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  • 1
    Keywords: CELLS ; tumor ; CELL ; imaging ; DEATH ; PROTEIN ; PROTEINS ; LINES ; DNA ; MARKER ; DYNAMICS ; BIOLOGY ; CELL-LINES ; SUPPRESSION ; NUCLEI ; CHROMATIN ; MEMBRANE ; STRESS ; REPAIR ; CELL-LINE ; leukemia ; REGION ; REGIONS ; RECRUITMENT ; TRACKING ; single-particle tracking ; DNA repair ; MITOSIS ; NUCLEAR-BODIES ; Sp100 ; TRANSCRIPTIONAL REGULATION ; nuclear envelope ; NUCLEAR-ENVELOPE ; BODIES ; TUMOR SUPPRESSION ; NUCLEAR ; ENVELOPE ; USA ; LOSSES ; ND10 ; ARSENIC TRIOXIDE ; viral ; SPLICING FACTORS ; three-dimensional ; CELL BIOLOGY ; dynamic ; DAUGHTER NUCLEI ; live cell imaging ; nuclear body ; NUCLEAR-MEMBRANE ; PML-RAR-ALPHA ; promyelocytic leukemia ; PROTEIN PML
    Abstract: Promyelocytic leukemia nuclear bodies (PML NBs) have been proposed to be involved in tumor suppression, viral defense, DNA repair, and/ or transcriptional regulation. To study the dynamics of PML NBs during mitosis, we developed several U2OS cell lines stably coexpressing PML-enhanced cyan fluorescent protein with other individual marker proteins. Using three-dimensional time-lapse live cell imaging and four-dimensional particle tracking, we quantitatively demonstrated that PML NBs exhibit a high percentage of directed movement when cells progressed from prophase to prometaphase. The timing of this increased dynamic movement occurred just before or upon nuclear entry of cyclin B1, but before nuclear envelope breakdown. Our data suggest that entry into prophase leads to a loss of tethering between regions of chromatin and PML NBs, resulting in their increased dynamics. On exit from mitosis, Sp100 and Fas death domain-associated protein (Daxx) entered the daughter nuclei after a functional nuclear membrane was reformed. However, the recruitment of these proteins to PML NBs was delayed and correlated with the timing of de novo PML NB formation. Together, these results provide insight into the dynamic changes associated with PML NBs during mitosis
    Type of Publication: Journal article published
    PubMed ID: 18480407
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  • 2
    Keywords: CELLS ; EXPRESSION ; CELL ; Germany ; MODEL ; GENE ; PROTEIN ; PROTEINS ; transcription ; MICE ; TRANSCRIPTION FACTOR ; DOMAIN ; CONTRAST ; BIOLOGY ; DELETION ; MOUSE ; MUTANT ; NERVOUS-SYSTEM ; TRANSCRIPTION FACTORS ; DIFFERENCE ; MIGRATION ; transactivation ; STEM-CELLS ; PHENOTYPE ; DEPENDENCE ; chromaffin cells ; MUTANTS ; USA ; Aorta ; TRANSCRIPTION FACTOR SOX10 ; CELL BIOLOGY ; HIRSCHSPRUNG-DISEASE ; NEURAL CREST DEVELOPMENT ; SYMPATHETIC NEURONS
    Abstract: Sry-box (Sox) 8, Sox9, and Sox10 are all strongly expressed in the neural crest. Here, we studied the influence of these closely related transcription factors on the developing adrenal medulla as one prominent neural crest derivative. Whereas Sox9 was not expressed, both Sox8 and Sox10 occurred widely in neural crest cells migrating to the adrenal gland and in the gland itself, and they were down-regulated in cells expressing catecholaminergic traits. Sox10-deficient mice lacked an adrenal medulla. The adrenal anlage was never colonized by neural crest cells, which failed to specify properly at the dorsal aorta and died apoptotically during migration. Furthermore, mutant neural crest cells did not express Sox8. Strong adrenal phenotypes were also observed when the Sox10 dimerization domain was inactivated or when a transactivation domain in the central portion was deleted. Sox8 in contrast had only minimal influence on adrenal gland development. Phenotypic consequences became only visible in Sox8-deficient mice upon additional deletion of one Sox10 allele. Replacement of Sox10 by Sox8, however, led to significant rescue of the adrenal medulla, indicating that functional differences between the two related Sox proteins contribute less to the different adrenal phenotypes of the null mutants than dependence of Sox8 expression on Sox10
    Type of Publication: Journal article published
    PubMed ID: 18272785
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  • 3
    Keywords: EXPRESSION ; GROWTH ; IN-VITRO ; CELL ; Germany ; PROTEIN ; PROTEINS ; INDUCTION ; BINDING ; BIOLOGY ; CELL-CYCLE ; DISRUPTION ; RATES ; DISPLAY ; MICROTUBULES ; PHENOTYPE ; OVEREXPRESSION ; ORGANIZATION ; CHROMOSOMES ; STRUCTURAL BASIS ; XENOPUS EGG EXTRACTS ; USA ; CENP-E ; CHROMOSOME SEGREGATION ; MITOTIC SPINDLE ; SEGREGATION ; ANAPHASE ; BIPOLAR SPINDLES ; KINETOCHORE MOTOR ; MEIOTIC SPINDLE ; PLUS-END TRACKING ; POLEWARD MICROTUBULE FLUX
