Biomedical subjects

J R Swedlow

Publications and source records attributed to J R Swedlow.

At least 19 recordsLinked to original sources

PubMed2001

Phosphorylation of threonine 156 of the mu2 subunit of the AP2 complex is essential for endocytosis in vitro and in vivo.

The clathrin-coated pit is the major port of entry for many receptors and pathogens and is the paradigm for membrane-based sorting events in higher cells [1]. Recently, it has been possible to reconstitute in vitro the events leading to assembly, invagination, and budding off of clathrin-coated vesicles, allowing dissection of the machinery required for sequestration of receptors into these structures [2-6]. The AP2 adaptor complex is a key element of this machinery linking receptors to the coat lattice, and it has previously been reported that AP2 can be phosphorylated both in vitro and in vivo [7-10]. However, the physiological significance of this has never been established. Here, we show that phosphorylation of a single threonine residue (Thr156) of the mu2 subunit of the AP2 complex is essential for efficient endocytosis of transferrin both in an in vitro coated-pit budding assay and in living cells.

Adaptor Protein Complex 1↗

PubMed2001

Chromatin-associated protein phosphatase 1 regulates aurora-B and histone H3 phosphorylation.

Proper chromosome condensation requires the phosphorylation of histone and nonhistone chromatin proteins. We have used an in vitro chromosome assembly system based on Xenopus egg cytoplasmic extracts to study mitotic histone H3 phosphorylation. We identified a histone H3 Ser(10) kinase activity associated with isolated mitotic chromosomes. The histone H3 kinase was not affected by inhibitors of cyclin-dependent kinases, DNA-dependent protein kinase, p90(rsk), or cAMP-dependent protein kinase. The activity could be selectively eluted from mitotic chromosomes and immunoprecipitated by specific anti-X aurora-B/AIRK2 antibodies. This activity was regulated by phosphorylation. Treatment of X aurora-B immunoprecipitates with recombinant protein phosphatase 1 (PP1) inhibited kinase activity. The presence of PP1 on chromatin suggested that PP1 might directly regulate the X aurora-B associated kinase activity. Indeed, incubation of isolated interphase chromatin with the PP1-specific inhibitor I2 and ATP generated an H3 kinase activity that was also specifically immunoprecipitated by anti-X aurora-B antibodies. Nonetheless, we found that stimulation of histone H3 phosphorylation in interphase cytosol does not drive chromosome condensation or targeting of 13 S condensin to chromatin. In summary, the chromosome-associated mitotic histone H3 Ser(10) kinase is associated with X aurora-B and is inhibited directly in interphase chromatin by PP1.

Animals↗

PubMed2001

Nuclear dynamics: where genes are and how they got there.

DNA is highly organized spatially, both within domains of chromatin along each chromosome and within the nucleus as a whole. Recent studies suggest that chromatin localization can affect transcriptional and replicational activity. The similarity between the movements of chromatin nuclear bodies suggests a common mechanism that regulates nuclear dynamics.

Active Transport, Cell Nucleus↗

PubMed2001

A role for the Adenomatous Polyposis Coli protein in chromosome segregation.

Mutations in the Adenomatous Polyposis Coli (APC) gene are responsible for familial colon cancer and also occur in the early stages of sporadic colon cancer. APC functions in the Wnt signalling pathway to regulate the degradation of beta-catenin (reviewed in refs 1-3). APC also binds to and stabilizes microtubules in vivo and in vitro, localizes to clusters at the ends of microtubules near the plasma membrane of interphase cells, and is an important regulator of cytoskeletal function. Here we show that cells carrying a truncated APC gene (Min) are defective in chromosome segregation. Moreover, during mitosis, APC localizes to the ends of microtubules embedded in kinetochores and forms a complex with the checkpoint proteins Bub1 and Bub3. In vitro, APC is a high-affinity substrate for Bub kinases. Our data are consistent with a role for APC in kinetochore-microtubule attachment and suggest that truncations in APC that eliminate microtubule binding may contribute to chromosomal instability in cancer cells.

Adenomatous Polyposis Coli Protein↗

PubMed2001

Regulation of clathrin-coated vesicle formation.

The formation of clathrin-coated pits at the plasma membrane requires the concerted action of many different molecules. The real challenge lies in determining the hierarchy of these interactions. We are using assays in both intact and permeabilized cells to dissect the temporal requirements for clathrin-coated vesicle formation, and also to examine the role of phosphorylation of the coat proteins.

Adaptor Proteins, Vesicular Transport↗

PubMed2001

A workingperson's guide to deconvolution in light microscopy.

