Lamin B Counteracts the Kinesin Eg5 to Restrain Spindle Pole Separation during Spindle Assembly*

Lamin B is a component of the membranous spindle matrix isolated from Xenopus egg extracts, and it is required for proper spindle morphogenesis. Besides lamin B, the spindle matrix contains spindle assembly factors (SAFs) such as Eg5 and dynein which are known to regulate microtubule organization and SAFs known to promote microtubule assembly such as Maskin and XMAP215. Because lamin B does not bind directly to microtubules, it must affect spindle morphogenesis indirectly by influencing the function of spindle matrix-associated SAFs. Using different assays in Xenopus egg extracts, we found that depleting lamin B caused formation of elongated and multipolar spindles, which could be reversed by partially inhibiting the kinesin Eg5, revealing an antagonistic relationship between Eg5 and lamin B. However, lamin B only very weakly antagonizes Eg5 in mediating poleward microtubule-flux based on fluorescence speckle microscopy. Depleting lamin B led to a very small but statistically significant increase in flux. Furthermore, flux reduction caused by partial Eg5 inhibition is only slightly reversed by removing lamin B. Because lamin B does not bind to Eg5, our studies suggest two nonexclusive mechanisms by which lamin B can indirectly antagonize Eg5. It could function in a network that restricts Eg5-driven microtubule sliding only when microtubules come into transient contact with the network. Lamin B could also function to sequester microtubule polymerization activities within the spindle. Without lamin B, increased microtubule assembly caused by the released SAFs would lead to excessive microtubule sliding that results in formation of elongated and multipolar spindles.