Principles and mechanisms of asymmetric cell division.

Asymmetric cell division (ACD) is an evolutionarily conserved mechanism used by prokaryotes and eukaryotes alike to control cell fate and generate cell diversity. A detailed mechanistic understanding of ACD is therefore necessary to understand cell fate decisions in health and disease. ACD can be manifested in the biased segregation of macromolecules, the differential partitioning of cell organelles, or differences in sibling cell size or shape. These events are usually preceded by and influenced by symmetry breaking events and cell polarization. In this Review, we focus predominantly on cell intrinsic mechanisms and their contribution to cell polarization, ACD and binary cell fate decisions. We discuss examples of polarized systems and detail how polarization is established and, whenever possible, how it contributes to ACD. Established and emerging model organisms will be considered alike, illuminating both well-documented and underexplored forms of polarization and ACD.

Dynamic centriolar localization of Polo and Centrobin in early mitosis primes centrosome asymmetry.

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 mechanisms regulating centrosome asymmetry and biased centrosome segregation are unclear. Using 3D-structured illumination microscopy (3D-SIM) and live-cell imaging, we show in fly neural stem cells (neuroblasts) that the mitotic kinase Polo and its centriolar protein substrate Centrobin (Cnb) accumulate on the daughter centriole during mitosis, thereby generating molecularly distinct mother and daughter centrioles before interphase. Cnb's asymmetric localization, potentially involving a direct relocalization mechanism, is regulated by Polo-mediated phosphorylation, whereas Polo's daughter centriole enrichment requires both Wdr62 and Cnb. Based on optogenetic protein mislocalization experiments, we propose that the establishment of centriole asymmetry in mitosis primes biased interphase MTOC activity, necessary for correct spindle orientation.

In Vivo Photocontrol of Microtubule Dynamics and Integrity, Migration and Mitosis, by the Potent GFP-Imaging-Compatible Photoswitchable Reagents SBTubA4P and SBTub2M.

Photoswitchable reagents are powerful tools for high-precision studies in cell biology. When these reagents are globally administered yet locally photoactivated in two-dimensional (2D) cell cultures, they can exert micron- and millisecond-scale biological control. This gives them great potential for use in biologically more relevant three-dimensional (3D) models and in vivo, particularly for studying systems with inherent spatiotemporal complexity, such as the cytoskeleton. However, due to a combination of photoswitch isomerization under typical imaging conditions, metabolic liabilities, and insufficient water solubility at effective concentrations, the in vivo potential of photoswitchable reagents addressing cytosolic protein targets remains largely unrealized. Here, we optimized the potency and solubility of metabolically stable, druglike colchicinoid microtubule inhibitors based on the styrylbenzothiazole (SBT) scaffold that are nonresponsive to typical fluorescent protein imaging wavelengths and so enable multichannel imaging studies. We applied these reagents both to 3D organoids and tissue explants and to classic model organisms (zebrafish, clawed frog) in one- and two-protein imaging experiments, in which spatiotemporally localized illuminations allowed them to photocontrol microtubule dynamics, network architecture, and microtubule-dependent processes in vivo with cellular precision and second-level resolution. These nanomolar, in vivo capable photoswitchable reagents should open up new dimensions for high-precision cytoskeleton research in cargo transport, cell motility, cell division, and development. More broadly, their design can also inspire similarly capable optical reagents for a range of cytosolic protein targets, thus bringing in vivo photopharmacology one step closer to general realization.

A spindle-independent cleavage furrow positioning pathway.

The mitotic spindle determines the cleavage furrow site during metazoan cell division, but whether other mechanisms exist remains unknown. Here we identify a spindle-independent mechanism for cleavage furrow positioning in Drosophila neuroblasts. We show that early and late furrow proteins (Pavarotti, Anillin, and Myosin) are localized to the neuroblast basal cortex at anaphase onset by a Pins cortical polarity pathway, and can induce a basally displaced furrow even in the complete absence of a mitotic spindle. Rotation or displacement of the spindle results in two furrows: an early polarity-induced basal furrow and a later spindle-induced furrow. This spindle-independent cleavage furrow mechanism may be relevant to other highly polarized mitotic cells, such as mammalian neural progenitors.

