William Sullivan

University of California, Santa Cruz, Santa Cruz, CA, United States

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Publications (72)516.78 Total impact

  • Barbara Fasulo, William Sullivan
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    ABSTRACT: The model organism Drosophila melanogaster is particularly well suited for live image analysis. The availability of GFP transgenic flies and a wide array of fluorescent probes, in conjunction with laser scanning confocal microscopy, allow us to image multiple aspects of the cell cycle simultaneously. Confocal microscopy provides the sensitivity and resolution to observe the dynamics of specific cellular events in real time. For example, GFP-histone and rhodamine-labeled tubulin enable one to follow specific nuclear and cytoskeletal events including nuclear envelope formation, nuclear envelope breakdown, spindle formation, centrosome duplication, separation and migration, chromosomes condensation, and segregation. This analysis permits a detailed morphological and temporal description of nuclear and cytoskeletal events in normal or drug-injected embryos.
    Methods in molecular biology (Clifton, N.J.) 01/2014; 1075:243-55. · 1.29 Impact Factor
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    ABSTRACT: The maternally inherited bacterium Wolbachia infects the germline of most arthropod species. Using D. simulans and D. melanogaster, we demonstrate that localization of Wolbachia to the fat bodies and adult brain is likely also a conserved feature of Wolbachia infection. Examination of three Wolbachia strains (WMel , WRiv , WPop ) revealed that the bacteria preferentially concentrate in the central brain with low titers in the optic lobes. Distribution within regions of the central brain is largely determined by the Wolbachia strain, while the titer is influenced by both, the host species and the bacteria strain. In neurons of the central brain and ventral nerve cord, Wolbachia preferentially localize to the neuronal cell bodies but not to axons. All examined Wolbachia strains are present intracellularly or in extracellular clusters, with the pathogenic WPop strain exhibiting the largest and most abundant clusters. We also discovered that 16 of 40 lines from the Drosophila Genetic Reference Panel are Wolbachia infected. Direct comparison of Wolbachia infected and cured lines from this panel reveals that differences in physiological traits (chill coma recovery, starvation, longevity) are likely due to host line influences. In addition, a tetracycline-induced increase in Drosophila longevity was detected many generations after treatment.
    Cellular Microbiology 03/2013; · 4.81 Impact Factor
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    ABSTRACT: Chromosome segregation must be coordinated with cell cleavage to ensure correct transmission of the genome to daughter cells. Here we identify a novel mechanism by which Drosophila melanogaster neuronal stem cells coordinate sister chromatid segregation with cleavage furrow ingression. Cells adapted to a dramatic increase in chromatid arm length by transiently elongating during anaphase/telophase. The degree of cell elongation correlated with the length of the trailing chromatid arms and was concomitant with a slight increase in spindle length and an enlargement of the zone of cortical myosin distribution. Rho guanine-nucleotide exchange factor (Pebble)-depleted cells failed to elongate during segregation of long chromatids. As a result, Pebble-depleted adult flies exhibited morphological defects likely caused by cell death during development. These studies reveal a novel pathway linking trailing chromatid arms and cortical myosin that ensures the clearance of chromatids from the cleavage plane at the appropriate time during cytokinesis, thus preserving genome integrity.
    The Journal of Cell Biology 11/2012; 199(5):745-53. · 10.82 Impact Factor
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    Justin Crest, Kirsten Concha-Moore, William Sullivan
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    ABSTRACT: Early Drosophila embryogenesis is characterized by shifting from astral microtubule-based to central spindle-based positioning of cleavage furrows. Before cellularization, astral microtubules determine metaphase furrow position by producing Rappaport-like furrows, which encompass rather than bisect the spindle. Their positioning is explained by our finding that the conserved central spindle components centralspindlin (mKLP1 and RacGAP50C), Polo, and Fascetto (Prc1) localize to the astral microtubule overlap region. These components and the chromosomal passenger complex localize to the central spindle, though no furrow forms there. We identify the maternally supplied RhoGEF2 as a key factor in metaphase furrow positioning. Unlike the zygotic, central spindle-localized RhoGEF (Pebble), RhoGEF2 localizes to metaphase furrows, a function distinct from RhoGEF/Pebble and likely due to the absence of a RacGAP50C binding domain. Accordingly, we find that ectopic activation of Rho GTPase generates furrows perpendicular to the central spindle during syncytial divisions. Whereas metaphase furrow formation is myosin independent, these ectopic furrows, like conventional furrows, require myosin as well as microtubules. These studies demonstrate that early Drosophila embryogenesis is primed to form furrows at either overlapping astral microtubules or the central spindle. We propose that the shift to the latter is driven by a corresponding shift from RhoGEF2 to Pebble in controlling furrow formation.
