Percentage Of Ciliated Cells Research Articles

A Review: Biomechanical Aspects of the Fallopian Tube Relevant to its Function in Fertility.

The fallopian tube (FT) plays a crucial role in the reproductive process by providing an ideal biomechanical and biochemical environment for fertilization and early embryo development. Despite its importance, the biomechanical functions of the FT that originate from its morphological aspects, and ultrastructural aspects, as well as the mechanical properties of FT, have not been studied nor used sufficiently, which limits the understanding of fertilization, mechanotrasduction, and mechanobiology during embryo development, as well as the replication of the FT in laboratory settings for infertility treatments. This paper reviews and revives valuable information on human FT reported in medical literature in the past five decades relevant to the biomechanical aspects of FT. In this review, we summarized the current state of knowledge concerning the morphological, ultrastructural aspects, and mechanical properties of the human FT. We also investigate the potential arising from a thorough consideration of the biomechanical functions and exploring often neglected mechanical aspects. Our investigation encompasses both macroscopic measurements (such as length, diameter, and thickness) and microscopic measurements (including the height of epithelial cells, the percentage of ciliated cells, cilia structure, and ciliary beat frequency). Our primary focus has been on healthy women of reproductive age. We have examined various measurement techniques, encompassing conventional metrology, 2D histological data as well as new spatial measurement techniques such as micro-CT.

Differential nasal swab cytology represents a valuable tool for therapy monitoring but not prediction of therapy response in chronic rhinosinusitis with nasal polyps treated with Dupilumab.

Chronic Rhinosinusitis with nasal polyps (CRSwNP) is a common chronic disease with a high impact on patients' quality of life. If conservative and surgical guideline treatment cannot sufficiently control disease burden, biologicals can be considered as a comparably new treatment option that has revolutionized CRSwNP therapy since the first approval of Dupilumab in 2019. With the aim to select patients who benefit from this new treatment and to find a marker for therapy monitoring, we investigated the cellular composition of nasal mucous membranes and inflammatory cells of patients suffering from CRSwNP and undergoing Dupilumab therapy using non-invasive nasal swab cytology. Twenty CRSwNP patients with the indication for Dupilumab therapy have been included in this prospective clinical study. In total, five study visits were conducted with ambulatory nasal differential cytology using nasal swabs starting with the beginning of therapy and followed by visits every 3 months for 12 months. First, these cytology samples were stained with the May-Grunwald-Giemsa method (MGG) and the percentage of ciliated cells, mucinous cells, eosinophil cells, neutrophil cells, and lymphocytes was analyzed. Secondly, an immunocytochemical (ICC) ECP-staining was performed to detect eosinophil granulocytes. Additionally, during each study visit the nasal polyp score, SNOT20 questionnaire, olfactometry, the total IgE concentration in peripheral blood as well as the eosinophil cell count in peripheral blood were recorded. The change of parameters was evaluated over one year and the correlation between clinical effectiveness and nasal differential cytology was analyzed. In both MGG (p<0.0001) and ICC analysis (p<0.001) a significant decrease of eosinophils was seen under Dupilumab treatment. When patients were divided into a Eo-low- (<21%) and Eo-high- (≥21%) group according to the percentage eosinophils in nasal swab catology in the first study visit, the Eo-high-group showed a greater change of eosinophils over time (Δ17.82) compared to the Eo-low-group (Δ10.67) but, however, no better response to therapy. The polyp score, SNOT20 questionnaire, and total IgE concentration in peripheral blood showed a significant decrease during the observation period (p<0.0001). Nasal swab cytology as an easy-to-apply diagnostic method allows detection and quantification of the different cell populations within the nasal mucosa at a given time. The nasal differential cytology showed a significant decrease of eosinophils during Dupilumab therapy and can therefore be used as non-invasvive method for monitoring therapy success of this cost intensive therapy and potentially can allow an optimized individual therapy planning and management for CRSwNP patients. Since the validity of initial nasal swab eosinophil cell count as a predictive biomarker for therapy response was limited in our study, additional studies including larger number of participants will be necessary to further evaluate the potential benefits for clinical practice of this new diagnostic method.

Abnormal accumulation of OFD1 in endometrial cancer with poor prognosis inhibits ciliogenesis.

