Visualization and Analysis of Microtubule Dynamics Using Dual Color-Coded Display of Plus-End Labels

Investigating spatial and temporal control of microtubule dynamics in live cells is critical to understanding cell morphogenesis in development and disease. Tracking fluorescently labeled plus-end-tracking proteins over time has become a widely used method to study microtubule assembly. Here, we report a complementary approach that uses only two images of these labels to visualize and analyze microtubule dynamics at any given time. Using a simple color-coding scheme, labeled plus-ends from two sequential images are pseudocolored with different colors and then merged to display color-coded ends. Based on object recognition algorithms, these colored ends can be identified and segregated into dynamic groups corresponding to four events, including growth, rescue, catastrophe, and pause. Further analysis yields not only their spatial distribution throughout the cell but also provides measurements such as growth rate and direction for each labeled end. We have validated the method by comparing our results with ground-truth data derived from manual analysis as well as with data obtained using the tracking method. In addition, we have confirmed color-coded representation of different dynamic events by analyzing their history and fate. Finally, we have demonstrated the use of the method to investigate microtubule assembly in cells and provided guidance in selecting optimal image acquisition conditions. Thus, this simple computer vision method offers a unique and quantitative approach to study spatial regulation of microtubule dynamics in cells.     

Testing procedures for non-stationarity and non-linearity in physiological signals

Most of the physiological signals (EEG, ECG, blood flow, human gait, etc.) characterize by complex dynamics including both non-stationarities and non-linearities. These time series resemble red noise with long-range correlation and 1/(f beta) power spectrum. A question arises as to how to distinguish the characteristics of the process underlying the signal dynamics from the properties of the observed time series. The classical methods to determine possible non-linear (chaotic) dynamics (e.g. correlation dimension) often fail in such signals because of relatively short data records containing stochastic components and non-stationarities. We report an application of several approaches, aimed at (1) determining of the non-stationarities in the signals and (2) testing whether non-linear dynamics exists. Assessment of the intrinsic correlation properties of the dynamic process and distinguishing the same from external trends was performed using singular spectra and detrended fluctuation analysis. The existence of non-linear dynamics was tested by correlation dimension (modified algorithm of re-embedding) and by correlation integrals of real and surrogate data. The correlation integrals of real signal and surrogate data sets were statistically compared using Kolmogorov-Smirnov (K-S) test. The procedures were tested on EEG and laser-Doppler (LD) blood flow. Our suggestion is that no one approach taken alone is the best for our aims. Instead, a battery of methods should be used.     

Flagellar protein dynamics in Chlamydomonas

Cilia and flagella appear to be stable, terminal, microtubule-containing organelles, but they also elongate and shorten in response to a variety of signals. To understand mechanisms that regulate flagellar dynamics, Chlamydomonas cells with nongrowing flagella were labeled with (35)S, and flagella and basal body components were examined for labeled polypeptides. Maximal incorporation of label into the flagella occurred within 3 h. Twenty percent of the flagellar polypeptides were exchanged. These included tubulins, dyneins, and 80 other axonemal and membrane plus matrix polypeptides. The most stable flagellar structure is the PF-ribbon, which comprises part of the wall of each doublet microtubule and is composed of tubulin and three other polypeptides. Most (35)S was incorporated into the high molecular weight ribbon polypeptide, rib240, and little, if any, (35)S is incorporated into PF-ribbon-associated tubulin. Both wild-type (9 + 2) and 9 + 0 flagella, which lack central microtubules, exhibited nearly identical exchange patterns, so labeling is not due to turnover of relatively labile central microtubules. To determine if flagellar length is balanced by protein exchange, (35)S incorporation into disassembling flagella was examined, as was exchange in flagella in which microtubule assembly was blocked by colchicine. Incorporation of (35)S-labeled polypeptides was found to occur into flagellar axonemes during wavelength-dependent shortening in pf18 and in fla10 cells induced to shorten flagella by incubation at 33 degrees C. Colchicine blocked tubulin addition but did not affect the exchange of the other exchangeable polypeptides; nor did it induce any change in flagellar length. Basal bodies also incorporated newly synthesized proteins. These data reveal that Chlamydomonas flagella are dynamic structures that incorporate new protein both during steady state and as flagella shorten and that protein exchange does not, alone, explain length regulation.     

