their axial position, suggesting that boundary cells contribute to refine the number and proportion of the distinct neuronal populations.

Centrosome linker protein C‐Nap1 maintains stem cells in mouse testes

The centrosome linker component C‐Nap1 (encoded by CEP250) anchors filaments to centrioles that provide centrosome cohesion by connecting the two centrosomes of an interphase cell into a single microtubule organizing unit. The role of the centrosome linker during development of an animal remains enigmatic. Here, we show that male CEP250−/− mice are sterile because sperm production is abolished. Premature centrosome separation means that germ stem cells in CEP250−/− mice fail to establish an E‐cadherin polarity mark and are unable to maintain the older mother centrosome on the basal site of the seminiferous tubules. This failure prompts premature stem cell differentiation in expense of germ stem cell expansion. The concomitant induction of apoptosis triggers the complete depletion of germ stem cells and consequently infertility. Our study reveals a role for centrosome cohesion in asymmetric cell division, stem cell maintenance, and fertility.

Distinct strengths of mTORC1 control T-cell memory via transcriptional FOXO1 and metabolic AMPKα1 pathways in linear cell differentiation and asymmetric cell division models.

Distinct strengths of mTORC1 control T-cell memory via transcriptional FOXO1 and metabolic AMPKα1 pathways in linear cell differentiation and asymmetric cell division models.

Cortical Cyclin A controls spindle orientation during asymmetric cell divisions in Drosophila

The coordination between cell proliferation and cell polarity is crucial to orient the asymmetric cell divisions to generate cell diversity in epithelia. In many instances, the Frizzled/Dishevelled planar cell polarity pathway is involved in mitotic spindle orientation, but how this is spatially and temporally coordinated with cell cycle progression has remained elusive. Using Drosophila sensory organ precursor cells as a model system, we show that Cyclin A, the main Cyclin driving the transition to M-phase of the cell cycle, is recruited to the apical-posterior cortex in prophase by the Frizzled/Dishevelled complex. This cortically localized Cyclin A then regulates the orientation of the division by recruiting Mud, a homologue of NuMA, the well-known spindle-associated protein. The observed non-canonical subcellular localization of Cyclin A reveals this mitotic factor as a direct link between cell proliferation, cell polarity and spindle orientation.

Spindle motility skews division site determination during asymmetric cell division in Physcomitrella.

Asymmetric cell division (ACD) underlies the development of multicellular organisms. In animal ACD, the cell division site is determined by active spindle-positioning mechanisms. In contrast, it is considered that the division site in plants is determined prior to mitosis by the microtubule-actin belt known as the preprophase band (PPB) and that the localization of the mitotic spindle is typically static and does not govern the division plane. However, in some plant species, ACD occurs in the absence of PPB. Here, we isolate a hypomorphic mutant of the conserved microtubule-associated protein TPX2 in the moss Physcomitrium patens (Physcomitrella) and observe spindle motility during PPB-independent cell division. This defect compromises the position of the division site and produces inverted daughter cell sizes in the first ACD of gametophore (leafy shoot) development. The phenotype is rescued by restoring endogenous TPX2 function and, unexpectedly, by depolymerizing actin filaments. Thus, we identify an active spindle-positioning mechanism that, reminiscent of acentrosomal ACD in animals, involves microtubules and actin filaments, and sets the division site in plants.

Polarity Events in the Drosophila melanogaster Oocyte.

Cell polarity is a pre-requirement for many fundamental processes in animal cells, such as asymmetric cell division, axon specification, morphogenesis and epithelial tissue formation. For all these different processes, polarization is established by the same set of proteins, called partitioning defective (Par) proteins. During development in Drosophila melanogaster, decision making on the cellular and organism level is achieved with temporally controlled cell polarization events. The initial polarization of Par proteins occurs as early as in the germline cyst, when one of the 16 cells becomes the oocyte. Another marked event occurs when the anterior–posterior axis of the future organism is defined by Par redistribution in the oocyte, requiring external signaling from somatic cells. Here, we review the current literature on cell polarity events that constitute the oogenesis from the stem cell to the mature egg.

