Regulation of protein transport from the Golgi complex to the endoplasmic reticulum by CDC42 and N-WASP.

Actin is involved in the organization of the Golgi complex and Golgi-to-ER protein transport in mammalian cells. Little, however, is known about the regulation of the Golgi-associated actin cytoskeleton. We provide evidence that Cdc42, a small GTPase that regulates actin dynamics, controls Golgi-to-ER protein transport. We located GFP-Cdc42 in the lateral portions of Golgi cisternae and in COPI-coated and noncoated Golgi-associated transport intermediates. Overexpression of Cdc42 and its activated form Cdc42V12 inhibited the retrograde transport of Shiga toxin from the Golgi complex to the ER, the redistribution of the KDEL receptor, and the ER accumulation of Golgi-resident proteins induced by the active GTP-bound mutant of Sar1 (Sar1[H79G]). Coexpression of wild-type or activated Cdc42 and N-WASP also inhibited Golgito-ER transport, but this was not the case in cells expressing Cdc42V12 and N-WASP(AWA), a mutant form of N-WASP that lacks Arp2/3 binding. Furthermore, Cdc42V12 recruited GFP-NWASP to the Golgi complex. We therefore conclude that Cdc42 regulates Golgi-to-ER protein transport in an N-WASP¿dependent manner

Estímulo del trabajo autónomo en el aprendizaje práctico de la Histología: una experiencia transversal en Ciencias de la salud

El estudio microscópico de tejidos y órganos constituye un aspecto muy importante del aprendizaje de la histología, pero no siempre se dispone de suficientes colecciones de muestras, sobre todo humanas, para que el alumnado pueda disponer de ellas de forma presencial durante las prácticas. Por ello, el portal virtual digital slidebox (DSB) permite al profesorado generar una colección de preparaciones histológicas virtuales, de manera que los estudiantes tienen acceso "online" a estas muestras y pueden visualizarlas a la misma escala que ofrece la observación directa del microscopio óptico, e incluso mayor. Para la realización de cada práctica en sus horas presenciales el alumno dispone previamente de un guión con los objetivos que debe cumplir al realizar la observación de cada preparación histológica. Al final de cada práctica presencial los objetivos son explicados por el alumno, y evaluados por el profesor, realizando una puesta en común. Una vez realizada la práctica, y en horario no presencial, el alumnado tiene la posibilidad de completar este trabajo usando el portal virtual DSB. Con las imágenes digitales captadas el alumno realiza su portafolio de prácticas incorporando dichas imágenes y rotulándolas. Al final del curso se realiza un examen práctico global y la evaluación final del portafolio

Role of age and comorbidities in mortality of patients with infective endocarditis

[Purpose]: The aim of this study was to analyse the characteristics of patients with IE in three groups of age and to assess the ability of age and the Charlson Comorbidity Index (CCI) to predict mortality. [Methods]: Prospective cohort study of all patients with IE included in the GAMES Spanish database between 2008 and 2015.Patients were stratified into three age groups:<65 years,65 to 80 years,and ≥ 80 years.The area under the receiver-operating characteristic (AUROC) curve was calculated to quantify the diagnostic accuracy of the CCI to predict mortality risk. [Results]: A total of 3120 patients with IE (1327 < 65 years;1291 65-80 years;502 ≥ 80 years) were enrolled.Fever and heart failure were the most common presentations of IE, with no differences among age groups.Patients ≥80 years who underwent surgery were significantly lower compared with other age groups (14.3%,65 years; 20.5%,65-79 years; 31.3%,≥80 years). In-hospital mortality was lower in the <65-year group (20.3%,<65 years;30.1%,65-79 years;34.7%,≥80 years;p < 0.001) as well as 1-year mortality (3.2%, <65 years; 5.5%, 65-80 years;7.6%,≥80 years; p = 0.003).Independent predictors of mortality were age ≥ 80 years (hazard ratio [HR]:2.78;95% confidence interval [CI]:2.32–3.34), CCI ≥ 3 (HR:1.62; 95% CI:1.39–1.88),and non-performed surgery (HR:1.64;95% CI:11.16–1.58).When the three age groups were compared,the AUROC curve for CCI was significantly larger for patients aged <65 years(p < 0.001) for both in-hospital and 1-year mortality. [Conclusion]: There were no differences in the clinical presentation of IE between the groups. Age ≥ 80 years, high comorbidity (measured by CCI),and non-performance of surgery were independent predictors of mortality in patients with IE.CCI could help to identify those patients with IE and surgical indication who present a lower risk of in-hospital and 1-year mortality after surgery, especially in the <65-year group

Focus on the Small GTPase Rab1: A Key Player in the Pathogenesis of Parkinson’s Disease

Parkinson’s disease (PD) is the second most frequent neurodegenerative disease. It is characterized by the loss of dopaminergic neurons in the substantia nigra and the formation of large aggregates in the survival neurons called Lewy bodies, which mainly contain α-synuclein (α-syn). The cause of cell death is not known but could be due to mitochondrial dysfunction, protein homeostasis failure, and alterations in the secretory/endolysosomal/autophagic pathways. Survival nigral neurons overexpress the small GTPase Rab1. This protein is considered a housekeeping Rab that is necessary to support the secretory pathway, the maintenance of the Golgi complex structure, and the regulation of macroautophagy from yeast to humans. It is also involved in signaling, carcinogenesis, and infection for some pathogens. It has been shown that it is directly linked to the pathogenesis of PD and other neurodegenerative diseases. It has a protective effect against α–σψν toxicity and has recently been shown to be a substrate of LRRK2, which is the most common cause of familial PD and the risk of sporadic disease. In this review, we analyze the key aspects of Rab1 function in dopamine neurons and its implications in PD neurodegeneration/restauration. The results of the current and former research support the notion that this GTPase is a good candidate for therapeutic strategies

Análisis de la biogénesis y estructura del acrosoma de rata mediante citoquímica ultraestructural y crioinmunotécnicas / José Angel Martínez Menárguez ; directores José Ballesta Germán, Hans J. Geuze.

