Single-Molecule Analysis of a Novel Kinesin Motor Protein Jeremy Meinke Advisor: Dr. Weihong Qiu Oregon State University, Department of Physics June 7, 2017 Abstract Kinesins are intracellular motor proteins that transform chemical energy into mechanical energy through ATP hydrolysis to move along microtubules. Kinesin roles can vary among transportation, regulation, and spindle alignment within most cells. Many kinesin have been found to move towards the plus end of microtubules at a steady velocity. For this experiment, we investigate BimC - a kinesin-5 as- sociated with mitotic spindle regulation - under high salt conditions. Using single molecule imaging with Total Internal Reflection Fluorescence Microscopy, we found BimC to be directed towards the minus end of microtubules at a high velocity of 597±214 nm/s (mean±S.D, n=124). BimC then joins the few other kinesin found so far to be minus-end-directed. However, preliminary results at low salt condi- tions suggest that BimC switches towards the plus end. BimC could very well be an early example of a kinesin motor protein that is directionally-dependent upon ionic strength. These results suggest multiple branches of further investigation into directionally-dependent kinesin proteins and what purposes they might have. Contents 1 Introduction 1 1.1 Kinesin Proteins . 1 1.2 Total Internal Reflection Fluorescence Microscopy . 3 1.3 Protein Motion Analysis . 3 2 Methods 5 2.1 Design, Expression, and Purification of BimC . 5 2.2 Single Molecule Imaging . 7 2.3 Analysis of Data/Results . 7 3 Results 9 3.1 Single Molecule Imaging of BimC . 9 3.2 Directionality of BimC in High Salt Conditions . 10 3.3 Velocity of BimC in High Salt Conditions . 12 3.4 Preliminary Low Salt Observations . 13 4 Discussion 14 5 Conclusion 16 6 Acknowledgments 17 7 Appendices 18 7.1 Appendix A: Protein Purification Buffers . 18 7.2 Appendix B: TIRF Buffers for High-Salt Analysis . 19 8 References 20 List of Figures 1.1 Kinesin Protein Schematic . 2 1.2 Single-Molecule Imaging Visualization . 3 2.1 BimC Protein Creation Flowchart . 5 2.2 BimC DNA Plasmid . 6 3.1 BimC Video Snapshots . 9 3.2 Kymograph of BimC on a Microtubule . 10 3.3 BimC Moves to and Stays at Microtubule Minus End . 11 3.4 BimC Velocity Histogram . 12 3.5 BimC Low Salt Microtubule Gliding . 13 4.1 Cin8 Kymograph Article Excerpt . 14 1. Introduction Intracellular motor proteins offer a glance into the inner mechanics of cells. They are commonly responsible for the movement and transportation of various cellular compo- nents, such as vesicles, chromosomes, and spindle structures [1]. Many motor proteins move along surfaces via ATP hydrolysis, a process that converts chemical energy into me- chanical. Of these proteins, most are grouped into three separate classifications: myosin, dynein, or kinesin. Different from the other proteins, myosin travels along actin micro-filaments and is often associated with the function/contraction of muscle cells. In comparison, the largest motor protein dynein moves across structures composed of alternating α and β tubulin, referred to as microtubules (MTs). Such MTs have minus and plus ends that correspond with which uncovered tubulin (α and β, respectively) is present on that side. MTs are asymmetric in nature and grow faster in the plus end direction. The complex dynein proteins are responsible for positioning organelles, chromosomes, and other structures within cells while commonly moving to the minus end of MTs. The specific roles of many individual motor proteins are still undetermined, but there are a diverse number that have been investigated. Some motor protein deficiencies have been linked to diseases or genetic syndromes in both plants and animals [2,3]. This thesis is focused on characterizing the motility of a protein-based molecular motor called BimC using single-molecule imaging. The goal is to determine how ionic strength affects the motility of BimC. 1.1 Kinesin Proteins Background Found inside individual cells of both plants and animals, kinesin proteins are one of the three categories of motor protein. Both kinesin and dynein utilize ATP hydrolysis to move along the train-track-like MTs. Kinesins though vary from dynein by both size (kinesin is much smaller and simpler), and direction of movement. Dynein is not present in land plants, which provokes further research into how kinesin motor proteins maintain the required cellular function without dynein. A key area of study for plant kinesins involves those that support plants during mitosis, specifically associated with the mitotic spindle and cell wall. Various kinesin motors have been shown to be active at these times, helping to transport wall material or maintain the structure of spindle MTs [4]. 