Primary Cilia, Ciliogenesis and the Actin Cytoskeleton: A Little Less Resorption, A Little More Actin Please

Primary cilia are microtubule-based organelles that extend from the apical surface of most mammalian cells, forming when the basal body (derived from the mother centriole) docks at the apical cell membrane. They act as universal cellular “antennae” in vertebrates that receive and integrate mechanical and chemical signals from the extracellular environment, serving diverse roles in chemo-, mechano- and photo-sensation that control developmental signaling, cell polarity and cell proliferation. Mutations in ciliary genes cause a major group of inherited developmental disorders called ciliopathies. There are very few preventative treatments or new therapeutic interventions that modify disease progression or the long-term outlook of patients with these conditions. Recent work has identified at least four distinct but interrelated cellular processes that regulate cilia formation and maintenance, comprising the cell cycle, cellular proteostasis, signaling pathways and structural influences of the actin cytoskeleton. The actin cytoskeleton is composed of microfilaments that are formed from filamentous (F) polymers of globular G-actin subunits. Actin filaments are organized into bundles and networks, and are attached to the cell membrane, by diverse cross-linking proteins. During cell migration, actin filament bundles form either radially at the leading edge or as axial stress fibers. Early studies demonstrated that loss-of-function mutations in ciliopathy genes increased stress fiber formation and impaired ciliogenesis whereas pharmacological inhibition of actin polymerization promoted ciliogenesis. These studies suggest that polymerization of the actin cytoskeleton, F-actin branching and the formation of stress fibers all inhibit primary cilium formation, whereas depolymerization or depletion of actin enhance ciliogenesis. Here, we review the mechanistic basis for these effects on ciliogenesis, which comprise several cellular processes acting in concert at different timescales. Actin polymerization is both a physical barrier to both cilia-targeted vesicle transport and to the membrane remodeling required for ciliogenesis. In contrast, actin may cause cilia loss by localizing disassembly factors at the ciliary base, and F-actin branching may itself activate the YAP/TAZ pathway to promote cilia disassembly. The fundamental role of actin polymerization in the control of ciliogenesis may present potential new targets for disease-modifying therapeutic approaches in treating ciliopathies.

Pleomorphic adenoma of the lacrimal gland: atypical location of a salivary tumor

Introduction: In daily ophthalmological practice, lacrimal gland tumors are rare. They represent 5 to 7.5% of all intraorbital tumors. The most common epithelial tumor of this gland is the pleomorphic adenoma, with a percentage of 25 to 50% of its tumor lesions. Objective: In this communication we presented a case of lacrimal gland pleomorphic adenoma and study the expression of Ki67 and the location and expression of MUC-1 and its correlation with tumor prognosis. We also carried out a retrospective descriptive study of the literature on the subject published between 1951 and 2020, using the MEDLINE database. Material and methods: The surgical piece examined, was processed according to the paraffin embedding technique, was performed with a histopathological diagnosis of pleomorphic lacrimal gland adenoma. Histological sections were stained with Hematoxylin / Eosin. Immunostaining with Ki67 and MUC-1 was performed with the DAKO LSAB + kit. Results: The diagnosis of hypercellular pleomorphic adenoma with areas of the remaining lacrimal gland was made. Ki67 labeling was low (≤15%). MUC-1 expression was intense, situated to the apical cell membrane of approximately 10% epitheliocytes from pseudoductal and cystic structures.Conclusions: Through histopathological evaluation, the correlation of Ki67 expression and the location and expression of MUC1, we verified that it is a non-recurrent pleomorphic adenoma without malignant transformation.

“Reversed polarization” of Na/K-ATPase—a sign of inverted transport in the human endolymphatic sac: a super-resolution structured illumination microscopy (SR-SIM) study

The human endolymphatic sac (ES) is believed to regulate inner ear fluid homeostasis and to be associated with Meniere’s disease (MD). We analyzed the ion transport protein sodium/potassium-ATPase (Na/K-ATPase) and its isoforms in the human ES using super-resolution structured illumination microscopy (SR-SIM). Human vestibular aqueducts were collected during trans-labyrinthine vestibular schwannoma surgery after obtaining ethical permission. Antibodies against various isoforms of Na/K-ATPase and additional solute-transporting proteins, believed to be essential for ion and fluid transport, were used for immunohistochemistry. A population of epithelial cells of the human ES strongly expressed Na/K-ATPase α1, β1, and β3 subunit isoforms in either the lateral/basolateral or apical plasma membrane domains. The β1 isoform was expressed in the lateral/basolateral plasma membranes in mostly large cylindrical cells, while β3 and α1 both were expressed with “reversed polarity” in the apical cell membrane in lower epithelial cells. The heterogeneous expression of Na/K-ATPase subunits substantiates earlier notions that the ES is a dynamic structure where epithelial cells show inverted epithelial transport. Dual absorption and secretion processes may regulate and maintain inner ear fluid homeostasis. These findings may shed new light on the etiology of endolymphatic hydrops and MD.

