Faithful propagation of life requires coordination of DNA replication and segregation with cell growth and division. In bacteria, this results in cell size homeostasis and periodicity in replication and division. The situation is perturbed under stress such as DNA damage, which induces filamentation as cell cycle progression is blocked to allow for repair. Mechanisms that release this morphological state for reentry into wild-type growth are unclear. Here we show that damage-induced Escherichia coli filaments divide asymmetrically, producing short daughter cells that tend to be devoid of damage and have wild-type size and growth dynamics. The Min-system primarily determines division site location in the filament, with additional regulation of division completion by chromosome segregation. Collectively, we propose that coordination between chromosome (and specifically terminus) segregation and cell division may result in asymmetric division in damage-induced filaments and facilitate recovery from a stressed state.

An intact actomyosin network is essential for anchoring polarity proteins to the cell cortex and maintaining cell size asymmetry during asymmetric cell division of Drosophila neuroblasts (NBs). However, the mechanisms that control changes in actomyosin dynamics during asymmetric cell division remain unclear. We find that the actin-binding protein, Moesin, is essential for NB proliferation and mitotic progression in the developing brain. During metaphase, phosphorylated Moesin (p-Moesin) is enriched at the apical cortex, and loss of Moesin leads to defects in apical polarity maintenance and cortical stability. This asymmetric distribution of p-Moesin is determined by components of the apical polarity complex and Slik kinase. During later stages of mitosis, p-Moesin localization shifts more basally, contributing to asymmetric cortical extension and myosin basal furrow positioning. Our findings reveal Moesin as a novel apical polarity protein that drives cortical remodeling of dividing NBs, which is essential for polarity maintenance and initial establishment of cell size asymmetry.

Many stem cells, including Drosophila germline stem cells (GSCs), divide asymmetrically, producing one stem cell and one differentiating daughter. Cytokinesis is often asymmetric, in that only one daughter cell inherits the midbody ring (MR) upon completion of abscission even in apparently symmetrically dividing cells. However, whether the asymmetry in cytokinesis correlates with cell fate or has functional relevance has been poorly explored. Here we show that the MR is asymmetrically segregated during GSC divisions in a centrosome age-dependent manner: male GSCs, which inherit the mother centrosome, exclude the MR, whereas female GSCs, which we here show inherit the daughter centrosome, inherit the MR. We further show that stem cell identity correlates with the mode of MR inheritance. Together our data suggest that the MR does not inherently dictate stem cell identity, although its stereotypical inheritance is under the control of stemness and potentially provides a platform for asymmetric segregation of certain factors.

The anterior-posterior axis of the Caenorhabditis elegans embryo is elaborated at the one-cell stage by the polarization of the partitioning (PAR) proteins at the cell cortex. Polarization is established under the control of the Rho GTPase RHO-1 and is maintained by the Rho GTPase CDC-42. To understand more clearly the role of the Rho family GTPases in polarization and division of the early embryo, we constructed a fluorescent biosensor to determine the localization of CDC-42 activity in the living embryo. A genetic screen using this biosensor identified one positive (putative guanine nucleotide exchange factor [GEF]) and one negative (putative GTPase activating protein [GAP]) regulator of CDC-42 activity: CGEF-1 and CHIN-1. CGEF-1 was required for robust activation, whereas CHIN-1 restricted the spatial extent of CDC-42 activity. Genetic studies placed CHIN-1 in a novel regulatory loop, parallel to loop described previously, that maintains cortical PAR polarity. We found that polarized distributions of the nonmuscle myosin NMY-2 at the cell cortex are independently produced by the actions of RHO-1, and its effector kinase LET-502, during establishment phase and CDC-42, and its effector kinase MRCK-1, during maintenance phase. CHIN-1 restricted NMY-2 recruitment to the anterior during maintenance phase, consistent with its role in polarizing CDC-42 activity during this phase.