Automated comparative proteomics based on multiplex tandem mass spectrometry and stable isotope labeling

Comparative proteomic approaches using isotopic labeling and MS have become increasingly popular. Conventionally quantification is based on MS or extracted ion chromatogram (XIC) signals of differentially labeled peptides. However, in these MS-based experiments, the accuracy and dynamic range of quantification are limited by the high noise levels of MS/XIC data. Here we report a quantitative strategy based on multiplex (derived from multiple precursor ions) MS/MS data. One set of proteins was metabolically labeled with [13C6]lysine and [15N4]arginine; the other set was unlabeled. For peptide analysis after tryptic digestion of the labeled proteins, a wide precursor window was used to include both the light and heavy versions of each peptide for fragmentation. The multiplex MS/MS data were used for both protein identification and quantification. The use of the wide precursor window increased sensitivity, and the y ion pairs in the multiplex MS/MS spectra from peptides containing labeled and unlabeled lysine or arginine offered more information for, and thus the potential for improving, protein identification. Protein ratios were obtained by comparing intensities of y ions derived from the light and heavy peptides. Our results indicated that this method offers several advantages over the conventional XIC-based approach, including increased sensitivity for protein identification and more accurate quantification with more than a 10-fold increase in dynamic range. In addition, the quantification calculation process was fast, fully automated, and independent of instrument and data type. This method was further validated by quantitative analysis of signaling proteins in the EphB2 pathway in NG108 cells.     

Bridging the Gap between Single Molecule and Ensemble Methods for Measuring Lateral Dynamics in the Plasma Membrane

The lateral dynamics of proteins and lipids in the mammalian plasma membrane are heterogeneous likely reflecting both a complex molecular organization and interactions with other macromolecules that reside outside the plane of the membrane. Several methods are commonly used for characterizing the lateral dynamics of lipids and proteins. These experimental and data analysis methods differ in equipment requirements, labeling complexities, and further oftentimes give different results. It would therefore be very convenient to have a single method that is flexible in the choice of fluorescent label and labeling densities from single molecules to ensemble measurements, that can be performed on a conventional wide-field microscope, and that is suitable for fast and accurate analysis. In this work we show that k-space image correlation spectroscopy (kICS) analysis, a technique which was originally developed for analyzing lateral dynamics in samples that are labeled at high densities, can also be used for fast and accurate analysis of single molecule density data of lipids and proteins labeled with quantum dots (QDs). We have further used kICS to investigate the effect of the label size and by comparing the results for a biotinylated lipid labeled at high densities with Atto647N-strepatvidin (sAv) or sparse densities with sAv-QDs. In this latter case, we see that the recovered diffusion rate is two-fold greater for the same lipid and in the same cell-type when labeled with Atto647N-sAv as compared to sAv-QDs. This data demonstrates that kICS can be used for analysis of single molecule data and furthermore can bridge between samples with a labeling densities ranging from single molecule to ensemble level measurements.     

Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy

Selective isotopic labeling provides an unparalleled window within which to study the structure and dynamics of RNAs by high resolution NMR spectroscopy. Unlike commonly used carbon sources, the asymmetry of ¹³C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using Escherichia coli variants, that until now were not feasible. Here we show that an E. coli mutant strain that lacks succinate and malate dehydrogenases (DL323) and grown on [3-¹³C]-pyruvate affords ribonucleotides with site specific labeling at C5′ (~95%) and C1′ (~42%) and minimal enrichment elsewhere in the ribose ring. Enrichment is also achieved at purine C2 and C8 (~95%) and pyrimidine C5 (~100%) positions with minimal labeling at pyrimidine C6 and purine C5 positions. These labeling patterns contrast with those obtained with DL323 E. coli grown on [1, 3-¹³C]-glycerol for which the ribose ring is labeled in all but the C4′ carbon position, leading to multiplet splitting of the C1′, C2′ and C3′ carbon atoms. The usefulness of these labeling patterns is demonstrated with a 27-nt RNA fragment derived from the 30S ribosomal subunit. Removal of the strong magnetic coupling within the ribose and base leads to increased sensitivity, substantial simplification of NMR spectra, and more precise and accurate dynamic parameters derived from NMR relaxation measurements. Thus these new labels offer valuable probes for characterizing the structure and dynamics of RNA that were previously limited by the constraint of uniformly labeled nucleotides.     

