scholarly journals PACRG and FAP20 form the inner junction of axonemal doublet microtubules and regulate ciliary motility

2019 ◽  
Vol 30 (15) ◽  
pp. 1805-1816 ◽  
Author(s):  
Erin E. Dymek ◽  
Jianfeng Lin ◽  
Gang Fu ◽  
Mary E. Porter ◽  
Daniela Nicastro ◽  
...  
Keyword(s):  
Cell Motility ◽  
Electron Tomography ◽  
Structural Studies ◽  
Ciliary Beating ◽  
Ciliary Motility ◽  
Functional Studies ◽  
Cryo Electron Tomography ◽  
Motile Cilia

We previously demonstrated that PACRG plays a role in regulating dynein-driven microtubule sliding in motile cilia. To expand our understanding of the role of PACRG in ciliary assembly and motility, we used a combination of functional and structural studies, including newly identified Chlamydomonas pacrg mutants. Using cryo-electron tomography we show that PACRG and FAP20 form the inner junction between the A- and B-tubule along the length of all nine ciliary doublet microtubules. The lack of PACRG and FAP20 also results in reduced assembly of inner-arm dynein IDA b and the beak-MIP structures. In addition, our functional studies reveal that loss of PACRG and/or FAP20 causes severe cell motility defects and reduced in vitro microtubule sliding velocities. Interestingly, the addition of exogenous PACRG and/or FAP20 protein to isolated mutant axonemes restores microtubule sliding velocities, but not ciliary beating. Taken together, these studies show that PACRG and FAP20 comprise the inner junction bridge that serves as a hub for both directly modulating dynein-driven microtubule sliding, as well as for the assembly of additional ciliary components that play essential roles in generating coordinated ciliary beating.

Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 null Tetrahymena cells reveals functional MIPs

2020 ◽  
Author(s):  
Amy S. Fabritius ◽  
Brian A. Bayless ◽  
Sam Li ◽  
Daniel Stoddard ◽  
Westley Heydeck ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Tetrahymena Thermophila ◽  
Electron Tomography ◽  
Functional Characterization ◽  
Ciliary Beating ◽  
Ciliary Function ◽  
Cryo Electron Tomography ◽  
Motile Cilia ◽  
Cilia And Flagella ◽  
Stable Populations

AbstractMotile cilia and flagella are built from stable populations of doublet microtubules that comprise their axonemes. Their unique stability is brought about, at least in part, by a network of Microtubule Inner Proteins (MIPs) found in the lumen of their doublet microtubules. Rib72A and Rib72B were identified as microtubule inner proteins (MIPs) in the motile cilia of Tetrahymena thermophila. Loss of these proteins leads to ciliary defects and loss of multiple MIPs. We performed mass spectrometry coupled with proteomic analysis and bioinformatics to identify the MIPs lost in RIB72A/B knockout (KO) Tetrahymena cells. From this analysis we identified a number of candidate MIPs and pursued one, Fap115, for functional characterization. We find that loss of Fap115 results in disrupted cell swimming and aberrant ciliary beating. Cryo-electron tomography reveals that Fap115 localizes to MIP6a in the A-tubule of the doublet microtubules. Overall, our results highlight the complex relationship between MIPs, ciliary structure, and ciliary function.

scholarly journals FAP206 is a microtubule-docking adapter for ciliary radial spoke 2 and dynein c

2015 ◽  
Vol 26 (4) ◽  
pp. 696-710 ◽  
Author(s):  
Krishna Kumar Vasudevan ◽  
Kangkang Song ◽  
Lea M. Alford ◽  
Winfield S. Sale ◽  
Erin E. Dymek ◽  
...  
Keyword(s):  
Cell Motility ◽  
Electron Tomography ◽  
Macromolecular Complexes ◽  
Radial Spoke ◽  
Ciliary Motility ◽  
Cryo Electron Tomography ◽  
Ciliary Protein ◽  
Radial Spokes ◽  
Dynein Arms

Radial spokes are conserved macromolecular complexes that are essential for ciliary motility. A triplet of three radial spokes, RS1, RS2, and RS3, repeats every 96 nm along the doublet microtubules. Each spoke has a distinct base that docks to the doublet and is linked to different inner dynein arms. Little is known about the assembly and functions of individual radial spokes. A knockout of the conserved ciliary protein FAP206 in the ciliate Tetrahymena resulted in slow cell motility. Cryo–electron tomography showed that in the absence of FAP206, the 96-nm repeats lacked RS2 and dynein c. Occasionally, RS2 assembled but lacked both the front prong of its microtubule base and dynein c, whose tail is attached to the front prong. Overexpressed GFP-FAP206 decorated nonciliary microtubules in vivo. Thus FAP206 is likely part of the front prong and docks RS2 and dynein c to the microtubule.

