preparation-prokaryotic-eukaryotic-organisms-using-chemical-drying

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

Central Michigan University

Tau is a microtubule binding protein implicated in a number of human neurodegenerative disorders, including Alzheimer's disease. Phosphorylation of serine-proline/threonine-proline sites, targeted by proline-directed kinases, coincides temporally with neurodegeneration in the human diseases. Recently, we demonstrated that this unique group of serines and threonines has a critical role in controlling tau toxicity in a Drosophila model of tauopathy. Here, we use a combination of genetic and biochemical approaches to examine these sites individually and to determine which of them is primarily responsible for controlling tau neurotoxicity. Despite the importance placed on individual phosphoepitopes and their contributions to disease pathogenesis, our results indicate that no single phosphorylation residue plays a dominant role in controlling tau toxicity. These findings suggest that serine-proline/threonine-proline sites cooperate to mediate neurodegeneration in vivo.

Tau phosphorylation sites work in concert to promote neurotoxicity in vivo.

Alzheimer's disease and other related neurodegenerative disorders known as tauopathies are characterized by the accumulation of abnormally phosphorylated and aggregated forms of the microtubule-associated protein tau. Several laboratories have identified a 17 kD proteolytic fragment of tau in degenerating neurons and in numerous cell culture models that is generated by calpain cleavage and speculated to contribute to tau toxicity. In the current study, we employed a Drosophila tauopathy model to investigate the importance of calpain-mediated tau proteolysis in contributing to tau neurotoxicity in an animal model of human neurodegenerative disease. We found that mutations that disrupted endogenous calpainA or calpainB activity in transgenic flies suppressed tau toxicity. Expression of a calpain-resistant form of tau in Drosophila revealed that mutating the putative calpain cleavage sites that produce the 17 kD fragment was sufficient to abrogate tau toxicity in vivo. Furthermore, we found significant toxicity in the fly retina associated with expression of only the 17 kD tau fragment. Collectively, our data implicate calpain-mediated proteolysis of tau as an important pathway mediating tau neurotoxicity in vivo.

Implicating calpain in tau-mediated toxicity in vivo.

La maladie d&#39;Alzheimer et autres troubles neurod&eacute;g&eacute;n&eacute;ratifs apparent&eacute;s appel&eacute;es tauopathies sont caract&eacute;ris&eacute;es par l&#39;accumulation d&#39;anormalement phosphoryl&eacute;e et agr&eacute;g&eacute;es des formes de la prot&eacute;ine associ&eacute;e aux microtubules tau. Plusieurs laboratoires ont identifi&eacute; un fragment prot&eacute;olytique de 17 kD de tau dans la d&eacute;g&eacute;n&eacute;rescence des neurones et dans nombreux mod&egrave;les de culture cellulaire qui est produite par clivage de la calpa&iuml;ne et sp&eacute;cul&eacute; &agrave; contribuer &agrave; la toxicit&eacute; du tau. Dans la pr&eacute;sente &eacute;tude, nous avons utilis&eacute; un mod&egrave;le de tauopathy de drosophile pour &eacute;tudier l&#39;importance de la prot&eacute;olyse de la calpa&iuml;ne-mediated tau en contribuant &agrave; la neurotoxicit&eacute; de tau dans un mod&egrave;le animal de maladie neurod&eacute;g&eacute;n&eacute;rative humaine. Nous avons d&eacute;couvert que des mutations qui ont perturb&eacute; l&#39;activit&eacute; endog&egrave;ne calpainA ou calpainB chez les mouches transg&eacute;niques supprim&eacute;e toxicit&eacute; tau. Expression d&#39;une forme r&eacute;sistante &agrave; la calpa&iuml;ne de tau chez la drosophile ont r&eacute;v&eacute;l&eacute; que les sites de clivage calpa&iuml;ne putatif qui produisent le fragment de kD 17 la mutation &eacute;tait suffisante pour abroger la toxicit&eacute; tau in vivo. En outre, nous avons trouv&eacute; une toxicit&eacute; importante dans la r&eacute;tine mouche associ&eacute;e &agrave; l&#39;expression du fragment de tau de kD 17 seulement. Collectivement, nos donn&eacute;es indiquent qu&#39;induite par la calpa&iuml;ne prot&eacute;olyse de tau comme une voie importante m&eacute;diation neurotoxicit&eacute; tau in vivo.

Impliquant calpa&iuml;ne dans la toxicit&eacute; induite par le tau in vivo.

Malattia di Alzheimer e altre patologie neurodegenerative correlate noti come tauopatie sono caratterizzati dall&#39;accumulo di anormalmente fosforilato e aggregati in forme di tau microtubule-associated protein. Diversi laboratori hanno identificato un frammento proteolitico di 17 kD di tau nella degenerazione dei neuroni e in numerosi modelli di coltura cellulare che &egrave; generato dalla scissione calpain e speculato per contribuire alla tossicit&agrave; del tau. In questo studio, abbiamo impiegato un modello di nota come taupatia drosofila per indagare l&#39;importanza della proteolisi mediata calpain tau contribuendo alla neurotossicit&agrave; tau in un modello animale della malattia neurodegenerativa umana. Abbiamo trovato che le mutazioni che interrotta attivit&agrave; endogena calpainA o calpainB in mosche transgeniche soppressa tossicit&agrave; tau. Espressione di una forma calpain-resistente di tau in Drosophila ha rivelato che mutando i siti di clivaggio calpain putativo che producono il frammento 17 kD &egrave; stato sufficiente per abrogare tossicit&agrave; tau in vivo. Inoltre, abbiamo trovato una significativa tossicit&agrave; nella retina vola associata all&#39;espressione del solo 17 kD tau frammento. Collettivamente, i nostri dati coinvolgono proteolisi calpain-mediata del tau come un importante percorso mediazione neurotossicit&agrave; tau in vivo.

Implicando calpain tau-mediata tossicit&agrave; in vivo.

The fruit fly, Drosophila melanogaster, is a powerful model system for applying molecular, cellular, and genetic approaches to understanding human tauopathies, including Alzheimer's disease. Here, we provide an introduction to using Drosophila as a tauopathy model system and describe several protocols that we use to analyze human tau protein expressed in flies. Methods to detect tau expression include light and scanning electron microscopy in the fly eye, confocal microscopy of primary neuronal cultures, and preparation of tissue homogenates for separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis with analysis by Western blotting.

Michelle L. Steinhilb

Department of Biology

Central Michigan University

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Michelle L. Steinhilb

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biology

Central Michigan University

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Department of Biology