Ionic strength-dependent persistence lengths of single-stranded RNA and DNA.

Dynamic RNA molecules carry out essential processes in the cell including translation and splicing. Base-pair interactions stabilize RNA into relatively rigid structures, while flexible non-base-paired regions allow RNA to undergo conformational changes required for function. To advance our understanding of RNA folding and dynamics it is critical to know the flexibility of these un-base-paired regions and how it depends on counterions. Yet, information about nucleic acid polymer properties is mainly derived from studies of ssDNA. Here we measure the persistence lengths (l(p)) of ssRNA. We observe valence and ionic strength-dependent differences in l(p) in a direct comparison between 40-mers of deoxythymidylate (dT(40)) and uridylate (rU(40)) measured using the powerful combination of SAXS and smFRET. We also show that nucleic acid flexibility is influenced by local environment (an adjoining double helix). Our results illustrate the complex interplay between conformation and ion environment that modulates nucleic acid function in vivo.

Compact and flexible raster scanning multiphoton endoscope capable of imaging unstained tissue.

We present a compact and flexible endoscope (3-mm outer diameter, 4-cm rigid length) that utilizes a miniaturized resonant/nonresonant fiber raster scanner and a multielement gradient-index lens assembly for two-photon excited intrinsic fluorescence and second-harmonic generation imaging of biological tissues. The miniaturized raster scanner is fabricated by mounting a commercial double-clad optical fiber (DCF) onto two piezo bimorphs that are aligned such that their bending axes are perpendicular to each other. Fast lateral scanning of the laser illumination at 4.1 frames/s (512 lines per frame) is achieved by simultaneously driving the DCF cantilever at its resonant frequency in one dimension and nonresonantly in the orthogonal axis. The implementation of a DCF into the scanner enables simultaneous delivery of the femtosecond pulsed 800-nm excitation source and epi-collection of the signal. Our device is able to achieve a field-of-view (FOV(xy)) of 110 µm by 110 µm with a highly uniform pixel dwell time. The lateral and axial resolutions for two-photon imaging are 0.8 and 10 µm, respectively. The endoscope's imaging capabilities were demonstrated by imaging ex vivo mouse tissue through the collection of intrinsic fluorescence and second-harmonic signal without the need for staining. The results presented here indicate that our device can be applied in the future to perform minimally invasive in vivo optical biopsies for medical diagnostics.

Spt6 enhances the elongation rate of RNA polymerase II in vivo.

Several eukaryotic transcription factors have been shown to modulate the elongation rate of RNA polymerase II (Pol II) on naked or chromatin-reconstituted templates in vitro. However, none of the tested factors have been shown to directly affect the elongation rate of Pol II in vivo. We performed a directed RNAi knock-down (KD) screen targeting 141 candidate transcription factors and identified multiple factors, including Spt6, that alter the induced Hsp70 transcript levels in Drosophila S2 cells. Spt6 is known to interact with both nucleosome structure and Pol II, and it has properties consistent with having a role in elongation. Here, ChIP assays of the first wave of Pol II after heat shock in S2 cells show that KD of Spt6 reduces the rate of Pol II elongation. Also, fluorescence recovery after photobleaching assays of GFP-Pol II in salivary gland cells show that this Spt6-dependent effect on elongation rate persists during steady-state-induced transcription, reducing the elongation rate from approximately 1100 to 500 bp/min. Furthermore, RNAi depletion of Spt6 reveals its broad requirement during different stages of development.

Miniature varifocal objective lens for endomicroscopy.

A miniature catadioptric lens for endoscopic imaging based on the principle of wavelength division multiplexing is presented. We demonstrate change of the magnification and the field of view (FOV) of the lens without any mechanical adjustment of the optical elements. The lens provides magnifications of ~-1.5× at 406-750 nm and ~-0.2× at 800 nm. The lens is used to demonstrate large-FOV (1.3 mm) reflectance imaging and high-resolution (0.57 µm) multiphoton fluorescence imaging of unstained mouse tissues.

Identification of a helical intermediate in trifluoroethanol-induced alpha-synuclein aggregation.

