Recent advances in genetically encoded fluorescent sensors enable the monitoring of cellular events from genetically defined groups of neurons in vivo. In this protocol, we describe how to use a time-correlated single-photon counting (TCSPC)-based fiber optics system to measure the intensity, emission spectra and lifetime of fluorescent biosensors expressed in deep brain structures in freely moving mice. When combined with Cre-dependent selective expression of genetically encoded Ca(2+) indicators (GECIs), this system can be used to measure the average neural activity from a specific population of cells in mice performing complex behavioral tasks. As an example, we used viral expression of GCaMPs in striatal projection neurons (SPNs) and recorded the fluorescence changes associated with calcium spikes from mice performing a lever-pressing operant task. The whole procedure, consisting of virus injection, behavior training and optical recording, takes 3-4 weeks to complete. With minor adaptations, this protocol can also be applied to recording cellular events from other cell types in deep brain regions, such as dopaminergic neurons in the ventral tegmental area. The simultaneously recorded fluorescence signals and behavior events can be used to explore the relationship between the neural activity of specific brain circuits and behavior.
MAP1LC3B, an ortholog of yeast Atg8 and a member of the family of proteins formerly also known as ATG8 in mammals (LC3B henceforth in the text), functions in autophagosome formation and autophagy substrate recruitment. LC3 exists in both a soluble (autophagosome-independent) form as well as a lipid modified form that becomes tightly incorporated into autophagosomal membranes. Although LC3 is known to associate with tens of proteins, relatively little is known about soluble LC3 aside from its interactions with the LC3 lipid conjugation machinery. In previous studies we found autophagosome-independent GFP-LC3B diffuses unusually slowly for a protein of its size, suggesting it may constitutively associate with a high molecular weight complex, form homo-oligomers or aggregates, or reversibly bind microtubules or membranes. To distinguish between these possibilities, we characterized the size, stoichiometry, and organization of autophagosome-independent LC3B in living cells and in cytoplasmic extracts using fluorescence recovery after photobleaching (FRAP) and fluorescence polarization fluctuation analysis (FPFA). We found that the diffusion of LC3B was unaffected by either mutational disruption of its lipid modification or microtubule depolymerization. Brightness and homo-FRET analysis indicate LC3B does not homo-oligomerize. However, mutation of specific residues on LC3B required for binding other proteins and mRNA altered the effective hydrodynamic radius of the protein as well as its stoichiometry. We conclude that when not bound to autophagosomes, LC3B associates with a multicomponent complex with an effective size of ~500 kDa in the cytoplasm. These findings provide new insights into the nature of soluble LC3B and illustrate the power of FRAP and FPFA to investigate the emergent properties of protein complexes in the autophagy pathway.
Dysferlinopathies, most commonly limb girdle muscular dystrophy 2B and Miyoshi myopathy, are degenerative myopathies caused by mutations in the DYSF gene encoding the protein dysferlin. Studies of dysferlin have focused on its role in the repair of the sarcolemma of skeletal muscle, but dysferlins association with calcium (Ca(2+)) signaling proteins in the transverse (t-) tubules suggests additional roles. Here, we reveal that dysferlin is enriched in the t-tubule membrane of mature skeletal muscle fibers. Following experimental membrane stress in vitro, dysferlin-deficient muscle fibers undergo extensive functional and structural disruption of the t-tubules that is ameliorated by reducing external [Ca(2+)] or blocking L-type Ca(2+) channels with diltiazem. Furthermore, we demonstrate that diltiazem treatment of dysferlin-deficient mice significantly reduces eccentric contraction-induced t-tubule damage, inflammation, and necrosis, which resulted in a concomitant increase in postinjury functional recovery. Our discovery of dysferlin as a t-tubule protein that stabilizes stress-induced Ca(2+) signaling offers a therapeutic avenue for limb girdle muscular dystrophy 2B and Miyoshi myopathy patients.