    Current biology: CB 09/2012; · 10.99 Impact Factor
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    ABSTRACT: Wolbachia endosymbionts carried by filarial nematodes give rise to the neglected diseases African river blindness and lymphatic filariasis afflicting millions worldwide. Here we identify new Wolbachia-disrupting compounds by conducting high-throughput cell-based chemical screens using a Wolbachia-infected, fluorescently labeled Drosophila cell line. This screen yielded several Wolbachia-disrupting compounds including three that resembled Albendazole, a widely used anthelmintic drug that targets nematode microtubules. Follow-up studies demonstrate that a common Albendazole metabolite, Albendazole sulfone, reduces intracellular Wolbachia titer both in Drosophila melanogaster and Brugia malayi, the nematode responsible for lymphatic filariasis. Significantly, Albendazole sulfone does not disrupt Drosophila microtubule organization, suggesting that this compound reduces titer through direct targeting of Wolbachia. Accordingly, both DNA staining and FtsZ immunofluorescence demonstrates that Albendazole sulfone treatment induces Wolbachia elongation, a phenotype indicative of binary fission defects. This suggests that the efficacy of Albendazole in treating filarial nematode-based diseases is attributable to dual targeting of nematode microtubules and their Wolbachia endosymbionts.
    PLoS Pathogens 09/2012; 8(9):e1002922. · 8.14 Impact Factor
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    ABSTRACT: Spatially and temporally dependent optical aberrations induced by the inhomogeneous refractive index of live samples limit the resolution of live dynamic imaging. We introduce an adaptive optical microscope with a direct wavefront sensing method using a Shack-Hartmann wavefront sensor and fluorescent protein guide-stars for live imaging. The results of imaging Drosophila embryos demonstrate its ability to correct aberrations and achieve near diffraction limited images of medial sections of large Drosophila embryos. GFP-polo labeled centrosomes can be observed clearly after correction but cannot be observed before correction. Four dimensional time lapse images are achieved with the correction of dynamic aberrations. These studies also demonstrate that the GFP-tagged centrosome proteins, Polo and Cnn, serve as excellent biological guide-stars for adaptive optics based microscopy.
    Optics Express 07/2012; 20(14):15969-82. · 3.55 Impact Factor
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    ABSTRACT: Parasitic filarial nematodes that belong to the Onchocercidae family live in mutualism with Wolbachia endosymbionts. We developed whole-mount techniques to follow the segregation patterns of Wolbachia through the somatic and germline lineages of four filarial species. These studies reveal multiple evolutionarily conserved mechanisms that are required for Wolbachia localization to the germline. During the initial embryonic divisions, Wolbachia segregate asymmetrically such that they concentrate in the posteriorly localized P(2) blastomere, a precursor to the adult germline and hypodermal lineages. Surprisingly, in the next division they are excluded from the germline precursor lineage. Rather, they preferentially segregate to the C blastomere, a source of posterior hypodermal cells. Localization to the germline is accomplished by a distinct mechanism in which Wolbachia invade first the somatic gonadal cells close to the ovarian distal tip cell, the nematode stem cell niche, from the hypodermis. This tropism is associated with a cortical F-actin disruption, suggesting an active engulfment. Significantly, germline invasion occurs only in females, explaining the lack of Wolbachia in the male germline. Once in the syncytial environment of the ovaries, Wolbachia rely on the rachis to multiply and disperse into the germ cells. The utilization of cell-to-cell invasion for germline colonization may indicate an ancestral mode of horizontal transfer that preceded the acquisition of the mutualism.
    Biology open. 06/2012; 1(6):536-47.
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    ABSTRACT: Defects in DNA replication and chromosome condensation are common phenotypes in cancer cells. A link between replication and condensation has been established, but little is known about the role of checkpoints in monitoring chromosome condensation. We investigate this function by live analysis, using the rapid division cycles in the early Drosophila embryo. We find that S-phase and topoisomerase inhibitors delay both the initiation and the rate of chromosome condensation. These cell cycle delays are mediated by the cell cycle kinases chk1 and wee1. Inhibitors that cause severe defects in chromosome condensation and congression on the metaphase plate result in delayed anaphase entry. These delays are mediated by wee1 and are not the result of spindle assembly checkpoint activation. In addition, we provide the first detailed live analysis of the direct effect of widely used anticancer agents (aclarubicin, ICRF-193, VM26, doxorubicin, camptothecin, aphidicolin, hydroxyurea, cisplatin, mechlorethamine and x-rays) on key nuclear and cytoplasmic cell cycle events.
