Osteoblast Recruitment and Bone Formation Enhanced by Cell Matrix–associated Heparin-binding Growth-associated Molecule (HB-GAM)

Bone has an enormous capacity for growth, regeneration, and remodeling. This capacity is largely due to induction of osteoblasts that are recruited to the site of bone formation. The recruitment of osteoblasts has not been fully elucidated, though the immediate environment of the cells is likely to play a role via cell– matrix interactions. We show here that heparin-binding growth-associated molecule (HB-GAM), an extracellular matrix–associated protein that enhances migratory responses in neurons, is prominently expressed in the cell matrices that act as target substrates for bone formation. Intriguingly, N-syndecan, which acts as a receptor for HB-GAM, is expressed by osteoblasts/osteoblast precursors, whose ultrastructural phenotypes suggest active cell motility. The hypothesis that HB-GAM/N-syndecan interaction mediates osteoblast recruitment, as inferred from developmental studies, was tested using osteoblast-type cells that express N-syndecan abundantly. These cells migrate rapidly to HB-GAM in a haptotactic transfilter assay and in a migration assay where HB-GAM patterns were created on culture wells. The mechanism of migration is similar to that previously described for the HB-GAM–induced migratory response of neurons. Our hypothesis that HB-GAM/N-syndecan interaction participates in regulation of osteoblast recruitment was tested using two different in vivo models: an adjuvant-induced arthritic model and a transgenic model. In the adjuvant-induced injury model, the expression of HB-GAM and of N-syndecan is strongly upregulated in the periosteum accompanying the regenerative response of bone. In the transgenic model, the HB-GAM expression is maintained in mesenchymal tissues with the highest expression in the periosteum. The HB-GAM transgenic mice develop a phenotype characterized by an increased bone thickness. HB-GAM may thus play an important role in bone formation, probably by mediating recruitment and attachment of osteoblasts/osteoblast precursors to the appropriate substrates for deposition of new bone.     

Reelin controls progenitor cell migration in the healthy and pathological adult mouse brain

Understanding the signals that control migration of neural progenitor cells in the adult brain may provide new therapeutic opportunities. Reelin is best known for its role in regulating cell migration during brain development, but we now demonstrate a novel function for reelin in the injured adult brain. First, we show that Reelin is upregulated around lesions. Second, experimentally increasing Reelin expression levels in healthy mouse brain leads to a change in the migratory behavior of subventricular zone-derived progenitors, triggering them to leave the rostral migratory stream (RMS) to which they are normally restricted during their migration to the olfactory bulb. Third, we reveal that Reelin increases endogenous progenitor cell dispersal in periventricular structures independently of any chemoattraction but via cell detachment and chemokinetic action, and thereby potentiates spontaneous cell recruitment to demyelination lesions in the corpus callosum. Conversely, animals lacking Reelin signaling exhibit reduced endogenous progenitor recruitment at the lesion site. Altogether, these results demonstrate that beyond its known role during brain development, Reelin is a key player in post-lesional cell migration in the adult brain. Finally our findings provide proof of concept that allowing progenitors to escape from the RMS is a potential therapeutic approach to promote myelin repair.     

Stable partial migration under a genetic threshold model of migratory behaviour

Many species show migratory behaviour in response to seasonal changes in environmental conditions. A peculiar, yet widespread phenomenon is partial migration, when a single population consists of both migratory and non‐migratory individuals. There are still many open questions regarding the stability and evolutionary significance of such populations. For passerines the inheritance of migratory activity is best described by the threshold model of quantitative genetics. Such a model has not yet been employed in theoretical studies, in which stability of partially migratory populations is usually linked to group differences in survival or reproduction. Here we develop a parsimonious model featuring a conditional genetic threshold for passerine migratory behaviour under which stable partial migration can be observed, and we explore the resulting selection landscape. Our model results show a cline in migratory behaviour across the landscape, from fully migratory populations to fully residential populations, with a fairly wide zone of partially migratory populations, which is stable in both time and space under a wide range of parameter settings. Temporal stability of the zone is linked with the yearly variance in both migration survival and resident winter survival. In contrast to other theoretical studies, we show that density dependence in winter survival is not essential for observing partially migratory populations. In addition, we observe that selection on the genetic threshold value occurs mainly at the borders of the zone of partial migration. This result suggests that fully migratory and fully residential populations in areas far from the zone of partial migration can harbour genetic diversity that allows the appearance of the alternative phenotype under (a wide range of) different conditions.     