Dynamic proteome analysis of Cyanothece sp. ATCC 51142 under constant light

Understanding the dynamic nature of protein abundances provides insights into protein turnover not readily apparent from conventional, static mass spectrometry measurements. This level of data is particularly informative when surveying protein abundances in biological systems subjected to large perturbations or alterations in environment such as cyanobacteria. Our current analysis expands upon conventional proteomic approaches in cyanobacteria by measuring dynamic changes of the proteome using a (13)C(15)N-l-leucine metabolic labeling in Cyanothece ATCC51142. Metabolically labeled Cyanothece ATCC51142 cells grown under nitrogen-sufficient conditions in continuous light were monitored longitudinally for isotope incorporation over a 48 h period, revealing 414 proteins with dynamic changes in abundances. In particular, proteins involved in carbon fixation, pentose phosphate pathway, cellular protection, redox regulation, protein folding, assembly, and degradation showed higher levels of isotope incorporation, suggesting that these biochemical pathways are important for growth under continuous light. Calculation of relative isotope abundances (RIA) values allowed the measurement of actual active protein synthesis over time for different biochemical pathways under high light exposure. Overall results demonstrated the utility of "non-steady state" pulsed metabolic labeling for systems-wide dynamic quantification of the proteome in Cyanothece ATCC51142 that can also be applied to other cyanobacteria.     

Monitoring aromatic picosecond to nanosecond dynamics in proteins via 13C relaxation: expanding perturbation mapping of the rigidifying core mutation, V54A, in eglin c

Long-range effects, such as allostery, have evolved in proteins as a means of regulating function via communication between distal sites. An NMR-based perturbation mapping approach was used to more completely probe the dynamic response of the core mutation V54A in the protein eglin c by monitoring changes in picosecond to nanosecond aromatic side-chain dynamics and H/D exchange stabilities. Previous side-chain dynamics studies on this mutant were limited to methyl-bearing residues, most of which were found to rigidify on the picosecond to nanosecond time scale in the form of a contiguous "network". Here, high precision (13)C relaxation data from 13 aromatic side chains were acquired by applying canonical relaxation experiments to a newly developed carbon labeling scheme [Teilum et al. (2006) J. Am. Chem. Soc. 128, 2506-2507]. The fitting of model-free parameters yielded S (2) variability which is intermediate with respect to backbone and methyl-bearing side-chain variability and tau e values that are approximately 1 ns. Inclusion of the aromatic dynamic response results in an expanded network of dynamically coupled residues, with some aromatics showing increases in flexibility, which partially offsets the rigidification in methyl side chains. Using amide hydrogen exchange, dynamic propagation on a slower time scale was probed in response to the V54A perturbation. Surprisingly, regional stabilization (slowed exchange) 10-12 A from the site of mutation was observed despite a global destabilization of 1.5 kcal x mol (-1). Furthermore, this unlikely pocket of stabilized residues colocalizes with increases in aromatic flexibility on the faster time scale. Because the converse is also true (destabilized residues colocalize with rigidification on the fast time scale), a plausible entropy-driven mechanism is discussed for relating colocalization of opposing dynamic trends on vastly different time scales.     

