Disturbed Cytoskeleton Research Articles

CO2-induced ocean acidification impairs the immune function of the Pacific oyster against Vibrio splendidus challenge: An integrated study from a cellular and proteomic perspective

Ocean acidification (OA) and pathogenic diseases pose a considerable threat to key species of marine ecosystem. However, few studies have investigated the combined impact of reduced seawater pH and pathogen challenge on the immune responses of marine invertebrates. In this study, Pacific oysters, Crassostrea gigas, were exposed to OA (~2000 ppm) for 28 days and then challenged with Vibrio splendidus for another 72 h. Hemocyte parameters showed that V. splendidus infection exacerbated the impaired oyster immune responses under OA exposure. An iTRAQ-based quantitative proteomic analysis revealed that C. gigas responded differently to OA stress and V. splendidus challenge, alone or in combination. Generally, OA appears to act via a generalized stress response by causing oxidative stress, which could lead to cellular injury and cause disruption to the cytoskeleton, protein turnover, immune responses and energy metabolism. V. splendidus challenge in oysters could suppress the immune system directly and lead to a disturbed cytoskeleton structure, increased protein turnover and energy metabolism suppression, without causing oxidative stress. The combined OA- and V. splendidus-treated oysters ultimately presented a similar, but stronger proteomic response pattern compared with OA treatment alone. Overall, the impaired oyster immune functions caused by OA exposure may have increased the risk of V. splendidus infection. These results have important implications for the impact of OA on disease outbreaks in marine invertebrates, which would have significant economic and ecological repercussions.

Mast cell activation is involved in stress-induced epithelial barrier dysfunction in the esophagus.

We aimed to investigate the role of mast cell in stress-induced barrier dysfunction in the esophagus and its possible pathway involved using mast cell-deficient (Ws/Ws) rats. Ws/Ws rats and normal (+/+) rats were submitted to chronic restraint stress (CRS) 2 h/day for 7 days. Tissues were obtained from distal esophagus. Mast cells were counted under Alcian blue-safranin O stain. Activation of mast cells was assessed using transmission electron microscope. Esophageal epithelial barrier dysfunction was evaluated by measuring intercellular spaces (ICS) and by quantifying tight junction (TJ) proteins. The localization and expression of mast cell-derived tryptase and proteinase activated receptor 2 (PAR-2) were assessed. A higher number of mast cells and higher proportion of activated mast cells were observed in CRS +/+ rats compared with non-stress controls. Increased ICS and decreased expression of some TJ proteins were observed in the CRS +/+ rats but not in the CRS Ws/Ws rats. Tryptase and its receptor PAR-2 were found elevated concomitantly by nearly 100% in CRS +/+ rats, but not in CRS Ws/Ws rats. Mast cells play an important role in stress-induced epithelial barrier dysfunction in esophagus. The mechanism may involve the activation of PAR-2 by mast cell-derived tryptase, causing proinflammatory responses and the subsequent disruption of the epithelial TJ proteins and a disturbed cytoskeleton function, resulting in dilated intercellular spaces.

Severe disruption of the cytoskeleton and immunologically relevant surface molecules in a human macrophageal cell line in microgravity—Results of an in vitro experiment on board of the Shenzhou-8 space mission

During spaceflight the immune system is one of the most affected systems of the human body. During the SIMBOX (Science in Microgravity Box) mission on Shenzhou-8, we investigated microgravity-associated long-term alterations in macrophageal cells, the most important effector cells of the immune system. We analyzed the effect of long-term microgravity on the cytoskeleton and immunologically relevant surface molecules. Human U937 cells were differentiated into a macrophageal phenotype and exposed to microgravity or 1g on a reference centrifuge on-orbit for 5 days. After on-orbit fixation, the samples were analyzed with immunocytochemical staining and confocal microscopy after landing. The unmanned Shenzhou-8 spacecraft was launched on board a Long March 2F (CZ-2F) rocket from the Jiuquan Satellite Launch Center (JSLC) and landed after a 17-day-mission. We found a severely disturbed actin cytoskeleton, disorganized tubulin and distinctly reduced expression of CD18, CD36 and MHC-II after the 5 days in microgravity. The disturbed cytoskeleton, the loss of surface receptors for bacteria recognition, the activation of T lymphocytes, the loss of an important scavenger receptor and of antigen-presenting molecules could represent a dysfunctional macrophage phenotype. This phenotype in microgravity would be not capable of migrating or recognizing and attacking pathogens, and it would no longer activate the specific immune system, which could be investigated in functional assays. Obviously, the results have to be interpreted with caution as the model system has some limitations and due to numerous technical and biological restrictions (e.g. 23°C and no CO2 supply during in-flight incubation). All parameter were carefully pre-tested on ground. Therefore, the experiment could be adapted to the experimental conditions available on Shenzhou-8.

