Department of Biological Sciences

Proper Localization of Borealin to the Centromere

Jordan J. Burlen

Department of Biological Sciences

Faculty Advisor: Dr. William R. Taylor

Cancer is characterized by uncontrolled cell proliferation. Greater understanding of nuclear and cytoplasmic division can identify cures for cancer and other diseases. The cell cycle is composed of interphase, mitosis, and cytokinesis. This process results in the formation of two identical daughter cells. Errors in cell cycle regulation can result in cancer. Incorrect microtubule attachment to kinetochores can cause separation errors of the sister chromatids, resulting in aneuploidy (an abnormal number of chromsomes). Oncogenesis is a possible result of aneuploidy. Incorrect microtubule attachment to kinetochores can cause separation errors of the sister chromatids.

The chromosomal passenger complex (CPC) prevents non-bipolar attachments by detecting and destabilizing them. The enzymatic subunit of the CPC that is involved in this process is the Aurora B kinase. The other members of the CPC include INCENP (an inner centromere protein), Survivin (a member of the inhibitor of apoptosis (IAP) family), and Borealin. The CPC localizes at the inner centromere early in mitosis. This localization is necessary for proper CPC function and chromosome segregation.

The role of Borealin is not fully understood. The function of the C-terminus of Borealin was studied by creating 124 AA and 110 AA truncations via polymerase chain reaction (PCR). Various members of Dr. William Taylor’s lab used these truncations to study the localization and function of Borealin. The 110 AA truncation localized to the centromere in immunoflorescence and cell cycle analysis. This suggests that the C-terminus, under the conditions analyzed, is nonessential for localization of Borealin to the centromere.

The Functional Regulation of Chromosomal Passenger Protein Borealin

Michael T. Borton

Department of Biological Sciences

Faculty Advisor: Dr William R. Taylor

The Spindle Assembly Checkpoint (SAC) is an evolutionarily conserved cell cycle control mechanism that prevents premature sister chromatid separation during mitosis.  The Chromosomal Passenger Complex (CPC) regulates SAC activation and consists of INCENP, Survivin, Borealin, and Aurora B Kinase. The CPC localizes to centromeres to ensure correct kinetochore-microtubule attachment.  Borealin localization to centromeres is regulated by phosphorylation; however, the opposing phosphatase that regulates Borealin dephosphorylation is unknown.  Mobility shift for Borealin in human cells containing knockouts for phosphatase Cdc14A or Cdc14B show no difference in Borealin expression.  In comparison, phosphatase inhibitor okadaic acid does delay the dephosphorylation of Borealin as cells exit mitosis suggesting that the phosphatase PP2A may be responsible for the dephosphorylation of Borealin. Additionally, Borealin protein stability is regulated; yet, the mechanisms that govern Borealin stability are unknown.  Increased levels of Borealin are found in samples containing proteasome inhibitor MG132, though the structurally different proteasome inhibitor Lactacystin did not suppress degradation suggesting that a protease other than the proteasome may be responsible for Borealin degradation.  Finally, Borealin localization to centromeres is mediated by phosphorylation of histone H2A (pH2A).  ChIP analysis of Borealin and pH2A indeed indicates that both localize primarily at the centromere of DNA. 

The tyraminergic inhibition of aversive responses in C. elegans requires neuropeptides and mimics noradrenergic anti-nociception in mammals

Andrew M. Stein

Department of Biological Sciences

Faculty Advisor: Dr. Richard Komuniecki

In the nematode model system, Caenorhabditis elegans, the monoamines, tyramine (TA) and octopamine (OA), invertebrate counterparts of epinephrine and norepinephrine, respectively, appear to independently activate global peptidergic signaling cascades that inhibit aversive responses to noxious stimuli mediated by two ASH sensory neurons. To better understand the complex role of peptidergic signaling in this monoamine-dependent inhibition, we examined the roles in individual neuropeptides expressed in the TA-sensitive ASI sensory neurons on various aspects of locomotory behavior. Peptides encoded by nlp-14were essential for the TA inhibition of aversive responses and animals overexpressing nlp-14 moved more slowly and reversed less frequently than wild type animals off but not on food. These results confirm that 1) the TA-dependent release of individual peptides from the ASIs is essential for the TA inhibition of aversive responses and 2) these peptides may have additional more wide-spread roles in modulating locomotory behavior. TA/OA inhibition of aversive responses mimics mammalian noradrenergic antinociception and suggests that C. elegans may be a useful model for understanding the noradrenergic modulation of chronic pain in mammals.

Regulation of MLK1 and MLK4 Protein Levels in T98G Glioblastoma cells

Lindsey A. Foster

Department of Biological Sciences

Faculty Advisor: Dr. Deborah N. Chadee

            Mixed-lineage kinases (MLKs) are serine/threonine protein kinases that are classified as mitogen-activated protein kinases (MAPKs) that function in signal transduction. Eukaryotic cells have conserved MAPK signaling pathways in order to respond to a variety of extracellular stimuli and control the regulation of biological processes. MLK3 is a client protein of the molecular chaperone Hsp90. Hsp90 interacts with and stabilizes client proteins.

Interaction Between Protein 2 and Protein 6 of Cauliflower mosaic virus

Vanessa Gantz

Advisor: Dr. Scott Leisner

Identification of genes that are essential for correct intermediate filament localization in C. elegans

Robert Puckett

Department of Biological Sciences

Faculty Advisor: Dr. John Plenefisch

            ifa-2 is a member of the mua (muscle attachment abnormal) family of genes of Caenorhabditiselegans and is known to encode the cytoplasmic intermediate filament IFA-2. This family of genes are involved in connecting skeletal muscle tissue to the cuticle across the intervening epidermis through hemidesmosomes and their associated intermediate filaments. Mutations in ifa-2 cause these connections to become fragile and fail, resulting in separation of the muscles from the body wall and locomotory defects in the worm. Proteins of the hemidesmosome are likely to be  involved in the proper localization of IFA-2. By using RNA interference (RNAi), a simple procedure that prevents the translation of a specific mRNAs, I can target specific hemidesmosome or other IFA-2 associated proteins for reduced expression. I am looking at both proteins known to be associated with muscle attachment and those suspected to be associated with IFA-2 based on the results of a yeast 2-hybrid study by graduate student Kyle Walker. These include KO4G7.1, let-805b, let-805c, let-805d, vab-10a, vab-10b, vab-19, pat-12j, and pat-12j. The feeding RNAi experiment is being performed on a line of worms that express an IFA-2::GFP fluorescent reporter protein, thereby allowing me to observe whether knock-down of expression of any of the proteins results in  mislocalization of IFA-2 and concomitant of muscle attachment defects using a fluorescent microscope.

Influence of Histone Deacetylases (HDACs) on NF-κB Activity

Amanda Kitson

Dr. Brian Ashburner

Dept. of Biological Sciences