Resume

• 1995

  Ph.D.

  My thesis probed the role of dynactin at the centrosome. Dynactin is the required cofactor of the microtubule motor

  cytoplasmic dynein. I found that dynactin is required for anchoring microtubules at the centrosome (and thus keeps them organized) and that this function was dynein-independent.

  Biology

• 1991

  B.S.

  Biology

• Protein Chemistry

  Biochemistry

  Microscopy

  Life Sciences

  Cell Signaling

  Protein Purification

  Immunohistochemistry

  PCR

  Biology

  Fluorescence Microscopy

  Cell Culture

  Teaching

  Lifesciences

  Transfection

  Tissue Culture

  Cell Biology

  Science

  Molecular Biology

  Cell

  Molecular Cloning

  Increased iron supplied through Fet3p results in replicative life span extension of Saccharomyces cerevisiae under conditions requiring respiratory metabolism.

  PA Kirchman

  CS Turn

  G Botta

  We have previously shown that copper supplementation extends the replicative life span of Saccharomyces cerevisiae when grown under conditions forcing cells to respire. We now show that copper's effect on life span is through Fet3p

  a copper containing enzyme responsible for high affinity transport of iron into yeast cells. Life span extensions can also be obtained by supplementing the growth medium with 1mM ferric chloride. Extension by high iron levels is still dependent on the presence of Fet3p. Life span extension by iron or copper requires growth on media containing glycerol as the sole carbon source

  which forces yeast to respire. Yeast grown on glucose containing media supplemented with iron show no extension of life span. The iron associated with cells grown in media supplemented with copper or iron is 1.4-1.8 times that of cells grown without copper or iron supplementation. As with copper supplementation

  iron supplementation partially rescues the life span of superoxide dismutase mutants. Cells grown with copper supplementation display decreased production of superoxide as measured by dihydroethidium staining.

  Increased iron supplied through Fet3p results in replicative life span extension of Saccharomyces cerevisiae under conditions requiring respiratory metabolism.

  M.M. Ivey

  In an undergraduate scientific curriculum

  it is important for students to develop the ability to read and discuss scientific literature

  and to effectively convey information to others. We present a stand-alone course that develops these skills

  using a series of independent modules all connected to a common theme of cancer. This theme serves as a foundation for discussions

  experiments

  and presentations. In this course

  students are exposed to four experimental techniques: viscometry

  plasmid-based assay

  metaphase spreads

  and ion chromatography. This course could be used as part of an honors curriculum

  or as a cohort-building course for a scholarship program. Individual modules could also be incorporated into a traditional chemistry or biology course.

  Discussion

  Implementation

  Presentation: A Standalone Course for High Ability Undergraduate Students

  WS Saunders

  SM Gollin

  DR Hoffelder

  JE Reing

  Most tumor cells are characterized by increased genomic instability and chromosome segregational defects

  often associated with hyperamplification of the centrosome and the formation of multipolar spindles. However

  extra centrosomes do not always lead to multipolarity. Here

  we describe a process of centrosomal clustering that prevented the formation of multipolar spindles in noncancer cells. Noncancer cells needed to overcome this clustering mechanism to allow multipolar spindles to form at a high frequency. The microtubule motor cytoplasmic dynein was a critical part of this coalescing machinery

  and in some tumor cells overexpression of the spindle protein NuMA interfered with dynein localization

  promoting multipolarity.

  Spindle multipolarity is prevented by centrosomal clustering.

  JT Washington

  Aims and background. \nThe pyruvate mimetic dichloroacetate (DCA) has been shown to induce cell death in cancer cells. A number of studies in vitro and in vivo have suggested this molecule may serve as an anticancer agent

  but some cells are resistant. Here we wanted to examine the effects of DCA on cancerous and noncancerous cells grown in culture for a prolonged period of exposure and at increasing concentrations. \nMethods. \nSix cancer cell lines (A549

  SK-HEP-1

  HCT116

  UPCI:SCC070

  HeLa and MES-SA) and three noncancerous lines (RPE

  GM03349B and HEK293) were exposed to 0.5 mM DCA for seven days and cell counts were taken every day to determine viability and cell cycle progression. The same cell lines were also exposed to higher doses of DCA up to 10 mM and viability was scored. \nResults. \nFive cancer cell lines showed high levels of cell death early in the trial

  but three of the lines showed a second delayed increase in cell death at later stages. HCT116 cells were unaffected by 0.5 mM DCA. GM03349B and RPE cells also died when treated with DCA. At high concentrations

  all cell lines exhibited high rates of death. No specific cell cycle arrest of the cells was observed.\nConclusion.\nWe found that there is considerable difference in the way cancer cells are affected by DCA. Some have populations that are highly resistant to treatment

  while others have stronger rates of death only after prolonged exposure. We also found noncancerous cells are not all resistant to DCA

  a significant finding that has not previously been observed in other in vitro DCA trials.

