Microbiology 204
Discussion Session #1 October 6, 2017
1-2:30PM CL220/223
Faculty leader: Cliff Lowell
Kayagaki et al, Liu et al. L-selectin mutations in neutrophil activation. Nat Commun, 8:15196, 2017
Everyone should read this paper before coming to class. You do not need to read the supplemental materials and supplemental figures, although the Discussion Leader may choose to have the class discuss one or a few supplemental figures.
See below if you have an assignment, in which case you have some additional preparation for class. Assignments rotate among the students.
REMEMBER: there are no “correct” answers to most of these questions. They are suppose to stimulate conversation.
LEARNING OBJECTIVES:
To learn about the leukocyte adhesion cascade and how L-selectin can serve as both an adhesive molecule and a signaling molecule, regulating both location and activation of inflammatory cells.
STUDENT ASSIGNMENTS
1. Discussion leader (see instruction page for advice on leading the discussion)
2. Review the leukocyte adhesion cascade, the types of selectins involved and what their ligands are. Find a picture for any number of review articles (some suggested in lecture) to outline the cascade.
3. Review the catch/slip bond transition of L-selectin. Data are mainly from ref #14 in the paper. Describe what the amino acid N138 does in L-selectin. This may require drawing a picture. A harder question: Where on the leukocyte body is L-selectin located (think about the surface of cells – they aren’t just round balls)? Does this subcellular localization affect the mechnochemistry of L-selectin?
4. Review Figures 1 a-I (panels a-c should be quick). Show how catch/slip bond transition is manifest in the WT cells and lost in the N138G mutant cells. These experiments are done with BM cells. Can you think of a limitation of using BM neutrophils? (fortunately, this is addressed in Sup Fig. 4)
5. Review Figure 2a-c. What do you think a consequence would be, in vivo, of forming all these neutrophil aggregates in the vasculature?
6. Review Figure2g-j. What is a Weibel-Palade body and what the heck are the authors talking about in this sentence?
7. Review Fig. 3. What does “priming” mean? Clearly there are all these changes in neutrophil surface proteins. Why is priming important? Supp Fig6 may seem counter-intuitive. This figure shows that Mac-1 (b2 integrin) mediated rolling and arrest is the same in WT versus N138G neutrophils. Yet Fig. 3b shows that N138G neutrophils express 3-fold higher levels of Mac-1. How can you reconcile these two observations?
8. Review Fig. 4. This is a very cool experiment! Why is the mixed chimera experiment so important?
9. Review Fig. 5. What is so weird about the body temp of mice DROPPING with sepsis? What do we humans do?
10. Review Fig 6. This is pretty easy and should be quick.
11. Review Fig 7. Why would the N138G mice have a larger IVC thrombus, but a lower number of Ly6G+ neutrophils per thrombus? Seems like they should have MORE neutrophils, not less. And why doesn’t the anti-L-selectin blocking mAb reduce the thrombus size in the WT mice? Shouldn’t it block neutrophil adhesion events?
12. This paper demonstrates that L-selectin signaling mediates neutrophil priming. But this is only demonstrated in the N138G mutant mice. Does that mean WT L-selectin also does this?
Student assignment #s
1. Camillia Azimi 10. Tara Mcintyre
2. Ian Boothby 11. Geil Merana
3. Casey Burnett 12. Andrew Ng (not here today)
4. Karen Chan 13. Ramiro Patino
5. Irene Chen 14. Anthony Venida
6. Antonia Gallman 15.
7. Kamir Hiam
8. Kristoffer Leon
9. Lauren Mchenry
(NOTE : if you will miss a discussion session, inform Dr. Lowell in advance; if assignments have already been made, you should additionally make a trade with one of your classmates who does not have an assignment that week so that your assignment is covered).