Nucleoplasmic β-actin exists in a dynamic equilibrium between low-mobility polymeric species and rapidly diffusing populations

Abstract

β-Actin, once thought to be an exclusively cytoplasmic protein, is now known to have important functions within the nucleus. Nuclear β-actin associates with and functions in chromatin remodeling complexes, ribonucleic acid polymerase complexes, and at least some ribonucleoproteins. Proteins involved in regulating actin polymerization are also found in the interphase nucleus. We define the dynamic properties of nuclear actin molecules using fluorescence recovery after photobleaching. Our results indicate that actin and actin-containing complexes are reduced in their mobility through the nucleoplasm diffusing at ∼0.5 μm2 s−1. We also observed that ∼20% of the total nuclear actin pool has properties of polymeric actin that turns over rapidly. This pool could be detected in endogenous nuclear actin by using fluorescent polymeric actin binding proteins and was sensitive to drugs that alter actin polymerization. Our results validate previous reports of polymeric forms of nuclear actin observed in fixed specimens and reveal that these polymeric forms are very dynamic.

Induction of HoxB Transcription by Retinoic Acid Requires Actin Polymerization

“…The observation in a recent FRAP analysis that ϳ20% of the total nuclear actin pool is in the polymeric state supports this idea (McDonald et al, 2006). Importantly transcription is affected by the down-regulation of N-WASP or by inhibiting actin polymerization either through the expression of polymerization-deficient actin mutants or by using high concentrations of specific drugs, such as cytochalasin D (CytD) or latrunculin A (LatA; McDonald et al, 2006;Wu et al, 2006;Yoo et al, 2007;Ye et al, 2008). These observations indicate that actin polymerization is necessary for gene transcription.…”

“…However, these data were obtained at a concentrations (1 M) of CytD 10-fold higher than those used in the present article, in which we nevertheless achieved a strong, but incomplete inhibition of actin polymerization (McDonald et al, 2006). The intriguing evidence that different subsets of genes could be differentially sensitive to the inhibition of actin polymerization is new.…”

Active maintenance of nuclear actin by importin 9 supports transcription

“…The first phase with the shortest half life of 7 s is likely to correspond to the rapidly moving actin monomers because it is present for both constructs. For GFP-actin, the half life of the second phase is 230 s and it corresponds in amplitude to the polymeric phase witnessed previously (20). The half life of the longest phase in the wild-type curve was 1,800 s, and thus demonstrates that a substantial population of actin (approximately 60%) is relatively tightly associated with nuclear complexes, and is relatively slowly exchanged from them.…”

“…Indeed, actin and nuclear myosins have been implicated in the movement of individual gene loci upon transcription activation (14)(15)(16) and even in rearranging whole chromosomes within the nucleus (17). Furthermore, although the functional conformation of nuclear actin is still under debate (18,19), many of the nuclear actin related functions mentioned above are disrupted upon treatment of cells with drugs affecting actin polymerization (15,20,21), suggesting that actin polymerization plays a role also in the cell nucleus.…”

Cleavage of DAP5 by coxsackievirus B3 2A protease facilitates viral replication and enhances apoptosis by altering translation of IRES-containing genes

“…Histone-1 was used as a nuclear purity control and β-actin was used as a loading control, which is present in both the nucleus and cytoplasm. 55,56 Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Amersham GE, Buckinghamshire, UK). Membranes were blocked in 5% skim milk in Tris-buffered saline 10% Tween (TBST) buffer for 1 h and subsequently incubated with one of the following primary antibodies against DAP5-N (Thermo Scientific), DAP5-C (Cell Signaling Technologies, Danvers, MA, USA), Oct-8 (FLAG), HA and p53 (Santa Cruz Biotechnology Inc.), Bcl-2 (Cell Signaling Technologies), caspase-3 (Cell Signaling Technologies), PARP (Santa Cruz Biotechnology Inc.), phospho-eIF4E (Ser209) (Santa Cruz Biotechnology Inc.), total eIF4E (Santa Cruz Biotechnology Inc.), VP1 (Dako, Santa Clara, CA, USA) and β-actin (Sigma) at 4°C overnight.…”