SIMULTANEOUS LABELING OF MICROTUBULES AND FIBRILLAR BUNDLES IN TOBACCO BY-2 CELLS BY THE ANTI-INTERMEDIATE FILAMENT ANTIBODY, ME-101

In previous studies on plant cells, antibodies directed against interm ediate filaments (Ifs) have shown that IF antigens are distributed in one of two quite distinct forms. The first co-distributes with each of the four microtubule arrays (cortical, preprophase band, spindle and phragmoplast), while the second form is associated with cytoplasmic pa racrystalline fibrillar bundles (FBs) of 10nm filaments. Conditions al lowing one form to be labelled with antibody have generally proved uns uitable for labelling of the other; this has prevented the simultaneou s visualization of the two forms of IF antigen in plants and the study of any possible physical relationships between them at the electron m icroscopic level. In this paper, we show that ME 101, which recognizes an epitope in the N-terminal portion of all classes of intermediate f ilaments, stains both forms of plant IF antigen simultaneously in toba cco suspension cells using immunofluorescence or immunogold labelling techniques. These cells contain in their cortex short (ca. 1 mu m) fib rillar bundles which stain with ME 101. These bundles appear to be ind ependent of the microtubule-associated epitope which stains in a conti nuous linear manner with ME 101. When protoplasts are either cleaved o pen on grids or sequentially extracted with detergents prior to critic al point drying, the short fibrillar bundles are specifically labelled by ME 101 tagged with colloidal gold. ME 101 also co-distributed with underlying linear filaments, which appeared to be microtubules. In ad dition to these structures, the cortex also contains a meshwork of var iably-sized fine filaments but these are not labelled with ME 101 nor with an antibody raised against the plant cytoskeleton, which recogniz es cytokeratin 8. These results confirm that the fibrillar bundles and the microtubule-associated form of plant IF antigens are present simu ltaneously rather than experimentally-interconvertible, and that they appear to be physically unconnected.