Förster resonance energy transfer (FRET) describes a physical phenomenon widely applied in biomedical research to estimate separations between biological molecules. Routinely, genetic engineering is used to incorporate spectral variants of the green fluorescent protein (GFPs), into cellular expressed proteins. The transfer efficiency or rate of energy transfer between donor and acceptor FPs is then assayed. As appreciable FRET occurs only when donors and acceptors are in close proximity (1-10nm), the presence of FRET may indicate that the engineered proteins associate as interacting species. For a homogeneous population of FRET pairs the separations between FRET donors and acceptors can be estimated from a measured FRET efficiency if it is assumed that donors and acceptors are randomly oriented and rotate extensively during their excited state (dynamic regime). Unlike typical organic fluorophores, the rotational correlation-times of FPs are typically much longer than their fluorescence lifetime; accordingly FPs are virtually static during their excited state. Thus, estimating separations between FP FRET pairs is problematic. To overcome this obstacle, we present here a simple method for estimating separations between FPs using the experimentally measured average FRET efficiency. This approach assumes that donor and acceptor fluorophores are randomly oriented, but do not rotate during their excited state (static regime). This approach utilizes a Monte-Carlo simulation generated look-up table that allows one to estimate the separation, normalized to the Förster distance, from the average FRET efficiency. Assuming a dynamic regime overestimates the separation significantly (by 10% near 0.5 and 30% near 0.75 efficiencies) compared to assuming a static regime, which is more appropriate for estimates of separations between FPs.
In the context of uncertainty about aetiology and prognosis, good clinical practice commonly recommends both affective (creating rapport, showing empathy) and cognitive reassurance (providing explanations and education) to increase self-management in groups with nonspecific pain conditions. The specific impact of each of these components in reference to patients outcomes has not been studied. This review aimed to systematically evaluate the evidence from prospective cohorts in primary care that measured patient-practitioner interactions with reference to patient outcomes. We carried out a systematic literature search and appraisal of study methodology. We extracted measures of affective and cognitive reassurance in consultations and their associations with consultation exit and follow-up measures of patients outcomes. We identified 16 studies from 16,059 abstracts. Eight studies were judged to be high in methodological quality. Pooling could not be achieved as a result of heterogeneity of samples and measures. Affective reassurance showed inconsistent findings with consultation exit outcomes. In 3 high-methodology studies, an association was found between affective reassurance and higher symptom burden and less improvement at follow-up. Cognitive reassurance was associated with higher satisfaction and enablement and reduced concerns directly after the consultations in 8 studies; with improvement in symptoms at follow-up in 7 studies; and with reduced health care utilization in 3 studies. Despite limitations, there is support for the notion that cognitive reassurance is more beneficial than affective reassurance. We present a tentative model based on these findings and propose priorities for future research.
The basal ganglia are subcortical nuclei that control voluntary actions, and they are affected by a number of debilitating neurological disorders. The prevailing model of basal ganglia function proposes that two orthogonal projection circuits originating from distinct populations of spiny projection neurons (SPNs) in the striatum--the so-called direct and indirect pathways--have opposing effects on movement: activity of direct-pathway SPNs is thought to facilitate movement, whereas activity of indirect-pathway SPNs is presumed to inhibit movement. This model has been difficult to test owing to the lack of methods to selectively measure the activity of direct- and indirect-pathway SPNs in freely moving animals. Here we develop a novel in vivo method to specifically measure direct- and indirect-pathway SPN activity, using Cre-dependent viral expression of the genetically encoded calcium indicator (GECI) GCaMP3 in the dorsal striatum of D1-Cre (direct-pathway-specific) and A2A-Cre (indirect-pathway-specific) mice. Using fibre optics and time-correlated single-photon counting (TCSPC) in mice performing an operant task, we observed transient increases in neural activity in both direct- and indirect-pathway SPNs when animals initiated actions, but not when they were inactive. Concurrent activation of SPNs from both pathways in one hemisphere preceded the initiation of contraversive movements and predicted the occurrence of specific movements within 500?ms. These observations challenge the classical view of basal ganglia function and may have implications for understanding the origin of motor symptoms in basal ganglia disorders.
The objectives of this study were to develop consensus on (i) the content of a clinical assessment for adults presenting to primary care with low back and leg pain, and (ii) the most important items for diagnosing spinal nerve root involvement.
Head blight, caused by Gibberella zeae, is a significant disease among cereal crops, including wheat, barley, and rice, due to contamination of grain with mycotoxins. G. zeae is spread by ascospores forcibly discharged from sexual fruiting bodies forming on crop residues. In this study, we characterized a novel gene, ROA, which is required for normal sexual development. Deletion of ROA (?roa) resulted in an abnormal size and shape of asci and ascospores but did not affect vegetative growth. The ?roa mutation triggered round ascospores and insufficient cell division after spore delimitation. The asci of the ?roa strain discharged fewer ascospores from the perithecia but achieved a greater dispersal distance than those of the wild-type strain. Turgor pressure within the asci was calculated through the analysis of osmolytes in the epiplasmic fluid. Deletion of the ROA gene appeared to increase turgor pressure in the mutant asci. The higher turgor pressure of the ?roa mutant asci and the mutant spore shape contributed to the longer distance dispersal. When the ?roa mutant was outcrossed with a ?mat1-2 mutant, a strain that contains a green fluorescence protein (GFP) marker in place of the MAT1-2 gene, unusual phenotypic segregation occurred. The ratio of GFP to non-GFP segregation was 1:1; however, all eight spores had the same shape. Taken together, the results of this study suggest that ROA plays multiple roles in maintaining the proper morphology and discharge of ascospores in G. zeae.