Evolutionary Divergence in Brain Size between Migratory and Resident Birds

Despite important recent progress in our understanding of brain evolution, controversy remains regarding the evolutionary forces that have driven its enormous diversification in size. Here, we report that in passerine birds, migratory species tend to have brains that are substantially smaller (relative to body size) than those of resident species, confirming and generalizing previous studies. Phylogenetic reconstructions based on Bayesian Markov chain methods suggest an evolutionary scenario in which some large brained tropical passerines that invaded more seasonal regions evolved migratory behavior and migration itself selected for smaller brain size. Selection for smaller brains in migratory birds may arise from the energetic and developmental costs associated with a highly mobile life cycle, a possibility that is supported by a path analysis. Nevertheless, an important fraction (over 68%) of the correlation between brain mass and migratory distance comes from a direct effect of migration on brain size, perhaps reflecting costs associated with cognitive functions that have become less necessary in migratory species. Overall, our results highlight the importance of retrospective analyses in identifying selective pressures that have shaped brain evolution, and indicate that when it comes to the brain, larger is not always better.     

Semaphorin-3A and its receptor neuropilin-1 are predominantly expressed in endothelial cells along the rostral migratory stream of young and adult mice

In the adult brain, neuroblasts originating in the subventricular zone migrate through the rostral migratory stream to the olfactory bulb. While migrating, neuroblasts undergo progressive differentiation until reaching their final locations and fates. Because molecules involved in migration may also exert differentiating effects on young neurons, the identification of factors that support migration could also shed light on the processes of adult neuroblast differentiation. This is the case for members of the family of semaphorins and of its cognate receptors, the neuropilins. Here, we have evaluated the presence of semaphorin-3A and of its receptor neuropilin-1 along the rostral migratory stream in young and adult mice by using immunocytochemical, histochemical, and in situ hybridization techniques. Our morphological studies show that semaphorin-3A and neuropilin-1 are both mainly expressed on endothelial cells along the rostral migratory stream during postnatal development. Our results suggest that endothelial cells constitute the primary source and target of semaphorin-3A along the rostral migratory stream. Moreover, the present work outlines the potential role of blood vessels on neuroblast migration in the postnatal rostral migratory stream.     

Migratory behavior of cells on embryonic retina basal lamina

In order to study cell translocation in vitro on a physiological substrate a novel cell migration assay was developed using the inner limiting membrane of the avian embryonic retina. The matrix sheet consists of a laminin-rich basal lamina covered by a dense layer of neuroepithelial endfeet. The retina basal lamina does not contain fibronectin. Cells translocating on this substrate displace the neuroepithelial endfeet, leaving behind tracks in the endfeet monolayer. Motility of cells and the relative forward to lateral migration can be quantitated by measuring lengths, widths, and areas of the tracks. Using this assay system, the conditions and patterns of cell migration for a variety of cells have been examined. In the absence of serum all cell types show only minor migratory activity and addition of serum to the culture medium always enhances the rate of cell migration in a saturable, dose-response manner. The serum cannot be replaced by fibronectin or vitronectin (serum spreading factor). For maximum cell migration, serum has to be constantly present in the medium; however, 58% cell migration is obtained in serum-free medium when the matrix is preincubated with serum. According to the area and linearity of the tracks, the migratory behavior of the different cells can be classified into three groups: (i) fibroblasts and the nonpigmented Bowes melanoma cells form straight and long tracks; (ii) glioma, sarcoma, and carcinoma cells from straight but short tracks, and (iii) neuronal tumor cells, epithelial cells, and pigmented B16 melanoma cells form wide and short tracks. Comparative studies with low and high metastatic clones of tumorgenic cell lines show that migratory activity and metastatic potential of cells do not necessarily correlate. Finally, we show that fibroblasts deposit fibronectin fibrils on their paths as they migrate on the basal lamina. Fibronectin trails are also seen when fibroblasts are cultured on plain basal laminae that are pretreated with detergent to remove the endfeet monolayer. Likewise, when fibroblasts are cultured in the presence of antifibronectin antibodies, the fibronectin secreted by cells is detectable. Due to antibody treatment the cellular fibronectin is precipitated and its normal fibril formation is inhibited; however, the translocation of fibroblasts is not impaired.     

