Express Journey through Research Odyssey

To study plant MAPs, these objectives were pursued:
(1) Preparative biochemical method to select for putative MAPs.
(2) In vitro assay to assess MT-binding of putative MAPs.
(3) Biochemical method to purify MT-binding, putative MAPs.
(4) In vitro assay to characterize MT-MAP interaction.
(5) Evidence that putative MAPs are MAPs in situ.

1) Identify carrot proteins that bind MTs in vitro MT cosedimentation assay Lane a: Total soluble proteins. Lane b: Tubulin-binding proteins (TBPs) selected by affinity chromatography. MT cosedimentation assay with TBPs Lane c (sup): TBPs that don't bind MTs. Lane d (pel): TBPs that do bind MTs.. The most prominent TBP that also binds MTs was designated pp50 (50kD; marked by the dash to its left).

2) Purify pp50 / confirm its MT-binding. Lane a: TBP loaded onto Ca2+/CaM column. Lane b (flow-through): TBPs that don't bind Ca2+/CaM. Lane c (EGTA eluate): TBPs that do bind Ca2+/CaM. MT cosedimentation assay with CaM eluate Lane d (sup): CaM eluate that doesn't bind MTs. Lane e (pel): CaM eluate that does bind MTs. Pp50 is a Ca2+/CaM-binding protein, and binds MTs directly (no other proteins present).

3) Pp50's MT and Ca2+/CaM interactions. Darkfield microscopy (direct observation - no fixation, no staining/labeling) A, (top): Pp50-induced MT bundles (laterally co-aligned). B, (bottom): Same sample after Ca2+/CaM added (both required). Bundles are inferred by brightness, which indicates greater light-scatter in darkfield. Moreover, figure A was imaged using 5X less the light intensity allowed in figure B. Pp50's MT-bundling property may be mediated by Ca2+/CaM signal events.

4) Pp50 co-localizes with extracted MT cytoskeleton in situ Protoplast footprints APE-50: Affinity-Purified anti-pp50 Eluted off Pp50. tubulin: monoclonal anti-carrot-tubulin. Protoplast treatments APM: MT-depolymerizing agent. taxol: MT-stabilizing agent. Treated protoplasts were extracted to yield footprints, then fixed, processed for immunofluorescence, and observed by conventional epifluorescence.

 

5) Pp50 co-localizes within 5nm of the MT cytoskeleton, fixed before extracted FRET The same specimen, field, and focal plane is shown in a-c. After fixation, the protoplast was processed for dual immunolocalization of MTs and pp50 (anti-tubulin and APE-50 as above). Imaged as diagrammed using confocal laser scanning microscopy. Because fixed before extraction, artificial re-distribution of pp50 to MTs is less likely. For 3rd case (Fluorescence Resonance Energy Transfer, FRET), pp50 and MTs must be within 50 Angstroms, in principle; note fibrous MT image is sharpened in c vs. a or b. Return to Publication Excerpts, In situ immunocytochemical evidence that a homolog of protein translation elongation factor EF-1-alpha is associated with microtubules in carrot cells [Durso, Leslie, & Cyr, 1996, Protoplasma 190, 141-150], Figure 6: Co-localization of affinity purified anti-pp50 and anti-tubulin antibodies in a carrot protoplast fixed prior to detergent extraction and examined by a fluorescence resonance energy transfer (FRET) technique.

Summary of Plant MAP Ph.D. work Some MT-binding proteins can be selected by tubulin affinity chromatography. EF-1-alpha homolog pp50 is a MT- and Ca2+/CaM-binding protein. EF-1-alpha induces MT bundles, the disruption of which Ca2+/CaM effects. A fraction of cellular EF-1-alpha is intimately associated with MTs in situ. EF-1-alpha = a plant MAP. Perhaps maintains bundled MTs common in the in situ cytoskeleton. Perhaps mediates the known response of MTs to Ca2+ signals via CaM.