微丝骨架和微管骨架系统在真核细胞的很多过程中发挥着重要的作用，大量研究结果表明微丝和微管在空间定位和功能上均存在密切的联系，而这种联系依赖于很多微丝和微管的交联蛋白来完成。近年来的研究发现，formin作为微丝骨架的调节因子，还可能影响微管骨架的动态，是一种潜在的微丝微管交联蛋白。AtFH14是一个典型的Ⅱ型Formin蛋白，本实验室前期的研究工作证明AtFH14定位于细胞分裂期的早前期带、纺锤体和成膜体上，通过与微丝和微管骨架相互作用参与植物细胞的分裂。我们以此为基础，进一步研究了AtFH14与微丝骨架和微管骨架互作的生化特性及其对两种骨架的交联过程。AtFH14含有两个formin同源结构域FH1和FH2以及一个PTEN结构域，为了研究AtFH14的功能，我们利用原核表达系统表达并纯化了两个包含AtFH14功能结构域的截短蛋白：AtFH14-FH1FH2和AtFH14-FH2。肌动蛋白聚合的动力学实验表明AtFH14-FH1FH2和AtFH14-FH2均能促进单体肌动蛋白成核，属于活性较强的成核因子，而且FH1结构域对于AtFH14介导的profilin-actin的成核是必需的。通过全内反射荧光显微镜技术我们发现AtFH14使肌动蛋白成核后不是结合微丝的正端，而是结合在微丝的负端区域，表明AtFH14是一个非持续性的formin蛋白。AtFH14-FH1FH2偶尔会形成聚集体，使多根微丝成核起始于同一位置，从而形成星状的结构。AtFH14-FH1FH2 和AtFH14-FH2均能结合微丝的正端，并抑制微丝正端的聚合和解聚以及微丝正端和负端的接合，而且FH1结构域只能很小程度地降低FH2结构域的微丝正端结合能力。另外，AtFH14-FH1FH2 和AtFH14-FH2结合在微丝的侧端可以诱导微丝形成微丝束，说明AtFH14的成束能力是依靠FH2结构域实现的，AtFH14诱导形成的微丝束是由同向或反向平行排列的微丝组成的。除了调节微丝的动态变化之外，AtFH14还能促进单体微管蛋白的聚合，这种促进作用是通过促进微管成束实现的。AtFH14-FH1FH2 和AtFH14-FH2均能够直接结合微管并使微管成束，说明AtFH14依靠FH2结构域结合微管。FH1结构域的存在只能很小程度地改变FH2结构域与微管的结合能力。当微管和单体肌动蛋白同时存在时，AtFH14-FH1FH2优先结合微管，结合了微管的AtFH14-FH1FH2不能促进单体肌动蛋白成核，说明微管抑制了AtFH14-FH1FH2的成核能力。当单体微管蛋白和单体肌动蛋白同时存在时，AtFH14-FH1FH2的肌动蛋白成核活性保持不变，表明微管蛋白单体对AtFH14-FH1FH2的成核能力没有影响。虽然结合在微管上的AtFH14-FH1FH2不能成核起始新的微丝，但是能够将靠近微管的短的微丝招募到微管上，而且刚结合到微管上的微丝会沿着微管滑动。被结合到微管上的微丝还能沿着微管继续聚合延伸，并形成长的微丝-微管束，说明结合在微管上的AtFH14-FH1FH2也不能结合微丝的正端从而不能抑制微丝正端的延伸。当微丝的延伸端逐渐延伸并远离微管时会形成微丝和微管的交叉连接。以上结果表明虽然AtFH14在结合了微管之后既不能使肌动蛋白成核，也不能结合微丝的正端，但是能够通过结合微丝侧端把微丝招募到微管上，并且不影响微丝正端的聚合。综上所述，一方面，AtFH14能够通过成核、封端和成束作用调节微丝骨架动态，另一方面，AtFH14能够结合微管并使微管成束，并且可以通过结合微丝的侧端将其招募到微管上，从而使微丝和微管发生交联。我们的工作呈现了AtFH14与微丝骨架和微管骨架互作的生化特性，这对于理解AtFH14在细胞内的特殊定位以及在细胞分裂过程中发挥的作用有重要的意义，同时也提供了一种微丝骨架和微管骨架互相联系的可能的方式。

The actin and microtubule cytoskeletal systems are well organized dynamic systems in plant cells, where they have crucial functions in many cellular processes, such as cell growth, cytoplasmic streaming and organelle movement, cell division, as well as cellular signaling. Microfilaments and microtubules often co-distribute in the cortical area in interphase cells and in the preprophase band, mitotic spindle, and phragmoplast in mitotic cells and meiotic cells, and the structural associations between the actin and microtubule cytoskeletons may be facilitated by some actin binding proteins like formins. AtFH14 was shown to decorate preprophase bands, spindles, and phragmoplasts to regulate plant cell division, as a result, a detailed analysis is needed to characterize the mechanism of how AtFH14 modulates actin and microtubule dynamics and crosslinks them together.AtFH14 is a class Ⅱ formin，consisting of formin homology domain FH1 and FH2, and a PTEN domain at the N-terminal. To characterize the functional properties of AtFH14, we expressed two constructs, AtFH14-FH1FH2 and AtFH14-FH2. Both AtFH14-FH1FH2 and AtFH14-FH2 were able to nucleate actin nucleation, and the nucleation efficiency of AtFH14 is close to that previously reported for AtFH1 and OsFH5, suggestting