In cultured mammalian cells, how dynein/dynactin contributes to spindle positioning is poorly understood. To assess the role of cortical dynein/dynactin in this process, we generated mammalian cell lines expressing localization and affinity purification (LAP)-tagged dynein/dynactin subunits from bacterial artificial chromosomes and observed asymmetric cortical localization of dynein and dynactin during mitosis. In cells with asymmetrically positioned spindles, dynein and dynactin were both enriched at the cortex distal to the spindle. NuMA, an upstream targeting factor, localized asymmetrically along the cell cortex in a manner similar to dynein and dynactin. During spindle motion toward the distal cortex, dynein and dynactin were locally diminished and subsequently enriched at the new distal cortex. At anaphase onset, we observed a transient increase in cortical dynein, followed by a reduction in telophase. Spindle motion frequently resulted in cells entering anaphase with an asymmetrically positioned spindle. These cells gave rise to symmetric daughter cells by dynein-dependent differential spindle pole motion in anaphase. Our results demonstrate that cortical dynein and dynactin dynamically associate with the cell cortex in a cell cycle-regulated manner and are required to correct spindle mispositioning in LLC-Pk1 epithelial cells.

Gene expression is restricted to specific times in cell division and differentiation through close control of both activation and inactivation of transcription. In budding yeast, strict spatiotemporal regulation of the transcription factor Ace2 ensures that it acts only once in a cell's lifetime: at the M-to-G1 transition in newborn daughter cells. The Ndr/LATS family kinase Cbk1, functioning in a system similar to metazoan hippo signaling pathways, activates Ace2 and drives its accumulation in daughter cell nuclei, but the mechanism of this transcription factor's inactivation is unknown. We found that Ace2's nuclear localization is maintained by continuous Cbk1 activity and that inhibition of the kinase leads to immediate loss of phosphorylation and export to the cytoplasm. Once exported, Ace2 cannot re-enter nuclei for the remainder of the cell cycle. Two separate mechanisms enforce Ace2's cytoplasmic sequestration: 1) phosphorylation of CDK consensus sites in Ace2 by the G1 CDKs Pho85 and Cdc28/CDK1 and 2) an unknown mechanism mediated by Pho85 that is independent of its kinase activity. Direct phosphorylation of CDK consensus sites is not necessary for Ace2's cytoplasmic retention, indicating that these mechanisms function redundantly. Overall, these findings show how sequential opposing kinases limit a daughter cell specific transcriptional program to a brief period during the cell cycle and suggest that CDKs may function as cytoplasmic sequestration factors.

During asymmetric cell division, the molecular motor dynein generates cortical pulling forces that position the spindle to reflect polarity and adequately distribute cell fate determinants. In Caenorhabditis elegans embryos, despite a measured anteroposterior force imbalance, antibody staining failed to reveal dynein enrichment at the posterior cortex, suggesting a transient localization there. Dynein accumulates at the microtubule plus ends, in an EBP-2EB-dependent manner. This accumulation, although not transporting dynein, contributes modestly to cortical forces. Most dyneins may instead diffuse to the cortex. Tracking of cortical dynein revealed two motions: one directed and the other diffusive-like, corresponding to force-generating events. Surprisingly, while dynein is not polarized at the plus ends or in the cytoplasm, diffusive-like tracks were more frequently found at the embryo posterior tip, where the forces are higher. This asymmetry depends on GPR-1/2LGN and LIN-5NuMA, which are enriched there. In csnk-1(RNAi) embryos, the inverse distribution of these proteins coincides with an increased frequency of diffusive-like tracks anteriorly. Importantly, dynein cortical residence time is always symmetric. We propose that the dynein-binding rate at the posterior cortex is increased, causing the polarity-reflecting force imbalance. This mechanism of control supplements the regulation of mitotic progression through the nonpolarized dynein detachment rate.

Positioning of microtubule-organizing centers (MTOCs) incorporates biochemical and mechanical cues for proper alignment of the mitotic spindle and cell division site. Current experimental and theoretical studies in the early Caenorhabditis elegans embryo assume remarkable changes in the origin and polarity of forces acting on the MTOCs. These changes must occur over a few minutes, between initial centration and rotation of the pronuclear complex and entry into mitosis, and the models do not replicate in vivo timing of centration and rotation. Here we propose a model that incorporates asymmetry in the microtubule arrays generated by each MTOC, which we demonstrate with in vivo measurements, and a similar asymmetric force profile to that required for posterior-directed spindle displacement during mitosis. We find that these asymmetries are capable of and important for recapitulating the simultaneous centration and rotation of the pronuclear complex observed in vivo. The combination of theoretical and experimental evidence provided here offers a unified framework for the spatial organization and forces needed for pronuclear centration, rotation, and spindle displacement in the early C. elegans embryo.

