scholarly journals Poly(ADP-ribosylation) is present in murine sciatic nerve fibers and is altered in a Charcot-Marie-Tooth-1E neurodegenerative model

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3318 ◽  
Author(s):  
Laura I. Lafon Hughes ◽  
Carlos J. Romeo Cardeillac ◽  
Karina B. Cal Castillo ◽  
Salomé C. Vilchez Larrea ◽  
José R. Sotelo Sosa ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Cortical Neurons ◽  
Cell Biology ◽  
Protein Modification ◽  
Nerve Fibers ◽  
Parp Inhibition ◽  
Sciatic Nerves ◽  
First Time ◽  
E Cadherin

BackgroundPoly-ADP-ribose (PAR) is a polymer synthesized by poly-ADP-ribose polymerases (PARPs) as a postranslational protein modification and catabolized mainly by poly-ADP-ribose glycohydrolase (PARG). In spite of the existence of cytoplasmic PARPs and PARG, research has been focused on nuclear PARPs and PAR, demonstrating roles in the maintenance of chromatin architecture and the participation in DNA damage responses and transcriptional regulation. We have recently detected non-nuclear PAR structurally and functionally associated to the E-cadherin richzonula adherensand the actin cytoskeleton of VERO epithelial cells. Myelinating Schwann cells (SC) are stabilized by E-cadherin rich autotypicadherens junctions (AJ). We wondered whether PAR would map to these regions. Besides, we have demonstrated an altered microfilament pattern in peripheral nerves of Trembler-J (Tr-J) model of CMT1-E. We hypothesized that cytoplasmic PAR would accompany such modified F-actin pattern.MethodsWild-type (WT) and Tr-J mice sciatic nerves cryosections were subjected to immunohistofluorescence with anti-PAR antibodies (including antibody validation), F-actin detection with a phalloidin probe and DAPI/DNA counterstaining. Confocal image stacks were subjected to a colocalization highlighter and to semi-quantitative image analysis.ResultsWe have shown for the first time the presence of PAR in sciatic nerves. Cytoplasmic PAR colocalized with F-actin at non-compact myelin regions in WT nerves. Moreover, in Tr-J, cytoplasmic PAR was augmented in close correlation with actin. In addition, nuclear PAR was detected in WT SC and was moderately increased in Tr-J SC.DiscussionThe presence of PAR associated to non-compact myelin regions (which constitute E-cadherin rich autotypicAJ/actin anchorage regions) and the co-alterations experienced by PAR and the actin cytoskeleton in epithelium and nerves, suggest that PAR may be a constitutive component ofAJ/actin anchorage regions. Is PAR stabilizing theAJ-actin complexes? This question has strong implications in structural cell biology and cell signaling networks. Moreover, if PAR played a stabilizing role, such stabilization could participate in the physiological control of axonal branching. PARP and PAR alterations exist in several neurodegenerative pathologies including Alzheimer’s, Parkinson’s and Hungtington’s diseases. Conversely, PARP inhibition decreases PAR and promotes neurite outgrowth in cortical neuronsin vitro. Coherently, the PARP inhibitor XAV939 improves myelinationin vitro,ex vivoandin vivo. Until now such results have been interpreted in terms of nuclear PARP activity. Our results indicate for the first time the presence of PARylation in peripheral nerve fibers, in a healthy environment. Besides, we have evidenced a PARylation increase in Tr-J, suggesting that the involvement of cytoplasmic PARPs and PARylation in normal and neurodegenerative conditions should be re-evaluated.

scholarly journals Innate lymphoid cells lacking surface expression of CD90 are functional

2021 ◽  
Author(s):  
J-H Schroeder ◽  
T Zabinski ◽  
J F Neves ◽  
GM Lord
Keyword(s):  
Steady State ◽  
Cell Biology ◽  
Surface Expression ◽  
Innate Lymphoid Cells ◽  
Lymphoid Cells ◽  
T Cell Biology ◽  
First Time ◽  
Made In

