LIS1 controls mitosis and mitotic spindle organization via the LIS1-NDEL1-dynein complex - PubMed (original) (raw)

. 2014 Jan 15;23(2):449-66.

doi: 10.1093/hmg/ddt436. Epub 2013 Sep 12.

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

Hyang Mi Moon et al. Hum Mol Genet. 2014.

Abstract

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.

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Figures

Figure 1.

Figure 1.

Loss of LIS1 results in a prolonged mitotic cell cycle to induce anaphase onset delay. (A) Frame series of time-lapse live imaging movies from _Lis1-_CKO-Cre MEFs (CreER ; Lis1hc/hc) and control WT-Cre MEFs (CreER; Lis1+/+). MEFs were co-infected with H2B-GFP (green) and mCherry–α-tubulin (red) retroviruses. Cre activation was induced by treatment of 4-hydroxy-TM for 12 h. Inset from WT-Cre; Metaphase—normal spindle morphology (mCherry–α-tubulin). Insets from _Lis1_-CKO-Cre; Late prometaphase—kinked and curved spindle morphology (mCherry–α-tubulin), Anaphase—arrowhead: unattached chromosomes (H2B-GFP), Telophase—arrow: lagging chromosomes (H2B-GFP). Minutes indicate the time from nuclear envelope breakdown. Scale bar: 10 μm. (B) Time from nuclear envelope breakdown to metaphase plate formation. (C) Time from metaphase plate formation to anaphase onset. (D) Time from nuclear envelope breakdown to anaphase onset. (E) Accumulated percentage of cells entered into anaphase from metaphase. Lines in (B–D): mean ± SD, asterisks: **P < 0.01, ***P < 0.001 by Student's _t_-test. ns, not significant.

Figure 2.

Figure 2.

Loss of LIS1 leads to extra centrosomes formation with centrosome number abnormality. (A) Time-lapse live cell imaging analysis reveals the centrosome number abnormality in _Lis1_-CKO-Cre MEFs compared with WT-Cre MEFs. (B) Multipolar division was found in time-lapse live cell imaging of _Lis1_-CKO-Cre MEFs (1/73). (C) Fixed sample analysis of centrosome number from WT and Lis1hc/ko MEFs. (D) WT MEFs with normal two centrosomes and Lis1hc/ko MEFs with extra centrosomes. MEFs were stained with γ-tubulin (green) and DAPI (blue). Scale bars: 10 μm. (E) Centrosome number distributions in WT and Lis1hc/ko MEFs. More than three slides were analyzed, n > 250 cells from each genotype. Bars: mean ± SEM, asterisk: ***P < 0.001 by the two-way ANOVA test.

Figure 3.

Figure 3.

Lis1 mutant MEFs exhibit centrosome clustering phenotype during mitosis. (A–D) Centrosome clustering phenotype with normal centrosome maturation in Lis1hc/ko MEFs. (A) Co-staining with pericentrin (pericentriolar material marker) and centrin (each centriole). Co-staining with centrin and several mature centriole markers: (B) cennexin/ODF2, (C) ninein, (D) Cep164, respectively. Insets in (A–D): high magnification images of centrosomes. Scale bars: 5 μm.

Figure 4.

Figure 4.

Loss of LIS1 induces chromosome misalignment in metaphase and chromosome missegregation in anaphase during mitosis. (A) Time-lapse live cell imaging analysis of chromosomal behavior (labeled with H2B-GFP, green) from _Lis1_-CKO-Cre MEFs and WT-Cre MEFs. Arrowhead: misaligned chromosomes in _Lis1_-CKO-Cre MEFs. Scale bars: 5 μm. (B) Chromosome missegregation phenotypes in mitosis of _Lis1_-CKO-Cre MEFs. Arrow: numerous lagging chromosomes; arrowhead: (left) the chromosomes stuck in intercellular chromatin bridge, (right) micronuclei formation in telophase. MT networks were visualized by mCherry–α-tubulin (red). (C) Quantification of lagging chromosome appearance in time-lapse live cell imaging of _Lis1-_CKO-Cre MEFs and WT-Cre MEFs. Numbers of lagging chromosomes—no lagging: 0, few lagging: 1–3, numerous lagging: ≥4. (D) Increased incidence of various chromosome segregation defects _Lis1-_CKO-Cre MEFs compared with WT-Cre MEFs.