Thefluorescence microscope is routinely used to study cellular structure in many biomedical research laboratories and is increasingly used as a quantitative assay system for cellular dynamics. One of the major causes of image degradation in the fluorescence microscope is blurring. Deconvolution algorithms use a model of the microscope imaging process to either subtract or reassign out-of-focus blur. A variety of algorithms are now commercially available, each with its own characteristic advantages and disadvantages. In this article, we review the imaging process in the fluorescence microscope and then discuss how the various deconvolution methods work. Finally, we provide a summary of practical tips for using deconvolution and discuss imaging artifacts and how to minimize them.

Algorithms↗

PubMed2000

In vivo analysis of Cajal body movement, separation, and joining in live human cells.

Cajal bodies (also known as coiled bodies) are subnuclear organelles that contain specific nuclear antigens, including splicing small nuclear ribonucleoproteins (snRNPs) and a subset of nucleolar proteins. Cajal bodies are localized in the nucleoplasm and are often found at the nucleolar periphery. We have constructed a stable HeLa cell line, HeLa(GFP-coilin), that expresses the Cajal body marker protein, p80 coilin, fused to the green fluorescent protein (GFP-coilin). The localization pattern and biochemical properties of the GFP-coilin fusion protein are identical to the endogenous p80 coilin. Time-lapse recordings on 63 nuclei of HeLa(GFP-coilin) cells showed that all Cajal bodies move within the nucleoplasm. Movements included translocations through the nucleoplasm, joining of bodies to form larger structures, and separation of smaller bodies from larger Cajal bodies. Also, we observed Cajal bodies moving to and from nucleoli. The data suggest that there may be at least two classes of Cajal bodies that differ in their size, antigen composition, and dynamic behavior. The smaller size class shows more frequent and faster rates of movement, up to 0.9 microm/min. The GFP-coilin protein is dynamically associated with Cajal bodies as shown by changes in their fluorescence intensity over time. This study reveals an unexpectedly high level of movement and interactions of nuclear bodies in human cells and suggests that these movements may be driven, at least in part, by regulated mechanisms.

Biological Transport↗

PubMed1997

Actin-dependent localization of an RNA encoding a cell-fate determinant in yeast.

The cytoplasmic localization of messenger RNA creates an asymmetric distribution of proteins that specify cell fate during development in multicellular eukaryotes. The protein Ash1 is a cell-fate determinant in budding yeast which localizes preferentially to the presumptive daughter nucleus, where it inhibits mating-type switching. Here we show that Ash1 mRNA is localized to the distal tip of daughter buds in post-anaphase cells. Three-dimensional imaging reveals that Ash1 mRNA is assembled into particles that associate with the cell cortex. To achieve this localization, Ash1 mRNA must have its 3' untranslated region and the actin cytoskeleton must be intact. Ash1 mRNA is not localized correctly in the absence of a myosin (Myo4) and is mislocalized to the mother-bud neck in the absence of a regulator of the actin cytoskeleton known as Bnil. We propose that Ash1 mRNA particles are transported into the daughter bud along actin filaments and are anchored at the distal tip. Thus, as in higher eukaryotes, Saccharomyces cerevisiae employs RNA localization to generate an asymmetric distribution of proteins and hence to determine cell fate.

Actins↗

PubMed1996

Fuzzy sequences, specific attachments? Chromosome dynamics.

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?

Cell-Free System↗

PubMed1996

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.

Algorithms↗

PubMed1995

c-Src enhances the spreading of src-/- fibroblasts on fibronectin by a kinase-independent mechanism.

We have explored the role of the tyrosine kinase c-Src in cellular adhesion. Fibroblasts derived from src-/- mice (src-/- fibroblasts) exhibit a reduced rate of spreading on fibronectin. These defect is rescued by expression of wild-type chicken c-Src. Analyses of mutants suggest that c-Src increases the rate of cell spreading in src-/- fibroblasts through a kinase-independent mechanism requiring both the SH3 and SH2 domains. To further address the role of c-Src in adhesion, we examined the activity and subcellular distribution of c-Src during the adhesion of fibroblasts on fibronectin. We observed a transient increase in the specific kinase activity of c-Src accompanied by the partial dephosphorylation of the negative regulatory site Y527. Activation of c-Src is followed by its redistribution to newly formed focal adhesions. These results suggest that the enzymatic activity and subcellular distribution of c-Src are coordinately regulated during cellular adhesion and that c-Src can affect adhesion by a kinase-independent mechanism.

Animals↗

PubMed1995

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.

Adenosine Triphosphatases↗

PubMed1994

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.

Amino Acid Sequence↗

PubMed1994

Germ cell-less encodes a cell type-specific nuclear pore-associated protein and functions early in the germ-cell specification pathway of Drosophila.

