Jason R. Swedlow

Interphase chromosomes undergo constrained diffusional motion in living cells

BACKGROUND Structural studies of fixed cells have revealed that interphase chromosomes are highly organized into specific arrangements in the nucleus, and have led to a picture of the nucleus as a static structure with immobile chromosomes held in fixed positions, an impression apparently confirmed by recent photobleaching studies. Functional studies of chromosome behavior, however, suggest that many essential processes, such as recombination, require interphase chromosomes to move around within the nucleus. RESULTS To reconcile these contradictory views, we exploited methods for tagging specific chromosome sites in living cells of Saccharomyces cerevisiae with green fluorescent protein and in Drosophila melanogaster with fluorescently labeled topoisomerase ll. Combining these techniques with submicrometer single-particle tracking, we directly measured the motion of interphase chromatin, at high resolution and in three dimensions. We found that chromatin does indeed undergo significant diffusive motion within the nucleus, but this motion is constrained such that a given chromatin segment is free to move within only a limited subregion of the nucleus. Chromatin diffusion was found to be insensitive to metabolic inhibitors, suggesting that it results from classical Brownian motion rather than from active motility. Nocodazole greatly reduced chromatin confinement, suggesting a role for the cytoskeleton in the maintenance of nuclear architecture. CONCLUSIONS We conclude that chromatin is free to undergo substantial Brownian motion, but that a given chromatin segment is confined to a subregion of the nucleus. This constrained diffusion is consistent with a highly defined nuclear architecture, but also allows enough motion for processes requiring chromosome motility to take place. These results lead to a model for the regulation of chromosome interactions by nuclear architecture.

Role of neurogenic genes in establishment of follicle cell fate and oocyte polarity during oogenesis in Drosophila

Oogenesis in Drosophila involves specification of both germ cells and the surrounding somatic follicle cells, as well as the determination of oocyte polarity. We found that two neurogenic genes, Notch and Delta, are required in oogenesis. These genes encode membrane proteins with epidermal growth factor repeats and are essential in the decision of an embryonic ectodermal cell to take on the fate of neuroblast or epidermoblast. In oogenesis, mutation in either gene leads to an excess of posterior follicle cells, a cell fate change reminiscent of the hyperplasia of neuroblasts seen in neurogenic mutant embryos. Furthermore, the Notch mutation in somatic cells causes mislocalization of bicoid in the oocyte. These results suggest that the neurogenic genes Notch and Delta are involved in both follicle cell development and the establishment of anterior-posterior polarity in the oocyte.

Association of the amino-terminal half of c-Src with focal adhesions alters their properties and is regulated by phosphorylation of tyrosine 527

We have characterized the mechanism by which the subcellular distribution of c‐Src is controlled by the phosphorylation of tyrosine 527. Mutation of this tyrosine dramatically redistributes c‐Src from endosomal membranes to focal adhesions. Redistribution to focal adhesions occurs independently of kinase activity and cellular transformation. In cells lacking the regulatory kinase (CSK) that phosphorylates tyrosine 527, c‐Src is also found predominantly in focal adhesions, confirming that phosphorylation of tyrosine 527 affects the location of c‐Src inside the cell. The first 251 amino acids of c‐Src are sufficient to allow association with focal adhesions, indicating that at least one signal for positioning c‐Src in focal adhesions resides in the amino‐terminal half. Point mutations and deletions in the first 251 amino acids of c‐Src reveal that association with focal adhesions requires the myristylation site needed for membrane attachment, as well as the SH3 domain. Expression of the amino‐terminal region alters both the structural and biochemical properties of focal adhesions. Focal adhesions containing this non‐catalytic portion of c‐Src are larger and exhibit increased levels of phosphotyrosine staining. Our results suggest that c‐Src may regulate focal adhesions and cellular adhesion by a kinase‐independent mechanism.

Multiple chromosomal populations of topoisomerase II detected in vivo by time-lapse, three-dimensional wide-field microscopy.

The localization of topoisomerase II (topo II) in vivo was studied by recording time-lapse, three-dimensional data sets of living Drosophila melanogaster embryos injected with rhodamine-labeled topo II. These images show that topo II is concentrated at specific sites within the interphase nucleus and that this localization is temporally regulated. The enzyme is not restricted to a central chromosome axis, but is distributed uniformly throughout the chromosome. During mitosis, the enzyme present in the early prophase chromosome is lost in two stages, following prophase and following anaphase. Overall, 70% of the enzyme leaves the nucleus and diffuses into the cytoplasm. The localization of the enzyme thus correlates with its role in chromosome condensation and segregation. Rather than being solely a structural protein, topo II appears to localize at the sites on the chromosome where it is required.

