Cell Polarity Axis Research Articles

Phosphatidylinositol transfer protein/planar cell polarity axis regulates neocortical morphogenesis by supporting interkinetic nuclear migration.

SUMMARYThe neocortex expands explosively during embryonic development. The earliest populations of neural stem cells (NSCs) form a thin pseudostratified epithelium whose contour determines that of the adult neocortex. Neocortical complexity is accompanied by disproportional expansion of the NSC layer in its tangential dimension to increase tissue surface area. How such disproportional expansion is controlled remains unknown. We demonstrate that a phosphatidylinositol transfer protein (PITP)/non-canonical Wnt planar cell polarity (ncPCP) signaling axis promotes tangential expansion of developing neocortex. PITP signaling supports trafficking of specific ncPCP receptors from the NSC Golgi system to potentiate actomyosin activity important for cell-cycle-dependent interkinetic nuclear migration (IKNM). In turn, IKNM promotes lateral dispersion of newborn NSCs and tangential growth of the cerebral wall. These findings clarify functional roles for IKNM in NSC biology and identify tissue dysmorphogenesis resulting from impaired IKNM as a factor in autism risk, developmental brain disabilities, and neural tube birth defects.

Determining protein polarization proteome-wide using physical dissection of individual Stentor coeruleus cells

Cellular components are non-randomly arranged with respect to the shape and polarity of the whole cell.1-4 Patterning within cells can extend down to the level of individual proteins and mRNA.5,6 But how much of the proteome is actually localized with respect to cell polarity axes? Proteomics combined with cellular fractionation7-11 has shown that most proteins localize to one or more organelles but does not tell us how many proteins have a polarized localization with respect to the large-scale polarity axes of the intact cell. Genome-wide localization studies in yeast12-15 found that only a few percent of proteins have a localized position relative to the cell polarity axis defined by sites of polarized cell growth. Here, we describe an approach for analyzing protein distribution within a cell with a visibly obvious global patterning-the giant ciliate Stentor coeruleus.16,17 Ciliates, including Stentor, have highly polarized cell shapes with visible surface patterning.1,18 A Stentor cell is roughly 2mm long, allowing a "proteomic dissection" in which microsurgery is used to separate cellular fragments along the anterior-posterior axis, followed by comparative proteomic analysis. In our analysis, 25% of the proteome, including signaling proteins, centrin/SFI proteins, and GAS2 orthologs, shows a polarized location along the cell's anterior-posterior axis. We conclude that a large proportion of all proteins are polarized with respect to global cell polarity axes and that proteomic dissection provides a simple and effective approach for spatial proteomics.

Protein phosphatase 1 in association with Bud14 inhibits mitotic exit in Saccharomyces cerevisiae.

Mitotic exit in budding yeast is dependent on correct orientation of the mitotic spindle along the cell polarity axis. When accurate positioning of the spindle fails, a surveillance mechanism named the spindle position checkpoint (SPOC) prevents cells from exiting mitosis. Mutants with a defective SPOC become multinucleated and lose their genomic integrity. Yet, a comprehensive understanding of the SPOC mechanism is missing. In this study, we identified the type 1 protein phosphatase, Glc7, in association with its regulatory protein Bud14 as a novel checkpoint component. We further showed that Glc7-Bud14 promotes dephosphorylation of the SPOC effector protein Bfa1. Our results suggest a model in which two mechanisms act in parallel for a robust checkpoint response: first, the SPOC kinase Kin4 isolates Bfa1 away from the inhibitory kinase Cdc5, and second, Glc7-Bud14 dephosphorylates Bfa1 to fully activate the checkpoint effector.

Auxin-dependent control of cytoskeleton and cell shape regulates division orientation in the Arabidopsis embryo

SummaryPremitotic control of cell division orientation is critical for plant development, as cell walls prevent extensive cell remodeling or migration. While many divisions are proliferative and add cells to existing tissues, some divisions are formative and generate new tissue layers or growth axes. Such formative divisions are often asymmetric in nature, producing daughters with different fates. We have previously shown that, in the Arabidopsis thaliana embryo, developmental asymmetry is correlated with geometric asymmetry, creating daughter cells of unequal volume. Such divisions are generated by division planes that deviate from a default “minimal surface area” rule. Inhibition of auxin response leads to reversal to this default, yet the mechanisms underlying division plane choice in the embryo have been unclear. Here, we show that auxin-dependent division plane control involves alterations in cell geometry, but not in cell polarity axis or nuclear position. Through transcriptome profiling, we find that auxin regulates genes controlling cell wall and cytoskeleton properties. We confirm the involvement of microtubule (MT)-binding proteins in embryo division control. Organization of both MT and actin cytoskeleton depends on auxin response, and genetically controlled MT or actin depolymerization in embryos leads to disruption of asymmetric divisions, including reversion to the default. Our work shows how auxin-dependent control of MT and actin cytoskeleton properties interacts with cell geometry to generate asymmetric divisions during the earliest steps in plant development.