Myosin-X is critical for migratory ability of Xenopus cranial neural crest cells

The neural crest is a highly migratory cell population, unique to vertebrates, that forms much of the craniofacial skeleton and peripheral nervous system. In exploring the cell biological basis underlying this behavior, we have identified an unconventional myosin, myosin-X (Myo10) that is required for neural crest migration. Myo10 is highly expressed in both premigratory and migrating cranial neural crest (CNC) cells in Xenopus embryos. Disrupting Myo10 expression using antisense morpholino oligonucleotides leads to impaired neural crest migration and subsequent cartilage formation, but only a slight delay in induction. In vivo grafting experiments reveal that Myo10-depleted CNC cells migrate a shorter distance and fail to segregate into distinct migratory streams. Finally, in vitro cultures and cell dissociation-reaggregation assays suggest that Myo10 may be critical for cell protrusion and cell-cell adhesion. These results demonstrate an essential role for Myo10 in normal cranial neural crest migration and suggest a link to cell-cell interactions and formation of processes.     

The Par-Tiam1 complex controls persistent migration by stabilizing microtubule-dependent front-rear polarity

BACKGROUND: The establishment and maintenance of cell polarity is crucial for many biological functions and is regulated by conserved protein complexes. The Par polarity complex consisting of Par3, Par6, and PKCzeta, in conjunction with Tiam1-mediated Rac signaling, controls apical-basal cell polarity in contacting epithelial cells. Here we tested the hypothesis that the Par complex, in conjunction with Tiam1, controls "front-rear" polarity during the persistent migration of freely migrating keratinocytes. RESULTS: Wild-type (WT) epidermal keratinocytes lacking cell-cell contacts are stably front-rear polarized and migrate persistently. In contrast, Tiam1-deficient (Tiam1 KO) and (si)Par3-depleted keratinocytes are generally unpolarized and migrate randomly because front-rear polarity is short lived. Immunoprecipitation experiments show that in migrating keratinocytes, Tiam1 associates with Par3 and PKCzeta. Moreover, Par3, PKCzeta, and Tiam1 proteins are enriched at the leading edges of polarized keratinocytes. Tiam1 KO keratinocytes are impaired in chemotactic migration toward growth factors, whereaes haptotactic migration is similar to WT. Par3 depletion or the blocking of PKCzeta signaling in WT keratinocytes impairs chemotaxis but has no additional effect on Tiam1 KO cells. The migratory and morphological defects in keratinocytes with impaired Par-Tiam1 function closely resemble cells with pharmacologically destabilized microtubules (MTs). Indeed, MTs in Tiam1 KO keratinocytes and WT cells treated with a PKCzeta inhibitor are unstable, thereby negatively influencing directional but not random migration. CONCLUSIONS: We conclude that the Par-Tiam1 complex stabilizes front-rear polarization of noncontacting migratory cells, thereby stimulating persistent and chemotactic migration, whereas in contacting keratinocytes, the same complex controls the establishment of long-lasting apical-basal polarity. These findings underscore a remarkable flexibility of the Par polarity complex that, depending on the biological context, controls distinct forms of cellular polarity.     