scholarly journals Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus

2019 ◽  
Vol 218 (12) ◽  
pp. 4236-4251 ◽  
Author(s):  
Gang Fu ◽  
Lei Zhao ◽  
Erin Dymek ◽  
Yuqing Hou ◽  
Kangkang Song ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Electron Tomography ◽  
3D Structure ◽  
Protein Composition ◽  
Ciliary Beating ◽  
Ciliary Motility ◽  
Cryo Electron Tomography ◽  
Central Apparatus ◽  
Subunit Organization ◽  
Insight Into

Nearly all motile cilia contain a central apparatus (CA) composed of two connected singlet microtubules with attached projections that play crucial roles in regulating ciliary motility. Defects in CA assembly usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of the CA projections are largely unknown. Here, we integrated biochemical and genetic approaches with cryo-electron tomography to compare the CA of wild-type Chlamydomonas with CA mutants. We identified a large (>2 MD) complex, the C1a-e-c supercomplex, that requires the PF16 protein for assembly and contains the CA components FAP76, FAP81, FAP92, and FAP216. We localized these subunits within the supercomplex using nanogold labeling and show that loss of any one of them results in impaired ciliary motility. These data provide insight into the subunit organization and 3D structure of the CA, which is a prerequisite for understanding the molecular mechanisms by which the CA regulates ciliary beating.

scholarly journals Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 null Tetrahymena cells reveals functional MIPs

2021 ◽  
pp. mbc.E20-12-0786
Author(s):  
Amy S. Fabritius ◽  
Brian A. Bayless ◽  
Sam Li ◽  
Daniel Stoddard ◽  
Westley Heydeck ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Tetrahymena Thermophila ◽  
Electron Tomography ◽  
Functional Characterization ◽  
Ciliary Beating ◽  
Ciliary Function ◽  
Luminal Side ◽  
Cryo Electron Tomography ◽  
Motile Cilia ◽  
Cilia And Flagella

The core structure of motile cilia and flagella, the axoneme, is built from a stable population of doublet microtubules. This unique stability is brought about, at least in part, by a network of Microtubule Inner Proteins (MIPs) that are bound to the luminal side of the microtubule walls. Rib72A and Rib72B were identified as MIPs in the motile cilia of the protist Tetrahymena thermophila. Loss of these proteins leads to ciliary defects and loss of additional MIPs. We performed mass spectrometry coupled with proteomic analysis and bioinformatics to identify the MIPs lost in RIB72A/B knockout Tetrahymena axonemes. We identified a number of candidate MIPs and pursued one, Fap115, for functional characterization. We find that loss of Fap115 results in disrupted cell swimming and aberrant ciliary beating. Cryo-electron tomography reveals that Fap115 localizes to MIP6a in the A-tubule of the doublet microtubules. Overall, our results highlight the complex relationship between MIPs, ciliary structure, and ciliary function.

scholarly journals Biallelic Mutations in ACACA Cause a Disruption in Lipid Homeostasis That Is Associated With Global Developmental Delay, Microcephaly, and Dysmorphic Facial Features

2021 ◽  
Vol 9 ◽  
Author(s):  
Xiaoting Lou ◽  
Xiyue Zhou ◽  
Haiyan Li ◽  
Xiangpeng Lu ◽  
Xinzhu Bao ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Cell Motility ◽  
Developmental Delay ◽  
Lipid Homeostasis ◽  
Global Developmental Delay ◽  
Facial Features ◽  
Functional Studies ◽  
Biallelic Mutations

