A Rac switch regulates random versus directionally persistent cell migration

Directional migration moves cells rapidly between points, whereas random migration allows cells to explore their local environments. We describe a Rac1 mechanism for determining whether cell patterns of migration are intrinsically random or directionally persistent. Rac activity promoted the formation of peripheral lamellae that mediated random migration. Decreasing Rac activity suppressed peripheral lamellae and switched the cell migration patterns of fibroblasts and epithelial cells from random to directionally persistent. In three-dimensional rather than traditional two-dimensional cell culture, cells had a lower level of Rac activity that was associated with rapid, directional migration. In contrast to the directed migration of chemotaxis, this intrinsic directional persistence of migration was not mediated by phosphatidylinositol 3′-kinase lipid signaling. Total Rac1 activity can therefore provide a regulatory switch between patterns of cell migration by a mechanism distinct from chemotaxis

Prediction analysis of networks and transcription factors regulating <i>IKBKAP</i> and <i>IKBKAP</i> co-regulated functional candidate genes.

<p>“String” analysis of protein networks showing the potential interaction between the majority of the <i>IKBKAP</i> co-regulated functional candidate genes. (B) Prediction of transcription factors (TF) related to <i>IKBKAP</i> and co-regulated functional candidate genes. (C) Shows relative quantification levels represented as mean ± s.d. of TF gene candidates taken from the total cDNA microarray analysis showing difference between WT and FD-hESC derived PNS neurons. * P<0.05 **P<0.01, Non statistical significance (NS).</p

IKAP expression in WT and FD early and mature neurons.

<p>(A) RT-PCR analysis of the expression of <i>IKBKAP</i> showing WT (upper lane) and FD (mis-spliced, lower lane) mRNA isoforms at the stage of early neuronal precursors, early and mature neurons. (B) qRT-PCR analysis of the levels of <i>IKBKAP</i> WT (blue) and FD (green) mRNA isoforms. (C) Western blot analysis of IKAP protein levels in WT and FD early and mature neurons. Actin served as loading control.</p

Comparative transcriptome analysis between human 12 weeks fetal WT and FD brains to WT and FD hESC derived neurons.

<p>Prior to cDNA microarray chip analysis mRNA and protein extracts from the brain samples were used to validate the FD phenotype of the FD splicing mutation in the <i>IKBKAP</i> gene at the transcriptional and translational levels by: (A) RT-PCR analysis of the expression of <i>IKBKAP</i> in human 12 weeks fetal WT and FD brains showing WT (upper lane) and FD (mis-spliced, lower lane) mRNA isoforms in the FD brain only. (B) Western blot analysis of IKAP protein levels in WT and FD brains. Note the almost complete absence of IKAP in the FD brain. β-Actin served as loading control. Comparative transcriptome analysis between data obtained from both cDNA microarray chips of WT and FD fetal brains and hESC-derived PNS neurons was performed by cross-referencing genes which their expression levels differ significantly (>2 fold) between FD and WT. (C) Venn Diagram representation of a wide genome transcriptome analysis of common genes that are differentially expressed in FD-hESC-derived PNS neurons (in green) and in two other known FD stem cell neural-derived models (FD fibroblasts derived iPSC (in yellow) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138807#pone.0138807.ref023" target="_blank">23</a>] and FD-hOE-MSC (in blue) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138807#pone.0138807.ref024" target="_blank">24</a>] and in FD Fetal brain (in red). Cross-referencing genes with their expression levels difference of >2 fold change for hESC p<0.05 or >1.5fold change p<0.05 for iPSC and hOE-MSC between WT and FD were considered for analysis. The number at the crossection between the diagrams represents the number of genes that are shared by these multiple analyses. For the list of genes see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138807#pone.0138807.s009" target="_blank">S3 Table</a>. (D) Gene sets were divided into downregulated and upregulated genes in both hESC-derived PNS neurons and Fetal brain biological systems. Venn diagram represents the results from this analysis showing in blue, the number of genes that differ in WT and FD in the fetal <i>in vivo</i> and in yellow, the number of genes that differ in WT and FD hESC derived neurons <i>in vitro</i>. The crossection between the diagrams represent the number of genes that are shared by both analyses. Upper diagrams represent the downregulated genes while the lower ones represent the upregulated genes.</p

Comparative analysis of expression differences in WT and FD hESC derived PNS differentiation process.

<p>(A) Venn diagram comparing the overall number of genes that showed > 2 fold expression differences between hNP and fully differentiated hESC-derived neurons in WT (in blue) and FD (in yellow) backgrounds. (B) The normalized relative quantification expression values are shown as mean ± s.d. of several developmental PNS markers and transcription factors in WT and FD hESC-derived neurons as obtained in the transcriptome cDNA chip analysis. * P<0.05 **P<0.01.</p

Characterization of IKAP localization in PNS WT and FD hESC-derived cultured neurons.

<p>Immunofluorescence confocal microscopy analysis was performed as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138807#pone.0138807.g003" target="_blank">Fig 3</a>. IKAP together with peripherin and Rab3a expression are shown within hESC derived neurons in WT (A-F) and FD (G-L) genetic backgrounds. Images B and H show the expression and localization of IKAP in WT and FD derived-neurons respectively. C and I, D and J show the expression of peripherin and Rab3a respectively. IKAP and Rab3a co-localization levels are shown in WT (F) and FD (L). Quantitative analysis of the mean intensity of IKAP and RAB3a colocalization levels in WT and FD axons is shown in M. 3D stacks of sequential confocal images were de-convolved using Huygens (scientific volume imaging-SVI) software and the analyzed for colocalization between the signals was performed using Imaris (Bitplan). Bar sizes are indicated in representative images. Mouse anti-hIKAP (BD Biosciences) was used in these experiments.</p