Zygote Activation: The Start of the New Generation in Angiosperms

In angiosperms, after the egg fuses with the sperm, many structural, physiological, and molecular biological changes occur in the fertilized egg. All of these changes facilitate the conversion of the haploid egg into the diploid zygote, a process known as the maternal-to-zygotic transition (MZT). In the egg, fertilized egg, and zygote, changes occur at each stage under the control of exact spatio-temporal regulation mechanisms. This review focuses on the molecular biological changes that occur during zygote activation in higher plants including the following: maturation and activation of intrinsic parental transcription; zygote genome activation (ZGA), changes in the expression levels of genes from the zygotic genome; the effect of parental genomic dosage; and cellular determination of zygotic asymmetrical division. It is these exact spatio-temporal regulation mechanisms that allow the egg to convert into the zygote, undergo asymmetrical cell division, and initiate embryogenesis. The results of recent studies have shown that the regulation of zygotic division is a complex process occurring in the cell (egg, fertilized egg, and zygote). The results so far have revealed just the tip of the iceberg of zygote activation. More research is required to explore the regulation of zygote activation.

Differential condensation of sister chromatids acts with Cdc6 to ensure asynchronous S-phase entry in Drosophila male germline stem cell lineage.

During Drosophila melanogaster male germline stem cell (GSC) asymmetric division, preexisting old versus newly synthesized histones H3 and H4 are asymmetrically inherited. However, the biological outcomes of this phenomenon have remained unclear. Here, we tracked old and new histones throughout the GSC cell cycle through the use of high spatial and temporal resolution microscopy. We found unique features that differ between old and new histone-enriched sister chromatids, including differences in nucleosome density, chromosomal condensation, and H3 Ser10 phosphorylation. These distinct chromosomal features lead to their differential association with Cdc6, a pre-replication complex component, and subsequent asynchronous DNA replication initiation in the resulting daughter cells. Disruption of asymmetric histone inheritance abolishes differential Cdc6 association and asynchronous S-phase entry, demonstrating that histone asymmetry acts upstream of these critical cell-cycle progression events. Furthermore, disruption of these GSC-specific chromatin features leads to GSC defects, indicating a connection between histone inheritance, cell-cycle progression, and cell fate determination.

An optogenetic GDI drives GPCR and Gα independent macrophage migration

Activators of G protein signaling (AGS) proteins play diverse functional roles in eukaryotic cells, including asymmetric cell division and membrane protein trafficking. The group II AGS (AGS3) is a guanine nucleotide dissociation inhibitor (GDI) consisting of four G‐protein regulatory (GPR) GoLoco motifs that bind to Gαi/o in the G protein heterotrimer. AGS3 binding should therefore release Gβγ from the heterotrimer. However, the ability of AGS3 to directly signal through Gβγ in a living cell has yet to be demonstrated. To investigate the role of AGS3 in Gβγ signaling independent of G protein‐coupled receptors, we engineered optogenetic AGS3 using one or more consensus GPR sequences. This consensus sequence has shown higher binding efficacy towards Gαi GDP comparative to the wild type. Upon expression, our Opto‐AGS3 showed localized PIP3 generation and directional migration in RAW 264.7 cells due to Gβγ generation, indicating non‐canonical Gβγ signaling of AGS3. Our results also provide GPCR and Gα independent optogenetic control of Gβγ signaling with subcellular spatial and millisecond temporal control.

Glial-secreted Netrins regulate Robo1/Rac1-Cdc42 signaling threshold levels during Drosophila asymmetric neural stem/progenitor cell division

Asymmetric stem cell division (ASCD) is a key mechanism in development, cancer, and stem cell biology. Drosophila neural stem cells, called neuroblasts (NBs), divide asymmetrically through intrinsic mechanisms. Here, we show that the extrinsic axon guidance cues Netrins, secreted by a glial niche surrounding larval brain neural stem cell lineages, regulate NB ASCD. Netrin-Frazzled/DCC signaling modulates, through Abelson kinase, Robo1 signaling threshold levels in Drosophila larval brain neural stem and progenitor cells of NBII lineages. Unbalanced Robo1 signaling levels induce ectopic NBs and progenitor cells due to failures in the ASCD process. Mechanistically, Robo1 signaling directly impinges on the intrinsic ASCD machinery, such as aPKC, Canoe/Afadin, and Numb, through the small GTPases Rac1 and Cdc42, which are required for the localization in mitotic NBs of Par-6, a Cdc42 physical partner and a core component of the Par (Par-6-aPKC-Par3/Bazooka) apical complex.