ObjectiveWe proposed that the deficit of ACC1 is the cause of patient symptoms including global developmental delay, microcephaly, hypotonia, and dysmorphic facial features. We evaluated the possible disease-causing role of the ACACA gene in developmental delay and investigated the pathogenesis of ACC1 deficiency.MethodsA patient who presented with global developmental delay with unknown cause was recruited. Detailed medical records were collected and reviewed. Whole exome sequencing found two variants of ACACA with unknown significance. ACC1 mRNA expression level, protein expression level, and enzyme activity level were detected in patient-derived cells. Lipidomic analysis, and in vitro functional studies including cell proliferation, apoptosis, and the migratory ability of patient-derived cells were evaluated to investigate the possible pathogenic mechanism of ACC1 deficiency. RNAi-induced ACC1 deficiency fibroblasts were established to assess the causative role of ACC1 deficit in cell migratory disability in patient-derived cells. Palmitate supplementation assays were performed to assess the effect of palmitic acid on ACC1 deficiency-induced cell motility deficit.ResultsThe patient presented with global developmental delay, microcephaly, hypotonia, and dysmorphic facial features. A decreased level of ACC1 and ACC1 enzyme activity were detected in patient-derived lymphocytes. Lipidomic profiles revealed a disruption in the lipid homeostasis of the patient-derived cell lines. In vitro functional studies revealed a deficit of cell motility in patient-derived cells and the phenotype was further recapitulated in ACC1-knockdown (KD) fibroblasts. The cell motility deficit in both patient-derived cells and ACC1-KD were attenuated by palmitate.ConclusionWe report an individual with biallelic mutations in ACACA, presenting global development delay. In vitro studies revealed a disruption of lipid homeostasis in patient-derived lymphocytes, further inducing the deficit of cell motility capacity and that the deficiency could be partly attenuated by palmitate.

scholarly journals Polymorphic Protective Dps–DNA Co-Crystals by Cryo Electron Tomography and Small Angle X-Ray Scattering

Biomolecules ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 39 ◽  
Author(s):  
Roman Kamyshinsky ◽  
Yury Chesnokov ◽  
Liubov Dadinova ◽  
Andrey Mozhaev ◽  
Ivan Orlov ◽  
...  
Keyword(s):  
Small Angle ◽  
Electron Tomography ◽  
Actual Structure ◽  
X Ray ◽  
Cell Parameters ◽  
X Ray Scattering ◽  
Cryo Electron Tomography ◽  
Dps Protein ◽  
Ray Scattering

Rapid increase of intracellular synthesis of specific histone-like Dps protein that binds DNA to protect the genome against deleterious factors leads to in cellulo crystallization—one of the most curious processes in the area of life science at the moment. However, the actual structure of the Dps–DNA co-crystals remained uncertain in the details for more than two decades. Cryo-electron tomography and small-angle X-ray scattering revealed polymorphous modifications of the co-crystals depending on the buffer parameters. Two different types of the Dps–DNA co-crystals are formed in vitro: triclinic and cubic. Three-dimensional reconstruction revealed DNA and Dps molecules in cubic co-crystals, and the unit cell parameters of cubic lattice were determined consistently by both methods.

scholarly journals Protective Dps–DNA co‐crystallization in stressed cells: an in vitro structural study by small‐angle X‐ray scattering and cryo‐electron tomography

FEBS Letters ◽  
2019 ◽  
Vol 593 (12) ◽  
pp. 1360-1371 ◽  
Author(s):  
Liubov A. Dadinova ◽  
Yurii M. Chesnokov ◽  
Roman A. Kamyshinsky ◽  
Ivan A. Orlov ◽  
Maxim V. Petoukhov ◽  
...  
Keyword(s):  
Structural Study ◽  
Small Angle ◽  
Electron Tomography ◽  
X Ray ◽  
X Ray Scattering ◽  
Cryo Electron Tomography ◽  
Stressed Cells ◽  
Ray Scattering

scholarly journals Role of the V1G1 subunit of V-ATPase in breast cancer cell migration

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maria De Luca ◽  
Roberta Romano ◽  
Cecilia Bucci
Keyword(s):  
Breast Cancer ◽  
Cell Migration ◽  
Cell Motility ◽  
Cancer Progression ◽  
Tumor Formation ◽  
Downstream Signaling ◽  
Late Endosomes ◽  
Intracellular Compartments