ABSTRACTHuge progress has been made in understanding the biology of innate lymphoid cells (ILC) by adopting several well-known concepts of T cell biology. As such flow cytometry gating strategies and markers, such as CD90, to identify ILC were discovered. Here we report that most non-NK intestinal ILC have a high expression of CD90 as expected, but surprisingly some have only a low or even no expression of this marker. CD90-negative CD127+ ILC were identified among all ILC subsets in the gut. CD90-negative cLP ILC2 were frequent at steady state. The frequency of CD90-negative CD127+ ILC was dependent on stimulatory cues in vitro and in vivo, and CD90-negative CD127+ ILC played a functional role as a source of IL-13, IFNγ and IL-17A at steady state and upon dextran sulphate sodium-elicited colitis. Hence, this study highlights for the first time that CD90 is not constitutively expressed by functional ILC in the gut.

scholarly journals Association of PDZ-containing protein PDZD11 with the human sodium-dependent multivitamin transporter

2011 ◽  
Vol 300 (4) ◽  
pp. G561-G567 ◽  
Author(s):  
Svetlana M. Nabokina ◽  
Veedamali S. Subramanian ◽  
Hamid M. Said
Keyword(s):  
Cell Biology ◽  
Protein S ◽  
Confocal Imaging ◽  
Intestinal Epithelial ◽  
Sodium Dependent ◽  
Interfering Rna ◽  
First Time ◽  
Two Hybrid

Intestinal absorption of biotin is mediated via the sodium-dependent multivitamin transporter (SMVT). Studies from our laboratory and others have characterized different aspects of the human SMVT (hSMVT), but nothing is currently known about protein(s) that may interact with hSMVT and affect its physiology/biology. In this study, a PDZ-containing protein PDZD11 was identified as an interacting partner with hSMVT using yeast two-hybrid screen of a human intestinal cDNA library. The interaction between hSMVT and PDZD11 was confirmed by in vitro GST-pull-down assay and in vivo in a mammalian cell environment by a two-hybrid luciferase and coimmunoprecipitation assays. Furthermore, confocal imaging of live human intestinal epithelial HuTu-80 cells expressing hSMVT-GFP and DsRed-PDZD11 demonstrated colocalization of these two proteins. We also examined the functional consequence of the interaction between hSMVT and PDZD11 in HuTu-80 cells and observed significant induction in [3H]biotin uptake upon coexpression of hSMVT and PDZD11. In contrast, knocking down of PDZD11 with gene-specific small interfering RNA led to a significant decrease in biotin uptake; biotinylation assay showed this to be associated with a marked decrease in level of expression of hSMVT at the cell membrane. By truncation approach, we also demonstrated that the PDZ binding domain that is located in the COOH-terminal tail of hSMVT polypeptide is involved in the interaction with PDZD11. These results demonstrate for the first time that PDZD11 is an interacting partner with hSMVT in intestinal epithelial cells and that this interaction affects hSMVT function and cell biology.

Application of a multiwire proportional chamber to the detection of axoplasmic transport

1976 ◽  
Vol 54 (3) ◽  
pp. 238-244 ◽  
Author(s):  
R. E. Snyder ◽  
R. A. Reynolds ◽  
R. S. Smith ◽  
W. S. Kendal
Keyword(s):  
Room Temperature ◽  
Nerve Fibers ◽  
Axoplasmic Transport ◽  
Nerve Activity ◽  
Proportional Chamber ◽  
Nerve Ganglion ◽  
Multiwire Proportional Chamber ◽  
Sciatic Nerves ◽  
A New Technique

A new technique for the detection of the axoplasmic transport of β-radioactively labelled materials is described wherein a multiwire proportional chamber is used to measure the distribution of activity along peripheral nerve fibers which are maintained in vitro. The operating principles of the chamber are described and basic construction parameters given. Potential radiolabels are discussed.Two types of studies were performed at room temperature in vitro using sciatic nerves of the amphibian Xenopus laevis: static and dynamic. In the static study the nerve ganglion was incubated for a suitable period of time in either L-[U-14C]leucine or L-[35S]methionine after which the ganglion was removed and the activity in the remaining nerve assayed with the chamber. In the dynamic study the nerve activity was assayed by the chamber while incubation proceeded so that a dynamic picture of transport could be observed. Using the second approach, transport rates were observed which are in agreement with others which have been reported in the literature.Some advantages and limitations of the technique are discussed.

