13 Jun BIO 350 What do the cells look like immediately after fusion
Question
BIOLOGY 350 – Section 1
CELL BIOLOGY
PROBLEM SET #1
(25 points)
Coworkers: ________________
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READ CAREFULLY!! This problem set is intended to help you determine your level of understanding of the material
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the problem set.
Question 1: [6 pts]
A rat cell and a chimpanzee cell are fused together. Antibodies to chimpanzee membrane anti gens are l abe l ed
with rhodamine (a red fluorescent molecule) and antibodies against rat antigens are labeled with fl uore sce in (a
green fluorescent molecule).
A. [2pts] What do the cells look like immediately after fusion? Explain and draw.
B. [2pts] What about 40 minutes later?
C. [2pts] If the temperature of the cell is increased, what should happen?
Question 2: [9 pts]
Two membrane preparations have been derived from the plasma membrane of erythrocytes and are present i n a
buffer solution: (1) right-side-out membrane vesicles, in which the exoplasmic leaflet of the vesicle faces the
buffer (equivalent in orientation to that of the exoplasmic leaflet of the intact plasma membrane); and (2) insi de out membrane vesicles in which the exoplasmic leaflet faces the interior of the vesicles.
A. [3pts] Annexin V is a protein that binds to phosphatidylserine, one of the phospho lipids present in cell
membranes. Fluorescently labeled annexin V is mixed with each vesicle preparation. The vesicles are washe d to
remove unbound annexin V and then examined by fluorescence microscopy. Presence of annexin V is detected by
green fluorescence, as shown above. What can you deduce about the precise location of phosphatidylserine on
the membrane these data? Explain
B. [2pts] You buy a compound that is known to recognize the caboxy terminus of the protein Erythrin, an integral
membrane protein of the erythrocyte plasma membrane. Mixing this compound with two membrane vesicle
preparations described above generates data similar to the one you saw for Annexin. What can you deduce about
the structure of Erythrin from these data? (Where is the C terminal in the normal cell?)
The two vesicle fractions are either untreated (–) or are incubated with the protease trypsin (+). Trypsin is an
enzyme that can cut proteins into aminoacids, but cannot cross membranes. After treatme nt with trypsin, the
membrane vesicles are washed to remove any lose aminoacid/peptide and the remaining proteins are re move d
from the membrane using detergents. You then run the proteins in a gel (SDS gel electrophoresis) and l abe l the
protein glycophorin A, an erythrocyte plasma membrane protein.
The following data are obtained: (ROV= Right Side out); The + or – indicate the presence or absence of trypsin.
To better understand this plot, watch this video about SDS gel electrophoresis.
(https://www.youtube.com/watch?v=3CrzY7jb9fQ )
(Remember that smaller peptides will migrate more, and are thus located towards the bottom. The thi ckne ss of
the bar and intensity indicate the amount of protein/peptides present.)
C. [4pts] What can be deduced from these data about the structure of glycophorin A? Explain
Question 3: [10 pts]
properties of a lipid bilayer are determined by the structures of its lipid molecules. Imagine that you modi fy your
membrane of study in the various ways listed below. For each case, decide whether the new membrane would be
more fluid, less fluid, or the same fluidity as a normal membrane. Provide a brief explanation for each answer.
A. [2 pts] The hydrocarbon chains were shorter than normal, say about 10 carbon atoms long.
B. [2 pts] All of the hydrocarbon chains were saturated.
C. [2 pts] All of the hydrocarbon chains were unsaturated.
D.[2 pts] Each lipid molecule were covalently linked through the end carbon atom of one of its hydrocarbon
chains to a lipid molecule in the opposite monolayer.
E. [2 pts] The bilayer contained a mixture of two kinds of lipid molecule, one with two saturated hydrocarbon tails
and the other with two unsaturated hydrocarbon tails.
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