Isolation of the Mitotic Spindle Matrix and its Methods of Use

Abstract

The invention encompasses a purified preparation of the mitotic spindle matrix essential for mitotic spindle assembly, which allows for identifying an agent that modulates a cell division and/or differentiation signaling pathway comprising determining the effect of the agent on spindle formation, MT nucleation, or lamin matrix assembly wherein the change in spindle formation, MT nucleation or lamin matrix assembly.

1 . A composition comprising an isolated mitotic spindle matrix capable of mediating mitotic spindle assembly and/or nucleating microtubule assembly and organization in vivo and in vitro, comprising a membrane-lamin-based assemblage containing one or more proteins and/or nucleic acids essential for a cell division and differentiation signaling pathway. 
     
     
         2 . A method for identifying an agent that modulates a cell division and/or differentiation signaling pathway comprising:
 (a) contacting a plurality of beads or dots each of said plurality of beads or dots comprising at least one protein with a biological sample in the presence or absence of said agent;   (b) determining the effect on spindle formation, MT nucleation, or lamin-membrane matrix assembly wherein the change in spindle formation, MT nucleation or lamin-membrane matrix assembly compared to a control indicates an agent capable of modulating a cell division and/or differentiation signaling pathway.   
     
     
         3 . The method of  claim 2 , wherein the cell division and/or differentiation signaling pathway is selected from the group consisting of a GTPase protein signaling pathway, a kinase signaling pathway, an ubiquitin signaling pathway, an apoptosis and capsase signaling pathway. 
     
     
         4 . The method of  claim 3 , wherein the GTPase protein is selected from the group consisting of Ran, Rab, Ras, Rho, Cdc, and Rac. 
     
     
         5 . The method of  claim 3 , wherein the kinase signaling pathway comprises a kinase protein is selected from the group consisting of Aurora A and B. 
     
     
         6 . The method of  claim 2 , wherein the biological sample is a cell lysate or extract. 
     
     
         7 . The method of  claim 6 , wherein the cells are HeLa cells, NIH3T3 cells or embryonic stem cells. 
     
     
         8 . The method of  claim 6 , wherein the cell extract is  Xenopus  egg extracts. 
     
     
         9 . The method of  claim 2 , wherein said beads or dots comprise agents which bind to the protein. 
     
     
         10 . The method of  claim 9 , wherein said protein is a wild-type protein. 
     
     
         11 . The method of  claim 10 , wherein said wild-type protein is Aurora A. 
     
     
         12 . The method of  claim 2 , wherein the control is a negative control. 
     
     
         13 . The method of  claim 12 , wherein the negative control does not comprise the agent. 
     
     
         14 . The method of  claim 12 , wherein the negative control comprises a mutant protein. 
     
     
         15 . The method of  claim 14 , wherein the mutant protein comprises one or more amino acid substitutions or deletions. 
     
     
         16 . The method of  claim 2 , wherein the control is a positive control. 
     
     
         17 . The method of  claim 16 , wherein the positive control comprises an inhibitor of MT nucleation, spindle formation or lamin formation. 
     
     
         18 . The method of  claim 2  further comprising determining the effect of the agent on post-translational modification of one or more proteins. 
     
     
         19 . The method of  claim 2  further comprising determining the effect of the agent on translation of one or more proteins. 
     
     
         20 . The method of  claim 2  further comprising determining the interaction of a lamin B matrix with a cellular component selected from the group consisting of: one or more RNA molecules, transcription factors, mitochondria, and membrane components. 
     
     
         21 - 33 . (canceled)