12 Jun BST5003 – Assignment 2 Cell and Molecular Biology
Question
Assignment 2
Deadline: 27th Sep 2016
Slot: G1
Course: Cell and Molecular Biology Code: BST5003
Analyze the Data: Actin Assembly
Understanding of actin filaments has been greatly facilitated by the ability of scientists to
purify actin and actin-binding proteins and the ability to assemble actin filaments in vitro.
Following are various experimental approaches designed to characterize actin assembly
and the effects of actin-binding proteins on actin assembly.
a.
The graph in part (a) of the figure depicts the actin polymerization rate at the plus
(+) and minus () ends of rabbit actin as a function of actin concentration. Assume that
you could add actin filaments of a predefined length to rabbit actin maintained at the
concentrations labeled A, B, and C in the figure. Diagram the appearance of the filaments
after a 10-minute incubation at each of the indicated actin concentrations, if the original
filaments are depicted as follows:
Originalfilament : __________________
Make sure to mark the location of the original (+) and () ends of the filament on your
diagrams.
(a)
b.
A novel actin-binding protein (X) is overexpressed in certain highly malignant
cancers. You wish to determine if protein X caps actin filaments at the (+) or () end.
You incubate an excess of protein X with various concentrations of G-actin under
conditions that induce polymerization. Control samples are incubated in the absence of
protein X. The results are shown in part (b) of the figure. How can you conclude from
these data that protein X binds to the (+) end of actin filaments? Design an experiment,
using myosin S1 fragments and electron microscopy, to corroborate the conclusion that
protein X binds to the (+) end. What results would you expect if this conclusion is
correct?
(b)
c.
An in vitro system was developed to study actin assembly and disassembly in
nonmuscle cells. In this study, tissue culture cells were incubated for several hours with
[35S]methionine so that all the actin monomers in each filament were labeled. Actin
filaments were then collected by differential centrifugation and put into a buffer
containing one of three different cytosolic extracts (A, B, or C). The amounts of soluble
actin in each sample were monitored over time (see part (c) of the figure). What do these
data indicate about the effects of A, B, and C on the assembly and disassembly of actin
filaments?
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