Regulation of Contractile Forces within CellsAvi Kandel, Ryan Frei, Ken PrehodaDepartment 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

Myosin is Involved with Contractile Force in the Cell

Acknowledgements:I would like to thank the entire Prehoda lab especially my summer mentor Ryan Frei.

Methods

ConclusionsCoexpression 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.

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

Purifying Protein

Future Experiments

The location of the tail that binds the heavy chain neck region can be determined by using a process of elimination of successful or un successful binding.

Results

RLC and questionably ELC coexpressed with the histidine tagged zipper neck. The zipper neck is confirmed by the bottom bands around 13 kD and the Flag tagged RLC is confirmed by the western blot. The proteins also appeared on the elutions that correspond to a protein complex that is around 50kD in size, the size of the three protein complex.

NM II shifts Conformation when Inhibited

Phosphorylation of the inhibited conformationcauses a conformational change that leads tounfolding into the active form of myosin. Ourcurrent 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

RLC and questionably ELC coexpressed with the histidine tagged zipper neck. The zipper neck is confirmed by the bottom bands around 13 kD and the Flag tagged RLC is confirmed by the western blot. The proteins also appeared on the elutions that correspond to a protein complex that is around 50kD in size, the size of the three protein complex.