A role for cell polarity proteins in mitotic exit

The budding yeast mitotic exit network (MEN) is a signal transduction cascade that controls exit from mitosis by facilitating the release of the cell cycle phosphatase Cdc14 from the nucleolus. The G protein Tem1 regulates MEN activity. The Tem1 guanine nucleotide exchange factor (GEF) Lte1 associates with the cortex of the bud and activates the MEN upon the formation of an anaphase spindle. Thus, the cell cortex has an important but ill-defined role in MEN regulation. Here, we describe a network of conserved cortical cell polarity proteins that have key roles in mitotic exit. The Rho-like GTPase Cdc42, its GEF Cdc24 and its effector Cla4 [a member of the p21-activated kinases (PAKs)] control the initial binding and activation of Lte1 to the bud cortex. Moreover, Cdc24, Cdc42 and Ste20, another PAK, probably function parallel to Lte1 in facilitating mitotic exit. Finally, the cell polarity proteins Kel1 and Kel2 are present in complexes with both Lte1 and Tem1, and negatively regulate mitotic exit.     

A role for Wnts in morpho-genesis and tissue polarity

Recent studies have shown that secreted Wnt proteins control morphogenetic movements in fish and frog embryos. The analysis of Dishevelled, a cytoplasmic mediator of Wnt signalling, reveals unexpected similarity between gastrulation in vertebrates and polarization of cells in Drosophila epithelia.     

Embryonic polarity: a role for microtubules

Researchers have suspected that initial polarization of the Caenorhabditis elegans embryo might be directed by microtubules, but demonstrating this has faced obstacles. A new study has cleverly bypassed these obstacles.     

A specific role for Arabidopsis TRAPPII in post-Golgi trafficking that is crucial for cytokinesis and cell polarity

Cytokinesis and cell polarity are supported by membrane trafficking from the trans-Golgi network (TGN), but the molecular mechanisms that promote membrane trafficking from the TGN are poorly defined in plant cells. Here we show that TRAPPII in Arabidopsis regulates the post-Golgi trafficking that is crucial for assembly of the cell plate and cell polarity. Disruptions of AtTRS120 or AtTRS130, two genes encoding two key subunits of TRAPPII, result in defective cytokinesis and cell polarity in embryogenesis and seedling development. In attrs120 and attrs130, the organization and trafficking in the endoplasmic reticulum (ER)-Golgi interface are normal. However, post-Golgi trafficking to the cell plate and to the cell wall, but not to the vacuole, is impaired. Furthermore, TRAPPII is required for the selective transport of PIN2, but not PIN1, to the plasma membrane. We revealed that AtTRS130 is co-localized with RAB-A1c. Expression of constitutively active RAB-A1c partially rescues attrs130. RAB-A1c, which resides at the TGN, is delocalized to the cytosol in attrs130. We propose that TRAPPII in Arabidopsis acts upstream of Rab-A GTPases in post-Golgi membrane trafficking in plant cells.     

Mechanisms and physiological role of polarity in plants

The concept of polarity was the starting point for the attempts of many investigators to understand the principles of differentiation, because the polar organization underlies specific three-dimensional structure of the organism and provides for the integrity and coordination of its functions. The polarity axes are established at the stage of zygote, extending to the developing embryo, and they ??vectorize?? subsequent plant growth and development. Polarization of cells and tissues is crucial for plant morphogenesis, because the emerging morphogenetic gradients provide the basis for differential genome activity at various stages of plant development. This review deals with the polarity phenomena and the mechanisms of symmetry axis formation at the level of cells and plant tissues. The roles of electrical gradients, Ca²⁺ ions, auxin, cytoskeleton, ROP-proteins, phosphoinositides, and microRNA in polarization of cells and tissues are considered.     

Intermediate filaments: a role in epithelial polarity

Intermediate filaments have long been considered mechanical components of the cell that provide resistance to deformation stress. Practical experimental problems, including insolubility, lack of good pharmacological antagonists, and the paucity of powerful genetic models have handicapped the research of other functions. In single-layered epithelial cells, keratin intermediate filaments are cortical, either apically polarized or apico-lateral. This review analyzes phenotypes of genetic manipulations of simple epithelial cell keratins in mice and Caenorhabditis elegans that strongly suggest a role of keratins in apico-basal polarization and membrane traffic. Published evidence that intermediate filaments can act as scaffolds for proteins involved in membrane traffic and signaling is also discussed. Such a scaffolding function would generate a highly polarized compartment within the cytoplasm of simple epithelial cells. While in most cases mechanistic explanations for the keratin-null or overexpression phenotypes are still missing, it is hoped that investigators will be encouraged to study these as yet poorly understood functions of intermediate filaments.     