1 Structure Kinesin motor proteins are made up of a specific sequence of amino acids divided into four distinct domains. These domains are the motor, neck linker, coil, and tail. The motor domain is the tightly bound portion of the protein that binds and releases to the MT via ATP hydrolysis to induce periodic movement. The neck linker connects the motor with the coil domain, and propagates tension as the protein moves. The coil domain is an alpha-helical portion of amino acids between the neck linker and tail. It contains at least one dimerization region that allows it to bind/connect with other proteins. The tail domain is thus at the end of the protein structure, and often responsible for other binding sites needed for transportation of the cellular cargo. The corresponding DNA sequence used to produce the amino acids creates one of each domain, ergo, a motor connected with a neck linker, coils, and then tail. Kinesin proteins are sometimes considered homo-dimers, where the dimerization re- gion of the coil domain connects to the coil domain of another of the same protein. This creates a wrapped coil-coil region with two motors, necks, and tails as depicted in Figure 1.1. Another common form of kinesin is a homo-tetramer, where two dimer constructs combine due to another binding site near the coil/tail domains. This creates a protein with two motor domains on either side, allowing for possible connection to two separate MTs [5]. (a) Kinesin homo-dimer schematic (b) Kinesin tetramer schematic Figure 1.1: Model of a generic kinesin motor protein, homo-dimer (a) such that it has two motor domains, connected by their neck-linkers and coil domains. For a natural homo-tetramer like BimC, another two motor domains exist opposite of the others, as in Figure (b). The BimC Motor Protein Although kinesin proteins have a variety of roles, our lab analyzes kinesin motor proteins primarily in plants, involved in the transportation and regulation of individual cells. These proteins are key in cellular function, and our study enables us to better understand the complexity of mitotic functions. The kinesin-5 family of proteins, which includes BimC, commonly establishes and maintains the spindle formation during mitosis. Found in the Aspergillus nidulans fungi, 2 BimC has been shown to bind to two separate MTs in order to balance parallel spindle MTs, maintaining important alignment throughout the process of mitosis [6]. As a natural tetramer, BimC's structure consists of four independent motor domains, two on each end which are used to \walk" along the MTs. Of the same kinesin-5 family, Cin8 is a natural tetramer that has been observed to exhibit bidirectional movement. Results issued showed that Cin8 directionality de- pended upon the number of MTs the proteins were on, while another case related the bi-directionality to possible ionic dependence [3,7]. 1.2 Total Internal Reflection Fluorescence Microscopy Total Internal Reflection Fluorescence (TIRF) Microscopy involves the use of a micro- scope to see matter on the surface of a sample slide with high sensitivity (S/N). This is done through total internal reflection, in which a beam of light is directed at the sample at an angle greater than the critical angle. This creates an evanescent wave that decays exponentially through the sample. The angle and intensity of the initial beam is adjusted such that the evanescent wave has enough energy to excite just the fluorophores near the cover-slip surface. Figure 1.2: Visualization of the MT being held in place on the cover slide, with BimC homo-dimer proteins binding and moving along the MT. Polyethylene glycol (PEG) is placed on the cover-slip followed by biotin, then streptavidin. These bind to and secure MTs, which allow accurate movement measurements of protein once added. *Edited ver- sion of an image by Allison Gicking, graduate student with the Qiu lab. For the analysis of kinesin proteins along MTs, a green fluorescent protein (GFP) is added to the protein. Another fluorescent marker is placed on MTs, with a higher concentration added to the plus end in order to mark the polarity of the MT. The TIRF microscopy depicted in Figure 1.2 excites the fluorophores and allows them to be analyzed over time. 1.3 Protein Motion Analysis In the analysis of a kinesin protein, there are a few key traits that need to be found. The two main ones are directionality and processivity, which are strong indicators of a highly functional motor protein. Directionality can be determined by an analysis of what 3 direction the protein moves along a MT. Although most kinesin travel to the plus end, there have been some that move at high speeds in the minus end direction. Processivity pertains to a few different characteristics including velocity, run length, and how diffusive the protein is (how long it is continually attached to a MT). A highly processive kinesin would be found with a high velocity and run length along a MT.