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1.
EMBO Rep ; 25(1): 144-167, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38177906

RESUMO

The tight junction (TJ) in epithelial cells is formed by integral membrane proteins and cytoplasmic scaffolding proteins. The former contains the claudin family proteins with four transmembrane segments, while the latter includes Par3, a PDZ domain-containing adaptor that organizes TJ formation. Here we show the single membrane-spanning protein TMEM25 localizes to TJs in epithelial cells and binds to Par3 via a PDZ-mediated interaction with its C-terminal cytoplasmic tail. TJ development during epithelial cell polarization is accelerated by depletion of TMEM25, and delayed by overexpression of TMEM25 but not by that of a C-terminally deleted protein, indicating a regulatory role of TMEM25. TMEM25 associates via its N-terminal extracellular domain with claudin-1 and claudin-2 to suppress their cis- and trans-oligomerizations, both of which participate in TJ strand formation. Furthermore, Par3 attenuates TMEM25-claudin association via binding to TMEM25, implying its ability to affect claudin oligomerization. Thus, the TJ protein TMEM25 appears to negatively regulate claudin assembly in TJ formation, which regulation is modulated by its interaction with Par3.


Assuntos
Claudinas , Junções Íntimas , Junções Íntimas/metabolismo , Claudinas/genética , Claudinas/metabolismo , Proteínas de Transporte/metabolismo , Células Epiteliais , Claudina-1/genética , Claudina-1/metabolismo
2.
Dev Biol ; 514: 37-49, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-38885804

RESUMO

The conserved bazooka (baz/par3) gene acts as a key regulator of asymmetrical cell divisions across the animal kingdom. Associated Par3/Baz-Par6-aPKC protein complexes are also well known for their role in the establishment of apical/basal cell polarity in epithelial cells. Here we define a novel, positive function of Baz/Par3 in the Notch pathway. Using Drosophila wing and eye development, we demonstrate that Baz is required for Notch signaling activity and optimal transcriptional activation of Notch target genes. Baz appears to act independently of aPKC in these contexts, as knockdown of aPKC does not cause Notch loss-of-function phenotypes. Using transgenic Notch constructs, our data positions Baz activity downstream of activating Notch cleavage steps and upstream of Su(H)/CSL transcription factor complex activity on Notch target genes. We demonstrate a biochemical interaction between NICD and Baz, suggesting that Baz is required for NICD activity before NICD binds to Su(H). Taken together, our data define a novel role of the polarity protein Baz/Par3, as a positive and direct regulator of Notch signaling through its interaction with NICD.


Assuntos
Proteínas de Drosophila , Receptores Notch , Transdução de Sinais , Asas de Animais , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Receptores Notch/metabolismo , Asas de Animais/metabolismo , Asas de Animais/embriologia , Asas de Animais/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Ligação Proteica , Drosophila melanogaster/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Olho/embriologia , Olho/metabolismo , Olho/crescimento & desenvolvimento , Drosophila/metabolismo , Drosophila/embriologia , Polaridade Celular , Peptídeos e Proteínas de Sinalização Intracelular
3.
Development ; 149(21)2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36218069

RESUMO

During brain development, neural precursor cells (NPCs) expand initially, and then switch to generating stage-specific neurons while maintaining self-renewal ability. Because the NPC pool at the onset of neurogenesis crucially affects the final number of each type of neuron, tight regulation is necessary for the transitional timing from the expansion to the neurogenic phase in these cells. However, the molecular mechanisms underlying this transition are poorly understood. Here, we report that the telencephalon-specific loss of PAR3 before the start of neurogenesis leads to increased NPC proliferation at the expense of neurogenesis, resulting in disorganized tissue architecture. These NPCs demonstrate hyperactivation of hedgehog signaling in a smoothened-dependent manner, as well as defects in primary cilia. Furthermore, loss of PAR3 enhanced ligand-independent ciliary accumulation of smoothened and an inhibitor of smoothened ameliorated the hyperproliferation of NPCs in the telencephalon. Thus, these findings support the idea that PAR3 has a crucial role in the transition of NPCs from the expansion phase to the neurogenic phase by restricting hedgehog signaling through the establishment of ciliary integrity.


