Abstract
Filamin A is a homodimeric actin-binding protein that crosslinks actin filaments to define cell shape and movement. In addition to cytoskeleton remodelling, Filamin A acts as a versatile molecular scaffold, known to interact with over a hundred diverse cellular proteins. Because of its extensive roles, the pathogenic variants of Filamin A can give rise to a spectrum of human disorders that can affect multiple body organ systems.
Originally identified as a cytoplasmic protein, later research revealed Filamin A as a nuclear protein too. The structure of Filamin A comprises of an N-terminal actin-binding domain and 24 immunoglobulin-like repeat units that are separated by two regions of low complexity known as Hinge-1 and Hinge-2 which adds mechanical flexibility to the otherwise rigid protein. Filamin A is susceptible to proteolytic cleavage by the calcium-dependent protease calpain at these hinge regions. It is proposed that nuclear translocation of Filamin A relies on calpain-dependent cleavage. However, reports of full-length Filamin A in the nucleus, suggests that cleavage might not be required for nuclear translocation. Thus, the subcellular localization of Filamin A remains controversial. This study aimed to determine the cell-cycle dependent localization of Filamin A and understand if nuclear entry is Hinge-1 independent.
Cells were synchronised to the early S phase using double thymidine block and collected at various time intervals following release. Immunofluorescence staining and confocal microscopy revealed that Filamin A localization is linked to progression through the cell-cycle. In interphase, Filamin A is largely cytoplasmic, with some amounts in the nucleus too. In prophase, Filamin A localises near the peripheral membrane to maintain cortical rigidity of rounding cells. During metaphase and anaphase a remarkable association of Filamin A and the mitotic spindles is observed, defining a potential role of Filamin A in orchestrating the organisation and distribution of microtubules and chromosomes. During cytokinesis, Filamin A is highly concentrated in both cytoplasm and nucleus, with nuclear levels declining towards the end of cytokinesis as the cell completes division. Overall, the results are consistent with the hypothesis that the subcellular localization of Filamin A is cell-cycle dependent. Understanding the localization of Filamin A could potentially explain biological mechanisms behind disease pathogenesis and support future research aiming to design Filamin A therapeutics.