Ena/VASP proteins regulate cortical neuronal positioning

Development of the multilayered cerebral cortex involves extensive regulated migration of neurons arising from the deeper germinative layers of the mammalian brain. The anatomy and formation of the cortical layers has been well characterized; however, the underlying molecular mechanisms that control the migration and the final positioning of neurons within the cortex remain poorly understood. Here, we report evidence for a key role of Ena/VASP proteins, a protein family implicated in the spatial control of actin assembly and previously shown to negatively regulate fibroblast cell speeds, in cortical development. Ena/VASP proteins are highly expressed in the developing cortical plate in cells bordering Reelin-expressing Cajal-Retzius cells and in the intermediate zone through which newly born cells migrate. Inhibition of Ena/VASP function through retroviral injections in utero led to aberrant placement of early-born pyramidal neurons in the superficial layers of both the embryonic and the postnatal cortex in a cell-autonomous fashion. The abnormally placed pyramidal neurons exhibited grossly normal morphology and polarity. Our results are consistent with a model in which Ena/VASP proteins function in vivo to control the position of neurons in the mouse neocortex.     

Radial glial dependent and independent dynamics of interneuronal migration in the developing cerebral cortex

Interneurons originating from the ganglionic eminence migrate tangentially into the developing cerebral wall as they navigate to their distinct positions in the cerebral cortex. Compromised connectivity and differentiation of interneurons are thought to be an underlying cause in the emergence of neurodevelopmental disorders such as schizophrenia. Previously, it was suggested that tangential migration of interneurons occurs in a radial glia independent manner. Here, using simultaneous imaging of genetically defined populations of interneurons and radial glia, we demonstrate that dynamic interactions with radial glia can potentially influence the trajectory of interneuronal migration and thus the positioning of interneurons in cerebral cortex. Furthermore, there is extensive local interneuronal migration in tangential direction opposite to that of pallial orientation (i.e., in a medial to lateral direction from cortex to ganglionic eminence) all across the cerebral wall. This counter migration of interneurons may be essential to locally position interneurons once they invade the developing cerebral wall from the ganglionic eminence. Together, these observations suggest that interactions with radial glial scaffold and localized migration within the expanding cerebral wall may play essential roles in the guidance and placement of interneurons in the developing cerebral cortex.     

Apico-basal polarity complex and cancer

Apico-basal polarity is a cardinal molecular feature of adult eukaryotic epithelial cells and appears to be involved in several key cellular processes including polarized cell migration and maintenance of tissue architecture. Epithelial cell polarity is maintained by three well-conserved polarity complexes, namely, PAR, Crumbs and SCRIB. The location and interaction between the components of these complexes defines distinct structural domains of epithelial cells. Establishment and maintenance of apico-basal polarity is regulated through various conserved cell signalling pathways including TGFβ, Integrin and WNT signalling. Loss of cell polarity is a hallmark for carcinoma, and its underlying molecular mechanism is beginning to emerge from studies on model organisms and cancer cell lines. Moreover, deregulated expression of apico-basal polarity complex components has been reported in human tumours. In this review, we provide an overview of the apico-basal polarity complexes and their regulation, their role in cell migration, and finally their involvement in carcinogenesis.     

Regulation of cell adhesions and motility during initiation of neural crest migration

Accurate neural crest cell (NCC) migration requires tight control of cell adhesions, cytoskeletal dynamics and cell motility. Cadherins and RhoGTPases are critical molecular players that regulate adhesions and motility during initial delamination of NCCs from the neuroepithelium. Recent studies have revealed multiple functions for these molecules and suggest that a precise balance of their activity is crucial. RhoGTPase appears to regulate both cell adhesions and protrusive forces during NCC delamination. Increasing evidence shows that cadherins are multi-functional proteins with novel, adhesion-independent signaling functions that control NCC motility during both delamination and migration. These functions are often regulated by specific proteolytic cleavage of cadherins. After NCC delamination, planar cell polarity signaling acts via RhoGTPases to control NCC protrusions and migration direction.     

