通过代谢点击介导的果胶标记对活的伸长细胞中的细胞壁多糖进行动态成像
Dynamic imaging of cell wall polysaccharides by metabolic click-mediated labeling of pectins in living elongating cells.
作者信息
Ropitaux Marc, Hays Quentin, Baron Aurélie, Fourmois Laura, Boulogne Isabelle, Vauzeilles Boris, Lerouge Patrice, Mollet Jean-Claude, Lehner Arnaud
机构信息
UNIROUEN, Laboratoire Glyco-MEV, Normandie Université, UR 4358, Structure Fédérative de Recherche Normandie Végétal FED 4277, Innovation Chimie Carnot, 76000, Rouen, France.
Université Paris-Saclay, CNRS, Institut de Chimie des Substances Naturelles, UPR 2301, 91198, Gif-sur-Yvette, France.
出版信息
Plant J. 2022 May;110(3):916-924. doi: 10.1111/tpj.15706. Epub 2022 Mar 6.
Protein tracking in living plant cells has become routine with the emergence of reporter genes encoding fluorescent tags. Unfortunately, this imaging strategy is not applicable to glycans because they are not directly encoded by the genome. Indeed, complex glycans result from sequential additions and/or removals of monosaccharides by the glycosyltransferases and glycosidases of the cell's biosynthetic machinery. Currently, the imaging of cell wall polymers mainly relies on the use of antibodies or dyes that exhibit variable specificities. However, as immunolocalization typically requires sample fixation, it does not provide access to the dynamics of living cells. The development of click chemistry in plant cell wall biology offers an alternative for live-cell labeling. It consists of the incorporation of a carbohydrate containing a bio-orthogonal chemical reporter into the target polysaccharide using the endogenous biosynthetic machinery of the cell. Once synthesized and deposited in the cell wall, the polysaccharide containing the analog monosaccharide is covalently coupled to an exogenous fluorescent probe. Here, we developed a metabolic click labeling approach which allows the imaging of cell wall polysaccharides in living and elongating cells without affecting cell viability. The protocol was established using the pollen tube, a useful model to follow cell wall dynamics due to its fast and tip-polarized growth, but was also successfully tested on Arabidopsis root cells and root hairs. This method offers the possibility of imaging metabolically incorporated sugars of viable and elongating cells, allowing the study of the long-term dynamics of labeled extracellular polysaccharides.
随着编码荧光标签的报告基因的出现,对活植物细胞中的蛋白质进行追踪已成为常规操作。不幸的是,这种成像策略不适用于聚糖,因为它们不是由基因组直接编码的。实际上,复杂聚糖是由细胞生物合成机制中的糖基转移酶和糖苷酶对单糖进行顺序添加和/或去除而产生的。目前,细胞壁聚合物的成像主要依赖于使用特异性各异的抗体或染料。然而,由于免疫定位通常需要对样品进行固定,因此无法获取活细胞的动态信息。植物细胞壁生物学中点击化学的发展为活细胞标记提供了一种替代方法。它包括利用细胞的内源性生物合成机制将含有生物正交化学报告基团的碳水化合物掺入目标多糖中。一旦合成并沉积在细胞壁中,含有类似单糖的多糖就会与外源性荧光探针共价偶联。在这里,我们开发了一种代谢点击标记方法,该方法能够对活的伸长细胞中的细胞壁多糖进行成像,而不会影响细胞活力。该方案是使用花粉管建立的,由于其快速且顶端极化生长,花粉管是研究细胞壁动态的有用模型,但该方法也在拟南芥根细胞和根毛上成功进行了测试。这种方法提供了对活的伸长细胞中代谢掺入的糖进行成像的可能性,从而能够研究标记的细胞外多糖的长期动态。