quantifying nuclear movement in wild type and mmb1-delta ... · mmb1-delta and klp4-delta fission...
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Quantifying Nuclear Movement in Wild Type and mmb1-delta and klp4-delta Fission Yeast Cells.
IISc Bangalore
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BackgroundFission 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/
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BackgroundMicrotubule 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 nuclearpositioning in fission yeast based on microtubule pushing. J Cell Biol.2001;153(2):397-411. doi:10.1083/jcb.153.2.397.
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BackgroundMicrotubules 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. Microtubuledynamics regulates mitochondrial fission. bioRxiv. 2017.doi:10.1101/178913.
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Klp4Kinesin protein that stabilizes microtubules
Deletion leads to short microtubules
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MethodsYeast strain with microtubules tagged with GFP with the nucleus stained with hoechst
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MethodsYeast Strain with microtubules tagged with mcherry with the nucleus stained with
hoechst
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Weak mcherry signal in stained cells
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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.
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MethodsUnable to visualize microtubules and nucleus simultaneously
Future possibility:
Yeast strain with the microtubules tagged with mcherry and the ER tagged with GFP
Requires cross
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The Lab
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Exploring Bangalore
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AcknowledgementsThank you to Vaishnavi, Leeba, all the lab, and the SN Bose Program!