The NuMA-related Mud protein binds Pins and regulates spindle orientation in Drosophila neuroblasts.

Asymmetric cell division generates cell diversity during development and regulates stem-cell self-renewal in Drosophila and mammals. In Drosophila, neuroblasts align their spindle with a cortical Partner of Inscuteable (Pins)-G alpha i crescent to divide asymmetrically, but the link between cortical polarity and the mitotic spindle is poorly understood. Here, we show that Pins directly binds, and coimmunoprecipitates with, the NuMA-related Mushroom body defect (Mud) protein. Pins recruits Mud to the neuroblast apical cortex, and Mud is also strongly localized to centrosome/spindle poles, in a similar way to NuMA. In mud mutants, cortical polarity is normal, but the metaphase spindle frequently fails to align with the cortical polarity axis. When spindle orientation is orthogonal to cell polarity, symmetric division occurs. We propose that Mud is a functional orthologue of mammalian NuMA and Caenorhabditis elegans Lin-5, and that Mud coordinates spindle orientation with cortical polarity to promote asymmetric cell division.

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains.

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a differentiating ganglion mother cell (GMC), which will undergo one additional division to give rise to two neurons or glia. Studies in NBs have uncovered the molecular mechanisms underlying cell polarity, spindle orientation, neural stem cell self-renewal, and differentiation. These asymmetric cell divisions are readily observable via live-cell imaging, making larval NBs ideally suited for investigating the spatiotemporal dynamics of asymmetric cell division in living tissue. When properly dissected and imaged in nutrient-supplemented medium, NBs in explant brains robustly divide for 12-20 h. Previously described methods are technically difficult and may be challenging to those new to the field. Here, a protocol is described for the preparation, dissection, mounting, and imaging of live third-instar larval brain explants using fat body supplements. Potential problems are also discussed, and examples are provided for how this technique can be used.

Fragile X protein controls neural stem cell proliferation in the Drosophila brain.

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and is caused by the loss of function for Fragile X protein (FMRP), an RNA-binding protein thought to regulate synaptic plasticity by controlling the localization and translation of specific mRNAs. We have recently shown that FMRP is required to control the proliferation of the germline in Drosophila. To determine whether FMRP is also required for proliferation during brain development, we examined the distribution of cell cycle markers in dFmr1 brains compared with wild-type throughout larval development. Our results indicate that the loss of dFmr1 leads to a significant increase in the number of mitotic neuroblasts (NB) and BrdU incorporation in the brain, consistent with the notion that FMRP controls proliferation during neurogenesis. Developmental studies suggest that FMRP also inhibits neuroblast exit from quiescence in early larval brains, as indicated by misexpression of Cyclin E. Live imaging experiments indicate that by the third instar larval stage, the length of the cell cycle is unaffected, although more cells are found in S and G2/M in dFmr1 brains compared with wild-type. To determine the role of FMRP in neuroblast division and differentiation, we used Mosaic Analysis with a Repressible Marker (MARCM) approaches in the developing larval brain and found that single dFmr1 NB generate significantly more neurons than controls. Our results demonstrate that FMRP is required during brain development to control the exit from quiescence and proliferative capacity of NB as well as neuron production, which may provide insights into the autistic component of FXS.

Asymmetric chromatin retention and nuclear envelopes separate chromosomes in fused cells in vivo.

Hybrid cells derived through fertilization or somatic cell fusion recognize and separate chromosomes of different origins. The underlying mechanisms are unknown but could prevent aneuploidy and tumor formation. Here, we acutely induce fusion between Drosophila neural stem cells (neuroblasts; NBs) and differentiating ganglion mother cells (GMCs) in vivo to define how epigenetically distinct chromatin is recognized and segregated. We find that NB-GMC hybrid cells align both endogenous (neuroblast-origin) and ectopic (GMC-origin) chromosomes at the metaphase plate through centrosome derived dual-spindles. Physical separation of endogenous and ectopic chromatin is achieved through asymmetric, microtubule-dependent chromatin retention in interphase and physical boundaries imposed by nuclear envelopes. The chromatin separation mechanisms described here could apply to the first zygotic division in insects, arthropods, and vertebrates or potentially inform biased chromatid segregation in stem cells.