Multicellular modeling of ciliopathy by combining iPS cells and microfluidic airway-on-a-chip technology.

Mucociliary clearance is an essential lung function that facilitates the removal of inhaled pathogens and foreign matter unidirectionally from the airway tract and is innately achieved by coordinated ciliary beating of multiciliated cells. Should ciliary function become disturbed, mucus can accumulate in the airway causing subsequent obstruction and potentially recurrent pneumonia. However, it has been difficult to recapitulate unidirectional mucociliary flow using human-derived induced pluripotent stem cells (iPSCs) in vitro and the mechanism governing the flow has not yet been elucidated, hampering the proper humanized airway disease modeling. Here, we combine human iPSCs and airway-on-a-chip technology, to demonstrate the effectiveness of fluid shear stress (FSS) for regulating the global axis of multicellular planar cell polarity (PCP), as well as inducing ciliogenesis, thereby contributing to quantifiable unidirectional mucociliary flow. Furthermore, we applied the findings to disease modeling of primary ciliary dyskinesia (PCD), a genetic disease characterized by impaired mucociliary clearance. The application of an airway cell sheet derived from patient-derived iPSCs and their gene-edited counterparts, as well as genetic knockout iPSCs of PCD causative genes, made it possible to recapitulate the abnormal ciliary functions in organized PCP using the airway-on-a-chip. These findings suggest that the disease model of PCD developed here is a potential platform for making diagnoses and identifying therapeutic targets and that airway reconstruction therapy using mechanical stress to regulate PCP might have therapeutic value.

Regulation of Cdc42 for polarized growth in budding yeast.

The Rho GTPase Cdc42 is a central regulator of cell polarity in diverse cell types. The activity of Cdc42 is dynamically controlled in time and space to enable distinct polarization events, which generally occur along a single axis in response to spatial cues. Our understanding of the mechanisms underlying Cdc42 polarization has benefited largely from studies of the budding yeast Saccharomyces cerevisiae, a genetically tractable model organism. In budding yeast, Cdc42 activation occurs in two temporal steps in the G1 phase of the cell cycle to establish a proper growth site. Here, we review findings in budding yeast that reveal an intricate crosstalk among polarity proteins for biphasic Cdc42 regulation. The first step of Cdc42 activation may determine the axis of cell polarity, while the second step ensures robust Cdc42 polarization for growth. Biphasic Cdc42 polarization is likely to ensure the proper timing of events including the assembly and recognition of spatial landmarks and stepwise assembly of a new ring of septins, cytoskeletal GTP-binding proteins, at the incipient bud site. Biphasic activation of GTPases has also been observed in mammalian cells, suggesting that biphasic activation could be a general mechanism for signal-responsive cell polarization. Cdc42 activity is necessary for polarity establishment during normal cell division and development, but its activity has also been implicated in the promotion of aging. We also discuss negative polarity signaling and emerging concepts of Cdc42 signaling in cellular aging.

In Situ Hybridization: Fruit Fly Embryos and Tissues

AbstractIt is well known that transcript localization controls important biological processes, including cell fate determination, cell polarity, cell migration, morphogenesis, neuronal function, and embryonic axis specification. Thus, the sub‐cellular visualization of transcripts in ‘their original place’ (in situ) is an important tool to infer and understand their trafficking, stability, translation, and biological functions. This has been made possible through the use of labeled ‘anti‐sense’ probes that can be readily detected after hybridization to their ‘sense’ counterparts. The following is a series of protocols for conducting in situ hybridization in Drosophila (fruit fly) embryos or tissues. Probe‐detection methods include a relatively simple alkaline phosphatase reaction, as well as higher‐resolution and higher‐throughput versions using fluorescence‐conjugated tyramide labeling. New modifications that enhance probe penetration and detection in various tissues are also provided. © 2017 by John Wiley & Sons, Inc.