Measuring fast dynamics in solutions and cells with a laser scanning microscope

Single-point fluorescence correlation spectroscopy (FCS) allows measurements of fast diffusion and dynamic processes in the microsecond-to-millisecond time range. For measurements on living cells, image correlation spectroscopy (ICS) and temporal ICS extend the FCS approach to diffusion times as long as seconds to minutes and simultaneously provide spatially resolved dynamic information. However, ICS is limited to very slow dynamics due to the frame acquisition rate. Here we develop novel extensions to ICS that probe spatial correlations in previously inaccessible temporal windows. We show that using standard laser confocal imaging techniques (raster-scan mode) not only can we reach the temporal scales of single-point FCS, but also have the advantages of ICS in providing spatial information. This novel method, called raster image correlation spectroscopy (RICS), rapidly measures during the scan many focal points within the cell providing the same concentration and dynamic information of FCS as well as information on the spatial correlation between points along the scanning path. Longer time dynamics are recovered from the information in successive lines and frames. We exploit the hidden time structure of the scan method in which adjacent pixels are a few microseconds apart thereby accurately measuring dynamic processes such as molecular diffusion in the microseconds-to-seconds timescale. In conjunction with simulated data, we show that a wide range of diffusion coefficients and concentrations can be measured by RICS. We used RICS to determine for the first time spatially resolved diffusions of paxillin-EGFP stably expressed in CHOK1 cells. This new type of data analysis has a broad application in biology and it provides a powerful tool for measuring fast as well as slower dynamic processes in cellular systems using any standard laser confocal microscope.     

Rotational diffusion of human lipoproteins and their receptors as determined by time-resolved phosphorescence anisotropy

Time-resolved phosphorescence anisotropy has been used to assess the rotational dynamics of human serum lipoproteins labeled with phosphorescent probes of high triplet yield. Labeling the lipid phase of low density, very low density, and high density lipoproteins with an eosinyl fatty acid revealed the existence of two motions. The shorter time constant was attributed to motion of the chromophore within the lipoprotein particle, while the longer time constant represented the global tumbling of the particles in solution. The measured correlation times for this global motion were about twice those predicted from the Stokes-Einstein relationship. Covalent labeling of the apolipoproteins of the low and high density lipoproteins with erythrosin revealed the existence of segmental motion of labeled domains of the apolipoprotein within their respective particles. The correlation times for this motion were within the range 10-50 microseconds. The binding of low density lipoproteins to receptors on membranes isolated from the adrenal cortex resulted in a freezing of the global motion, but maintenance of the faster segmental motion of the labeled domains of the apolipoprotein. The experiments imply that in these membranes there is no global motion of the low density lipoprotein-receptor complex on the phosphorescence time scale. Similar results were found for the binding of high density lipoproteins to liver plasma membranes. The contributions of nonspecific binding of the labeled lipoproteins to the measured phosphorescence anisotropy were carefully assessed.     

Asymmetry of <Superscript>13</Superscript>C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy

Selective isotopic labeling provides an unparalleled window within which to study the structure and dynamics of RNAs by high resolution NMR spectroscopy. Unlike commonly used carbon sources, the asymmetry of ¹³C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using E. coli variants, that until now were not feasible. Here we show that an E. coli mutant strain that lacks succinate and malate dehydrogenases (DL323) and grown on [3-¹³C]-pyruvate affords ribonucleotides with site specific labeling at C5′ (~95%) and C1′ (~42%) and minimal enrichment elsewhere in the ribose ring. Enrichment is also achieved at purine C2 and C8 (~95%) and pyrimidine C5 (~100%) positions with minimal labeling at pyrimidine C6 and purine C5 positions. These labeling patterns contrast with those obtained with DL323 E. coli grown on [1, 3-¹³C]-glycerol for which the ribose ring is labeled in all but the C4′ carbon position, leading to multiplet splitting of the C1′, C2′ and C3′ carbon atoms. The usefulness of these labeling patterns is demonstrated with a 27-nt RNA fragment derived from the 30S ribosomal subunit. Removal of the strong magnetic coupling within the ribose and base leads to increased sensitivity, substantial simplification of NMR spectra, and more precise and accurate dynamic parameters derived from NMR relaxation measurements. Thus these new labels offer valuable probes for characterizing the structure and dynamics of RNA that were previously limited by the constraint of uniformly labeled nucleotides.     