Because oligomers and aggregates of the protein α-synuclein (αS) are implicated in the initiation and progression of Parkinson's disease, investigation of various αS aggregation pathways and intermediates aims to clarify the etiology of this common neurodegenerative disorder. Here, we report the formation of short, flexible, ß-sheet-rich fibrillar species by incubation of αS in the presence of intermediate (10-20% v/v) concentrations of 2,2,2-trifluoroethanol (TFE). We find that efficient production of these TFE fibrils is strongly correlated with the TFE-induced formation of a monomeric, partly helical intermediate conformation of αS, which exists in equilibrium with the natively disordered state at low [TFE] and with a highly α-helical conformation at high [TFE]. This partially helical intermediate is on-pathway to the TFE-induced formation of both the highly helical monomeric conformation and the fibrillar species. TFE-induced conformational changes in the monomer protein are similar for wild-type αS and the C-terminal truncation mutant αS1-102, indicating that TFE-induced structural transitions involve the N terminus of the protein. Moreover, the secondary structural transitions of three Parkinson's disease-associated mutants, A30P, A53T, and E46K, are nearly identical to wild-type αS, but oligomerization rates differ substantially among the mutants. Our results add to a growing body of evidence indicating the involvement of helical intermediates in protein aggregation processes. Given that αS is known to populate both highly and partially helical states upon association with membranes, these TFE-induced conformations imply relevant pathways for membrane-induced αS aggregation both in vitro and in vivo.

A desolvation model for trifluoroethanol-induced aggregation of enhanced green fluorescent protein.

Studies of amyloid disease-associated proteins in aqueous solutions containing 2,2,2-trifluoroethanol (TFE) have shown that the formation of structural intermediates is often correlated with enhanced protein aggregation. Here, enhanced green fluorescent protein (EGFP) is used as a model protein system to investigate the causal relationship between TFE-induced structural transitions and aggregation. Using circular dichroism spectroscopy, light scattering measurements, and transmission electron microscopy imaging, we demonstrate that population of a partially α-helical, monomeric intermediate is roughly correlated with the growth of ß-sheet-rich, flexible fibrils for acid-denatured EGFP. By fitting our circular dichroism data to a model in which TFE-water mixtures are assumed to be ideal solutions, we show that increasing entropic costs of protein solvation in TFE-water mixtures may both cause the population of the intermediate state and increase aggregate production. Tertiary structure and electrostatic repulsion also impede aggregation. We conclude that initiation of EGFP aggregation in TFE likely involves overcoming of multiple protective factors, rather than stabilization of aggregation-prone structural elements.

Use of a lensed fiber for a large-field-of-view, high-resolution, fiber-scanning microendoscope.

We report the application of a lensed fiber to a miniaturized fiber raster scanner in order to reduce the fiber's output beam size, thereby allowing for a compact and flexible endoscope capable of a large field of view (FOV) and high spatial resolution. For a proof of principle, the fabricated lensed fiber scanner is paired with a miniaturized gradient-index assembly to achieve a one-photon lateral resolution of 1.1 µm with a FOV that has a diameter of 440 µm.

Multifocal multiphoton endoscope.

We report a miniaturized resonant/non-resonant multi-fiber raster scanner that is paired with a gradient-index lens assembly to achieve a compact and flexible multifocal multiphoton endoscope capable of longitudinal parallel image acquisition. Multiphoton images are obtained simultaneously at three axial depths, separated by ≥4.8 µm, by incorporating three axially offset double clad optical fibers into the miniaturized scanner. The fabricated endoscope has an outer diameter of 3 mm, a rigid length of 4 cm, and acquires images at 4 frames/s per focal plane, with lateral and axial resolutions for two-photon imaging of 0.8 and 10 µm, respectively.

Dynamics of heat shock factor association with native gene loci in living cells.

Direct observation of transcription factor action in the living cell nucleus can provide important insights into gene regulatory mechanisms. Live-cell imaging techniques have enabled the visualization of a variety of intranuclear activities, from chromosome dynamics to gene expression. However, progress in studying transcription regulation of specific native genes has been limited, primarily as a result of difficulties in resolving individual gene loci and in detecting the small number of protein molecules functioning within active transcription units. Here we report that multiphoton microscopy imaging of polytene nuclei in living Drosophila salivary glands allows real-time analysis of transcription factor recruitment and exchange on specific native genes. After heat shock, we have visualized the recruitment of RNA polymerase II (Pol II) to native hsp70 gene loci 87A and 87C in real time. We show that heat shock factor (HSF), the transcription activator of hsp70, is localized to the nucleus before heat shock and translocates from nucleoplasm to chromosomal loci after heat shock. Assays based on fluorescence recovery after photobleaching show a rapid exchange of HSF at chromosomal loci under non-heat-shock conditions but a very slow exchange after heat shock. However, this is not a consequence of a change of HSF diffusibility, as shown here directly by fluorescence correlation spectroscopy. Our results provide strong evidence that activated HSF is stably bound to DNA in vivo and that turnover or disassembly of transcription activator is not required for rounds of hsp70 transcription. This and previous studies indicate that transcription activators display diverse dynamic behaviours in their associations with targeted loci in living cells. Our method can be applied to study the dynamics of many factors involved in transcription and RNA processing, and in their regulation at native heat shock genes in vivo.