Assuntos
Proteínas Hedgehog , Células-Tronco Neurais , Células-Tronco Neurais/fisiologia , Neurônios , Neurogênese , Transdução de Sinais/fisiologia
4.
Development ; 149(22)2022 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-36264257

RESUMO

Apico-basolateral polarization is essential for epithelial cells to function as selective barriers and transporters, and to provide mechanical resilience to organs. Epithelial polarity is established locally, within individual cells to establish distinct apical, junctional and basolateral domains, and globally, within a tissue where cells coordinately orient their apico-basolateral axes. Using live imaging of endogenously tagged proteins and tissue-specific protein depletion in the Caenorhabditiselegans embryonic intestine, we found that local and global polarity establishment are temporally and genetically separable. Local polarity is initiated prior to global polarity and is robust to perturbation. PAR-3 is required for global polarization across the intestine but local polarity can arise in its absence, as small groups of cells eventually established polarized domains in PAR-3-depleted intestines in a HMR-1 (E-cadherin)-dependent manner. Despite the role of PAR-3 in localizing PKC-3 to the apical surface, we additionally found that PAR-3 and PKC-3/aPKC have distinct roles in the establishment and maintenance of local and global polarity. Taken together, our results indicate that different mechanisms are required for local and global polarity establishment in vivo.


Assuntos
Polaridade Celular , Células Epiteliais , Células Epiteliais/metabolismo , Junções Intercelulares , Mucosa Intestinal , Intestinos , Epitélio
5.
Dev Biol ; 499: 59-74, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37172642

RESUMO

The molecular links between tissue-level morphogenesis and the differentiation of cell lineages in the pancreas remain elusive despite a decade of studies. We previously showed that in pancreas both processes depend on proper lumenogenesis. The Rab GTPase Rab11 is essential for epithelial lumen formation in vitro, however few studies have addressed its functions in vivo and none have tested its requirement in pancreas. Here, we show that Rab11 is critical for proper pancreas development. Co-deletion of the Rab11 isoforms Rab11A and Rab11B in the developing pancreatic epithelium (Rab11pancDKO) results in ∼50% neonatal lethality and surviving adult Rab11pancDKO mice exhibit defective endocrine function. Loss of both Rab11A and Rab11B in the embryonic pancreas results in morphogenetic defects of the epithelium, including defective lumen formation and lumen interconnection. In contrast to wildtype cells, Rab11pancDKO cells initiate the formation of multiple ectopic lumens, resulting in a failure to coordinate a single apical membrane initiation site (AMIS) between groups of cells. This results in an inability to form ducts with continuous lumens. Here, we show that these defects are due to failures in vesicle trafficking, as apical and junctional components remain trapped within Rab11pancDKO cells. Together, these observations suggest that Rab11 directly regulates epithelial lumen formation and morphogenesis. Our report links intracellular trafficking to organ morphogenesis in vivo and presents a novel framework for decoding pancreatic development.


Assuntos
Pâncreas , Proteínas rab de Ligação ao GTP , Camundongos , Animais , Epitélio/metabolismo , Membrana Celular/metabolismo , Isoformas de Proteínas/metabolismo , Pâncreas/metabolismo , Morfogênese , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
6.
J Biol Chem ; 299(3): 102947, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36707051

RESUMO

Animal cells establish polarity via the partitioning-defective protein system. Although the core of this system comprises only four proteins, a huge number of reported interactions between these members has made it difficult to understand how the system is organized and functions at the molecular level. In a recent JBC article, the Prehoda group has succeeded in reconstituting some of these interactions in vitro, resulting in a much clearer and simpler picture of partitioning-defective complex assembly.