Brain regions associated with visual cues are important for bird migration

Long-distance migratory birds have relatively smaller brains than short-distance migrants or residents. Here, we test whether reduction in brain size with migration distance can be generalized across the different brain regions suggested to play key roles in orientation during migration. Based on 152 bird species, belonging to 61 avian families from six continents, we show that the sizes of both the telencephalon and the whole brain decrease, and the relative size of the optic lobe increases, while cerebellum size does not change with increasing migration distance. Body mass, whole brain size, optic lobe size and wing aspect ratio together account for a remarkable 46% of interspecific variation in average migration distance across bird species. These results indicate that visual acuity might be a primary neural adaptation to the ecological challenge of migration.     

Phosphatidylinositol 3-kinase in the G protein-coupled receptor-induced chemokinesis and chemotaxis of MDA-MB-468 breast carcinoma cells: a comparison with leukocytes

The polarization of tumor cells and leukocytes into a front end and a rear end is a crucial prerequisite for their autonomous, directed movement. Phosphatidylinositol 3-kinase (PI3K) is assumed to play an important role in this polarization process, whereas the results obtained with different cell types and different migration assays widely vary. Thus, we conducted a comparative study on the role of the PI3K in the locomotor activity and directionality of the migration of tumor cells on the example of MDA-MB-468 breast carcinoma cells in comparison with CTLs and neutrophil granulocytes. We used our well-established, collagen-based, three-dimensional migration assay for the investigation of the chemokinesis and chemotaxis of these cells. Our results show that the role of the PI3K in the regulation of migratory activity is distinct between the investigated cell types: the migration of CTLs and MDA-MB-468 cells was impaired by the inhibition of the PI3K with wortmannin, whereas neutrophil granulocytes were only slightly affected. However, neither cell type was impaired in the ability to respond chemotactically to gradients of ligands to G protein-coupled receptors. Thus, the PI3K contributes to the regulation of migratory activity but not to the directionality of migration of MDA-MB-468 breast carcinoma cells. As a further conclusion with regard to cancer treatment, the PI3K is not a suitable target for the inhibition of metastasis formation, because the migration of leukocytes is also affected, which leads to a dysfunction of the immune defense.     

Distinct regulation of mitogen-activated protein kinases and p27Kip1 in smooth muscle cells from different vascular beds. A potential role in establishing regional phenotypic variance

Excessive proliferation and migration of vascular smooth muscle cells (SMCs) participate in atherosclerotic plaque growth. In this study, we investigated whether SMCs from vessels with different atherogenicity exhibit distinct growth and migratory potential and investigated the underlying mechanisms. In fat-fed rabbits, we found increased cell proliferation and atheroma formation in the aortic arch versus the femoral artery. When examined in culture, SMCs isolated from the aortic arch (ASMCs) displayed a greater capacity for inducible proliferation and migration than paired cultures of femoral artery SMCs. Two lines of evidence suggested that distinct regulation of the growth suppressor p27(Kip1) (p27) contributes to establishing these phenotypic dissimilarities. First, p27 expression was comparably lower in ASMCs, which exhibited a higher fraction of p27 phosphorylated on Thr-187 and ubiquitinated. Second, forced p27 overexpression in ASMCs impaired their proliferative and migratory potential. We found that platelet-derived growth factor-BB-dependent induction of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway was comparably higher in ASMCs. Importantly, pharmacological inhibition of MAPKs increased p27 expression and attenuated ASMC proliferation and migration. In contrast, forced MAPK activation diminished p27 expression and markedly augmented femoral artery SMC proliferation and migration. We propose that intrinsic differences in the regulation of MAPKs and p27 play an important role in creating variance in the proliferative and migratory capacity of vascular SMCs, which might in turn contribute to establishing regional variability in atherogenicity.     

Modularity of trophic network is driven by phylogeny and migration in a steppe ecosystem

Evidence is mounting that the structures of trophic networks are governed by migratory movements of interacting species and also by their phylogenetic relationships. Using the largest available trophic network of a large steppe ecosystem, we tested that steppe trophic networks including migratory species are associated with (i) migratory strategy and (ii) phylogenetic relatedness of interacting species: (1) whole graph-level metrics, estimated as modularity, and (2) species-level network metrics, measured as node degree (number of interacting partners), and centrality metrics. We found that (1) a substantial number of links were established by migrant taxa; (2) the phylogenetic signal in network structure was moderate for both consumer and prey nodes; (3) both consumer and prex phylogenies affected modularity, which was modulated by migration strategy; and (4) all species-level graph properties significantly differed between networks including and excluding migratory taxa. In sum, here we show that the structure of steppe trophic networks is primarily governed by migratory strategies and to a lesser extent, by phylogenetic relatedness, using the largest available food web representative for steppe ecology and migration biology.     

