Transcript PowerPoint Presentation - University of Oregon (SPUR)

Regulation of Contractile Forces within Cells
Avi Kandel, Ryan Frei, Ken Prehoda
Department of Chemistry, Institute of Molecular Biology, University of Oregon, Eugene, OR, United States of America, 97403
Abstract
Myosin is a motor protein that has many different functions, including muscle contraction, cell
transport, and cell adhesion. There are of course several different subtypes and isoforms of
myosin proteins so the project will focus on a single type of myosin, non-muscle myosin II B (NM II
B) in Drosophila. Many of the cell functions that require mechanical force rely on myosin to
provide the force. To do this, myosin forms filaments that connect with polarized actin filaments.
The myosin filaments then shrink to pull the actin filaments together to cause tension in the actin
filaments. Myosin is made of heavy chains and light chains. There are two identical heavy chains
that each contain an N-terminal globular head region, neck region, and a C-terminal long α-helical
tail region. There are also two types of light chains, the regulatory light chain (RLC) and essential
light chain (ELC). The phosphorylation state of the RLC regulates whether myosin is in an active or
inactive position. In myosin’s inactive position, one head region folds onto the other and it is
believed that part of the tail region binds to the neck region. In this project we will determine
where exactly the neck region binds to the tail region. Different lengths of the tail region will be
expressed in order to systematically narrow down the location of binding on the primary structure
of the tail region. The information gained from this experiment will help us better understand the
regulation of NM II B.
NM II Mediated Contraction is
Essential for Life
NM II shifts Conformation
when Inhibited
Methods
Made three gene coexpression insert imbedded with ribosmal binding
sites. The genes were put together by using a three step overlapping
PCR. The proteins were purified by the histidine tag from the pBH
plasmid. The Ni bead purification method was used to isolate tagged
protein.
Making Expression Vector
Phosphorylation of the inhibited conformation
causes a conformational change that leads to
unfolding into the active form of myosin. Our
current model hypothesizes that it is
phosphorylation of the regulatory light
chain that causes the shift to the active
conformation.
Coexpression Method must
be Used to Study Regulation
of NM II
Purifying Protein
Conclusions
Myosin is Involved with
Contractile Force in the Cell
Results
Coexpression of three NM II genes is possible. Not only were all three
proteins expressed but they were successfully formed the semi-native
complex. This means this coexpression method can be utilized for several
future experiments that will assay the regulation of NM II including
locating self-inhibitory sites and the role of the phosphorylation of RLC
on NM II regulation.
Future Experiments
Acknowledgements:
I would like to thank the entire Prehoda lab especially my
summer mentor Ryan Frei.
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