We tested the classical hypothesis that astral prometaphase bipolar mitotic spindles are maintained by balanced outward and inward makes exerted on spindle poles by kinesin-5 and -14 using modeling of in vitro and in vivo data from embryos. incorporating motor kinetics and load-dependent detachment. Motor parameters from this model were applied to a new stochastic force-balance model for prometaphase spindles providing a good fit to data from embryos. Maintenance of virtual spindles required dynamic ipMTs and a narrow range of kinesin-5 to kinesin-14 ratios matching that found in embryos. Functional perturbation and modeling suggest that this range can be extended significantly by a disassembling lamin-B envelope that surrounds the prometaphase spindle and augments the finely tuned antagonistic kinesin pressure balance to maintain strong prometaphase spindles as MTs assemble and chromosomes are pushed to the equator. Introduction Mitosis depends on the mitotic spindle a subcellular machine which uses microtubule (MT)-based pressure generators to assemble itself and to segregate Skepinone-L chromosomes (Walczak et al. 1998 Sharp et al. 2000 Mitchison and Salmon 2001 Wadsworth and Khodjakov 2004 Brust-Mascher and Scholey 2007 Walczak and Heald 2008 During prometaphase i.e. the period between nuclear envelope breakdown (NEB) and metaphase bipolar astral spindles use dynamically unstable MTs and motors to capture replicated condensed chromosomes and to maneuver them onto the spindle Skepinone-L equator (Mitchison and Kirschner 1984 Alexander and Rieder 1991 Echeverri et al. 1996 Wollman et al. 2005 In many systems an antagonistic sliding filament Skepinone-L mechanism driven by MT plus end-directed kinesin-5 and minus end-directed kinesin-14 motors is usually proposed to produce balanced outward and inward forces on spindle poles to maintain these prometaphase spindles as they capture chromosomes (for reviews see Hoyt and Geiser 1996 Kashina et al. 1997 Civelekoglu-Scholey and Scholey 2007 For example cells contain two members of the kinesin-5 family (Hoyt et al. 1992 which are required for spindle pole parting during preliminary spindle set up and withstand a kinesin-14-generated spindle collapse during prometaphase (Saunders and Hoyt 1992 embryo mitotic spindles contain only 1 kinesin-5 KLP61F (Heck et al. 1993 Cole et al. 1994 which is certainly dispensable for preliminary spindle set up but must prevent prometaphase spindle collapse powered with a kinesin-14 Ncd (Clear et al. 1999 Brust-Mascher Skepinone-L et al. 2009 Particularly time-lapse imaging and useful perturbation from Skepinone-L the living embryo early prometaphase spindle claim that it is taken care of at a steady-state amount of ～8 μm with a power stability generated by KLP61F and Ncd (Clear et al. 1999 2000 Tao et al. 2006 which balance is certainly tipped with the up-regulation of various other outward pressure generators e.g. cortical dynein and KLP3A causing the spindle to elongate to its new steady-state metaphase length of 10-12 μm (Sharp et al. 2000 Kwon et al. 2004 This model was supported by observations that genetic or antibody-mediated inhibition of KLP61F caused wild-type prometaphase spindles to collapse and that loss of Ncd function in null mutants caused prometaphase spindles to elongate prematurely but bipolar spindle length was managed after the inhibition of both KLP61F and Ncd (Sharp et al. 1999 2000 Brust-Mascher et al. 2009 However several uncertainties remain because it is not known precisely when these motors take action and whether they actually localize to interpolar MTs (ipMTs) during prometaphase or whether antagonistic MT sliding driven by KLP61F and Ncd can take action alone to maintain the early prometaphase spindle. Moreover a computational model questioned the validity of SPP1 the antagonistic motor-dependent sliding filament model (Nédélec 2002 and you will find suggestions that other mechanisms may be involved (Johansen and Johansen 2007 Gardner et al. 2008 Wollman et al. 2008 Brust-Mascher et al. 2009 In this context it is significant that recent work suggests that a membranous lamin-B envelope derived from the nuclear membrane could contribute to spindle assembly and stability in some systems (Tsai et al. 2006 The KLP61F-Ncd force-balance hypothesis gains further support from observations that purified KLP61F which.