The aim of the present study was to examine primary cilia in endometrial tissue during the menstrual cycle and to clarify their morphological changes with different grades of endometrial cancer. Images of fluorescence immunostaining taken by confocal microscopy were used to count the number of primary cilia in normal endometrium and endometrioid carcinoma Grade 1 and Grade 3 specimens. To examine the association between autophagy and ciliogenesis in endometrioid carcinoma, the expression of p62/Sequestosome-1, a selective substrate for autophagy, and oral-facial-digital syndrome 1 protein (OFD1), a protein associated with ciliogenesis, were examined using images of fluorescence immunostaining taken by confocal microscopy. The level of p62 expression was confirmed by western blotting. In proliferative and secretory endometrial stromal cells, the percentage of cells that were ciliated was 7.2 and 32.7% (95% confidence interval=21.61-39.79; P<0.01), and the length of the primary cilia was 1.24 µm and 2.34 µm (0.92-1.26; P<0.01), respectively. In stromal cells of endometrioid carcinoma Grade 1 and Grade 3, the percentage of ciliated cells was 13.5 and 2.9% (7.89-15.05; P<0.001), and the length of the primary cilia was 2.02 and 1.14 µm (0.76-0.99; P<0.001), respectively. In both normal menstrual cycle tissue and endometrial carcinomas, the percentage of primary cilia was lower and their length was shorter in tissues with higher proliferative potential. The expression of OFD1 was significantly higher in Grade 3 compared with Grade 1 as indicated by quantifying the intensity of the fluorescence images (133–12248; P=0.046). To the best of our knowledge, this is the first study concerning the distribution of primary cilia in normal endometrium and endometrial cancer tissues. Overall, fewer ciliated cells in the highly malignant endometrial cancer tissues may be associated not only to the proliferation of cancer cells, but also to the excessive accumulation of OFD1 due to dysfunctional autophagy.

AAV-mediated gene editing lights up the lung

AAV-mediated gene editing lights up the lung

Identification of Cilia in Different Mouse Tissues.

Cilia are microtubule-based hair-like organelles that extend from the cell surface. However, the existence and distribution of cilia in each organ and tissue at the postnatal stage in vivo remain largely unknown. In this study, we defined cilia distribution and arrangement and measured the ciliary lengths and the percentage of ciliated cells in different organs and tissues in vivo by using cilium dual reporter-expressing transgenic mice. Cilia were identified by the presence of ARL13B with an mCherry+ signal, and the cilium basal body was identified by the presence of Centrin2 with a GFP+ signal. Here, we provide in vivo evidence that chondrocytes and cells throughout bones have cilia. Most importantly, we reveal that: 1. primary cilia are present in hepatocytes; 2. no cilia but many centrioles are distributed on the apical cell surface in the gallbladder, intestine, and thyroid epithelia; 3. cilia on the cerebral cortex are well oriented, pointing to the center of the brain; 4. ARL13B+ inclusion is evident in the thyroid and islets of Langerhans; and 5. approximately 2% of cilia show irregular movement in nucleus pulposus extracellular fluid. This study reveals the existence and distribution of cilia and centrioles in different tissues and organs, and provides new insights for further comprehensive study of ciliary function in these organs and tissues.

Primary Cilia Formation Does Not Rely on WNT/β-Catenin Signaling

Primary cilia act as crucial regulators of embryo development and tissue homeostasis. They are instrumental for modulation of several signaling pathways, including Hedgehog, WNT, and TGF-β. However, gaps exist in our understanding of how cilia formation and function is regulated. Recent work has implicated WNT/β-catenin signaling pathway in the regulation of ciliogenesis, yet the results are conflicting. One model suggests that WNT/β-catenin signaling negatively regulates cilia formation, possibly via effects on cell cycle. In contrast, second model proposes a positive role of WNT/β-catenin signaling on cilia formation, mediated by the re-arrangement of centriolar satellites in response to phosphorylation of the key component of WNT/β-catenin pathway, β-catenin. To clarify these discrepancies, we investigated possible regulation of primary cilia by the WNT/β-catenin pathway in cell lines (RPE-1, NIH3T3, and HEK293) commonly used to study ciliogenesis. We used WNT3a to activate or LGK974 to block the pathway, and examined initiation of ciliogenesis, cilium length, and percentage of ciliated cells. We show that the treatment by WNT3a has no- or lesser inhibitory effect on cilia formation. Importantly, the inhibition of secretion of endogenous WNT ligands using LGK974 blocks WNT signaling but does not affect ciliogenesis. Finally, using knock-out cells for key WNT pathway components, namely DVL1/2/3, LRP5/6, or AXIN1/2 we show that neither activation nor deactivation of the WNT/β-catenin pathway affects the process of ciliogenesis. These results suggest that WNT/β-catenin-mediated signaling is not generally required for efficient cilia formation. In fact, activation of the WNT/β-catenin pathway in some systems seems to moderately suppress ciliogenesis.