Engineered kinases as a tool for phosphorylation of selected targets in vivo.

Reversible protein phosphorylation by kinases controls a plethora of processes essential for the proper development and homeostasis of multicellular organisms. One main obstacle in studying the role of a defined kinase-substrate interaction is that kinases form complex signaling networks and most often phosphorylate multiple substrates involved in various cellular processes. In recent years, several new approaches have been developed to control the activity of a given kinase. However, most of them fail to regulate a single protein target, likely hiding the effect of a unique kinase-substrate interaction by pleiotropic effects. To overcome this limitation, we have created protein binder-based engineered kinases that permit a direct, robust, and tissue-specific phosphorylation of fluorescent fusion proteins in vivo. We show the detailed characterization of two engineered kinases based on Rho-associated protein kinase (ROCK) and Src. Expression of synthetic kinases in the developing fly embryo resulted in phosphorylation of their respective GFP-fusion targets, providing for the first time a means to direct the phosphorylation to a chosen and tagged target in vivo. We presume that after careful optimization, the novel approach we describe here can be adapted to other kinases and targets in various eukaryotic genetic systems to regulate specific downstream effectors.

Stem cell self-renewal: centrosomes on the move.

Three recent studies show that centrosome asymmetry correlates with self-renewal of Drosophila neural and germline stem cells and that equalizing centrosomes disrupts asymmetric cell division.

Distinct roles for two receptor tyrosine kinases in epithelial branching morphogenesis in Drosophila.

Branching morphogenesis is a widespread mechanism used to increase the surface area of epithelial organs. Many signaling systems steer development of branched organs, but it is still unclear which cellular processes are regulated by the different pathways. We have used the development of the air sacs of the dorsal thorax of Drosophila to study cellular events and their regulation via cell-cell signaling. We find that two receptor tyrosine kinases play important but distinct roles in air sac outgrowth. Fgf signaling directs cell migration at the tip of the structure, while Egf signaling is instrumental for cell division and cell survival in the growing epithelial structure. Interestingly, we find that Fgf signaling requires Ras, the Mapk pathway, and Pointed to direct migration, suggesting that both cytoskeletal and nuclear events are downstream of receptor activation. Ras and the Mapk pathway are also needed for Egf-regulated cell division/survival, but Pointed is dispensable.

Mechanical regulation of cell size, fate, and behavior during asymmetric cell division.

Asymmetric cell division (ACD) is an evolutionary conserved mechanism used by prokaryotes and eukaryotes alike to generate cell diversity. ACD can be manifested in biased segregation of macromolecules or differential partitioning of cell organelles. Cells are also constantly subject to extrinsic or intrinsic mechanical forces, influencing cell behavior and fate. During ACD, cell intrinsic forces generated through the spatiotemporal regulation of the actomyosin cytoskeleton can influence sibling cell size. External mechanical stresses are further translated by transcriptional coactivators or mechanically gated ion channels. Here, we will discuss recent literature, exploring how mechanical cues influence various aspects of ACD and stem cell behavior, and how these mechanical cues contribute to cell fate decisions.

Spatiotemporally Controlled Myosin Relocalization and Internal Pressure Generate Sibling Cell Size Asymmetry.

Metazoan cells can generate unequal-sized sibling cells during cell division. This form of asymmetric cell division depends on spindle geometry and Myosin distribution, but the underlying mechanics are unclear. Here, we use atomic force microscopy and live cell imaging to elucidate the biophysical forces involved in the establishment of physical asymmetry in Drosophila neural stem cells. We show that initial apical cortical expansion is driven by hydrostatic pressure, peaking shortly after anaphase onset, and enabled by a relief of actomyosin contractile tension on the apical cell cortex. An increase in contractile tension at the cleavage furrow combined with the relocalization of basally located Myosin initiates basal and sustains apical extension. We propose that spatiotemporally controlled actomyosin contractile tension and hydrostatic pressure enable biased cortical expansion to generate sibling cell size asymmetry. However, dynamic cleavage furrow repositioning can compensate for the lack of biased expansion to establish physical asymmetry.

