LV-pIN-KDEL: a novel lentiviral vector demonstrates the morphology, dynamics and continuity of the endoplasmic reticulum in live neurones.
LV-pIN-KDEL: a novel lentiviral vector demonstrates the morphology, dynamics and continuity of the endoplasmic reticulum in live neurones.
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DOI：
10.1186/1471-2202-9-10
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发表时间：
2008-01-23
期刊：
影响因子：
2.4
通讯作者：
Jones, Owen T.
中科院分区：
文献类型：
--
作者：
Jones, Vicky C.;McKeown, Lynn;Verkhratsky, Alexei;Jones, Owen T.
神经元内质网（ER）是一个广泛的，复杂的内膜系统，含有Ca 2+泵，和Ca 2+通道，使其能够作为一个动态的钙库。目前，关于神经元中ER的连续性，如何与钙信号交叉以及物理区室化的可能性存在争议。不幸的是，ER结构的可用探针如活体染料受到其膜特异性的限制。ER靶向GFP质粒的引入已经向前迈出了相当大的一步，但是这些质粒难以通过常规转染方法在神经元中表达。为了避免这些问题，我们已经设计了一种新的ER靶向GFP构建体，称为pIN-KDEL，进入第三代复制缺陷型自失活慢病毒载体系统，该系统能够介导不同分裂和有丝分裂后哺乳动物细胞（包括神经元）中的基因转导。在HEK 293（或COS-7）细胞中表达后，LV-pIN-KDEL产生的荧光模式仅与ER标记物sec 61 β共定位，但不与其他主要细胞器共定位。我们没有发现细胞毒性的证据，只有很少的包涵体形成。为了探索探针在分辨活细胞中的ER中的效用，用LV-pIN-KDEL转导HEK 293或COS-7细胞，并在48小时后，以1分钟至数小时的间隔直接成像。LV-pIN-KDEL荧光显示内质网为管状晶格结构，其形态可在数秒内显著改变。虽然GFP可能是光毒性的，但细胞和ER的完整性保留了数周，甚至在光暴露长达24小时后。使用LV-pIN-KDEL，我们已经成像的ER在不同的固定的神经元培养，并使用实时成像，发现证据广泛的，动态重塑的神经元ER在活海马培养，脑切片，外植体和神经胶质细胞。最后，通过光漂白中的荧光损失（FLIP）方法，在单个感兴趣区域的连续照射去除了外植体培养物中LV-pIN-KDEL转导的神经细胞的所有荧光，从而提供了令人信服的证据，即在神经元中，内质网不仅是动态的，而且是连续的。基于慢病毒的ER靶向报告基因LV-pIN-KDEL在定义ER的结构方面提供了优于现有系统的相当大的优势，特别是在原代细胞如众所周知难以转染的神经元中。LV-pIN-KDEL转导的神经元的图像和连续光漂白实验表明，内质网是一个动态的结构与一个单一的连续管腔。预计LV-pIN-KDEL的引入将极大地促进健康和患病脑组织中神经元ER的结构可塑性和连续性质的实时可视化。
The neuronal endoplasmic reticulum (ER) is an extensive, complex endomembrane system, containing Ca2+ pumps, and Ca2+ channels that permit it to act as a dynamic calcium store. Currently, there is controversy over the continuity of the ER in neurones, how this intersects with calcium signalling and the possibility of physical compartmentalisation. Unfortunately, available probes of ER structure such as vital dyes are limited by their membrane specificity. The introduction of ER-targeted GFP plasmids has been a considerable step forward, but these are difficult to express in neurones through conventional transfection approaches. To circumvent such problems we have engineered a novel ER-targeted GFP construct, termed pIN-KDEL, into a 3rd generation replication-defective, self-inactivating lentiviral vector system capable of mediating gene transduction in diverse dividing and post-mitotic mammalian cells, including neurones. Following its expression in HEK293 (or COS-7) cells, LV-pIN-KDEL yielded a pattern of fluorescence that co-localised exclusively with the ER marker sec61β but with no other major organelle. We found no evidence for cytotoxicity and only rarely inclusion body formation. To explore the utility of the probe in resolving the ER in live cells, HEK293 or COS-7 cells were transduced with LV-pIN-KDEL and, after 48 h, imaged directly at intervals from 1 min to several hours. LV-pIN-KDEL fluorescence revealed the endoplasmic reticulum as a tubular lattice structure whose morphology can change markedly within seconds. Although GFP can be phototoxic, the integrity of the cells and ER was retained for several weeks and even after light exposure for periods up to 24 h. Using LV-pIN-KDEL we have imaged the ER in diverse fixed neuronal cultures and, using real-time imaging, found evidence for extensive, dynamic remodelling of the neuronal ER in live hippocampal cultures, brain slices, explants and glia. Finally, through a Fluorescence Loss in Photobleaching (FLIP) approach, continuous irradiation at a single region of interest removed all the fluorescence of LV-pIN-KDEL-transduced nerve cells in explant cultures, thus, providing compelling evidence that in neurons the endoplasmic reticulum is not only dynamic but also continuous. The lentiviral-based ER-targeted reporter, LV-pIN-KDEL, offers considerable advantages over present systems for defining the architecture of the ER, especially in primary cells such as neurones that are notoriously difficult to transfect. Images and continuous photobleaching experiments of LV-pIN-KDEL-transduced neurones demonstrate that the endoplasmic reticulum is a dynamic structure with a single continuous lumen. The introduction of LV-pIN-KDEL is anticipated to greatly facilitate a real-time visualisation of the structural plasticity and continuous nature of the neuronal ER in healthy and diseased brain tissue.
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DOI：
10.1083/jcb.131.6.1715
发表时间：
1995-12
期刊：
The Journal of cell biology
影响因子：
--
作者：
Nakamura N;Rabouille C;Watson R;Nilsson T;Hui N;Slusarewicz P;Kreis TE;Warren G
通讯作者：
Warren G
影响因子：
4.2
作者：
Jakobsson, J;Ericson, C;Lundberg, C
通讯作者：
Lundberg, C
影响因子：
7.8
作者：
Kalies, KU;Rapoport, TA;Hartmann, E
通讯作者：
Hartmann, E
影响因子：
7.5
作者：
Levine, T;Rabouille, C
通讯作者：
Rabouille, C
影响因子：
64.5
作者：
Mogami, H;Nakano, K;Petersen, OH
通讯作者：
Petersen, OH