PubMed Health⌕ Search

Biomedical subjects

Yi Rao

Publications and source records attributed to Yi Rao.

At least 19 recordsLinked to original sources
PubMed2006

Absolute orientation of molecules at interfaces.

A method to determine the absolute orientation of molecules at liquid interfaces by sum frequency generation (SFG) is reported. It is based on measurements of the orientations of two nonparallel vibrationally active chromophores in the molecule of interest combined with a rotation matrix formulation to obtain the absolute molecular orientation. We chose m-tolunitrile, a planar molecule adsorbed to the air/water interface, as a proof-of-method experiment. Quantitative analysis of different polarization sum frequency intensities facilitate unique peak assignments of the methyl and nitrile groups of m-tolunitrile. The SFG analysis of the measurement yields a nitrile group tilting at 53 degrees to the surface normal, and the C3 axis of the methyl group is almost upright at 23 degrees with respect to the surface normal. Using a rotation matrix formulation, we found that the angle between the surface plane and the m-tolunitrile molecular plane is 70 degrees.

Journal Article↗
PubMed2006

FAK and Src kinases are required for netrin-induced tyrosine phosphorylation of UNC5.

During neuronal development, netrin and its receptors UNC5 and DCC (deleted in colorectal cancer) guide axonal growth cones in navigating to their targets. Netrin also plays important roles in the regulation of cell migration, tissue morphogenesis and tumor growth. Here, we show that netrin induces UNC5 tyrosine phosphorylation and that this effect of netrin is dependent on its co-receptor DCC. UNC5 tyrosine phosphorylation is known to be important for netrin to induce cell migration and axonal repulsion. Src tyrosine kinase activity is required for netrin to stimulate UNC5 tyrosine phosphorylation in neurons and transfected cells. The SH2 domain of Src kinase directly interacts with the cytosolic domain of UNC5 in a tyrosine-phosphorylation-dependent manner. Furthermore, the tyrosine kinase focal adhesion kinase (FAK) is also involved in netrin-induced UNC5 tyrosine phosphorylation. Both Src and FAK can phosphorylate UNC5. Our data suggest a model in which netrin stimulates UNC5 tyrosine phosphorylation and signaling in a manner dependent on the co-receptor DCC, through the recruitment of Src and FAK kinases.

Animals↗
PubMed2005

Regulated formation and selection of neuronal processes underlie directional guidance of neuronal migration.

Axon guidance and neuronal migration are critical features of neural development, and it is believed that extracellular gradients of secreted guidance cues play important roles in pathfinding. It has been well documented that the growth cones of extending axons respond to such extracellular gradients by growing toward or away from the source of the secreted cue via asymmetrical extension of a single growth cone. However, it is unclear whether migrating neurons change direction in response to guidance molecules using the same mode of turning as extending axons. In this study, we demonstrate that migrating neurons turn away from the chemo-repellent Slit through repeated rounds of process extension and retraction and do not turn through the reorientation of a single growth cone. We further show that Slit increases the rate of somal process formation and that these processes form preferentially on the side of the cell body furthest away from the Slit source. In addition, Slit causes cell turning through asymmetric process selection. Finally, we show that multiple types of migrating neurons employ this mode of cell turning in response to a variety of guidance cues. These results show that migrating neurons employ a unique type of turning when faced with secreted guidance cues that is distinct from the type employed by axons.

Animals↗
PubMed2005

Ventral migration of early-born neurons requires Dcc and is essential for the projections of primary afferents in the spinal cord.

Neuronal migration and lamina-specific primary afferent projections are crucial for establishing spinal cord circuits, but the underlying mechanisms are poorly understood. Here, we report that in mice lacking Dcc (deleted in colorectal cancer), some early-born neurons could not migrate ventrally in the spinal cord. Conversely, forced expression of Dcc caused ventral migration and prevented dorsolateral migration of late-born spinal neurons. In the superficial layer of the spinal cord of Dcc-/- mutants, mislocalized neurons are followed by proprioceptive afferents, while their presence repels nociceptive afferents through Sema3a. Thus, our study has shown that Dcc is a key molecule required for ventral migration of early-born neurons, and that appropriate neuronal migration is a prerequisite for, and coupled to, normal projections of primary afferents in the developing spinal cord.

Animals↗
PubMed2005

Both the establishment and the maintenance of neuronal polarity require active mechanisms: critical roles of GSK-3beta and its upstream regulators.

