Robert Amélie, Rossow Molly J, Hookway Caroline, Adam Stephen A, Gelfand Vladimir I
Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611.
Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611
Proc Natl Acad Sci U S A. 2015 Jul 7;112(27):E3505-14. doi: 10.1073/pnas.1505303112. Epub 2015 Jun 24.
Intermediate filaments (IFs) are a component of the cytoskeleton capable of profound reorganization in response to specific physiological situations, such as differentiation, cell division, and motility. Various mechanisms were proposed to be responsible for this plasticity depending on the type of IF polymer and the biological context. For example, recent studies suggest that mature vimentin IFs (VIFs) undergo rearrangement by severing and reannealing, but direct subunit exchange within the filament plays little role in filament dynamics at steady state. Here, we studied the dynamics of subunit exchange in VIF precursors, called unit-length filaments (ULFs), formed by the lateral association of eight vimentin tetramers. To block vimentin assembly at the ULF stage, we used the Y117L vimentin mutant (vimentin(Y117L)). By tagging vimentin(Y117L) with a photoconvertible protein mEos3.2 and photoconverting ULFs in a limited area of the cytoplasm, we found that ULFs, unlike mature filaments, were highly dynamic. Subunit exchange among ULFs occurred within seconds and was limited by the diffusion of soluble subunits in the cytoplasm rather than by the association and dissociation of subunits from ULFs. Our data demonstrate that cells expressing vimentin(Y117L) contained a large pool of soluble vimentin tetramers that was in rapid equilibrium with ULFs. Furthermore, vimentin exchange in ULFs required ATP, and ATP depletion caused a dramatic reduction of the soluble tetramer pool. We believe that the dynamic exchange of subunits plays a role in the regulation of ULF assembly and the maintenance of a soluble vimentin pool during the reorganization of filament networks.
中间丝（IFs）是细胞骨架的一个组成部分，能够在响应特定生理情况（如分化、细胞分裂和运动）时进行深刻的重组。根据中间丝聚合物的类型和生物学背景，人们提出了各种机制来解释这种可塑性。例如，最近的研究表明，成熟的波形蛋白中间丝（VIFs）通过切断和重新退火进行重排，但在稳态下，丝状物内的直接亚基交换在丝状物动力学中作用不大。在这里，我们研究了由八个波形蛋白四聚体横向结合形成的VIF前体（称为单位长度丝（ULFs））中亚基交换的动力学。为了在ULF阶段阻断波形蛋白的组装，我们使用了Y117L波形蛋白突变体（波形蛋白（Y117L））。通过用可光转换蛋白mEos3.2标记波形蛋白（Y117L）并在细胞质的有限区域内对ULFs进行光转换，我们发现与成熟丝状物不同，ULFs具有高度的动态性。ULFs之间的亚基交换在几秒钟内发生，并且受到细胞质中可溶性亚基扩散的限制，而不是受亚基与ULFs的缔合和解离的限制。我们的数据表明，表达波形蛋白（Y117L）的细胞含有大量与ULFs处于快速平衡状态的可溶性波形蛋白四聚体。此外，ULFs中的波形蛋白交换需要ATP，ATP耗尽会导致可溶性四聚体库显著减少。我们认为，亚基的动态交换在ULF组装的调节以及丝状物网络重组过程中可溶性波形蛋白库的维持中发挥作用。
