ABSTRACT
SUMMARY: In order to further understand the production and intracellular trafficking of pharmaceutical proteins in plants, the light and heavy chains (LC and HC) of the human immunodeficiency virus neutralizing monoclonal antibody 2G12 were fused to fluorescent proteins [Venus and monomeric red fluorescent protein (mRFP)] to enable the visualization of their passage through the plant cell. Co-expression of LC and HC with various markers of the endomembrane system demonstrated that LC fusions were found in mobile punctate structures, which are likely to be pre-vacuolar compartments (PVCs) as a proportion of the LC fusions were found to be located in the vacuole. In addition, apoplast labelling was also observed with a 2G12LC-RFP fusion. The HC fusion expressed alone was found only in the endoplasmic reticulum (ER). When the LC and HC fusions were expressed together, they were found to co-locate to larger punctate structures, which were morphologically distinct from any observed on expression of LC or HC alone. These structures appeared to be in close association with the ER and their labelling partially overlapped with PVC marker fluorescence, but no increase in apoplast labelling was observed. Co-immunoprecipitation data demonstrated that the presence of the fluorescent proteins did not affect the assembly of the antibody, and also showed the association of BiP with the antibody chains. The antigen-binding activity of the Venus-fused 2G12 antibody was confirmed by enzyme-linked immunosorbent assay.
Subject(s)
Antibodies, Monoclonal/analysis , HIV Antibodies/analysis , Nicotiana/genetics , Recombinant Fusion Proteins/analysis , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Binding Sites, Antibody , Broadly Neutralizing Antibodies , Enzyme-Linked Immunosorbent Assay , HIV Antibodies/chemistry , HIV Antibodies/metabolism , Immunohistochemistry , Luminescent Proteins/analysis , Pharmaceutical Preparations/analysis , Pharmaceutical Preparations/chemistry , Pharmaceutical Preparations/metabolism , Plants, Genetically Modified/metabolism , Protein Transport , Recombinant Fusion Proteins/metabolism , Rhizobium , Nicotiana/metabolism , Nicotiana/ultrastructure , Transformation, Genetic , Red Fluorescent ProteinABSTRACT
Controlling microtubule dynamics and spatial organization is a fundamental requirement of eukaryotic cell function. Members of the ORBIT/MAST/CLASP family of microtubule-associated proteins associate with the plus ends of microtubules, where they promote the addition of tubulin subunits into attached kinetochore fibers during mitosis and stabilize microtubules in the vicinity of the plasma membrane during interphase. To date, nothing is known about their function in plants. Here, we show that the Arabidopsis thaliana CLASP protein is a microtubule-associated protein that is involved in both cell division and cell expansion. Green fluorescent protein-CLASP localizes along the full length of microtubules and shows enrichment at growing plus ends. Our analysis suggests that CLASP promotes microtubule stability. clasp-1 T-DNA insertion mutants are hypersensitive to microtubule-destabilizing drugs and exhibit more sparsely populated, yet well ordered, root cortical microtubule arrays. Overexpression of CLASP promotes microtubule bundles that are resistant to depolymerization with oryzalin. Furthermore, clasp-1 mutants have aberrant microtubule preprophase bands, mitotic spindles, and phragmoplasts, indicating a role for At CLASP in stabilizing mitotic arrays. clasp-1 plants are dwarf, have significantly reduced cell numbers in the root division zone, and have defects in directional cell expansion. We discuss possible mechanisms of CLASP function in higher plants.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/cytology , Arabidopsis/metabolism , Cell Division , Microtubule-Associated Proteins/genetics , Arabidopsis/drug effects , Arabidopsis/genetics , Arabidopsis Proteins/chemistry , Cell Division/drug effects , Cell Proliferation/drug effects , Cell Size/drug effects , Cytokinesis/drug effects , Dinitrobenzenes/pharmacology , Gene Expression Regulation, Plant/drug effects , Green Fluorescent Proteins/metabolism , Interphase/drug effects , Microtubule-Associated Proteins/chemistry , Microtubules/drug effects , Microtubules/metabolism , Mitosis/drug effects , Mutation/genetics , Phylogeny , Plant Epidermis/cytology , Plant Epidermis/drug effects , Plant Epidermis/ultrastructure , Plant Leaves/cytology , Plant Leaves/drug effects , Plant Leaves/ultrastructure , Plant Roots/cytology , Plant Roots/drug effects , Plant Roots/growth & development , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Sulfanilamides/pharmacologyABSTRACT
Rab GTPases are universal key regulators of intracellular secretory trafficking events. In particular, Rab 5 homologues have been implicated in endocytic events and in the vacuolar pathway. In this study, we investigate the location and function of a member of this family, AtRabF2b (Ara7) in tobacco (Nicotiana tabacum) leaf epidermal cells using a live cell imaging approach. Fluorescent-tagged AtRabF2b[wt] localized to the prevacuolar compartment and Golgi apparatus, as determined by coexpression studies with fluorescent markers for these compartments. Mutations that impair AtRabF2b function also alter the subcellular location of the GTPase. In addition, coexpression studies of the protein with the vacuole-targeted aleurain-green fluorescent protein (GFP) and rescue experiments with wild-type AtRabF2b indicate that the dominant-negative mutant of AtRabF2b causes the vacuolar marker to be secreted to the apoplast. Our results indicate a clear role of AtRabF2b in the vacuolar trafficking pathway.
