RESUMO
Recent reports show that, after nuclear envelope breakdown, lamin-B, a component of the nuclear lamina in interphase, localizes around the mitotic spindle as a membranous network. How this process occurs, however, and how it influences mitotic spindle morphogenesis is unclear. Here, we develop a computational model based on a continuum description to represent the abundance and location of various molecular species involved during mitosis, and use the model to test a number of hypotheses regarding the formation of the mitotic matrix. Our model illustrates that freely diffusible nuclear proteins can be captured and transported to the spindle poles by minus-end-directed microtubule (MT) motors. Moreover, simulations show that these proteins can be used to build a shell-like region that envelopes the mitotic spindle, which helps to improve the focusing of the mitotic spindle by spatially restricting MT polymerization and limiting the effective diffusion of the free MTs. Simulations also confirm that spatially dependent regulation of the spindle network through the Ran system improves spindle focusing and morphology. Our results agree with experimental observations that lamin-B reorganizes around the spindle and helps to maintain spindle morphology.
RESUMO
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.
Assuntos
Cinesinas/metabolismo , Lamina Tipo B/metabolismo , Microtúbulos/metabolismo , Oócitos/metabolismo , Fuso Acromático/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Dineínas/genética , Dineínas/metabolismo , Cinesinas/genética , Lamina Tipo B/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/genética , Oócitos/citologia , Fuso Acromático/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
The mitotic spindle plays an essential role in chromosome segregation during cell division. Spindle formation and proper function require that microtubules with opposite polarity overlap and interact. Previous computational simulations have demonstrated that these antiparallel interactions could be created by complexes combining plus- and minus-end-directed motors. The resulting spindles, however, exhibit sparse antiparallel microtubule overlap with motor complexes linking only a nominal number of antiparallel microtubules. Here we investigate the role that spatial differences in the regulation of microtubule interactions can have on spindle morphology. We show that the spatial regulation of microtubule catastrophe parameters can lead to significantly better spindle morphology and spindles with greater antiparallel MT overlap. We also demonstrate that antiparallel microtubule overlap can be increased by having new microtubules nucleated along the length of existing astral microtubules, but this increase negatively affects spindle morphology. Finally, we show that limiting the diffusion of motor complexes within the spindle region increases antiparallel microtubule interaction.