Repair of the injured vascular intima requires a series of coordinated events that mediate both endothelial regeneration and reannealing of adherens junctions (AJs) to form a restrictive endothelial barrier. The forkhead transcription factor FoxM1 is essential for endothelial proliferation after vascular injury. However, little is known about mechanisms by which FoxM1 regulates endothelial barrier reannealing. Here, using a mouse model with endothelial cell (EC)-restricted disruption of FoxM1 (FoxM1 CKO) and primary cultures of ECs with small interfering RNA (siRNA)-mediated knockdown of FoxM1, we demonstrate a novel requisite role of FoxM1 in mediating endothelial AJ barrier repair through the transcriptional control of beta-catenin. In the FoxM1 CKO lung vasculature, we observed persistent microvessel leakage characterized by impaired reannealing of endothelial AJs after endothelial injury. We also showed that FoxM1 directly regulated beta-catenin transcription and that reexpression of beta-catenin rescued the defective AJ barrier-reannealing phenotype of FoxM1-deficient ECs. Knockdown of beta-catenin mimicked the phenotype of defective barrier recovery seen in FoxM1-deficient ECs. These data demonstrate that FoxM1 is required for reannealing of endothelial AJs in order to form a restrictive endothelial barrier through transcriptional control of beta-catenin expression. Therefore, means of activating FoxM1-mediated endothelial repair represent a new therapeutic strategy for the treatment of inflammatory vascular diseases associated with persistent vascular barrier leakiness such as acute lung injury.
Dysferlin is a Ca(2+)-binding protein found in many different cell types. It is required for membrane wound repair in muscle, but it is not known whether it has the same function in other cells. Here we report the activation of an intercellular signaling pathway in sea urchin embryos by membrane wounding that evokes Ca(2+) spikes in neighboring cells. This pathway was mimicked by ATP application, and inhibited by apyrase, cadmium, and omega-agatoxin-IVA. Microinjection of dysferlin antisense phosphorodiamidate morpholino oligonucleotides blocked this pathway, whereas control morpholinos did not. Co-injection of mRNA encoding human dysferlin with the inhibitory morpholino rescued signaling activity. We conclude that in sea urchin embryos dysferlin mediates Ca(2+)-triggered intercellular signaling in response to membrane wounding.
Cell surface area is maintained in most cells by coupling exocytotic activity to compensatory endocytosis, a process that specifically retrieves membrane inserted by exocytosis. Although such coupling mechanisms seem to be ubiquitous, the mechanisms through which these membrane trafficking events are linked have remained elusive. A mechanism for coupling exocytosis to endocytosis in fruit fly nerve terminals that depends on the exocytotic insertion of vesicular calcium channels into the plasma membrane has recently been identified. This coupling mechanism resembles one previously described in sea urchin eggs. Here, I compare the similarities and differences of the processes involved in linking exocytosis to endocytosis in these two invertebrate systems and speculate on whether the vertebrate coupling mechanism might also depend on vesicular channels.
Small birds and mammals commonly minimize respiratory heat loss with reciprocating counterflow exchangers in their nasal passageways. These animals extract heat from the air in an exhalation to warm those passageways and then use that heat to warm the subsequent inhalation. Although the near-constant volume of buildings precludes direct application of the device, a pair of such exchangers located remotely from each other circumvents that problem. A very simple and crudely constructed small-scale physical model of the device worked well enough as a heat conserver to suggest utility as a ventilator for buildings.
