Kerivan Emily M, Amari Victoria N, Weeks William B, Hardin Leigh H, Tobin Lyle, Al Azzam Omayma Y, Reinemann Dana N
Department of Biomedical Engineering, University of Mississippi, University, MS, USA 38677.
Department of Chemical Engineering, University of Mississippi, University, MS, USA 38677.
bioRxiv. 2024 Nov 19:2024.02.26.582155. doi: 10.1101/2024.02.26.582155.
Cytoskeletal protein ensembles exhibit emergent mechanics where behavior exhibited in teams is not necessarily the sum of the components' single molecule properties. In addition, filaments may act as force sensors that distribute feedback and influence motor protein behavior. To understand the design principles of such emergent mechanics, we developed an approach utilizing QCM-D to measure how actomyosin bundles respond mechanically to environmental variables that alter constituent myosin II motor behavior.
QCM-D is used for the first time to probe alterations in actin-myosin bundle viscoelasticity due to changes in skeletal myosin II concentration and motor nucleotide state. Actomyosin bundles were constructed on a gold QCM-D sensor using a microfluidic setup, and frequency and dissipation change measurements were recorded for each component addition to decipher which assay constituents lead to changes in bundle structural compliancy.
Lowering myosin concentration is detected as lower shifts in frequency and dissipation, while the relative changes in frequency and dissipation shifts for both the first and second actin additions are relatively similar. Strikingly, buffer washes with different nucleotides (ATP vs. ADP) yielded unique signatures in frequency and dissipation shifts. As myosin II's ADP-bound state tightly binds actin filaments, we observe an increase in frequency and decrease in dissipation change, indicating a decrease in viscoelasticity, likely due to myosin's increased affinity for actin, conversion from an active motor to a static crosslinker, and ability to recruit additional actin filaments from the surface, making an overall more rigid sensor coating. However, lowering the ADP concentration results in increased system compliancy, indicating that transient crosslinking and retaining a balance of motor activity perhaps results in a more cooperative and productive force generating system.
QCM-D can detect changes in actomyosin viscoelasticity due to molecular-level alterations, such as motor concentration and nucleotide state. These results provide support for actin's role as a mechanical force-feedback sensor and demonstrate a new approach for deciphering the feedback mechanisms that drive emergent cytoskeletal ensemble crosstalk and intracellular mechanosensing. This approach can be adapted to investigate environmental influences on more complex cytoskeletal ensemble mechanics, including addition of other motors, crosslinkers, and filament types.
细胞骨架蛋白集合体表现出涌现力学，即团队中表现出的行为不一定是各组分单分子特性的总和。此外，细丝可能充当力传感器，分布反馈并影响运动蛋白的行为。为了理解这种涌现力学的设计原理，我们开发了一种利用石英晶体微天平耗散技术（QCM-D）的方法，以测量肌动球蛋白束如何对改变组成型肌球蛋白II运动行为的环境变量作出机械响应。
首次使用QCM-D来探究由于骨骼肌肌球蛋白II浓度和运动核苷酸状态的变化而导致的肌动蛋白-肌球蛋白束粘弹性的改变。使用微流体装置在金质QCM-D传感器上构建肌动球蛋白束，并记录每次添加组分时的频率和耗散变化测量值，以解读哪些检测成分会导致束结构顺应性的变化。
检测到肌球蛋白浓度降低时频率和耗散的变化较小，而第一次和第二次添加肌动蛋白时频率和耗散变化的相对变化较为相似。引人注目的是，用不同核苷酸（ATP与ADP）进行缓冲液冲洗会在频率和耗散变化中产生独特的特征。由于肌球蛋白II的ADP结合状态紧密结合肌动蛋白丝，我们观察到频率增加而耗散变化减小，这表明粘弹性降低，可能是由于肌球蛋白对肌动蛋白的亲和力增加、从活性运动蛋白转变为静态交联剂以及从表面募集额外肌动蛋白丝的能力，从而形成了整体更刚性的传感器涂层。然而，降低ADP浓度会导致系统顺应性增加，这表明瞬时交联和保持运动活性的平衡可能会导致产生更协同且高效的力的系统。
QCM-D可以检测由于分子水平改变（如运动蛋白浓度和核苷酸状态）而导致的肌动球蛋白粘弹性变化。这些结果支持了肌动蛋白作为机械力反馈传感器的作用，并展示了一种新方法，用于解读驱动细胞骨架集合体涌现串扰和细胞内机械传感的反馈机制。这种方法可用于研究环境对更复杂的细胞骨架集合体力学的影响，包括添加其他运动蛋白、交联剂和细丝类型。
