Guidelines and assignments for Mol Biol class on Dec 15, 2015
Correspondence: Dr. Shainn-Wei Wang & Dr. Kung-Chia Young
Reading material: Mitochondria DNA stress primes the antiviral innate immune
response. A. P. West, et al., Nature. 2015 Apr 23;520(7548):553-7. doi:
10.1038/nature14156. Epub 2015 Feb 2.
Instructions:
1. Grouping: IMM and MT students are divided into 3 groups each (i.e., IMM1,
IMM2, IMM3, MT1, MT2, and MT3)
2. Prepare ppt slides (the whole group, each slides with names of the contributors)
for no more than 20 min presentation time; turn in by e-mail to Dr. Wang in IMM
(swwang@mail.ncku.edu.tw) and Dr. Young in DMLSB (t7908077@mail.ncku.edu.tw)
no later than 4 pm on Monday (Dec. 14 )
3. Problem based learning (PBL) for each group: Each group needs to search more
information from the PUBMED to clarify the assigned problems/topics in the list
below. Please simplify complicated background with cartoons and some text
explanations. If possible, each member in the group needs to present a portion of
the prepared materials.
Background (20 min/each group):
MT1: (You need to answer Q1 and Q2 after briefly summarizing the abstract section)
Q1: What is the role of mitochondria to general cell metabolism?
Q2: What is the role of TFAM to mitochondria and the possible outcomes of mtDNA
stress to cells?
IMM1: (You need to answer Q3 and Q4 after briefly summarizing the abstract
section)
Q3: How would viral infection affect mitochondria function?
Q4: How would mitochondria affect the cytosolic antiviral signaling in addition to the
mtDNA stress mentioned in the paper?
Materials/Methods and strategy: (20 min/each group).
MT2
Q5: What is nucleoids?
Q6: What is oxidative phosphorylation in mitochondria
Q7: Why the authors studied the cellular responses to mtDNA stress in the absence
of oxidative phosphorylation deficiency by using TFAM heterozygous knockout
(Tfam+/-) mouse model?
Q8: Try your best to explain the animal model used in this study, including the
Tfam+/- and Tfamfl/fl mice, as well as the knockout strategy using Estrogen
receptor(ER)-Cre transgenic mice.
Q9: Try your best to introduce Oxygen consumption analysis.
IMM2:
Q10: Briefly introduce all the viruses used in the study
Q11: Describe how to perform in vivo HSV-1 infection, dorsal root ganglia isolation
and viral titration
Q12: What is cGAS-HA and UL12 M185? Describe how the authors use retroviral
vectors to express cGAS-HA and UL12 M185.
Q13: Describe how authors perform immunofluorescence microscopy and how they
quantify nucleoids.
Q14: Describe how the authors perform nuclear fractionation and how they detect
mtDNA in cytosolic extracts.
Results and Discussions: (20 min/each group)
MT3 (presenting Fig. 1, 2 & 3 and focused on the following questions):
Q15: How did authors define mtDNA stress and perform the microarray analysis (Fig.
1)?
Q16: What is ISGs and poly (I:C)? Why did the authors analyze ISG expression after
poly(I:C) stimulation of the MEF lines and what is the importance of increased
expression of cytoplasmic RNA and DNA sensors? (Fig.1)
Q17: What is the advantage and disadvantage of using inducible TFAM depletion
(TFD) model to support the authors’ results on (Tfam+/-) mouse model? Describe
their method and critical data from the TFD model. (Extended figs, selective data will
be fine)
Q18: Why the authors assayed for extra-mitocondria mtDNA in TFD cells? Briefly
describe the possible implications of mitochondria stress to the response of NLRP3
inflammasosome (Extended fig 2).
Q19: Why the authors investigated the cytosolic DNA sensor cGAS and downstream
STING-associated pathway in mtDNA stress signaling? What was their findings (Fig 2)
Q20: Why the authors used Herpes simplex virus 1 to demonstrate the significance of
mtDNA stress-induced antiviral priming? What evidence the authors had to indicate
that HSV-1 induced mtDNA stress? (Fig. 3)
IMM3 (presenting Fig. 3 and 4, and focused on the following questions)
Q21: What is the evidence and implications that the mtDNA stress potentiates viral
resistance (Figs. 3 and 4)
Q22. How did the authors distinguish that the antiviral response was stemmed from
mitochondria but not just merely due to the sensing of viral DNA/RNA form other
cytosolic DNA/RNA sensor? (Figs.3 and 4, discussion and the comparison of WT and
Tfam+/-)
Q23: What is ddC? How ddC diminished mtDNA stress? Why the application of ddC
to the MEF cells is an effective way to probe a direct requirement for mtDNA stress in
antiviral priming in Tfam-deficient cells? (Fig 3 and extended Fig. 5)
Q24: What are the arguments and the evidence of nucleoid stress the authors had to
support that the virus-induced mtDNA stress may boost host antiviral response? (Figs
4 and 6 plus extended Fig 5)
Q25. Does induction of mtDNA stress and TFAM depletion a general consequence of
viral infection?(Fig. 4 and extended Fig. 6)
Q26. Why the authors sought to determine that HSV-1-induced mtDNA dysregulation
is necessary to fully engage antiviral signaling? What evidence they provided? (Fiig. 4
and extended Fig. 7)
Final comments: What have you learned from the paper and your study group?
MT1: One to two conclusion slides.
IMM1: One to two conclusion slides.