Centro de Biologia Ambiental/Departamento de Biologia Animal, Faculdade de Ciências, Universidade de Lisboa, Lisbon, Portugal; Instituto Gulbenkian de Ciência, Oeiras, Portugal.
Differentiation. 2013 Jul-Sep;86(1-2):48-56. doi: 10.1016/j.diff.2013.07.001. Epub 2013 Aug 20.
Akt is a highly conserved serine-threonine protein kinase which has been implicated in a wide variety of cellular functions, from the regulation of growth and metabolism, to activation of pro-survival pathways and cell proliferation, and promotion of differentiation in specific cell types. However, very little is known about the spatial and temporal pattern of Akt activity within cells and whether this pattern changes as cells enter and proceed in their differentiation programs. To address this issue we profiled Akt activation in E8.5-E13.5 mouse embryos and in C2C12 cells. We used a commercial antibody against Akt, phosphorylated on one of its activating residues, Thr-308, and performed high resolution confocal imaging of the immunofluorescence in labeled embryos. We observe strong Akt activity during mitosis in the dermomyotome, the neuroepithelium and some mesenchymal cells. This burst of activity fills the whole cell except for heterochromatin-positive areas in the nucleus. A surge in activity during mitosis is also observed in subconfluent C2C12 cells. Later on in the differentiation programs of skeletal muscle and neural cells, derivatives of the dermomyotome and neuroepithelium, respectively, we find robust, sustained Akt activity in the cytoplasm, but not in the nucleus. Concomitantly with skeletal muscle differentiation, Akt activity becomes concentrated in the sarcomeric Z-disks whereas developing neurons maintain a uniform cytoplasmic pattern of activated Akt. Our findings reveal unprecedented cellular and subcellular details of Akt activity during mouse embryo development, which is spatially and temporally consistent with proposed functions for Akt in mitosis and myogenic and neural differentiation and/or survival. Our results thus demonstrate a subcellular change in the pattern of Akt activation when skeletal muscle and neural progenitor cells cease dividing and progress in their differentiation programs.
Akt 是一种高度保守的丝氨酸/苏氨酸蛋白激酶，涉及多种细胞功能，从调节生长和代谢，到激活促生存途径和细胞增殖，以及促进特定细胞类型的分化。然而，关于 Akt 在细胞内的时空活性模式以及这种模式是否随着细胞进入并进行分化程序而改变，我们知之甚少。为了解决这个问题，我们对 E8.5-E13.5 天小鼠胚胎和 C2C12 细胞中的 Akt 激活进行了分析。我们使用了一种针对 Akt 的商业抗体，该抗体磷酸化其一个激活残基 Thr-308，并对标记胚胎中的免疫荧光进行了高分辨率共聚焦成像。我们观察到在真皮肌和神经上皮以及一些间质细胞的有丝分裂过程中，Akt 活性很强。这种活性爆发充满了整个细胞，除了核内异染色质阳性区域。在未完全汇合的 C2C12 细胞中也观察到有丝分裂过程中的活性激增。在真皮肌和神经细胞的分化程序后期，分别为真皮肌和神经上皮的衍生物，我们在细胞质中发现了强大而持续的 Akt 活性，但在核内没有。随着骨骼肌分化，Akt 活性集中在肌节的 Z 盘上，而发育中的神经元则保持激活的 Akt 的均匀细胞质模式。我们的发现揭示了在小鼠胚胎发育过程中 Akt 活性的前所未有的细胞和亚细胞细节，这与 Akt 在有丝分裂、肌生成和神经分化和/或存活中的功能是空间和时间一致的。因此，我们的研究结果表明，当骨骼肌和神经祖细胞停止分裂并进入分化程序时，Akt 激活的模式发生了亚细胞变化。
