Biochemistry, Biophysics, and Structural Biology Commons^™

Novel Mechanistic Insight Into Ciliary Regulation: Old Pathways Yield New Mechanisms, Larissa L. Dougherty

Dartmouth College Ph.D Dissertations

Cilia are structures present on most eukaryotic cells which provide important signaling and motile components to cells from early development to fully differentiated and matured cells. Regulation of these structures is critical to proper functioning of the cell and is known to be tied to the cell cycle. Preparation for ciliary assembly following cell cycle exit and ciliary disassembly following cell cycle reentry requires components throughout the cell body and within the cilium to facilitate this process. Here I identify how the cell adapts to ensure modifications to cilia occur for assembly or disassembly using the model organism Chlamydomonas reinhardtii. …

In Vivo Evaluation Of (-)-Zampanolide Demonstrates Potent And Persistent Antitumor Efficacy When Targeted To The Tumor Site., Leila Takahashi-Ruiz, Joseph D Morris, Phillip Crews, Tyler A Johnson, April L Risinger

Natural Sciences and Mathematics | Faculty Scholarship

Microtubule-stabilizing agents (MSAs) are a class of compounds used in the treatment of triple-negative breast cancer (TNBC), a subtype of breast cancer where chemotherapy remains the standard-of-care for patients. Taxanes like paclitaxel and docetaxel have demonstrated efficacy against TNBC in the clinic, however new classes of MSAs need to be identified due to the rise of taxane resistance in patients. (-)-Zampanolide is a covalent microtubule stabilizer that can circumvent taxane resistance in vitro but has not been evaluated for in vivo antitumor efficacy. Here, we determine that (-)-zampanolide has similar potency and efficacy to paclitaxel in TNBC cell lines, but …

The Effects Of Acetylenic Tricyclic Bis-(Cyano Enone) On Cell Migration, Eddie Chan

Electronic Thesis and Dissertation Repository

Although cancer survival rates have significantly improved over the past few decades, the improvements are primarily due to early diagnosis and inhibiting cancer growth. Limited progress has been made in the treatment of cancer metastasis, which contributes to 90% of cancer related deaths, and therapeutic agents targeting the various aspects of metastasis are lacking. One potential approach is to utilize small pharmacological compounds to inhibit tumour cell motility, as a strategy against tumour cell migration, invasion, and metastasis. The acetylenic tricyclic bis-(cyano enone), TBE-31, has been shown to be a promising chemopreventative compound. However, its effects on cell migration are …

Microtubule Severing At Crossover Sites By Katanin Generates Ordered Cortical Microtubule Arrays In Arabidopsis, Quan Zhang, Erica Fishel, Tyler Bertroche, Ram Dixit

Biology Faculty Publications & Presentations

Highlights

• Severing primarily depolymerizes the overlying CMT at crossover sites
• Severing probability increases nonlinearly with crossover time
• Katanin localizes to crossover sites and is required for severing
• Loss of katanin activity prevents the formation of coaligned CMT arrays

Summary
The noncentrosomal cortical microtubules (CMTs) of land plants form highly ordered parallel arrays that mediate cell morphogenesis by orienting cellulose deposition [1, 2 and 3]. Since new CMTs initiate from dispersed cortical sites at random orientations [4], parallel array organization is hypothesized to require selective pruning of CMTs that are not in the dominant orientation. Severing of CMTs at crossover sites …

Single-Molecule Analysis Of The Microtubule Cross-Linking Protein Map65-1 Reveals A Molecular Mechanism For Contact-Angle-Dependent Microtubule Bundling, Amanda Tulin, Sheri Mcclerklin, Yue Huang, Ram Dixit

Biology Faculty Publications & Presentations

Bundling of microtubules (MTs) is critical for the formation of complex MT arrays. In land plants, the interphase cortical MTs form bundles specifically following shallow-angle encounters between them. To investigate how cells select particular MT contact angles for bundling, we used an in vitro reconstitution approach consisting of dynamic MTs and the MT-cross-linking protein MAP65-1. We found that MAP65-1 binds to MTs as monomers and inherently targets antiparallel MTs for bundling. Dwell-time analysis showed that the affinity of MAP65-1 for antiparallel overlapping MTs is about three times higher than its affinity for single MTs and parallel overlapping MTs. We also …

Cdk1 And Plk1 Mediate A Clasp2 Phospho-Switch That Stabilizes Kinetochore–Microtubule Attachments, Ana R. R. Maia, Zaira Garcia, Lilian Kabeche, Marin Barisic

Dartmouth Scholarship

Accurate chromosome segregation during mitosis relies on a dynamic kinetochore (KT)-microtubule (MT) interface that switches from a labile to a stable condition in response to correct MT attachments. This transition is essential to satisfy the spindle-assembly checkpoint (SAC) and couple MT-generated force with chromosome movements, but the underlying regulatory mechanism remains unclear. In this study, we show that during mitosis the MT- and KT-associated protein CLASP2 is progressively and distinctively phosphorylated by Cdk1 and Plk1 kinases, concomitant with the establishment of KT-MT attachments. CLASP2 S1234 was phosphorylated by Cdk1, which primed CLASP2 for association with Plk1. Plk1 recruitment to KTs …

A Conserved Cam- And Radial Spoke–Associated Complex Mediates Regulation Of Flagellar Dynein Activity, Erin E. Dymek, Elizabeth F. Smith

Dartmouth Scholarship

For virtually all cilia and eukaryotic flagella, the second messengers calcium and cyclic adenosine monophosphate are implicated in modulating dynein- driven microtubule sliding to regulate beating. Calmodulin (CaM) localizes to the axoneme and is a key calcium sensor involved in regulating motility. Using immunoprecipitation and mass spectrometry, we identify members of a CaM-containing complex that are involved in regulating dynein activity. This complex includes flagellar-associated protein 91 (FAP91), which shares considerable sequence similarity to AAT-1, a protein originally identified in testis as an A-kinase anchor protein (AKAP)- binding protein. FAP91 directly interacts with radial spoke protein 3 (an AKAP), which …

Minus-End Capture Of Preformed Kinetochore Fibers Contributes To Spindle Morphogenesis, Alexey Khodjakov, Lily Copenagle, Michael B. Gordon, Duane A. Compton, Tarun M. Kapoor

Dartmouth Scholarship

Near-simultaneous three-dimensional fluorescence/differential interference contrast microscopy was used to follow the behavior of microtubules and chromosomes in living alpha-tubulin/GFP-expressing cells after inhibition of the mitotic kinesin Eg5 with monastrol. Kinetochore fibers (K-fibers) were frequently observed forming in association with chromosomes both during monastrol treatment and after monastrol removal. Surprisingly, these K-fibers were oriented away from, and not directly connected to, centrosomes and incorporated into the spindle by the sliding of their distal ends toward centrosomes via a NuMA-dependent mechanism. Similar preformed K-fibers were also observed during spindle formation in untreated cells. In addition, upon monastrol removal, centrosomes established a transient …