Dokument

Summary:
Nuclear actin and its biological relevance raised much interest in the past few years. Although its existence was initially discussed controversially, more recent data provided insights into its functions and address stimuli as well as dynamics of nuclear actin filaments. Hence, our group recently showed that nuclear volume expansion in early G1 of mammalian cells required a network of transient and dynamic F-actin inside the daughter nuclei. However, only few regulatory pathways in nuclear F-actin formation were identified, while higher organization of actin filaments remained mostly uninvestigated. As such, the roles of actin crosslinking proteins were less well explored, but members of the spectrin repeat family were proposed to have nuclear functions. For instance, nucleocytoplasmic shuttling of the actin-bundling protein Alpha actinin-4 was associated with invasive cancer phenotypes. We therefore decided to investigate non-muscle Alpha actinins and their roles in early G1 nuclei. We initially confirmed cytoplasmic and nuclear localization of Alpha actinin-4 in interphase and early G1 cells and its interaction with nuclear F-actin by immunostaining and phalloidin pulldown experiments. Subcellular fractionations and subsequent Co-immunoprecipitations revealed the presence of Alpha actinin-1 SNAP Flag in the nuclear compartment. Imaging of histone 2B mCherry expressing cells during early G1 and following 3D reconstructions allowed for nuclear volume analysis and revealed that Alpha actinin-4, but not Alpha actinin-1 was required for proper nuclear volume expansion. We further visualized nuclear F-actin (bundles) in fixed postmitotic cells by super-resolution microscopy. Expression of a nuclear targeted dominant negative mutant of Alpha actinin-4 exhibited a significant reduction of phalloidin-AlexaFluor647 localizations per nucleus compared to the wild-type construct. These nuclei further represented fewer actin filament bundles with reduced widths, suggesting that Alpha actinin-4 is a key regulator of nuclear F-actin bundling and proper nuclear volume expansion in early G1. Further investigations revealed an additional involvement of nuclear Alpha actinin-4 in other cellular processes such as proliferation. Together, this work provides information about crucial functions of Alpha actinin-4 in the nuclear compartment of postmitotic mammalian cells and might therefore aid future investigations into its role for tumorigenesis and metastasis.

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