Plant cell development: Forcing cell polarity.

Mechanical tension is shown to contribute to the establishment of the cell polarity axis in plant epidermal stem cells, which is important for regulating asymmetric cell division.

A tectorin-based matrix and planar cell polarity genes are required for normal collagen-fibril orientation in the developing tectorial membrane.

The tectorial membrane is an extracellular structure of the cochlea. It develops on the surface of the auditory epithelium and contains collagen fibrils embedded in a tectorin-based matrix. The collagen fibrils are oriented radially with an apically directed slant - a feature considered crucial for hearing. To determine how this pattern is generated, collagen-fibril formation was examined in mice lacking a tectorin-based matrix, epithelial cilia or the planar cell polarity genes Vangl2 and Ptk7 In wild-type mice, collagen-fibril bundles appear within a tectorin-based matrix at E15.5 and, as fibril number rapidly increases, become co-aligned and correctly oriented. Epithelial width measurements and data from Kif3acKO mice suggest, respectively, that radial stretch and cilia play little, if any, role in determining normal collagen-fibril orientation; however, evidence from tectorin-knockout mice indicates that confinement is important. PRICKLE2 distribution reveals the planar cell polarity axis in the underlying epithelium is organised along the length of the cochlea and, in mice in which this polarity is disrupted, the apically directed collagen offset is no longer observed. These results highlight the importance of the tectorin-based matrix and epithelial signals for precise collagen organisation in the tectorial membrane.

The polarity of myxobacterial gliding is regulated by direct interactions between the gliding motors and the Ras homolog MglA

Gliding motility in Myxococcus xanthus is powered by flagella stator homologs that move in helical trajectories using proton motive force. The Frz chemosensory pathway regulates the cell polarity axis through MglA, a Ras family GTPase; however, little is known about how MglA establishes the polarity of gliding, because the gliding motors move simultaneously in opposite directions. Here we examined the localization and dynamics of MglA and gliding motors in high spatial and time resolution. We determined that MglA localizes not only at the cell poles, but also along the cell bodies, forming a decreasing concentration gradient toward the lagging cell pole. MglA directly interacts with the motor protein AglR, and the spatial distribution of AglR reversals is positively correlated with the MglA gradient. Thus, the motors moving toward lagging cell poles are less likely to reverse, generating stronger forward propulsion. MglB, the GTPase-activating protein of MglA, regulates motor reversal by maintaining the MglA gradient. Our results suggest a mechanism whereby bacteria use Ras family proteins to modulate cellular polarity.

The 14-3-3 protein Bmh1 functions in the spindle position checkpoint by breaking Bfa1 asymmetry at yeast centrosomes.

In addition to their well-known role in microtubule organization, centrosomes function as signaling platforms and regulate cell cycle events. An important example of such a function is the spindle position checkpoint (SPOC) of budding yeast. SPOC is a surveillance mechanism that ensures alignment of the mitotic spindle along the cell polarity axis. Upon spindle misalignment, phosphorylation of the SPOC component Bfa1 by Kin4 kinase engages the SPOC by changing the centrosome localization of Bfa1 from asymmetric (one centrosome) to symmetric (both centrosomes). Here we show that, unexpectedly, Kin4 alone is unable to break Bfa1 asymmetry at yeast centrosomes. Instead, phosphorylation of Bfa1 by Kin4 creates a docking site on Bfa1 for the 14-3-3 family protein Bmh1, which in turn weakens Bfa1-centrosome association and promotes symmetric Bfa1 localization. Consistently, BMH1-null cells are SPOC deficient. Our work thus identifies Bmh1 as a new SPOC component and refines the molecular mechanism that breaks Bfa1 centrosome asymmetry upon SPOC activation.

A function for the midbody remnant in embryonic patterning

Asymmetric cell divisions combine cell division with fate specification and one general model of how this is achieved was proposed already decades ago1,2: During interphase, the cell polarity axis is specified, followed by orientation of the spindle along the polarity axis and segregation of fate determinants along the polarity axis during mitosis. In most cells, the polarity axis and the spindle will usually align with the long axis that the cell had before division, also called Hertwig’s rule3–6. In the C. elegans embryo, the first polarity axis also forms along the long axis of the embryo by enrichment of myosin in the anterior7 and formation of mutually exclusive anterior and posterior cortical polarity domains, mediated through directional cortical contractile flow8–10. The directionality of this flow is determined by an extrinsic cue, the entry of the sperm, which inhibits Rho-dependent myosin activation at the future posterior pole by bringing with it the Rho GTPase activating protein CYK-411,12. Moreover, since there is no previous division ‘history’ before the first cleavage, mechanisms have to ensure that the nucleus-centrosome complex undergoes a 90 degree rotation so that the spindle can subsequently elongate along the long axis13–15. Additional mechanisms ensure that the site of cleavage is perpendicular to the long axis16,17. Hence, tight coupling of an extrinsic cue to intrinsic polarity formation and spindle elongation enables alignment of the division orientation with the long axis of the organism and successful segregation of fate determinants.

