FRET Imaging in Three-dimensional Hydrogels.

Imaging of Förster resonance energy transfer (FRET) is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

IL-6 and Akt are involved in muscular pathogenesis in myasthenia gravis.

INTRODUCTION: Anti-acetylcholine receptor (AChR) autoantibodies target muscles in spontaneous human myasthenia gravis (MG) and its induced experimental autoimmune model MG (EAMG). The aim of this study was to identify novel functional mechanisms occurring in the muscle pathology of myasthenia. RESULTS: A transcriptome analysis performed on muscle tissue from MG patients (compared with healthy controls) and from EAMG rats (compared with control rats) revealed a deregulation of genes associated with the Interleukin-6 (IL-6) and Insulin-Like Growth Factor 1 (IGF-1) pathways in both humans and rats. The expression of IL-6 and its receptor IL-6R transcripts was found to be altered in muscles of EAMG rats and mice compared with control animals. In muscle biopsies from MG patients, IL-6 protein level was higher than in control muscles. Using cultures of human muscle cells, we evaluated the effects of anti-AChR antibodies on IL-6 production and on the phosphorylation of Protein Kinase B (PKB/Akt). Most MG sera and some monoclonal anti-AChR antibodies induced a significant increase in IL-6 production by human muscle cells. Furthermore, Akt phosphorylation in response to insulin was decreased in the presence of monoclonal anti-AChR antibodies. CONCLUSIONS: Anti-AChR antibodies alter IL-6 production by muscle cells, suggesting a putative novel functional mechanism of action for the anti-AChR antibodies. IL-6 is a myokine with known effects on signaling pathways such as Akt/mTOR (mammalian Target of Rapamycin). Since Akt plays a key role in multiple cellular processes, the reduced phosphorylation of Akt by the anti-AChR antibodies may have a significant impact on the muscle fatigability observed in MG patients.

EBP50 promotes focal adhesion turnover and vascular smooth muscle cells migration.

The ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a PDZ-containing scaffolding protein that regulates a variety of physiological functions. In the vasculature, EBP50 promotes neointima formation following arterial injury. In this study the role of EBP50 on vascular smooth muscle cell (VSMC) migration was characterized. The spreading and motility of primary VSMC isolated from EBP50 knockout (KO) mice were significantly reduced compared to wild-type (WT) cells. EBP50-null VSMC had fewer and larger focal adhesions than wild-type cells. Assembly and disassembly of focal adhesion-assessed by live-cell total internal reflection fluorescence imaging-in response to epidermal growth factor (EGF) were significantly reduced in KO cells. Immunoprecipitation experiments showed that EBP50 interacts with EGF receptor via the PDZ2 domain and with focal adhesion kinase (FAK) via the C-terminal ERM domain. EBP50 promoted the formation of a complex containing both EGF receptor and FAK. Phosphorylation of Tyr-925 of FAK in response to EGF was significantly reduced in KO cell compared to WT cells. The residence time of FAK in focal adhesions-determined by fluorescence recovery after photobleaching-was increased in WT cells. Collectively, these studies indicate that EBP50, by scaffolding EGF receptor and FAK, facilitates activation of FAK, focal adhesion turnover, and migration of VSMC.

Store-operated Ca(2+) entry (SOCE) contributes to normal skeletal muscle contractility in young but not in aged skeletal muscle.

Muscle atrophy alone is insufficient to explain the significant decline in contractile force of skeletal muscle during normal aging. One contributing factor to decreased contractile force in aging skeletal muscle could be compromised excitation-contraction (E-C) coupling, without sufficient available Ca(2+) to allow for repetitive muscle contractility, skeletal muscles naturally become weaker. Using biophysical approaches, we previously showed that store-operated Ca(2+) entry (SOCE) is compromised in aged skeletal muscle but not in young ones. While important, a missing component from previous studies is whether or not SOCE function correlates with contractile function during aging. Here we test the contribution of extracellular Ca(2+) to contractile function of skeletal muscle during aging. First, we demonstrate graded coupling between SR Ca(2+) release channel-mediated Ca(2+) release and activation of SOCE. Inhibition of SOCE produced significant reduction of contractile force in young skeletal muscle, particularly at high frequency stimulation, and such effects were completely absent in aged skeletal muscle. Our data indicate that SOCE contributes to the normal physiological contractile response of young healthy skeletal muscle and that defective extracellular Ca(2+) entry through SOCE contributes to the reduced contractile force characteristic of aged skeletal muscle.