that AtFH14 is an efficient actin nucleator. Furthermore, the polyproline-rich FH1 domain is necessary for the nucleation from the profilin-actin complex. AtFH14 is probably a nonprocessive formin, as the pointed end region, instead of the barbed end of actin filament bound to the AtFH14-FH1FH2-coated beads after nucleation, and no buckle events were detected in all the independent experiments. What’s more, actin filaments sometimes initiated from one origin, forming stellar like structures, perhaps as a result of multimerization or clustering of AtFH14. Both AtFH14-FH1FH2 and AtFH14-FH2 inhibit elongation and depolymerization at the barbed ends of actin filaments efficiently, indicating that AtFH14 is a tight barbed end capper, and FH1 slightly reduces the affinity of FH2 for binding the barbed end. This result can be further confirmed by the end-to-end annealing assay, as both AtFH14-FH1FH2 and AtFH14-FH2 inhibit the annealing reaction strongly. AtFH14 can also bind to the side of actin filaments and organize actin filaments into antiparallel and parallel bundles, and FH2 domain is essential for binding and bundling activity.In addition to regulating microfilament dynamics, AtFH14 can promote polymerization of monomeric tubulin through bundling microtubules. Both AtFH14-FH1FH2 and AtFH14-FH2 can directly bind and bundle microtubules, indicating that FH2 domain of AtFH14 is essential for binding and bundling activity of microtubules. Microtubules inhibit actin nucleation activity of AtFH14-FH1FH2, but the same concentration of monomeric tubulin had no effect on the actin polymerization induced by AtFH14-FH1FH2, implying that AtFH14 prefers binding microtubules to binding actin monomers, and the microtubule-binding AtFH14 could not nucleate actin polymerization. However, when an actin filament got close to the microtubules，AtFH14-FH1FH2 was able to recruit the actin filament to microtubules. The newly recruited actin filament could slide and elongate along the microtubules, inducing the formation of microfilament-microtubule bundles, and when the growing actin filament elongated away from the microtubules, it would form crosslinking of microfilaments and microtubules.In brief, on one hand, AtFH14 plays an important role in stabilizing actin filaments through nucleation, barbed ends capping and bundling. On the other hand, AtFH14 is able to stabilize microtubules through organize microtubules into bundles, and recruit actin filaments to microtubules, inducing the formation of bundles or crosslinking of microfilaments and microtubules. This will help us to better understand how AtFH14 function as a crosslinking protein between microfilaments and microtubules in preprophase bands, spindles, and phragmoplasts to participate in the process of plant cell division, and provide a possible way for the interactions between microtubules and microfilaments.