The Ran GTPase mediates chromatin signaling to control cortical polarity during polar body extrusion in mouse oocytes

The molecular basis for asymmetric meiotic divisions in mammalian oocytes that give rise to mature eggs and polar bodies remains poorly understood. Previous studies demonstrated that the asymmetrically positioned meiotic chromosomes provide the cue for cortical polarity in mouse oocytes. Here we show that the chromatin-induced cortical response can be fully reconstituted by injecting DNA-coated beads into metaphase II-arrested eggs. The injected DNA beads induce a cortical actin cap, surrounded by a myosin II ring, in a manner that depends on the number of beads and their distance from the cortex. The Ran GTPase plays a critical role in this process, because dominant-negative and constitutively active Ran mutants disrupt DNA-induced cortical polarization. The Ran-mediated signaling to the cortex is independent of the spindle but requires cortical myosin II assembly. We hypothesize that a Ran(GTP) gradient serves as a molecular ruler to interpret the asymmetric position of the meiotic chromatin.     

Lis1/dynactin regulates metaphase spindle orientation in Drosophila neuroblasts

Mitotic spindle orientation in polarized cells determines whether they divide symmetrically or asymmetrically. Moreover, regulated spindle orientation may be important for embryonic development, stem cell biology, and tumor growth. Drosophila neuroblasts align their spindle along an apical/basal cortical polarity axis to self-renew an apical neuroblast and generate a basal differentiating cell. It is unknown whether spindle alignment requires both apical and basal cues, nor have molecular motors been identified that regulate spindle movement. Using live imaging of neuroblasts within intact larval brains, we detect independent movement of both apical and basal spindle poles, suggesting that forces act on both poles. We show that reducing astral microtubules decreases the frequency of spindle movement, but not its maximum velocity, suggesting that one or few microtubules can move the spindle. Mutants in the Lis1/dynactin complex strongly decrease maximum and average spindle velocity, consistent with this motor complex mediating spindle/cortex forces. Loss of either astral microtubules or Lis1/dynactin leads to spindle/cortical polarity alignment defects at metaphase, but these are rescued by telophase. We propose that an early Lis1/dynactin-dependent pathway and a late Lis1/dynactin-independent pathway regulate neuroblast spindle orientation.     

PACAP decides neuronal laminar fate via PKA signaling in the developing cerebral cortex

Laminar formation in the developing cerebral cortex requires the precisely regulated generation of phenotype-specified neurons. To test the possible involvement of pituitary adenylate cyclase-activating polypeptide (PACAP) in this formation, we investigated the effects of PACAP administered into the telencephalic ventricular space of 13.5-day-old mouse embryos. PACAP partially inhibited the proliferation of cortical progenitors and altered the position and gene-expression profiles of newly generated neurons otherwise expected for layer IV to those of neurons for the deeper layers, V and VI, of the cerebral cortex. The former and latter effects were seen only when the parent progenitor cells were exposed to PACAP in the later and in earlier G1 phase, respectively; and these effects were suppressed by co-treatment with a protein kinase A (PKA) inhibitor. These observations suggest that PACAP participates in the processes forming the neuronal laminas in the developing cortex via the intracellular PKA pathway.     

Planar polarity of ependymal cilia

Ependymal cells, epithelial cells that line the cerebral ventricles of the adult brain in various animals, extend multiple motile cilia from their apical surface into the ventricles. These cilia move rapidly, beating in a direction determined by the ependymal planar cell polarity (PCP). Ciliary dysfunction interferes with cerebrospinal fluid circulation and alters neuronal migration. In this review, we summarize recent studies on the cellular and molecular mechanisms underlying two distinct types of ependymal PCP. Ciliary beating in the direction of fluid flow is established by a combination of hydrodynamic forces and intracellular planar polarity signaling. The ciliary basal bodies' anterior position on the apical surface of the cell is determined in the embryonic radial glial cells, inherited by ependymal cells, and established by non-muscle myosin II in early postnatal development.     

Cellular and molecular mechanisms of neuronal migration in neocortical development

The complicated mammalian brain structure arises from accurate movements of neurons from their birthplace to their final locations. Detailed observation of this migration process by various methods revealed that neuronal migration is highly motile and that there are different modes of migration. Moreover, mouse mutants or human disorders that disrupt normal migration have provided significant insights into molecular pathways that control the neuronal migration. Although our knowledge is still fragmentary, it is becoming clear that various molecules are participating in this process. In this review, we outline about the cellular and molecular mechanisms of neuronal migration in the cerebral cortex.     