  Dichloroacetate induces different rates of cell death in cancer and noncancer cell lines in vitro.

  T. A. Schroer

  D. M. Eckley

  B. R. Scipioni

  T-Y. Yeh

  Dynactin is an essential part of the cytoplasmic dynein motor that enhances motor processivity and serves as an adaptor that allows dynein to bind cargoes. Much is known about dynactin's interaction with dynein and microtubules

  but how it associates with its diverse complement of subcellular binding partners remains mysterious. It has been suggested that cargo specification involves a group of subunits referred to as the \"pointed end complex\". We used chemical cross-linking

  RNAi and protein overexpression to characterize interactions within the pointed end complex and explore how it contributes to dynactin's interactions with endomembranes. The Arp11 subunit

  which caps one end of dynactin's Arp1 filament

  and p62

  which binds Arp11 and Arp1

  are necessary for dynactin stability. These subunits also allow dynactin to bind the nuclear envelope prior to mitosis. p27 and p25

  by contrast

  are peripheral components that can be removed without any obvious impact on dynactin integrity. Dynactin lacking these subunits shows reduced membrane binding. Depletion of p27 and p25 results in impaired early and recycling endosome movement

  but late endosome movement is unaffected and mitotic spindles appear normal. We conclude that the pointed end complex is a bipartite structural domain that stabilizes dynactin and supports its binding to different subcellular structures.

  Dynactin's pointed end complex is a cargo-targeting module.

  TA Schroer

  KC Maier

  CL Brown

  SJ King

  Cytoplasmic dynein and dynactin are megadalton-sized multisubunit molecules that function together as a cytoskeletal motor. In the present study

  we explore the mechanism of dynein-dynactin binding in vitro and then extend our findings to an in vivo context. Solution binding assays were used to define binding domains in the dynein intermediate chain (IC) and dynactin p150Glued subunit. Transient overexpression of a series of fragments of the dynein IC was used to determine the importance of this subunit for dynein function in mammalian tissue culture cells. Our results suggest that a functional dynein-dynactin interaction is required for proper microtubule organization and for the transport and localization of centrosomal components and endomembrane compartments. The dynein IC fragments have different effects on endomembrane localization

  suggesting that different endomembranes may bind dynein via distinct mechanisms.

  Analysis of the dynein-dynactin interaction in vitro and in vivo.

  TA Schroer

  Centrosomal dynactin is required for normal microtubule anchoring and/or focusing independently of dynein. Dynactin is present at centrosomes throughout interphase

  but dynein accumulates only during S and G2 phases. Blocking dynein-based motility prevents recruitment of dynactin and dynein to centrosomes and destabilizes both centrosomes and the microtubule array

  interfering with cell cycle progression during mitosis. Destabilization of the centrosomal pool of dynactin does not inhibit dynein-based motility or dynein recruitment to centrosomes

  but instead causes abnormal G1 centriole separation and delayed entry into S phase. The correct balance of centrosome-associated dynactin subunits is apparently important for satisfaction of the cell cycle mechanism that monitors centrosome integrity before centrosome duplication and ultimately governs the G1 to S transition. Our results suggest that

  in addition to functioning as a microtubule anchor

  dynactin contributes to the recruitment of important cell cycle regulators to centrosomes.