Cadherin-2 Controls Directional Chain Migration of Cerebellar Granule Neurons

Long distance migration of differentiating granule cells from the cerebellar upper rhombic lip has been reported in many vertebrates. However, the knowledge about the subcellular dynamics and molecular mechanisms regulating directional neuronal migration in vivo is just beginning to emerge. Here we show by time-lapse imaging in live zebrafish (Danio rerio) embryos that cerebellar granule cells migrate in chain-like structures in a homotypic glia-independent manner. Temporal rescue of zebrafish Cadherin-2 mutants reveals a direct role for this adhesion molecule in mediating chain formation and coherent migratory behavior of granule cells. In addition, Cadherin-2 maintains the orientation of cell polarization in direction of migration, whereas in Cadherin-2 mutant granule cells the site of leading edge formation and centrosome positioning is randomized. Thus, the lack of adhesion leads to impaired directional migration with a mispositioning of Cadherin-2 deficient granule cells as a consequence. Furthermore, these cells fail to differentiate properly into mature granule neurons. In vivo imaging of Cadherin-2 localization revealed the dynamics of this adhesion molecule during cell locomotion. Cadherin-2 concentrates transiently at the front of granule cells during the initiation of individual migratory steps by intramembraneous transport. The presence of Cadherin-2 in the leading edge corresponds to the observed centrosome orientation in direction of migration. Our results indicate that Cadherin-2 plays a key role during zebrafish granule cell migration by continuously coordinating cell-cell contacts and cell polarity through the remodeling of adherens junctions. As Cadherin-containing adherens junctions have been shown to be connected via microtubule fibers with the centrosome, our results offer an explanation for the mechanism of leading edge and centrosome positioning during nucleokinetic migration of many vertebrate neuronal populations.     

Lipoic acid affects cellular migration into the central nervous system and stabilizes blood-brain barrier integrity

Reactive oxygen species (ROS) play an important role in various events underlying multiple sclerosis (MS) pathology. In the initial phase of lesion formation, ROS are known to mediate the transendothelial migration of monocytes and induce a dysfunction of the blood-brain barrier (BBB). In this study, we describe the beneficial effect of the antioxidant alpha-lipoic acid (LA) on these phenomena. In vivo, LA dose-dependently prevented the development of clinical signs in a rat model for MS, acute experimental allergic encephalomyelitis (EAE). Clinical improvement was coupled to a decrease in leukocyte infiltration into the CNS, in particular monocytes. Monocytes isolated from the circulation of LA-treated rats revealed a reduced migratory capacity to cross a monolayer of rat brain endothelial cells in vitro compared with monocytes isolated from untreated EAE controls. Using live cell imaging techniques, we visualized and quantitatively assessed that ROS are produced within minutes upon the interaction of monocytes with brain endothelium. Monocyte adhesion to an in vitro model of the BBB subsequently induced enhanced permeability, which could be inhibited by LA. Moreover, administration of exogenous ROS to brain endothelial cells induced cytoskeletal rearrangements, which was inhibited by LA. In conclusion, we show that LA has a protective effect on EAE development not only by affecting the migratory capacity of monocytes, but also by stabilization of the BBB, making LA an attractive therapeutic agent for the treatment of MS.     

