ABSTRACT
We have probed single kinetochore microtubule (k-MT) dynamics in budding yeast in the G1 phase of the cell cycle by automated tracking of a green fluorescent protein tag placed proximal to the centromere on chromosome IV and of a green fluorescent protein tag fused to the spindle pole body protein Spc42p. Our method reliably distinguishes between different dynamics in wild-type and mutant strains and under different experimental conditions. Using our methods we established that in budding yeast, unlike in metazoans, chromosomes make dynamic attachments to microtubules in G1. This makes it possible to interpret measurements of centromere tag dynamics as reflecting k-MT dynamics. We have examined the sensitivity of our assay by studying the effect of temperature, exposure to benomyl, and a tubulin mutation on k-MT dynamics. We have found that lowering the temperature and exposing cells to benomyl attenuate k-MT dynamics in a similar manner. We further observe that, in contrast to previous reports, the mutant tub2-150 forms k-MTs that depolymerize faster than wild type. Based on these findings, we propose high-resolution light microscopy of centromere dynamics in G1 yeast cells as a sensitive assay for the regulation of single k-MT dynamics.
Subject(s)
Image Enhancement/methods , Image Interpretation, Computer-Assisted/methods , Imaging, Three-Dimensional/methods , Kinetochores/metabolism , Microscopy, Fluorescence/methods , Microtubules/metabolism , Saccharomycetales/metabolism , Algorithms , Kinetics , Kinetochores/ultrastructure , Microtubules/ultrastructure , Protein Transport/physiology , Saccharomycetales/cytologySubject(s)
BRCA1 Protein/genetics , BRCA2 Protein/genetics , DNA Mutational Analysis/methods , Breast Neoplasms/genetics , Breast Neoplasms/prevention & control , DNA Mutational Analysis/economics , Electrophoresis, Gel, Two-Dimensional/methods , Female , Genetic Testing/economics , Genetic Testing/methods , Humans , Mutation , Ovarian Neoplasms/genetics , Ovarian Neoplasms/prevention & control , Reproducibility of ResultsABSTRACT
In this paper, we describe an algorithmic framework for the automatic detection of diffraction-limited fluorescent spots in 3D optical images at a separation below the Rayleigh limit, i.e. with super-resolution. We demonstrate the potential of super-resolution detection by tracking fluorescently tagged chromosomes during mitosis in budding yeast. Our biological objective is to identify and analyse the proteins responsible for the generation of tensile force during chromosome segregation. Dynamic measurements in living cells are made possible by green fluorescent protein (GFP)-tagging chromosomes and spindle pole bodies to generate cells carrying four fluorescent spots, and observe the motion of the spots over time using 3D-fluorescence microscopy. The central problem in spot detection arises with the partial or complete overlap of spots when tagged objects are separated by distances below the resolution of the optics. To detect multiple spots under these conditions, a set of candidate mixture models is built, and the best candidate is selected from the set based on chi2-statistics of the residuals in least-square fits of the models to the image data. Even with images having a signal-to-noise ratio (SNR) as low as 5-10, we are able to increase the resolution two-fold below the Rayleigh limit. In images with a SNR of 5-10, the accuracy with which isolated tags can be localized is less than 5 nm. For two tags separated by less than the Rayleigh limit, the localization accuracy is found to be between 10 and 20 nm, depending on the effective point-to-point distance. This indicates the intimate relationship between resolution and localization accuracy.
Subject(s)
Algorithms , Chromosomes, Fungal/physiology , Imaging, Three-Dimensional/methods , Microscopy, Fluorescence/methods , Yeasts/cytology , Fluorescent Dyes/analysis , Green Fluorescent Proteins , Luminescent Proteins , Spindle ApparatusABSTRACT
Two-dimensional gene scanning (TDGS) is a method for analyzing multiple DNA fragments in parallel for all possible sequence variations, using extensive multiplex PCR and two-dimensional electrophoretic separation on the basis of size and melting temperature. High throughput application of TDGS is limited by the prolonged time periods necessary to complete the second-dimension electrophoretic separation step--denaturing gradient gel electrophoresis--and the current need for gel staining. To address these problems, we constructed a high-voltage, automatic, two-dimensional electrophoresis system and used this in combination with thinner gels to reduce two-dimensional electrophoresis time about 80%. Instead of gel staining, we used three different fluorophores to simultaneously analyze three samples in the same gel. These improvements greatly increase TDGS speed and throughput and make the method highly suitable for large-scale single-nucleotide polymorphism discovery and genetic testing.
Subject(s)
DNA/analysis , Electrophoresis, Gel, Two-Dimensional/methods , Polymerase Chain Reaction/methods , Adaptor Proteins, Signal Transducing , BRCA1 Protein/genetics , Carrier Proteins , DNA/genetics , Humans , MutL Protein Homolog 1 , Neoplasm Proteins/genetics , Nuclear Proteins , Sensitivity and Specificity , Tumor Suppressor Protein p53/geneticsABSTRACT
The complex series of movements that mediates chromosome segregation during mitosis is dependent on the attachment of microtubules to kinetochores, DNA-protein complexes that assemble on centromeric DNA. We describe the use of live-cell imaging and chromatin immunoprecipitation in S. cerevisiae to identify ten kinetochore subunits, among which are yeast homologs of microtubule binding proteins in animal cells. By analyzing conditional mutations in several of these proteins, we show that they are required for the imposition of tension on paired sister kinetochores and for correct chromosome movement. The proteins include both molecular motors and microtubule associated proteins (MAPs), implying that motors and MAPs function together in binding chromosomes to spindle microtubules.