scholarly journals Identification and Characterization of a CandidateWolbachia pipientisType IV Effector That Interacts with the Actin Cytoskeleton

mBio ◽  
2016 ◽  
Vol 7 (4) ◽  
Author(s):  
Kathy B. Sheehan ◽  
MaryAnn Martin ◽  
Cammie F. Lesser ◽  
Ralph R. Isberg ◽  
Irene L. G. Newton
Keyword(s):  
Actin Cytoskeleton ◽  
Cell Biology ◽  
Direct Interaction ◽  
Growth Defect ◽  
Rhodamine Phalloidin ◽  
Wolbachia Pipientis ◽  
Persistent Infections ◽  
Host Development

ABSTRACTMany bacteria live as intracellular symbionts, causing persistent infections within insects. One extraordinarily common infection is that ofWolbachia pipientis, which infects 40% of insect species and induces reproductive effects. The bacteria are passed from generation to generation both vertically (through the oocyte) and horizontally (by environmental transmission). Maintenance of the infection withinDrosophila melanogasteris sensitive to the regulation of actin, asWolbachiainefficiently colonizes strains hemizygous for the profilin or villin genes. Therefore, we hypothesized thatWolbachiamust depend on the host actin cytoskeleton. In this study, we identify and characterize aWolbachiaprotein (WD0830) that is predicted to be secreted by the bacterial parasite. Expression of WD0830 in a model eukaryote (the yeastSaccharomyces cerevisiae) induces a growth defect associated with the appearance of aberrant, filamentous structures which colocalize with rhodamine-phalloidin-stained actin. Purified WD0830 bundles actinin vitroand cosediments with actin filaments, suggesting a direct interaction of the two proteins. We characterized the expression of WD0830 throughoutDrosophiladevelopment and found it to be upregulated in third-instar larvae, peaking in early pupation, during the critical formation of adult tissues, including the reproductive system. In transgenic flies, heterologously expressed WD0830 localizes to the developing oocyte. Additionally, overexpression of WD0830 results in increasedWolbachiatiters in whole flies, in stage 9 and 10 oocytes, and in embryos, compared to controls, suggesting that the protein may facilitateWolbachia’s replication or transmission. Therefore, this candidate secreted effector may play a role inWolbachia’s infection of and persistence within host niches.IMPORTANCEThe obligate intracellularWolbachia pipientisis a ubiquitous alphaproteobacterial symbiont of arthropods and nematodes and is related to the rickettsial pathogensEhrlichiaspp. andAnaplasmaspp. Studies ofWolbachiacell biology suggest that this bacterium relies on host actin for efficient proliferation and transmission between generations. Here, we identified and characterized aWolbachiaprotein that localizes to and manipulates the eukaryotic actin cytoskeleton, is expressed byWolbachiaduring host development, and altersWolbachiatiters and localization in transgenic fruit flies. We hypothesize that WD0830 may be utilized by the bacterium to facilitate replication in or invasion of different niches during host development.

scholarly journals BrainPhys neuronal medium optimized for imaging and optogenetics in vitro

2020 ◽  
Author(s):  
Michael Zabolocki ◽  
Kasandra McCormack ◽  
Mark van den Hurk ◽  
Bridget Milky ◽  
Andrew Shoubridge ◽  
...  
Keyword(s):  
Neuronal Activity ◽  
Cortical Neurons ◽  
Cell Biology ◽  
Cellular Reprogramming ◽  
Synaptic Activity ◽  
Neuronal Cultures ◽  
Light Spectrum ◽  
Human Neurons ◽  
Wide Range

AbstractThe capabilities of imaging technologies, fluorescent sensors, and optogenetics tools for cell biology have improved exponentially in the last ten years. At the same time, advances in cellular reprogramming and organoid engineering have quickly expanded the use of human neuronal models in vitro. Altogether this creates an increasing need for tissue culture conditions better adapted to live-cell imaging. Here, we identified multiple caveats of traditional media when used for live imaging and functional assays on neuronal cultures (e.g., phototoxicity, suboptimal fluorescence signals, and unphysiological neuronal activity). To overcome these issues, we developed a new neuromedium, “BrainPhys™ Imaging”, in which we adjusted fluorescent and phototoxic compounds. The new medium is based on the formulation of the original BrainPhys medium, which we designed to better support the neuronal activity of human neurons in vitro1. We tested the new imaging-optimized formulation on human neurons cultured in monolayers or organoids, and rat primary neurons. BrainPhys Imaging enhanced fluorescence signals and reduced phototoxicity throughout the entire light spectrum. Importantly, consistent with standard BrainPhys, we showed that the new imaging medium optimally supports the electrical and synaptic activity of midbrain and human cortical neurons in culture. We also benchmarked the capacity of the new medium for functional calcium imaging and optogenetic control of human neurons. Altogether, our study shows that the new BrainPhys Imaging improves the quality of a wide range of fluorescence imaging applications with live neurons in vitro while supporting cell viability and neuronal functions.