AbstractV-ATPase is a large multi-subunit complex that regulates acidity of intracellular compartments and of extracellular environment. V-ATPase consists of several subunits that drive specific regulatory mechanisms. The V1G1 subunit, a component of the peripheral stalk of the pump, controls localization and activation of the pump on late endosomes and lysosomes by interacting with RILP and RAB7. Deregulation of some subunits of the pump has been related to tumor invasion and metastasis formation in breast cancer. We observed a decrease of V1G1 and RAB7 in highly invasive breast cancer cells, suggesting a key role of these proteins in controlling cancer progression. Moreover, in MDA-MB-231 cells, modulation of V1G1 affected cell migration and matrix metalloproteinase activation in vitro, processes important for tumor formation and dissemination. In these cells, characterized by high expression of EGFR, we demonstrated that V1G1 modulates EGFR stability and the EGFR downstream signaling pathways that control several factors required for cell motility, among which RAC1 and cofilin. In addition, we showed a key role of V1G1 in the biogenesis of endosomes and lysosomes. Altogether, our data describe a new molecular mechanism, controlled by V1G1, required for cell motility and that promotes breast cancer tumorigenesis.

Effects of ethanol on in vitro ciliary motility

1988 ◽  
Vol 65 (4) ◽  
pp. 1617-1620 ◽  
Author(s):  
D. R. Maurer ◽  
J. Liebman
Keyword(s):  
Beat Frequency ◽  
Slow Motion ◽  
Ciliary Beat Frequency ◽  
Blood Levels ◽  
Ciliary Beating ◽  
Ciliary Motility ◽  
Social Drinking ◽  
High Concentrations ◽  
Ciliary Beat

Consumption of ethanol can impair lung function and slow total lung clearance. High concentrations of ethanol have been shown to slow or arrest ciliary beating. This study examined the effects of concentrations of alcohol comparable to blood levels achieved from social drinking on ciliary beat frequency. We obtained ciliated cells by brushing the trachea of unanesthetized sheep during fiber-optic bronchoscopy. The cells were suspended in a perfusion chamber and physiological conditions were maintained in vitro. Ciliary beat frequency and synchrony were determined by slow-motion analysis of video images obtained by interference contrast microscopy. Metachronal ciliary coordination was observed in all preparations. The ciliary beat frequency was stimulated at ethanol concentrations from 0.01 up to but not including 0.1%, unchanged at 0.5 and 1%, and slowed at 2%. While confirming inhibition of ciliary motility at very high ethanol levels, we observed no acute impairment of ciliary function at ethanol concentrations comparable to those achieved from social drinking. Indeed, we found an unexpected stimulation of ciliary beating at low levels of ethanol. How this alteration in ciliary beating would affect pulmonary clearance remains unknown at this time.

scholarly journals Krüppel-Like Factor 7 is a Marker of Aggressive Gastric Cancer and Poor Prognosis

2017 ◽  
Vol 43 (3) ◽  
pp. 1090-1099 ◽  
Author(s):  
Zhonghua Jiang ◽  
Tingting Yu ◽  
Zhining Fan ◽  
Hongmei Yang ◽  
Xin Lin
Keyword(s):  
Gastric Cancer ◽  
Disease Free Survival ◽  
Clinicopathological Characteristics ◽  
The Cancer Genome Atlas ◽  
Strong Negative Correlation ◽  
Functional Studies ◽  
Expression Of Genes ◽  
Cancer Genome Atlas

Background/Aims: Krüppel-like factor (KLF) 7 protein is a member of the KLF transcription factor family, which plays important roles in regulating the expression of genes involved in cell growth, proliferation, differentiation and metabolism. However, the role of KLF7 in gastric cancer (GC) is unknown. The aim of this study is to explore the role of KLF7 in GC and its correlation with clinicopathological characteristics and prognosis of GC patients. Methods: We first systematically evaluated dysregulation of the KLF family in The Cancer Genome Atlas (TCGA) GC database. Then, 252 patients who underwent surgery for GC were enrolled to validate the results from the TCGA. Functional studies were also used to explore the role of KLF7 in GC. Results: In the TCGA database, we found that KLF7 was an independent predictor for survival by both univariate and multivariate analysis (P<0.05). In a validation cohort, KLF7 expression was significantly increased in GC tissues compared with adjacent normal controls (P=0.013). High KLF7 expression correlated with inferior prognostic factors, such as T stage (P=0.022), N stage (P =0.005) and lymphovascular invasion (P=0.009). Furthermore, we observed a strong negative correlation between KLF7 expression and 5-year overall survival and disease-free survival in GC patients (P<0.05). Moreover, our in vitro studies showed a notable decrease in migration in KLF7 knockdown cells. Conclusion: KLF7 has an important role in GC progression, as it inhibits GC cell migration and may serve as a prognostic marker.

Sign in / Sign up

Export Citation Format

Share Document