Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy

The self-assembly of bacterial 30S ribosomes involves a large number of RNA folding and RNA-protein binding steps. The sequence of steps determines the overall assembly mechanism and the structure of the mechanism has ramifications for the robustness of biogenesis and resilience against kinetic traps. Thermodynamic interdependencies of protein binding inferred from omission-reconstitution experiments are thought to preclude certain assembly pathways and thus enforce ordered assembly, but this concept is at odds with kinetic data suggesting a more parallel assembly landscape. A major challenge is deconvolution of the statistical distribution of intermediates that are populated during assembly at high concentrations approaching in vivo assembly conditions. To specifically resolve the intermediates formed by binding of three ribosomal proteins to the full length 16S rRNA, we introduce Fluorescence Triple-Correlation Spectroscopy (F3CS). F3CS identifies specific ternary complexes by detecting coincident fluctuations in three-color fluorescence data. Triple correlation integrals quantify concentrations and diffusion kinetics of triply labeled species, and F3CS data can be fit alongside auto-correlation and cross-correlation data to quantify the populations of 10 specific ribosome assembly intermediates. The distribution of intermediates generated by binding three ribosomal proteins to the entire native 16S rRNA included significant populations of species that were not previously thought to be thermodynamically accessible, questioning the current interpretation of the classic omission-reconstitution experiments. F3CS is a general approach for analyzing assembly and function of macromolecular complexes, especially those too large for traditional biophysical methods.     

Stoichiometry of molecular complexes at adhesions in living cells

We describe a method to detect molecular complexes and measure their stoichiometry in living cells from simultaneous fluctuations of the fluorescence intensity in two image channels, each detecting a different kind of protein. The number and brightness (N&B) analysis, namely, the use of the ratio between the variance and the average intensity to obtain the brightness of molecules, is extended to the cross-variance of the intensity fluctuations in two channels. We apply the cross-variance method to determine the stoichiometry of complexes containing paxillin and vinculin or focal adhesion kinase (FAK) in disassembling adhesions in mouse embryo fibroblasts expressing FAK, vinculin, and paxillin-tagged with EGFP and mCherry. We found no complexes of these proteins in the cytoplasm away from the adhesions. However, at the adhesions, large aggregates leave, forming a hole, during their disassembly. This hole shows cross-correlation between FAK and paxillin and vinculin and paxillin. From the amplitude of the correlated fluctuations we determine the composition of the aggregates leaving the adhesions. These aggregates disassemble rapidly in the cytoplasm because large complexes are found only in very close proximity to the adhesions or at their borders.     

Specifically associated PCR products probed by coincident detection of two-color cross-correlated fluorescence intensities in human gene polymorphisms of methylene tetrahydrofolate reductase at site C677T: a novel measurement approach without follow-up mathematical analysis

Whole blood samples of known methylene tetrahydrofolate reductase (MTHFR) genotypes from 24 individuals were examined at site C677T. Their amplified DNA products were assessed by two-color fluorescence cross-correlation measurements and agarose gel electrophoresis/capillary gel electrophoresis. DNA subpopulations were identified which were not associated with the proper genotype by primer combinations and cycling conditions called multiplexes. We confirmed that DNA analysis by two-color fluorescence cross-correlation measurements allowed the detection of fluorescence signals specifically associated with the proper genotypes in a mixture of amplified nontarget DNA molecules without DNA sizing. The measurement approach does not require complex, follow-up mathematical analysis and is applicable to any single nucleotide polymorphisms. The simple immunogenetic model showed how the approach works to reveal specific DNA target by preventing detection of nontarget DNA. Under those experimental conditions, a new ultrasensitive, and specific method for clinical immunologists is born.     

Auditory nerve inputs to cochlear nucleus neurons studied with cross-correlation

The strength of synapses between auditory nerve (AN) fibers and ventral cochlear nucleus (VCN) neurons is an important factor in determining the nature of neural integration in VCN neurons of different response types. Synaptic strength was analyzed using cross-correlation of spike trains recorded simultaneously from an AN fiber and a VCN neuron in anesthetized cats. VCN neurons were classified as chopper, primarylike, and onset using previously defined criteria, although onset neurons usually were not analyzed because of their low discharge rates. The correlograms showed an excitatory peak (EP), consistent with monosynaptic excitation, in AN-VCN pairs with similar best frequencies (49% 24/49 of pairs with best frequencies within +/-5%). Chopper and primarylike neurons showed similar EPs, except that the primarylike neurons had shorter latencies and shorter-duration EPs. Large EPs consistent with end bulb terminals on spherical bushy cells were not observed, probably because of the low probability of recording from one. The small EPs observed in primarylike neurons, presumably spherical bushy cells, could be derived from small terminals that accompany end bulbs on these cells. EPs on chopper or primarylike-with-notch neurons were consistent with the smaller synaptic terminals on multipolar and globular bushy cells. Unexpectedly, EPs were observed only at sound levels within about 20 dB of threshold, showing that VCN responses to steady tones shift from a 1:1 relationship between AN and VCN spikes at low sound levels to a more autonomous mode of firing at high levels. In the high level mode, the pattern of output spikes seems to be determined by the properties of the postsynaptic spike generator rather than the input spike patterns. The EP amplitudes did not change significantly when the presynaptic spike was preceded by either a short or long interspike interval, suggesting that synaptic depression and facilitation have little effect under the conditions studied here.     