    Abstract: In metaphase Xenopus egg extracts, global microtubule growth is mainly promoted by two unrelated microtubule stabilizers, end-binding protein 1 (EB1) and XMAP215. Here, we explore their role and potential redundancy in the regulation of spindle assembly and function. We find that at physiological expression levels, both proteins are required for proper spindle architecture: Spindles assembled in the absence of EB1 or at decreased XMAP215 levels are short and frequently multipolar. Moreover, the reduced density of microtubules at the equator of Delta EB1 or Delta XMAP215 spindles leads to faulty kinetochore-microtubule attachments. These spindles also display diminished poleward flux rates and, upon anaphase induction, they neither segregate chromosomes nor reorganize into interphasic microtubule arrays. However, EB1 and XMAP215 nonredundantly regulate spindle assembly because an excess of XMAP215 can compensate for the absence of EB1, whereas the overexpression of EB1 cannot substitute for reduced XMAP215 levels. Our data indicate that EB1 could positively regulate XMAP215 by promoting its binding to the microtubules. Finally, we show that disruption of the mitosis-specific XMAP215-EB1 interaction produces a phenotype similar to that of either EB1 or XMAP215 depletion. Therefore, the XMAP215-EB1 interaction is required for proper spindle organization and chromosome segregation in Xenopus egg extracts
    Type of Publication: Journal article published
    PubMed ID: 19369422
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  • 4
    Keywords: ASSOCIATION, BIOLOGY, CELL, CELLS, CHROMATIN, COMPLEX, COMPLEXES, DIFFUSION, DNA-REPLICATION, DYNAMI
    Abstract: Telomerase-negative tumor cells maintain their telomeres via an alternative lengthening of telomeres (ALT) mechanism. This process involves the association of telomeres with promyelocytic leukemia nuclear bodies (PML-NBs). Here, the mobility of both telomeres and PML-NBs as well as their interactions were studied in human U2OS osteosarcoma cells, in which the ALT pathway is active. A U2OS cell line was constructed that had lac operator repeats stably integrated adjacent to the telomeres of chromosomes 6q, 11p, and 12q. By fluorescence microscopy of autofluorescent LacI repressor bound to the lacO arrays the telomere mobility during interphase was traced and correlated with the telomere repeat length. A confined diffusion model was derived that describes telomere dynamics in the nucleus on the time scale from seconds to hours. Two telomere groups were identified that differed with respect to the nuclear space accessible to them. Furthermore, translocations of PML-NBs relative to telomeres and their complexes with telomeres were evaluated. Based on these studies, a model is proposed in which the shortening of telomeres results in an increased mobility that could facilitate the formation of complexes between telomeres and PML-NBs
    Type of Publication: Journal article published
    PubMed ID: 19211845
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  • 5
    Keywords: EXPRESSION ; SACCHAROMYCES-CEREVISIAE ; METABOLISM ; ACTIVATED PROTEIN-KINASE ; PHOSPHORYLATION ; YEAST ; ENDOPLASMIC-RETICULUM ; P-BODIES ; LOW-TEMPERATURE ; MAMMALIAN HIBERNATION
    Abstract: Cells respond to different types of stress by inhibition of protein synthesis and subsequent assembly of stress granules (SGs), cytoplasmic aggregates that contain stalled translation preinitiation complexes. Global translation is regulated through the translation initiation factor eukaryotic initiation factor 2 alpha (eIF2 alpha) and the mTOR pathway. Here we identify cold shock as a novel trigger of SG assembly in yeast and mammals. Whereas cold shock-induced SGs take hours to form, they dissolve within minutes when cells are returned to optimal growth temperatures. Cold shock causes eIF2 alpha phosphorylation through the kinase PERK in mammalian cells, yet this pathway is not alone responsible for translation arrest and SG formation. In addition, cold shock leads to reduced mitochondrial function, energy depletion, concomitant activation of AMP-activated protein kinase (AMPK), and inhibition of mTOR signaling. Compound C, a pharmacological inhibitor of AMPK, prevents the formation of SGs and strongly reduces cellular survival in a translation-dependent manner. Our results demonstrate that cells actively suppress protein synthesis by parallel pathways, which induce SG formation and ensure cellular survival during hypothermia.