Dynamic MAPK signaling activity underlies a transition from growth arrest to proliferation in Drosophila scribble mutant tumors.

Human tumors exhibit plasticity and evolving capacity over time. It is difficult to study the mechanisms of how tumors change over time in human patients, in particular during the early stages when a few oncogenic cells are barely detectable. Here, we used a Drosophila tumor model caused by loss of scribble (scrib), a highly conserved apicobasal cell polarity gene, to investigate the spatial-temporal dynamics of early tumorigenesis events. The fly scrib mutant tumors have been successfully used to model many aspects of tumorigenesis processes. However, it is still unknown whether Drosophila scrib mutant tumors exhibit plasticity and evolvability along the temporal axis. We found that scrib mutant tumors displayed different growth rates and cell cycle profiles over time, indicative of a growth arrest-to-proliferation transition as the scrib mutant tumors progress. Longitudinal bulk and single-cell transcriptomic analysis of scrib mutant tumors revealed that the MAPK pathway, including JNK and ERK signaling activities, showed quantitative changes over time. We found that high JNK signaling activity caused G2/M cell cycle arrest in early scrib mutant tumors. In addition, JNK signaling activity displayed a radial polarity with the JNKhigh cells located at the periphery of scrib mutant tumors, providing an inherent mechanism that leads to an overall decrease in JNK signaling activity over time. We also found that ERK signaling activity, in contrast to JNK activity, increased over time and promoted growth in late-stage scrib mutant tumors. Furthermore, high JNK signaling activity repressed ERK signaling activity in early scrib mutant tumors. Together, these data demonstrate that dynamic MAPK signaling activity, fueled by intratumor heterogeneity derived from tissue topological differences, drives a growth arrest-to-proliferation transition in scrib mutant tumors.This article has an associated First Person interview with the joint first authors of the paper.

A genetic mosaic analysis with a repressible cell marker screen to identify genes involved in tracheal cell migration during Drosophila air sac morphogenesis.

Branching morphogenesis of the Drosophila tracheal system relies on the fibroblast growth factor receptor (FGFR) signaling pathway. The Drosophila FGF ligand Branchless (Bnl) and the FGFR Breathless (Btl/FGFR) are required for cell migration during the establishment of the interconnected network of tracheal tubes. However, due to an important maternal contribution of members of the FGFR pathway in the oocyte, a thorough genetic dissection of the role of components of the FGFR signaling cascade in tracheal cell migration is impossible in the embryo. To bypass this shortcoming, we studied tracheal cell migration in the dorsal air sac primordium, a structure that forms during late larval development. Using a mosaic analysis with a repressible cell marker (MARCM) clone approach in mosaic animals, combined with an ethyl methanesulfonate (EMS)-mutagenesis screen of the left arm of the second chromosome, we identified novel genes implicated in cell migration. We screened 1123 mutagenized lines and identified 47 lines displaying tracheal cell migration defects in the air sac primordium. Using complementation analyses based on lethality, mutations in 20 of these lines were genetically mapped to specific genomic areas. Three of the mutants were mapped to either the Mhc or the stam complementation groups. Further experiments confirmed that these genes are required for cell migration in the tracheal air sac primordium.

Sibling cell size matters.

A motor protein called Klp10A ensures that germline stem cells in male fruit flies divide to produce two sibling cells that are equal in size.

Drosophila melanogaster Neuroblasts: A Model for Asymmetric Stem Cell Divisions.