Abstract 3813: Activation of EGFR induces loss of primary cilia in oral mucosa carcinogenesis

Abstract Objective: Primary cilia are evolutionally conserved cell structures involving multiple cell functions by sensing extracellular signals. Loss of primary cilia is frequently observed in various cancer types, suggesting that the absence of this organelle may promote tumorigenesis. However, little is known about the role of primary cilia in oral premalignant lesions and oral squamous cell carcinoma (OSCC). Materials and Methods: Immunofluosense staining was carried out to detect the incidence of primary cilia in every stages of oral carcinogenesis from normal to OSCC. Immunohistochemical analysis was used for validating the correlation between the ciliary incidence and the expression level of EGFR in the oral carcinogenesis. Furthermore, the change of primary cilia was detected by activating and inhibiting the EGFR pathway activity in vitro. Results: A significant decrease in the percentage of ciliated cells was found on both oral leukoplakia (OLK), as well as OSCC tissues, compared to normal oral mucosa epithelium, and this loss of cilia was independent of the increased proliferative index. A parallel increase of EGFR activity was observed in OLK and OSCC tissues. In addition, EGF stimulation induced obvious primary cilia absorption, whereas inhibition of EGFR by gefitinib significantly restored cilia formation in vitro. We further found that the activation of EGFR resulted in the phosphorylation of AURKA, a pivotal kinase promoting disassembly of primary cilia. Conclusion: EGFR-AURKA pathway might be a critical regulator of ciliary loss in the carcinogenesis of oral mucosa. This may provide insight into the development of new therapeutic strategies to prevent oral premalignant lesions from becoming malignant. Citation Format: Fengg yin, Zihao Wei, Jing Li, Liang Xie, Qianming Chen. Activation of EGFR induces loss of primary cilia in oral mucosa carcinogenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3813.

Low fluid shear stress promoted ciliogenesis via Dvl2 in hUVECs

This study aims to explore the mechanism of fluid shear stress in regulating the primary cilia assembly or disassembly in human umbilical vein endothelial cells (hUVECs) using microfluidic chamber experiments. Immunofluorescence analysis showed that primary cilia assembled under disturbed fluid shear stress (DF) of 1dyne/cm2, while disassembled under unidirectional shear stress (USS) of 15dynes/cm2. Disheveled (Dvl2) in Wnt signaling pathway was effectively co-immunoprecipitated with Bardet-Biedl syndrome proteins 8 (Bbs8) and γ-tubulin. Compared with those in the control group, the percentages of ciliated cells with Dvl2 overexpression were found to be 67% and 59.667%, respectively, under USS and DF (an increment of 21-38.7%); while, those with Dvl2 silencing were 16% and 32.667%, respectively, under USS and DF (a decrement of 23-30%). Further, the expression of Bbs8 and γ-tubulin was decreased by RNA interference of Dvl2 but increased with Dvl2 overexpression. The results indicated that Dvl2 played a pivotal role during DF-induced primary cilia assembly, and was important for apical docking of basal bodies through Bbs8 and γ-tubulin.

How do elevated levels of testosterone affect the function of the human fallopian tube and fertility?—New insights

Excess testosterone levels affect up to 20% of the female population worldwide and are a key component in the pathogenesis of polycystic ovary syndrome. However, little is known about how excess testosterone affects the function of the human fallopian tube-the site of gamete transport, fertilization, and early embryogenesis. Therefore, this study aimed to characterize alterations caused by long-term exposure to male testosterone levels. For this purpose, the Fallopian tubes of nine female-to-male transsexuals, who had been undergoing testosterone treatment for 1-3 years, were compared with the tubes of 19 cycling patients. In the ampulla, testosterone treatment resulted in extensive luminal accumulations of secretions and cell debris which caused ciliary clumping and luminal blockage. Additionally, the percentage of ciliated cells in the ampulla was significantly increased. Transsexual patients, who had had sexual intercourse before surgery, showed spermatozoa trapped in mucus. Finally, in the isthmus complete luminal collapse occurred. Our results imply that fertility in women with elevated levels of testosterone is altered by tubal luminal obstruction resulting in impaired gamete transport and survival.