    Type of Publication: Journal article published
    PubMed ID: 22875991
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  • 6
    Keywords: IN-VITRO ; PROTEIN ; INTERMEDIATE-FILAMENTS ; vimentin ; SKELETAL-MUSCLE ; ARCHITECTURE ; STRIATED-MUSCLE ; Z-DISC ; THIN-FILAMENTS ; MOTILE PROPERTIES
    Abstract: Desmin intermediate filaments (DIFs) form an intricate meshwork that organizes myofibers within striated muscle cells. The mechanisms that regulate the association of desmin to sarcomeres and their role in desminopathy are incompletely understood. Here we compare the effect nebulin binding has on the assembly kinetics of desmin and three desminopathy-causing mutant desmin variants carrying mutations in the head, rod, or tail domains of desmin (S46F, E245D, and T453I). These mutants were chosen because the mutated residues are located within the nebulin-binding regions of desmin. We discovered that, although nebulin M160-164 bound to both desmin tetrameric complexes and mature filaments, all three mutants exhibited significantly delayed filament assembly kinetics when bound to nebulin. Correspondingly, all three mutants displayed enhanced binding affinities and capacities for nebulin relative to wild-type desmin. Electron micrographs showed that nebulin associates with elongated normal and mutant DIFs assembled in vitro. Moreover, we measured significantly delayed dynamics for the mutant desmin E245D relative to wild-type desmin in fluorescence recovery after photobleaching in live-cell imaging experiments. We propose a mechanism by which mutant desmin slows desmin remodeling in myocytes by retaining nebulin near the Z-discs. On the basis of these data, we suggest that for some filament-forming desmin mutants, the molecular etiology of desminopathy results from subtle deficiencies in their association with nebulin, a major actin-binding filament protein of striated muscle.
    Type of Publication: Journal article published
    PubMed ID: 23615443
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  • 7
    Keywords: MONOCLONAL-ANTIBODIES ; SIALIC-ACID ; ADHESION MOLECULE ; CRYOELECTRON MICROSCOPY ; CAPSID PROTEIN MU-1 ; NONENVELOPED VIRUS ; MAMMALIAN REOVIRUS ; SIGMA-1 PROTEIN ; MEMBRANE PENETRATION ; TYPE-1 REOVIRUS
    Abstract: Polarized epithelial cells that line the digestive, respiratory, and genitourinary tracts form a barrier that many viruses must breach to infect their hosts. Current understanding of cell entry by mammalian reovirus (MRV) virions and infectious subvirion particles (ISVPs), generated from MRV virions by extracellular proteolysis in the digestive tract, are mostly derived from in vitro studies with nonpolarized cells. Recent live-cell imaging advances allow us for the first time to visualize events at the apical surface of polarized cells. In this study, we used spinning-disk confocal fluorescence microscopy with high temporal and spatial resolution to follow the uptake and trafficking dynamics of single MRV virions and ISVPs at the apical surface of live polarized Madin-Darby canine kidney cells. Both types of particles were internalized by clathrin-mediated endocytosis, but virions and ISVPs exhibited strikingly different trafficking after uptake. While virions reached early and late endosomes, ISVPs did not and instead escaped the endocytic pathway from an earlier location. This study highlights the broad advantages of using live-cell imaging combined with single-particle tracking for identifying key steps in cell entry by viruses.
