Microtubule minus ends can be labelled with a phage display antibody specific to alpha-tubulin.
To investigate the orientation of alpha- and beta-tubulin heterodimers within microtubules, we cloned a phage display antibody to alpha-tubulin. The N-terminal 100 residues of alpha-tubulin were bacterially expressed and used to select clones from a large repertoire of antibody-expressing phagemid particles. One clone reacted with the expressed alpha-tubulin N terminus and native tubulin dimer but not with the expressed beta-tubulin N terminus. Electron microscopy showed 30 nm gold beads coated with the antibody binding to one end of brain microtubules. The beads bound to the minus ends of axonemes but not to the brain tubulin extensions from their plus ends. In sliding motility assays with a plus end directed motor, beads were pushed ahead of the microtubules. Our results indicate that an N-terminal epitope of alpha-tubulin is exposed only at the minus ends of microtubules.
Fan
J
J
MRC Laboratory of Molecular Biology, Cambridge, UK.
Griffiths
A D
AD
Lockhart
A
A
Cross
R A
RA
Amos
L A
LA
D000818:Animals; D000906:Antibodies; D001435:Bacteriophages / Q000276:immunology*; D001921:Brain / Q000648:ultrastructure; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D005407:Flagella / Q000737:chemistry / Q000648:ultrastructure; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000276:immunology; D008854:Microscopy, Electron / Q000379:methods; D008857:Microscopy, Interference / Q000379:methods; D008863:Microspheres; D008870:Microtubules / Q000737:chemistry* / Q000276:immunology / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012617:Sea Urchins / Q000737:chemistry / Q000648:ultrastructure; D014404:Tubulin / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology*
D000906:Antibodies; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; D014404:Tubulin; C055966:immunoglobulin Fv