N-cadherin mediates cortical organization in the mouse brain

The cerebral cortex is a complex laminated structure generated by the sequential migration of developing neurons from the ventricular zone. One of the molecules that may play a role in cortical morphogenesis is N-cadherin since its blocking causes disruption of the ordered arrangement of cells in other neural tissues, such as the neural retina. Here, we show that when the N-cadherin gene had been conditionally deleted in the mouse cerebral cortex, the intra-cortical structures were nearly completely randomized; e.g., mitotic cells and postmitotic cells were scattered throughout the cortex without any order. These defects seemed to mainly originate from the disruption of the adherens junctions (AJs) localized in the apical end of neuroepithelial cells, where N-cadherin is normally most highly concentrated. In the absence of N-cadherin, neuroepithelial or radial glial cells could not expand their bodies or processes to span the distance between the ventricular and pial surfaces and therefore terminated them in the middle zone of the cortex. These results demonstrate that N-cadherin is essential for maintaining the normal architecture of neuroepithelial or radial glial cells and that their disruption randomizes the internal structures of the cortex.     

Fission yeast cytoskeletons and cell polarity factors: connecting at the cortex

Cell polarity is a fundamental property of cells from unicellular to multicellular organisms. Most of the time, it is essential so that the cells can achieve their function. The fission yeast Schizosaccharomyces pombe is a powerful genetic model organism for studying the molecular mechanisms of the cell polarity process. Indeed, S. pombe cells are rod-shaped and cell growth is restricted at the poles. The accurate localization of the cell growth machinery at the cell cortex, which involves the actin cytoskeleton, depends on cell polarity pathways that are temporally and spatially regulated. The importance of interphase microtubules and cell polarity factors acting at the cortex of cell ends in this process has been shown. Here, we review recent advances in knowledge of molecular pathways leading to the establishment of a cellular axis in fission yeast. We also describe the role of cortical proteins and mitotic cytoskeletal rearrangements that control the symmetry of cell division.     

Centrosomes can initiate a polarity axis from any position within one-cell C. elegans embryos

The stereotyped asymmetry of one-cell C. elegans embryos has proven to be an important model for identifying molecular determinants of cell polarity. How polarity is initiated is less well understood. Polarity establishment depends on centrosomes, which use two molecularly distinct pathways to break symmetry. In both, the centrosome's position adjacent to the cell cortex is thought to determine where polarization starts. Defects in centrosome-cortex juxtaposition correlate with defects in polarity establishment in several mutants, suggesting that these processes may be linked, but there is no direct test of this. Here we assess how centrosome position relative to the cortex affects polarity establishment. We find that centrosomes can initiate polarity from any position within the embryo volume, but centrosome-cortex proximity decreases the time required to initiate polarity. Polarization itself brings about close centrosome-cortex proximity. Prior to polarization, cytoplasmic microtubules constrain centrosome movement near the cortex, expanding the controversial role of microtubules during polarity establishment. The ability of centrosomes to induce a single polarity axis from any position within the egg emphasizes the flexible, self-organizing properties of polarization in C. elegans embryos and contrasts the common view of C. elegans development as invariant.     

The unfolding story of two lissencephaly genes and brain development

Formation of our highly structured human brain involves a cascade of events, including differentiation, fate determination, and migration of neural precursors. In humans, unlike many other organisms, the cerebral cortex is the largest component of the brain. As in other mammals, the human cerebral cortex is located on the surface of the telencephalon and generally consists of six layers that are formed in an orderly fashion. During neuronal development, newly born neurons, moving in a radial direction, must migrate through previously formed layers to reach their proper cortical position. This is one of several neuronal migration routes that takes place in the developing brain; other modes of migration are tangential. Abnormal neuronal migration may in turn result in abnormal development of the cortical layers and deleterious consequences, such as Lissencephaly. Lissencephaly, a severe brain malformation, can be caused by mutations in one of two known genes:LIS1 anddoublecortin (DCX). Recent in vitro and in vivo studies, report on possible functions for these gene products.     