Time course of conjugated neuronal firing in the motor cortex of conditioned cats

The activity of neurons able to generate impulses synergistically at intervals of not more than 0.5 msec (conjugated neuronal firing) was analyzed in the motor cortex of cats with two forms of learned behavior (a conditioned response to time and a conditioned defensive response to sound). It is shown that conjugated neuronal firing is a process based on spontaneous impulse traffic and that its nonuniform distribution in time is determined by changes occurring in cellular structures during adaptive behavior. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, No. 6, pp. 619–622, June, 1996     

Reduced Synchronization in the Visual Cortex of Cats with Strabismic Amblyopia

Synchronous firing of spatially separate neurons was studied with multi-electrode recordings in area 17 of the visual cortex of strabismic cats which had developed behaviourally verified amblyopia of the deviated eye. Responses of neurons were evoked with moving light bars or gratings of different spatial frequency. Neurons driven by the normal eye displayed stronger synchronization of their responses than neurons dominated by the amblyopic eye. These interocular differences were highly significant and particularly pronounced for grating stimuli of high spatial frequency. No interocular differences were noted with respect to the amplitudes of responses to the light bars and gratings. These results suggest reduced synchronization of population responses as a neurophysiological correlate of strabismic amblyopia and underline the importance of correlated firing of spatially separate cortical neurons for normal processing of visual information.     

Mapping dynamic protein interactions in MAP kinase signaling using live-cell fluorescence fluctuation spectroscopy and imaging

Fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS), and photon counting histograms (PCH) are fluctuation methods that emerged recently as potentially useful tools for obtaining parameters of molecular dynamics, interactions, and oligomerization in vivo. Here, we report the successful implementation of FCS, FCCS, and PCH in live yeast cells using fluorescent protein-tagged proteins expressed from their native chromosomal loci, examining cytosolic dynamics and interactions among components of the mitogen activated protein kinase (MAPK) cascade, a widely occurring signaling motif, in response to mating pheromone. FCS analysis detailed the diffusion characteristics and mobile concentrations of MAPK proteins. FCCS analysis using EGFP and mCherry-tagged protein pairs observed the interactions of Ste7 (MAPK kinase) with the MAPKs, Fus3 or Kss1, and of the scaffold protein, Ste5, with Ste7 and Ste11 (MAPK kinase kinase) in the cytosol, providing in vivo constants of their binding equilibrium. The interaction of Ste5 with Fus3 in the cytosol was below the limit of detection, suggesting a weak interaction, if it exists, with K(d) >400-500 nM. Using PCH, we show that cytosolic Ste5 were mostly monomers. Artificial dimerization of Ste5, as confirmed by PCH, using a dimerizing tag, stimulated the interaction between Ste5 and Fus3. Native Ste5 was found to bind Fus3 preferentially at the cortex in pheromone-treated cells, as detected by fluorescence resonance energy transfer (FRET). These results provide a quantitative spatial map of MAPK complexes in vivo and directly support the model that membrane association and regulation of the Ste5 scaffold are critical steps in MAPK activation.     

Reliability of measuring sciatic and tibial nerve movement with diagnostic ultrasound during a neural mobilisation technique

Diagnostic ultrasound provides a technique whereby real-time, in vivo analysis of peripheral nerve movement is possible. This study measured sciatic nerve movement during a "slider" neural mobilisation technique (ankle dorsiflexion/plantar flexion and cervical extension/flexion). Transverse and longitudinal movement was assessed from still ultrasound images and video sequences by using frame-by-frame cross-correlation software. Sciatic nerve movement was recorded in the transverse and longitudinal planes. For transverse movement, at the posterior midthigh (PMT) the mean value of lateral sciatic nerve movement was 3.54 mm (standard error of measurement [SEM] +/- 1.18 mm) compared with anterior-posterior/vertical (AP) movement of 1.61 mm (SEM +/- 0.78 mm). At the popliteal crease (PC) scanning location, lateral movement was 6.62 mm (SEM +/- 1.10 mm) compared with AP movement of 3.26 mm (SEM +/- 0.99 mm). Mean longitudinal sciatic nerve movement at the PMT was 3.47 mm (SEM +/- 0.79 mm; n = 27) compared with the PC of 5.22 mm (SEM +/- 0.05 mm; n = 3). The reliability of ultrasound measurement of transverse sciatic nerve movement was fair to excellent (Intraclass correlation coefficient [ICC] = 0.39-0.76) compared with excellent (ICC = 0.75) for analysis of longitudinal movement. Diagnostic ultrasound presents a reliable, noninvasive, real-time, in vivo method for analysis of sciatic nerve movement.     

基于短时傅里叶变换的胎心音瞬时心率检测算法

目的：针对母亲的活动和胎儿在子宫内的活动都会导致胎心音信号的幅度出现较大波动,传统自相关算法难以准确测量胎儿瞬时心率的问题,提出一种基于短时傅里叶变换的胎心音瞬时心率检测新方法,．方法：对胎心音进行短时傅里叶变换,从二维时频图中提取出胎心音特征模板并与目标图像进行归一化互相关匹配,绘制出互相关曲线,根据互相关曲线中的相邻峰值点时间间隔,计算出胎儿的瞬时心率。结果：实验结果表明,该算法的识别准确率比传统算法高5％。结论：该算法克服了胎心音易受噪声干扰的弱点,实现了胎心音瞬时心率的计算,而且还提高了胎心率计算的准确性,为胎心音波形分析和胎心率监护提供了一种新的方法。