Effects of pelvic endometriosis and adenomyosis on ciliary beat frequency and muscular contractions in the human fallopian tube

BackgroundPelvic endometriosis (EM) and adenomyosis (AM) have different effects on the fallopian tube. This study aimed to assess the transport capability of the fallopian tube in women with pelvic EM or AM.MethodsTwenty women with uterine leiomyoma (control group), 20 with adenomyosis without pelvic EM (AM group) and 35 with pelvic EM without AM (EM group) were included. EM cases were further divided into the tubal EM and non-tubal EM subgroups. Ciliary beat frequency (CBF), percentage of ciliated cells, and smooth muscle contraction were measured.ResultsCBFs of the ampulla in EM cases were significantly lower than those of control and AM cases; CBFs of the ampulla and isthmus in tubal EM cases were significantly lower than those of the control group and non-tubal EM subgroup. In both the ampulla and isthmus segment, percentages of ciliated cells in EM patients were significantly lower than those of AM and control patients; the tubal EM subgroup showed significantly lower values than the control group and non-tubal EM subgroup. Amplitude-to-weight ratios of longitudinal muscular contractility in EM cases were significantly lower than control values; tubal EM cases showed significantly lower values than controls and the non-tubal EM subgroup. Contraction frequencies in EM cases were significantly lower than those of control and AM cases, in both longitudinal and circular muscles; tubal EM cases showed significantly lower values than controls and the non-tubal EM subgroup.ConclusionEM with tubal EM damaged transport function of the fallopian tube, to varying degrees, whereas tubal function in EM without tubal EM and in AM is not altered.

687 Autophagy and ciliogenesis are coordinately regulated by EphA2 in epidermis

Autophagy and ciliogenesis are required for proper epidermal differentiation; however, the link between epidermal autophagy and ciliogenesis remains unclear. Knockdown of the receptor tyrosine kinase EphA2 in a human keratinocyte cell line (hTCEpi) resulted in an increase in: (i) large vacuoles; (ii) LC3II, a mature form of LC3 protein; and (iii) p62, a negative marker of autophagy flux. Loss of EphA2 in human epidermal keratinocytes (HEKs) decreased LC3 turnover, another measure of autophagy flux. This establishes a positive role for EphA2 in autophagy. BioID assays combined with unbiased proteomic analyses reveal that in HEK 3-D raft cultures, 10% of proteins interacting with EphA2 involved autophagy. Among them was phospholipase D1 (PLD1), a critical regulator of end-stage autophagy. Pulldown and co-immunoprecipitation assays confirmed that EphA2 binds to PLD1. Pharmacologic and genetic inhibition of PLD1 signaling reversed vacuole accumulation and increased LC3II in hTCEpi cells lacking EphA2. This is compelling evidence that EphA2 regulates autophagy via PLD1. Impaired autophagy also impacted ciliogenesis in epithelial cells lacking EphA2, as the percentage of ciliated cells were decreased in hTCEpi cells and HEKs threated with siEphA2. Such a decrease was reversed by knockdown of PLD1. Embryonic skin of Xenopus, a well-established in vivo model of ciliogenesis, was treated with a Morpholino oligo targeting Epha2, which impaired ciliogenesis, clearly establishing a role for EphA2 in this process. We show that EphA2, in part, coordinates autophagy and ciliogenesis, processes essential for proper epidermal differentiation. An abnormality in EphA2 signaling has been demonstrated in psoriasis; a disease characterized by impaired epidermal differentiation. Thus, understanding the relationships between EphA2 signaling, autophagy and ciliogenesis on the background of psoriasis is warranted.

