Spontaneous Cdc42 polarization independent of GDI-mediated extraction and actin-based trafficking.
Spontaneous Cdc42 polarization independent of GDI-mediated extraction and actin-based trafficking.
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DOI：
10.1371/journal.pbio.1002097
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发表时间：
2015-04
期刊：
影响因子：
9.8
通讯作者：
Martin SG
中科院分区：
文献类型：
--
作者：
Bendezú FO;Vincenzetti V;Vavylonis D;Wyss R;Vogel H;Martin SG
小的rho家族GTPase Cdc42对细胞极化至关重要，并且在没有上游空间信号的情况下自发极化。自发极化被认为需要通过鸟嘌呤核苷酸解离抑制剂（GDI）介导的膜提取和囊泡运输来动态回收Cdc42。在这里，我们描述了一个功能性的Cdc42荧光等位基因在裂变酵母中，它证明了Cdc42的动力学和极化独立于这些途径。此外，一个经过改造的Cdc42等位基因通过两亲性螺旋独立于这些循环途径靶向膜是可行的，并且在裂变和出芽酵母中自发地极化到多个位点。我们发现Cdc42在膜上高度移动，并在活性位点积聚，在那里它表现出较慢的移动。相比之下，含有Cdc42等位基因的几乎不移动的跨膜结构域支持活力和极化活性，但不会在活性位点积累。我们提出Cdc42激活，通过正反馈增强，通过捕获快速扩散的非活性分子导致其局部积累。这项对裂变酵母的研究揭示了小GTPase Cdc42的活性和非活性形式在膜中的横向扩散速率不同，这为它如何自发极化提供了见解，从而决定了细胞的极性。细胞极化是大多数细胞的关键特征，是其功能组织的基础。一种名为Cdc42的中心极性因子，是一种通过戊烯酰化靶向质膜的小GTPase，以活性gtp结合的形式促进细胞极化。Cdc42是一个关键的极性因子，因为它在假定的极性位点积累，先前的研究表明，这涉及到Cdc42在质膜上和膜外的再循环。此外，即使在没有任何预先定位的标志的情况下，其活性也可以通过自我增强的正反馈机制自发地使单个位置的细胞极化。在这项研究中，我们构建了Cdc42的第一个功能性荧光标记等位基因，取代了pombe Schizosaccharomyces的内源性基因组拷贝。这允许通过活体显微镜测量质膜上Cdc42的动力学。出乎意料的是，这种方法揭示了Cdc42主要通过横向扩散，而不是在质膜上和细胞膜上移动。具有替代膜靶向机制的工程Cdc42等位基因表明，Cdc42活性确实在缺乏已知的膜上和膜外循环Cdc42途径的情况下发生极化。我们进一步表明，活性形式Cdc42-GTP比Cdc42-GDP更具流动性。因此，我们提出Cdc42极化是其局部激活的结果-通过自我增强反馈或响应上游线索-通过活性Cdc42扩散速率的降低。
The small Rho-family GTPase Cdc42 is critical for cell polarization and polarizes spontaneously in absence of upstream spatial cues. Spontaneous polarization is thought to require dynamic Cdc42 recycling through Guanine nucleotide Dissociation Inhibitor (GDI)-mediated membrane extraction and vesicle trafficking. Here, we describe a functional fluorescent Cdc42 allele in fission yeast, which demonstrates Cdc42 dynamics and polarization independent of these pathways. Furthermore, an engineered Cdc42 allele targeted to the membrane independently of these recycling pathways by an amphipathic helix is viable and polarizes spontaneously to multiple sites in fission and budding yeasts. We show that Cdc42 is highly mobile at the membrane and accumulates at sites of activity, where it displays slower mobility. By contrast, a near-immobile transmembrane domain-containing Cdc42 allele supports viability and polarized activity, but does not accumulate at sites of activity. We propose that Cdc42 activation, enhanced by positive feedback, leads to its local accumulation by capture of fast-diffusing inactive molecules. This study of fission yeast reveals that the active and inactive forms of the small GTPase Cdc42 have different rates of lateral diffusion in the membrane, providing insights into how it becomes spontaneously polarized, thereby determining the polarity of the cell. Cell polarization is a critical feature of most cells that underlies their functional organization. A central polarity factor called Cdc42, a small GTPase targeted to the plasma membrane by prenylation, promotes cell polarization in its active GTP-bound form. Cdc42 is a key polarity factor because it accumulates at presumptive sites of polarity, which previous work suggested involves Cdc42 recycling on and off the plasma membrane. In addition, its activity can spontaneously polarize cells in a single location by self-enhancing positive feedback mechanisms, even in the absence of any pre-localized landmarks. In this study, we constructed the first functional fluorescently tagged allele of Cdc42 that replaces the endogenous genomic copy in Schizosaccharomyces pombe. This allowed measurements of Cdc42 dynamics at the plasma membrane by live microscopy. Unexpectedly, this approach revealed that Cdc42 primarily moves through lateral diffusion, rather than on and off the plasma membrane. Engineered Cdc42 alleles with alternative membrane-targeting mechanisms demonstrated that Cdc42 activity, indeed, polarizes in the absence of known pathways that recycle Cdc42 on and off the membrane. We further show that the active form, Cdc42-GTP, is less mobile than Cdc42-GDP. We thus propose that Cdc42 polarization occurs as a consequence of its local activation—either through self-enhanced feedback or in response to upstream cues—by a reduction in the active Cdc42 diffusion rate.
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影响因子：
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作者：
Hazan, I;Liu, HP
通讯作者：
Liu, HP
影响因子：
64.5
作者：
Etienne-Manneville, S;Hall, A
通讯作者：
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影响因子：
9.2
作者：
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通讯作者：
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DOI：
10.1073/pnas.182370299
发表时间：
2002-09-17
影响因子：
11.1
作者：
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通讯作者：
Bi, E
影响因子：
64.5
作者：
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通讯作者：
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