ABSTRACT Myosin VI (MVI) is a unique actin-based motor protein moving towards the minus end of actin filaments, in the opposite direction than other known myosins. Besides well described functions of MVI in endocytosis and maintenance of Golgi apparatus, there are few reports showing its involvement in transcription. We previously demonstrated that in neurosecretory PC12 cells MVI was present in the cytoplasm and nucleus, and its depletion caused substantial inhibition of cell migration and proliferation. Here, we show an increase in nuclear localization of MVI upon cell stimulation, and identification of potential nuclear localization (NLS) and nuclear export (NES) signals within MVI heavy chain. These signals seem to be functional as the MVI nuclear presence was affected by the inhibitors of nuclear import (ivermectin) and export (leptomycin B). In nuclei of stimulated cells, MVI colocalized with active RNA polymerase II, BrUTP-containing transcription sites and transcription factor SP1 as well as SC35 and PML proteins, markers of nuclear speckles and PML bodies, respectively. Mass spectrometry analysis of samples of a GST-pull-down assay with the MVI tail domain as a “bait” identified several new potential MVI binding partners. Among them are proteins involved in transcription and post-transcriptional processes. We confirmed interaction of MVI with heterogeneous nuclear ribonucleoprotein U (hnRNPU) and nucleolin, proteins involved in pre-mRNA binding and transport, and nucleolar function, respectively. Our data provide an insight into mechanisms of involvement of MVI in nuclear processes via interaction with nuclear proteins and support a notion for important role(s) for MVI in gene expression.

中文翻译：

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神经分泌 PC12 细胞核中的肌球蛋白 VI：刺激依赖性核易位和与核蛋白的相互作用

摘要 肌球蛋白 VI (MVI) 是一种独特的基于肌动蛋白的运动蛋白，向肌动蛋白丝的负端移动，与其他已知肌球蛋白的方向相反。除了已充分描述的 MVI 在高尔基体的内吞作用和维持中的功能外，很少有报告显示其参与转录。我们之前已经证明，在神经分泌型 PC12 细胞中，MVI 存在于细胞质和细胞核中，其消耗导致细胞迁移和增殖的显着抑制。在这里，我们展示了细胞刺激后 MVI 核定位的增加，以及 MVI 重链内潜在核定位 (NLS) 和核输出 (NES) 信号的识别。这些信号似乎是有功能的，因为 MVI 核的存在受到核输入（伊维菌素）和输出（细霉素 B）抑制剂的影响。在受刺激细胞的细胞核中，MVI 与活性 RNA 聚合酶 II、含 BrUTP 的转录位点和转录因子 SP1 以及 SC35 和 PML 蛋白、核斑点和 PML 体的标记物共定位。以 MVI 尾部结构域作为“诱饵”的 GST 下拉测定样品的质谱分析确定了几个新的潜在 MVI 结合伙伴。其中包括参与转录和转录后过程的蛋白质。我们证实了 MVI 与异质核核糖核蛋白 U (hnRNPU) 和核仁蛋白、分别参与前 mRNA 结合和转运的蛋白质以及核仁功能的相互作用。