TI - Results and discussion Mtor localizes to a nuclear derived spindle matrix in living cells . AB - To investigate the localization of Mtor in living cells , we generated a Drosophila S2 cell line stably coexpressing Mtor-mCherry and GFP-alpha-tubulin . Mtor-mCherry is nuclear in interphase and at nuclear envelope breakdown ( NEB ) reorganizes into a fusiform structure coalescent with spindle MTs ( Fig. 1 A ; and Video 1 , available at . http : . . . . . . /www.jcb.org/cgi/content/full/jcb. lt @@@@@ gt 200811012/DC ) . Mtor-mCherry shows a highly adaptable morphology in response to changes in spindle shape and dynamics throughout mitosis , which is inconsistent with a static structure . Similar to endogenous Mtor , Mtor-mCherry retracts and loses the fusiform shape upon MT depolymerization but is retained in a conspicuous milieu around chromosomes ( Fig 1 , B-D and F ; and Video 2 ) , suggesting that MTs exert a pushing force on the Mtor-defined matrix . Previous electron microscopy analysis revealed the existence of a membranous network surrounding the spindle from prophase to metaphase in S2 cells . In this study , we used immunofluorescence to show that lamin B is not fully disintegrated at this stage ( Fig 1 G ) . Similar results have recently been reported in living Drosophila embryos and neuroblasts , where a spindle envelope was proposed to limit the diffusion of nuclear derived Nup107 before anaphase . To test whether this membranous network works as a diffusion barrier around the spindle , we compared the dynamic behavior of Mtor-mCherry relative to GFP-alpha-tubulin and a known MT-associated protein , Jupiter , upon colchicine addition . GFP-alpha-tubulin or Jupiter-GFP fluorescence is gradually lost from the spindle region with an equivalent gain in the cytoplasm ( Fig 1 , C and E ) . In contrast , Mtor-mCherry remains confined to the spindle region with no detectable fluorescence gain in the cytoplasm ( Fig 1 D ) . These results argue against the existence of a diffusion barrier around the metaphase spindle in Drosophila S2 cells and suggest that Mtor is being selectively retained in this region . To shed light on the dynamic properties of Mtor , we used FRAP . In interphase nuclei , there is ~50% recovery of fluorescence in the bleached region with an equivalent loss from a similar unbleached region and undetectable cytoplasmic exchange ( Fig 2 , A and A' ) , suggesting that Mtor in the nucleoplasm is mobile . In mitosis , FRAP of Mtor-mCherry in one half-spindle is mirrored by an equivalent loss of fluorescence from the unbleached half-spindle as if Mtor exchanges between half-spindles ( Fig 2 , B and B' ) . However , this recovery was slower ( t1/2 = 187 + - 43 s , n = 9 cells ) than in interphase nuclei ( t1/2 = 90 + - 51 s , n = 3 cells ; Fig 2 , A' and B' ) and had a minor contribution from a cytoplasmic pool ( Fig 2 , B'-C' ) . In both interphase and mitosis , the recovery curves of Mtor-mCherry fitted a single exponential , suggesting affinity to a yet unidentified SUBstrate , whereas GFP-alpha-tubulin in the spindle displayed biphasic recovery kinetics and best fit the sum of two exponentials as result of a rapid diffusion phase ( t1/2 = 042 + - 039 s , n = 3 cells ) followed by a slower recovery phase ( t1/2 = 286 + - 67 s ) associated with MT turnover ( Fig 2 , D-D'' ) . Finally , in S2 cells that sporadically form two spindles in the same cytoplasm , we found fluorescence exchange within the same spindle and from surrounding cytoplasm with no apparent loss from the neighboring unbleached spindle ( Fig 2 , E and E' ) , supporting that Mtor is unable to exchange between two spindles located <10 um apart . Collectively , these data indicate that Mtor is part of a dynamic , nuclear derived spindle matrix surrounded by a fenestrated membranous system containing lamin B and shows mobility properties that are distinct from MTs and associated proteins .