CSB428H1F FINAL EXAMINATION 2009 ANSWER KEY
#1: Langevin et al.
What is the role of the exocyst complex, specifically Sec5, in delivery of DE-Cad/Arm to the plasma
membrane from the recycling endosome and how do they interact with RE components such as Rab11?
Drosophila Sec5, Sec6 and Sec15 tethers DE-Cad vesicles originating from the RE to lateral
membrane for exocytosis. Sec5 also mediates transcytosis of DE-Cad. In addition, Arm interact with
Sec10 at PM and Arm act as a landmark for DE-Cad delivery. In the absence of Sec5, Sec6 and Sec15
function, DE-Cad accumulates in an enlarged RE.
sec5 mutant notum cells generated using FLP/FRT were analyzed for different intracellular markers to
investigate the nature of the enlarged intracellular compartment. Golgi marker Lava Lamp, late
endosome marker Hepatocyte growth factor-Regulated tyrosine kinase Substrate, early endosome
marker Rab5 was distributed normally in mutant cells. But Rab11 distribution was disrupted in mutant
cells: they were found in large punctate structures with higher intensity of Rab11 and colocalized with
DE-Cad in RE. Additional RE maker Nuclear Fallout confirmed this result. This shows Sec5 is
necessary for the recycling of DE-Cad from RE to the lateral membrane
Investigate possible interaction between components of the exocyst complex with the cell cytoskeleton.
Specifically, MT have been known to play a role in vesicular trafficking, and the authors could
investigate if disruption of MT also disrupts delivery of DE-Cad to lateral membrane and
establishment of AJs. MT could be disrupted by drugs such as nocodazole or knockdown of
#2: Chaung et al.
What is the role of the FYVE-containing protein SARA in vesicular trafficking of rhodopsin to the OS
of rods and how does this contribute to disc biogenesis in OS?
SARA interacts with PI3P that is abundant on nascent discs for tethering of rhodopsin-laden vesicles.
SARA also interacts with rhodopsin C’ tail and Syntaxin3, which interacts with the SNARE complex
on nascent discs during this process to mediate rhodopsin delivery.
A competition experiment was done since they hypothesized that FYVE domain of SARA binds PI3P.
Overexpression of FYVESARA-RFP or 3XFYVEEEA1-RFP caused mislocalization of h-rhodopsin to cell
body/synapses. It also reduced endogenous rhodopsin in OS of rods. As a control, FYVESARA-RFP or
3XFYVEEEA1-RFP also caused mislocalization of the rhodopsin surrogate Arrestin into cell
bodies/synapses in rods harvested in light. This showed SARA-PI3P interaction is necessary for
delivery of rhodopsin to the OS.
Investigate the role of MT structure axoneme in disc biogenesis. Try disrupting axoneme using a drug
to see if rhodopsin can still be delivered to OS. Also investigate the interaction of SARA with motor
proteins that are responsible for delivery of rhodopsin. What are the actual motors?
#3: Benton et al
What protein substrate is phosphorylated by PAR-1 that binds 14-3-3 and how does this contribute to
exclusion of apical proteins at the lateral side and establishment of lateral polarity?
PAR-1 phosphorylates Baz on two conserved serines to generate 14-3-3 binding sites. Binding of 14-3-
3 inhibits Baz self-oligomerization, binding to aPKC and formation of Par-6/aPKC complex at the
lateral side. At the same time, Crb-Sdt also recruits Baz to the apical side to further exclude it from the
lateral side and help establish lateral polarity.
The GAL4-UAS system was used to express mutant Baz in the follicular epithelium. BAZS151A, S1085A-
GFP and BAZS1085A-GFP was strongly mislocalized and sometimes exclusively found in lateral domain.
They also showed strong overlap with PAR-1 at lateral cortex and recruited aPKC and PAR-6 as well.
However, lateral marker Dlg and Crb-Sdt complex was normal. This showed that phosphorylation of
S151 and S1085, as well as the binding of 14-3-3 the Baz on these sites is necessary for the lateral
exclusion of Baz and prevention of PAR-6/aPKC complex formation at the lateral side.
The authors did not elaborate further as to why the S151 site seems to be less important than the S1805
or S1085 and S151 combined in terms of phosphorylation and 14-3-3 binding. For example, it is not
clear why BAZS151A, S1085A-GFP and BAZS1085A-GFP were more strongly mislocalized than BAZS151A-GFP.
Could this affect Baz binding to other proteins such as aPKC and PAR-6?
#4: Martin-Belmonte et al.
How does PI45P2 distribution affect the localization of apical complexes in MDCK cells and what is
the role of PTEN, Anx2, Cdc42 and aPKC in this pathway?
PTEN is localized to the apical domain and creates PI45P2 at the apical side, which then recruits
Annexin 2 via direct binding. Anx2 then binds Cdc42 and Cdc42 then bind aPKC, and aPKC help
establish apical polarity. The establishment of apical polarity through this pathway is central to lumen
formation in MDCK cysts and loss of any component of this pathway inhibits lumen formation.
In cysts depleted of Cdc42 using siRNA, central lumen formation was inhibited. Instead, multiple