Monoclonal antibodies to plant nuclear matrix reveal intermediate filament- related components within the nucleus

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Abstract
We have prepared a nuclear matrix fraction from purified nuclei of carrot (Daucus carota L.) suspension culture cells, and used this fraction to produce a library of monoclonal antibodies. We report the preliminary characterisation of two antibodies-JIM 62 and JIM 63. The antibodies recognise a polypeptide doublet band at 92xl03Mr, which has been partially purified by differential urea extraction. Other intermediate filament antibodies-ME101, which recognises an epitope conserved among many intermediate filament proteins, and AFB, a monoclonal antibody to plant intermediate filament proteins, and an autoimmune serum directed against human lamina A and C (LSI), also label these bands, suggesting they are related to the intermediate filament/lamin family. IFA, another intermediate filament antibody, labels a band at approximately 60x103Mr, which is also enriched in the urea extracts of nuclear matrices. Immunofluorescence microscopy with JIM 63, ME 101, AFB and LSI shows network-like staining, often extending around the nucleolus. In many cases the staining reveals structures that appear to be bundles of fibres. JIM 63 also shows a weak staining of the nuclear rim in carrot nuclei, which can be greatly enhanced by treatment of the specimen with cold methanol after fixation. JIM 63 cross-reacts with all the other plant species we have tested. Vibratome sections of pea roots, extracted as for nuclear matrix preparation and stained with JIM 63 show a clear, strong nuclear rim labelling. Furthermore, JIM 63 strongly labels the nuclear lamina in rat liver nuclei. We suggest that the 92x103Mr protein(s) are related to intermediate filaments and/or lamins, and are distributed both within the nucleus and at the nuclear periphery.