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. 2015 Sep 23;10(9):e0138763.
doi: 10.1371/journal.pone.0138763. eCollection 2015.

LGN Directs Interphase Endothelial Cell Behavior via the Microtubule Network

Catherine E Wright  1 , Erich J Kushner  2 , Quansheng Du  3 , Victoria L Bautch  4
Affiliations

LGN Directs Interphase Endothelial Cell Behavior via the Microtubule Network

Catherine E Wright et al. PLoS One. .

Abstract

Angiogenic sprouts require coordination of endothelial cell (EC) behaviors as they extend and branch. Microtubules influence behaviors such as cell migration and cell-cell interactions via regulated growth and shrinkage. Here we investigated the role of the mitotic polarity protein LGN in EC behaviors and sprouting angiogenesis. Surprisingly, reduced levels of LGN did not affect oriented division of EC within a sprout, but knockdown perturbed overall sprouting. At the cell level, LGN knockdown compromised cell-cell adhesion and migration. EC with reduced LGN levels also showed enhanced growth and stabilization of microtubules that correlated with perturbed migration. These results fit a model whereby LGN influences interphase microtubule dynamics in endothelial cells to regulate migration, cell adhesion, and sprout extension, and reveal a novel non-mitotic role for LGN in sprouting angiogenesis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Loss of LGN perturbs sprouting angiogenesis.
(A, B) Confocal images (compressed z-stacks) of representative beads with sprouting HUVEC and indicated viral infection. Green, GFP-labeled infected HUVEC. (C-E) Quantification of indicated parameters. Statistics, unpaired student’s t-test, two-tailed. Error bars, SEM; n = 5 experiments; **, p<0.01. ****; p<0.0001.
Fig 2
Fig 2. Cell-cell adhesions are destabilized in LGN KD HUVEC.
(A) Representative images of HUVEC monolayers stained for VE-cadherin (green) and nucleus (DRAQ, pink) between indicated groups. (B) Scatter plot of % VE-cadherin area in HUVEC monolayers between indicated groups. Statistics, one-way ANOVA with Tukey’s test; n = 2 experiments; ***, p<0.001.(C) Representative images of HUVEC monolayers stained for VE-cadherin (green) and nucleus (DRAQ, pink) pre- and post- EDTA washout. After washout, junctions were allowed to reform for 1 hr. (D) Scatter plot of % VE-cadherin area in EC monolayers between indicated groups. White boxes, areas of higher magnification. Control, HUVEC; NT RNA, HUVEC +non-targeting RNA; LGN KD, HUVEC + LGN siRNA. Statistics, one-way ANOVA with Tukey’s test. Error bars, mean and 95% confidence intervals (CI); n = 2 experiments; ***, p≤ 0.001; ns, not significant. Scale bars, 10μm.
Fig 3
Fig 3. LGN KD HUVEC have reduced migration and perturbed focal adhesion turnover.
(A) Plots of individual cell migration tracks, axes in μm. (B) Quantification of total distance traveled over 12 hr. Statistics, unpaired student’s t-test, two-tailed. Error bars, average and 95% CI; n = 3 experiments; *, p<0.05. (C) Schematic showing directional change measurement. (D) Percentage of EC angle changes that averaged less than 30° over 12 hr for a given cell. Statistics, unpaired student’s t-test, two-tailed. Error bars, average and 95% CI; n = 3 experiments; ***, p<0.001. (E) Distribution of focal adhesion (FA) length in control and LGN KD HUVEC 20 min after nocodazole washout. Statistics, unpaired student’s t-test, two-tailed. Error bars, SEM; n = 3 experiments; *, p<0.05; **, p<0.01; ***, p<0.001. (F) Scatter plot of longest FAs in control and LGN KD HUVEC 20 min after nocozadole washout. FAs were visualized by vinculin staining and analyzed using FAAS (http://faas.bme.unc.edu/) and parameters defined in Methods. Statistics, unpaired student’s t-test, two-tailed. Error bars, mean and 95% CI; n = 3 experiments; ***, p<0.001.
Fig 4
Fig 4. Abnormal MT dynamics and stability in HUVEC with reduced LGN.
(A) Representative images of EV and LGN KD HUVEC stained with α-tubulin 1 min post-nocodazole washout. (B) Scatter plot of MT nucleations post-nocodazole washout. Statistics, unpaired student’s t-test, two-tailed. Error bars, mean and 95% CI; n = 3 experiments; NS, not significant. (C) Distributions of MT length 1 min post-nocodazole washout; Statistics, unpaired student’s t-test, two-tailed; n = 3 experiments; EV, n = 210; LGN KD, n = 188; p<0.0001. (D) Time projections of 60 sec movies of EB1-GFP labeled MT plus-ends in EV and LGN KD HUVEC. (E-F) Quantification of indicated parameters. EV, n = 10 cells; LGN KD, n = 11 cells. Statistics, unpaired student’s t-test, two-tailed. Error bars, mean and 95% CI; n = 2 experiments; *, p = 0.01; NS, not significant. (G) Distribution of MT plus ends based on lifetime and growth speed. Statistics, unpaired student’s t-test, two-tailed; n = 2 experiments; ***, p<0.001. (H) Representative images of HUVEC 1 hr post cold washout stained for MTs (α-tubulin, red), detyrosinated MTs (green), and nuclei (blue) between control (Cont); non targeting siRNA (NT RNA) and LGN KD siRNA treated cells. (I) Scatter plot comparing detyrosinated tubulin levels as a ratio of detyrosinated tubulin/α-tubulin between groups. Statistics, one-way ANOVA with Tukey’s test; n = 2 experiments; ***, p<0.001; ****, p<0.0001. (J) Representative images of HUVEC 1 hr post cold washout stained for MTs (α-tubulin, red), acetylated MTs (green), and nuclei (blue) between indicated groups. (K) Scatter plot comparing acetylated tubulin levels as a ratio of acetylated tubulin/α-tubulin between groups. White boxes, areas of higher magnification. Statistics, one-way ANOVA with Tukey’s test. Error bars, mean and 95% CI; n = 2 experiments; ns, not significant; *, p≤0.05; **, p≤0.01. Scale bars, 10μm.
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