Assuntos
Polaridade Celular , Proteínas , Animais , Proteínas/metabolismo , Proteínas de Ciclo Celular/metabolismo
7.
Development ; 148(13)2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34104942

RESUMO

Epithelial cilia, whether motile or primary, often display an off-center planar localization within the apical cell surface. This form of planar cell polarity (PCP) involves the asymmetric positioning of the ciliary basal body (BB). Using the monociliated epithelium of the embryonic zebrafish floor-plate, we investigated the dynamics and mechanisms of BB polarization by live imaging. BBs were highly motile, making back-and-forth movements along the antero-posterior (AP) axis and contacting both the anterior and posterior membranes. Contacts exclusively occurred at junctional Par3 patches and were often preceded by membrane digitations extending towards the BB, suggesting focused cortical pulling forces. Accordingly, BBs and Par3 patches were linked by dynamic microtubules. Later, BBs became less motile and eventually settled at posterior apical junctions enriched in Par3. BB posterior positioning followed Par3 posterior enrichment and was impaired upon Par3 depletion or disorganization of Par3 patches. In the PCP mutant vangl2, BBs were still motile but displayed poorly oriented membrane contacts that correlated with Par3 patch fragmentation and lateral spreading. Thus, we propose an unexpected function for posterior Par3 enrichment in controlling BB positioning downstream of the PCP pathway.


Assuntos
Corpos Basais/metabolismo , Proteínas de Transporte/metabolismo , Cílios/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Proteínas de Transporte/genética , Polaridade Celular , Feminino , Masculino , Proteínas de Membrana/metabolismo , Microtúbulos/metabolismo , Transcriptoma , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
8.
Acta Pharmacol Sin ; 45(1): 112-124, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37731037

RESUMO

Proinflammatory M1 macrophages are critical for the progression of atherosclerosis. The Par3-like protein (Par3L) is a homolog of the Par3 family involved in cell polarity establishment. Par3L has been shown to maintain the stemness of mammary stem cells and promote the survival of colorectal cancer cells. In this study, we investigated the roles of the polar protein Par3L in M1 macrophage polarization and atherosclerosis. To induce atherosclerosis, Apoe-/- mice were fed with an atherosclerotic Western diet for 8 or 16 weeks. We showed that Par3L expression was significantly increased in human and mouse atherosclerotic plaques. In primary mouse macrophages, oxidized low-density lipoprotein (oxLDL, 50 µg/mL) time-dependently increased Par3L expression. In Apoe-/- mice, adenovirus-mediated Par3L overexpression aggravated atherosclerotic plaque formation accompanied by increased M1 macrophages in atherosclerotic plaques and bone marrow. In mouse bone marrow-derived macrophages (BMDMs) or peritoneal macrophages (PMs), we revealed that Par3L overexpression promoted LPS and IFNγ-induced M1 macrophage polarization by activating p65 and extracellular signal-regulated kinase (ERK) rather than p38 and JNK signaling. Our results uncover a previously unidentified role for the polarity protein Par3L in aggravating atherosclerosis and favoring M1 macrophage polarization, suggesting that Par3L may serve as a potential therapeutic target for atherosclerosis.


Assuntos
Aterosclerose , Placa Aterosclerótica , Camundongos , Humanos , Animais , Placa Aterosclerótica/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Aterosclerose/metabolismo , Macrófagos/metabolismo , Apolipoproteínas E/metabolismo , Ativação de Macrófagos , Camundongos Endogâmicos C57BL
9.
J Neurosci ; 42(24): 4774-4793, 2022 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-35705493

RESUMO

Normal neural circuits and functions depend on proper neuronal differentiation, migration, synaptic plasticity, and maintenance. Abnormalities in these processes underlie various neurodevelopmental, neuropsychiatric, and neurodegenerative disorders. Neural development and maintenance are regulated by many proteins. Among them are Par3, Par6 (partitioning defective 3 and 6), and aPKC (atypical protein kinase C) families of evolutionarily conserved polarity proteins. These proteins perform versatile functions by forming tripartite or other combinations of protein complexes, which hereafter are collectively referred to as "Par complexes." In this review, we summarize the major findings on their biophysical and biochemical properties in cell polarization and signaling pathways. We next summarize their expression and localization in the nervous system as well as their versatile functions in various aspects of neurodevelopment, including neuroepithelial polarity, neurogenesis, neuronal migration, neurite differentiation, synaptic plasticity, and memory. These versatile functions rely on the fundamental roles of Par complexes in cell polarity in distinct cellular contexts. We also discuss how cell polarization may correlate with subcellular polarization in neurons. Finally, we review the involvement of Par complexes in neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease. While emerging evidence indicates that Par complexes are essential for proper neural development and maintenance, many questions on their in vivo functions have yet to be answered. Thus, Par3, Par6, and aPKC continue to be important research topics to advance neuroscience.


