Bio 362 Human Genetics Spring 2008
Polycystic kidney disease study sheet for March 11th
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Background reading:
What is the anatomy of a kidney?
How does a nephron work?
Which cells in the nephron contain the primary cilia?

Hildebrandt and Otto, 2005:
What is the cilia hypothesis?
What are primary cilia, and how are they different from motile cilia?
What does the two-hit hypothesis have to do with autosomal dominant PKD?
Thought question with no obvious answer: why doesn't the two-hit hypothesis apply for the genes associated with recessive cystic kidney disease?
Skim the portion of this paper that describes all the types of cystic kidney disease and their associated genes. Then focus more on the pathogenic mechanisms sections. What's the common thread among the genes associated with cystic kidney disease?
Explain the different hypotheses concerning the pathogenic mechanisms for cystic kidney disease. Are these hypotheses mutually exclusive?
What is the mechanosensory hypothesis, and what sorts of experiments could be done to test it?
What data do the authors say are hard to reconcile with the mechanosensory hypothesis? Do you agree?
Look up focal adhesions and adherens junctions in table 3.4 on p. 69 of the Strachan textbook. Look at Figure 1 in the Vasioukhin and Fuchs paper in the supplementary folder for this week on Louise. This figure depicts the proteins in a typical adherens junction, on top of which additional tissue-specific proteins (as in the kidney) exist. Note that the beta catenin protein in the adherens junction plays not only a structural role but also a signalling role in the "Wnt" pathway mentioned in the Hildebrandt and Otto paper.
What other tissues are affected in some syndromes that include cystic kidney disease? Why are these other tissues affected?
What is situs inversus, and what does left-right asymmetry have to do with cilia? Glance at the remaining papers in the reference folder on Louise to get a sense of the mechanistic link.
What is the prognosis for treatments of cystic kidney disease?

Delmas, 2004:
What phenomenon is the author trying to explain?
On the second page, the author asserts that PKD2 is held in a high-energy closed state that relaxes into the open state upon stimulation, but that certain data are at odds with that idea. What are those data, and do you agree with the original high-energy closed state idea?
What experiment with antibodies provides insight into the role of PKD1? (see Fig.1)
Using the models drawn in Figure 2 as your basis, speculate on how PKD1 and PKD2 might interact upon bending of cilia or stress on the junctions between cells.
What role might the cytoskeleton play in mechanosensory gating?
What is the conclusion of the paper? Is the polycystin complex the mechanosensory gate?
Is this paper well-written?

Bhunia et al, 2002:
What cell types are used in FIg 1A? How are they different from the cells in part B? Make a chart for yourself to keep track of which cell lines express which form of PKD1, and whether the expression is permanent or only transient after transfection.
What common theme is demonstrated by the FACS traces for different cells types in 1A and 1B? (How can FACS analysis sort cells by cell cycle stage?)
In Figure 2, sections A and B represent Western blots, but how is the source of the protein different?
In 2A, what was done differently to show "activity" versus "protein" for each CDK?
In 2A, why are the p21 panels different-- aren't the samples from the same cell lines? What do these panels show regarding the mechanism of PKD1-induced cell cycle arrest?
In Figure 2C, how is the role of p21 further confirmed?
Are p21 levels (in response to PKD1) induced at the transcriptional level? What transcription factor is involved? (Figure 2D)
In Figure 3A, what do we presume the IRF-1 Luc represents (i.e. what do you infer the promoter is)?
Does PKD1 expression affect phosphorylation levels of STAT1? (Figure 3B)
In Figure 3C, the "hSIE" probe is an oligonucleotide to which STAT proteins can bind. The mobility of this oligonucleotide is different depending on whether there are actually any STAT proteins bound to it. Focus on lanes 7 and 8. What happens when excess PKD1 is present? What are the controls in lanes 9 and 10?
Is STAT1 crucial for p21 induction? Focus on Figure 4A and C.
So, p21 is turned on by STAT1, but what phosphorylates STAT1 to allow it to be active? PKD1 and PKD2 don't have kinase activity. JAK proteins typically turn on STAT transcription factors. What did the researchers do to see if JAK1 or JAK2 is involved?
In Figure 5A, anti-FLAG binds the transfected (and epitope tagged) PKD protein, and anti-HA binds the transfected, epitope tagged JAK2 protein. What was immunoprecipitated in each panel? What was detected within each clump of immunoprecipitate? What is required for JAK2 to be phosphorylated?
Figure 5B: does PKD1 associate with JAK1?
In Figure 6, part A shows you that the cell line CB33 is naturally low in PKD2, so that it is a useful cell line for part B. In part B, what must be true for all the events leading to p21 induction (and cell cycle arrest) to occur?
Why show the experiments in Figure 7?

Chauvet et al., 2004 (questions from Cecie and Chris):

Figure 1
What do the different colors represent?
What is the difference between the wild-type and transgenic cells? What are the implications of this?
Why might there be no visible localization in wildtype cells?
Figure 2
Generally, how does figure 2 differ from figure 1?
What can we determine from comparing section C to F?
Figure 3
How could the researchers identify the different sections of the polycystin-1 protein in the cells?
What do sections E through I tell us about the localizaiton of the different sections of the pc-1 protein?
Figure 4
How do the p200 and p200Δ constructs localize in the cells?
What is the AP-1 pathway and how do the researchers measure its activity? What effects on the AP-1 pathway do the researchers see with the different constructs?
Figure 5
How does this data help us determine a role for polycystin-2?
Figure 6
What is UUO? 
What is Mex-46, and what does it bind to? 
What does it mean that there was no nuclear staining seen in panels G-I? 
Figure 7
What does this data demonstrate about the C terminus of polycystine-1?