Copy - MODERN ZEN PRESENTATION
Heritage A
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Transcript
PRESENTATION ON
SHARK KINASE
IM group project
- Shark (SH2 domain ankyrin repeat kinase) is a Drosophila non-receptor tyrosine kinase [1]
- Essential for Draper-mediated signalling events in vivo [2]
- Shark has a role in the phagocytosis of axonal debris and neuronal cell corpses by the glia
- Want to see how it effects phagocytosis by the muscles
SHARK KINASE
- Ligand is recognised by draper and binds eg. phosphatidylserine [3]
- Recruitment of Shark - Shark binds to the phosphorylated ITAM motif of Draper [4]
- Src phosphorylates the ITAM motif of Draper
SHARK IS REQUIRED FOR DRAPER MEDIATED Phagocytosis
- Draper interacts with CED-6 to promote phagocytosis
- CED-6 is a protein involved in regulation and execution of apoptosis [5]
- Know that the pathway plays a key role in Glial cells
- Want to quantify its role in muscles
- Is involved at the start of the endocytic pathway so would expect significant affects from knock down
- Has a human homologue SYK - so is applicable to human disease
WHY ARE WE RESEARCHING THIS ?
HYPOTHESIS
Knock down of Shark will decrease neuromuscular junction pruning
- Use Shark RNAi lines to knock down Shark in muscle cells
- Gal-4 -> under the control of Bg57 gene trap, drives expression in muscle cells
- UAS (upstream activating sequence) fused to the RNAi line in separate fly with GFP (reporter sequence) downstream
- Compare to CHMP2B mutant
Methods
Methods - A problem
- Possibility that Shark RNAi lines could cause the embryos to be very ill / death of embryos
- Shark RNAi was used in a recent project by Wang et al, embryos survived [6]
- May use drug inhibitor to knock it out
- Using Image J
- Bouton size and number
- Neuron length
- Measure amount of synaptic puncta
data collection
Wild Type
Knock down
Expecting to see
[1]R. Fernandez, F. Takahashi, Z. Liu, R. Steward, D. Stein, and E. R. Stanley, “The Drosophila shark tyrosine kinase is required for embryonic dorsal closure,” Genes & development, vol. 14, no. 5, pp. 604–14, 2000, Accessed: Dec. 06, 2023. [Online]. Available: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC316420/[2]J. S. Ziegenfuss et al., “Draper-dependent glial phagocytic activity is mediated by Src and Syk family kinase signalling,” Nature, vol. 453, no. 7197, pp. 935–939, Apr. 2008, doi: https://doi.org/10.1038/nature06901.[3]E. A. Britt, V. Gitau, A. Saha, and A. P. Williamson, “Modular Organization of Engulfment Receptors and Proximal Signaling Networks: Avenues to Reprogram Phagocytosis,” Frontiers in Immunology, vol. 12, Apr. 2021, doi: https://doi.org/10.3389/fimmu.2021.661974.[4]S. B. Serizier, J. S. Peterson, and K. McCall, “Non-autonomous cell death induced by the Draper phagocytosis receptor requires signaling through the JNK and SRC pathways,” Journal of Cell Science, vol. 135, no. 20, Oct. 2022, doi: https://doi.org/10.1242/jcs.250134.[5]Q. A. Liu and M. O. Hengartner, “Candidate Adaptor Protein CED-6 Promotes the Engulfment of Apoptotic Cells in C. elegans,” Cell, vol. 93, no. 6, pp. 961–972, Jun. 1998, doi: https://doi.org/10.1016/s0092-8674(00)81202-7.[6]Y. Wang et al., “Glial Draper signaling triggers cross-neuron plasticity in bystander neurons after neuronal cell death in Drosophila,” Nature Communications, vol. 14, no. 1, p. 4452, Jul. 2023, doi: https://doi.org/10.1038/s41467-023-40142-y.
REfrences
Thank You!