Transient arrest of 3T3 cells in mitosis and inhibition of nuclear lamin reassembly around chromatin induced by anti-vimentin antibodies.
We have microinjected anti-vimentin antibodies into nocodazole-arrested, prometaphasic 3T3 cells. Exit of injected and control cells from mitosis has been assessed at various time points after release from nocodazole by examining the distribution of nuclear lamins, the degree of chromatin condensation and the appearance of daughter cells. It is shown here that 1 h after release from nocodazole, the vast majority of cells injected with control antibodies enter interphase, whereas most of the cells injected with antivimentin antibodies remain prometaphasic. Microinjection of vimentin-free (MCF-7) cells with anti-vimentin antibodies does not affect the normal completion of mitosis in the majority of the cells. The arresting effect on 3T3 cells is dependent on the concentration of the microinjected anti-vimentin antibodies and can also be detected when Fabs are injected instead of intact IgG. Sampling at later time points and monitoring of individual microinjected cells indicate that the antibody-induced effect lasts for 1.5 to 4 h, i.e., up to 400% longer than the normal mitotic cycle of 3T3 cells. However, 4 h after release from nocodazole, most of the antibody-arrested cells recover and divide successfully. Electron microscopy shows that the IFs of the microinjected cells are thicker than normal and tend to anastomose. Interestingly, the nuclei of some of the cells that escape the antibody-induced arrest and successfully divide appear segmented or lobulated. Using a mitotic cell-free system, we further demonstrate that anti-vimentin antibodies interfere with nuclear lamin assembly around chromatin particles. In agreement to the in vivo results, the inhibitory effect of the antibodies appears to subside with time. Taken together, these data suggest that the remodelling of the vimentin network which normally takes place during mitosis is an important rearrangement of the cytoplasm required for efficient nuclear reassembly at the end of cell division.
Kouklis
P D
PD
Cell Biology Programme, European Molecular Biology Laboratory, Heidelberg/Germany.
Merdes
A
A
Papamarcaki
T
T
Georgatos
S D
SD
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry*; D002843:Chromatin / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D007399:Interphase; D034882:Lamins; D051379:Mice; D008845:Microinjections; D008854:Microscopy, Electron; D008938:Mitosis; D009687:Nuclear Proteins / Q000032:analysis / Q000378:metabolism*; D013997:Time Factors; D014746:Vimentin / Q000276:immunology*
D002843:Chromatin; D034882:Lamins; D009687:Nuclear Proteins; D014746:Vimentin