Formation of inner and outer cells of the mouse embryo distinguishes pluripotent inner cell mass (ICM) from differentiating trophectoderm (TE). Carm1, which methylates histone H3R17 and R26, directs cells to ICM rather that TE. To understand the mechanism by which this epigenetic modification directs cell fate, we generated embryos with in vivo-labeled cells of different Carm1 levels, using time-lapse imaging to reveal dynamics of their behavior, and related this to cell polarization. This shows that Carm1 affects cell fate by promoting asymmetric divisions, that direct one daughter cell inside, and cell engulfment, where neighboring cells with lower Carm1 levels compete for outside positions. This is associated with changes to the expression pattern and spatial distribution of cell polarity proteins: Cells with higher Carm1 levels show reduced expression and apical localization of Par3 and a dramatic increase in expression of PKCII, antagonist of the apical protein aPKC. Expression and basolateral localization of the mouse Par1 homologue, EMK1, increases concomitantly. Increased Carm1 also reduces Cdx2 expression, a transcription factor key for TE differentiation. These results demonstrate how the extent of a specific epigenetic modification could affect expression of cell polarity and fate-determining genes to ensure lineage allocation in the mouse embryo.

Proper spindle orientation is required for asymmetric cell division and the establishment of complex tissue architecture. In the developing epidermis, spindle orientation requires a conserved cortical protein complex of LGN/NuMA/dynein-dynactin. However, how microtubule dynamics are regulated to interact with this machinery and properly position the mitotic spindle is not fully understood. Furthermore, our understanding of the processes that link spindle orientation during asymmetric cell division to cell fate specification in distinct tissue contexts remains incomplete. We report a role for the microtubule catastrophe factor KIF18B in regulating microtubule dynamics to promote spindle orientation in keratinocytes. During mitosis, KIF18B accumulates at the cell cortex, colocalizing with the conserved spindle orientation machinery. In vivo we find that KIF18B is required for oriented cell divisions within the hair placode, the first stage of hair follicle morphogenesis, but is not essential in the interfollicular epidermis. Disrupting spindle orientation in the placode, using mutations in either KIF18B or NuMA, results in aberrant cell fate marker expression of hair follicle progenitor cells. These data functionally link spindle orientation to cell fate decisions during hair follicle morphogenesis. Taken together, our data demonstrate a role for regulated microtubule dynamics in spindle orientation in epidermal cells. This work also highlights the importance of spindle orientation during asymmetric cell division to dictate cell fate specification.

Cells actively position their nuclei based on their activity. In fission yeast, microtubule-dependent nuclear centering is critical for symmetrical cell division. After spindle disassembly at the end of anaphase, the nucleus recenters over an ∼90-min period, approximately half of the duration of the cell cycle. Live-cell and simulation experiments support the cooperation of two distinct microtubule competition mechanisms in the slow recentering of the nucleus. First, a push-push mechanism acts from spindle disassembly to septation and involves the opposing actions of the mitotic spindle pole body microtubules that push the nucleus away from the ends of the cell, while a postanaphase array of microtubules baskets the nucleus and limits its migration toward the division plane. Second, a slow-and-grow mechanism slowly centers the nucleus in the newborn cell by a combination of microtubule competition and asymmetric cell growth. Our work underlines how intrinsic properties of microtubules differently impact nuclear positioning according to microtubule network organization and cell size.

Drosophila male germline stem cells (GSCs) divide asymmetrically, balancing self-renewal and differentiation. Although asymmetric stem cell division balances between self-renewal and differentiation, it does not dictate how frequently differentiating cells must be produced. In male GSCs, asymmetric GSC division is achieved by stereotyped positioning of the centrosome with respect to the stem cell niche. Recently we showed that the centrosome orientation checkpoint monitors the correct centrosome orientation to ensure an asymmetric outcome of the GSC division. When GSC centrosomes are not correctly oriented with respect to the niche, GSC cell cycle is arrested/delayed until the correct centrosome orientation is reacquired. Here we show that induction of centrosome misorientation upon culture in poor nutrient conditions mediates slowing of GSC cell proliferation via activation of the centrosome orientation checkpoint. Consistently, inactivation of the centrosome orientation checkpoint leads to lack of cell cycle slowdown even under poor nutrient conditions. We propose that centrosome misorientation serves as a mediator that transduces nutrient information into stem cell proliferation, providing a previously unappreciated mechanism of stem cell regulation in response to nutrient conditions.