Yorkie controls tube length and apical barrier integrity during airway development

Epithelial organ size and shape depend on cell shape changes, cell–matrix communication, and apical membrane growth. The Drosophila melanogaster embryonic tracheal network is an excellent model to study these processes. Here, we show that the transcriptional coactivator of the Hippo pathway, Yorkie (YAP/TAZ in vertebrates), plays distinct roles in the developing Drosophila airways. Yorkie exerts a cytoplasmic function by binding Drosophila Twinstar, the orthologue of the vertebrate actin-severing protein Cofilin, to regulate F-actin levels and apical cell membrane size, which are required for proper tracheal tube elongation. Second, Yorkie controls water tightness of tracheal tubes by transcriptional regulation of the δ-aminolevulinate synthase gene (Alas). We conclude that Yorkie has a dual role in tracheal development to ensure proper tracheal growth and functionality.

Yorkie controls tube length and apical barrier integrity in the developing Drosophila airways

Epithelial organ size and shape depend on cell shape changes, cell-matrix communication and apical membrane growth. The Drosophila embryonic tracheal network is an excellent model to study these processes. Here, we show that the transcriptional co-activator of the Hippo pathway, Yorkie (YAP in vertebrates), plays distinct roles in the developing Drosophila airways. Yorkie exerts a cytoplasmic function by binding Drosophila Twinstar, the orthologue of the vertebrate actin-severing protein Cofilin, to regulate F-actin levels and apical cell membrane size, which are required for proper tracheal tube elongation. Second, Yorkie controls water-tightness of tracheal tubes by transcriptional regulation of the enzyme δ-aminolevulinate synthase (Alas). We conclude that Yorkie has a dual role in tracheal development to ensure proper tracheal growth and functionality.Short SummaryThis work identified an alternative role of the transcriptional co-activator Yorkie (Yki) in controlling water impermeability and tube size of the developing Drosophila airways. Tracheal impermeability is triggered by Yki-mediated transcriptional regulation of δ-aminolevulinate synthase, Alas, whereas tube elongation is controlled by binding of Yki to the actin severing factor Twinstar.

Corrigendum to: Cytoplasmic polyadenylation element binding protein 2 (CPEB2) is required for tight-junction assembly for establishment of porcine trophectoderm epithelium

Cytoplasmic polyadenylation element binding protein (CPEB) is an RNA-binding protein that promotes elongation of poly(A) tails and regulates mRNA translation. CPEB depletion in mammary epithelium is known to disrupt tight-junction (TJ) assembly via mislocalisation of tight junction protein 1 (TJP1), but the role of CPEB in the biological functions associated with TJs has not yet been studied. The objective of this study was to investigate the roles of CPEB2 during porcine parthenote development. CPEB2 was detected in both the nuclei and apical cytoplasm at the 4- and 8-cell stages and was localised to cell–cell contact after the initiation of the morula stage. Its depletion led to retarded blastocyst formation caused by impaired TJ assembly. Moreover, transcription of TJ-associated genes, including TJP1, Coxsackie virus and adenovirus receptor (CXADR) and occludin (OCLN), was not affected, but the corresponding proteins were not properly localised at the apical cell membrane in morulae, suggesting that CPEB2 confers mRNA stability or determines subcellular localisation for translation. Remarkably reduced relative levels of TJP1 transcripts bearing the 3′-untranslated region were noted, indicating that CPEB2 mediates TJP1 mRNA stability. In conclusion, our findings demonstrate that because of its regulation of TJP1, CPEB2 is required for TJ assembly during porcine blastocyst development.

Cytoplasmic polyadenylation element binding protein 2 (CPEB2) is required for tight-junction assembly for establishment of porcine trophectoderm epithelium

Cytoplasmic polyadenylation element binding protein (CPEB) is an RNA-binding protein that promotes elongation of poly(A) tails and regulates mRNA translation. CPEB depletion in mammary epithelium is known to disrupt tight-junction (TJ) assembly via mislocalisation of tight junction protein 1 (TJP1), but the role of CPEB in the biological functions associated with TJs has not yet been studied. The objective of this study was to investigate the roles of CPEB2 during porcine parthenote development. CPEB2 was detected in both the nuclei and apical cytoplasm at the 4- and 8-cell stages and was localised to cell–cell contact after the initiation of the morula stage. Its depletion led to retarded blastocyst formation caused by impaired TJ assembly. Moreover, transcription of TJ-associated genes, including TJP1, Coxsackie virus and adenovirus receptor (CXADR) and occludin (OCLN), was not affected, but the corresponding proteins were not properly localised at the apical cell membrane in morulae, suggesting that CPEB2 confers mRNA stability or determines subcellular localisation for translation. Remarkably reduced relative levels of TJP1 transcripts bearing the 3′-untranslated region were noted, indicating that CPEB2 mediates TJP1 mRNA stability. In conclusion, our findings demonstrate that because of its regulation of TJP1, CPEB2 is required for TJ assembly during porcine blastocyst development.

