RESUMEN
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.
Asunto(s)
Aumento de la Imagen/métodos , Interpretación de Imagen Asistida por Computador/métodos , Imagenología Tridimensional/métodos , Cinetocoros/metabolismo , Microscopía Fluorescente/métodos , Microtúbulos/metabolismo , Saccharomycetales/metabolismo , Algoritmos , Cinética , Cinetocoros/ultraestructura , Microtúbulos/ultraestructura , Transporte de Proteínas/fisiología , Saccharomycetales/citologíaRESUMEN
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.
Asunto(s)
Algoritmos , Cromosomas Fúngicos/fisiología , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , Levaduras/citología , Colorantes Fluorescentes/análisis , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes , Huso AcromáticoRESUMEN
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.
Asunto(s)
Cromosomas Fúngicos/metabolismo , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Núcleo Celular/metabolismo , Cromátides/química , Cromátides/genética , Cromátides/metabolismo , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Segregación Cromosómica , Cromosomas Fúngicos/química , Cromosomas Fúngicos/genética , ADN de Hongos/genética , ADN de Hongos/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cinetocoros/química , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/química , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/genética , Proteínas Motoras Moleculares/metabolismo , Mutación/genética , Pruebas de Precipitina , Unión Proteica , Saccharomyces cerevisiae/genética , Huso Acromático/química , Huso Acromático/metabolismoRESUMEN
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.