The spindle assembly checkpoint (SAC) is a conserved mitotic regulator that preserves genome stability by monitoring kinetochore-microtubule attachments and blocking anaphase onset until chromosome biorientation is achieved. Despite its central role in maintaining mitotic fidelity, the ability of the SAC to delay mitotic exit in the presence of kinetochore-microtubule attachment defects (SAC "strength") appears to vary widely. How different cellular aspects drive this variation remains largely unknown. Here we show that SAC strength is correlated with cell fate during development of Caenorhabditis elegans embryos, with germline-fated cells experiencing longer mitotic delays upon spindle perturbation than somatic cells. These differences are entirely dependent on an intact checkpoint and only partially attributable to differences in cell size. In two-cell embryos, cell size accounts for half of the difference in SAC strength between the larger somatic AB and the smaller germline P1 blastomeres. The remaining difference requires asymmetric cytoplasmic partitioning downstream of PAR polarity proteins, suggesting that checkpoint-regulating factors are distributed asymmetrically during early germ cell divisions. Our results indicate that SAC activity is linked to cell fate and reveal a hitherto unknown interaction between asymmetric cell division and the SAC.

Asymmetric cell division is the primary mechanism to generate cellular diversity, and it relies on the correct partitioning of cell fate determinants. However, the mechanism by which these determinants are delivered and positioned is poorly understood, and the upstream signal to initiate asymmetric cell division is unknown. Here we report that the endoplasmic reticulum (ER) is asymmetrically partitioned during mitosis in epithelial cells just before delamination and selection of a proneural cell fate in the early Drosophila embryo. At the start of gastrulation, the ER divides asymmetrically into a population of asynchronously dividing cells at the anterior end of the embryo. We found that this asymmetric division of the ER depends on the highly conserved ER membrane protein Jagunal (Jagn). RNA inhibition of jagn just before the start of gastrulation disrupts this asymmetric division of the ER. In addition, jagn-deficient embryos display defects in apical-basal spindle orientation in delaminated embryonic neuroblasts. Our results describe a model in which an organelle is partitioned asymmetrically in an otherwise symmetrically dividing cell population just upstream of cell fate determination and updates previous models of spindle-based selection of cell fate during mitosis.

The Drosophila tumor suppressor Lethal (2) giant larvae (Lgl) regulates the apical-basal polarity in epithelia and asymmetric cell division. However, little is known about the role of Lgl in cell polarity in migrating cells. In this study we show direct physiological interactions between the mammalian homologue of Lgl (Lgl1) and the nonmuscle myosin II isoform A (NMII-A). We demonstrate that Lgl1 and NMII-A form a complex in vivo and provide data that Lgl1 inhibits NMII-A filament assembly in vitro. Furthermore, depletion of Lgl1 results in the unexpected presence of NMII-A in the cell leading edge, a region that is not usually occupied by this protein, suggesting that Lgl1 regulates the cellular localization of NMII-A. Finally, we show that depletion of Lgl1 affects the size and number of focal adhesions, as well as cell polarity, membrane dynamics, and the rate of migrating cells. Collectively these findings indicate that Lgl1 regulates the polarity of migrating cells by controlling the assembly state of NMII-A, its cellular localization, and focal adhesion assembly.

Phosphatidylinositol (PI), an important constituent of membranes, contains stearic acid as the major fatty acid at the sn-1 position. This fatty acid is thought to be incorporated into PI through fatty acid remodeling by sequential deacylation and reacylation. However, the genes responsible for the reaction are unknown, and consequently, the physiological significance of the sn-1 fatty acid remains to be elucidated. Here, we identified acl-8, -9, and -10, which are closely related to each other, and ipla-1 as strong candidates for genes involved in fatty acid remodeling at the sn-1 position of PI. In both ipla-1 mutants and acl-8 acl-9 acl-10 triple mutants of Caenorhabditis elegans, the stearic acid content of PI is reduced, and asymmetric division of stem cell-like epithelial cells is defective. The defects in asymmetric division of these mutants are suppressed by a mutation of the same genes involved in intracellular retrograde transport, suggesting that ipla-1 and acl genes act in the same pathway. IPLA-1 and ACL-10 have phospholipase A(1) and acyltransferase activity, respectively, both of which recognize the sn-1 position of PI as their substrate. We propose that the sn-1 fatty acid of PI is determined by ipla-1 and acl-8, -9, -10 and crucial for asymmetric divisions.