Assuntos
Polaridade Celular , Proteína Quinase C , Proteínas de Ciclo Celular/metabolismo , Polaridade Celular/fisiologia , Proteína Quinase C/metabolismo , Proteínas , Transdução de Sinais
10.
Mol Carcinog ; 62(12): 1990-2004, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37702006

RESUMO

Partitioning defective 3 (Par3) is a polarity protein critical in establishing epithelial cell polarity and tight junctions (TJs). Impaired intestinal epithelial barrier integrity is closely associated with colitis-associated colorectal cancer (CRC) progression. According to the GEO and TCGA database analyses, we first observed that the expression of Par3 was reduced in CRC patients. To understand how Par3 is related to CRC, we investigated the role of Par3 in the development of CRC using an in vivo genetic approach. Our results show that the intestinal epithelium-specific PAR3 deletion mice demonstrated a more severe CRC phenotype in the context of azoxymethane/dextran sodium sulfate (AOM/DSS) treatment, with a corresponding increase in tumor number and inflammatory cytokines profile. Mechanistically, loss of Par3 disrupts the TJs of the intestinal epithelium and increases mucosal barrier permeability. The interaction of Par3 with ZO-1 prevents intramolecular interactions within ZO-1 protein and facilitates the binding of occludin to ZO-1, hence preserving TJs integrity. Our results suggest that Par3 deficiency permits pathogenic bacteria and their endotoxins to penetrate the intestinal submucosa and activate TLR4/MyD88/NF-κB signaling, promoting inflammation-driven CRC development and that Par3 may be a novel potential molecular marker for the diagnosis of early-stage CRC.


Assuntos
Neoplasias Associadas a Colite , Colite , Humanos , Camundongos , Animais , Colite/induzido quimicamente , Colite/complicações , Colite/metabolismo , Neoplasias Associadas a Colite/complicações , Neoplasias Associadas a Colite/metabolismo , Neoplasias Associadas a Colite/patologia , Junções Íntimas/metabolismo , Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL
11.
Cancer Cell Int ; 23(1): 79, 2023 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-37095487

RESUMO

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a highly malignant tumor that is frequently associated with lymph node metastasis, resulting in poor prognosis and survival in patients. In the tumor microenvironment, hypoxia plays an important role in regulating cellular responses such as progressive and rapid growth and metastasis. In these processes, tumor cells autonomously undergo diverse transitions and acquire functions. However, hypoxia-induced transition of OSCC and the involvement of hypoxia in OSCC metastasis remain unclear. Therefore, in this study, we aimed to elucidate the mechanism of hypoxia-induced OSCC metastasis and particularly, its impact on tight junctions (TJs). METHODS: The expression of hypoxia-inducible factor 1-alpha (HIF-1α) was detected in tumor tissues and adjacent normal tissues from 29 patients with OSCC using reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry (IHC). The migration and invasion abilities of OSCC cell lines treated with small interfering (si)RNA targeting HIF-1α or cultured in hypoxic conditions were analyzed using Transwell assays. The effect of HIF-1α expression on in vivo tumor metastasis of OSCC cells was evaluated using lung metastasis model. RESULTS: HIF-1α was overexpressed in patients with OSCC. OSCC metastasis was correlated with HIF-1α expression in OSCC tissues. Hypoxia increased the migration and invasion abilities of OSCC cell lines by regulating the expression and localization of partitioning-defective protein 3 (Par3) and TJs. Furthermore, HIF-1α silencing effectively decreased the invasion and migration abilities of OSCC cell lines and restored TJ expression and localization via Par3. The expression of HIF-1α was positively regulated the OSCC metastasis in vivo. CONCLUSIONS: Hypoxia promotes OSCC metastasis by regulating the expression and localization of Par3 and TJ proteins. HIF-1α positively correlates to OSCC metastasis. Lastly, HIF-1α expression could regulate the expression of Par3 and TJs in OSCC. This finding may aid in elucidating the molecular mechanisms of OSCC metastasis and progression and developing new diagnostic and therapeutic approaches for OSCC metastasis.