Maintenance of epithelial surface membrane lipid polarity: A role for differing phospholipid translocation rates

Summary Large differences in lipid composition of apical and basolateral membranes from epithelial cells exist. To determine the responsible mechanism(s), rat renal cortical brush border and basolateral membrane phospholipids were labeled using³²P and either [³H]-glycerol or [2-³H] acetate for incorporation and degradation studies, respectively. Brush border and basolateral membrane fractions were isolated simultaneously from the same cortical homogenate. Different phospholipid classes were degraded at variable rates with phosphatidylcholine having the fastest decay rate. Decay rates for individual phospholipid classes were, however, similar in both brush border and basolateral membrane fractions. In phospholipid incorporation studies again, large variations existed between individual phospholipid classes with phosphatidylcholine and phosphatidylinositol showing the most rapid rates of incorporation. Sphingomyelin and phosphatidylserine showed extremely slow incorporation rates and did not enter into the isotopic decay phase for 48 hr. In contrast to degradation studies, however, the same phospholipid class labeled the two surface membrane domains at highly variable rates. The difference in these rates, with the exception of phosphatidylinositol, were identical to the differences in phospholipid compositions between the two membranes. For example, phosphatidylcholine was incorporated into the basolateral membrane 2.5 × faster than into the brush border membrane and its relative composition was 2.5 × greater in the basolateral membrane. The opposite was true for sphingomyelin. These results indicate incorporation and not degradation rates of individual phospholipids play a major role in regulating the differing phospholipid composition of brush border and basolateral membranes.     

Ezrin/moesin in motile Walker 256 carcinosarcoma cells: signal-dependent relocalization and role in migration

Rat Walker 256 carcinosarcoma cells spontaneously develop front-tail polarity and migrate in the absence of added stimuli. Constitutive activation of phosphatidylinositol-3 kinase (PI 3-kinase), Rac, Rho and Rho kinase are essential for these processes. Ezrin and moesin are putative targets of these signaling pathways leading to spontaneous migration. To test this hypothesis, we used specific siRNA probes that resulted in a downregulation of ezrin and moesin by about 70% and in a similar reduction in the fraction of migrating cells. Spontaneous polarization however was not affected, indicating a more subtle role of ezrin and moesin in migration. We provide furthermore evidence that endogenous ezrin and moesin colocalize with F-actin at the contracted tail of polarized cells, similar to ectopically expressed green fluorescent protein-tagged ezrin. Our results suggest that myosin light chain and ezrin are markers of front and tail, respectively, even in the absence of morphological polarization. We further show that endogenous ezrin and moesin are phosphorylated and that activities of PI-3 kinase, Rho and Rac, but not of Rho-kinase, are required for this C-terminal phosphorylation. Activation of protein kinase C in contrast suppressed phosphorylation of ezrin and moesin. Inhibition of ezrin phosphorylation prevented its membrane association.     

A role for the segment polarity gene shaggy/GSK-3 in the Drosophila circadian clock.

Tissue-specific overexpression of the glycogen synthase kinase-3 (GSK-3) ortholog shaggy (sgg) shortens the period of the Drosophila circadian locomotor activity cycle. The short period phenotype was attributed to premature nuclear translocation of the PERIOD/TIMELESS heterodimer. Reducing SGG/GSK-3 activity lengthens period, demonstrating an intrinsic role for the kinase in circadian rhythmicity. Lowered sgg activity decreased TIMELESS phosphorylation, and it was found that GSK-3 beta specifically phosphorylates TIMELESS in vitro. Overexpression of sgg in vivo converts hypophosphorylated TIMELESS to a hyperphosphorylated protein whose electrophoretic mobility, and light and phosphatase sensitivity, are indistinguishable from the rhythmically produced hyperphosphorylated TIMELESS of wild-type flies. Our results indicate a role for SGG/GSK-3 in TIMELESS phosphorylation and in the regulated nuclear translocation of the PERIOD/TIMELESS heterodimer.     