Dense fibrillar collagen matrices to analyse extracellular matrix receptor function

AimThe goal of this study was to understand whether dense fibrillar collagen matrices, with a hierarchical structure resembling native collagen matrices, could be useful to study collagen receptor function, in a more physiological context. The receptor analysed here was integrin α11β1, already shown to be involved in cell attachment and migration on collagen-coated plastic, and also in contraction of loose fibrillar collagen hydrogels. Materials and methodsCollagen matrices prepared here corresponded to dense fibrillar hydrogels concentrated at 5mg/ml. The behaviour of α11β1 deficient fibroblasts seeded on these concentrated matrices was assessed in terms of adhesion, morphology and migration, then compared to that observed on classical hydrogels at 1mg/ml, corresponding to loose collagen matrices. ResultsShort-term attachment assays showed disturbed interactions between α11β1 deficient cells and collagen matrices in a concentration-dependent manner. Long-term assays revealed reduced cell spreading of alpha 11−/− cells on the dense collagen matrices, associated with a disturbed cytoskeleton network. Moreover, anoikis was observed when alpha 11−/− cells were seeded on 5mg/ml matrices, and not on looser 1mg/ml matrices. In scratch wound in vitro assays, carried out with cells on 5mg/ml fibrillar collagen matrices, alpha 11−/− cells migrated much better than their wild-type counterparts. In contrast, no significant difference was observed between wild and knock-out cells seeded on plastic. ConclusionsThe present study demonstrates the validity of in vivo-like dense fibrillar collagen matrices to evaluate cell receptor functions more significantly than with 2D cell cultures or loose hydrogels.

Growth Factor Independence 1b (Gfi1b) Is An Essential Regulator of Late Stage Megakaryocyte Maturation and Platelet Production