12.
J Biol Chem ; 297(6): 101354, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34717957

RESUMO

Hepatocytes differ from columnar epithelial cells by their multipolar organization, which follows the initial formation of central lumen-sharing clusters of polarized cells as observed during liver development and regeneration. The molecular mechanism for hepatocyte polarity establishment, however, has been comparatively less studied than those for other epithelial cell types. Here, we show that the tight junction protein Par3 organizes hepatocyte polarization via cooperating with the small GTPase Cdc42 to target atypical protein kinase C (aPKC) to a cortical site near the center of cell-cell contacts. In 3D Matrigel culture of human hepatocytic HepG2 cells, which mimics a process of liver development and regeneration, depletion of Par3, Cdc42, or aPKC results in an impaired establishment of apicobasolateral polarity and a loss of subsequent apical lumen formation. The aPKC activity is also required for bile canalicular (apical) elongation in mouse primary hepatocytes. The lateral membrane-associated proteins Lgl1 and Lgl2, major substrates of aPKC, seem to be dispensable for hepatocyte polarity establishment because Lgl-depleted HepG2 cells are able to form a single apical lumen in 3D culture. On the other hand, Lgl depletion leads to lateral invasion of aPKC, and overexpression of Lgl1 or Lgl2 prevents apical lumen formation, indicating that they maintain proper lateral integrity. Thus, hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC; Par3 cooperates with Cdc42 to recruit aPKC, which plays a crucial role in apical membrane development and regulation of the lateral maintainer Lgl.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas de Ciclo Celular/análise , Proteínas do Citoesqueleto/análise , Hepatócitos/citologia , Isoenzimas/análise , Proteína Quinase C/análise , Proteína cdc42 de Ligação ao GTP/análise , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Polaridade Celular , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteína Quinase C/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo
13.
Kidney Int ; 101(4): 733-751, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34929254

RESUMO

Glomerular diseases are a major cause for chronic kidney disorders. In most cases podocyte injury is causative for disease development. Cytoskeletal rearrangements and morphological changes are hallmark features of podocyte injury and result in dedifferentiation and loss of podocytes. Here, we establish a link between the Par3 polarity complex and actin regulators necessary to establish and maintain podocyte architecture by utilizing mouse and Drosophila models to characterize the functional role of Par3A and Par3B and its fly homologue Bazooka in vivo. Only simultaneous inactivation of both Par3 proteins caused a severe disease phenotype. Rescue experiments in Drosophila nephrocytes revealed atypical protein kinase C (aPKC)-Par6 dependent and independent effects. While Par3A primarily acts via aPKC-Par6, Par3B function was independent of Par6. Actin-associated synaptopodin protein levels were found to be significantly upregulated upon loss of Par3A/B in mouse podocytes. Tropomyosin2, which shares functional similarities with synaptopodin, was also elevated in Bazooka depleted nephrocytes. The simultaneous depletion of Bazooka and Tropomyosin2 resulted in a partial rescue of the Bazooka knockdown phenotype and prevented increased Rho1-GTP, a member of a GTPase protein family regulating the cytoskeleton. The latter contribute to the nephrocyte phenotype observed upon loss of Bazooka. Thus, we demonstrate that Par3 proteins share a high functional redundancy but also have specific functions. Par3A acts in an aPKC-Par6 dependent way and regulates RhoA-GTP levels, while Par3B exploits Par6 independent functions influencing synaptopodin localization. Hence, Par3A and Par3B link elements of polarity signaling and actin regulators to maintain podocyte architecture.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Drosophila , Podócitos , Actinas/metabolismo , Animais , Polaridade Celular , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Membrana/genética , Camundongos , Podócitos/metabolismo , Proteína Quinase C
14.
J Cell Sci ; 133(22)2020 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-33093242