  Distinct cell cycle-dependent roles for dynactin and dynein at centrosomes

  SJ King

  AD Stephens

  SA Lex

  TL Culver-Hanlon

  Microtubule-associated proteins (MAPs) use particular microtubule-binding domains that allow them to interact with microtubules in a manner specific to their individual cellular functions. Here

  we have identified a highly basic microtubule-binding domain in the p150 subunit of dynactin that is only present in the dynactin members of the CAP–Gly family of proteins. Using single-particle microtubule-binding assays

  we found that the basic domain of dynactin moves progressively along microtubules in the absence of molecular motors — a process we term 'skating'. In contrast

  the previously described CAP–Gly domain of dynactin remains firmly attached to a single point on microtubules. Further analyses showed that microtubule skating is a form of one-dimensional diffusion along the microtubule. To determine the cellular function of the skating phenomenon

  dynein and the dynactin microtubule-binding domains were examined in single-molecule motility assays. We found that the basic domain increased dynein processivity fourfold whereas the CAP–Gly domain inhibited dynein motility. Our data show that the ability of the basic domain of dynactin to skate along microtubules is used by dynein to maintain longer interactions for each encounter with microtubules.

  A microtubule-binding domain in dynactin increases dynein processivity by skating along microtubules.

  TA Schroer

  DA Compton

  CL Crego

  DM Eckley

  SR Gill

  The multiprotein complex

  dynactin

  is an integral part of the cytoplasmic dynein motor and is required for dynein-based motility in vitro and in vivo. In living cells

  perturbation of the dynein–dynactin interaction profoundly blocks mitotic spindle assembly

  and inhibition or depletion of dynein or dynactin from meiotic or mitotic cell extracts prevents microtubules from focusing into spindles. In interphase cells

  perturbation of the dynein–dynactin complex is correlated with an inhibition of ER-to-Golgi movement and reorganization of the Golgi apparatus and the endosome–lysosome system

  but the effects on microtubule organization have not previously been defined. To explore this question

  we overexpressed a variety of dynactin subunits in cultured fibroblasts. Subunits implicated in dynein binding have effects on both microtubule organization and centrosome integrity. Microtubules are reorganized into unfocused arrays. The pericentriolar components

  γ tubulin and dynactin

  are lost from centrosomes

  but pericentrin localization persists. Microtubule nucleation from centrosomes proceeds relatively normally

  but microtubules become disorganized soon thereafter. Overexpression of some

  but not all

  dynactin subunits also affects endomembrane localization. These data indicate that dynein and dynactin play important roles in microtubule organization at centrosomes in fibroblastic cells and provide new insights into dynactin–cargo interactions.

  Dynactin is required for microtubule anchoring at centrosomes.

  I am a Cell Biologist; I mostly teach undergraduate courses and mentor undergraduate research with an interest in ensuring that students can make the leap from their undergraduate studies to more advanced studies in graduate or professional schools. My goal is for students to see the links between research and what is learned in class and get a sense of how scientists work to solve biological problems (to use the title of the course we used to have to register for at JHU). In doing so

  students are better prepared to think as scientists and have a great advantage in making links between disparate fields and improve their critical thinking and hypothesis generating skills.\nMy elective courses are also offered for Master's degree candidates.\n\nSpecialties: I am an expert in fluorescence microscopy techniques

  tissue culture

  molecular biology techniques; I have experience in protein purification. My main focus is on cytoskeletal regulation

  both in interphase and mitosis

  examining how microtubules direct various cellular functions and how they themselves are controlled. The best protein ever is dynactin.

  Nick

  Quintyne

  State University of New York at Fredonia

  University of Pittsburgh

  Florida Atlantic University

  Johns Hopkins University

  Fredonia

  NY

  I am a member of the faculty of the biology department at The State University of New York at Fredonia. I teach upper-level courses in Cell Biology

  Cancer Biology and Cell Signaling. The latter two courses are cross-listed for graduate students. I also teach Introduction to Cell and Molecular Biology for majors and Human Biology for non-majors.\nIn addition

  I have an active research program examining various aspects of cytoskeletal regulation in cancerous and noncancerous cells

  with an emphasis on motor proteins and their accessories.\nI have mentored 17 undergraduate students in my laboratory at Fredonia.

  Assistant Professor of Biology

  State University of New York at Fredonia

  Worked in the laboratory of Bill Saunders

  examining multipolar spindle formation in oral cancer cells.

  University of Pittsburgh

  Postdoctoral Fellow

  Worked in the lab of Trina Schroer on cell cycle roles for dynactin

  Johns Hopkins University

  Florida Atlantic University

  Jupiter

  FL

  I taught several courses at different levels of the honors biology curriculum

  mostly in molecular biology. This included introductory biology as well as cell biology

  developmental biology and cancer biology.\nMy research examines the role of various motor proteins in cell cycle progress and cytoskeletal organization.\nI supervised 41 undergraduate theses.

  Assistant Professor of Biology