In many organisms, like
Caenorhabditis elegans and
Drosophila melanogaster, establishment of spatial patterns and definition of cell fate are driven by the segregation of determinants in response to ...spatial cues, as early as oogenesis or fertilization. In these organisms, a family of conserved proteins, the PAR proteins, is involved in the asymmetric distribution of cytoplasmic determinants and in the control of asymmetric divisions. In the mouse embryo, it is only at the 8-cell stage during compaction that asymmetries, leading to cellular diversification and blastocyst morphogenesis, are first observed. However, it has been suggested that developmentally relevant asymmetries could be established already in the oocyte and during fertilization. This led us to study the PAR proteins during the early stages of mouse development. We observed that the homologues of the different members of the PAR/aPKC complex and PAR1 are expressed in the preimplantation mouse embryo. During the first embryonic cleavages, before compaction, PARD6b and EMK1 are observed on the spindle. The localization of these two proteins becomes asymmetric during compaction, when blastomeres flatten upon each other and polarize. PARD6b is targeted to the apical pole, whereas EMK1 is distributed along the baso-lateral domain. The targeting of EMK1 is dependent upon cell–cell interactions while the apical localization of PARD6b is independent of cell contacts. At the 16-cell stage, aPKCζ colocalizes with PARD6b and a colocalization of the three proteins (PARD6b/PARD3/aPKCζ can occur in blastocysts, only at tight junctions. This choreography suggests that proteins of the PAR family are involved in the setting up of blastomere polarity and blastocyst morphogenesis in the early mammalian embryo although the interactions between the different players differ from previously studied systems. Finally, they reinforce the idea that the first developmentally relevant asymmetries are set up during compaction.
Lethal giant larvae (Lgl) is an evolutionarily conserved tumor suppressor whose loss of function causes disrupted epithelial architecture with enhanced cell proliferation and defects in cell ...polarity. A role for Lgl in the establishment and maintenance of cell polarity via suppression of the PAR-aPKC polarity complex is established; however, the mechanism by which Lgl regulates cell proliferation is not fully understood. Here we show that depletion of Lgl1 and Lgl2 in MDCK epithelial cells results in overproliferation and overproduction of Lgl2 causes G1 arrest. We also show that Lgl associates with the VprBP-DDB1 complex independently of the PAR-aPKC complex and prevents the VprBP-DDB1 subunits from binding to Cul4A, a central component of the CRL4 VprBP ubiquitin E3 ligase complex implicated in G1- to S-phase progression. Consistently, depletion of VprBP or Cul4 rescues the overproliferation of Lgl-depleted cells. In addition, the affinity between Lgl2 and the VprBP-DDB1 complex increases at high cell density. Further, aPKC-mediated phosphorylation of Lgl2 negatively regulates the interaction between Lgl2 and VprBP-DDB1 complex. These results suggest a mechanism protecting overproliferation of epithelial cells in which Lgl plays a critical role by inhibiting formation of the CRL4 VprBP complex, resulting in G1 arrest.
The basolateral tumor suppressor protein Lgl is important for the regulation of epithelial cell polarity and tissue morphology. Recent studies have shown a physical and functional interaction of Lgl ...with another polarity-regulating protein machinery, the apical PAR-3-aPKC-PAR-6 complex, in epithelial cells. However, the mechanism of Lgl-mediated regulation of epithelial cell polarity remains obscure. By an siRNA method, we here show that endogenous Lgl is required for the disassembly of apical membrane domains in depolarizing MDCK cells induced by Ca²⁺ depletion. Importantly, this Lgl function is mediated by the suppression of the apical PAR-3-aPKC-PAR-6 complex activity. Analysis using 2D- or 3D-cultured cells in collagen gel suggests the importance of this suppressive regulation of Lgl on the collagen-mediated re-establishment of apical membrane domains and lumen formation. These results indicate that basolateral Lgl plays a crucial role in the disassembly of apical membrane domains to induce the orientation of apical membrane polarity, which is mediated by the suppression of apical PAR-3-aPKC-PAR-6 complex activity.
The slit diaphragm (SD), the specialized intercellular junction between renal glomerular epithelial cells (podocytes), provides a selective-filtration barrier in renal glomeruli. Dysfunction of the ...SD results in glomerular diseases that are characterized by disappearance of SD components, such as nephrin, from the cell surface. Although the importance of endocytosis and degradation of SD components for the maintenance of SD integrity has been suggested, the dynamic nature of the turnover of intact cell-surface SD components remained unclear. Using isolated rat glomeruli we show that the turnover rates of cell-surface SD components are relatively high; they almost completely disappear from the cell surface within minutes. The exocytosis, but not endocytosis, of heterologously expressed nephrin requires the kinase activity of the cell polarity regulator atypical protein kinase C (aPKC). Consistently, we demonstrate that podocyte-specific deletion of aPKCλ resulted in a decrease of cell-surface localization of SD components, causing massive proteinuria. In conclusion, the regulation of SD turnover by aPKC is crucial for the maintenance of SD integrity and defects in aPKC signalling can lead to proteinuria. These findings not only reveal the pivotal importance of the dynamic turnover of cell-surface SD components but also suggest a novel pathophysiological basis in glomerular disease.