Electrophysiological characterization of V2a interneurons and their locomotor-related activity in the neonatal mouse spinal cord.

The V2a class of Chx10-expressing interneurons has been implicated in frequency-dependent control of left-right phase during locomotion in the mouse. We have used the Chx10::CFP mouse line to further investigate the properties and locomotion-related activity of V2a interneurons in the isolated neonatal spinal cord. V2a interneurons can be divided into three classes, based on their tonic, phasic, or delayed-onset responses to step depolarization. Electrical coupling is found only between neurons of same class and helps to synchronize neuronal activity within the class. Serotonin (5-HT) excites isolated tonic V2a interneurons by depolarizing the neurons and increasing their membrane input resistance, with no significant effects on action potential properties, a mechanism distinct from 5-HT excitation of commissural interneurons. During NMDA-/5-HT-induced locomotor-like activity, patch-clamp recordings and two-photon calcium imaging experiments show that approximately half of V2a interneurons fire rhythmically with ventral root-recorded motor activity; the rhythmic V2a interneurons fired during one half of the cycle, in phase with either the ipsilateral or the contralateral L2 ventral root bursts. The percentage of rhythmically firing V2a interneurons increases during higher-frequency fictive locomotion, and they become significantly more rhythmic in their firing during the locomotor cycle; this may help to explain the frequency-dependent shift in left-right coupling in Chx10::DTA mice, which lack these neurons. Our results together with data from the accompanying paper (Dougherty and Kiehn, 2009) reinforce earlier proposals that the V2a interneurons are components of the hindlimb central pattern generator, helping to organize left-right locomotor coordination in the neonatal mouse spinal cord.

GUV preparation and imaging: minimizing artifacts.

The components of biological membranes are present in a physical mixture. The nonrandom ways that the molecules of lipids and proteins mix together can strongly influence the association of proteins with each other, and the chemical reactions that occur in the membrane, or that are mediated by the membrane. A particular type of nonrandom mixing is the separation of compositionally distinct phases. Any such phase separation would result in preferential partition of some proteins and lipids between the coexisting phases, and thus would influence which proteins could be in contact, and whether a protein could find its target. Phase separation in a plasma membrane would also influence the binding of molecules from outside the cell to the membrane, including recognition proteins on viruses, bacteria, and other cells. The concept of these and other events associated with membrane phase separation are sometimes grouped together as the "raft model" of biological membranes. Several types of experiments are aimed at detecting and characterizing membrane phase separation. Visualizing phase separation has special value, both because the immiscibility is so decisively determined, and also because the type of phase can often be identified. The fluorescence microscope has proven uniquely useful for yielding images of separated phases, both in certain cell preparations, and especially in models of cell membranes. Here we discuss ways to prepare useful model membranes for image studies, and how to avoid some of the artifacts that can plague these studies.

Dopamine-induced oscillations of the pyloric pacemaker neuron rely on release of calcium from intracellular stores.

Endogenously bursting neurons play central roles in many aspects of nervous system function, ranging from motor control to perception. The properties and bursting patterns generated by these neurons are subject to neuromodulation, which can alter cycle frequency and amplitude by modifying the properties of the neuron's ionic currents. In the stomatogastric ganglion (STG) of the spiny lobster, Panulirus interruptus, the anterior burster (AB) neuron is a conditional oscillator in the presence of dopamine (DA) and other neuromodulators and serves as the pacemaker to drive rhythmic output from the pyloric network. We analyzed the mechanisms by which DA evokes bursting in the AB neuron. Previous work showed that DA-evoked bursting is critically dependent on external calcium (Harris-Warrick RM, Flamm RE. J Neurosci 7: 2113-2128, 1987). Using two-photon microscopy and calcium imaging, we show that DA evokes the release of calcium from intracellular stores well before the emergence of voltage oscillations. When this release from intracellular stores is blocked by antagonists of ryanodine or inositol trisphosphate (IP(3)) receptor channels, DA fails to evoke AB bursting. We further demonstrate that DA enhances the calcium-activated inward current, I(CAN), despite the fact that it significantly reduces voltage-activated calcium currents. This suggests that DA-induced release of calcium from intracellular stores activates I(CAN), which provides a depolarizing ramp current that underlies endogenous bursting in the AB neuron.

