Tubulin conjugates

Tubulin conjugates are used for observation of cell cycle-dependent microtubule dynamics, mitotic spindle morphogenesis and visualisation of tubulin transport in neurons. 

Robust decreases in the expression of the various proteasome subunits and ubiquitin-conjugating enzymes have been described in prefrontal cortex in schizophrenia. Neuronal ubiquitin and proteasomes play an important role in the assembly, function and plasticity of the synapse. Structural proteins including tubulin and a-spectrin also show decreased expression in prefrontal cortex. 

Fig. 3. Immunofluorescence localization of tubulin in microtubules in Swiss 3T3 cultured fibroblasts. Swiss 3T3 cells were fixed in glutaraldehyde followed by treatment with sodium borohydride (4). Tubulin was detected using a rat monoclonal antitubulin antibody (YLl/2), followed by goat antirat IgG conjugated to rhodamine, all steps in the continuous presence of 0.1% saponin. Note the individual microtubules visible under the dark nuclear region (bar = 4 im).

Fig. 13.8. Effect of paclitaxel on the morphology of C6 cells in culture. Cells were treated with LCM only (C,D) or paclitaxel-LCM (A,B) for 8 hours. The cells were stained with fluorescein-conjugated wheat germ agglutinin (WGA) to reveal the cell surface, and with Texas red-conjugated secondary antibody to rabbit anti-tubulin to reveal the presence of microtubules. Both channels were recorded simultaneously. Panels A and C show the presence of WGA, while B and D reveal the presence of tubulin. (Taken from ref. 532.)...

Figure 2. Cultured pulmonary artery endothelial cells stained for tubulin (red), actin (green) and DNA (blue). The dual immunofluorescence procedure used rabbit anti-actin IgG and mouse anti-alpha tubulin IgG as primary antibodies. The secondary antibodies used were Texas Red-conjugated goat, anti-rabbit IgG and FITC-conjugated goat, anti-mouse IgG. The sample was also stained with the DNA-specific dye Hoechst 33342. Scale bar is equal to 20 microns.

Singer and colleagues (68, 69) have successfully visualized cytoskeletal mRNAs and their associate proteins using biotinylated cDNA probes followed by antibodies to biotin and collodial gold-conjugated antibodies. Their method used in situ hybridization followed by whole mount TEM of Triton-extracted chicken embryo fibroblasts. Cytoskeletal mRNAs were found in close proximity to actin protein and further from tubulin filaments. While the whole mount technology does limit the technique to extracted cells, applications to thin sections will allow greater resolution. 

Fluoresceine, Alexa Fluor, BODIPY and other conjugates of actin, tubulin and phallotoxins... 

Several additional compounds derived from S. ceUulosum species are currently under study for pharmaceutical development (see Figure 12.6) Chlorotoiul was recently shown to exhibit impressive antimalarial activity in mouse models [83] and the tubulin binder disorazole has demonstrated imposing activity in cancer models in mice when administered as peptide conjugate targeting... 

The maytansines are a family of very potent (sub-nanomolar) antimitotic agents that inhibit tubulin polymerization. Attempts to use maytansine derivatives themselves in clinical trials have shown them to possess very narrow therapeutic windows." To overcome this limitation, derivatives have been made that allow for conjugation to antibodies. 

Figure 3 Double labelling of a- and p-tubulin components of microtubules labelled with 5 nm and 10 nm gold conjugates in a section of a PLT embedded cell. Whilst lines of >ld marker can be seen occasionally following the profile of a microtubule (arrow) this figure illustrates the point that although the microtubules can be seen in the section, only those antigens accessible at the section surface can be labelled. Magnification x 74000.

Figure 4. MC3T3 cells on the active area of the device immediately after mechanical stimulation. Indirect immunofluorescence using primary antibody against actin (Tubulin 3, Thermo Scientific, used at 1 50) and secondary antibody (Chromeo 488 conjugated Coat anti-Rabbit IgG, Active Motif 1 500) (400X, microscope Olympus BX41, Olympus Cell A Imaging Software).

Staining Applications Axonemal tubulins biomole-cules elastin fibers eye lens proteins crystaUins fim-gi ° protozoa glycosaminoglycans neiirofila-ments nucleic acids proteins conjugated proteins polar lipids phosphorpoteins ... 

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