Förster resonance energy transfer (FRET) is a mechanism where energy is transferred from an excited donor fluorophore to adjacent chromophores via non-radiative dipole-dipole interactions. FRET theory primarily considers the interactions of a single donor-acceptor pair. Unfortunately, it is rarely known if only a single acceptor is present in a molecular complex. Thus, the use of FRET as a tool for measuring protein-protein interactions inside living cells requires an understanding of how FRET changes with multiple acceptors. When multiple FRET acceptors are present it is assumed that a quantum of energy is either released from the donor, or transferred in toto to only one of the acceptors present. The rate of energy transfer between the donor and a specific acceptor (k(D-->A)) can be measured in the absence of other acceptors, and these individual FRET transfer rates can be used to predict the ensemble FRET efficiency using a simple kinetic model where the sum of all FRET transfer rates is divided by the sum of all radiative and non-radiative transfer rates.
Cerulean and Venus are recently developed fluorescent proteins, often used as a donor-acceptor pair by researchers in Forster resonance energy transfer-based colocalization studies. We characterized the fluorescent properties of these two proteins in a broad spectral range (form ultraviolet to visible region). Excitation spectra, lifetimes, and polarization spectra show significant energy transfer from aromatic amino acids to the fluorescent protein chromophore. High steady-state anisotropy values and the lack of a fast component in anisotropy decays show that the fluorescent protein chromophore is rigidly fixed within the protein structure. Furthermore, we show that the chromophores are not accessible to external quenchers, such as acrylamide or potassium iodide (KI), allowing the removal of "unwanted" background in the environment with external quencher, while leaving the Cerulean/Venus fluorescence unchanged.
Climatic extremes can be as significant as averages in setting the conditions for successful organismal function and in determining the distribution of different forms. For lightweight, flexible structures such as leaves, even extremes lasting a few seconds can matter. The present review considers two extreme situations that may pose existential risks. Broad leaves heat rapidly when ambient air flows drop below c. 0.5 m s(-1). Devices implicated in minimizing heating include: reduction in size, lobing, and adjustments of orientation to improve convective cooling; low near-infrared absorptivity; and thickening for short-term heat storage. Different features become relevant when storm gusts threaten to tear leaves and uproot trees with leaf-level winds of 20 m s(-1) or more. Both individual leaves and clusters may curl into low-drag, stable cones and cylinders, facilitated by particular blade shapes, petioles that twist readily, and sufficient low-speed instability to initiate reconfiguration. While such factors may have implications in many areas, remarkably little relevant experimental work has addressed them.
Pulmonary hypertension (PH) is an unremitting disease defined by a progressive increase in pulmonary vascular resistance leading to right-sided heart failure. Using mice with genetic deletions of caveolin 1 (Cav1) and eNOS (Nos3), we demonstrate here that chronic eNOS activation secondary to loss of caveolin-1 can lead to PH. Consistent with a role for eNOS in the pathogenesis of PH, the pulmonary vascular remodeling and PH phenotype of Cav1-/- mice were absent in Cav1-/-Nos3-/- mice. Further, treatment of Cav1-/- mice with either MnTMPyP (a superoxide scavenger) or l-NAME (a NOS inhibitor) reversed their pulmonary vascular pathology and PH phenotype. Activation of eNOS in Cav1-/- lungs led to the impairment of PKG activity through tyrosine nitration. Moreover, the PH phenotype in Cav1-/- lungs could be rescued by overexpression of PKG-1. The clinical relevance of the data was indicated by the observation that lung tissue from patients with idiopathic pulmonary arterial hypertension demonstrated increased eNOS activation and PKG nitration and reduced caveolin-1 expression. Together, these data show that loss of caveolin-1 leads to hyperactive eNOS and subsequent tyrosine nitration-dependent impairment of PKG activity, which results in PH. Thus, targeting of PKG nitration represents a potential novel therapeutic strategy for the treatment of PH.
At its fundamental level, human memory is thought to occur at individual synaptic contact sites and manifest as persistent changes in synaptic efficacy. In digital electronics, the fundamental structure for implementing memory is the flip-flop switch, a circuit that can be triggered to flip between two stable states. Recently, crystals of Ca(2+)/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha) catalytic domains, the enzymatic portion of a dodecameric holoenzyme involved in memory, were found to form dimers [Rosenberg OS, Deindl S, Sung RJ, Nairn AC, Kuriyan J (2005) Structure of the autoinhibited kinase domain of CaMKII and SAXS analysis of the holoenzyme. Cell 123:849-860]. Although the formation of dimers in the intact holoenzyme has not been established, several features of the crystal structure suggest that dimers could act as a synaptic switch. ATP-binding sites were occluded, and the T286 autophosphorylation site responsible for persistent kinase activation was buried. These features would act to stabilize an autoinhibited "paired"-enzyme state. Ca(2+)-calmodulin binding was postulated to trigger the formation of an active state with unpaired catalytic domains. This conformation would allow ATP access and expose T286, autophosphorylation of which would act to maintain the "unpaired" conformation. We used fluorescence anisotropy and FRET imaging of Venus-tagged CaMKIIalpha to test the hypothesis that neuronal CaMKIIalpha can flip between two stable conformations in living cells. Our data support the existence of catalytic domain pairs, and glutamate receptor activation in neurons triggered an increase in anisotropy consistent with a structural transition from a paired to unpaired conformation.