Transmission electron microscopy characterization of fluorescently labelled amyloid ß 1-40 and α-synuclein aggregates.

BACKGROUND: Fluorescent tags, including small organic molecules and fluorescent proteins, enable the localization of protein molecules in biomedical research experiments. However, the use of these labels may interfere with the formation of larger-scale protein structures such as amyloid aggregates. Therefore, we investigate the effects of some commonly used fluorescent tags on the morphologies of fibrils grown from the Alzheimer's disease-associated peptide Amyloid ß 1-40 (Aß40) and the Parkinson's disease-associated protein α-synuclein (αS). RESULTS: Using transmission electron microscopy (TEM), we verify that N-terminal labeling of Aß40 with AMCA, TAMRA, and Hilyte-Fluor 488 tags does not prevent the formation of protofibrils and amyloid fibrils of various widths. We also measure the two-photon action cross-section of Aß40 labelled with Hilyte Fluor 488 and demonstrate that this tag is suitable for use with two-photon fluorescence techniques. Similarly, we find that Alexa Fluor 488 labelling of αS variant proteins near either the N or C terminus (position 9 or 130) does not interfere with the formation of amyloid and other types of αS fibrils. We also present TEM images of fibrils grown from αS C-terminally labelled with enhanced green fluorescent protein (EGFP). Near neutral pH, two types of αS-EGFP fibrils are observed via TEM, while denaturation of the EGFP tag leads to the formation of additional species. CONCLUSIONS: We demonstrate that several small extrinsic fluorescent tags are compatible with studies of amyloid protein aggregation. However, although fibrils can be grown from αS labelled with EGFP, the conformation of the fluorescent protein tag affects the observed aggregate morphologies. Thus, our results should assist researchers with label selection and optimization of solution conditions for aggregation studies involving fluorescence techniques.

Multiphoton microscopy for structure identification in human prostate and periprostatic tissue: implications in prostate cancer surgery.

OBJECTIVE: • To test whether multiphoton microscopy (MPM) might allow identification of prostatic and periprostatic structures with magnification and resolution similar to gold standard histopathology. MATERIAL AND METHODS: • The present study included 95 robotic radical prostatectomy patients who consented to participate in an Institutional Review Board-approved study starting in 2007. • The types of specimens used for imaging were excised surgical margins and biopsies, and sections obtained from the excised prostate. • The specimens were imaged with a custom-built MPM system. • All images were compared with haematoxylin/eosin histopathology of the same specimen. RESULTS: • MPM of freshly excised, unprocessed and unstained tissue can identify all relevant prostatic and periprostatic structures, such as nerves, blood vessels, capsule, underlying acini and also pathological changes, including prostate cancer. • Histological confirmation and correlation of these structures and pathologies have validated the findings of MPM. CONCLUSIONS: • MPM shows great promise as a tool for real-time intra-surgical histopathology without needing excision or administration of contrast agents. • The results will, however, need to be confirmed in true surgical settings using a miniaturized MPM microendoscope.

Polarized microtubule arrays in apical dendrites and axons.

The polarization of microtubules within neurons in vivo is crucial in their role of determining the directions and speeds of intracellular transport. More than a decade ago, electron microscopy studies of mature hippocampal cultures indicated that their axons contained microtubules of uniform polarity and that dendrites contained microtubules of mixed polarity. Here, we evaluated polarity distributions in native brain tissues and in cultures by using multiphoton microscopy and second-harmonic generation from microtubules. We confirmed the expected polarized microtubule arrays in axons; however, we also unexpectedly found them ubiquitously in apical dendrites of mature hippocampal CA1 and cortical layer V pyramidal neurons. Some of these organized dendritic microtubule arrays extended for >270 microm with overall polarity of >80%. Our research indicates neurite-specific and age-dependent microtubule organizations that have direct implications for neuronal cargo transport.

Conformational changes of calmodulin upon Ca2+ binding studied with a microfluidic mixer.

