The nuclear envelope (NE) consists of two adjacent membranes, the inner (INM) and the outer nuclear membrane (ONM). The ONM is functionally connected to the endoplasmic-reticulum (ER) and contains numerous ribosomes, while the INM contains a unique set of transmembrane proteins and maintains close contact with chromatin in the nuclear matrix through the nuclear lamina network [1,2]. The ONM/ER comprising a single continuous membrane system is connected to the INM through nuclear pore complexes (NPC), which constitute the unique gateway for macromolecular transport across the nuclear-cytoplasmic boundary [3,4]. The cytoskeletal protein actin may play a role in NE function. This view has received support from recent evidence suggesting that the NE-membrane system is physically connected to the cytoplasmic microfilament network. For example, ONM proteins containing giant spectrin repeats or SUN (for Sad1p, UNC-84 homology) domains are involved in nuclear anchorage and migration, probably via actin filament interactions [5,6]. In metazoan cells, the integral ONM protein nesprin (member of the Syne/ANC-1 protein family) contains an α-actinin like actin-binding domain potentially capable to link the NE-membrane to the cytoplasmic actin cytoskeleton .
Actin is a highly conserved cytoskeletal protein that is distributed in a dynamic equilibrium between the ~42 kD monomeric (G-actin) and the filamentous polymerised (F-actin) forms. Actin's endogenous, polymeric properties are preserved in vitro and have been extensively characterised [8,9]. Polymerisation occurs at the so-called critical concentration, where non-covalent interactions overcome a rate limiting step yielding trimeric nucleation complexes to which monomers associate spontaneously and seed filament growth . Inside living cells, the non-steady-state dynamics of actin polymerisation are highly regulated by an array of actin binding proteins (for review see [11-13]). Actin binding proteins regulate changes in actin's polymerisation state within functional sub-cellular domains or compartments allowing for actin's role in a variety of cellular processes as diverse as signal transduction and cell motility. Importantly, the ONM protein nesprin enhances actin polymerisation rates, shortens filament elongation times, and increases filament bundling in vitro. Therefore, it has been hypothesised that nesprin (or other cytoskeletal microfilament binding ONM proteins) can recruit actin to the cytoplasmic ONM interface .
Previously, ultra-structural studies using electron microscopy in fixed material have demonstrated "filament-like" networks continuous with the NE-membrane and NPC [14,15]. Moreover, light microscopic studies have reported a fine perinuclear "shell" of actin filaments around the nuclei of fixed cultured cells . In the current study we visualised actin polymerisation in proximity to the NE-membrane inside living cells. This approach has been technically challenging, in part because the compartmentalised gradient is expected to be small (compared with large backgrounds coming from actin involved in other processes). We overcame these difficulties, using a variety of fluorescent probes for actin and confocal fluorescence imaging methods. We report perinuclear actin inside intact living cells during interphase, and we provide evidence that perinuclear actin polymerisation involves binding at the ONM of the NE-membrane.