Single Molecule Study On Motion And Mechanism Of Kinesin Motors

With the complication functions of the living cell,various transportation systems and organelles have been formed to take charge of the intracellular substances transportation in cell.This transportation is closely related to cytoskeleton?microtubules and microfilaments?,motor proteins and so on.One of the most important and basic mechanisms is the movement of motor proteins:they move on the appropriate substrate surface to transport various substances and organelles in cell.Motor proteins are divided into microtubule motor proteins and microfilament motor proteins?myosin?.Microtubule motor proteins include Kinesin and Dynein.Kinesin interact with the microtubules by utilizing adenosine triphosphate?ATP?hydrolysis to provide energy to accomplish the transportation of various intracellular.As important molecule motors in organisms,motor protein such asproteins play very important physiological functions in various physiological processes.For example,the contraction of muscle is completed by the energy from the coarse myosin filament and the fine actin filament.At present,the study of kinematics mechanism of motor proteins such as kinesins is one of the hot topices in intracellular movement.Single-molecule technology is one of the most basic techniques for studying organisms.Compared with traditional physiological research methods,single molecule technology has many significant advantages.Such as,the directly observation of the behavior,trajectory and dynamic characteristics of a single molecule,alsothe can discovery of deeper scientific information that is hidden by the average effect.Single-molecule total internal reflection fluorescence microscopy?SMTIRF?is one of the most widely important techniques in studying single-molecule motion behavior and mechanism both in vivo and in vitro.In this thesis,the kinematics characteristics and mechanism of three different kineisn proteins were studied by using SMTIRF,which combined with molecular cloning,point mutation,single-molecule tracking and single-molecule photobleaching.The main research contents include the following three parts:In the first part we studied Os KCH₂,which is a member of the kinesin-14 family from O.sativa with a special CH domain.We revealed the different states of motion on microtubules and various functions and roles of differenet domains in different motion.?1?Firstly,we validated Os KCH₂ was the first kineisn that replaced the function of dynein in higher plants.It is because Os KCH₂ has a special direction of movement on microtubules.?2?We also showed Os KCH₂ had similar physiological functions as Os KCH₁?binding microtubules and microfilaments at the same time?,and drove the microfilaments to the minus-end of microtubules.?3?Then we found the two helical domains of Os KCH₂?CC1 and CC2?had important functions.CC1 domain determines the aggregation of Os KCH₂,while the CC2 domain is the main determinant for the processive movement of Os KCH₂.These studies provide most favorable evidences and experimental supportings for the existence of kinesin proteins with special direction of motion,and lay a basis foundation for the future research of these kinesin motors.Second part:Klp A in Aspergillus Niger is belonged to kineisn-14 family has the ability to move processively to the plus-end of the microtubules by single homodimers.However,the aggregations of Klp A moved towards the minus-end of the microtubules at the same time.Why is Klp A so special?In order to understand the kinetics of this protein and to explain why Ncd?from Drosophila melanogaster?from kinesin-14 family cannot move to the minus-end of the microtubules.We studieddid researchs on them carefully.and got the results,they showed:?1?when a soft amino acid link?-3x GS?was inserted into the helical domains of Klp A and Ncd,it completely changed the movement direction of Klp A?from plus to minus?and Ncd?from non-processive to processive?.?2?we also proved that the insertion released the tail domain of Klp A and Ncd.?3?Functionally,the insertion weakened the ability of mutants?Klp A-3x GS,Ncd-3x GS?to bind different microtubules.These results not only dchange the traditional understanding of helical domains,but also greatly enhancedenhance the current thinking and assumptions on the direction change of Kinesin.This provide a firm basis for us to further study about the theoretical mechanism of the kinesin motors motors.It also provides reliable experimental supports and theoretical basis for completing totally control of the kinesin orientation.The last part:we studied about the kinetic characteristics of an orphan kinesin KINID1b from moss Physcomitrella.We found that:?1?the motor domain of KINID1b was a plus-end directed kinesin motor domain;?2?the helical domain of KINID1b was an additional microtubule binding domain and promotes the crosslinking with different microtubules;?3?KINID1b was an autoinhibition orphan kinesin?the motor domain interact with the tail domain?which makes the full-length of KINID1b impossible to move on microtubules.The results also showe that KINID1b is the first autoinhibaition orphan kinesin that cross-linking different microtubules.This makes an outstanding contribution to understand the characteristics of orphan kinesin in the future,also fully grasp the functions of orphan kinesin in plants and provide some theoretical support for the future study of pharmacology of orphan kinesin proteins.In this thesis,TIRF technology was mainly used to study the kinetics of kinesins.Firstly,we revealed the dynamic characteristics of Os KCH₂ in higher plants.Then we expanded the reasons for the directional movement of the kinesin-14 family,and found the key factors for how to change and control the directional movement of Klp A and Ncd.Finally,we h revealed the characteristics of autoinhibition of KINID1b kinesin and foudfound the reasons for crosslinking and sliding different microtubules.These results laid solid foundations for studying the kinetics and mechanism of kinesin in vitro,offered new possibilities for the study of kinesin family in flowering plants,and provided a new understanding of kinematics of orphan kinesin.It also idlay a solid foundation for comprehensively understanding and mastering the dynamic characteristics of kinesins,and the in-depth study of the mechanism and function of kinesins.These results provide important theoretical basis for studying the biological activities and the mechanism of human diseases in the future.