Axon-dendrite polarity is a cardinal feature of neuronal morphology essential for information flow. Here we report a differential distribution of GSK-3beta activity in the axon versus the dendrites. A constitutively active GSK-3beta mutant inhibited axon formation, whereas multiple axons formed from a single neuron when GSK-3beta activity was reduced by pharmacological inhibitors, a peptide inhibitor, or siRNAs. An active mechanism for maintaining neuronal polarity was revealed by the conversion of preexisting dendrites into axons upon GSK-3 inhibition. Biochemical and functional data show that the Akt kinase and the PTEN phosphatase are upstream of GSK-3beta in determining neuronal polarity. Our results demonstrate that there are active mechanisms for maintaining as well as establishing neuronal polarity, indicate that GSK-3beta relays signaling from Akt and PTEN to play critical roles in neuronal polarity, and suggest that application of GSK-3beta inhibitors can be a novel approach to promote generation of new axons after neural injuries.

Animals↗
PubMed2005

Netrin-1 and slit-2 regulate and direct neurite growth of ventral midbrain dopaminergic neurons.

We investigated the roles of netrin-1 and slit-2 in regulation and navigation of dopamine (DA) axon growth using an explant culture preparation of embryonic ventral midbrain (embryonic day 14) and a co-culture system. We found that netrin-1 protein significantly enhanced DA axonal outgrowth and promoted DA axonal outgrowth in a co-culture system of netrin-1 expressing cells. Such effects were mediated by the receptor DCC as demonstrated by antibody perturbation of the DCC receptor. In contrast, slit-2 inhibited DA neuron extensions and repelled DA neurite growth. These slit-2 activities required robo receptors since the reduced neurite extension was abolished by addition of excess robo receptors. In this system, netrin-1 stimulated and slit-2 opposed DA neurite growth. Such regulation may be important for DA axonal maintenance, regeneration, and phenotypic target recognition.

Animals↗
PubMed2005

Netrin induces down-regulation of its receptor, Deleted in Colorectal Cancer, through the ubiquitin-proteasome pathway in the embryonic cortical neuron.

The proper regulation of temporal and spatial expression of the axon guidance cues and their receptors is critical for the normal wiring of nervous system during development. Netrins, a family of secreted guidance cues, are involved in the midline crossing of spinal commissural axons and in the guidance of cortical efferents. Axons normally lose the responsiveness to their attractants when they arrive at their targets, where the attractant is produced. However the molecular mechanism is still unknown. We investigated the molecular mechanism of down-regulation of netrin-1 signaling in the embryonic cortical neurons. Netrin-1 induced the ubiquitination and proteolytic cleavage of Deleted in Colorectal Cancer (DCC), a transmembrane receptor for netrin, in dissociated cortical neurons. A dramatic decrease of DCC level particularly on the cell surface was also observed after netrin-1 stimulation. Specific ubiquitin-proteasome inhibitors prevented the netrin-induced DCC cleavage and decrease of cell surface DCC. We suggest that the ligand-mediated down-regulation of DCC might participate in the loss of netrin-responsiveness in the developing nervous system.

Animals↗
PubMed2005

Investigations of neuronal migration in the central nervous system.

The migration of neuronal precursor cells is essential for the formation of the embryonic nervous system and for the maintenance of the adult nervous system. Modern approaches have greatly facilitated molecular and cellular studies of mechanisms underlying neuronal migration. Here we use the cells migrating from the anterior subventricular zone to the olfactory bulb as a model to discuss in some detail how neuronal migration can be studied. These methods can be adapted to other models of neuronal (or somatic cell) migration.

Animals↗
PubMed2004

Activation of FAK and Src are receptor-proximal events required for netrin signaling.

The axon guidance cue netrin is importantly involved in neuronal development. DCC (deleted in colorectal cancer) is a functional receptor for netrin and mediates axon outgrowth and the steering response. Here we show that different regions of the intracellular domain of DCC directly interacted with the tyrosine kinases Src and focal adhesion kinase (FAK). Netrin activated both FAK and Src and stimulated tyrosine phosphorylation of DCC. Inhibition of Src family kinases reduced DCC tyrosine phosphorylation and blocked both axon attraction and outgrowth of neurons in response to netrin. Mutation of the tyrosine phosphorylation residue in DCC abolished its function of mediating netrin-induced axon attraction. On the basis of our observations, we suggest a model in which DCC functions as a kinase-coupled receptor, and FAK and Src act immediately downstream of DCC in netrin signaling.

Animals↗
PubMed2004

Netrin requires focal adhesion kinase and Src family kinases for axon outgrowth and attraction.