A microfluidic mixer is applied to study the kinetics of calmodulin conformational changes upon Ca2+ binding. The device facilitates rapid, uniform mixing by decoupling hydrodynamic focusing from diffusive mixing and accesses time scales of tens of microseconds. The mixer is used in conjunction with multiphoton microscopy to examine the fast Ca2+-induced transitions of acrylodan-labeled calmodulin. We find that the kinetic rates of the conformational changes in two homologous globular domains differ by more than an order of magnitude. The characteristic time constants are approximately 490 micros for the transitions in the C-terminal domain and approximately 20 ms for those in the N-terminal domain of the protein. We discuss possible mechanisms for the two distinct events and the biological role of the stable intermediate, half-saturated calmodulin.

Activity of Hb9 interneurons during fictive locomotion in mouse spinal cord.

Hb9 interneurons (Hb9 INs) are putative components of the mouse spinal locomotor central pattern generator (CPG) and candidates for the rhythm-generating kernel. Studies in slices and hemisected spinal cords showed that Hb9 INs display TTX-resistant membrane potential oscillations, suggesting a role in rhythm generation. To further investigate the roles of Hb9 INs in the locomotor CPG, we used two-photon calcium imaging in the in vitro isolated whole neonatal mouse spinal cord preparation to record the activity of Hb9 INs, which were subsequently stained for unambiguous genetic identification. We elicited fictive locomotion by transmitter application or by electrically stimulating the caudal tip of the spinal cord. Although most Hb9 INs were rhythmically active during fictive locomotion, their activity was sparse and they failed to fire with each cycle of the episode. If Hb9 INs are the principal pacemakers of the CPG in the hemisegment in which they are located, they should direct the firing of motor neurons, with their activity preceding that of their ipsilateral segmental ventral roots. Instead, during each locomotor cycle, onset of Hb9 IN activity lagged behind the onset of the ipsilateral ventral root burst by a mean phase of 0.21 during electrical stimulation and 0.28 during transmitter application. Whole-cell recordings in intact and hemisected spinal cords confirmed the imaging results. Our data suggest that Hb9 INs participate in fictive locomotion, but the delayed onset of activity relative to ipsilateral motoneurons suggests that Hb9 INs are unlikely to be the sole intrasegmental rhythm-generating kernel of the CPG.

Mechanisms of quenching of Alexa fluorophores by natural amino acids.

Quenching of fluorophores by the same proteins that they covalently label is a phenomenon that is neither well-known nor well-characterized. It is often assumed that fluorophores are unperturbed by their target proteins. However, it has been observed that attached fluorophores can be quenched by contact with amino acids within the same protein, and this property has been exploited to report on changing conformational states or intramolecular dynamics of proteins. We show in this communication that fluorescence of Alexa dyes is, in fact, quenched by interactions with Trp, Tyr, Met, and His residues through a combination of static and dynamic quenching mechanisms. In light of this finding, the potential effect of intramolecular quenching should be considered in the interpretation of data that involves quantitative measurements of fluorescence intensity in proteins.

Spatiotemporal dynamics of rhythmic spinal interneurons measured with two-photon calcium imaging and coherence analysis.

In rhythmic neural circuits, a neuron often fires action potentials with a constant phase to the rhythm, a timing relationship that can be functionally significant. To characterize these phase preferences in a large-scale, cell type-specific manner, we adapted multitaper coherence analysis for two-photon calcium imaging. Analysis of simulated data showed that coherence is a simple and robust measure of rhythmicity for calcium imaging data. When applied to the neonatal mouse hindlimb spinal locomotor network, the phase relationships between peak activity of >1,000 ventral spinal interneurons and motor output were characterized. Most interneurons showed rhythmic activity that was coherent and in phase with the ipsilateral motor output during fictive locomotion. The phase distributions of two genetically identified classes of interneurons were distinct from the ensemble population and from each other. There was no obvious spatial clustering of interneurons with similar phase preferences. Together, these results suggest that cell type, not neighboring neuron activity, is a better indicator of an interneuron's response during fictive locomotion. The ability to measure the phase preferences of many neurons with cell type and spatial information should be widely applicable for studying other rhythmic neural circuits.

Endoscope lens with dual fields of view and resolutions for multiphoton imaging.

We report the development of a miniaturized dual-optical-zone endoscope objective lens. The lens has two foci, with 0.18 and 0.50 NAs. We demonstrate multiphoton imaging with dual fields of view and resolutions using the new lens. A combination of multiphoton and single-photon microscopic imaging is also demonstrated.