Abstract 2358Absence of Gfi1b in mice is embryonically lethal and causes failure to produce functional megakaryocytes and platelets. Thrombopoiesis, the production of platelets by megakaryocytes, is an essential process in hemostasis that needs to be well controlled. Too many platelets can cause thrombosis, too few cause excessive bleeding. How terminal megakaryocyte maturation and platelet release works is incompletely understood but requires many factors such as Fli1, Gata1, MyH9, p45-NFE2, or c-Myc. Expression array analysis of hematopoietic cells from conditionally Gfi1b deficient mice (Gfi1bfl/fl Mx-Cre) revealed an up-regulation of many factors important for megakaryocyte function like Itga2b, Itgb3, CD9, CD41, CD61, PF4 and Ppbp. Gfi1b ablation in adult Gfi1bfl/fl Mx-Cre mice leads to a severe drop in platelet counts to less than 20% of wt mice with an increase in mean platelet volume (MPV) by 40%. However, megakaryocyte numbers rise up to 100 fold over normal levels when Gfi1b is absent. FACS analysis of bone marrow cells of Gfi1b deficient mice showed a higher number of MEPs, a higher proportion of smaller megakaryocytes and an aberrant population of cKithiCD41hiCD9veryhi cells, which are not present in wt animals. Gfi1b−/− megakaryocytes can reach wt size and normal ploidy as shown by FACS analysis and immunofluorescence microscopy. Transmission electron microscopy (TEM) of pIpC induced Gfi1bfl/fl Mx-Cre megakaryocytes revealed an excess number and larger size of so called “demarcation membranes” in unusual parallel layers and a strongly reduced number of dense granula. Strikingly, both intact and fragmented megakaryocytes were frequently found within bone marrow blood vessels in Gfi1b−/− mice. In addition, a high percentage of megakaryocytes were found in a state of disintegration, without signs of proper platelet release. These features are rarely seen in wt mice. It is known that Gfi1b is required for erythropoiesis and Gfi1bfl/fl Mx-Cre mice show signs of anemia and stress erythropoiesis, which might explain high MEP numbers, which could explain the high numbers of megakaryocytes.To better define the function of Gfi1b in late stage megakaryocyte development, we decided to abrogate Gfi1b expression more specifically by using mice that express Cre recombinase under the megakaryocyte specific promoter of the PF4 gene (PF4-Cre). We observed that Gfi1bfl/fl PF4-Cre mice develop a very severe thrombocytopenia reaching only 2% of wt platelet counts in peripheral blood accompanied by an increase of MPV by 80% over wt levels. Most of Gfi1bfl/fl PF4-Cre mice died at 6–8 weeks of age from severe internal bleedings. Megakaryocyte numbers increase in these mice by 5 to 10 fold and they also reach high ploidy, but their morphology is highly disturbed. Gfi1bfl/fl PF4-Cre megakaryocytes contain less cytoplasm, few dense granula organized in a small patch and a lobulated, ring-shaped nucleus localized close to the cell membrane giving the cells an almost “inside-out” appearance and indicating a disturbed cytoskeleton organization. Gfi1bfl/flPF4-Cre mice show a stress induced splenic erythropoiesis and an increase in MEP numbers, probably a consequence of their substantial hemorrhaging owing to the low platelet counts. The high MEPs number might explain the increase in megakaryocytes in these mice compared to wt controls. Immunofluorescence analysis of Gfi1bfl/fl PF4-Cre megakaryocytes compared to wt counterparts showed less expression of van Willebrand factor (vWF), an important regulator of thrombopoiesis. Q-PCR analysis on mRNA from sorted Gfi1bfl/fl PF4-Cre wt megakaryocytes revealed a lower expression of vWF, but higher PF4, very high Mpl and high CCNE1 expression.Our data show that Gfi1b controls the production of platelets from megakaryocytes, but does not affect the maturation of megakaryocytes as such. However, Gfi1b is required to maintain the cellular organelle structure in megakaryocytes and, more specifically is required to control the formation of dense granula and demarcation membrane formation. Gfi1b ablation in megakaryocytes results in a phenotype with high similarities to Gata1-low mice and syndromes involving mutations in the Gata1 target Gpib, the receptor for vWF, causing Bernhard-Soulier Syndrome (BBS). It is thus possible that Gfi1b is another candidate gene involved in megakaryocyte related diseases. Disclosures:No relevant conflicts of interest to declare.

Enzyme-histochemical studies of griseofulvin-intoxicated mouse livers.

Enzyme-histochemical studies were conducted on livers of mice chronically fed griseofulvin (GF) in order to produce Mallory bodies (MBs) in hepatocytes. The development of MBs is associated with derangement of the immunohistochemically detectable intermediate filament (IF) cytoskeleton of the cytokeratin (CK) type, although no strict correlation between appearance or involution of MBs and the cytoskeletal alterations exists. Since the function of the IF cytoskeleton and the relationship of its disturbance to cell injury is unknown, the aim of the present study was to correlate the activities of several key enzymes of cellular metabolic pathways with the disturbance of the cytoskeleton architecture. For that purpose enzyme-histochemistry in combination with immunohistochemical CK-IF stainings were performed on identical sections. In GF-intoxicated mouse livers the normal topography of enzyme activities was disturbed, but no strict colocalization of enzymatic and cytoskeletal changes was found. Glucose-6-phosphatase, a microsomal enzyme involved in glucose output and gluconeogenesis, showed elevated activity in MB-free hepatocytes with diminished immunostainable CK-IF cytoskeleton refuting the concept of a disability of those cells to export glucose. It could indeed indicate that those cells without MBs are in the state of recovery. However, these cells could also resemble "hyperactive foci". Glycogen was decreased in MB-containing hepatocytes with disturbed cytoskeleton, and this feature favours the assumption of cell degeneration. On the other hand, the mitochondrial marker enzymes, i.e. succinate dehydrogenase, cytochrome-c-oxidase and 3-hydroxybutyrate dehydrogenase, remained unchanged in altered hepatocytes. Alkaline phosphatase activity at the canalicular pole of GF-intoxicated hepatocytes was elevated, indicating cholestatic features associated with this disorder. However, since altered hepatocytes did not show impairment of oxido-reductase activities, a severe impairment of bile secretion as a consequence of cell damage is unlikely. Unchanged or even increased ATPase activity of altered hepatocytes also indicated their sustained metabolic abilities. The results presented provide indirect evidence that hepatocytes with disturbed IF cytoskeleton do not significantly differ from normal cells with respect to oxidative metabolism, fatty acid synthesis and gluconeogenesis. This suggests that alterations of the IF cytoskeleton associated with GF intoxication and MB formation have no significant adverse influence on the metabolic functions of liver cells, as far as can be assessed by evaluation by enzyme-histochemical staining of several key enzymes.

