Quantifying Nuclear Movement in Wild Type and mmb1-delta and - PowerPoint PPT Presentation

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Quantifying Nuclear Movement in Wild Type and mmb1-delta and klp4-delta Fission Yeast Cells. IISc Bangalore Background Fission yeast typically divide symmetrically after a period of growth. Microtubules contribute to this process through

Quantifying Nuclear Movement in Wild Type and mmb1-delta and klp4-delta Fission Yeast Cells. IISc Bangalore
Background Fission yeast typically divide symmetrically after a period of growth. Microtubules contribute to this process through positioning the nucleus at the center of the cell, which becomes the site of cell division. http://www.labex-celtisphybio.fr/cytoskeleton-architecture-and-cellular-morphogenesis/
Background Microtubule pushing: Growth and catastrophe of microtubules helps to center the nucleus of the cell. Tran PT, Marsh L, Doye V, Inoué S, Chang F. A mechanism for nuclear positioning in fission yeast based on microtubule pushing. J Cell Biol. 2001;153(2):397-411. doi:10.1083/jcb.153.2.397.
Background Microtubules and mitochondria are associated through the protein mmb1 during interphase. During mitosis mitochondria undergo fission by dnm1. Mehta K, Chug MK, Jhunjhunwala S, Ananthanarayanan V. Microtubule dynamics regulates mitochondrial fission. bioRxiv. 2017. doi:10.1101/178913.
Klp4 Kinesin protein that stabilizes microtubules Deletion leads to short microtubules
Methods Yeast strain with microtubules tagged with GFP with the nucleus stained with hoechst
Methods Yeast Strain with microtubules tagged with mcherry with the nucleus stained with hoechst
Weak mcherry signal in stained cells
Attempted Protocols Stain concentration: 2, 5, and 10 microgram/mL hoechst stain ● Time: 15 and 20 minutes of staining ● ● Location: staining in the hood, staining in the incubator Imaging: confocal dishes, slides, and slides with agarose pads ● Imaging media: EMM+N, EMM+N and amino acids, hoechst stain ● Cells: grown overnight in YEA, taken directly from plate ● Optimized protocol: 2 microgram/mL stain for 15 minutes in the incubator, image on slides with agarose pads in EMM+N and amino acids, with cells taken directly from the plate.
Methods Unable to visualize microtubules and nucleus simultaneously Future possibility: Yeast strain with the microtubules tagged with mcherry and the ER tagged with GFP Requires cross
The Lab
Exploring Bangalore
Acknowledgements Thank you to Vaishnavi, Leeba, all the lab, and the SN Bose Program!

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