Structure And Function Of Unconventional Myosins

我们已经开始研究肌球蛋白18A(M18A)的功能。为此，我们培育了M18A条件性基因敲除小鼠，用于从特定组织中移除M18A。我们还产生了M18A特异性抗体和荧光标记的M18A质粒，用于研究M18A在普通细胞中的亚细胞定位。使用不同的成像技术，我们已经定位了M18A到肌球蛋白丰富的区域，包括内应力纤维和板层突起。我们已经开始开发siRNA和shRNA技术来抑制M18A在这些细胞中的表达，以帮助揭示其细胞功能。我们将继续研究M18A在整个动物和培养细胞中的作用。 为了帮助我们了解这些肌球蛋白富集区，我们已经开始了对非肌肉肌球蛋白II(NM II)的进一步研究。NM II为多种发育和细胞过程提供动力，包括黏附、迁移和分裂。NM II的单个单体是由两条肌球蛋白重链(MHC)、两条基本轻链和两条调节轻链组成的六聚体。三个不同的基因编码MHC蛋白，导致三个不同的单体群体，称为NM IIA，NM IIB和NM IIC。小鼠的基因敲除和拯救实验表明，具有不同生物物理特性的个体亚型既有独特的功能，也有多余的功能。为了发挥它们的功能，NM II单体聚合成反平行的细丝，与肌动蛋白细胞骨架接合。目前尚不清楚这三种NMII亚型是否共聚合以产生混合细丝，或者细丝是否完全由单一亚型组成。澄清这一问题对于理解和解释研究单个NM II亚型的作用的实验至关重要。分子生物学方法产生了相互矛盾的结果，部分原因是下拉和IP可能产生假阳性，因为多个细丝通过肌动蛋白细胞骨架连接。虽然体外实验提供了对NM II亚型的不同生物物理特征的洞察，但它们不能再现活细胞中蛋白质所经历的生理条件。最后，虽然很明显NM II亚型在细胞的某些区域表现出重叠的定位，但这并不能证明是共聚的，因为这种成像通常远低于区分单丝和多丝所需的分辨率。NM II细丝的长度通常为300纳米，类似于标准光学显微镜的分辨率极限。因此，为了确定NM II亚型是否可以在活细胞中共聚合，我们使用了荧光团标记的MHC亚型与双色超分辨率TIRF结构照明显微镜(TIRF-SIM)相结合。这项技术提供了大约100纳米的横向分辨率，比标准光学显微镜获得的分辨率高出近2.5倍。使用TIRF-SIM和MHC结构，在头部或尾部都有荧光团，我们可以明确地识别单个NM II细丝，以及每个细丝的方向。利用这些技术，我们证明了外源表达的NM IIA和NM IIB在活细胞中共聚合，产生不同种类的细丝。令人惊讶的是，即使在异构体被认为是分离的区域，例如在极化细胞的前沿，NM IIA被认为是唯一局域的，我们仍然在几乎每一根细丝中看到共聚的证据。我们已经使用异构体特异性抗体用内源性蛋白证实了这些结果。这些结果表明，NM II异构体在与其他异构体共聚合时，既发挥了异构体特有的作用，又发挥了多余的作用，并表明微丝中每种异构体的比例决定了它的生物物理性质。在解释单个NM II亚型的实验时，应该考虑这些结果。

We have begun work investigating the function of myosin 18A (M18A). To this end, we have generated M18A conditional knockout mice that will be used to remove M18A from specific tissues. We have also generated M18A-specific antibodies and fluorescently-tagged M18A plasmids that are being used to investigate the subcellular localization of M18A in generic cells. Using various imaging techniques, we have localized M18A to acto-myosin rich regions, including internal stress fibers and lamellar protrusions. We have begun developing siRNA and shRNA technologies to suppress M18A expression in these cells to help reveal its cellular function. We will continue to investigate the role of M18A in both the whole animal and in cultured cells. To aid in our understanding of these acto-myosin rich regions, we have begun further investigation into non-muscle myosin II (NM II). NM II powers a variety of developmental and cellular processes, including adhesion, migration, and division. A single monomer of NM II is a hexamer consisting of two myosin heavy chains (MHC), two essential light chains, and two regulatory light chains. Three distinct genes encode MHC proteins, resulting in three distinct monomer populations, termed NM IIA, NM IIB and NM IIC. Knockout and rescue experiments in mice have demonstrated both unique and redundant functions for individual isoforms, which possess distinct biophysical properties. To exert their function, NM II monomers polymerize into anti-parallel filaments that engage the actin cytoskeleton. It is unclear whether or not the three NMII isoforms co-polymerize to produce mixed filaments or if filaments consist entirely of a single isoform. Clarifying this issue is critical for understanding and interpreting experiments investigating the roles of individual NM II isoforms. Molecular biological approaches have produced conflicting results, in part because pull-downs and IPs can yield false positives due to multiple filaments being connected through the actin cytoskeleton. While in vitro experiments have provided insights into the distinct biophysical characteristics of the NM II isoforms, they do not reproduce the physiological conditions experienced by the proteins in living cells. Finally, while it is clear that NM II isoforms display overlapping localization in some areas of the cell, this does not prove co-polymerization, as this imaging is typically well below the resolution required to distinguish a single filament from multiple filaments. NM II filaments are typically 300 nm in length, similar to the resolution limit of standard light microscopy. Therefore, to determine if NM II isoforms can co-polymerize in live cells, we used fluorophore-tagged MHC isoforms in conjunction with two-color super-resolution TIRF structured illumination microscopy (TIRF-SIM). This technique provides a lateral resolution of approximately 100 nm, nearly 2.5x higher than the resolution achieved with standard light microscopy. Using TIRF-SIM and MHC constructs with a fluorophore on either the head or the tail, we can unequivocally identify individual NM II filaments, along with the orientation of each filament. Using these techniques, we demonstrate that exogenously expressed NM IIA and NM IIB co-polymerize in live cells to produce heterogeneous filaments. Surprisingly, even in areas where the isoforms are thought to be segregated, such as at the leading edge of a polarized cell where NM IIA is thought to be uniquely localized, we still see evidence of co-polymerization in nearly every filament. We have confirmed these results with endogenous proteins using isoform-specific antibodies. These results argue that NM II isoforms are performing both their isoform-specific and redundant roles while co-polymerized with other isoforms, and it suggests that it is the ratio of each isoform within the filament that provides it with its biophysical properties. These results should be considered whenever interpreting experiments on individual NM II isoforms.