Signaling and cytoskeletal requirements in erythroblast enucleation
Signaling and cytoskeletal requirements in erythroblast enucleation
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DOI：
10.1182/blood-2011-09-379263
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发表时间：
2012-06-21
期刊：
影响因子：
20.3
通讯作者：
Kalfa, Theodosia A.
中科院分区：
文献类型：
--
作者：
Konstantinidis, Diamantis G.;Pushkaran, Suvarnamala;Kalfa, Theodosia A.
为了了解细胞骨架和膜信号分子在成红细胞去核中的作用，我们开发了一种新的多参数高速细胞流动成像分析方案。该方案使我们能够观察到F-肌动蛋白和磷酸化肌球蛋白调节轻链（pMRLC）组装成一个收缩的肌动球蛋白环（CAR）之间的新生网织红细胞和核，在人口的去核成红细胞。CAR形成和随后的去核在Rac 1和Rac 2 GTP酶基因缺失的小鼠成红细胞中不受影响，因为Rac 3的补偿。所有Rac GTP酶的药理学抑制或基因缺失改变了F-肌动蛋白和pMRLC的分布，并抑制去核。用NSC 23766、细胞松弛素-D、秋水仙碱、ML 7或分别抑制Rac活性、肌动蛋白或微管蛋白聚合、MRLC磷酸化或脂筏组装的菲律宾蛋白处理的成红细胞表现出降低的去核效率，如通过流式细胞术定量的。如通过高速流动成像分析所评估的，秋水仙碱抑制成红细胞极化，在去核的准备阶段涉及微管，而NSC 23766导致在网织红细胞-蛋白核细胞边界中不存在脂筏组装。总之，去核是一个多步骤的过程，类似于胞质分裂，需要通过微管功能建立细胞极性，然后形成收缩性肌动球蛋白环，以及网织红细胞和蛋白核细胞之间的脂筏聚结。（血。2012;119（25）：6118-6127）
To understand the role of cytoskeleton and membrane signaling molecules in erythroblast enucleation, we developed a novel analysis protocol of multiparameter high-speed cell imaging in flow. This protocol enabled us to observe F-actin and phosphorylated myosin regulatory light chain (pMRLC) assembled into a contractile actomyosin ring (CAR) between nascent reticulocyte and nucleus, in a population of enucleating erythroblasts. CAR formation and subsequent enucleation were not affected in murine erythroblasts with genetic deletion of Rac1 and Rac2 GTPases because of compensation by Rac3. Pharmacologic inhibition or genetic deletion of all Rac GTPases altered the distribution of F-actin and pMRLC and inhibited enucleation. Erythroblasts treated with NSC23766, cytochalasin-D, colchicine, ML7, or filipin that inhibited Rac activity, actin or tubulin polymerization, MRLC phosphorylation, or lipid raft assembly, respectively, exhibited decreased enucleation efficiency, as quantified by flow cytometry. As assessed by high-speed flow-imaging analysis, colchicine inhibited erythroblast polarization, implicating microtubules during the preparatory stage of enucleation, whereas NSC23766 led to absence of lipid raft assembly in the reticulocyte-pyrenocyte border. In conclusion, enucleation is a multistep process that resembles cytokinesis, requiring establishment of cell polarity through microtubule function, followed by formation of a contractile actomyosin ring, and coalescence of lipid rafts between reticulocyte and pyrenocyte. (Blood. 2012;119(25):6118-6127)