scholarly journals ENKD1 is a centrosomal and ciliary microtubule-associated protein important for primary cilium assembly and Hedgehog signaling

2021 ◽  
Author(s):  
Fatmanur Tiryaki ◽  
Jovana Deretic ◽  
Elif Nur Firat-Karalar
Keyword(s):  
Primary Cilium ◽  
Mammalian Cells ◽  
Hedgehog Signaling ◽  
Developmental Disorders ◽  
Cell Function ◽  
Microtubule Organization ◽  
Regulatory Relationship ◽  
Cellular Assays ◽  
And Function

Centrioles and cilia are conserved, microtubule-based structures critical for cell function and development. Their structural and functional defects cause cancer and developmental disorders. How microtubules are organized into ordered structures by microtubule-associated proteins (MAPs) and tubulin modifications is best understood during mitosis but is largely unexplored for the centrioles and the ciliary axoneme, which are composed of remarkably stable microtubules that maintain their length at steady state. In particular, we know little about the identity of the centriolar and ciliary MAPs and how they work together during the assembly and maintenance of the cilium and centriole. Here, we identified Enkurin domain containing 1 (ENKD1) as a component of the centriole wall and the axoneme in mammalian cells, and showed that it has extensive proximity interactions with these compartments and MAPs. Using in vitro and cellular assays, we found that ENKD1 is a new MAP that promotes microtubule polymerization and regulates microtubule organization and stability. Consistently, overexpression of ENKD1 increased tubulin polymerization and acetylation and disrupted microtubule organization. Cells depleted for ENKD1 were defective in ciliary length and content regulation and failed to respond to Hedgehog pathway activation. Together, our results establish ENKD1 as a new centriolar and ciliary MAP that regulate primary cilium structure and function, and advances our understanding of the functional and regulatory relationship between MAPs and the primary cilium.

scholarly journals Primary Cilium in Cancer Hallmarks

2019 ◽  
Vol 20 (6) ◽  
pp. 1336 ◽  
Author(s):  
Lucilla Fabbri ◽  
Frédéric Bost ◽  
Nathalie Mazure
Keyword(s):  
Cancer Progression ◽  
Primary Cilium ◽  
Mammalian Cells ◽  
Current Knowledge ◽  
Wide Spectrum ◽  
Cancer Hallmarks ◽  
Mutual Regulation ◽  
Ultrastructure And Function ◽  
And Function ◽  
External Stimuli

The primary cilium is a solitary, nonmotile and transitory appendage that is present in virtually all mammalian cells. Our knowledge of its ultrastructure and function is the result of more than fifty years of research that has dramatically changed our perspectives on the primary cilium. The mutual regulation between ciliogenesis and the cell cycle is now well-recognized, as well as the function of the primary cilium as a cellular “antenna” for perceiving external stimuli, such as light, odorants, and fluids. By displaying receptors and signaling molecules, the primary cilium is also a key coordinator of signaling pathways that converts extracellular cues into cellular responses. Given its critical tasks, any defects in primary cilium formation or function lead to a wide spectrum of diseases collectively called “ciliopathies”. An emerging role of primary cilium is in the regulation of cancer development. In this review, we seek to describe the current knowledge about the influence of the primary cilium in cancer progression, with a focus on some of the events that cancers need to face to sustain survival and growth in hypoxic microenvironment: the cancer hallmarks.

scholarly journals Circulating miRNA 887 is differentially expressed in ARDS and modulates endothelial function

2020 ◽  
Vol 318 (6) ◽  
pp. L1261-L1269 ◽  
Author(s):  
Andrew J. Goodwin ◽  
Pengfei Li ◽  
Perry V. Halushka ◽  
James A. Cook ◽  
Aman S. Sumal ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Function ◽  
In Silico Analysis ◽  
Leukocyte Migration ◽  
Differentially Expressed ◽  
Expression Of Genes ◽  
Cell Gene Expression ◽  
And Function ◽  
The Impact

Circulating microRNAs (miRNAs) can be taken up by recipient cells and have been recently associated with the acute respiratory distress syndrome (ARDS). Their role in host predisposition to the syndrome is unknown. The objective of the study was to identify circulating miRNAs associated with the development of sepsis-related ARDS and examine their impact on endothelial cell gene expression and function. We determined miRNA levels in plasma collected from subjects during the first 24 h of admission to a tertiary intensive care unit for sepsis. A miRNA that was differentially expressed between subjects who did and did not develop ARDS was identified and was transfected into human pulmonary microvascular endothelial cells (HPMECs). RNA sequencing, in silico analysis, cytokine expression, and leukocyte migration assays were used to determine the impact of this miRNA on gene expression and cell function. In two cohorts, circulating miR-887-3p levels were elevated in septic patients who developed ARDS compared with those who did not. Transfection of miR-887-3p into HPMECs altered gene expression, including the upregulation of several genes previously associated with ARDS (e.g., CXCL10, CCL5, CX3CL1, VCAM1, CASP1, IL1B, IFNB, and TLR2), and activation of cellular pathways relevant to the response to infection. Functionally, miR-887-3p increased the endothelial release of chemokines and facilitated trans-endothelial leukocyte migration. Circulating miR-887-3p is associated with ARDS in critically ill patients with sepsis. In vitro, miR-887-3p regulates the expression of genes relevant to ARDS and neutrophil tracking. This miRNA may contribute to ARDS pathogenesis and could represent a novel therapeutic target.

