LIS1 controls mitosis and mitotic spindle organization via the LIS1-NDEL1-dynein complex.

Heterozygous LIS1 mutations are responsible for the human neuronal migration disorder lissencephaly. Mitotic functions of LIS1 have been suggested from many organisms throughout evolution. However, the cellular functions of LIS1 at distinct intracellular compartments such as the centrosome and the cell cortex have not been well defined especially during mitotic cell division. Here, we used detailed cellular approaches and time-lapse live cell imaging of mitosis from Lis1 mutant mouse embryonic fibroblasts to reveal critical roles of LIS1 in mitotic spindle regulation. We found that LIS1 is required for the tight control of chromosome congression and segregation to dictate kinetochore-microtubule (MT) interactions and anaphase progression. In addition, LIS1 is essential for the establishment of mitotic spindle pole integrity by maintaining normal centrosome number. Moreover, LIS1 plays crucial roles in mitotic spindle orientation by increasing the density of astral MT plus-end movements toward the cell cortex, which enhances cortical targeting of LIS1-dynein complex. Overexpression of NDEL1-dynein and MT stabilization rescues spindle orientation defects in Lis1 mutants, demonstrating that mouse LIS1 acts via the LIS1-NDEL1-dynein complex to regulate astral MT plus-ends dynamics and establish proper contacts of MTs with the cell cortex to ensure precise cell division.

Novel embryonic neuronal migration and proliferation defects in Dcx mutant mice are exacerbated by Lis1 reduction.

Heterozygous LIS1 mutations and males with loss of the X-linked DCX result in lissencephaly, a neuronal migration defect. LIS1 regulates nuclear translocation and mitotic division of neural progenitor cells, while the role of DCX in cortical development remains poorly understood. Here, we uncovered novel neuronal migration and proliferation defects in the Dcx mutant embryonic brains. Although cortical organization was fairly well preserved, Dcx(ko/Y) neurons displayed defective migration velocities similar to Lis1(+/ko) neurons when characterized by time-lapse video-microscopy of embryonic cortical slices. Dcx(ko/Y) migrating neurons displayed novel multidirectional movements with abnormal morphology and increased branching. Surprisingly, Dcx(ko/Y) radial glial cells displayed spindle orientation abnormalities similar to Lis1(+/ko) cells that in turn lead to moderate proliferation defects both in vivo and in vitro. We found functional genetic interaction of the two genes, with the combined effects of Lis1 haploinsufficiency and Dcx knock-out leading to more severe neuronal migration and proliferation phenotypes in the Lis1(+/ko);Dcx(ko/Y) male double mutant compared with the single mutants, resulting in cortical disorganization and depletion of the progenitor pool. Thus, we provide definitive evidence for a critical role for Dcx in neuronal migration and neurogenesis, as well as for the in vivo genetic interaction of the two genes most commonly involved in human neuronal migration defects.

Neuroepithelial stem cell proliferation requires LIS1 for precise spindle orientation and symmetric division.

Mitotic spindle orientation and plane of cleavage in mammals is a determinant of whether division yields progenitor expansion and/or birth of new neurons during radial glial progenitor cell (RGPC) neurogenesis, but its role earlier in neuroepithelial stem cells is poorly understood. Here we report that Lis1 is essential for precise control of mitotic spindle orientation in both neuroepithelial stem cells and radial glial progenitor cells. Controlled gene deletion of Lis1 in vivo in neuroepithelial stem cells, where cleavage is uniformly vertical and symmetrical, provokes rapid apoptosis of those cells, while radial glial progenitors are less affected. Impaired cortical microtubule capture via loss of cortical dynein causes astral and cortical microtubules to be greatly reduced in Lis1-deficient cells. Increased expression of the LIS/dynein binding partner NDEL1 restores cortical microtubule and dynein localization in Lis1-deficient cells. Thus, control of symmetric division, essential for neuroepithelial stem cell proliferation, is mediated through spindle orientation determined via LIS1/NDEL1/dynein-mediated cortical microtubule capture.

Role of 14-3-3 proteins in eukaryotic signaling and development.

14-3-3 genes encode a ubiquitous family of highly conserved eukaryotic proteins from fungi to humans and plants with several molecular and cellular functions. Most notably, 14-3-3 proteins bind to phosphoserine/phosphothreonine motifs in a sequence-specific manner. More than 100 14-3-3 binding partners involved in signal transduction, cell cycle regulation, apoptosis, stress responses, and malignant transformation have been identified. The 14-3-3 proteins form homodimers and heterodimers, and there is redundancy of the binding specificity and function of different 14-3-3 proteins because of their highly similar amino acid sequence and tertiary structure. 14-3-3 proteins can regulate target protein function by several mechanisms. Although the molecular and cellular functions of 14-3-3 proteins have been well studied, there have been fewer studies addressing the in vivo role of 14-3-3s. Here we review what is known about 14-3-3 proteins during eukaryotic development.

Multiple dose-dependent effects of Lis1 on cerebral cortical development.

Humans with heterozygous inactivating mutations of the Lis1 gene display type I lissencephaly, a severe form of cortical dysplasia hypothesized to result from abnormal neuronal migration. Previously we reported the construction of an allelic series of the Lis1 gene in mice to analyze the effects of graded reduction of LIS1 protein on the pathogenesis of this disorder and demonstrated a cell autonomous defect in neuronal migration (Hirotsune et al., 1998). Here we report the systematic examination of the consequences of dosage reduction of LIS1 on neocortical development using wild-type, null heterozygous (45% LIS1 protein), and compound null/hypomorphic (35% LIS1 protein) mice. The development of the preplate, Cajal-Retzius cells, and the radial glial scaffold appeared unaffected by LIS1 levels. However, a dose-dependent morphologic change in disorganization of the subplate was noted. LIS1 dose-dependent defects in neuronal migration were found in vivo and in vitro. The position and number of mitotic cells in the ventricular zone were more abnormal as LIS1 levels decreased, suggesting defects in interkinetic nuclear migration and neuroblast proliferation. LIS1 dose-dependent progressive thinning of the cortex and ventricular zone occurred by programmed cell death. Thus, in addition to its requirement for cell autonomous neuronal migration, LIS1 influences the generation and survival of cortical ventricular zone neuroblasts. These studies reveal the importance of LIS1 levels in orderly cerebral cortical morphogenesis and suggest new insights into the pathogenesis of type I lissencephaly.