    Type of Publication: Journal article published
    PubMed ID: 23427267
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  • 8
    Keywords: TRANSPORT ; BEHAVIOR ; BINDING PROTEIN ; GUIDANCE ; CELL CORTEX ; PLUS ENDS ; GROWING NERVE PROCESSES ; NEURONAL GROWTH CONES ; DYNAMIC MICROTUBULES ; ACTIN-FILAMENTS
    Abstract: Dynamic microtubules (MTs) are required for neuronal guidance, in which axons extend directionally toward their target tissues. We found that depletion of the MT-binding protein Xenopus cytoplasmic linker-associated protein 1 (XCLASP1) or treatment with the MT drug Taxol reduced axon outgrowth in spinal cord neurons. To quantify the dynamic distribution of MTs in axons, we developed an automated algorithm to detect and track MT plus ends that have been fluorescently labeled by end-binding protein 3 (EB3). XCLASP1 depletion reduced MT advance rates in neuronal growth cones, very much like treatment with Taxol, demonstrating a potential link between MT dynamics in the growth cone and axon extension. Automatic tracking of EB3 comets in different compartments revealed that MTs increasingly slowed as they passed from the axon shaft into the growth cone and filopodia. We used speckle microscopy to demonstrate that MTs experience retrograde flow at the leading edge. Microtubule advance in growth cone and filopodia was strongly reduced in XCLASP1-depleted axons as compared with control axons, but actin retrograde flow remained unchanged. Instead, we found that XCLASP1-depleted growth cones lacked lamellipodial actin organization characteristic of protrusion. Lamellipodial architecture depended on XCLASP1 and its capacity to associate with MTs, highlighting the importance of XCLASP1 in actin-microtubule interactions.
    Type of Publication: Journal article published
    PubMed ID: 23515224
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  • 9
    Keywords: GENES ; SACCHAROMYCES-CEREVISIAE ; BINDING ; BUDDING YEAST ; DNA-DAMAGE ; MITOSIS ; IN-VITRO REGULATION ; MITOTIC EXIT ; ANAPHASE SPINDLE ; KINASE KIN4
    Abstract: In addition to their well-known role in microtubule organization, centrosomes function as signaling platforms and regulate cell cycle events. An important example of such a function is the spindle position checkpoint (SPOC) of budding yeast. SPOC is a surveillance mechanism that ensures alignment of the mitotic spindle along the cell polarity axis. Upon spindle misalignment, phosphorylation of the SPOC component Bfa1 by Kin4 kinase engages the SPOC by changing the centrosome localization of Bfa1 from asymmetric (one centrosome) to symmetric (both centrosomes). Here we show that, unexpectedly, Kin4 alone is unable to break Bfa1 asymmetry at yeast centrosomes. Instead, phosphorylation of Bfa1 by Kin4 creates a docking site on Bfa1 for the 14-3-3 family protein Bmh1, which in turn weakens Bfa1-centrosome association and promotes symmetric Bfa1 localization. Consistently, BMH1-null cells are SPOC deficient. Our work thus identifies Bmh1 as a new SPOC component and refines the molecular mechanism that breaks Bfa1 centrosome asymmetry upon SPOC activation.
    Type of Publication: Journal article published
    PubMed ID: 24850890
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  • 10
    Keywords: ANGIOGENESIS ; GROWTH-FACTOR ; IN-VITRO ; KINASE ; ACTIVATION ; CELL-ADHESION ; KAPPA-B ; TRANSCRIPTIONAL REGULATION ; TUMOR-GROWTH ; E-SELECTIN
    Abstract: Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular physiology. VEGF-A stimulates signal transduction pathways that modulate endothelial outputs such as cell migration, proliferation, tubulogenesis, and cell-cell interactions. Multiple VEGF-A isoforms exist, but the biological significance of this is unclear. Here we analyzed VEGF-A isoform-specific stimulation of VCAM-1 gene expression, which controls endothelial-leukocyte interactions, and show that this is dependent on both ERK1/2 and activating transcription factor-2 (ATF-2). VEGF-A isoforms showed differential ERK1/2 and p38 MAPK phosphorylation kinetics. A key feature of VEGF-A isoform-specific ERK1/2 activation and nuclear translocation was increased phosphorylation of ATF-2 on threonine residue 71 (T71). Using reverse genetics, we showed ATF-2 to be functionally required for VEGF-A-stimulated endothelial VCAM-1 gene expression. ATF-2 knockdown blocked VEGF-A-stimulated VCAM-1 expression and endothelial-leukocyte interactions. ATF-2 was also required for other endothelial cell outputs, such as cell migration and tubulogenesis. In contrast, VCAM-1 was essential only for promoting endothelial-leukocyte interactions. This work presents a new paradigm for understanding how soluble growth factor isoforms program complex cellular outputs and responses by modulating signal transduction pathways.
    Type of Publication: Journal article published
    PubMed ID: 24966171
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