Ninein (Nin) is a centrosomal protein whose gene is mutated in Seckel syndrome (SCKL, MIM 210600), an inherited recessive disease that results in primordial dwarfism, cognitive deficiencies, and increased sensitivity to genotoxic stress. Nin regulates neural stem cell self-renewal, interkinetic nuclear migration, and microtubule assembly in mammals. Nin is evolutionarily conserved, yet its role in cell division and development has not been investigated in a model organism. Here we characterize the single Nin orthologue in Drosophila Drosophila Nin localizes to the periphery of the centrosome but not at centriolar structures as in mammals. However, Nin shares the property of its mammalian orthologue of promoting microtubule assembly. In neural and germline stem cells, Nin localizes asymmetrically to the younger (daughter) centrosome, yet it is not required for the asymmetric division of stem cells. In wing epithelia and muscle, Nin localizes to noncentrosomal microtubule-organizing centers. Surprisingly, loss of nin expression from a nin mutant does not significantly affect embryonic and brain development, fertility, or locomotor performance of mutant flies or their survival upon exposure to DNA-damaging agents. Although it is not essential, our data suggest that Nin plays a supportive role in centrosomal and extracentrosomal microtubule organization and asymmetric stem cell division.

Stem cell division is tightly controlled via secreted signaling factors and cell adhesion molecules provided from local niche structures. Molecular mechanisms by which each niche component regulates stem cell behaviors remain to be elucidated. Here we show that heparan sulfate (HS), a class of glycosaminoglycan chains, regulates the number and asymmetric division of germline stem cells (GSCs) in the Drosophila testis. We found that GSC number is sensitive to the levels of 6-O sulfate groups on HS. Loss of 6-O sulfation also disrupted normal positioning of centrosomes, a process required for asymmetric division of GSCs. Blocking HS sulfation specifically in the niche, termed the hub, led to increased GSC numbers and mispositioning of centrosomes. The same treatment also perturbed the enrichment of Apc2, a component of the centrosome-anchoring machinery, at the hub-GSC interface. This perturbation of the centrosome-anchoring process ultimately led to an increase in the rate of spindle misorientation and symmetric GSC division. This study shows that specific HS modifications provide a novel regulatory mechanism for stem cell asymmetric division. The results also suggest that HS-mediated niche signaling acts upstream of GSC division orientation control.

The anaphase spindle determines the position of the cytokinesis furrow, such that the contractile ring assembles in an equatorial zone between the two spindle poles. Contractile ring formation is mediated by RhoA activation at the equator by the centralspindlin complex and midzone microtubules. Astral microtubules also inhibit RhoA accumulation at the poles. In the Caenorhabditis elegans one-cell embryo, the astral microtubule-dependent pathway requires anillin, NOP-1, and LET-99. LET-99 is well characterized for generating the asymmetric cortical localization of the Gα-dependent force-generating complex that positions the spindle during asymmetric division. However, whether the role of LET-99 in cytokinesis is specific to asymmetric division and whether it acts through Gα to promote furrowing are unclear. Here we show that LET-99 contributes to furrowing in both asymmetrically and symmetrically dividing cells, independent of its function in spindle positioning and Gα regulation. LET-99 acts in a pathway parallel to anillin and is required for myosin enrichment into the contractile ring. These and other results suggest a positive feedback model in which LET-99 localizes to the presumptive cleavage furrow in response to the spindle and myosin. Once positioned there, LET-99 enhances myosin accumulation to promote furrowing in both symmetrically and asymmetrically dividing cells.

Cdc42, a conserved Rho GTPase, plays a central role in polarity establishment in yeast and animals. Cell polarity is critical for asymmetric cell division, and asymmetric cell division underlies replicative aging of budding yeast. Yet how Cdc42 and other polarity factors impact life span is largely unknown. Here we show by live-cell imaging that the active Cdc42 level is sporadically elevated in wild type during repeated cell divisions but rarely in the long-lived bud8 deletion cells. We find a novel Bud8 localization with cytokinesis remnants, which also recruit Rga1, a Cdc42 GTPase activating protein. Genetic analyses and live-cell imaging suggest that Rga1 and Bud8 oppositely impact life span likely by modulating active Cdc42 levels. An rga1 mutant, which has a shorter life span, dies at the unbudded state with a defect in polarity establishment. Remarkably, Cdc42 accumulates in old cells, and its mild overexpression accelerates aging with frequent symmetric cell divisions, despite no harmful effects on young cells. Our findings implicate that the interplay among these positive and negative polarity factors limits the life span of budding yeast.