Subject(s)
Chromosomes, Fungal/metabolism , Kinetochores/metabolism , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Biological Transport , Cell Nucleus/metabolism , Chromatids/chemistry , Chromatids/genetics , Chromatids/metabolism , Chromatin/chemistry , Chromatin/genetics , Chromatin/metabolism , Chromosome Segregation , Chromosomes, Fungal/chemistry , Chromosomes, Fungal/genetics , DNA, Fungal/genetics , DNA, Fungal/metabolism , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/metabolism , Kinetochores/chemistry , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/genetics , Microtubules/chemistry , Molecular Motor Proteins/chemistry , Molecular Motor Proteins/genetics , Molecular Motor Proteins/metabolism , Mutation/genetics , Precipitin Tests , Protein Binding , Saccharomyces cerevisiae/genetics , Spindle Apparatus/chemistry , Spindle Apparatus/metabolismABSTRACT
Several kinesin holoenzymes, including the heterotrimeric kinesin-II and bipolar KLP61F complexes described here, are being purified in our laboratory using microtubule affinity precipitation and conventional biochemical fractionation procedures. These protocols have been optimized by using pan-kinesin peptide antibodies and subunit-specific antibodies to monitor the enrichment of kinesin-related polypeptides in particular fractions by immunoblotting. Protein purification represents the most direct route available for determining the oligomeric state and subunit composition of a kinesin holoenzyme, for identifying tightly associated accessory subunits such as SpKAP115, and for determining the molecular architecture and functional properties of native kinesin motors. Protein purification methods therefore represent an important complementary approach to molecular genetic approaches that are being pursued in many other laboratories.
Subject(s)
Drosophila Proteins , Embryo, Nonmammalian/chemistry , Kinesins/isolation & purification , Microtubules/metabolism , Molecular Motor Proteins/isolation & purification , Animals , Blotting, Western , Calcium-Binding Proteins/isolation & purification , Calcium-Binding Proteins/metabolism , Centrifugation, Density Gradient , Chemical Precipitation , Chromatography, Gel , Chromatography, Ion Exchange , Cytosol/chemistry , Drosophila melanogaster/chemistry , Drosophila melanogaster/embryology , Embryo, Nonmammalian/physiology , Holoenzymes/isolation & purification , Kinesins/metabolism , Microtubule-Associated Proteins/isolation & purification , Microtubule-Associated Proteins/metabolism , Molecular Motor Proteins/metabolism , Muscle Proteins/isolation & purification , Muscle Proteins/metabolism , Ovum/chemistry , Polymers/isolation & purification , Polymers/metabolism , Sea Urchins/chemistry , Sea Urchins/embryologyABSTRACT
A CdSe optical parametric oscillator (OPO) pumped by a 2.79-mum , Cr, Er:YSGG laser yielded a 59% signal-plus-idler slope efficiency (eta), a total idler output of 1.2-2.4mJ between 8.5 and 12.3 mum , and an idler beam that was 2.2-2.5 times the diffraction limit. A ZnGeP(2) OPO operated with a lower threshold, eta = 29% , and a forward idler output of 0.7-2.4 mJ from 6.9 to 9.9 microm . The signal and idler bandwidths were typically 4 cm(-1) for each OPO.
ABSTRACT
We have generated output energies of 900 mJ at 2050 nm from a pulsed, room-temperature, normal-mode Co:MgF(2) laser, with average powers as high as 6.5 W. Over the range 1800 to 2450 nm we have obtained pulse energies of at least 100 mJ.
ABSTRACT
A cw Ti:A1(2)O(3) ring laser with a threshold power of 119 mW is demonstrated. It provides a tunable source of single-frequency, diffraction-limited radiation that is suitable for injection seeding. The Ti:A1(2)O(3) laser is operated with a diode-laser-pumped, frequency-doubled, Nd:YAG laser as the sole pump source.
ABSTRACT
We report operation of a laser-diode side-pumped Nd: YAG laser with a novel pumping geometry that ensures efficient conversion of pump energy into the TEM(00) mode. Of the 1064-nm output, 11.8 mJ of energy was obtained in a 200-micros pulse with 64 mJ of pump energy at 808 nm. The overall conversion and slope efficiencies were 18% and 23%, respectively.
ABSTRACT
This study was designed to investigate the reliability of real-ear measurements of sound pressure level (SPL) and to compare these values with two coupler measures of SPL. A commercially available probe tube microphone system was used to measure real ear SPL in both children and adults. Test-retest reliability decreased as a function of frequency for both groups and, in general, was slightly poorer for the children. For both groups, coupler to real ear differences were larger for the 2 cm3 coupler than for the reduced volume coupler; however, no significant differences were observed between groups. In addition, a measure of ear canal volume was not found to be a good predictor of coupler to real ear discrepancies.
Subject(s)
Hearing Aids , Hearing Loss, Sensorineural/rehabilitation , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Humans , Loudness Perception , Middle Aged , Pitch DiscriminationABSTRACT
The functional gain of a hearing aid typically is determined by comparing aided and unaided behavioral thresholds. With this method, however, true gain may be underestimated in frequency regions of normal or near-normal hearing sensitivity (i.e., in cases of sloping, rising, or trough-shaped audiograms). Internal hearing-aid noise and/or amplified room noise imposes a lower limit on obtainable aided thresholds. In these cases, comparing aided and unaided acoustic-reflex thresholds may be a valuable clinical alternative to traditional means of determining real-ear gain. This study compared sound-field behavioral threshold and acoustic-reflex threshold estimates of functional gain for individuals with a variety of audiometric configurations. The sound-field behavioral threshold measurements were found to underestimate functional gain if unaided thresholds approached the normal hearing range. In regions of greater hearing loss, behavioral and acoustic-reflex estimates of functional gain were in good agreement.