The Ubiquitin-Proteasome Pathway Mediates Gelsolin Protein Downregulation in Pancreatic Cancer

A well-known observation with respect to cancer biology is that transformed cells display a disturbed cytoskeleton. The underlying mechanisms, however, remain only partly understood. In an effort to identify possible mechanisms, we compared the proteome of pancreatic cancer with matched normal pancreas and observed diminished protein levels of gelsolin--an actin filament severing and capping protein of crucial importance for maintaining cytoskeletal integrity--in pancreatic cancer. Additionally, pancreatic ductal adenocarcinomas displayed substantially decreased levels of gelsolin as judged by Western blot and immunohistochemical analyses of tissue micoarrays, when compared with cancerous and untransformed tissue from the same patients (P < 0.05). Importantly, no marked downregulation of gelsolin mRNA was observed (P > 0.05), suggesting that post-transcriptional mechanisms mediate low gelsolin protein levels. In apparent agreement, high activity ubiquitin-proteasome pathway in both patient samples and the BxPC-3 pancreatic cancer cell line was detected, and inhibition of the 26s proteasome system quickly restored gelsolin protein levels in the latter cell line. The status of ubiquitinated gelsolin is related to lymph node metastasis of pancreatic cancer. In conclusion, gelsolin levels are actively downregulated in pancreatic cancer and enhanced targeting of gelsolin to the ubiquitin-proteasome pathway is an important contributing factor for this effect.

Increased Gs signalling in platelets and impaired collagen activation, due to a defect in the dystrophin gene, result in increased blood loss during spinal surgery

Controversy exists regarding the cause of the significantly increased blood loss during spinal surgery in Duchenne muscular dystrophy (DMD) patients compared with similar surgery in other patients. DMD is caused by a mutation in the cytoskeletal dystrophin, which binds to extracellular matrix laminin and which has been described as a G-protein-coupled receptor. We hypothesized that disturbed cytoskeleton organization in DMD patients would alter Gs protein and collagen signalling in platelets, leading to dysfunctional platelets and a haemorrhagic tendency during surgery. In the present study, we found that platelets and skin fibroblasts, respectively, express the Dp71 and Dp116 dystrophin isoforms. Absent or decreased expression of these isoforms in DMD patients correlates with significant Gs alpha upregulation. Moreover, dysfunctional dystrophin in these cells is accompanied with increased Gs signalling and higher cAMP levels after Gs stimulation. Functional analysis showed that DMD platelets responded slower to collagen with an extensive shape change in the aggregometer and with a significantly reduced platelet adhesion to coated collagen under flow. The decreased collagen activation was shown to result from both Gs activation and cytoskeletal disruption and not from decreased expression of platelet membrane receptors or impaired von Willebrand factor (vWF) activity. In conclusion, DMD platelets have a disorganized cytoskeleton and manifest Gs hyperactivity and reduced platelet collagen reactivity. Their increased bleeding during surgery will, at least partly, result from the increased platelet Gs activity after the release of natural Gs agonists as prostacyclin during surgery and an ineffective reactivity to collagen.

Alzheimer's disease: an intracellular movement disorder?

Axonal transport is essential for maintaining the structure and function of nerve cells. Deficient axonal transport has been implicated in several neurodegenerative diseases, including Alzheimer's disease (AD). In addition to a disturbed cytoskeleton and other abnormalities observed in AD that are suggestive of axonal transport deficits, several AD-related proteins are implicated in the regulation of axonal transport. A recent study has demonstrated that the axonal transport deficit occurs early in the course of AD, preceding amyloid pathology substantially in mouse models of AD; more importantly, the study showed that reduced axonal transport leads to increased amyloid beta production and deposition. These data place axonal transport deficits at a central point in the pathogenesis of AD.