Cell Biology of Laryngeal Epithelial Defenses in Health and Disease: Further Studies

2003 ◽  
Vol 112 (6) ◽  
pp. 481-491 ◽  
Author(s):  
Nikki Johnston ◽  
David Bulmer ◽  
Peter E. Ross ◽  
Sophie E. Axford ◽  
Gulnaz A. Gill ◽  
...  
Keyword(s):  
Cell Biology ◽  
Laryngopharyngeal Reflux ◽  
Cell Damage ◽  
Experimental Studies ◽  
Posterior Commissure ◽  
Health And Disease ◽  
Vitro Model ◽  
International Collaborative Research ◽  
E Cadherin

This is the second annual report of an international collaborative research group that is examining the cellular impact of laryngopharyngeal reflux (LPR) on laryngeal epithelium. The results of clinical and experimental studies are presented. Carbonic anhydrase (CA), E-cadherin, and MUC gene expression were analyzed in patients with LPR, in controls, and in an in vitro model. In patients with LPR, we found decreased levels of CAIII in vocal fold epithelium and increased levels in posterior commissure epithelium. The experimental studies confirm that laryngeal CAIII is depleted in response to reflux. Also, cell damage does occur well above pH 4.0. In addition, E-cadherin (transmembrane cell surface molecules, which have a key function in epithelial cell adhesion) was not present in 37% of the LPR laryngeal specimens. In conclusion, the laryngeal epithelium lacks defenses comparable to those in esophageal epithelium, and these differences may contribute to the increased susceptibility of laryngeal epithelium to reflux-related injury.

scholarly journals Calpain activation and disturbance of autophagy are induced in cortical neurons in vitro by exposure to HA/β-Ga2O3:Cr3+ nanoparticles

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4365 ◽  
Author(s):  
Yu Lei ◽  
Chengkun Wang ◽  
Quan Jiang ◽  
Xiaoyi Sun ◽  
Yongzhong Du ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Engineered Nanoparticles ◽  
Catalytic Reactions ◽  
Inflammatory Signaling ◽  
Primary Cortical Neurons ◽  
Calpain Activation ◽  
Dose Dependent Decrease ◽  
First Time ◽  
Dose Dependent

The toxicity of engineered nanoparticles remains a concern. The knowledge of biohazards associated with particular nanoparticles is crucial to make this cutting-edge technology more beneficial and safe. Here, we evaluated the toxicity of Ga2O3 nanoparticles (NPs), which are frequently used to enhance the performance of metal catalysts in a variety of catalytic reactions. The potential inflammatory signaling associated with the toxicity of HA/β-Ga2O3:Cr3+ NPs in primary cortical neurons was examined. We observed a dose-dependent decrease in cell viability and an increase in apoptosis in neurons following various concentrations (0, 1, 5, 25, 50, 100 µg/ml) of HA/β-Ga2O3:Cr3+ NPs treatment. Consistently, constitutively active forms of calcineurin (48 kDa) were significantly elevated in cultured primary cortical neurons, which was consistent with calpain activation indicated by the breakdown products of spectrin. Moreover, HA/β-Ga2O3:Cr3+ NPs result in the elevation of LC3-II formation, SQSTM/p62, and Cathepsin B, whereas phosphorylation of CaMKII (Thr286) and Synapsin I (Ser603) were downregulated in the same context. Taken together, these results demonstrate for the first time that calpain activation and a disturbance of autophagy signaling are evoked by exposure to HA/β-Ga2O3:Cr3+ NPs, which may contribute to neuronal injury in vitro.