    Molecular biology of the cell 01/2012; 23(6):1047-57. · 5.98 Impact Factor
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    ABSTRACT: RNA interference (RNAi) is an efficient reverse genetics technique for investigating gene function in eukaryotes. The method has been widely used in model organisms, such as the free-living nematode Caenorhabditis elegans, where it has been deployed in genome-wide high throughput screens to identify genes involved in many cellular and developmental processes. However, RNAi techniques have not translated efficiently to animal parasitic nematodes that afflict humans, livestock and companion animals across the globe, creating a dependency on data tentatively inferred from C. elegans. We report improved and effective in vitro RNAi procedures we have developed using heterogeneous short interfering RNA (hsiRNA) mixtures that when coupled with optimized immunostaining techniques yield detailed analysis of cytological defects in the human parasitic nematode, Brugia malayi. The cellular disorganization observed in B. malayi embryos following RNAi targeting the genes encoding γ-tubulin, and the polarity determinant protein, PAR-1, faithfully phenocopy the known defects associated with gene silencing of their C. elegans orthologs. Targeting the B. malayi cell junction protein, AJM-1 gave a similar but more severe phenotype than that observed in C. elegans. Cellular phenotypes induced by our in vitro RNAi procedure can be observed by immunofluorescence in as little as one week. We observed cytological defects following RNAi targeting all seven B. malayi transcripts tested and the phenotypes mirror those documented for orthologous genes in the model organism C. elegans. This highlights the reliability, effectiveness and specificity of our RNAi and immunostaining procedures. We anticipate that these techniques will be widely applicable to other important animal parasitic nematodes, which have hitherto been mostly refractory to such genetic analysis.
    Parasites & Vectors 01/2012; 5:16. · 3.25 Impact Factor
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    ABSTRACT: Although much is known about interactions between bacterial endosymbionts and their hosts, little is known concerning the host factors that influence endosymbiont titer. Wolbachia endosymbionts are globally dispersed throughout most insect species and are the causative agent in filarial nematode-mediated disease. Our investigation indicates that gurken (grk), a host gene encoding a crucial axis determinant, has a cumulative, dosage-sensitive impact on Wolbachia growth and proliferation during Drosophila oogenesis. This effect appears to be mediated by grk mRNA and its protein-binding partners Squid and Hrp48/Hrb27C, implicating the grk mRNA-protein (mRNP) complex as a rate-limiting host factor controlling Wolbachia titer. Furthermore, highly infected flies exhibit defects that match those occurring with disruption of grk mRNPs, such as nurse cell chromatin disruptions and malformation of chorionic appendages. These findings suggest a feedback loop in which Wolbachia interaction with the grk mRNP affects both Wolbachia titer and grk mRNP function.
    Journal of Cell Science 12/2011; 124(Pt 24):4299-308. · 5.88 Impact Factor
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    ABSTRACT: Filarial nematodes maintain a mutualistic relationship with the endosymbiont Wolbachia. Depletion of Wolbachia produces profound defects in nematode development, fertility and viability and thus has great promise as a novel approach for treating filarial diseases. However, little is known concerning the basis for this mutualistic relationship. Here we demonstrate using whole mount confocal microscopy that an immediate response to Wolbachia depletion is extensive apoptosis in the adult germline, and in the somatic cells of the embryos, microfilariae and fourth-stage larvae (L4). Surprisingly, apoptosis occurs in the majority of embryonic cells that had not been infected prior to antibiotic treatment. In addition, no apoptosis occurs in the hypodermal chords, which are populated with large numbers of Wolbachia, although disruption of the hypodermal cytoskeleton occurs following their depletion. Thus, the induction of apoptosis upon Wolbachia depletion is non-cell autonomous and suggests the involvement of factors originating from Wolbachia in the hypodermal chords. The pattern of apoptosis correlates closely with the nematode tissues and processes initially perturbed following depletion of Wolbachia, embryogenesis and long-term sterilization, which are sustained for several months until the premature death of the adult worms. Our observations provide a cellular mechanism to account for the sustained reductions in microfilarial loads and interruption of transmission that occurs prior to macrofilaricidal activity following antibiotic therapy of filarial nematodes.
