Romagnoli Silvia, Cai Giampiero, Cresti Mauro
Dipartimento Scienze Ambientali G. Sarfatti, Università di Siena, via Mattioli 4, 53100 Siena, Italy.
Plant Cell. 2003 Jan;15(1):251-69. doi: 10.1105/tpc.005645.
The movement of pollen tube organelles relies on cytoskeletal elements. Although the movement of organelles along actin filaments in the pollen tube has been studied widely and is becoming progressively clear, it remains unclear what role microtubules play. Many uncertainties about the role of microtubules in the active transport of pollen tube organelles and/or in the control of this process remain to be resolved. In an effort to determine if organelles are capable of moving along microtubules in the absence of actin, we extracted organelles from tobacco pollen tubes and analyzed their ability to move along in vitro-polymerized microtubules under different experimental conditions. Regardless of their size, the organelles moved at different rates along microtubules in the presence of ATP. Cytochalasin D did not inhibit organelle movement, indicating that actin filaments are not required for organelle transport in our assay. The movement of organelles was cytosol independent, which suggests that soluble factors are not necessary for the organelle movement to occur and that microtubule-based motor proteins are present on the organelle surface. By washing organelles with KI, it was possible to release proteins capable of gliding carboxylated beads along microtubules. Several membrane fractions, which were separated by Suc density gradient centrifugation, showed microtubule-based movement. Proteins were extracted by KI treatment from the most active organelle fraction and then analyzed with an ATP-sensitive microtubule binding assay. Proteins isolated by the selective binding to microtubules were tested for the ability to glide microtubules in the in vitro motility assay, for the presence of microtubule-stimulated ATPase activity, and for cross-reactivity with anti-kinesin antibodies. We identified and characterized a 105-kD organelle-associated motor protein that is functionally, biochemically, and immunologically related to kinesin. This work provides clear evidence that the movement of pollen tube organelles is not just actin based; rather, they show a microtubule-based motion as well. This unexpected finding suggests new insights into the use of pollen tube microtubules, which could be used for short-range transport, as actin filaments are in animal cells.
花粉管细胞器的移动依赖于细胞骨架成分。尽管花粉管中细胞器沿肌动蛋白丝的移动已得到广泛研究且逐渐明晰，但微管发挥何种作用仍不清楚。关于微管在花粉管细胞器主动运输及/或该过程控制中的作用，仍有许多不确定因素有待解决。为了确定在没有肌动蛋白的情况下细胞器是否能够沿微管移动，我们从烟草花粉管中提取了细胞器，并分析了它们在不同实验条件下沿体外聚合微管移动的能力。无论大小如何，细胞器在ATP存在的情况下沿微管以不同速率移动。细胞松弛素D不抑制细胞器移动，这表明在我们的实验中肌动蛋白丝对于细胞器运输不是必需的。细胞器的移动不依赖于胞质溶胶，这表明可溶性因子对于细胞器移动的发生不是必需的，并且基于微管的运动蛋白存在于细胞器表面。通过用KI洗涤细胞器，可以释放能够使羧化珠子沿微管滑动的蛋白质。通过蔗糖密度梯度离心分离的几个膜组分显示出基于微管的移动。通过KI处理从活性最高的细胞器组分中提取蛋白质，然后用ATP敏感的微管结合试验进行分析。通过与微管的选择性结合分离的蛋白质在体外运动试验中测试其使微管滑动的能力、微管刺激的ATP酶活性的存在以及与抗驱动蛋白抗体的交叉反应性。我们鉴定并表征了一种105-kD的细胞器相关运动蛋白，其在功能、生化和免疫方面与驱动蛋白相关。这项工作提供了明确的证据，表明花粉管细胞器的移动不仅仅基于肌动蛋白；相反，它们也显示出基于微管的运动。这一意外发现为花粉管微管的用途提供了新的见解，花粉管微管可用于短距离运输，就像动物细胞中的肌动蛋白丝一样。