RESUMO

Cell polarity is essential for various asymmetric cellular events, and the partitioning defective (PAR) protein PAR3 (encoded by PARD3 in mammals) plays a unique role as a cellular landmark to establish polarity. In epithelial cells, PAR3 localizes at the subapical border, such as the tight junction in vertebrates, and functions as an apical determinant. Although we know a great deal about the regulators of PAR3 localization, how PAR3 is concentrated and localized to a specific membrane domain remains an important question to be clarified. In this study, we demonstrate that ASPP2 (also known as TP53BP2), which controls PAR3 localization, links PAR3 and protein phosphatase 1 (PP1). The ASPP2-PP1 complex dephosphorylates a novel phosphorylation site, Ser852, of PAR3. Furthermore, Ser852- or Ser889-unphosphorylatable PAR3 mutants form protein clusters, and ectopically localize to the lateral membrane. Concomitance of clustering and ectopic localization suggests that PAR3 localization is a consequence of local clustering. We also demonstrate that unphosphorylatable forms of PAR3 exhibited a low molecular turnover and failed to coordinate rapid reconstruction of the tight junction, supporting that both the phosphorylated and dephosphorylated states are essential for the functional integrity of PAR3.


Assuntos
Polaridade Celular , Proteína Quinase C , Animais , Proteínas de Ciclo Celular/metabolismo , Análise por Conglomerados , Fosforilação , Proteína Quinase C/metabolismo , Junções Íntimas/metabolismo
15.
Development ; 146(15)2019 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-31405903

RESUMO

Cdc42 regulates epithelial morphogenesis together with the Par complex (Baz/Par3-Par6-aPKC), Crumbs (Crb/CRB3) and Stardust (Sdt/PALS1). However, how these proteins work together and interact during epithelial morphogenesis is not well understood. To address this issue, we used the genetically amenable Drosophila pupal photoreceptor and follicular epithelium. We show that during epithelial morphogenesis active Cdc42 accumulates at the developing apical membrane and cell-cell contacts, independently of the Par complex and Crb. However, membrane localization of Baz, Par6-aPKC and Crb all depend on Cdc42. We find that although binding of Cdc42 to Par6 is not essential for the recruitment of Par6 and aPKC to the membrane, it is required for their apical localization and accumulation, which we find also depends on Par6 retention by Crb. In the pupal photoreceptor, membrane recruitment of Par6-aPKC also depends on Baz. Our work shows that Cdc42 is required for this recruitment and suggests that this factor promotes the handover of Par6-aPKC from Baz onto Crb. Altogether, we propose that Cdc42 drives morphogenesis by conferring apical identity, Par-complex assembly and apical accumulation of Crb.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Membrana/metabolismo , Células Fotorreceptoras/citologia , Proteína Quinase C/metabolismo , Animais , Polaridade Celular/fisiologia , Drosophila melanogaster/metabolismo , Epitélio/crescimento & desenvolvimento , Morfogênese/fisiologia , Ligação Proteica/fisiologia
16.
Development ; 146(6)2019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30814118

RESUMO

The MARK/PAR-1 family of kinases are conserved regulators of cell polarity that share a conserved C-terminal kinase-associated domain (KA1). Localization of MARK/PAR-1 kinases to specific regions of the cell cortex is a hallmark of polarized cells. In Caenorhabditiselegans zygotes, PAR-1 localizes to the posterior cortex under the influence of another polarity kinase, aPKC/PKC-3. Here, we report that asymmetric localization of PAR-1 protein is not essential, and that PAR-1 kinase activity is regulated spatially. We find that, as in human MARK1, the PAR-1 KA1 domain is an auto-inhibitory domain that suppresses kinase activity. Auto-inhibition by the KA1 domain functions in parallel with phosphorylation by PKC-3 to suppress PAR-1 activity in the anterior cytoplasm. The KA1 domain also plays an additional role that is essential for germ plasm maintenance and fertility. Our findings suggest that modular regulation of kinase activity by redundant inhibitory inputs contributes to robust symmetry breaking by MARK/PAR-1 kinases in diverse cell types.


Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Polaridade Celular , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Serina-Treonina Quinases/fisiologia , Alelos , Animais , Linhagem da Célula , Citoplasma/metabolismo , Proteínas de Fluorescência Verde , Humanos , Microscopia Confocal , Mutação , Fosforilação , Domínios Proteicos , Proteína Quinase C/fisiologia , Interferência de RNA , Treonina/química
17.
Breast Cancer Res Treat ; 192(1): 75-87, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35079981

RESUMO

PURPOSE: Paclitaxel, belongs to tubulin-binding agents (TBAs), shows a great efficacy against breast cancer via stabilizing microtubules. Drug resistance limits its clinical application. Here we aimed to explore a role of Polarity protein Par3 in improving paclitaxel effectiveness. METHODS: Breast cancer specimens from 45 patients were collected to study the relationship between Par3 expression and paclitaxel efficacy. The Kaplan-Meier method was used for survival analysis. Cell viability was measured in breast cancer cells (SK-BR-3 and T-47D) with Par3 over-expression or knockdown. The flow cytometry assays were performed to measure cell apoptosis and cell cycle. BrdU incorporation assay and Hoechst 33,258 staining were performed to measure cell proliferation and cell apoptosis, respectively. Immunofluorescence was used to detect microtubule structures. RESULTS: Par3 expression was associated with good response of paclitaxel in breast cancer patients. Consistently, Par3 over-expression significantly sensitized breast cancer cells to paclitaxel by promoting cell apoptosis and reducing cell proliferation. In Par3 overexpressing cells upon paclitaxel treatment, we observed intensified cell cycle arrests at metaphase. Further exploration showed that Par3 over-expression stabilized microtubules of breast cancer cells in response to paclitaxel and resists to microtubules instability induced by nocodazole, a microtubule-depolymerizing agent. CONCLUSION: Par3 facilitates polymeric forms of tubulin and stabilizes microtubule structure, which aggravates paclitaxel-induced delay at the metaphase-anaphase transition, leading to proliferation inhibition and apoptosis of breast cancer cells. Par3 has a potential role in sensitizing breast cancer cells to paclitaxel, which may provide a more precise assessment of individual treatment and novel therapeutic targets.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos Fitogênicos , Neoplasias da Mama , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Paclitaxel , Antineoplásicos Fitogênicos/uso terapêutico , Apoptose , Neoplasias da Mama/tratamento farmacológico , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Microtúbulos , Paclitaxel/uso terapêutico , Tubulina (Proteína)
18.
Cell Immunol ; 373: 104496, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35259602

RESUMO

OBJECTIVE: This research aimed to investigate the mechanism in psoriasis with the involvement of Par3-containing exosomes from macrophages by regulating the asymmetric division of basal stem cells. METHODS: BrdU labeling and double immunofluorescence assays were conducted to detect the proportion of asymmetric division in psoriasis mice. Western blot assay was conducted to examine the expression of Par3/mInsc/LGN signaling pathway-related proteins in psoriasis mice. Next, the asymmetric division of keratinocytes in normal mice treated with macrophages and their secreted exosomes were determined, together with the related protein detection. After establishing a macrophage-specific Par3 knockout mouse model, the asymmetric division of isolated keratinocytes and the related proteins were measured. An epidermal-specific mInsc, LGN, or NuMA knockout mouse model was induced, followed by the determination of the asymmetric division of isolated keratinocytes. RESULTS: The asymmetric division of basal stem cells was increased, and the expression of Par3/mInsc/LGN signaling pathway-related proteins was elevated in psoriasis. Par3-containing macrophage-derived exosomes enhanced asymmetric division of basal stem cells and expression of Par3/mInsc/LGN signaling pathway-related proteins in mice. However, mice with Par3 loss presented opposite trends. There was a decreased asymmetric division of basal stem cells in epidermal-specific mInsc, LGN, and NUMA knockout mice. CONCLUSION: Our study suggests that macrophage-derived exosomes-shuttled Par3 are absorbed by the basal stem cells and regulate the asymmetric division of cells to produce a large number of transit-amplifying cells, thus causing psoriasis-related symptoms in conjunction with various other factors.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Psoríase , Animais , Epiderme , Queratinócitos/metabolismo , Camundongos , Psoríase/metabolismo , Transdução de Sinais , Células-Tronco
19.
Cell Mol Life Sci ; 78(7): 3657-3672, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33651172