Epithelial polarity and spindle orientation: intersecting pathways

During asymmetric stem cell divisions, the mitotic spindle must be correctly oriented and positioned with respect to the axis of cell polarity to ensure that cell fate determinants are appropriately segregated into only one daughter cell. By contrast, epithelial cells divide symmetrically and orient their mitotic spindles perpendicular to the main apical–basal polarity axis, so that both daughter cells remain within the epithelium. Work in the past 20 years has defined a core ternary complex consisting of Pins, Mud and Gαi that participates in spindle orientation in both asymmetric and symmetric divisions. As additional factors that interact with this complex continue to be identified, a theme has emerged: there is substantial overlap between the mechanisms that orient the spindle and those that establish and maintain apical–basal polarity in epithelial cells. In this review, we examine several factors implicated in both processes, namely Canoe, Bazooka, aPKC and Discs large, and consider the implications of this work on how the spindle is oriented during epithelial cell divisions.

Establishment of a robust single axis of cell polarity by coupling multiple positive feedback loops

Establishment of cell polarity—or symmetry breaking—relies on local accumulation of polarity regulators. Although simple positive feedback is sufficient to drive symmetry breaking, it is highly sensitive to stochastic fluctuations typical for living cells. Here, by integrating mathematical modelling with quantitative experimental validations, we show that in the yeast Saccharomyces cerevisiae a combination of actin- and guanine nucleotide dissociation inhibitor-dependent recycling of the central polarity regulator Cdc42 is needed to establish robust cell polarity at a single site during yeast budding. The guanine nucleotide dissociation inhibitor pathway consistently generates a single-polarization site, but requires Cdc42 to cycle rapidly between its active and inactive form, and is therefore sensitive to perturbations of the GTPase cycle. Conversely, actin-mediated recycling of Cdc42 induces robust symmetry breaking but cannot restrict polarization to a single site. Our results demonstrate how cells optimize symmetry breaking through coupling between multiple feedback loops.

Phosphorylation‐dependent localization of the response regulator FrzZ signals cell reversals in Myxococcus xanthus

The life cycle of Myxococcus xanthus includes co-ordinated group movement and fruiting body formation, and requires directed motility and controlled cell reversals. Reversals are achieved by inverting cell polarity and re-organizing many motility proteins. The Frz chemosensory pathway regulates the frequency of cell reversals. While it has been established that phosphotransfer from the kinase FrzE to the response regulator FrzZ is required, it is unknown how phosphorylated FrzZ, the putative output of the pathway, targets the cell polarity axis. In this study, we used Phos-tag SDS-PAGE to determine the cellular level of phospho-FrzZ under different growth conditions and in Frz signalling mutants. We detected consistent FrzZ phosphorylation, albeit with a short half-life, in cells grown on plates, but not from liquid culture. The available pool of phospho-FrzZ correlated with reversal frequencies, with higher levels found in hyper-reversing mutants. Phosphorylation was not detected in hypo-reversing mutants. Fluorescence microscopy revealed that FrzZ is recruited to the leading cell pole upon phosphorylation and switches to the opposite pole during reversals. These results are consistent with the hypothesis that the Frz pathway modulates reversal frequency through a localized response regulator that targets cell polarity regulators at the leading cell pole.

Anthrax receptors position the spindle

Spindle orientation plays a pivotal role in tissue morphogenesis. An asymmetric anthrax receptor cap is revealed to promote activation of a formin to orient the spindle along the planar cell polarity (PCP) axis in zebrafish dorsal epiblast cells.

