A monoclonal antibody raised against cytoplasmic fibrillar bundles from carrot cells, and its cross-reaction with animal intermediate filaments

Abstract

ABSTRACT Carrot suspension cells contain cytoplasmic bundles of fibrils that are distinct from F-actin and microtubules and have some of the characteristics of intermediate filaments. In characterizing these fibrillar bundles further, we have raised a monoclonal antibody against them. This anti-fibrillar bundle antibody (AFB) immunoblots vimentin from a range of animal cells and tissues, as well as glial fibrillary acidic protein in brain and desmin in BHK fibroblasts, which are representatives of the type III intermediate filaments. Immunofluorescence staining of PtK2 cells indicates that AFB recognizes a network co-distributing with cytoplasmic microtubules. When this co-alignment is disturbed with the anti-microtubule agent, colcemid, the AFB staining segregates with the collapsed perinuclear whorls of vimentin. In carrot, AFB immunoblots the major bundle proteins but not plant tubulin. In plant as in animal cells, AFB immunofluorescently labels antigens that co-distribute with microtubules. In onion cells (which, unlike carrot, do not possess paracrystalline arrays of fibrils) AFB labels all four microtubule arrays throughout the cell cycle. The antigens do not, however, collapse around the spindle poles during mitosis. Double immunofluorescence, using anti-dog brain tubulin, indicates that the FB antigen is more diffusely distributed than tubulin; it is patchy and co-alignment is not exact, particularly during early preprophase band formation. Antigens in detergent-insoluble fibrils of carrot cells therefore exist both in animal type III intermediate filaments (IF), and in a more dispersed, microtubule-associated manner in onion meristematic cells. This constitutes an independent line of evidence for the existence of IF antigens in plants.