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Structural protein 4.1, which has crucial interactions within the spectrin-actin lattice of the human red cell membraneskeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higher-resolution detergent-extracted cell whole-mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin during nuclear assembly. By directly imaging actin under nonperturbing conditions, the total nuclear actin population is retained and visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As nuclear lamina assembled, but preceding DNA synthesis, actin distributed in a reticulated pattern throughout the nucleus. Protein 4.1 epitopes also were detected when actin began to accumulate in nuclei, producing a diffuse coincident pattern. As nuclei matured, actin was detected both coincident with and also independent of 4.1 epitopes. To test whether acquisition of nuclear actin is required for nuclear assembly, the actin inhibitor latrunculin A was added to Xenopus egg extracts during nuclear assembly. Latrunculin A strongly perturbed nuclear assembly and produced distorted nuclear structures containing neither actin nor protein 4.1. Our results suggest that actin as well as 4.1 is necessary for nuclear assembly and that 4.1-actin interactions may be critical.
Fig. 1. Colocalization of nuclear actin and 4.1 in human diploid fibroblasts. (A) Double-label immunofluorescence of 4.1 SABD epitopes (green) and actin (red) in WI38 cells extracted in situ to prepare nuclear matrix. The merged image shows a high degree of coincidence (yellow). (B) Transient cotransfection of WI38 cells with YFP-actin (red) and CFP-80kD4.1R (green). Cells were imaged 4–72 h posttransfection. The 24-h image presented shows expression of YFP-actin in both cytoplasm and nucleus, whereas CFP-4.1 is exclusively nuclear, generating coincident nuclear signals (yellow). (Bar, 10 μm.)
Fig. 2. Immunolocalization of nuclear actin and 4.1 SABD epitopes by double-label immuno-EM of WI38 human fibroblasts. In detergent-extracted cell whole mounts, actin epitopes (10-nm beads) and 4.1 SABD epitopes (5-nm beads) decorated fibrous structures near dense structures in nuclear matrix. At the top is a stereo-pair image of a nuclear matrix region with boxes AâC indicating areas presented as stereo pairs at higher magnification in AâC, respectively. The arrow in C points to intermingled 5- and 10-nm beads.
Fig. 3. Acquisition of actin during nuclear assembly in vitro. Fluorescent actin (red) was imaged as nuclei assembled in Xenopus egg extract after the addition of demembranated sperm (DNA, blue) in a representative experiment (n = 4). Diffuse actin was detected in assembling pronuclei only after 30 min. As mature nuclei assembled (50–70 min), an intranuclear actin network-like pattern became apparent.
Fig. 4. Acquisition of nuclear actin relative to nuclear pores, lamina formation, and DNA synthesis during nuclear assembly in vitro. Nuclei from Xenopus egg extract reactions spiked with fluorescent actin (red) were isolated at the times indicated, fixed, and probed with mAb414 against nuclear pore proteins (A, green) or antibody L046F7 against lamin (B, green). DNA synthesis was detected with anti-BrdUrd (C, green). Controls without BrdUrd had no detectable green signals. In merged images of mature nuclei, BrdUrd signals coincide with DNA (blue) as expected (aqua), and actin appears noncoincident. Diffuse actin and irregularly distributed pores are detected at 30 min, lamin epitopes at 40 min, and BrdUrd at 50 min. Assembly times vary with extract, but the relative sequence of actin acquisition, pore assembly, lamina formation, and DNA synthesis was maintained in three independent experiments.
Fig. 5. Nuclear actin and 4.1. (A) Acquisition of actin relative to 4.1 SABD epitopes during nuclear assembly in vitro. Nuclei (DNA, blue) assembled in Xenopus egg extract spiked with fluorescent actin (red) were fixed and probed with anti-4.1 SABD (green). Data presented, from one of three independent experiments, used the same extract as described for Fig. 4. Diffuse actin and SABD epitopes detected at 30 min in the merge were largely coincident (yellow). At later times, coincidence of actin with 4.1 foci was apparent along with some areas of noncoincidence. (B) Latrunculin A (Lat A) inhibition of nuclear assembly in vitro (Right). Nuclei assembled in Xenopus egg extracts containing fluorescent actin (red) and 0.1 mM latrunculin A had highly aberrant chromatin morphology (94%; DNA, blue), and actin was not detected by fluorescence microscopy. (Left) A control nucleus. An equal concentration of DMSO (latrunculin vehicle) had no effect on nuclear assembly. (C) By Western blot analysis of equivalent numbers of nuclei from reactions with 0.1 mM latrunculin A or dominant negative 4.1 SABD peptides, actin was not detected, whereas it was readily detected in controls (Left). Nuclei perturbed by 0.1 mM latrunculin A also had no detectable 4.1 epitopes when probed with anti-4.1 SABD or anti-4.1 C terminus (αCTD) (Right).
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