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There exists abundant molecular and ultra-structural evidence to suggest that cytoplasmic actin …

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- Actin polymerisation at the cytoplasmic face of eukaryotic nuclei

Previous studies using conventional approaches in fixed cells have described evidence for perinuclear actin in a variety of cell types including giant polyploid silk gland epithelial cells [25], insect cells [26], amobea [27] and fungi [28]. In vertebrate tissue cells a perinuclear actin basket, revealed by phalloidin has also been reported [29,30]. However, these studies were limited to a visualisation of actin after cell fixation, and lacked information on actin polymerisation, in situ, ie inside living cells. In the current study, we provide evidence for perinuclear actin polymerisation using spinning disk confocal microscopy. Our results show that inside intact living cells actin polymerises at NE-membranes, in a manner involving free-barbed-end protomers and through yet unidentified molecular components at the cytoplasmic face of the NE-membrane.

Actin is one of the most abundant proteins in eukaryotic cells and this exacerbates detection of specifically localised subcellular compartments or gradients, because the vast majority of measured signal is derived from actin bound in very bright structures (e.g.: stress fibers) or G-actin. Evidently, this is less problematic for studies investigating actin polymerisation in more accessible regions for imaging e.g. actin polymerisation at the leading edge of the plasma membrane [8,31-33]. By contrast, in the current study our interest was focused on perinuclear actin. To visualise actin in the perinuclear cytoplasm we used spinning disk confocal microscopy in order to maximise three-dimensional resolution. With this approach it was striking that a nuclear actin ring was observed using several types of probes for visualisation, but was not readily observed using conventional wide-field imaging. The need for spinning disk confocal microscopy followed by deconvolution can explain why the perinuclear actin ring has until now not been described inside living cells.

Using confocal imaging our results reveal that cytoplasmic micro-injection led, within minutes, to accumulation of fluorescent actin at the NE-membrane. This rapid accumulation of micro-injected actin fits with the idea that the perinuclear pool of actin and cytoplasmic actin are in dynamic equilibrium. One mechanism for cytoplasmic G-actin to interact with the perinuclear actin pool could be that the latter concentrates actin binding sites that present free-barbed-ends to which free cytoplasmic G-actin may further associate. Certainly, our results using CD-BODIPY are highly consistent with this view. We have shown that CD-BODIPY that binds free-barbed-end actin with high affinity in vitro, yields a labelling pattern coincident with the NE-membrane in situ. Pertinently, the NE-membrane associated CD-BODIPY signal was observed to extend into the perinuclear cytoplasm, but never into the nuclear matrix, suggesting that the signal is continuous with the cytoplasmic actin cytoskeleton. However, given the sensitivity and resolution limits of optical microscopy this does not rule out the possibility that actin polymerisation could be physically located at the inner-face of the NE-membrane.

A recent study has shown that microinjected actin is localised to the NE-membrane of starfish oocytes, in a ring pattern around the nucleus [34] analogous to our findings presented in the current work. This study showed that a transient burst of actin polymerisation occurs upon NE-membrane fenestration marking the beginning of nuclear envelope breakdown (NEBD), and M-phase progression. Using ultra-structural electron microscopy Lenard et al. localised actin polymerisation to the inner face of the NE-membrane in accordance with their model. Our current study using high-resolution multidimensional light microscopy raised the same question: i.e. whether polymerisation occurs at the inner-(nuclear matrix) or outer-(cytoplasmic) face of the NE-membrane. By contrast to the actin polymerisation upon NEBD, our results suggest that perinuclear actin polymerisation in interphase nuclei of metazoan cells is driven from physical interactions at the cytoplasmic side of the NE-membrane. In vitro, we observed long actin filaments propagating outwards from intact nuclei isolated from differentiated rat liver cells, and incubated under conditions favouring actin polymerisation. Furthermore, nuclei were intact and apparently impermeable (in the absence of GTP) to actin because we never observed fluorescent actin inside the nuclear matrix. Additional argument supporting the view that actin binds the cytoplasmic face of isolated nuclei was derived from our results indicating that actin polymerisation was strongly diminished in the presence of antibodies (or lectin) targeting ONM proteins, but not INM. Both antibody (mAb414) and lectin targeting ONM nucleoporins inhibited actin polymerisation as strongly as anti-nesprin (a non-nucleoporin target). Therefore we hypothesise that these effects were due to molecular steric hindrance of a cytoplasmic ONM actin binding site closely juxtaposed with nucleoporins and/or non-nucleoporins. This interpretation is also consistent with our observation that no actin labelling method yielded precise co-localisation with the nucleoporin POM121. Despite the uncertainty concerning whether nucleoporins and/or non-nucleoporins provide the actin binding site, our results do demonstrate that intact interphase nuclei support actin polymerisation at the cytoplasmic face of ONM. Interestingly, previous studies on isolated patch-clamped nuclei have provided evidence that NPC ionic permeability measured by electrophysiology can be modulated by actin added to the ionic bathing solution suggesting that the effect involved cytoplasmic ONM interactions [35]. Moreover, a role for NPC-associated cytoplasmic cytoskeleton filaments in regulation of NPC transport functions has been postulated [14]. In either case, taken together with our current results, these hypotheses provide for a plausible functional impact linking the signalling milieu of the cytoplasmic actin cytoskeleton with NPC structure and function.

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