scholarly journals Interaction of Aurora-A and centrosomin at the microtubule-nucleating site in Drosophila and mammalian cells

2003 ◽  
Vol 162 (5) ◽  
pp. 757-764 ◽  
Author(s):  
Yasuhiko Terada ◽  
Yumi Uetake ◽  
Ryoko Kuriyama
Keyword(s):  
Mammalian Cells ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Mitotic Spindles ◽  
Microtubule Nucleation ◽  
Microtubule Organization ◽  
Centrosomal Protein ◽  
Spindle Poles

A mitosis-specific Aurora-A kinase has been implicated in microtubule organization and spindle assembly in diverse organisms. However, exactly how Aurora-A controls the microtubule nucleation onto centrosomes is unknown. Here, we show that Aurora-A specifically binds to the COOH-terminal domain of a Drosophila centrosomal protein, centrosomin (CNN), which has been shown to be important for assembly of mitotic spindles and spindle poles. Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of γ-tubulin and other centrosomal components to the centrosome. The NH2-terminal half of CNN interacts with γ-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells. These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring γ-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN.

scholarly journals PRMT5 Is Upregulated in Activated T Cells and Is a Novel Therapeutic Target for Acute Gvhd

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2034-2034
Author(s):  
Parvathi Ranganathan ◽  
Katiri Snyder ◽  
Nina Zizter ◽  
Hannah K. Choe ◽  
Robert A Baiocchi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
T Cells ◽  
T Cell ◽  
Western Blot ◽  
Cell Function ◽  
T Cell Proliferation ◽  
Activated T Cells ◽  
And Function

Abstract Introduction: Acute graft-versus-host disease (aGVHD), a T cell-mediated immunological disorder is the leading cause of non-relapse mortality in patients receiving allogeneic bone marrow transplants. Protein arginine methyltransferase 5 (PRMT5) catalyzes symmetric dimethylation (me2s) of arginine (R) residues on histones (primarily H3R8 and H3R4) and other proteins. PRMT5 is overexpressed in many leukemias and lymphomas, and epigenetic changes driven by PRMT5 lead to repression of tumor suppressors and promote growth and survival of cancer cells. Recently it was shown that T cells are sensitive to R-methylation and PRMT5 promotes activation of memory T helper cells. Here we investigate: 1) mechanisms by which PRMT5 regulates T cell function; and 2) PRMT5 inhibition as a therapeutic strategy for aGVHD. Materials and Methods: Splenic T cells were isolated from lethally irradiated B6D2F1 mice that received either T cell depleted bone marrow (TCD-BM) or TCD-BM with C57/BL6 (B6) allogeneic splenocytes on day 21 post-transplant. In vitro activation of B6 T cells was achieved with CD3/CD28 Dynabeads or co-culture with allogeneic BM-derived dendritic cells. PRMT5 expression (RT-PCR, western blot) and function (H3R8me2s western blot) were evaluated. PRT220, a novel inhibitor of PRMT5, was used to evaluate PRMT5 inhibition on T cell function in vitro and in vivo. We assessed T cell proliferation (Cell Trace Violet, Ki67), apoptosis (Annexin V), cytokine secretion (ELISA, flow cytometry), cell cycle (PI incorporation), and cell signaling (western blot). Lethally irradiated F1 recipients received TCD-BM only (10x106 cells) or TCD-BM + B6 splenocytes (20 x 106). Recipients of allogeneic splenocytes were treated with PRT220 (2mg/kg) or vehicle by oral gavage once weekly starting day 7 post-transplant. Mice were monitored for survival and clinical aGVHD scores. Results: PRMT5 expression and function is upregulated following T cell activation. Inhibition of PRMT5 reduces T cell proliferation and IFN-g secretion. PRMT5 inhibition in CD3/CD28 stimulated T cells results in disruption of multiple histone epigenetic marks, cell-cycle progression (via G1 arrest) and perturbation of ERK-MAPK signaling cascades. Finally, administration of PRT220 resulted in significantly prolonging the survival of allo-transplanted recipient mice (median survival, PRT220 vs. vehicle, 36.5 vs. 26 days, p=0.01). PRT220-treated recipients also exhibited significant lower aGVHD clinical (p<0.05), pathological scores (p<0.05) and lower serum TNF-a (p<0.05) and IFN-g (p<0.05) than vehicle-treated recipients. Conclusions: PRMT5 expression and function are upregulated in activated T cells. Inhibition of PRMT5 function using a novel and specific small-molecule inhibitor, PRT220, down-regulates T cells proliferative and effector response, induces cell-cycle arrest and perturbs signaling pathways. PRT220 shows potent biological activity in vivo by reducing aGVHD clinical severity and significantly prolonging survival in mouse models of aGVHD. Therefore, PRMT5 is a novel and druggable target for aGVHD. Disclosures No relevant conflicts of interest to declare.