A role for gibberellic acid in orienting microtubules and regulating cell growth polarity in the maize root cortex

The role of gibberellins and cortical microtubules in determining the polarity of cell growth in the root cortex of maize (Zea mays L.) was examined. Inhibition of gibberellin biosynthesis, either naturally through mutation (d5 mutant) or by means of chemicals such as 2S,3S paclobutrazol, caused thickening of root apices and increased their starch content. Immunofluorescence microscopy of cortical microtubules, coupled with a comparison of cell widhts, lengths and shapes, indicated that the meristem and immediate post-mitotic zone were the targets of gibberellin deficiency. Cortical cells in these regions were impaired in their ability to develop highly ordered transversal arrays of cortical microtubules. Consequently, the cells became wider and shorter. Application of gibberellic acid re-established the arrangements of cortical microtubules and the polarity of cell growth characteristic for roots having normal levels of gibberellins, it also decreased the starch content. These results indicate that gibberellins are morphogenetically active substances, not only in shoots but also in roots of maize.Abbreviations CMT cortical microtubule - GA gibberellin - GA₃ gibberellic acid - MT microtubule - PIG postmitotic isodiametric growth The authors acknowledge the support to F.B. from the Royal Society (London UK). We also thank Dr. J. Lenton (University of Bristol, Long Ashton Research Station) who kindly supplied us with 2S,3S paclobutrazol and grains of the GA-deficient d5 mutant of maize.     

A role for the polarity complex and PI3 kinase in branch formation within retinotectal arbors of zebrafish

The developing zebrafish retinotectal arbors make many trial branches with synapses but most are retracted. With NMDA blockers, branches are withdrawn at a higher rate, and a synapse on a branch not only stabilizes that branch, but biases new branches to form nearby. Here, we tested whether new branch formation requires the polarity complex, which is essential for organizing the cytoskeleton in initial axon formation. The complex (PAR3, PAR6, and atypical protein kinase C [aPKC]) is downstream of phosphatidyl‐inositol‐3‐kinase (PI3K), and its aPKC could be activated by retrograde arachidonic acid synaptic signaling. DiO‐labeled arbors in zebrafish were imaged on day 3 (before treatment) and 1–2 days after treatment to suppress or inhibit PAR3, PAR6, or PI3K. Intraocular antisense (AS) oligos to PAR3 or PAR6 both severely limited branch addition, which was most evident in arbors with few branches before treatment. As a result of the inability to branch, arbor segments grew longer than in controls. Both PI3K inhibition (LY294002) and AS suppression of PI3Kα and PI3Kδ isoforms likewise limited branch addition but also decreased growth, as the sum of segment lengths was below normal after 2 days. Both the results support the idea that the polarity complex and PI3K participate in arbor branch formation. The PKC inhibitor Go6983 also severely restricted branch addition and growth, as did bisindolyl‐maleimide and calphostin C reported previously, consistent with PKCζ, but not PKCµ, participation. These experiments suggest a mechanism whereby activity signaling could affect the branching of retinotectal arbors. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 591–601, 2014     

Revisiting the role of microtubules in C. elegans polarity

Cells must break symmetry to acquire polarity. Microtubules have been implicated in the induction of asymmetry in several cell types, but their role in the Caenorhabditis elegans zygote, a classic polarity model, has remained uncertain. One study (see Tsai and Ahringer on p. 397 of this issue) brings new light to this problem by demonstrating that severe loss of microtubules impairs polarity onset in C. elegans.     

Drosophila Patj plays a supporting role in apical-basal polarity but is essential for viability

Patj has been characterized as one of the so-called polarity proteins that play essential and conserved roles in regulating cell polarity in many different cell types. Studies of Drosophila and mammalian cells suggest that Patj is required for the apical polarity protein complex Crumbs-Stardust (Pals1 or Mpp5 in mammalian cells) to establish apical-basal polarity. However, owing to the lack of suitable genetic mutants, the exact in vivo function of Patj in regulating apical-basal polarity and development remains to be elucidated. Here, we generated molecularly defined null mutants of Drosophila Patj (dPatj). Our data show conclusively that dPatj only plays supporting and non-essential roles in regulating apical-basal polarity, although such a supporting role may become crucial in cells such as photoreceptors that undergo complex cellular morphogenesis. In addition, our results confirm that dPatj possesses an as yet unidentified function that is essential for pupal development.