Cells were microinjected with four mouse monoclonal antibodies that were directed against either alpha-or beta-tubulin subunits, one monoclonal with activity against both subunits, and a guinea pig polyclonal antibody with activity directed against both subunits, to determine the effects on the distribution of cytoplasmic microtubules and 10-nm filaments . The specificities of the antibodies were confirmed by Western blots, solid phase radioimmunoassay, and Western blot analysis of alpha-and beta-tubulin peptide maps. Two monoclonals DM1A and DM3B3, an anti-alpha-and anti-beta-tubulin respectively, and the guinea pig polyclonal anti-alpha/beta-tubulin antibody (GP1T4) caused the 10-nm filaments to collapse into large lateral aggregates collecting in the cell periphery or tight juxtanuclear caps; the other monoclonal antibodies had no effect when microinjected into cells . The filament collapsing was observed to be complete at 1 .5-2 h after injection . During the first 30 min after injection a few cytoplasmic microtubules near the cell periphery could be observed by fluorescence microscopy. This observation was confirmed by electron microscopy, which also demonstrated assembled microtubules in the juxtanuclear region . By 1 .5 h, when most of the 10-nm filaments were collapsed, the complete cytoplasmic array of microtubules was observed . Cells injected in prophase were able to assemble a mitotic spindle, suggesting that the antibody did not block microtubule assembly . Metabolic labeling with [35S]methionine of microinjected cells revealed that total protein synthesis was the same as that observed in uninfected cells. This indicated that the microinjected antibody apparently did not produce deleterious effects on cellular metabolism . These results suggest that through a direct interaction of antibodies with either alpha-or beta-tubulin subunits, 10-nm filaments were dissociated from their normal distribution . It is possible that the antibodies disrupted postulated 10-nm filament-microtubule interactions. l0-nm filament-microtubule interaction have been postulated based on studies by electron microscopy (4,17,26,32,53,55,58), immunofluorescence microscopy (8, 24), and biochemistry (43,54,55) in many eucaryotic cells. When cells are incubated in the presence of antimicrotubule drugs, such as colchicine, colcemid, or vinblastine, and cytoplasmic microtubules depolymerize (51) and the 10-nm filaments subsequently coil into juxtanuclear caps (9, 26) or collapse into large aggregates of filaments in the cytoplasm (12,28,33,35). Upon release from these drugs, the microtubules rapidly reassemble into their original cytoplasmic array (51) and the 10-nm filaments then uncoil and redistribute with the majority of microtubules (26). Although it is not known if these THE JOURNAL OF CELL BIOLOGY " VOLUME 9S MARCH 1984 847-858 ® The Rockefeller University Press " 0021-9525/84/03/0847/12 $1 .00 drugs directly affect the 10-nm filaments, it is thought that as a result of microtubule depolymerization, 10-nm filaments c...

The adenovirus early-region lB 19,000-molecular-weight tumor antigen is required for oncogenic transformation of cells by adenovirus. We have demonstrated that this tumor antigen is located in the nuclear envelope of infected and transformed cells and that a fraction of the protein within the nuclear envelope is associated with the nuclear lamina. During cell division in the transformed cells, the nuclear envelope containing the tumor antigen dissociates at metaphase and then reforms around the separated daughter chromosomes at telophase. Adenovirus mutants carrying lesions in the gene encoding this tumor antigen cause degradation of host cell chromosomal DNA, and in these mutants, the intracellular localization of the 19,000-dalton protein is altered. These results demonstrate that components of the nuclear envelope function in the organization of chromatin in infected and transformed cells and that a virus-encoded protein plays a critical role in this process.

Vascular endothelial cells cultured from guinea pig aorta or portal vein contain naturally occurring bundles of 100 A (diameter) filaments that completely encircle the nucleus. These rings are phase lucent and birefringent when examined with the light microscope. Perinuclear bundles of 100 A filaments were also seen in endothelial cells in vivo, indicating that they are a normal cytoplasmic component. These filaments did not decorate with S-1, and were not disrupted by glyceination. With these cells, experiments were designed to answer the following questions: (a) does Colcemid have an effect on these naturally occuring bundles? And (b) do these filaments remain during cell division? Endothelial cells grown in the presence of Colcemid were followed over 24 h. The perinuclear ring coiled into a juxtanuclear cap that consisted of disorganized arrays of 100 A filaments. This "coiling" effect was not blocked by cycloheximide, an inhibitor of protein synthesis. In another experiment, dividing cells were examined. During division the bundle of filaments is passively pulled in half into the daughter cells. These bundles did not disappear during the mitosis when mitotic spindle microtubules assemble. These studies suggest that Colcemid may exert a direct effect on 100 A filaments, independent of microtubules. Since these filaments do not disappear during mitosis, it is possible that in these cells the 100 A filaments and tubulin do not share a common pool of precursor proteins.

By indirect immunofluorescence the behavior of the 10-nm filaments was studied at various stages of mitosis in guinea pig vascular endothelial cells. Interphase cells contain a ring of 10-nm filaments that encircles the nucleus and is maintained in a plane parallel to the substrate. During prophase and metaphase the cells round up and the 10-nm filament ring becomes wavy though still a closed structure. As anaphase progresses the ring then elongates into a rectangle that contains the spindle apparatus and chromosomes. In late telophase, cytokinesis cleaves the 10-nm filaments into crescents at the site of the contractile ring. These crescents then close into rings in the daughter cells. If cytokinesis is inhibited with 5 #g of cytochalasin B per ml, then cleavage of the 10-nm filaments is blocked and the daughter nuclei remain surrounded by the parent ring. At no point during mitosis does the array of 10-nm filaments undergo major disassembly. These results indicate that, in contrast to the other major cytoplasmic structures, ventral microfilament bundles and cytoplasmic microtubules, which disassemble and reassemble during mitosis, 10-nm filaments remain intact throughout this process. The possibility is discussed that these filaments may function in transport of organelles and structural proteins, and provide the daughter cells with topological information about placement and assembly of these elements within the microtrabecular lattice.

No abstract