Dispersion, aberration and deconvolution in multi‐wavelength fluorescence images

The wavelength dependence of the incoherent point spread function in a wide‐field microscope was investigated experimentally. Dispersion in the sample and optics can lead to significant changes in the point spread function as wavelength is varied over the range commonly used in fluorescence microscopy. For a given sample, optical conditions can generally be optimized to produce a point spread function largely free of spherical aberration at a given wavelength. Unfortunately, deviations in wavelength from this value will result in spherically aberrated point spread functions. Therefore, when multiple fluorophores are used to localize different components in the same sample, the image of the distribution of at least one of the fluorophores will be spherically aberrated. This aberration causes a loss of intensity and resolution, thereby complicating the localization and analysis of multiple components in a multi‐wavelength image. We show that optimal resolution can be restored to a spherically aberrated image by constrained, iterative deconvolution, as long as the spherical aberration in the point spread function used for deconvolution matches the aberration in the image reasonably well. The success of this method is essentially independent of the initial degree of spherical aberration in the image. Deconvolution of many biological images can be achieved by collecting a small library of spherically aberrated and unaberrated point spread functions, and then choosing a point spread function appropriate for deconvolving each image. The co‐localization and relative intensities of multiple components can then be accurately studied in a multi‐wavelength image.

The SMC family: from chromosome condensation to dosage compensation

Recent genetic analyses in yeasts and biochemical studies in vertebrate cells have led to the discovery of a family of putative ATPases that play a fundamental role in chromosome condensation and segregation in mitosis. One of the members was also found to be involved in dosage compensation in Caenorhabditis elegans, providing a new link between global regulation of gene expression and chromosome structure. This unique family of proteins may control higher-order chromosome dynamics by regulated self-assembly or mechanochemical activity.

The Collection, Processing, and Display of Digital Three-Dimensional Images of Biological Specimens

In general, the study and analysis of biological structure requires a three-dimensional (3D) imaging capability. Dramatic technical advances have now made it possible to record 3D microscopic images of biological specimens using either electron or light microscopy. While the collection of 3D data sets has now become routine, the analysis and interpretation of these images generally require significant time and effort. This is true, in part, because each type of image seems to require a specific set of processing algorithms and parameters. In addition, the software tools required for extracting useful information from the resulting complicated multidimensional data sets (e.g., three spatial dimensions, time, different components) are not completely developed. Computational image processing provides a powerful approach for reducing the systematic errors present in any 3D data set and enhancing the clarity and contrast of relevant features.

Chromosome structure inside the nucleus

Recent in situ three-dimensional structural studies have provided a new model for the 30 nm chromatin fiber. In addition, research during the past year has revealed some of the molecular complexity of non-histone chromosomal proteins. Still to come is the unification of molecular insights with chromosomal architecture.

Chromosome dynamics: Fuzzy sequences, specific attachments?

The assembly of condensed chromosomes in a cell-free system is inhibited by the addition of proteins that bind AT-rich DNA. Does this implicate the AT-rich scaffold attachment regions (SARs) in the formation of chromosomes?

Deglycosylation of Gonadotropins with an Endoglycosidase

Abstract A commercially available endoglycosidase (N-glycanase, Genzyme, Boston, Mass.) purified from Flavobacterium meningosepticum with a specificity for cleaving asparagine-linked carbohydrate moieties in glycoproteins was tested on several pituitary and chorionic gonadotropins as substrates. All intact hormones tested were resistant to the action of the enzyme as were all β subunits from the respective gonadotropins. All α subunits, however, were susceptible to the enzyme as evidenced by a decrease in molecular size when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE). Preparative experiments with ovine luteinizing hormone subunit (oLHα) indicated that only 35-40% of the carbohydrate was removed after N-glycanase treatment, suggesting that perhaps only one of the two carbohydrate moieties was cleavable under the conditions employed. The enzyme-modified subunit (DG-oLHα) was able to recombine with untreated oLHβ. An in vitro steroidogenic bioassay (rat Leydig cell) showed that the recombinant (DG-oLHα-oLHβ) was about 22% as potent as the native oLH, but in a testicular membrane binding assay for LH, it was equal in potency to the native hormone in competing with the radioligand.