Major players involved
dGRASP-Mediated Noncanonical Integrin
Secretion
Is Required for Drosophila Epithelial
Remodeling
• Integrins – Integral plasma membrane αβ
heterodimers that mediate basal adhesion
to ECM components
• dGRASP
Hans Schotman,1 Leena Karhinen,1 and Catherine
Rabouille1,*
1The Cell Microscopy Centre, Department of Cell Biology, Institute of Biomembranes,
University Medical Centre Utrecht
February, 2008
Nelleke Spruijt
Sacha Dalhuijsen
Nick Sander
GRASP proteins
Aim
• Myristoylated peripheral cis-Golgi proteins
• Along with GM130 - implicated in
formation and maintenance of the Golgi –
also cell cycle checkpoints and spindle
dynamics
• Ortholog in Dictyostelium, GrhA, shown to
mediate secretion of AcbA by
unconventional pathway – completely
bypasses ER-Golgi-Plasma membrane
exocytic pathway
• What triggers the deposition of integrins to
the basal side?
• How are they transported?
Stages of Drosophila oocyte
development
10A – 10B Morphology / open
ZOC
Schematic representation – flattening of follicle cells
(Nature protocols, 2007)
Electron microscopy
α-Spectrin – plasma membrane marker - IF
http://dev.biologists.org/cgi/content-nw/full/128/16/3209/FIG1
Figure 1
dGRASP Localization in Follicle
Cells
What is the dGRASP phenotype?
10A
10B
> Disrupted structure of
follicle epithelial cells
Stage 10A: dGRASP localizes to the golgi
Stage 10B: dGRASP localizes to the open ZOC
Figure 1
Is the phenotype caused by a defect in
formation of junctions or adhesion?
Figure 3
Conclusions (1)
Stage 11
• Open ZOCs form at stage 10B
• dGRASP localizes to the open ZOC
• Epithelial integrity is disrupted in dGRASP
mutant
Adherin Junctions
Tricellular Septate Junctions
> No! The junctions still behave normal
Figure 3
Could a defect in integrins cause
the phenotype?
What is the localization of αPS1 in stage
10A and B?
> Same expression pattern as dGRASP
www.scq.ubc.ca/.../uploads/2006/08/integrins.gif
Figure 3
Does a mutation in dGRASP
disrupt αPS1 localization?
> αPS1 deposition is dGRASP-dependent
Does a mutation in dGRASP
disrupt αPS1 localization?
> αPS1 deposition is dGRASP-dependent
Figure 4 A,C
Is dGRASP involved in localization
of βPS?
Figure 4 B
Conclusions (2)
• βPS deposition is dGRASP independent
• dGRASP is required for the transport of
αPS1 to the open ZOC
> dGRASP is not involved in
βPS localization
• αPS1 in newly synthesized (fig. 5,
puromycine)
• Golgi-proteins in open ZOC
Figure 4 D,E
Is αPS1 transported to the Golgi?
Brefeldin A: Inhibits transport from ER to Golgi
– what is the role of the golgi in transport of
αPS1?
Is transport of αPS1 bypassing the
Golgi?
dSyntaxin5: t-SNARE essential
for transport to and through
the Golgi
Stage 10B
> αPS1 bypasses the
golgi at stage 10B
> Deposition of αPS1 is not Golgi-dependent
Figure 6 A
Figure 6 B, C
Conclusion
• βPS deposition is dGRASP independent
Schematic Model
dgos28; specific for
ER-to-Golgi
transport
• αPS1 transport to open ZOC at stage 10B
is:
– dGRASP dependent
– bypassing the Golgi
Figure 7
Some questions…
• Is αPS1 the only substrate?
• Why unconventional secrection?
• Why not also βPS?