Figure 5.

Figure 5.

Loss of LIS1 impairs spindle orientation during mitosis of MEFs. (A) Confocal Z-stack image series of mitotic spindles stained with pericentrin (centrosomal marker, green), α-tubulin (mitotic spindle, red) and DAPI (blue). MEFs were arrested in metaphase with MG132 treatment for 2 h. Scale bars: 5 μm. (B) Schematic representation of spindle orientation in mitotic cells. Spindle angle (α°) and distance between spindle poles (D, μm) were measured by taking Z-stack confocal images from 0.5 μm thick sections. Centrosomes were identified with co-localization of pericentrin and α-tubulin (marked in yellow). (C) Average spindle angles of Lis1hc/ko MEFs and WT MEFs. (D) Average distance between spindle poles. Bars in (C) and (D): mean ± SEM, Asterisks in (C) and (D): ***P < 0.001 by Student's _t_-test.

Figure 6.

Figure 6.

Loss of LIS1 results in less cortical dynein/dynactin complex recruitment to the cell cortex during mitosis. (A) Distribution of a dynactin subunit, p150glued (green) in mitosis of WT and Lis1hc/ko MEFs. Chromosomes were stained with DAPI (blue). Scale bars: 5 μm. (B) Relative fluorescence intensity of cortically located dynactin p150glued in WT and Lis1hc/ko MEFs. (C) Ratio of polar cortex-associated dynactin normalized by equatorial cortex-associated dynactin p150glued (more than eight cells were analyzed from each genotype). Asterisk in (A): *P < 0.05, (B) ns: not significant by Student's _t_-test.

Figure 7.

Figure 7.

Loss of LIS1 causes aberrant interactions between astral MT plus-ends and the cell cortex. (A) The astral MTs in early anaphase from Lis1hc/ko MEFs and WT MEFs stained with α-tubulin after glutaraldehyde fixation. Arrow: aberrant astral MT tips in Lis1hc/ko MEFs. (Right panel) Misattachment of astral MTs to the opposite polar cortex in Lis1hc/ko MEFs harboring extra centrosomes (four centrosomes). (B) Schematic representation of interaction between astral MTs and the cell cortex–end-on interaction (θ° ≥ 60) versus lateral interaction (θ° < 60). (C) Quantification of types of astral MT interaction with the cell cortex in MEFs. (D) Localization of MT plus-ends stained with EB1 (MT plus-end binding protein) in metaphase of Lis1hc/ko MEFs and WT MEFs. Blue dashed lines indicate the cell membranes. Scale bars: 5 μm. (E) Relative intensity of astral MTs labeled with EB1 in metaphase-arrested MEFs (more than eight cells were analyzed from each genotype). Asterisk in (E): ***P < 0.001 by Student's _t_-test.

Figure 8.

Figure 8.

Reduced frequency of movements of EB1-labeled astral MT plus-ends near the cell cortex. (A) Frame series of time-lapse live cell imaging movies from WT MEF infected with EB1-GFP adenovirus. (B) Frame series of time-lapse live cell imaging movies from Lis1hc/ko MEF. (A′, B′) High magnification images from insets in (A) and (B). Scale bars: 10 μm. These montages are generated from the representative movies of dividing MEFs from each genotype (more than ten cells were imaged for live cell imaging).

Figure 9.

Figure 9.

Spindle misorientation phenotype in Lis1 mutant MEFs is rescued by MT stabilization and overexpression of several components of the LIS1–NDEL1–dynein complex. (A) Average spindle angles in WT and Lis1hc/ko MEFs treated with DMSO and taxol. (B) MEFs infected with retroviruses encoding GFP, GFP-LIS1, GFP-NDEL1, GFP-NDE1 and GFP-DIC1. (C) Average spindle angles in NDE1 KO MEFs (Nde1ko/ko), NDEL1 CKO MEFs (Ndel1hc/hc + Cre) and GFP-LGN-C overexpressing WT MEFs. Bars in (A)–(C): mean ± SEM, Asterisks in (A)–(C): *P < 0.05, **P < 0.01, ***P < 0.001 by ANOVA with Bonferroni's post hoc test. ns, not significant.

Figure 10.

Figure 10.

Proposed working model of LIS1 function in mitotic cell divisions. See text in discussion.

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