RESUMO

Apical-basal polarity is a key feature of most epithelial cells and it is regulated by highly conserved protein complexes. In mammalian podocytes, which emerge from columnar epithelial cells, this polarity is preserved and the tight junctions are converted to the slit diaphragms, establishing the filtration barrier. In Drosophila, nephrocytes show several structural and functional similarities with mammalian podocytes and proximal tubular cells. However, in contrast to podocytes, little is known about the role of apical-basal polarity regulators in these cells. In this study, we used expansion microscopy and found the apical polarity determinants of the PAR/aPKC and Crb-complexes to be predominantly targeted to the cell cortex in proximity to the nephrocyte diaphragm, whereas basolateral regulators also accumulate intracellularly. Knockdown of PAR-complex proteins results in severe endocytosis and nephrocyte diaphragm defects, which is due to impaired aPKC recruitment to the plasma membrane. Similar, downregulation of most basolateral polarity regulators disrupts Nephrin localization but had surprisingly divergent effects on endocytosis. Our findings suggest that morphology and slit diaphragm assembly/maintenance of nephrocytes is regulated by classical apical-basal polarity regulators, which have distinct functions in endocytosis.


Assuntos
Polaridade Celular , Proteínas de Drosophila/metabolismo , Endocitose , Junções Intercelulares/fisiologia , Proteínas de Membrana/metabolismo , Podócitos/fisiologia , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Podócitos/citologia , Podócitos/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismo
20.
Proc Natl Acad Sci U S A ; 116(11): 4999-5008, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30814219

RESUMO

In the inner ear sensory epithelia, stereociliary hair bundles atop sensory hair cells are mechanosensory apparatus with planar polarized structure and orientation. This is established during development by the concerted action of tissue-level, intercellular planar cell polarity (PCP) signaling and a hair cell-intrinsic, microtubule-mediated machinery. However, how various polarity signals are integrated during hair bundle morphogenesis is poorly understood. Here, we show that the conserved cell polarity protein Par3 is essential for planar polarization of hair cells. Par3 deletion in the inner ear disrupted cochlear outgrowth, hair bundle orientation, kinocilium positioning, and basal body planar polarity, accompanied by defects in the organization and cortical attachment of hair cell microtubules. Genetic mosaic analysis revealed that Par3 functions both cell-autonomously and cell-nonautonomously to regulate kinocilium positioning and hair bundle orientation. At the tissue level, intercellular PCP signaling regulates the asymmetric localization of Par3, which in turn maintains the asymmetric localization of the core PCP protein Vangl2. Mechanistically, Par3 interacts with and regulates the localization of Tiam1 and Trio, which are guanine nucleotide exchange factors (GEFs) for Rac, thereby stimulating Rac-Pak signaling. Finally, constitutively active Rac1 rescued the PCP defects in Par3-deficient cochleae. Thus, a Par3-GEF-Rac axis mediates both tissue-level and hair cell-intrinsic PCP signaling.


Assuntos
Moléculas de Adesão Celular/metabolismo , Polaridade Celular , Células Ciliadas Auditivas Internas/citologia , Células Ciliadas Auditivas Internas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular , Feminino , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Masculino , Camundongos , Microtúbulos/metabolismo , Mosaicismo , Órgão Espiral/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo
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