The maternally supplied plasm at the posterior pole of a Drosophila embryo contains determinants that specify both the germ-cell precursors (pole cells) and the posterior axis. One pole plasma component, the product of the germ cell-less gene, has been found to be required for specification of pole cells, but not posterior somatic cells. Mothers with reduced levels of gcl give rise to progeny that lack pole cells, but are otherwise normal. Mothers overexpressing gcl, on the other hand, produce progeny exhibiting a transient increase of pole cells. Ectopic localization of gcl to the anterior pole of the embryo causes nuclei at that location to adopt characteristics of pole cell nuclei, with concurrent loss of somatic cells. We also present evidence indicating that the gcl protein associates specifically with the nuclear pores of the pole cell nuclei. This localization suggests a novel mechanism in the specification of cell fate for the germ line.

Animals↗

PubMed1994

Defining interactions and distributions of cadherin and catenin complexes in polarized epithelial cells.

The cadherin/catenin complex plays important roles in cell adhesion, signal transduction, as well as the initiation and maintenance of structural and functional organization of cells and tissues. In the preceding study, we showed that the assembly of the cadherin/catenin complex is temporally regulated, and that novel combinations of catenin and cadherin complexes are formed in both Triton X-100-soluble and -insoluble fractions; we proposed a model in which pools of catenins are important in regulating assembly of E-cadherin/catenin and catenin complexes. Here, we sought to determine the spatial distributions of E-cadherin, alpha-catenin, beta-catenin, and plakoglobin, and whether different complexes of these proteins accumulate at steady state in polarized Madin-Darby canine kidney cells. Protein distributions were visualized by wide field, optical sectioning, and double immunofluorescence microscopy, followed by reconstruction of three-dimensional images. In cells that were extracted with Triton X-100 and then fixed (Triton X-100-insoluble fraction), more E-cadherin was concentrated at the apical junction relative to other areas of the lateral membrane. alpha-Catenin and beta-catenin colocalize with E-cadherin at the apical junctional complex. There is some overlap in the distribution of these proteins in the lateral membrane, but there are also areas where the distributions are distinct. Plakoglobin is excluded from the apical junctional complex, and its distribution in the lateral membrane is different from that of E-cadherin. Cells were also fixed and then permeabilized to reveal the total cellular pool of each protein (Triton X-100-soluble and -insoluble fractions). This analysis showed lateral membrane localization of alpha-catenin, beta-catenin, and plakoglobin, and it also revealed that they are distributed throughout the cell. Chemical cross-linking of proteins and analysis with specific antibodies confirmed the presence at steady state of E-cadherin/catenin complexes containing either beta-catenin or plakoglobin, and catenin complexes devoid of E-cadherin. Complexes containing E-cadherin/beta-catenin and E-cadherin/alpha-catenin are present in both the Triton X-100-soluble and -insoluble fractions, but E-cadherin/plakoglobin complexes are not detected in the Triton X-100-insoluble fraction. Taken together, these results show that different complexes of cadherin and catenins accumulate in fully polarized epithelial cells, and that they distribute to different sites. We suggest that cadherin/catenin and catenin complexes at different sites have specialized roles in establishing and maintaining the structural and functional organization of polarized epithelial cells.

Animals↗

PubMed1994

Characterization of nuclear polyadenylated RNA-binding proteins in Saccharomyces cerevisiae.

To study the functions of heterogeneous nuclear ribonucleoproteins (hnRNPs), we have characterized nuclear polyadenylated RNA-binding (Nab) proteins from Saccharomyces cerevisiae. Nab1p, Nab2p, and Nab3p were isolated by a method which uses UV light to cross-link proteins directly bound to poly(A)+ RNA in vivo. We have previously characterized Nab2p, and demonstrated that it is structurally related to human hnRNPs. Here we report that Nab1p is identical to the Np13p/Nop3p protein recently implicated in both nucleocytoplasmic protein shuttling and pre-rRNA processing, and characterize a new nuclear polyadenylated RNA-binding protein, Nab3p. The intranuclear distributions of the Nab proteins were analyzed by three-dimensional immunofluorescence optical microscopy. All three Nab proteins are predominantly localized within the nucleoplasm in a pattern similar to the distribution of hnRNPs in human cells. The NAB3 gene is essential for cell viability and encodes an acidic ribonucleoprotein. Loss of Nab3p by growth of a GAL::nab3 mutant strain in glucose results in a decrease in the amount of mature ACT1, CYH2, and TPI1 mRNAs, a concomitant accumulation of unspliced ACT1 pre-mRNA, and an increase in the ratio of unspliced CYH2 pre-mRNA to mRNA. These results suggest that the Nab proteins may be required for packaging pre-mRNAs into ribonucleoprotein structures amenable to efficient nuclear RNA processing.

Amino Acid Sequence↗

PubMed1993

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.

Animals↗

PubMed1993

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.

Animals↗