Vasa nucleates asymmetric translation along the mitotic spindle during unequal cell divisions

mRNA translation on the spindle is hypothesized to be an essential strategy for the localized production of cell regulators. This mechanism may be important particularly in early embryonic cells, which have a large diffusion volume and that undergo rapid cell divisions. Evidence to test such a hypothesis has been, however, limited. Here, we use an embryo with both symmetric and asymmetric cell divisions and manipulate Vasa protein, an RNA-helicase, on the spindle in live sea urchin embryos. We learned that the spindle serves as a major site of translation and that protein synthesis within a single spindle can be unequal and help drive asymmetric cell divisions during embryogenesis. Recruiting Vasa to the ectopic sub-cellular region induced a new site of translation, disturbed asymmetric translation on the spindle, and changed the cell fate. Based on these observations, we conclude that Vasa functions in localized translation, which provides a spatiotemporal control in protein synthesis and is essential for rapidly developing embryonic cells.

Mitotic drive in asymmetric epigenetic inheritance.

Asymmetric cell division (ACD) produces two daughter cells with distinct cell fates. This division mode is widely used during development and by adult stem cells during tissue homeostasis and regeneration, which can be regulated by both extrinsic cues such as signaling molecules and intrinsic factors such as epigenetic information. While the DNA replication process ensures that the sequences of sister chromatids are identical, how epigenetic information is re-distributed during ACD has remained largely unclear in multicellular organisms. Studies of Drosophila male germline stem cells (GSCs) have revealed that sister chromatids incorporate pre-existing and newly synthesized histones differentially and segregate asymmetrically during ACD. To understand the underlying molecular mechanisms of this phenomenon, two key questions must be answered: first, how and when asymmetric histone information is established; and second, how epigenetically distinct sister chromatids are distinguished and segregated. Here, we discuss recent advances which help our understanding of this interesting and important cell division mode.

Integrative genome-wide analysis reveals EIF3A as a key downstream regulator of translational repressor protein Musashi 2 (MSI2).

Musashi 2 (MSI2) is an RNA binding protein (RBP) that regulates asymmetric cell division and cell fate decisions in normal and cancer stem cells. MSI2 appears to repress translation by binding to 3′ untranslated regions (3′UTRs) of mRNA, but the identity of functional targets remains unknown. Here, we used individual nucleotide resolution cross-linking and immunoprecipitation (iCLIP) to identify direct RNA binding partners of MSI2 and integrated these data with polysome profiling to obtain insights into MSI2 function. iCLIP revealed specific MSI2 binding to thousands of mRNAs largely in 3′UTRs, but translational differences were restricted to a small fraction of these transcripts, indicating that MSI2 regulation is not triggered by simple binding. Instead, the functional targets identified here were bound at higher density and contain more ‘UAG’ motifs compared to targets bound nonproductively. To further distinguish direct and indirect targets, MSI2 was acutely depleted. Surprisingly, only 50 transcripts were found to undergo translational induction on acute loss. Using complementary approaches, we determined eukaryotic translation initiation factor 3A (EIF3A) to be an immediate, direct target. We propose that MSI2 downregulation of EIF3A amplifies these effects on translation. Our results also underscore the challenges in defining functional targets of RBPs since mere binding does not imply a discernible functional interaction.

Wnt3a loaded deformable hydrogel acts as a 3D culture platform for in situ recruitment of stem cells to efficiently repair bone defects via the asymmetric division

Biomaterial-based tissue engineering has emerged as a hotspot in the field of osteanagenesis. Due to invasive operation in the transplantation process always bring irreparable damage to patients, approaches that enable innate repair mechanisms hold considerable promise for bone repair. To this end, a rational design based on in situ recruitment of stem cells is proposed to circumvent the troublesome problem. In this study, an interpenetration network hydrogel is developed utilizing chitosan (CS), in which the enriched bone marrow mesenchymal stem cells (BMSCs) can undergo a tridimensional and freely proliferation. Moreover, the encapsulation of biological growth factor Wnt3a promotes the differentiation of osteoprogenitor cells with an asymmetric cell divisions (ACD) manner, thereby accelerating bone formation. With this “smart biomaterials”, a robust instant stem cell ingrowth within the deformable hydrogel and a high efficiency of bone regeneration on a murine skull are observed, both of which are vital for clinical applications. Such an osteoinductive system represents the advanced design concept to repair bone defects effectively, and offers a promising strategy for the development of future bone tissue engineering.