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Final

2009 EXAM ANSWER

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Department
Cell and Systems Biology
Course Code
CSB428H1
Professor
U.Tepass

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CSB428H1F FINAL EXAMINATION 2009 ANSWER KEY
#1: Langevin et al.
Central question/hypothesis
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?
Main conclusion
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.
Key experiment
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
Criticism/future direction
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
XMAP215.
#2: Chaung et al.
1
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Central question/hypothesis
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?
Main conclusion
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.
Key experiment
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.
Criticism/future direction
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
Central question/hypothesis
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?
Main conclusion
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
2
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Description
CSB428H1F FINAL EXAMINATION 2009 ANSWER KEY #1: Langevin et al. Central questionhypothesis What is the role of the exocyst complex, specifically Sec5, in delivery of DE-CadArm to the plasma membrane from the recycling endosome and how do they interact with RE components such as Rab11? Main conclusion 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. Key experiment sec5 mutant notum cells generated using FLPFRT 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 Criticismfuture direction 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 XMAP215. #2: Chaung et al. 1 www.notesolution.com
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