dGRASP-Mediated Noncanonical Integrin Secretion

Is Required for Drosophila Epithelial Remodeling

Hans Schotman, Leena Karhinen, and Catherine Rabouille

Developmental Cell 14, 171–182, February 2008

Presented by Ian M. Parnham & Steven de Maat, supervised by Dr. Catherine Rabouille

Oogenesis

• Divided in to early (stages 1–6), mid- (stages 7–10a), and late (stages 10b–14) oogenesis.

• During oogenesis, the 15 nurse cells, the oocyte, and a surrounding monolayer of somatically derived follicle cells together comprise the egg chamber

2

Follicle Epithelium (Stage 10)

3

• BC: Border cells• NC: Nurse cells• CC: Centripetal

Cells• CFC: Cylindrical

Follicular Cells

• In stage 10A the Follicular cells migrate, covering the oocyte and changing there shape from cubical to columnar.

• In stage 10B follicle cells completely cover oocyte separating nurse cells from the oocyte, elongating there shape

What are we going to look at?

4

Immunofluoresence (IF) Immunoelectron microscopy (IEM)

ZOC: zone of contactFigure 1

Observations

• Architecture of epithelial tissue depends intercellular junctions and adhesion

• Adhesion with extracellular matrix components at the basal side is mediated by integrins and integral plasma membrane αβ heterodimers

• In the wing imaginal discs of Dropsophila larvae, where αPS1βPS(PS1) and αPS2βPS (PS2) maintain integrity of the developing wing.

5

dGRASP

• dGRASP has been considered bona fide Golgi proteins.

• Involved in formation and maintenance of the Golgi

• GrhA (GRASP65/55 homologue) has been shown to mediate Golgi independent secretion of AcbA in Dictyostelium.

6

Where does dGRASP localize to, during Oocyte development?

7Figure 1

Aim

Investigate the role of dGRASP in follicular epithelium

surrounding the developing oocyte

8

Do dGRASP binding partners also translocate towards the open ZOC during oocyte development?

9Figure 1

Where does the newly localized dGRASP come from?

10Figure 2

Do dgrasp RNA & dGRASP translocate to the same place?

11Figure 2

Recap

• Stage 10A to 10B: formation of open ZOC

• Stage 10B dGRASP localizes to the open ZOC, almost no co-localisation with Golgi

• Stage 10B dgrasp mRNA also localizes to the open ZOC

What does dGRASP do at the open ZOC?

13

Wild Type – Stage 10B

dgrasp100.2 – Stage 10B

dGRASP is important in maintaining follicular epithelium integrity

Figure 3

How are integrins deposited during the progression from stage 10A -> 10B

14

Integrin αPS1 Is deposited at the open ZOC in stage 10B

Figure 3

Is Integrin αPS1 also deposited at the open ZOC of dgrasp100.2 in stage 10B?

15Figure 4

Is Integrin αPS1 also deposited at the open ZOC of dgrasp100.2 in stage 10B?

16Figure 4

Is βPS also effected by dgrasp100.2?

17Figure 4

Recap• dGRASP mutant leads to disrupted follicular

epithelium• αPS1 is deposited at the open ZOC• αPS1 deposition at the open ZOC is dependent on

dGRASP• βPS and other adherens junction proteins were

dGRASP independent

• How does dGRASP enable αPS1 deposition?

Is αPS1 deposition Golgi dependent?

19Figure 6

Is αPS1 deposition dSyntaxin-5 dependent?

Figure 6

Conclusion

• In stage 10B dGRASP localizes to the open ZOC, almost no co-localisation with Golgi

• dGRASP mutant leads to disrupted follicular epithelium, by interfering with αPS1 deposition at the open ZOC

• dGRASP mediated αPS1 deposition is Golgi independent

21

Discussion

22

Is αPS1 newly synthesized at the open ZOC?

Figure 5

Discussion

23

Is αPS1 newly synthesized at the open ZOC?

Figure 5

The End

24