1. A kinase anchor protein 200 Why chose Akap200? In situ Hybridization. Autophagic Vacuoles Detection. Future Directions.

2. Genetics Screens Red-ambiguous

3. A kinase anchoring protein 200 Molecular Function: protein kinase A anchor protein. Expression Level: 16APF: 3 tags, 23APF: 67tags. Akaps function in cyclic AMP –dependent protein kinase (PKA) signal transduction, targeting bound PKA to docking sites in organelles or the cytoskeleton. Genetics studies have implicated akap200 as a negative regulator of Ras pathway.

4. akap200 embryonic expression Hypothesis: Is the Expression pattern of akap200 similar to the known pro- apoptotics genes? Methods: 1.In situ hybridization of rpr and hid. 2.Double in situ hybridization. Akap200 S12 Akap200 S15/16 haemocytes midline glial cells

5. 1.In situ Hybridization Previous Method: Short DNA probes, Proteinase K treatment. New Method: Longer RNA probes, acetone and xylene treatment. Improvements 1. Reduce the background level. 2. Clear and sharp signal detection. 3. Maintain the embryo integrity.

6. Improvement of in situ hybridization on embryos

7. Example: No signals of CG4091 were detected in embryos by using old method. Clear and sharp signals on the digestive system and nerve system were detected by using the new method.

8. Different embryonic stages of Akap200, Rpr and Hid expression

9. 2.Double in situ hybridization: Expression of pro-apoptotic genes, reaper (RPR), grim, and head involution defective (HID) is confined only to those cells destined to die. Double in situ hybridization technique was used to identify gene whose expression is localized similar to pro-apoptotic genes and might have roles in embryonic programmed cell death.

10. Double in situ hybridization: Result: Can’t distinguish between single signals (Hid) or overlay (Akap200 + Hid). Brown: Akap200, Purple: Hid

11. Retinal Phenotype in EP2265 EP2565 has extra secondary pigment cells. EP2256

12. In situ hybridization on retina

13. Autophagic Vacuoles Detection Monodansylcadaverine MDC

14. Apoptosis v.s. Autophagic cell death Apoptosis is characterized by early condensation of the chromatin and then portions of cell pinch off to form apoptotic bodies that are phagocytosed by surrounding cells. Autophagic vacuoles do not increase in number. In contrast, during autophagic cell death, the number and size of autophagic vacuoles increase and cellular content are destroyed by the cell’s own lysosomes after being sequestered within autophagic vacuoles.

15. What is autophagic vacuoles? During autophagic cell death, segment of ER envelop cytoplasmic components and pinch off to form autophagosome, which are bound by double membranes. Autophagosome fuse with lysosomes to form autolysosomes or autophagic vacuoles which have a single limiting membrane. The contents of autolysosomes are broken down by acid phosphatase and after hydrolysis of their contents, autolysosomes are termed “residual bodies”.

17. Monodansylcadaverine MDC A lysosomotropic agent, it is concentrated into acid compartments by an ion trapping mechanism. A solvent polarity probe, it increases its relative fluorescence intensity by interacting with membrane lipids. MDC only accumulates in mature autophagic vscuoles, such as late autophagosome and autolysosome.

18. MDC Label Mature Autophagic Vacuoles on Salivary Gland

19. 26hr APF OreR Autophagic vacuoles Nucleus

20. Future Directions Functional studies on the Akap200 excision lines from S. Jackson’s Lab.

21. Akap200 excision lines (A, B) wild type. (C, D) Akap200 k07118 /Df(2L) N22-14. (E, F) Akap200 ix4 /Df(2L) N22-14. (G,H) Akap ^7 /Df(2L) N22-14. Akap200 k07118: P element insert into 1 st intron. 4kb internal deletion in P element. Akap200 ix4: 4kb internal deletion in P element. Akap ^7: P element + 1.2kb of 1 st Akap200 intron removed.

22. Adult Phenotype in Akap200 hemizygous mutants

23. Next? Use the TUNEL technique to detect DNA fragmentation on retina for Akap200^7/Df(2L)N22-14. Which cells expressed akap200? Where is the Akap200 and Pka-RII localization on retina and salivary glands?

24. Red depicts filamentous actin in subcortical regions of nurse cells and ring canals, green symbolizes Akap200, dark blue is Pka-RII, pink is Pka-C; yellow circles and orange blobs are other proteins that regulate actin filament polymerization or function. From S.M Jackson and C.A. Berg. Development 2002 Model for localized regulation of PKA activity by Akap200

25. Staining of Actin Filaments and Nuclei From J.Jochova, Z. Zakeri and R. Lockshin (1997) Cell Death and Differentiation. 4,140-149

26. Acid Phosphatase Detection in Drosophila Salivary Glands

27. Akap200; Actin; Autophagic Vacuoles; PCD??? Akap200 is required for PKA RII localization and morphology of actin structures during oogenesis. Actin filament coated organelles in the cells of salivary gland may be related or identical to autophagic vacuoles. What will be the role of akap200 in salivary gland? ( formation and localization of autophagic vacuoles by regulating actin filament??)