Dynamics of DNA replication: an ultrastructural study

DNA replication in cells takes place in domains scattered throughout the nucleoplasm. We have characterized the dynamics of DNA synthesis in synchronized mid-S-phase HeLa cells. Saponin-permeabilized cells were allowed to elongate nascent DNA chains in presence of biotin-dUTP for 5, 15, and 30 min (a pulse experiment), or for 5 min followed by an incubation with unlabeled precursors for 10 or 25 min (a pulse-and-chase experiment). The replication foci were then identified in ultrathin sections using immunogold labeling of the incorporated biotin. Total number of particles per nucleus, total scanned area of the nucleus, size, shape, and gold particle number of each labeled cluster, and the density of clusters per nucleus were evaluated. We have demonstrated that as replication proceeds, the labeled sites increase in size up to 240 nm (30 min incorporation) while maintaining a broadly round shape. In pulse-and-chase experiments the labeled DNA was shown to spread to occupy DNA foci of approximately 400 nm in diameter. These results demonstrate that DNA replication is compartmentalized within cell nuclei at the level of DNA foci and support the view that the synthetic centers are spatially constrained while the chromatin loops are dynamic during DNA synthesis.     

A model of interorganelle monoclonal antibody transport and secretion in mouse hybridoma cells

A three compartment model (ER --> Golgi --> extracellular medium) is used here to describe the interorganelle transport and final secretion of an IgG(2a) monoclonal antibody (MAb) in 9.2.27 murine hybridoma cells. Model simulations of pulse-chase and continuous labeling experiments are used to gain a better understanding of the kinetics of MAb interorganelle traffic. Simulation results for the continuous labeling case compare well with experimental data obtained during continuous labeling of 9.2.27 hybridoma cells. Incorporation of this compartmental transport model into our previously developed model of MAb synthesis and assembly can provide a useful tool for analyzing the dynamics and regulation of the complete antibody secretory pathway under different growth and/or nutritional conditions.     

Investigating macromolecules inside cultured and injected cells by in-cell NMR spectroscopy

The noninvasive character of NMR spectroscopy, combined with the sensitivity of the chemical shift, makes it ideally suited to investigate the conformation, binding events and dynamics of macromolecules inside living cells. These 'in-cell NMR' experiments involve labeling the macromolecule of interest with a nonradioactive but NMR-active isotope (15N or 13C). Cellular samples are prepared either by selectively overexpressing the protein in suitable cells (e.g., bacterial cells grown on isotopically labeled media), or by injecting isotopically labeled proteins directly into either cells or cell extracts. Here we provide detailed protocols for in-cell NMR experiments in the prokaryotic organism Escherichia coli, as well as eukaryotic cells and extracts employing Xenopus laevis oocytes or egg extracts. In-cell NMR samples with proteins overexpressed in E. coli can be produced within 13-14 h. Preparing Xenopus oocyte samples for in-cell NMR experiments takes 6-14 h depending on the oocyte preparation scheme and the injection method used.     

A fluorescent imaging method for analyzing the biodistribution of therapeutic monoclonal antibodies that can distinguish intact antibodies from their breakdown products

Many monoclonal antibodies have been developed for therapy over the last 2 decades. In the development of therapeutic antibodies, the preclinical assessment of an antibody's biodistribution is important for the prediction of the antibody's efficacy and safety. For imaging analyses of such biodistributions, radioisotope (RI) labeling and fluorescence labeling methods are typically used, but the resulting data are limited because these methods cannot distinguish breakdown products from intact antibodies. To resolve this problem, we developed a novel method using fluorescent resonance energy transfer (FRET)-type labeling and a spectral unmixing tool. With FRET-type labeling (labeling with 2 species of fluorophore), different fluorescence properties of labeled intact antibodies and their breakdown products (the hydrolyzed/digested type of breakdown products) are made visible. With the spectral unmixing tool, the fluorescence of a solution containing the intact antibody and its breakdown products could be unmixed in proportion to their contents. Moreover, when labeled antibodies that targeted either human epidermal growth factor receptor-2 or epidermal growth factor receptor were injected into nude mice implanted subcutaneously with tumor cells, the accumulation of the injected labeled antibodies and their breakdown products in the tumor could be separately analyzed by both whole-mouse imaging and a tumor homogenate analysis. These results suggest that our method using FRET-type labeling and a spectral unmixing tool could be useful in distinguishing breakdown products from intact antibodies.     

Antibody labeling of bovine neurofilaments: implications on the structure of neurofilament sidearms.