Coordinating mitosis with cell polarity: Molecular motors at the cell cortex

In many cell divisions, the position of the spindle apparatus is coordinated with polarity signals at the cell cortex so that copies of the genome are delivered to regions of the cell that are designated for differential inheritance by the two progeny. To coordinate spindle position with cell polarity, the spindle interfaces with elements on the cortex, where molecular motors often produce the forces that power displacement. Here we describe the molecular pathways by which cortical motors translocate the spindle in budding yeast, where the mechanisms are understood relatively well, and we compare these pathways to spindle positioning processes in metazoan systems, where the molecular details are less well understood.     

Centrosome positioning in polarized cells: Common themes and variations

The centrosome position is tightly regulated during the cell cycle and during differentiated cellular functions. Because centrosome organizes the microtubule network to coordinate both intracellular organization and cell signaling, centrosome positioning is crucial to determine either the axis of cell division, the direction of cell migration or the polarized immune response of lymphocytes. Since alteration of centrosome positioning seems to promote cell transformation and tumor spreading, the molecular mechanisms controlling centrosome movement in response to extracellular and intracellular cues are under intense investigation. Evolutionary conserved pathways involving polarity proteins and cytoskeletal rearrangements are emerging as common regulators of centrosome positioning in a wide variety of cellular contexts.     

Cdc42 protein acts upstream of IQGAP1 and regulates cytokinesis in mouse oocytes and embryos

Cdc42 and Rac1 Rho family GTPases, and their interacting protein IQGAP1 are the key regulators of cell polarity. We examined the role of Cdc42 and IQGAP1 in establishing the polarity of mouse oocyte and regulation of meiotic and mitotic divisions. We showed that Cdc42 was localized on the microtubules of meiotic and mitotic spindle and in the cortex of mouse oocytes and cleaving embryos. IQGAP1 was present in the cytoplasm and cortex of growing and fully-grown oocytes. During maturation it disappeared from the cortex and during meiotic and mitotic cytokinesis it concentrated in the contractile ring. Toxin B inhibition of the binding activity of Cdc42 changed the localization of IQGAP1, inhibited emission of the first polar body, and caused disappearance of the cortical actin without affecting the migration of meiotic spindle. This indicates, that in maturing oocytes accumulation of cortical actin is not indispensable for spindle migration. In zygotes treated with toxin B actin cytoskeleton was rearranged and the first and/or subsequent cytokinesis were inhibited. Our results indicate that Cdc42 acts upstream of IQGAP1 and is involved in regulation of cytokinesis in mouse oocytes and cleaving embryos, rather than in establishing the polarity of the oocyte.     

The mechanism of axon growth

Cell adhesion molecules (CAMs) are not just an inert glue that mediates static cell-cell and cell-extracellular matrix (ECM) adhesion; instead, their adhesivity is dynamically controlled to enable a cell to migrate through complex environmental situations. Furthermore, cell migration requires distinct levels of CAM adhesivity in various subcellular regions. Recent studies on L1, a CAM in the immunoglobulin superfamily, demonstrate that cell adhesion can be spatially regulated by the polarized internalization and recycling of CAMs. This article examines the molecular mechanism of axon growth, with a particular focus on the role of L1 trafficking in the polarized adhesion and migration of neuronal growth cones.     

Staurosporine induces ganglion cell differentiation in part by stimulating urokinase-type plasminogen activator expression and activation in the developing chick retina

The plus-ends of microtubules target the cell cortex to modulate actin protrusion dynamics and polarity, but little is known of the molecular mechanism that couples the interaction. EB1 protein associates with the plus-ends of microtubules, placing EB1 in an ideal spatial position to mediate microtubule-actin cross talk. The objective of the current study was to further understand intracellular signaling involved in EB1-dependent cell polarity and motility. B16F10 mouse melanoma cells were depleted of EB1 protein using short hair-pin RNA interference. Correlative live cell-immunofluorescence microscopy was performed to determine localization of WAVE2 and IQGAP1 to protruding versus retracting edges. EB1 knock down caused poor subcellular separation of WAVE2 and IQGAP1, and overall decreased localization. Activation of PKC corrected defects in WAVE2 and IQGAP1 localization, cell spreading and cell shape to levels observed in control cells, but did not correct defects in cell migration. Consistent with these findings, decreased PKC phosphorylation was observed in EB1 knock down cells. These findings support a model where EB1 protein links microtubules to actin protrusion and cell polarity through signaling pathways involving PKC.