Assays for the detection of microtubule depolymerization inhibitors

Abstract

This invention provides methods for the screening and identification of agents having potent effects on the progression of the cell cycle. In one embodiment, the methods involve contacting a polymerized microtubule with a microtubule severing protein or a microtubule depolymerizing protein in the presence of an ATP or a GTP and a test agent; and (ii) detecting the formation of tubulin monomers, dimers or oligomers. The p60 subunit of katanin provides a particularly preferred microtubule severing protein possessing both ATPase and microtubule severing activities.

1 - 25 . (canceled)  
     
     
         26 . An isolated polypeptide having microtubule severing activity, said polypeptide comprising an amino acid sequence that is encoded by a nucleic acid sequence that hybridizes under stringent conditions with a nucleic acid sequence that encodes SEQ ID NO:1.  
     
     
         27 . The polypeptide of  claim 26 , wherein said polypeptide comprises SEQ ID NO:1.  
     
     
         28 . The polypeptide of  claim 26 , wherein said polypeptide: 
 1) comprises at least 8 contiguous amino acids of SEQ ID NO: 1;    2) elicits the production of an antibody that specifically binds to SEQ ID NO:1; and    3) does not bind to antiserum that is raised against SEQ ID NO:1, and that has been fully immunosorbed with SEQ ID NO:1.    
     
     
         29 . The polypeptide of  claim 26 , wherein said polypeptide is the pelypeptide consists of SEQ ID NO:1.  
     
     
         30 - 33 . (canceled)  
     
     
         34 . A kit for screening for agents that modulate microtubule depolymerization, said kit comprising one or more containers containing one or more of an isolated microtubule severing protein and a microtubule depolymerizing protein.  
     
     
         35 . The kit of  claim 34 , further comprising a polymerized microtubule labeled with 4′-6-diamidino-2-phenylindole (DAPI).  
     
     
         36 . The kit of  claim 34 , wherein said microtubule is stabilized by contact with an agent chosen from one or more of paclitaxel, paclitaxel analogue, and non-hydrolyzable nucleotide GTP analogue.  
     
     
         37 . The kit of  claim 36 , wherein said microtubule is attached to a solid surface.  
     
     
         38 . The kit of  claim 37 , wherein said microtubule is attached to said surface by binding with a motor protein.  
     
     
         39 . The kit of  claim 34 , wherein said microtubule severing protein or microtubule depolymerizing protein is selected from the group consisting of katanin polypeptide, p60 subunit of katanin polypeptide,  Xenopus  kinesin central motor 1 (XKCM1) polypeptide, and a stathmin (OP18) polypeptide.  
     
     
         40 - 41 . (canceled)  
     
     
         42 . The kit of  claim 34 , wherein said one or more of microtubule severing protein and microtubule depolymerizing protein is attached to a solid surface.  
     
     
         43 - 59 . (canceled)  
     
     
         60 . The polypeptide of  claim 26 , wherein said nucleic acid sequence that encodes SEQ ID NO:1 is listed in Genbank AF052191.  
     
     
         61 . The polypeptide of  claim 26 , wherein said polypeptide comprises SEQ ID NO:1 having conservative substitutions.  
     
     
         62 . The polypeptide of  claim 26 , wherein said polypeptide consists of SEQ ID NO:1 having conservative substitutions.  
     
     
         63 . The polypeptide of  claim 26 , wherein said hybridization conditions comprise hybridization at 42° C. overnight in 50% formamide.  
     
     
         64 . The polypeptide of  claim 26 , wherein said isolated polypeptide is recombinant.  
     
     
         65 . An isolated polypeptide comprising SEQ ID NO:1 and having microtubule severing activity.  
     
     
         66 . An isolated polypeptide consisting of SEQ ID NO:1.  
     
     
         67 . The kit of  claim 34 , further comprising tubulin that is labeled with a label chosen from one or more of 4′-6-diamidino-2-phenylindole (DAPI), anilinonapthalene sulfonate (ANS), bis-ANS (Bis-anilinonapthalene sulfonate), N-phenyl-1-naphthylene (NPN), ruthernium red, cresol violet, and 4-(dicyanovinyl)julolidine (DCVJ).  
     
     
         68 . The kit of  claim 34 , further comprising tubulin that is labeled with 4′-6-diamidino-2-phenylindole (DAPI).  
     
     
         69 . The kit of  claim 34 , wherein said microtubule depolymerizing protein comprises  Xenopus  kinesin central motor 1 (XKCM1) polypeptide.  
     
     
         70 . The kit of  claim 34 , wherein said microtubule depolymerizing protein comprises stathmin (OP18) polypeptide.  
     
     
         71 . The kit of  claim 34 , wherein said microtubule depolymerizing protein comprises katanin polypeptide.  
     
     
         72 . The kit of  claim 34 , wherein said microtubule depolymerizing protein comprises a p60 subunit of katanin polypeptide.  
     
     
         73 . The kit of  claim 37 , wherein said microtubule is attached to said surface by binding with a molecule chosen from one or more of inactivated microtubule motor protein, avidin-biotin linkage, anti-tubulin antibody, microtubule binding protein (MAP), polyarginine, polyhistidine, and polylysine.  
     
     
         74 . A kit comprising the polypeptide of  claim 26.