Tesis-Universidad de Murcia.Bibliografia: p. 234-272.Consulte la tesis en: BCA. GENERAL. ARCHIVO UNIVERSITARIO. D 463.Consulte la tesis en: BCA. GENERAL. ARCHIVO UNIVERSITARIO. T.M.-1342

Caracterización citoquímica, distribución y modelo de formación de cadenas oligasocarídicas en el acrosoma de rata / José Angel Martínez Menárguez ; José Ballesta Germán, director.

Tesis - Universidad de Murcia.Consulte la tesis en: BCA. GENERAL. ARCHIVO UNIVERSITARIO. TL 242

Structure and Dynamics of the Golgi Complex at 15ºC: Low Temperature Induces the Formation of Golgi-Derived Tubules

Immunofluorescence and cryoimmunoelectron microscopy were used to examine the morphological and functional effects on the Golgi complex when protein transport is blocked at the ERGIC (ER-Golgi intermediate compartment) in HeLa cells incubated at low temperature (15ºC). At this temperature, the Golgi complex showed long tubules containing resident glycosylation enzymes but not matrix proteins. These Golgi-derived tubules also lacked anterograde (VSV-G) or retrograde (Shiga toxin) cargo. The formation of tubules was dependent on both energy and intact microtubule and actin cytoskeletons. Conversely, brefeldin A or cycloheximide treatments did not modify the appearance. When examined at the electron microscope, Golgi stacks were long and curved and appeared connected to tubules immunoreactive to galactosyltransferase antibodies but devoid of Golgi matrix proteins. Strikingly, COPI proteins moved from membranes to the cytosol at 15ºC which could explain the formation of tubules

Low temperature (15ºC) induces COPII dissociation from membranes and slow exit from the endoplasmic reticulum in HeLa cells

Low temperature induces a transport blockade at the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) in cultured cells. Our previous studies support that the primary effect of low temperature is the detachment of COPI complexes from membranes. In the present study, we have used immunofluorescence and cryoimmunoelectron microscopy to investigate the effects of low temperature on both COPII and clathrin coat complexes in HeLa cells. Strikingly, COPII proteins moved from membranes to the cytosol at 15ºC, accumulating into electron-dense areas. In agreement with this observation, we also showed that ER exit is delayed in cells cultured at this temperature. By contrast, clathrin coat is not affected. Together, our results demonstrate that low temperature induces COPII dissociation from membranes and slow exit from the endoplasmic reticulum

Molecular and morphological analysis of the golgi apparatus of dopaminergic neurons in human substantia nigra: new citopathological findings in Parkinson's disease

Fragmentation of the Golgi ribbon is a common feature of many neurodegenerative diseases, as a Parkinson´s disease, but little is known about the causes of this alteration. It is believed it could be the consequence of an ER-Golgi transport imbalance and/or cytoskeleton alterations. Usually, cytopathological findings of the disease have been described using different experimental models of Parkinson´s disease but it do not exist enough studies in human tissue that help to understand the cell biology of this pathology. In the present study we analyze the morphological characteristics of Golgi apparatus and possible molecules involved in Golgi fragmentation in human samples of substantia nigra of Parkinson´s disease necropsies. Our previous studies, using a cellular model of Parkinson´s disease, demonstrated that the homeostasis of a limited number of proteins could be directly related with Golgi fragmentation, alterations in intracellular trafficking and Lewy body aggregation. We have also confirmed, using necropsies of nigra substantia Parkinson´s patients, changes in the levels Golgi reassembly-stacking GRASP65 and golgin 84 which could be implicated in golgi apparatus fragmentation. We have also demonstrated, that exists main alterations in the distribution and levels of Rab1 and Syntaxin 5. In Parkinson´s disease necropsies, interestly, Syntaxin 5 is accumulated mainly out of the cell in extracellular inclusions. These differences found in Parkinson´s samples in comparison with control samples become in important hallmarks for understanding cytopathology of Parkinson´s disease

Golgi Fragmentation in Neurodegenerative Diseases: Is There a Common Cause?

In most mammalian cells, the Golgi complex forms a continuous ribbon. In neurodegenerative diseases, the Golgi ribbon of a specific group of neurons is typically broken into isolated elements, a very early event which happens before clinical and other pathological symptoms become evident. It is not known whether this phenomenon is caused by mechanisms associated with cell death or if, conversely, it triggers apoptosis. When the phenomenon was studied in diseases such as Parkinson's and Alzheimer's or amyotrophic lateral sclerosis, it was attributed to a variety of causes, including the presence of cytoplasmatic protein aggregates, malfunctioning of intracellular tra c and/or alterations in the cytoskeleton. In the present review, we summarize the current findings related to these and other neurodegenerative diseases and try to search for clues on putative common causes