scholarly journals 3D FIB-SEM reconstruction of microtubule–organelle interaction in whole primary mouse β cells

2020 ◽  
Vol 220 (2) ◽  
Author(s):  
Andreas Müller ◽  
Deborah Schmidt ◽  
C. Shan Xu ◽  
Song Pang ◽  
Joyson Verner D’Costa ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cell Function ◽  
Secretory Granules ◽  
Detailed Knowledge ◽  
Β Cells ◽  
Microtubule Organization ◽  
Supportive Role ◽  
Primary Mouse ◽  
Microtubule Networks ◽  
Insulin Secretory Granule

Microtubules play a major role in intracellular trafficking of vesicles in endocrine cells. Detailed knowledge of microtubule organization and their relation to other cell constituents is crucial for understanding cell function. However, their role in insulin transport and secretion is under debate. Here, we use FIB-SEM to image islet β cells in their entirety with unprecedented resolution. We reconstruct mitochondria, Golgi apparati, centrioles, insulin secretory granules, and microtubules of seven β cells, and generate a comprehensive spatial map of microtubule–organelle interactions. We find that microtubules form nonradial networks that are predominantly not connected to either centrioles or endomembranes. Microtubule number and length, but not microtubule polymer density, vary with glucose stimulation. Furthermore, insulin secretory granules are enriched near the plasma membrane, where they associate with microtubules. In summary, we provide the first 3D reconstructions of complete microtubule networks in primary mammalian cells together with evidence regarding their importance for insulin secretory granule positioning and thus their supportive role in insulin secretion.

scholarly journals The primary cilium dampens proliferative signaling and represses a G2/M transcriptional network in quiescent myoblasts

2020 ◽  
Author(s):  
Nisha Venugopal ◽  
Ananga Ghosh ◽  
Hardik Gala ◽  
Ajoy Aloysius ◽  
Neha Vyas ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Primary Cilium ◽  
Developmental Disorders ◽  
Adult Stem Cells ◽  
Primary Cilia ◽  
Translational Repressor ◽  
Elevated Protein ◽  
Proliferative Signaling

Abstract Background Reversible cell cycle arrest (quiescence/G0) is characteristic of adult stem cells and is actively controlled at multiple levels. Quiescent cells also extend a primary cilium, which functions as a signaling hub. Primary cilia have been shown to be important in multiple developmental processes, and are implicated in numerous developmental disorders. Although the association of the cilium with G0 is established, the role of the cilium in the control of the quiescence program is still poorly understood. Results Primary cilia are dynamically regulated across different states of cell cycle exit in skeletal muscle myoblasts: quiescent myoblasts elaborate a primary cilium in vivo and in vitro , but terminally differentiated myofibers do not. Myoblasts where ciliogenesis is ablated using RNAi against a key ciliary assembly protein (IFT88) can exit the cell cycle but display an altered quiescence program and impaired self-renewal. Specifically, the G0 transcriptome in IFT88 knockdown cells is aberrantly enriched for G2/M regulators, suggesting a focused repression of this network by the cilium. Cilium-ablated cells also exhibit features of activation including enhanced activity of Wnt and mitogen signaling and elevated protein synthesis via inactivation of the translational repressor 4E-BP1. Conclusions Taken together, our results show that the primary cilium integrates and dampens proliferative signaling, represses translation and G2/M genes, and is integral to the establishment of the quiescence program.

scholarly journals Specific effects of antitumor active norspermidine on the structure and function of DNA

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Takashi Nishio ◽  
Yuko Yoshikawa ◽  
Chwen-Yang Shew ◽  
Naoki Umezawa ◽  
Tsunehiko Higuchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Function ◽  
Specific Effect ◽  
Underlying Mechanism ◽  
Chemical Structures ◽  
Specific Effects ◽  
Methylene Groups ◽  
And Function ◽  
Dna Measurements

