The Role of Mud in Regulating Kinesin Protein Function

1. Vincent Cutillas Johnston Lab Department of Biology University of New Mexico The Role of Mud in RegulatingKinesin Protein Function Source: Cell Image Library, doi:10.7295/W9CIL42515

2. Mitosis Overview Source: https://www.bioexplorer.net/divisions_of_biology/cell_biology/mitosis/

3. What is Asymmetric Cell Division? • Cortical cues on either side of the spindle determine cell fate • Insc-Pins-Mud complex previously known to be highly important in correct spindle positioning Source: https://www.researchgate.net/figure/Asymmetric-division-in-Drosophila-neuroblasts-Polarized-localization-of-apical-complexes_fig1_239075988

4. Mud involvement in Spindle Orientation Force Generation • Mud is involved in two pathways that maintain spindle orientation • When coupled to dynein, centrioles are pulled up towards the membrane Source: Lu, M. S., and C. A. Johnston. “Molecular Pathways Regulating Mitotic Spindle Orientation in Animal Cells.” Development, vol. 140, no. 9, Sept. 2013, pp. 1843–1856., doi:10.1242/dev.087627.

5. Mud/Pins Pathway and the role of Warts kinase • Previous work in the lab has shown that Warts will phosphorylate Mud in order for Mud to translocate and bind Pins Source: Dewey, Evan B., et al. “Warts Phosphorylates Mud to Promote Pins-Mediated Mitotic Spindle Orientation in Drosophila , Independent of Yorkie.” Current Biology, vol. 25, no. 21, 2015, pp. 2751–2762., doi:10.1016/j.cub.2015.09.025.

6. Mass Spectrometry of Mud Revealed Two Important Motor Proteins Pavarotti (Pav) • Member of the Kinesin-6 family • Known to transport important cargo to cell cortex • Plus-end-directed kinesin

7. Mass Spectrometry of Mud Revealed Two Important Motor Proteins Non-claret disjunctional (Ncd) • Member of the Kinesin-14 family • A “backwards” kinesin in almost every sense • Motor domain on C-terminus • Is a minus-end-directed kinesin • In acentriolar cells: important in spindle focusing • In multipolar cells: important in pole focusing to rescue spindle morphology

8. The Three Kinesin Domains • Motor Domain: Catalyzes ATP in order to create the motile function of the kinesin • -Neck Linker region responsible for linking both the motor domain dimer together • Stalk/Coiled Coil Domain: Stabilizes the dimerization of the protein • Tail/Cargo Domain: Binds cargo of the kinesin, usually for transport of materials from one region of the cell to another Source: http://www.davidmoore.org.uk/21st_Century_Guidebook_to_Fungi_PLATINUM/Ch05_13.htm ; http://www.science20.com/run_and_tumble/walk_it_motor_proteins

9. Ncd: Minus-end-directed kinesin (Kinesin 14 family)Pav: Plus-end-directed kinesin (Kinesin 6 family) Ncd Pav

10. GST Fusion Proteins Sources: https://www.abbkine.com/featured-purkine-gst-tag-protein-purification-kit-glutathione-released/ ; https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-cloning-tutorial/a/overview-dna-cloning

11. Mud binds Pav on Cargo and Coiled Coil Domain CT/Cargo Domain ? CC/Stalk Domain NT/Motor Domain Microtubule Source: http://pdb101.rcsb.org/motm/64

12. Mud binds Ncd on Cargo and Motor Domains ? NT/Cargo Domain CC/Stalk Domain ? CT/Motor Domain Microtubule

13. Ncd Intra-Molecular Interaction

14. Updated Hypothesis: Pav binds Mud to translocate it from the centrosome to the membrane, Mud facilitates NcdCT/NcdNT auto-inhibition Mud Mud “Open”, Non-Inhibited Conformation Closed, Inhibited Conformation

15. 14-3-3ε and Aurora B localization mechanism • Pav and Ncd both contain a highly conserved HRRSRSA amino acid sequence in their cargo domain • This sequence is also conserved among species and can be found in human MKLP1, the human ortholog of Pav • This site has been shown to bind 14-3-3 and Aurora B • Both in Ncd and in MKLP1, it has been shown that phosphorylation of the first serine causes dissociation of 14-3-3 from the second serine to localize the kinesin specifically to the mitotic spindle • Dissociation of 14-3-3 from the cargo domain may be a regulatory mechanism for the binding of Mud

16. Mud Preferentially Binds Second Phosphorylation Site on Pav

17. Future/Planned Directions • TIRF Microscopy • Determine if kinesin is responsible for movement/bundling after Mud binds • Echinoid Induced-Polarity Assay • Determine cellular significance of loss of Mud/Pav/Ncd/14-3-3 • In vivo Experiment using Confocal Microscopy • Determine effects of Mud localization and tissue morphology with loss of Pav/Ncd/14-3-3

18. TIRF Microscopy – Motility Assay GFP Mud Microtubule RFP RFP RFP RFP RFP RFP RFP RFP Source: http://people.umass.edu/rossj/Motors.html

19. Echinoid Induced-Polarity Assay • Echinoid tag expresses ectopically and allows for cell-cell adhesion with a GFP tag • After transfection into S2 cells, the cells can be treated with RNAi, and spindle axis can be measured relative to the arc created by Ed::GFP-Pins Source: Johnston, Christopher A., et al. “Identification of an Aurora-A/PinsLINKER/ Dlg Spindle Orientation Pathway Using Induced Cell Polarity in S2 Cells.” Cell, vol. 138, no. 6, 2009, pp. 1150–1163., doi:10.1016/j.cell.2009.07.041.

20. Drosophila Genetics 101 One fly carries a transcriptionally activated gene of interest One fly carries a tissue-specific Expression of GAL4 When crossed, the tissue-specific GAL4 will activate the transcription of the gene of interest We can use this to then generate flies containing RNAi or overexpression of genes in any tissue we select in vivo Source: Nature Reviews Genetics 3, 176-188 (March 2002)

21. Drosophila as a Model to Study Asymmetric, Oriented Cell Division Oocytes Wing Disks Neuroblasts Source: http://rusty.fhl.washington.edu/celldynamics/events/workshops/archive/2003/cytomod_abstracts/ABrand/index.html ; http://jcs.biologists.org/content/118/15/3363 ; https://www.flickr.com/photos/thejcb/4118265744

22. Confocal Microscopy • Use of immunofluorescence to tag proteins of interest in order to determine effect of motor protein RNAi on spindle morphology • Spindle angle can be measured against the Miranda (Mira) crest or the surrounding tissue morphology in vivo Source: https://www.researchgate.net/figure/Asymmetric-division-in-Drosophila-neuroblasts-Polarized-localization-of-apical-complexes_fig1_239075988

23. Thank you! Questions? Source: https://www.artofthecell.com/animation/well-you-can-tell-by-the-way-i-use-my-walk