•Transgenic rats expressing the oxytocin-mRFP1 fusion gene were generated.•The mRFP1 fluorescence was increased in the hypothalamus of aged transgenic rats.•The increased urocortin-LI cells ...co-existed in mRFP1 cells of aged transgenic rats.
To visualize oxytocin in the hypothalamo-neurohypophysial system, we generated a transgenic rat that expresses the oxytocin-monomeric red fluorescent protein 1 (mRFP1) fusion gene. In the present study, we examined the age-related changes of oxytocin-mRFP1 fluorescent intensity in the posterior pituitary (PP), the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) of transgenic rats. The mRFP1 fluorescent intensities were significantly increased in the PP, the SON and the PVN of 12-, 18- and 24-month-old transgenic rats in comparison with 3-month-old transgenic rats. Immunohistochemical staining for urocortin, which belongs to the family of corticotropin-releasing factor family, revealed that the numbers of urocortin-like immunoreactive (LI) cells in the SON and the PVN were significantly increased in 12-, 18- and 24-month-old transgenic rats in comparison with 3-month-old transgenic rats. Almost all of urocortin-LI cells co-exist mRFP1-expressing cells in the SON and the PVN of aged transgenic rats. These results suggest that oxytocin content of the hypothalamo-neurohypophysial system may be modulated by age-related regulation. The physiological role of the co-existence of oxytocin and urocortin in the SON and PVN of aged rats remains unclear.
Eukaryotic mRNAs containing premature termination codons (PTCs) are degraded by a process known as nonsense-mediated mRNA decay (NMD). NMD has been suggested to require the recognition of PTC by an ...mRNA surveillance complex containing UPF1/SMG-2. In multicellular organisms, UPF1/SMG-2 is a phosphoprotein, and its phosphorylation contributes to NMD. Here we show that phosphorylated hUPF1, the human ortholog of UPF1/SMG-2, forms a complex with human orthologs of the
C. elegans NMD proteins SMG-5 and SMG-7. The complex also associates with protein phosphatase 2A (PP2A), resulting in dephosphorylation of hUPF1. Overexpression of hSMG-5 mutants that retain interaction with P-hUPF1 but which cannot induce its dephosphorylation impair NMD, suggesting that NMD requires P-hUPF1 dephosphorylation. We also show that P-hUPF1 forms distinct complexes containing different isoforms of hUPF3A. We propose that sequential phosphorylation and dephosphorylation of hUPF1 by hSMG-1 and PP2A, respectively, contribute to the remodeling of the mRNA surveillance complex.
Atypical protein kinase C (aPKC) generally plays crucial roles in the establishment of cell polarity in various biological contexts. In mammalian epithelial cells, aPKC essentially works towards the ...transition of primordial spot-like adherens junctions (AJs) into continuous belt-like AJs, also called zonula adherens, lined with perijunctional actin belts. To reveal the mechanism underlying this aPKC function, we investigated the functional relationship between aPKC and myosin II, the essential role of which in epithelial-junction development was recently demonstrated. Despite its deleterious effects on junction formation, overexpression of a dominant-negative mutant of aPKC (aPKCλ kn) did not interfere with the initial phase of myosin-II activation triggered by the formation of Ca²⁺-switch-induced cell-cell contacts. Furthermore, cells overexpressing aPKCλ kn exhibited myosin-II-dependent asymmetric organization of F-actin along the apicobasal axis, suggesting that aPKC contributes to junction development without affecting the centripetal contraction of the circumferential actomyosin cables. Time-lapse analyses using GFP-actin directly revealed that the circumferential actomyosin cables were centrifugally expanded and developed into perijunctional actin belts during epithelial polarization, and that aPKCλ kn specifically compromised this process. Taken together, we conclude that aPKC is required for antagonizing the myosin-II-driven centripetal contraction of the circumferential actin cables, thereby efficiently coupling the myosin-II activity with junction development and cell polarization. The present results provide novel insights into not only the site of action of aPKC kinase activity but also the role of actomyosin contraction in epithelial polarization.
We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for ...subsequent asymmetric cleavage by interacting with PAR-3. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells, this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that the over-expression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or non-ionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na+, K+-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3-PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.
Cell polarity plays a critical role in neuronal differentiation during development of the central nervous system (CNS). Recent studies have established the significance of atypical protein kinase C ...(aPKC) and its interacting partners, which include PAR-3, PAR-6 and Lgl, in regulating cell polarization during neuronal differentiation. However, their roles in neuronal maintenance after CNS development remain unclear. Here we performed conditional deletion of aPKCλ, a major aPKC isoform in the brain, in differentiated neurons of mice by camk2a-cre or synapsinI-cre mediated gene targeting. We found significant reduction of aPKCλ and total aPKCs in the adult mouse brains. The aPKCλ deletion also reduced PAR-6β, possibly by its destabilization, whereas expression of other related proteins such as PAR-3 and Lgl-1 was unaffected. Biochemical analyses suggested that a significant fraction of aPKCλ formed a protein complex with PAR-6β and Lgl-1 in the brain lysates, which was disrupted by the aPKCλ deletion. Notably, the aPKCλ deletion mice did not show apparent cell loss/degeneration in the brain. In addition, neuronal orientation/distribution seemed to be unaffected. Thus, despite the polarity complex disruption, neuronal deletion of aPKCλ does not induce obvious cell loss or disorientation in mouse brains after cell differentiation.
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