We carried out immunolabeling studies of purified bovine spinal cord neurofilaments (NFs) and filaments reconstituted from several combinations of the NF triplet polypeptides, NF-H, NF-M, and NF-L. Six antibodies with known epitopes in either the rod domains or the tailpiece extensions of the NF triplet were used in these studies, and the immune complexes were visualized directly by the glycerol-spray, rotary shadowing technique, which permitted unambiguous identification of the NF sidearms. Antibodies directed against the tailpiece extensions of NF-H and NF-M labeled the sidearms of native NFs and reconstituted filaments containing those two polypeptides, but not the backbone of the filaments. Combining these two antibodies in the same labeling experiment resulted in more intense labeling than either of the antibodies alone, indicating that both NF-H and NF-M are capable of forming sidearms. The anti-NF-L tailpiece antibody recognized only a limited number of sites along native NFs, but labeled reconstituted NF-L homopolymers uniformly and heavily. This suggests that the NF-L tailpiece extension is relatively inaccessible in native filaments, but is accessible in reconstituted homopolymers. One possible explanation is that, in native NFs, the NF-H- and NF-M-containing sidearms curtailed antibody access to NF-L. A second possibility that is not mutually exclusive with the first is that, when both NF-L and another triplet polypeptide are present, they preferentially form heterodimers such that the NF-L tailpiece epitope becomes hidden. Taken collectively, and in combination with published structural information, our data are consistent with a subunit packing scheme in which an NF-L-containing dimer serves as the fundamental building block of most mammalian NFs, such that their sidearms consist of pairs of NF-H/NF-L, NF-M/NF-L, or NF-L/NF-L tailpiece extensions.     

Investigation of the dynamics underlying periodic complexes in the EEG

Periodic complexes (PC), occurring lateralised or diffuse, are relatively rare EEG phenomena which reflect acute severe brain disease. The pathophysiology is still incompletely understood. One hypothesis suggested by the alpha rhythm model of Lopes da Silva is that periodic complexes reflect limit cycle dynamics of cortical networks caused by excessive excitatory feedback. We examined this hypothesis by applying a recently developed technique to EEGs displaying periodic complexes and to periodic complexes generated by the model. The technique, non-linear cross prediction, characterises how well a time series can be predicted, and how much amplitude and time asymmetry is present. Amplitude and time asymmetry are indications of non-linearity. In accordance with the model, most EEG channels with PC showed clear evidence of amplitude and time asymmetry, pointing to non-linear dynamics. However, the non-linear predictability of true PC was substantially lower than that of PC generated by the model. Furthermore, no finite value for the correlation dimension could be obtained for the real EEG data, whereas the model time series had a dimension slighter higher than one, consistent with a limit cycle attractor. Thus we can conclude that PC reflect non-linear dynamics, but a limit cycle attractor is too simple an explanation. The possibility of more complex (high dimensional and spatio-temporal) non-linear dynamics should be investigated. Received: 26 February 1998 / Accepted in revised form: 24 August 1998     

Patterning of the head in hydra as visualized by a monoclonal antibody: III. The dynamics of head regeneration

The dynamics of the early patterning processes leading to the regeneration of a head in tissue excised from the body column of Hydra oligactis were examined by using a monoclonal antibody, CP8. This antibody displays position-specific binding, labeling the head ectodermal epithelial cells. During regeneration of a head, antibody labeling is present well before morphological signs of the head, at a time correlated with the determination of the tissue (Javois et al., Dev. Biol., 117:607-618, '86). By quantifying antibody labeling during regeneration of three different pieces of tissue excised from the body column, it was found that the dynamics of the early patterning processes as visualized by CP8 labeling varied. The pattern of labeling observed as well as the spread of labeled tissue suggested that the amount and geometry of apical tissue in the regenerate played a critical role in the patterning processes. Contrary to the labeling pattern observed in heads which formed during bud development or which regenerated following decapitation (Javois et al., '86), not all the CP8+ tissue was confined to the head structures in these regenerates. Several alternative explanations for this surprising result are presented. The usefulness of these data in refining pattern formation models by more explicitly constraining their parameters is discussed.