Copy - MODERN ZEN PRESENTATION

PRESENTATION ON

IM group project

SHARK KINASE

SHARK KINASE

• 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 IS REQUIRED FOR DRAPER MEDIATED Phagocytosis

- Recruitment of Shark - Shark binds to the phosphorylated ITAM motif of Draper [4]

- Ligand is recognised by draper and binds eg. phosphatidylserine [3]

- Src phosphorylates the ITAM motif of Draper

• Draper interacts with CED-6 to promote phagocytosis
• CED-6 is a protein involved in regulation and execution of apoptosis [5]

WHY ARE WE RESEARCHING THIS ?

• 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

HYPOTHESIS

Knock down of Shark will decrease neuromuscular junction pruning

Methods

• 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 - 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

data collection

• Using Image J
• Bouton size and number

• Neuron length
• Measure amount of synaptic puncta

-> expect increased boutons and debris as there would be no absorption of the boutons

Expecting to see

Knock down

Wild Type

REfrences

[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.

Thank You!