Aquaporin 2 Expression in Human Fetal Kidney Development

Introduction: The metanephrogenic zone, renal cortex and renal pyramids develop into their final form by week 13. The metanephric kidney produces large quantities of diluted urine in order to maintain volumes of amniotic fluid. Aquaporins are transmembrane protein channels that enable water transport through biological membranes. Aquaporin 2 (AQP2) is a water channel found in the supranuclear region and apical area of the cell membrane of the kidneys collecting tubule cells. Its main function is reabsorption of water through vasopressin stimulation. Materials and methods: Immunohistochemistry was used to study fetal renal tissue of 34 post-mortem fetuses of 9 weeks to 24 weeks gestational age. Results: AQP2 expression is present in connecting tubules and collecting tubules during the targeted time period. From week 9 to 12, the expression is cytoplasmic. From week 13 to 20 the enhancement of expression in the apical cell membrane occurs with the advancement of fetal age. At the end of the studied period, from week 21 to 24, both cytoplasmic and apical expression were observed. In animal studies AQP2 expression has an increasing trend during development. In contradiction with these results, other authors described low AQP2 levels in the human fetal kidney. Conclusions: This study helps to understand the amniotic fluid’s homeostasis during pregnancy. In the beginning of the fetal period AQP2 protein is present in the cytoplasm of epithelial cells of the collecting duct and distal connecting duct. During the fetal period, AQP2 expression in collecting ducts becomes more enhanced in the apical membrane of the cells.

Depletion of Cholesterol Reduces ENaC Activity by Decreasing Phosphatidylinositol-4,5-Bisphosphate in Microvilli

Background/Aims: The epithelial sodium channel (ENaC) in cortical collecting duct (CCD) principal cells plays a critical role in regulating systemic blood pressure. We have previously shown that cholesterol (Cho) in the apical cell membrane regulates ENaC; however, the underlying mechanism remains unclear. Methods: Patch-clamp technique and confocal microscopy were used to evaluate ENaC activity and density. Results: Here we show that extraction of membrane Cho with methyl-β-cyclodextrin (MβCD) significantly reduced amiloride-sensitive current and ENaC single-channel activity. The effects were reproduced by inhibition of Cho synthesis in the cells with lovastatin. We have previously shown that phosphatidylinositol-4,5-bisphosphate (PIP2), an ENaC activator, is predominantly located in the microvilli, a specialized apical membrane domain. Here, our confocal microscopy data show that α-ENaC was co-localized with PIP2 in the microvilli and that Cho was also co-localized with PIP2 in the microvilli. Either extraction of Cho with MβCD or inhibition of Cho synthesis with lovastatin consistently reduced the levels of Cho, PIP2, and ENaC in the microvilli. Conclusions: Since PIP2 can directly stimulate ENaC and also affect ENaC trafficking, these data suggest that depletion of Cho reduces ENaC apical density and activity at least in part by decreasing PIP2 in the microvilli.

ZnT4 (SLC30A4)-null (“lethal milk”) mice have defects in mammary gland secretion and hallmarks of precocious involution during lactation

During lactation, highly specialized secretory mammary epithelial cells (MECs) produce and secrete huge quantities of nutrients and nonnutritive factors into breast milk. The zinc (Zn) transporter ZnT4 ( SLC30A4) transports Zn into the trans-Golgi apparatus for lactose synthesis, and across the apical cell membrane for efflux from MECs into milk. This is consistent with observations in “lethal milk” ( lm/lm) mice, which have a truncation mutation in SLC30A4, and present with not only low milk Zn concentration, but also smaller mammary glands, decreased milk volume, and lactation failure by lactation day 2. However, the molecular underpinnings of these defects are not understood. Here, we used lactating C57BL/6J lm/lm (ZnT4-null) mice to explore the consequences of a ZnT4-null phenotype on mammary gland function during early lactation. Lactating C57BL/6J lm/lm mice had significantly fewer, smaller, and collapsed alveoli comprising swollen, lipid-filled MECs during early lactation. These defects were associated with decreased Akt expression and STAT5 activation, indicative of defects in MEC secretion. In addition, increased expression of ZnT2, TNF-α, and cleaved e-cadherin concomitant with increased activation of STAT3 implicated the loss of ZnT4 in precocious activation of involution. Collectively, our study indicates that the loss of ZnT4 has profound consequences on MEC secretion and may promote tissue remodeling in the mammary gland during early lactation.