The function and assembly of the lamin-B spindle sheath

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DESCRIPTION (provided by applicant): This proposal aims to study how the mitotic membrane network that surrounds the spindle microtubules regulates spindle morphogenesis and spindle orientation. The assembly and maintenance of mitotic spindle morphology requires balancing of forces generated by microtubule-based motor proteins such as dynein and the kinesin Eg5, and proper regulation of microtubule dynamics. Our studies of the lamin-B-containing mitotic membrane network, which we refer to as the lamin-B spindle sheath, have shown that this spindle-associated structure regulates spindle morphology and spindle orientation. Since lamin-B interacts with the dynein regulator NudEL and the microtubule depolymerase MCAK, we hypothesize that the lamin-B spindle sheath that surrounds the body of the spindle microtubules functions as a barrier to limit microtubule growth within the spindle boundary through MCAK and/or NudEL. Moreover, we propose that the lamin-B spindle sheath surrounding the spindle poles regulates the astral microtubules to ensure proper search and capture of the cortical spindle orientation cues. We will test these ideas in Aim 1 and Aim 2 using a number of assays and tools we have generated. We have shown previously that RanGTP, microtubules, and dynein are all required for the lamin-B spindle sheath assembly. More recently, we have found that lamin-B binds to several nucleoporins in mitosis. In Aim 3, we will test the hypothesis that the interactions between lamin-B and nucleoporins are required for the assembly of the spindle sheath. These studies will make a significant contribution toward understanding how non-microtubule cellular structures regulate spindle assembly and orientation in mitosis. PUBLIC HEALTH RELEVANCE: Most studies of spindle assembly have focused on activities and forces within the spindle microtubules. However, many observations have shown that structures outside of spindle microtubules also contribute toward maintaining spindle morphology and position. Our studies have uncovered that a lamin-B-containing membrane network organized around the spindle microtubules plays a role in spindle morphogenesis. With the newly identified molecular handles, we propose to further dissect the mechanism by which the lamin- membrane network interacts with spindle microtubules to facilitate their organization and how this would in turn regulate spindle orientation during cell division. This proposal addresses an area of cell division that has received relatively little attention and it aims to uncover novel insights about spindle assembly mechanisms that had not been considered previously. Since the mitotic lamin-containing membrane network contains signaling molecules and cell fate determinants, we believe that understanding how this structure interacts with spindle microtubules during cell division will open new avenues to explore therapeutic targets for halting uncontrolled cell divisions.