Abstract We compared the effects of trivalent polyamines, spermidine (SPD) and norspermidine (NSPD), a chemical homologue of SPD, on the structure of DNA and gene expression. The chemical structures of SPD and NSPD are different only with the number of methylene groups between amine groups, [N-3-N-4-N] and [N-3-N-3-N], respectively. SPD plays vital roles in cell function and survival, including in mammals. On the other hand, NSPD has antitumor activity and is found in some species of plants, bacteria and algae, but not in humans. We found that both polyamines exhibit biphasic effect; enhancement and inhibition on in vitro gene expression, where SPD shows definitely higher potency in enhancement but NSPD causes stronger inhibition. Based on the results of AFM (atomic force microscopy) observations together with single DNA measurements with fluorescence microscopy, it becomes clear that SPD tends to align DNA orientation, whereas NSPD induces shrinkage with a greater potency. The measurement of binding equilibrium by NMR indicates that NSPD shows 4–5 times higher affinity to DNA than SPD. Our theoretical study with Monte Carlo simulation provides the insights into the underlying mechanism of the specific effect of NSPD on DNA.

scholarly journals Exhausted CD8+ T cells exhibit low and strongly inhibited TCR signaling during chronic LCMV infection

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ioana Sandu ◽  
Dario Cerletti ◽  
Manfred Claassen ◽  
Annette Oxenius
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Viral Infections ◽  
Cell Receptor ◽  
Target Cells ◽  
Tcr Signaling ◽  
And Function

Abstract Chronic viral infections are often associated with impaired CD8+ T cell function, referred to as exhaustion. Although the molecular and cellular circuits involved in CD8+ T cell exhaustion are well defined, with sustained presence of antigen being one important parameter, how much T cell receptor (TCR) signaling is actually ongoing in vivo during established chronic infection is unclear. Here, we characterize the in vivo TCR signaling of virus-specific exhausted CD8+ T cells in a mouse model, leveraging TCR signaling reporter mice in combination with transcriptomics. In vivo signaling in exhausted cells is low, in contrast to their in vitro signaling potential, and despite antigen being abundantly present. Both checkpoint blockade and adoptive transfer of naïve target cells increase TCR signaling, demonstrating that engagement of co-inhibitory receptors curtails CD8+ T cell signaling and function in vivo.

Effect of Epidermal Growth Factor on Adult Rat Hepatocyte Monolayer Cultures

1980 ◽  
Vol 38 ◽  
pp. 778-779
Author(s):  
W. A. Samsonoff ◽  
R. Jansing
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Function ◽  
Parenchymal Cell ◽  
Male Rats ◽  
Monolayer Cultures ◽  
Epidermal Growth ◽  
And Function ◽  
Hepatocyte Monolayer

Primary hepatocyte monolayer cultures have proven useful for in vitro study of parenchymal cell function. Unfortunately these cultures lose liver function rapidly and are often short-lived. Modified subtrates (1,2) can overcome some of these problems, but they produce fewer viable cells. Specific media supplements can also prolong cellular function. Epidermal growth factor (EGF) for example, significantly affects the structure and function of cells in culture (3). In this study we determined the effect of EGF on the ultrastructure and function of hepatocytes from Fisher 344 adult male rats. They were maintained as primary monolayer cultures in Leibovitz L-15 medium with supplements.

Disruption of Golgi structure and function in mammalian cells expressing a mutant dynamin

2000 ◽  
Vol 113 (11) ◽  
pp. 1993-2002 ◽  
Author(s):  
H. Cao ◽  
H.M. Thompson ◽  
E.W. Krueger ◽  
M.A. McNiven
Keyword(s):  
Mammalian Cells ◽  
Living Cells ◽  
Dominant Negative ◽  
Coated Vesicle ◽  
Large Numbers ◽  
Golgi Structure ◽  
And Function ◽  
Golgi Network ◽  
Mutant Cells

The large GTPase dynamin is a mechanoenzyme that participates in the scission of nascent vesicles from the plasma membrane. Recently, dynamin has been demonstrated to associate with the Golgi apparatus in mammalian cells by morphological and biochemical methods. Additional studies using a well characterized, cell-free assay have supported these findings by demonstrating a requirement for dynamin function in the formation of clathrin-coated, and non-clathrin-coated vesicles from the trans-Golgi network (TGN). In this study, we tested if dynamin participates in Golgi function in living cells through the expression of a dominant negative dynamin construct (K44A). Cells co-transfected to express this mutant dynamin and a GFP-tagged Golgi resident protein (TGN38) exhibit Golgi structures that are either compacted, vesiculated, or tubulated. Electron microscopy of these mutant cells revealed large numbers of Golgi stacks comprised of highly tubulated cisternae and an extraordinary number of coated vesicle buds. Cells expressing mutant dynamin and GFP-tagged VSVG demonstrated a marked retention (8- to 11-fold) of the nascent viral G-protein in the Golgi compared to control cells. These observations in living cells are consistent with previous morphological and in vitro studies demonstrating a role for dynamin in the formation of secretory vesicles from the TGN.

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