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- Persson, Malin
(författare)
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Characterization and optimization of the in vitro motility assay for fundamental studies of myosin II
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Myosin II is the molecular motor responsible for muscle contraction. It transforms the chemical energy in ATP into mechanical work while interacting with actin filaments in so called cross-bridge cycles. Myosin II or its proteolytic fragments e.g., heavy meromyosin (HMM) can be adsorbed to moderately hydrophobic surfaces in vitro, while maintaining their ability to translocate actin filaments. This enables observation of myosin-induced actin filament sliding in a microscope. This “in vitro motility assay” (IVMA) is readily used in fundamental studies of actomyosin, including studies of muscle contraction. The degree of correlation of the myosin II function in the IVMA with its function in muscle depends on how the myosin molecules are arranged on the surface. Therefore a multi-technique approach, including total internal reflection spectroscopy, fluorescence interference contrast microscopy and quartz crystal microbalance with dissipation, was applied to characterize the HMM surface configurations. Several configurations with varying distributions were identified depending on the surface property. The most favorable HMM configurations for actin binding were observed on moderately hydrophobic surfaces.The effects on actomyosin function of different cargo sizes and amount of cargo loaded on an actin filament were also investigated. No difference in sliding velocities could be observed, independent of cargo size indicating that diffusional processive runs of myosin II along an actin filament are not crucial for actomyosin function in muscle. Furthermore, a tool for accurate velocity measurements appropriate for IVMAs at low [MgATP] was developed by utilizing the actin filament capping protein CapZ. These improvements allowed an investigation of the [MgATP]-velocity relationship to study possible processivity in fast skeletal muscle myosin II. It is shown that the [MgATP]–velocity relationship is well described by a Michaelis-Menten hyperbola. In addition, statistical cross-bridge modeling showed that the experimental results are in good agreement with recent findings of actomyosin cross-bridge properties, e.g., non-linear cross-bridge elasticity. However, no effect of inter-head cooperativity could be observed. In conclusion, the described results have contributed to in-depth understanding of the actomyosin cross-bridge cycle in muscle contraction.
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| 2. |
- Rahman, Mohammad A.
(författare)
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Biophysical studies of the actin-myosin motor system and applications in nanoscience
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The actin-myosin motor system plays important roles in cellular processes. In addition, actin and myosin have been used for developments towards nanotechnological applications in recent years. Therefore, fundamental biophysical studies of actin and myosin and the actomyosin force generating cycle are important both in biology and for nanotechnology where the latter applications require methodological insights for optimization. This dual goal is central in the present thesis with major focus on factors that control the function (e.g. velocity) and the effectiveness of transport of filaments (e.g. filament flexural rigidity) through nanoscale channels with supplementation of methodological insights. The thesis thus provides evidence that actin is a dynamic filament whose flexural rigidity is different at different MgATP concentrations as well as in the presence or absence of myosin binding. Furthermore, probing the myosin ATPase cycle with the myosin inhibitor blebbistatin revealed that velocity is easily modified by this drug. Our detailed studies also suggest that actin-myosin force generation is preceded by Pi release and that blebbistatin changes the rate limiting transition in the cycle from the attachment step to a step between weakly attached states. The studies of actin dynamics and of the actomyosin force generating cycle were largely performed using in vitro motility assay (IVMA) where surface adsorbed myosin motor or its proteolytic fragments propel fluorescently labeled actin filaments. The IVMA is often taken as the basis for developments towards different nanotechnological applications. However, in the IVMA, actomyosin motility is often negatively affected by the presence of “dead”, non-functional myosin heads. Therefore, in this thesis, two popular methods, that are often used to remove dead myosin heads, are analyzed and compared. It was found that after affinity purification, the in vitro actin sliding velocity is reduced compared to the control conditions, something that was not seen with the use of blocking actin. Therefore, the effects of the affinity purification method should be considered when interpreting IVMA data. This is important while using IVMA both for fundamental studies and for nanotechnological applications. Another issue in the use of IVMAs in nanotechnological applications is the requirement for expensive and time-consuming fabrication of nanostructured devices. We therefore developed a suitable method for regenerating molecular motor based bionanodevices without a need to disassemble the flow cell. Evidence is presented that, use of proteinase K with a suitable detergent (SDS or Triton X100) lead to successful regeneration of devices where both actin-myosin and microtubule-kinesin motility are used. Lastly, this thesis presents efforts to immobilize engineered light sensitive myosin motors on trimethyl chlorosilane (TMCS) derivatized surfaces for light operated switching of myosin motor in order to control actin movement in nano-networks. This has potential for developing a programmable junction in a biocomputation network. In brief, the described results have contributed both to the fundamental understanding of actin and myosin properties and the actomyosin interaction mechanisms. They have also given technical insights for molecular motor based bionanotechnology.
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| 3. |
- ten Siethoff, Lasse, 1981-
(författare)
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Towards Myosin Powered Lab-on-a-Chip Devices
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Myosins are protein motors that use chemical energy in the form of adenosinetriphosphate to produce force and motion. These molecular motors might be usedto power transportation in Lab-on-a-chip devices where a series of laboratory tasks(e.g. separation, concentration and detection) are performed in one sequence on asmall chip. Because of the small size, lab-on-a-chip devices are predicted to befaster and more sensitive than conventional systems. Further potential advantagesinclude cost efficiency and the possibility to perform many analyzes in parallel.Substituting microfluidics with myosin based transport would allow furtherminiaturization and make lab-on-a-chip devices more readily portable by reducingthe need for external power supplies. However, there are also limitations thathamper the development of such devices. Here we investigate several aspects of amyosin powered lab-on-a-chip device and present ways to overcome criticallimitations. First we demonstrate covalent attachment of antibodies to actinfilament shuttles with retained ability of the filaments to be propelled by myosinfragments, previously believed to be difficult. Secondly we develop a separationmethod to overcome the deleterious effects of body fluids on the actomyosinsystem. Thirdly, we explore the possibility to concentrate actin shuttles on ananostructured surface and achieve >20 times concentration in <1 min. Monte-Carlo simulations of the concentration process suggest further room forimprovement. Fourth, we develop novel techniques for fast and automaticdetection of fluorescence at certain check points which improves S/N ratio >20times. Finally, we take the first steps towards the development of threedimensional,nanowire-based transport systems, important both for lab-on-a-chipapplications and fundamental studies. Our results demonstrate the potential of amyosin based lab-on-a-chip device and lay the foundation for furtherdevelopments. Thus, we anticipate that this work will influence future studiestowards a complete diagnostic lab-on-a-chip work-up based on molecular motors.In addition, the work might also have implications for the development of futurebiocomputation and drug screening devices as well as novel biophysical studies ofthe actomyosin system.
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