Asymmetric cell division (ACD) is a fundamental mechanism to generate cell diversity, giving rise to daughter cells with different developmental potentials. ACD is manifested in the asymmetric segregation of proteins or mRNAs, when the two daughter cells differ in size or are endowed with different potentials to differentiate into a particular cell type (Horvitz and Herskowitz, Cell 68:237-255, 1992). Drosophila neuroblasts, the neural stem cells of the developing fly brain, are an ideal system to study ACD since this system encompasses all of these characteristics. Neuroblasts are intrinsically polarized cells, utilizing polarity cues to orient the mitotic spindle, segregate cell fate determinants asymmetrically, and regulate spindle geometry and physical asymmetry. The neuroblast system has contributed significantly to the elucidation of the basic molecular mechanisms underlying ACD. Recent findings also highlight its usefulness to study basic aspects of stem cell biology and tumor formation. In this review, we will focus on what has been learned about the basic mechanisms underlying ACD in fly neuroblasts.

Cell Polarity Regulates Biased Myosin Activity and Dynamics during Asymmetric Cell Division via Drosophila Rho Kinase and Protein Kinase N.

Cell and tissue morphogenesis depends on the correct regulation of non-muscle Myosin II, but how this motor protein is spatiotemporally controlled is incompletely understood. Here, we show that in asymmetrically dividing Drosophila neural stem cells, cell intrinsic polarity cues provide spatial and temporal information to regulate biased Myosin activity. Using live cell imaging and a genetically encoded Myosin activity sensor, we found that Drosophila Rho kinase (Rok) enriches for activated Myosin on the neuroblast cortex prior to nuclear envelope breakdown (NEB). After NEB, the conserved polarity protein Partner of Inscuteable (Pins) sequentially enriches Rok and Protein Kinase N (Pkn) on the apical neuroblast cortex. Our data suggest that apical Rok first increases phospho-Myosin, followed by Pkn-mediated Myosin downregulation, possibly through Rok inhibition. We propose that polarity-induced spatiotemporal control of Rok and Pkn is important for unequal cortical expansion, ensuring correct cleavage furrow positioning and the establishment of physical asymmetry.

Myosin efflux promotes cell elongation to coordinate chromosome segregation with cell cleavage.

Chromatid segregation must be coordinated with cytokinesis to preserve genomic stability. Here we report that cells clear trailing chromatids from the cleavage site by undergoing two phases of cell elongation. The first phase relies on the assembly of a wide contractile ring. The second phase requires the activity of a pool of myosin that flows from the ring and enriches the nascent daughter cell cortices. This myosin efflux is a novel feature of cytokinesis and its duration is coupled to nuclear envelope reassembly and the nuclear sequestration of the Rho-GEF Pebble. Trailing chromatids induce a delay in nuclear envelope reassembly concomitant with prolonged cortical myosin activity, thus providing forces for the second elongation. We propose that the modulation of cortical myosin dynamics is part of the cellular response triggered by a "chromatid separation checkpoint" that delays nuclear envelope reassembly and, consequently, Pebble nuclear sequestration when trailing chromatids are present at the midzone.Chromatid segregation must be coordinated with cytokinesis to preserve genomic stability. Here the authors show that cells clear trailing chromatids from the cleavage site in a two-step cell elongation and demonstrate the role of myosin efflux in the second phase.

Spatio-temporally separated cortical flows and spindle geometry establish physical asymmetry in fly neural stem cells.

Asymmetric cell division, creating sibling cells with distinct developmental potentials, can be manifested in sibling cell size asymmetry. This form of physical asymmetry occurs in several metazoan cells, but the underlying mechanisms and function are incompletely understood. Here we use Drosophila neural stem cells to elucidate the mechanisms involved in physical asymmetry establishment. We show that Myosin relocalizes to the cleavage furrow via two distinct cortical Myosin flows: at anaphase onset, a polarity induced, basally directed Myosin flow clears Myosin from the apical cortex. Subsequently, mitotic spindle cues establish a Myosin gradient at the lateral neuroblast cortex, necessary to trigger an apically directed flow, removing Actomyosin from the basal cortex. On the basis of the data presented here, we propose that spatiotemporally controlled Myosin flows in conjunction with spindle positioning and spindle asymmetry are key determinants for correct cleavage furrow placement and cortical expansion, thereby establishing physical asymmetry.