Although netrins are an important family of neuronal guidance proteins, intracellular mechanisms that mediate netrin function are not well understood. Here we show that netrin-1 induces tyrosine phosphorylation of proteins including focal adhesion kinase (FAK) and the Src family kinase Fyn. Blockers of Src family kinases inhibited FAK phosphorylation and axon outgrowth and attraction by netrin. Dominant-negative FAK and Fyn mutants inhibited the attractive turning response to netrin. Axon outgrowth and attraction induced by netrin-1 were significantly reduced in neurons lacking the FAK gene. Our results show the biochemical and functional links between netrin, a prototypical neuronal guidance cue, and FAK, a central player in intracellular signaling that is crucial for cell migration.

Analysis of Variance↗
PubMed2004

Temporal regulation of cerebellar EGL migration through a switch in cellular responsiveness to the meninges.

We have studied the temporal and spatial control of cell migration from the external germinal layer (EGL) in the mammalian cerebellum as a model for cortical migration. Our results have demonstrated that embryonic EGL cells do not migrate into internal layers because they respond to a diffusible attractant in the meninges, the nonneural tissues covering the nervous system, and to a repellent in the neuroepithelium. Two developmental changes are important for postnatal EGL migration: the disappearance of the repellent in the inner layers and a switch in cellular responsiveness of EGL cells so that the postnatal EGL cells respond to the repellent, but not the attractant in the meninges. Besides revealing the signaling role of meninges in cortical development, our study suggests that an active mechanism is required to prevent cell migration, and that mechanisms of cell migration should be studied even in the absence of apparent changes in cell positions. We propose a model for the developmental control of neuronal migration in the cerebellar cortex.

Animals↗
PubMed2004

Visualization of spatially and temporally regulated N-WASP activity during cytoskeletal reorganization in living cells.

Members of the WASP/WAVE family of proteins are key regulators of cytoskeletal reorganization across a diverse range of cellular processes. Despite a wealth of biochemical data about WASP/WAVE regulation in vitro, our understanding of the in vivo regulation of these proteins is hampered by the inability to monitor subcellular regulation of their activities in living cells. Here we establish a fluorescence resonance energy transfer-based approach to visualize spatial and temporal regulation of neuronal Wiskott-Aldrich syndrome protein (N-WASP) activity in living cells. Using time-lapse microscopy, we characterize the activation of N-WASP in response to growth-factor stimulation, and we reveal that N-WASP is activated inside extending filopodia. Furthermore, we suggest a role of N-WASP in regulating membrane ruffling downstream of phosphatidylinositol 4,5-bisphosphate and Cdc42.

Animals↗
PubMed2004

Local inhibition guides the trajectory of early longitudinal tracts in the developing chick brain.

During development of the chick central nervous system, the trajectories of the descending medial and lateral longitudinal fascicles (MLF and LLF) are pioneered by axons originating from the interstitial nucleus of Cajal (INC) and the mesencephalic trigeminal nucleus (MTN), respectively. Both tracts cross rhombomere 1 at two specific locations in the basal plate. In this study, we have investigated the molecular properties of these crossing points and find that they are permissive regions situated in an otherwise inhibitory boundary region. We show that the dorsal part of rhombomere 1 is inhibitory for the growth of both MTN and INC axons. Ventrally, MLF and LLF axons are repelled from the midline by Slit proteins. Our results reveal the existence of a new repulsive/inhibitory mechanism for axons in the alar plate in addition to the ventral repulsion by Slit proteins. This suggests a model where MLF and LLF axons are channeled longitudinally within the neural tube by both dorsal and ventral constraints.

Animals↗
PubMed2004

Modulation of inflammation by slit protein in vivo in experimental crescentic glomerulonephritis.

A basic conservation of cell migration guidance mechanisms in the nervous and immune systems was proposed when Slit, known for its role in axon guidance, was found to inhibit chemokine-induced leukocyte chemotaxis in vitro. These studies examined the role of Slit2 in modulating inflammation in vivo. In a rat model of glomerulonephritis, endogenous glomerular Slit2 expression fell after disease induction, and its inhibition during the early disease period accelerated inflammation. Ex vivo glomerular leukocytes showed decreased chemokine and chemoattractant-induced chemotaxis in response to Slit2, suggesting an anti-inflammatory role for glomerular Slit2. In contrast to the effect of inhibition, glomerulonephritis was ameliorated by systemic Slit2 administration. Slit2 treatment improved disease histologically and also improved renal function when given early in the disease course. Leukocytes harvested from rats receiving Slit2 showed decreased monocyte chemoattractant protein-1 (MCP)-1-mediated migration, consistent with a peripheral Slit2 effect. In keeping with this functional alteration, Slit2-mediated inhibition of RAW264.7 cell chemotaxis was associated with decreased levels of active cdc42 and Rac1, implicating GTPases in leukocyte Slit2 signaling. These findings suggest a role for endogenous Slit2 in the inhibition of chemoattractant-mediated signals, demonstrate a potentially important anti-inflammatory effect for Slit2 in vivo, and provide further evidence for conserved mechanisms guiding the process of migration in distinct cell types.

