"Caged cytoskeletons": a rapid method for the isolation of microtubule-associated proteins from synchronized plant suspension cells.
In the cytoskeleton method for isolating microtubule-associated proteins MAP65, DcKRP120-1 and DcKRP120-2, carrot cells are first converted to protoplasts but this method cannot be used to isolate mitotic MAPs as mitotic synchrony is eroded during lengthy cellulase treatment. Anti-microtubule cycle blocks would also be unsuitable. We report here a method for overcoming these problems. Cellulase degradation of tobacco BY-2 cells for only several minutes allows extraction of detergent-soluble proteins, leaving synchronized "caged cytoskeletons" for depolymerization and enabling affinity purification of MAPs on neurotubules. This rapid and simple method should be of general utility: it can be bulked up, avoids anti-microtubule blocks, and is applicable to other cell suspensions. The effectiveness of the caged cytoskeleton method is demonstrated by comparing known MAPs (the 65 kDa structural MAPs and the kinesin-related protein, TKRP125) in synchronized cells taken at the mitotic peak with those in unsynchronized cells.
McCutcheon
S
S
Department of Cell and Developmental Biology, John Innes Centre, Colney, Norwich NR4 7UH, UK.
Hemsley
R J
RJ
Jopson
M F
MF
Lloyd
C W
CW
D016590:Aphidicolin / Q000494:pharmacology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003599:Cytoskeleton / Q000737:chemistry* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism; D008938:Mitosis / Q000187:drug effects; D008969:Molecular Sequence Data; D011485:Protein Binding; D011523:Protoplasts; D014026:Nicotiana / Q000166:cytology* / Q000187:drug effects; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism
D008869:Microtubule-Associated Proteins; D014404:Tubulin; D016590:Aphidicolin