INTRANUCLEAR NETWORK OF 3–5nm AND 8–10nm FIBERS IN EL‐4 LYMPHOMA CELLS

Abstract

While much evidence indicates a high degree of spatial organization in the nucleus, the underlying molecular structures that support it remain poorly characterized. By extracting with high concentrations of RNase A in a modification of the sequential extraction protocol of Penman, we have identified a novel intranuclear network in the mouse lymphoma cell line, EL-4. Micrographs of embedment-free sections of extracted cells reveal anastomosing filaments of two different diameters: 3-5 nm and 8-10 nm. The 3-5-nm filaments are interconnected in many junctions and appear to blend smoothly into each other. The 8-10-nm fibers frequently split into two 3-5-nm filaments. Some 3-5-nm fibers appear to be connected at 90 degrees angles with the 8-10-nm fibers. All junctions are smooth with no apparent junction protein. Flow cytometric analysis of RNase A- (and DNase I-) extracted nuclear matrices indicates that they do not contain significant amounts of protein that react with anti-actin and anti-vimentin monoclonal antibodies. Extraction of EL-4 nuclear matrices with high salt does not reveal 8-10-nm core filaments described after similar treatment of tumor cell lines of cervical and mammary origin. The novel characteristics of the core filaments in EL-4 lymphoma cells may reflect cell-type specificity of the nuclear matrix.

Amino-terminal Polypeptides of Vimentin Are Responsible for the Changes in Nuclear Architecture Associated with Human Immunodeficiency Virus Type 1 Protease Activity in Tissue Culture Cells

“…Although the effects on the cytoplasmic cytoskeletal structures are readily explained by the cleavage of their integral or associated proteins (Shoeman et al, 1990a(Shoeman et al, , 1993, the events giving rise to the nuclear alterations have remained an enigma. These nuclear changes occur most rapidly at the lowest concentrations of HIV-1 PR tested and closely resemble those described for a variety of tissues examined in HIV-1-infected individuals (Shoeman et al, 1992, for discussion and references).Because several IF subunit proteins are excellent substrates for HIV-1 PR (Shoeman et al, 1990a) and because the nuclear matrix core filaments are morphologically indistinguishable from the cytoplasmic IFs (Jackson and Cook, 1988;He et al, 1990;Wang and Traub, 1991;Padros et al, 1997), it seemed likely that one or more components of the nuclear matrix might be cleaved by HIV-1 PR and be responsible for the alterations in nuclear structure. Although it was possible to visualize alterations in residual cellular and nuclear structures in HIV-1 PR-treated cells, it was not possible to correlate the cleavage of a nuclear protein (e.g., NuMA; the nuclear mitotic apparatus protein) with the changes in nuclear architecture.…”

“…Although many mammalian cells are known to contain deep tubular invaginations of the nuclear envelope (Fricker et al, 1997), some of which are associated with perinuclear rings of IFs (Kamei, 1994), and nuclei of the cell line SW 13 are generally irregular in shape (Sarria et al, 1994), the invaginations described in this article in HSF and SW 13 T3 M [vimentin ϩ ] cells correlate with treatment with the HIV-1 PR. Given the propensity for HIV-1 PR to cleave IF proteins (Shoeman et al, 1990a), we had originally thought that the nuclear matrix core filaments, which are morphologically indistinguishable from the cytoplasmic IFs (Jackson and Cook, 1988;He et al, 1990;Wang and Traub, 1991;Padros et al, 1997), or other compo- Figure 4. Microinjection of the mixture of vimentin cleavage products produced in vitro by the action of HIV-1 PR or a defined vimentin peptide encompassing the entire amino-terminal head domain into SW 13 [vimentin Ϫ ] cells also produces nuclear chromatin condensation and redistribution, but has no effect on nuclear shape.…”

“…Because several IF subunit proteins are excellent substrates for HIV-1 PR (Shoeman et al, 1990a) and because the nuclear matrix core filaments are morphologically indistinguishable from the cytoplasmic IFs (Jackson and Cook, 1988;He et al, 1990;Wang and Traub, 1991;Padros et al, 1997), it seemed likely that one or more components of the nuclear matrix might be cleaved by HIV-1 PR and be responsible for the alterations in nuclear structure. Although it was possible to visualize alterations in residual cellular and nuclear structures in HIV-1 PR-treated cells, it was not possible to correlate the cleavage of a nuclear protein (e.g., NuMA; the nuclear mitotic apparatus protein) with the changes in nuclear architecture.…”

MODY1 mutation Q268X in hepatocyte nuclear factor 4alpha allows for dimerization in solution but causes abnormal subcellular localization.

“…pMT7.HNF4.wt and pMT7.HNF4.Q268X were overexpressed in COS-7 cells, and nuclear extracts were prepared using hypotonic lysis followed by extraction in 0.43 mmol/l KCl in buffer H for 45 min at 4°C as previously described (20). Where indicated, subsequent DNase I and RNase A treatment conditions were adapted from previously described protocols (23,24). Briefly, after salt extraction, the remains of the nuclei were rinsed in DNase I buffer (10 mmol/l Pipes, pH 7.0, 300 mmol/l sucrose, 50 mmol/l NaCl, 3 mmol/l MgCl 2 , 1 mmol/l EGTA) and then treated with 16 units/µl DNase I (Boehringer Mannheim) for 50 min at 32°C.…”

RNA‐dependent nuclear matrix contains a 33 kb globin full domain transcript as well as prosomes but no 26S proteasomes

“…On the other hand, early work had already shown that up to 300 different proteins are constituents of this nuclear backbone, and that protein composition of the nuclear matrix changed in relation with species and cell differentiation [Capco et al, 1982]. In the last 10 years, electron micrographs from several laboratories have demonstrated more precisely filamentous networks of the nuclear matrix which were found to contain RNP particles as well as actin and lamins [Gounon and Karsenti, 1981; Hozak et al, 1995; Padros et al, 1997; Rando et al, 2000; Okorokov et al, 2002; Andrin and Hendzel, 2004].…”