    PLoS Pathogens 11/2011; 7(11):e1002351. · 8.14 Impact Factor
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    ABSTRACT: Wolbachia is a globally distributed bacterial endosymbiont present in arthropods and nematodes. The advent of sensitive PCR-based approaches has greatly facilitated the identification of Wolbachia-infected individuals and analysis of population infection levels. Here, a complementary visual fluorescence-based Wolbachia screening approach is described. Through the use of the fluorescent dye Syto-11, Wolbachia can be efficiently detected in various Drosophila tissues, including ovaries. Syto-11 also stains Wolbachia in other insects. Because Wolbachia is inherited through the maternal germ line, bacteria reside in the ovaries of flies in infected populations. An advantage of this staining approach is that it informs about Wolbachia titer as well as its tissue and cellular distribution. Using this method, the infection status of insect populations in two central California locations was determined, and variants with unusually low or high Wolbachia titers were isolated. In addition, a variant with ovarioles containing both infected and uninfected egg chambers was identified. Syto-11 staining of Cardinium- and Spiroplasma-infected insects was also analyzed.
    Applied and environmental microbiology 05/2011; 77(14):4788-94. · 3.69 Impact Factor
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    ABSTRACT: We report a technique for measuring and correcting the wavefront aberrations introduced by a biological sample using a Shack-Hartmann wavefront sensor, a fluorescent reference source, and a deformable mirror. The reference source and sample fluorescence are at different wavelengths to separate wavefront measurement and sample imaging. The measurement and correction at one wavelength improves the resolving power at a different wavelength, enabling the structure of the sample to be resolved.
    Optics Letters 03/2011; 36(6):825-7. · 3.39 Impact Factor
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    ABSTRACT: We demonstrated the used of an adaptive optic system in biological imaging to improve the imaging characteristics of a wide field microscope. A crimson red fluorescent bead emitting light at 650 nm was used together with a Shack-Hartmann wavefront sensor and deformable mirror to compensate for the aberrations introduce by a Drosophila embryo. The measurement and correction at one wavelength improves the resolving power at a different wavelength, enabling the structure of the sample to be resolved (510 nm). The use of the crimson beads allow for less photobleaching to be done to the science object of the embryo, in this case our GFP model (green fluorescent beads), and allows for the science object and wavefront reference to be spectrally separated. The spectral separation allows for single points sources to be used for wavefront measurements, which is a necessary condition for the Shack-Hartmann Wavefront sensor operation.
    Proc SPIE 02/2011;
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    09/2010; , ISBN: 9780470015902
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    ABSTRACT: We present a new method to directly measure and correct the aberrations introduced when imaging through thick biological tissue. A Shack-Hartmann wavefront sensor is used to directly measure the wavefront error induced by a Drosophila embryo. The wavefront measurements are taken by seeding the embryo with fluorescent microspheres used as "artificial guide-stars." The wavefront error is corrected in ten millisecond steps by applying the inverse to the wavefront error on a micro-electro-mechanical deformable mirror in the image path of the microscope. The results show that this new approach is capable of improving the Strehl ratio by 2 times on average and as high as 10 times when imaging through 100 microm of tissue. The results also show that the isoplanatic half-width is approximately 19 microm resulting in a corrected field of view 38 microm in diameter around the guide-star.
    Optics Express 08/2010; 18(16):17521-32. · 3.55 Impact Factor
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    Jian Cao, Justin Crest, Barbara Fasulo, William Sullivan
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    ABSTRACT: Proper centrosome separation is a prerequisite for positioning the bipolar spindle. Although studies demonstrate that microtubules (MTs) and their associated motors drive centrosome separation [1], the role of actin in centrosome separation remains less clear. Studies in tissue culture cells indicate that actin- and myosin-based cortical flow is primarily responsible for driving late centrosome separation [2], whereas other studies suggest that actin plays a more passive role by serving as an attachment site for astral MTs to pull centrosomes apart [3-6]. Here we demonstrate that prior to nuclear envelope breakdown (NEB) in Drosophila embryos, proper centrosome separation does not require myosin II but requires dynamic actin rearrangements at the growing edge of the interphase cap. Both Arp2/3- and Formin-mediated actin remodeling are required for separating the centrosome pairs before NEB. The Apc2-Armadillo complex appears to link cap expansion to centrosome separation. In contrast, the mechanisms driving centrosome separation after NEB are independent of the actin cytoskeleton and compensate for earlier separation defects. Our studies show that the dynamics of actin polymerization drive centrosome separation, and this has important implications for centrosome positioning during processes such as cell migration [7, 8], cell polarity maintenance [9, 10], and asymmetric cell division [11, 12].