Turnover rate of cerebrospinal fluid in female sheep: changes related to different light-dark cycles.

BACKGROUND: Sheep are seasonal breeders. The key factor governing seasonal changes in the reproductive activity of the ewe is increased negative feedback of estradiol at the level of the hypothalamus under long-day conditions. It has previously been demonstrated that when gonadotropin secretions are inhibited during long days, there is a higher concentration of estradiol in the cerebrospinal fluid (CSF) than during short days. This suggests an involvement of the CSF and choroid plexus in the neuroendocrine regulatory loop, but the mechanisms underlying this phenomenon remain unknown. One possible explanation of this difference in hormonal content is an effect of concentration or dilution caused by variations in CSF secretion rate. The aim of this study was thus to investigate changes in the CSF turnover rate related to light-dark cycles. METHODS: The turnover rate of the CSF was estimated by measuring the time taken for the recovery of intraventricular pressure (IVP) after removal of a moderate volume (0.5 to 2 ml) of CSF (slope in mmHg/min). The turnover rate was estimated three times in the same group of sheep: during a natural period of decreasing day-length corresponding to the initial period when gonadotropin activity is stimulated (SG1), during a long-day inhibitory period (IG), and finally during a short-day stimulatory period (SG2). RESULTS: The time taken and the speed of recovery of initial IVP differed between groups: 8 min 30 sec, 0.63 +/- 0.07 mmHg/min(SG1), 11 min 1 sec, 0.38 +/- 0.06 mmHg/min (IG) and 9 min 0 sec, 0.72 +/- 0.15 mmHg/min (SG2). Time changes of IVP differed between groups (ANOVA, p < 0.005, SG1 different from IG, p < 0.05). The turnover rate in SG2: 183.16 +/- 23.82 mul/min was not significantly different from SG1: 169. 23 +/- 51.58 mul/min (Mann-Whitney test, p = 0.41), but was significantly different from IG: 71.33 +/- 16.59 mul/min (p = 0.016). CONCLUSION: This study shows that the turnover rate of CSF in ewes changes according to the light-dark cycle; it is increased during short day periods and reduced in long day periods. This phenomenon could account for differences in hormonal concentrations in the CSF in this seasonal species.

Evidence that the choroids plexus in female sheep express P-glycoprotein.

OBJECTIVES: Previous studies in the female sheep have shown that photoperiod modulates the passage of sex steroids between the blood and the cerebrospinal fluid (CSF) and have suggested the involvement of efflux transport. The objective of the present work was to assess the presence of P-glycoprotein (Pgp), which could be responsible for this transport at the level of the choroid plexuses (CP). METHODS: We used flow cytometry and the UIC2 monoclonal antibody against Pgp, to demonstrate the presence of Pgp in the epithelial cell fraction isolated from the sheep choroid plexuses (CPEC). Thanks to the size of the brain structures in sheep, we analyse separately the CP from 4th ventricle (4V) and the CP from the lateral ventricle (LV). RESULTS: In the whole population isolated from the CP, the 4V contained a higher percentage of living, epithelial cells than the LV. The immunoreactive cells to the UIC2 antibody i.e. bearing activated form of Pgp, represented 26.8% of the CPEC in the 4V, and 39.3% in the LV (P<0.05). CONCLUSION: Pgp is expressed in the CPEC from the sheep. Differences in the expression of Pgp between CPEC from LV and 4V might suggest in vivo functional differences between LV and 4V CP in sheep.