Cross-talk unfolded: MARCKS proteins

2002 ◽  
Vol 362 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Anna ARBUZOVA ◽  
Arndt A. P. SCHMITZ ◽  
Guy VERGÈRES
Keyword(s):  
Actin Cytoskeleton ◽  
Cross Talk ◽  
Protein Modification ◽  
Membrane Binding ◽  
Point Of View ◽  
Cellular Migration ◽  
Effector Domain ◽  
Kinase Substrate ◽  
Natively Unfolded

The proteins of the MARCKS (myristoylated alanine-rich C kinase substrate) family were first identified as prominent substrates of protein kinase C (PKC). Since then, these proteins have been implicated in the regulation of brain development and postnatal survival, cellular migration and adhesion, as well as endo-, exo- and phago-cytosis, and neurosecretion. The effector domain of MARCKS proteins is phosphorylated by PKC, binds to calmodulin and contributes to membrane binding. This multitude of mutually exclusive interactions allows cross-talk between the signal transduction pathways involving PKC and calmodulin. This review focuses on recent, mostly biophysical and biochemical results renewing interest in this protein family. MARCKS membrane binding is now understood at the molecular level. From a structural point of view, there is a consensus emerging that MARCKS proteins are ‘natively unfolded'. Interestingly, domains similar to the effector domain have been discovered in other proteins. Furthermore, since the effector domain enhances the polymerization of actin in vitro, MARCKS proteins have been proposed to mediate regulation of the actin cytoskeleton. However, the recent observations that MARCKS might serve to sequester phosphatidylinositol 4,5-bisphosphate in the plasma membrane of unstimulated cells suggest an alternative model for the control of the actin cytoskeleton. While myristoylation is classically considered to be a co-translational, irreversible event, new reports on MARCKS proteins suggest a more dynamic picture of this protein modification. Finally, studies with mice lacking MARCKS proteins have investigated the functions of these proteins during embryonic development in the intact organism.

scholarly journals REGENERATION OF AXIS CYLINDERS IN VITRO

1913 ◽  
Vol 18 (4) ◽  
pp. 412-415 ◽  
Author(s):  
Ragnvald Ingebrigtsen
Keyword(s):  
Guinea Pigs ◽  
Nerve Fibers ◽  
Spinal Ganglia ◽  
First Time

It has been shown for the first time that nerve fibers grow out from pieces of cerebellum of young cats and guinea pigs, when cultivated in coagulated plasma. The same phenomenon has been observed in cultures of spinal ganglia. The nerve fibers do not anastomose and they extend into the plasma unaccompanied by structures of any kind.

scholarly journals The melibiose-derived glycation product mimics a unique epitope present in human and animal tissues

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Magdalena Staniszewska ◽  
Agnieszka Bronowicka-Szydełko ◽  
Kinga Gostomska-Pampuch ◽  
Jerzy Szkudlarek ◽  
Arkadiusz Bartyś ◽  
...  
Keyword(s):  
Cell Biology ◽  
Resonance Spectroscopy ◽  
Open Chain ◽  
Glycation Product ◽  
Glycation End Products ◽  
Patients With Diabetes ◽  
Living Organisms ◽  
First Time

AbstractNon-enzymatic modification of proteins by carbohydrates, known as glycation, leads to generation of advanced glycation end-products (AGEs). In our study we used in vitro generated AGEs to model glycation in vivo. We discovered in vivo analogs of unusual melibiose-adducts designated MAGEs (mel-derived AGEs) synthesized in vitro under anhydrous conditions with bovine serum albumin and myoglobin. Using nuclear magnetic resonance spectroscopy we have identified MAGEs as a set of isomers, with open-chain and cyclic structures, of the fructosamine moiety. We generated a mouse anti-MAGE monoclonal antibody and show for the first time that the native and previously undescribed analogous glycation product exists in living organisms and is naturally present in tissues of both invertebrates and vertebrates, including humans. We also report MAGE cross-reactive auto-antibodies in patients with diabetes. We anticipate our approach for modeling glycation in vivo will be a foundational methodology in cell biology. Further studies relevant to the discovery of MAGE may contribute to clarifying disease mechanisms and to the development of novel therapeutic options for diabetic complications, neuropathology, and cancer.

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