A Novel Role of Primary Cilia in Breast Cancer Recurrence

Recurrent tumors are the predominant cause of breast cancer‐related deaths, therefore there is an urgent need for more effective therapeutic strategies. However, a critical barrier to their development is a lack of understanding of the molecular mechanisms involved in breast tumor recurrence. Recently, primary cilia, microtubule‐based organelles that project from the surface of vertebrate cells, have emerged into the spotlight for their link to numerous human diseases, including cancer. However, the relationship between primary cilia and breast cancer recurrence remains unknown. Here, using a genetically engineered mouse model of breast cancer, which closely recapitulates the clinical phenomenon of breast cancer recurrence, we have uncovered that primary cilia are more highly enriched in recurrent mammary tumors than in both primary mammary tumors and asymptomatic/contralateral mouse mammary tissues. Congruently, we found that the percentage of ciliated cells was substantially elevated in recurrent breast cancer cells compared to primary breast cancer cells. Moreover, RNA‐sequence analysis also revealed that GLI2 and GLI3, which are major downstream effectors of the morphogenic Sonic Hedgehog signaling pathway and are also well‐known to localize to primary cilia, were upregulated in recurrent breast cancer cells compared to primary breast cancer cells. Collectively, these data suggest that primary cilia may have a role in breast cancer recurrence. Our study also indicates that, at least within the recurrent stages of the disease, the primary cilium may serve as a specialized oncogenic signaling organelle.Support or Funding InformationTo Sarah C. Goetz: R00 HD076444 (from the National Institute of Child Health and Human Development).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Missense mutations in the WD40 domain of AHI1 cause non-syndromic retinitis pigmentosa

BackgroundRecent findings suggesting that Abelson helper integration site 1 (AHI1) is involved in non-syndromic retinal disease have been debated, as the functional significance of identified missense variants was uncertain. We...

(Pro)renin receptor (ATP6AP2) depletion arrests As4.1 cells in the G0/G1 phase thereby increasing formation of primary cilia.

The (pro)renin receptor [(P)RR, ATP6AP2] is a multifunctional transmembrane protein that activates local renin–angiotensin systems, but also interacts with Wnt pathways and vacuolar H+‐ATPase (V‐ATPase) during organogenesis. The aim of this study was to characterize the role of ATP6AP2 in the cell cycle in more detail. ATP6AP2 down‐regulation by siRNA in renal As4.1 cells resulted in a reduction in the rate of proliferation and a G0/G1 phase cell cycle arrest. We identified a number of novel target genes downstream of ATP6AP2 knock‐down that were related to the primary cilium (Bbs‐1, Bbs‐3, Bbs‐7, Rabl5, Ttc26, Mks‐11, Mks‐5, Mks‐2, Tctn2, Nme7) and the cell cycle (Pierce1, Clock, Ppif). Accordingly, the number of cells expressing the primary cilium was markedly increased. We found no indication that these effects were dependent of V‐ATPase activity, as ATP6AP2 knock‐down did not affect lysosomal pH and bafilomycin A neither influenced the ciliary expression pattern nor the percentage of ciliated cells. Furthermore, ATP6AP2 appears to be essential for mitosis. ATP6AP2 translocated from the endoplasmatic reticulum to mitotic spindle poles (pro‐, meta‐ and anaphase) and the central spindle bundle (telophase) and ATP6AP2 knock‐down results in markedly deformed spindles. We conclude that ATP6AP2 is necessary for cell division, cell cycle progression and mitosis. ATP6AP2 also inhibits ciliogenesis, thus promoting proliferation and preventing differentiation.

Nerve growth factor-promotes primary cilium assembly in cholinergic neurons from the human basal forebrain

The primary cilium is a non-motile sensory antenna protruding from the surface of nearly all cells of the body, able to mediate the cellular response to extracellular signals. Although many of its functions remain to be clarified, it has been recently shown a role in neurogenesis [1]. In this study we evaluated the presence of the primary cilium in neurons isolated from the human fetal nucleus basalis of Meynert (hfnbM), a basal forebrain region crucially involved in the cholinergic transmission required for learning and memory. The hfnbM cells are characterized by the expression of cholinergic markers, such as choline acetyl transferase (ChAT) and also express the primary cilium, which, in basal conditions, was detected in the 17% of cells. It is known that nerve growth factor (NGF) supports survival, maintenance, connectivity and function of the brain cholinergic neurons. Indeed, we demonstrated that hfnbM cells respond to NGF in terms of proliferation, neurite formation and ChAT expression. Interestingly, NGF exposure significantly increased the percentage of ciliated cells (34.9%±1.8%). Given the known adverse effect of systemic chronic inflammation in the pathogenesis of neurodegenerative diseases, such as Alzheimer’s disease, characterized by the loss of nbM neurons, we exposed our cells to tumor necrosis factor-α (TNF-α). We observed that TNF-α significantly reduced the number of ciliated cells (4.3%±2%). Our results strongly suggest for the first time that primary cilia may be involved in the NGF-driven maturation of human nbM cholinergic neurons and suggest that the deleterious effects of neuroinflammation may be linked to an altered formation of the primary cilium.