3D culture model and computer-assisted videomicroscopy to analyze migratory behavior of noninvasive and invasive bronchial epithelial cells

To date, most of the studies in the field of cell migration have been applied to two-dimensional (2D) models. To mimic the three-dimensional (3D) conditions similar to those observed in vivo during tumor invasion, we developed a 3D model of cell migration in which cells were embedded in a collagen I matrix placed in a double-compartment chamber. Using time-lapse videomicroscopy and interactive cell tracking in a four-dimensional data set, we determined the cell trajectories and their migration kinetics. We compared the 2D and 3D migratory behavior of a noninvasive cell line (16HBE) with the migratory behavior of an invasive cell line (BZR). Our results show that the 3D migration kinetics of the noninvasive cell line were lower than the migration kinetics of the invasive cell line. In contrast, in 2D models, no significant difference was observed between the two cell lines. To validate our 3D model, we further investigated the effect of epidermal growth factor (EGF), a promoter of tumor cell motility and invasion on the noninvasive cell line (16HBE). EGF increased significantly the migration kinetics of the noninvasive cell line. Our results show that the 3D model of cell migration allowed us to differentiate the migratory behavior of invasive and noninvasive cells and that such a model can help in the development of molecular targeted therapy as it approaches the in vivo conditions. tumor invasion; metastasis; image analysis; kinetic migration; epidermal growth factor     

Multi-cellular logistics of collective cell migration

During development, the formation of biological networks (such as organs and neuronal networks) is controlled by multicellular transportation phenomena based on cell migration. In multi-cellular systems, cellular locomotion is restricted by physical interactions with other cells in a crowded space, similar to passengers pushing others out of their way on a packed train. The motion of individual cells is intrinsically stochastic and may be viewed as a type of random walk. However, this walk takes place in a noisy environment because the cell interacts with its randomly moving neighbors. Despite this randomness and complexity, development is highly orchestrated and precisely regulated, following genetic (and even epigenetic) blueprints. Although individual cell migration has long been studied, the manner in which stochasticity affects multi-cellular transportation within the precisely controlled process of development remains largely unknown. To explore the general principles underlying multicellular migration, we focus on the migration of neural crest cells, which migrate collectively and form streams. We introduce a mechanical model of multi-cellular migration. Simulations based on the model show that the migration mode depends on the relative strengths of the noise from migratory and non-migratory cells. Strong noise from migratory cells and weak noise from surrounding cells causes "collective migration," whereas strong noise from non-migratory cells causes "dispersive migration." Moreover, our theoretical analyses reveal that migratory cells attract each other over long distances, even without direct mechanical contacts. This effective interaction depends on the stochasticity of the migratory and non-migratory cells. On the basis of these findings, we propose that stochastic behavior at the single-cell level works effectively and precisely to achieve collective migration in multi-cellular systems.     

Novel role of Giα2 in cell migration: Downstream of PI3-kinase–AKT and Rac1 in prostate cancer cells

Tumor cell motility is the essential step in cancer metastasis. Previously, we showed that oxytocin and epidermal growth factor (EGF) effects on cell migration in prostate cancer cells require Giα2 protein. In the current study, we investigated the interactions among G-protein coupled receptor (GPCR), Giα2, PI3-kinase, and Rac1 activation in the induction of migratory and invasive behavior by diverse stimuli. Knockdown and knockout of endogenous Giα2 in PC3 cells resulted in attenuation of transforming growth factor β1 (TGFβ1), oxytocin, SDF-1α, and EGF effects on cell migration and invasion. In addition, knockdown of Giα2 in E006AA cells attenuated cell migration and overexpression of Giα2 in LNCaP cells caused significant increase in basal and EGF-stimulated cell migration. Pretreatment of PC3 cells with Pertussis toxin resulted in attenuation of TGFβ1- and oxytocin-induced migratory behavior and PI3-kinase activation without affecting EGF-induced PI3-kinase activation and cell migration. Basal- and EGF-induced activation of Rac1 in PC3 and DU145 cells were not affected in cells after Giα2 knockdown. On the other hand, Giα2 knockdown abolished the migratory capability of PC3 cells overexpressing constitutively active Rac1. The knockdown or knockout of Giα2 resulted in impaired formation of lamellipodia at the leading edge of the migrating cells. We conclude that Giα2 protein acts at two different levels which are both dependent and independent of GPCR signaling to induce cell migration and invasion in prostate cancer cells and its action is downstream of PI3-kinase–AKT–Rac1 axis.