Molecular Basis of Dynamic Localization of Class-I Myosins

去年，我们通过固定细胞的抗体染色显示，内源性DMIB均匀地定位于静止细胞的质膜、随机运动细胞的活跃突起和细胞-细胞接触处，以及运动极化细胞的前部。尾部的磷脂结合BH位点是DMIB与质膜结合的必要条件和充分条件，DMIB中富含PIP2和PIP3的所有膜区域也都富含PIP2和PIP3。 然而，尽管质膜结合是必需的，但在随机细胞运动和饥饿诱导的细胞极化过程中，仅有BH位点并不足以正确地重新定位DMIB，因为单独定位Tail和定位全长肌球蛋白是不同的。在我们所研究的所有情况下，决定DMIB定位的主要因素有两个：通过尾部的BH位点与质膜脂结合，以及通过头部的ATP敏感的肌动蛋白结合位点与细胞质F-肌动蛋白结合。这两种主要的相互作用相互竞争，可以解释表达的尾巴和全长DMIB之间的大部分差异。 去年，我们观察到并发表了(Brzeska等人，2012年)，将强肌动蛋白结合突变体(DMIB-N154A)与类似肌动蛋白波的结构紧密联系在一起。肌动蛋白波是一种F-肌动蛋白结构，通过一种踏步机制在细胞内自我传播，即将肌动蛋白单体添加到细胞的正端，并将单体从肌动蛋白细丝的负端解离。DMIB是肌动蛋白波的已知成分，但DMIB如何与肌动蛋白波结合以及DMIB在肌动蛋白波的形成和功能中的作用尚不清楚。 我们现在已经开发了一种系统，用于将GFP标记的DMIB突变体和RFP标记的F-肌动蛋白标记(Actlife)共转染Dictyostelials。这使我们能够监测F-肌动蛋白和DMIB在活的野生型和DMIB缺失细胞中的动态定位。我们通过TIRF显微镜证实，强结合肌动蛋白的突变体DMIB-N154A与肌动蛋白波相关。我们发现，由于弱结合肌动蛋白的DMIB突变体(DMIB-E407K)不与波结合，所以DMIB与肌动蛋白波的结合绝对需要DMIB头部的肌动蛋白结合。我们还发现，在一定条件下，DMIB-N154A(强肌动蛋白结合突变体)可以促进肌动蛋白波的形成。然而，DMIB对于波形的形成并不是绝对必需的，因为DMIB-零单元形成波形。 在目前的肌动蛋白波功能模型中，DMIB通过其头部的脂结合部位与质膜结合，并通过其尾部将肌动蛋白波锚定到质膜(也可能招募参与肌动蛋白聚合的蛋白质)。我们正在通过在DMIB缺失的细胞中表达头部具有受损的脂结合部位和/或尾部具有受损的SH3结构域(与参与肌动蛋白聚合的其他蛋白质相互作用)的DMIB突变体来验证这一假设。实验正在进行中，目前还没有明确的结果。也有人提出，DMIB在肌动蛋白波中作为一个加端马达取代质膜上的肌动蛋白细丝，从而允许将肌动蛋白单体添加到延长的肌动蛋白细丝中。我们对没有运动活性的强肌动蛋白结合突变体的结果与这一提议不一致。

Last year we showed by antibody-staining of fixed cells that endogenous DMIB is localized uniformly on the plasma membrane of resting cells, at active protrusions and cell-cell contacts of randomly moving cells, and at the front of motile polarized cells. The phospohlipid-binding BH site in the tail is both required and sufficient for association of DMIB with the plasma membrane at all stages and all the membrane regions enriched in DMIB were also enriched in either or both PIP2 and PIP3. However, although required for plasma membrane association, the BH-site alone is not sufficient for proper relocalization of DMIB during random cell movement and starvation-induced cell polarization since localization of tail alone was different than localization of full length myosin. In all situations that we have studied, there were two main factors determining DMIB localization: binding to plasma membrane lipids through the BH-site in the tail and binding to cytoplasmic F-actin through the ATP-sensitive actin-binding site in the head. These two major interactions competing with each can explain most of the differences between the localization of expressed tail and full-length DMIB. Last year, we observed, and published (Brzeska et al., 2012), a strong association of the strong actin-binding mutant (DMIB-N154A) with structures resembling actin waves. Actin waves are F-actin structures self-propagating through the cell by a treadmilling mechanism, i.e. addition of actin monomers to the plus end and dissociation of monomers from the minus end of actin filaments. DMIB is a known component of actin waves but how DMIB associates with actin waves and DMIBs role in the formation and function of actin waves are not known. We have now developed a system for co-transfection of Dictyostelium with GFP-tagged DMIB mutants and RFP-labeled F-actin marker (actlife). This allows us to monitor the dynamic localization of F-actin and DMIB in live wild-type and DMIB-null cells. We confirmed by TIRF microscopy that the strong actin-binding mutant DMIB-N154A associates with actin waves. We found that actin-binding by the DMIB head is absolutely required for association of DMIB with actin waves since the weak actin-binding DMIB mutant (DMIB-E407K) does not associate with waves. We also found that, under certain conditions, DMIB-N154A (the strong actin-binding mutant) enhances formation of actin waves. However, DMIB is not absolutely required for wave formation since DMIB-null cells form waves. In one current model of the function of actin waves, DMIB binds to the plasma membrane via the lipid-binding site in its head and anchors the actin wave to the plasma membrane (and also possibly recruits proteins involved in actin polymerization) via its tail. We are testing this hypothesis by expressing in DMIB-null cells DMIB mutants with a compromised lipid-binding site in the head and/or a compromised SH3 domain (that interacts with other proteins involved in actin polymerization) in the tail. The experiments are in progress with no clear results at this time. It has also been proposed that DMIB acts within the actin wave as a plus-end motor displacing actin filaments from the plasma membrane thus allowing addition of actin monomers to the elongating actin filament. Our results with the strong actin binding mutant, which does not have motor activity, are not consistent with this proposal.