Role of IFN-γ, IL-13, and IL-17 on mucociliary differentiation of nasal epithelial cells in chronic rhinosinusitis with nasal polyps.

Mucociliary dysfunction is a prominent pathophysiological feature of chronic rhinosinusitis with nasal polyps (CRSwNP); however, the precise mechanisms underlying mucociliary dysfunction are still unclear. The aim of this study was therefore to evaluate the effects of IFN-γ, IL-13, and IL-17 on human nasal mucociliary differentiation and ciliary beat frequency (CBF) in patients with CRSwNP. Human nasal epithelial cells from tissue of patients with CRSwNP and control subjects were established as air-liquid interface (ALI) primary cultures. Confluent cultures were incubated with10 ng/mL each of IFN-γ, IL-13, or IL-17 for 14 days and assessed for expression of specific morphological markers and factors associated with mucociliary differentiation, the percentage of ciliated and goblet cells, and CBF. In comparison with control subjects, percentage of ciliated cells and CBF were decreased; while percentage of goblet cells, FOXJ1, and MUC5AC mRNA expression were increased in nasal polyp-derived epithelial cultures. Treatment with IFN-γ and IL-13 significantly decreased the expression of β-tubulin IV (specific cilia marker), ciliated cell number, and expression of FOXJ1 and DNAI2, in epithelial cultures derived from both CRSwNP patients and control subjects. Furthermore, while both IFN-γ and IL-13 treatment significantly decreased the CBF of cells from both CRSwNP patients and control subjects, IL-13 additionally significantly increased goblet cell number and the expression of MUC5AC and CLCA1, in these cultures. IL-17 treatment did not significantly affect ciliated or goblet cell differentiation, CBF, nor MUC5AC and CLCA1 expression, but increased both MUC5B mRNA and protein expression in these cultures. The demonstration that IFN-γ and IL-13 both significantly reduce ciliated cell differentiation and CBF in CRSwNP patients, and IL-13 additionally induces significant goblet cell hyperplasia and MUC5AC mucin expression, as well as IL-17 significantly increases MUC5B mucin expression, suggests that these inflammatory cytokines may be potential therapeutic targets in the management of CRSwNP.

Culture and detection of primary cilia in endothelial cell models.

Background The primary cilium is a sensor of blood-induced forces in endothelial cells (ECs). Studies that have examined EC primary cilia have reported a wide range of cilia incidence (percentage of ciliated cells). We hypothesise that this variation is due to the diversity in culture conditions in which the cells are grown. We studied two EC types: human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMEC-1s). Both cell types were grown in media containing foetal bovine serum (FBS) at high (20 % FBS and 10 % FBS for HUVECs and HMEC-1s, respectively) or low (2 % FBS) concentrations. Cells were then either fixed at confluence, serum-starved or grown post-confluence for 5 days in corresponding expansion media (cobblestone treatment). For each culture condition, we quantified cilia incidence and length.ResultsHUVEC ciliogenesis is dependent on serum concentration during the growth phase; low serum (2 % FBS) HUVECs were not ciliated, whereas high serum (20 % FBS) confluent HUVECs have a cilia incidence of 2.1 ± 2.2 % (median ± interquartile range). We report, for the first time, the presence of cilia in the HMEC-1 cell type. HMEC-1s have between 2.2 and 3.5 times greater cilia incidence than HUVECs (p < 0.001). HMEC-1s also have shorter cilia compared to HUVECs (3.0 ± 1.0 μm versus 5.1 ± 2.4 μm, at confluence, p = 0.003).Conclusions We demonstrate that FBS plays a role in determining the prevalence of cilia in HUVECs. In doing so, we highlight the importance of considering a commonly varied parameter (% FBS), in the experimental design. We recommend that future studies examining large blood vessel EC primary cilia use confluent HUVECs grown in high serum medium, as we found these cells to have a higher cilia incidence than low serum media HUVECs. For studies interested in microvasculature EC primary cilia, we recommend using cobblestone HMEC-1s grown in high serum medium, as these cells have a 19.5 ± 6.2 % cilia incidence.Electronic supplementary materialThe online version of this article (doi:10.1186/s13630-015-0020-2) contains supplementary material, which is available to authorized users.