Inscuteable Regulates the Pins-Mud Spindle Orientation Pathway

During asymmetric cell division, alignment of the mitotic spindle with the cell polarity axis ensures that the cleavage furrow separates fate determinants into distinct daughter cells. The protein Inscuteable (Insc) is thought to link cell polarity and spindle positioning in diverse systems by binding the polarity protein Bazooka (Baz; aka Par-3) and the spindle orienting protein Partner of Inscuteable (Pins; mPins or LGN in mammals). Here we investigate the mechanism of spindle orientation by the Insc-Pins complex. Previously, we defined two Pins spindle orientation pathways: a complex with Mushroom body defect (Mud; NuMA in mammals) is required for full activity, whereas binding to Discs large (Dlg) is sufficient for partial activity. In the current study, we have examined the role of Inscuteable in mediating downstream Pins-mediated spindle orientation pathways. We find that the Insc-Pins complex requires Gαi for partial activity and that the complex specifically recruits Dlg but not Mud. In vitro competition experiments revealed that Insc and Mud compete for binding to the Pins TPR motifs, while Dlg can form a ternary complex with Insc-Pins. Our results suggest that Insc does not passively couple polarity and spindle orientation but preferentially inhibits the Mud pathway, while allowing the Dlg pathway to remain active. Insc-regulated complex assembly may ensure that the spindle is attached to the cortex (via Dlg) before activation of spindle pulling forces by Dynein/Dynactin (via Mud).

The Rap1–Rgl–Ral signaling network regulates neuroblast cortical polarity and spindle orientation

A crucial first step in asymmetric cell division is to establish an axis of cell polarity along which the mitotic spindle aligns. Drosophila melanogaster neural stem cells, called neuroblasts (NBs), divide asymmetrically through intrinsic polarity cues, which regulate spindle orientation and cortical polarity. In this paper, we show that the Ras-like small guanosine triphosphatase Rap1 signals through the Ral guanine nucleotide exchange factor Rgl and the PDZ protein Canoe (Cno; AF-6/Afadin in vertebrates) to modulate the NB division axis and its apicobasal cortical polarity. Rap1 is slightly enriched at the apical pole of metaphase/anaphase NBs and was found in a complex with atypical protein kinase C and Par6 in vivo. Loss of function and gain of function of Rap1, Rgl, and Ral proteins disrupt the mitotic axis orientation, the localization of Cno and Mushroom body defect, and the localization of cell fate determinants. We propose that the Rap1-Rgl-Ral signaling network is a novel mechanism that cooperates with other intrinsic polarity cues to modulate asymmetric NB division.

Motility and Segregation of Hsp104-Associated Protein Aggregates in Budding Yeast

During yeast cell division, aggregates of damaged proteins are segregated asymmetrically between the bud and the mother. It is thought that protein aggregates are cleared from the bud via actin cable-based retrograde transport toward the mother and that Bni1p formin regulates this transport. Here, we examined the dynamics of Hsp104-associated protein aggregates by video microscopy, particle tracking, and image correlation analysis. We show that protein aggregates undergo random walk without directional bias. Clearance of heat-induced aggregates from the bud does not depend on formin proteins but occurs mostly through dissolution via Hsp104p chaperon. Aggregates formed naturally in aged cells also exhibit random walk but do not dissolve during observation. Although our data do not disagree with a role for actin or cell polarity in aggregate segregation, modeling suggests that their asymmetric inheritance can be a predictable outcome of aggregates' slow diffusion and the geometry of yeast cells.

LGN/mInsc and LGN/NuMA Complex Structures Suggest Distinct Functions in Asymmetric Cell Division for the Par3/mInsc/LGN and Gαi/LGN/NuMA Pathways

Asymmetric cell division requires the establishment of cortical cell polarity and the orientation of the mitotic spindle along the axis of cell polarity. Evidence from invertebrates demonstrates that the Par3/Par6/aPKC and NuMA/LGN/Gαi complexes, which are thought to be physically linked by the adaptor protein mInscuteable (mInsc), play indispensable roles in this process. However, the molecular basis for the binding of LGN to NuMA and mInsc is poorly understood. The high-resolution structures of the LGN/NuMA and LGN/mInsc complexes presented here provide mechanistic insights into the distinct and highly specific interactions of the LGN TPRs with mInsc and NuMA. Structural comparisons, together with biochemical and cell biology studies, demonstrate that the interactions of NuMA and mInsc with LGN are mutually exclusive, with mInsc binding preferentially. Our results suggest that the Par3/mInsc/LGN and NuMA/LGN/Gαi complexes play sequential and partially overlapping roles in asymmetric cell division.