BIOL 3410: Cell Biology Fall 2021 Section 3.

Assignment 2 (34 points)

Question 1

To stimulate an immune response, a T lymphocyte must come into physical contact with an antigen-presenting cell. At the site of contact, the T-lymphocyte rearranges multiple proteins to form an immune synapse. Panels A and B (left) summarize FRAP experiments exploring the lateral mobility of the membrane-bound protein CD81. To do this, the authors tagged CD81 with the auto-fluorescent protein mCherry and used fluorescence microscopy to image the cells. Panel A (top row) shows T lymphocytes alone. The middle and bottom rows show T lymphocytes in contact with a antigen-presenting cell (not fluorescent, but annotated with an asterisk). The white circles show the regions bleached in the FRAP experiment (the green regions are unbleached control).

(a) What does FRAP stand for? (2 point)

(b) Describe CD81 fluorescence in pre-bleach unstimulated T lymphocytes (2 points).

(c) What happens to CD81 when the T lymphocyte is in contact with an antigen presenting cell? (2 points)

Panel B shows FRAP data on the above cells.

(d) After 200 seconds of recovery, what percentage of CD81 fluorescence is mobile in an unstimulated cell? (2 points)

(e) After 200 seconds of recovery, what percentage of CD81 fluorescence is mobile in the center of the cell-to-cell contact zone? (2 points)

(f) What do you think is happening to CD81 at the cell-to-cell contact zone? (4 points)

(g) What is the purpose of measuring fluorescence in the red circles (panels A)? (2 points)

Question 2.

CD81 is a membrane-associated protein required for B cell function in the immune system.

A. Using your favorite interweb search engine, find and download the human CD81 protein sequence and paste it below. (4 points)

B. Using the online tool EMBOSS (see URL below), copy/paste the above sequence into the submission window then hit “Submit”. (2 points)

https://www.ebi.ac.uk/Tools/seqstats/emboss_pepwindow/

C. Using your scientific spidey sense, label what you think are candidate transmembrane domains. Hint: take a look at the accompanying paper by Zimmerman et al. (2016) and use this to refine your answer. (4 points)

D. Are the N and C-termini of CD81 on the intracellular or extracellular face of the plasma membrane? (2 points)

E. Based on the CD81 crystal structure and the CD81 FRAP data from question 1, what is likely to be happening to CD81 at an immune synapse. Use one or more diagrams to illustrate your answer. (4 points).

F. CD81 can bind to small molecules. What’s its ligand? (2 points)