    Current biology: CB 04/2010; 20(8):770-6. · 10.99 Impact Factor
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    ABSTRACT: Centrioles are cylinders made of nine microtubule (MT) triplets present in many eukaryotes. Early studies, where centrosomes were seen at the poles of the mitotic spindle led to their coining as "the organ for cell division". However, a variety of subsequent observational and functional studies showed that centrosomes might not always be essential for mitosis. Here we review the arguments in this debate. We describe the centriole structure and its distribution in the eukaryotic tree of life and clarify its role in the organization of the centrosome and cilia, with an historical perspective. An important aspect of the debate addressed in this review is how centrioles are inherited and the role of the spindle in this process. In particular, germline inheritance of centrosomes, such as their de novo formation in parthenogenetic species, poses many interesting questions. We finish by discussing the most likely functions of centrioles and laying out new research avenues.
    Cellular and Molecular Life Sciences CMLS 03/2010; 67(13):2173-94. · 5.62 Impact Factor
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    ABSTRACT: Adaptive optics (AO) improves the quality of astronomical imaging systems by using real time measurement of the turbulent medium in the optical path using a guide star (natural or artificial) as a point source reference beacon [1]. AO has also been applied to vision science to improve the view of the human eye. This paper will address our current research focused on the improvement of fluorescent microscopy for biological imaging utilizing current AO technology. A Shack-Hartmann wavefront sensor (SHWS) is used to measure the aberration introduced by a Drosophila Melanogaster embryo with an implanted 1 micron fluorescent bead that serves as a point source reference beacon. Previous measurements of the wavefront aberrations have found an average peak-to-valley and root-mean-square (RMS) wavefront error of 0.77 micrometers and 0.15 micrometers, respectively. Measurements of the Zernike coefficients indicated that the correction of the first 14 Zernike coefficients is sufficient to correct the aberrations we measured. Here we show that a MEMS deformable mirror with 3.5 microns of stroke and 140 actuators is sufficient to correct these aberrations. The design, assembly and initial results for the use of a MEMS deformable mirror, SHWS and implanted fluorescent reference beacon for wavefront correction are discussed.
    Proc SPIE 02/2010;
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    Anne Royou, Mary E Gagou, Roger Karess, William Sullivan
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    ABSTRACT: The mechanisms that safeguard cells against chromosomal instability (CIN) are of great interest, as CIN contributes to tumorigenesis. To gain insight into these mechanisms, we studied the behavior of cells entering mitosis with damaged chromosomes. We used the endonuclease I-CreI to generate acentric chromosomes in Drosophila larvae. While I-CreI expression produces acentric chromosomes in the majority of neuronal stem cells, remarkably, it has no effect on adult survival. Our live studies reveal that acentric chromatids segregate efficiently to opposite poles. The acentric chromatid poleward movement is mediated through DNA tethers decorated with BubR1, Polo, INCENP, and Aurora-B. Reduced BubR1 or Polo function results in abnormal segregation of acentric chromatids, a decrease in acentric chromosome tethering, and a great reduction in adult survival. We propose that BubR1 and Polo facilitate the accurate segregation of acentric chromatids by maintaining the integrity of the tethers that connect acentric chromosomes to their centric partners.
    Cell 01/2010; 140(2):235-45. · 31.96 Impact Factor

Publication Stats

2k Citations
516.78 Total Impact Points

Institutions

  • 1993–2014
    • University of California, Santa Cruz
      • • Department of Molecular Cell & Developmental Biology
      • • Jack Baskin School of Engineering
      Santa Cruz, CA, United States
  • 2011
    • Lewis & Clark College
      Portland, Oregon, United States
  • 2009
    • Albion College
      • Biology
      Albion, MI, United States
  • 2007
    • University of Minnesota Duluth
      Duluth, Minnesota, United States
  • 2005
    • Harvard Medical School
      • Department of Systems Biology
      Boston, MA, United States
  • 1996
    • Pierre and Marie Curie University - Paris 6
      Lutetia Parisorum, Île-de-France, France
  • 1990–1996
    • University of California, San Francisco
      • Department of Biochemistry and Biophysics
      San Francisco, CA, United States