Levonorgestrel decreases cilia beat frequency of human fallopian tubes and rat oviducts without changing morphological structure.

SummaryLevonorgestrel, a derivative of progesterone, effectively protects women against unwanted pregnancy as an emergency contraceptive. Previous studies have not been successful in determining the mechanism by which levonorgestrel acts. In the present study we analysed cilia beat action and cilia morphology following levonorgestrel exposure in vitro and in vivo using both light and electron microscopy. There was a significant decrease in the ciliary beat frequency (CBF) of human fallopian tubes between mucosal explants bathed in 5 μmol/L levonorgestrel and those bathed in medium alone (P < 0.05). There was a tendency for CBF to decrease more in the ampulla than in isthmus, but there were no differences between the proliferative and secretory phases. In rat oviducts, levonorgestrel produced a similar reduction in CBF (~ 10%) compared with the saline control group (P < 0.05). Histological and ultrastructural analysis demonstrated no changes in the percentage of ciliated cells or in the classic ‘9 + 2’ structure of cilia following levonorgestrel treatment in either system. Thus, levonorgestrel reduces CBF without damaging cilia morphology. Decreases in CBF may indicate a pathological role for levonorgestrel in the transportation of the ovum and zygote in the fallopian tube.

The MAL protein is crucial for proper membrane condensation at the ciliary base, which is required for primary cilium elongation.

The base of the primary cilium contains a zone of condensed membranes whose importance is not known. Here, we have studied the involvement of MAL, a tetraspanning protein that exclusively partitions into condensed membrane fractions, in the condensation of membranes at the ciliary base and investigated the importance of these membranes in primary cilium formation. We show that MAL accumulates at the ciliary base of epithelial MDCK cells. Knockdown of MAL expression resulted in a drastic reduction in the condensation of membranes at the ciliary base, the percentage of ciliated cells and the length of the cilia, but did not affect the docking of the centrosome to the plasma membrane or produce missorting of proteins to the pericentriolar zone or to the membrane of the remaining cilia. Rab8 (for which there are two isoforms, Rab8A and Rab8b), IFT88 and IFT20, which are important components of the machinery of ciliary growth, were recruited normally to the ciliary base of MAL-knockdown cells but were unable to elongate the primary cilium correctly. MAL, therefore, is crucial for the proper condensation of membranes at the ciliary base, which is required for efficient primary cilium extension.

Loss of primary cilia occurs early in breast cancer development.

BackgroundPrimary cilia are microtubule-based organelles that protrude from the cell surface. Primary cilia play a critical role in development and disease through regulation of signaling pathways including the Hedgehog pathway. Recent mouse models have also linked ciliary dysfunction to cancer. However, little is known about the role of primary cilia in breast cancer development. Primary cilia expression was characterized in cancer cells as well as their surrounding stromal cells from 86 breast cancer patients by counting cilia and measuring cilia length. In addition, we examined cilia expression in normal epithelial and stromal cells from reduction mammoplasties as well as histologically normal adjacent tissue for comparison.ResultsWe observed a statistically significant decrease in the percentage of ciliated cells on both premalignant lesions as well as in invasive cancers. This loss of cilia does not correlate with increased proliferative index (Ki67-positive cells). However, we did detect rare ciliated cancer cells present in patients with invasive breast cancer and found that these express a marker of basaloid cancers that is associated with poor prognosis (Cytokeratin 5). Interestingly, the percentage of ciliated stromal cells associated with both premalignant and invasive cancers decreased when compared to stromal cells associated with normal tissue. To understand how cilia may be lost during cancer development we analyzed the expression of genes required for ciliogenesis and/or ciliary function and compared their expression in normal versus breast cancer samples. We found that expression of ciliary genes were frequently downregulated in human breast cancers.ConclusionsThese data suggest that primary cilia are lost early in breast cancer development on both the cancer cells and their surrounding stromal cells.