Förster resonance energy transfer (FRET) microscopy is widely used to study protein interactions in living cells. Typically, spectral variants of the Green Fluorescent Protein (FPs) are incorporated into proteins expressed in cells, and FRET between donor and acceptor FPs is assayed. As appreciable FRET occurs only when donors and acceptors are within 10 nm of each other, the presence of FRET can be indicative of aggregation that may denote association of interacting species. By monitoring the excited-state (fluorescence) decay of the donor in the presence and absence of acceptors, dual-component decay analysis has been used to reveal the fraction of donors that are FRET positive (i.e., in aggregates)._However, control experiments using constructs containing both a donor and an acceptor FP on the same protein repeatedly indicate that a large fraction of these donors are FRET negative, thus rendering the interpretation of dual-component analysis for aggregates between separately donor-containing and acceptor-containing proteins problematic. Using Monte-Carlo simulations and analytical expressions, two possible sources for such anomalous behavior are explored: 1) conformational heterogeneity of the proteins, such that variations in the distance separating donor and acceptor FPs and/or their relative orientations persist on time-scales long in comparison with the excited-state lifetime, and 2) FP dark states.
Tissue edema and endothelial barrier dysfunction as observed in sepsis and acute lung injury carry high morbidity and mortality, but currently lack specific therapy. In a recent case report, we described fast resolution of pulmonary edema on treatment with the tyrosine kinase inhibitor imatinib through an unknown mechanism. Here, we explored the effect of imatinib on endothelial barrier dysfunction and edema formation.
Förster resonance energy transfer (FRET) microscopy is frequently used to study protein interactions and conformational changes in living cells. The utility of FRET is limited by false positive and negative signals. To overcome these limitations we have developed Fluorescence Polarization and Fluctuation Analysis (FPFA), a hybrid single-molecule based method combining time-resolved fluorescence anisotropy (homo-FRET) and fluorescence correlation spectroscopy. Using FPFA, homo-FRET (a 1-10 nm proximity gauge), brightness (a measure of the number of fluorescent subunits in a complex), and correlation time (an attribute sensitive to the mass and shape of a protein complex) can be simultaneously measured. These measurements together rigorously constrain the interpretation of FRET signals. Venus based control-constructs were used to validate FPFA. The utility of FPFA was demonstrated by measuring in living cells the number of subunits in the ?-isoform of Venus-tagged calcium-calmodulin dependent protein kinase-II (CaMKII?) holoenzyme. Brightness analysis revealed that the holoenzyme has, on average, 11.9 ± 1.2 subunit, but values ranged from 10-14 in individual cells. Homo-FRET analysis simultaneously detected that catalytic domains were arranged as dimers in the dodecameric holoenzyme, and this paired organization was confirmed by quantitative hetero-FRET analysis. In freshly prepared cell homogenates FPFA detected only 10.2 ± 1.3 subunits in the holoenzyme with values ranging from 9-12. Despite the reduction in subunit number, catalytic domains were still arranged as pairs in homogenates. Thus, FPFA suggests that while the absolute number of subunits in an auto-inhibited holoenzyme might vary from cell to cell, the organization of catalytic domains into pairs is preserved.
Low-back related leg pain with or without nerve root involvement is associated with a poor prognosis compared to low back pain (LBP) alone. Compared to the literature investigating prognostic indicators of outcome for LBP, there is limited evidence on prognostic factors for low back-related leg pain including the group with nerve root pain. This 1 year prospective consultation-based observational cohort study will describe the clinical, imaging, demographic characteristics and health economic outcomes for the whole cohort, will investigate differences and identify prognostic indicators of outcome (i.e. change in disability at 12 months), for the whole cohort and, separately, for those classified with and without nerve root pain. In addition, nested qualitative studies will provide insights on the clinical consultation and the impact of diagnosis and treatment on patients symptom management and illness trajectory.
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