Animals↗
PubMed2003

Neuronal repellent Slit2 inhibits dendritic cell migration and the development of immune responses.

One of the essential functions of dendritic cells is to take up Ags in peripheral tissues and migrate into secondary lymphoid organs to present Ags to lymphocytes for the induction of immune responses. Although many studies have demonstrated that the migration of dendritic cells is closely associated with the development of immune responses, little is known about factors that inhibit dendritic cell migration and control the extent of immune responses to Ag stimulation. We show that Slit2, a neuronal repellent factor, is up-regulated in the skin by allergen sensitization and down-regulates the migration of Langerhans cells. The effect is mediated by direct interaction of Slit2 with cells that express a Slit-specific receptor, Robo1. Slit2-mediated inhibition of Langerhans cell migration results in suppression of contact hypersensitivity responses. These findings provide insights into a novel mechanism by which Slit2 functions as an anti-inflammatory factor for the initiation of immune responses.

Animals↗
PubMed2003

Slit protein-mediated inhibition of CXCR4-induced chemotactic and chemoinvasive signaling pathways in breast cancer cells.

Slit, which mediates its function by binding to the Roundabout (Robo) receptor, has been shown to regulate neuronal and CXCR4-mediated leukocyte migration. Slit-2 was shown to be frequently inactivated in lung and breast cancers because of hypermethylation of its promoter region. Furthermore, the CXCR4/CXCL12 axis has been reported recently to be actively involved in breast cancer metastasis to target organs such as lymph nodes, lung, and bone. In this study, we sought to characterize the effect of Slit (=Slit-2) on the CXCL12/CXCR4-mediated metastatic properties of breast cancer cells. We demonstrate here that breast cancer cells and tissues derived from breast cancer patients express Robo 1 and 2 receptors. We also show that Slit treatment inhibits CXCL12/CXCR4-induced breast cancer cell chemotaxis, chemoinvasion, and adhesion, the fundamental components that promote metastasis. Slit had no significant effect on the CXCL12-induced internalization process of CXCR4. In addition, characterization of signaling events revealed that Slit inhibits CXCL12-induced tyrosine phosphorylation of focal adhesion components such as RAFTK/Pyk2 at residues 580 and 881, focal adhesion kinase at residue 576, and paxillin. We found that Slit also inhibits CXCL12-induced phosphatidylinositol 3-kinase, p44/42 MAP kinase, and metalloproteinase 2 and 9 activities. However, it showed no effect on JNK and p38 MAP kinase activities. To our knowledge, this is the first report to analyze in detail the effect of Slit on breast cancer cell motility as well as its effect on the critical components of the cancer cell chemotactic machinery. Studies of the Slit-Robo complex may foster new anti-chemotactic approaches to block cancer cell metastasis.

Breast Neoplasms↗
PubMed2003

Robo4 is a vascular-specific receptor that inhibits endothelial migration.

Guidance and patterning of axons are orchestrated by cell-surface receptors and ligands that provide directional cues. Interactions between the Robo receptor and Slit ligand families of proteins initiate signaling cascades that repel axonal outgrowth. Although the vascular and nervous systems grow as parallel networks, the mechanisms by which the vascular endothelial cells are guided to their appropriate positions remain obscure. We have identified a putative Robo homologue, Robo4, based on its differential expression in mutant mice with defects in vascular sprouting. In contrast to known neuronal Robo family members, the arrangement of the extracellular domains of Robo4 diverges significantly from that of all other Robo family members. Moreover, Robo4 is specifically expressed in the vascular endothelium during murine embryonic development. We show that Robo4 binds Slit and inhibits cellular migration in a heterologous expression system, analogous to the role of known Robo receptors in the nervous system. Immunoprecipitation studies indicate that Robo4 binds to Mena, a known effector of Robo-Slit signaling. Finally, we show that Robo4 is the only Robo family member expressed in primary endothelial cells and that application of Slit inhibits their migration. These data demonstrate that Robo4 is a bona fide member of the Robo family and may provide a repulsive cue to migrating endothelial cells during vascular development.

Activin Receptors, Type I↗