Imaging endogenous synaptic proteins in primary neurons at single-cell resolution using CRISPR/Cas9.

Fluorescence imaging at single-cell resolution is a crucial approach to analyzing the spatiotemporal regulation of proteins within individual cells of complex neural networks. Here we present a nonviral strategy that enables the tagging of endogenous loci by CRISPR/Cas9-mediated genome editing combined with a nucleofection technique. The method allowed expression of fluorescently tagged proteins at endogenous levels, and we successfully achieved tagging of a presynaptic protein, synaptophysin (Syp), and a postsynaptic protein, PSD-95, in cultured postmitotic neurons. Superresolution fluorescence microscopy of fixed neurons confirmed the identical localization patterns of the tagged proteins to those of endogenous ones verified by immunohistochemistry. The system is also applicable for multiplexed labeling and live-cell imaging. Live imaging with total internal reflection fluorescence microscopy of a single dendritic process of a neuron double-labeled with Syp-mCherry and PSD-95-EGFP revealed the previously undescribed dynamic localization of the proteins synchronously moving along dendritic shafts. Our convenient and versatile strategy is potent for analysis of proteins whose ectopic expressions perturb cellular functions.

Optimized CRISPR/Cas9-mediated in vivo genome engineering applicable to monitoring dynamics of endogenous proteins in the mouse neural tissues.

To analyze the expression, localization, and functional dynamics of target proteins in situ, especially in living cells, it is important to develop a convenient, versatile, and efficient method to precisely introduce exogenous genes into the genome, which is applicable for labeling and engineering of the endogenous proteins of interest. By combining the CRISPR/Cas9 genome editing technology with an electroporation technique, we succeeded in creating knock-in alleles, from which GFP (RFP)-tagged endogenous proteins are produced, in neurons and glial cells in vivo in the developing mouse retina and brain. Correct gene targeting was confirmed by single-cell genotyping and Western blot analysis. Several gene loci were successfully targeted with high efficiency. Moreover, we succeeded in engineering the mouse genome to express foreign genes from the endogenous gene loci using a self-cleaving 2A peptide. Our method could be used to monitor the physiological changes in localization of endogenous proteins and expression levels of both mRNA and protein at a single cell resolution. This work discloses a powerful and widely applicable approach for visualization and manipulation of endogenous proteins in neural tissues.

Dynamic spatiotemporal patterns of alternative splicing of an F-actin scaffold protein, afadin, during murine development.

An F-actin scaffold protein, afadin, comprises two splice variants called l-afadin (a long isoform) and s-afadin (a short isoform). It is known that in adult tissues, l-afadin is ubiquitously expressed while s-afadin is restrictedly expressed in brain. In cultured cortical neurons, l-afadin potentiates axonal branching whereas s-afadin blocks axonal branching by functioning as a naturally occurring dominant-negative isoform that forms a heterodimer with l-afadin. However, the temporal and spatial expression pattern of s-afadin during development or across multiple tissues and organs has not been fully understood. In this study, using Western blotting and RT-qPCR techniques and the fluorescent splicing reporters, we examined the detailed expression patterns of l- and s-afadin isoforms across various tissues and cell types, including rat organs at developmental stages, primary cultured neuronal and non-neuronal cells prepared from the developing rat brain, and in neurons in vitro generated from P19 embryonal carcinoma (EC) cells. Both mRNA and protein of s-afadin were abundantly expressed in various regions of rat neuronal tissues, and their expression dynamically changed during development in vivo. The expression of s-afadin was also detected in primary rat cortical neurons, but not in astrocytes or fibroblasts, and the neuronal expression increased during neuronal maturation in vitro. The dynamic alternative splicing event of afadin during development was successfully visualized with the newly developed fluorescent splicing reporter plasmids at a single cell level. Moreover, s-afadin was undetectable in undifferentiated EC cells, and the expression was induced upon differentiation of the cells into neurons. These data suggest that spatiotemporal and dynamic alternative splicing produces differential expression patterns of afadin isoforms and that alternative splicing of afadin is controlled by the neuron-specific regulator(s) whose activity is triggered and dynamically altered during neuronal differentiation and maturation.

Differential regional and subcellular localization patterns of afadin splice variants in the mouse central nervous system.

AF6/afadin is an F-actin scaffold protein that plays essential roles in the organization of cell-cell junctions of polarized epithelia. Afadin comprises two major isoforms produced by alternative splicing - a longer isoform l-afadin, having the F-actin-binding domain, and a shorter isoform s-afadin, harboring the amino acid sequences unique to the isoform but lacking the F-actin-binding domain. We recently identified functional differences between l- and s-afadin isoforms in the regulation of axon branching in primary cultured cortical neurons; the former potentiates and the latter blocks axon branching. Previous biochemical reports indicate differences in tissue and cell-type distributions of isoforms, and it was shown that l-afadin is ubiquitously expressed in various tissues and cell-types, while s-afadin is predominantly expressed in neuronal tissues and cultured neurons. However, the spatial expression pattern of s-afadin across neuronal tissues or within neurons has not been revealed because no antibody specific for s-afadin is yet available. In this study, we report the generation and characterization of an antibody that specifically distinguishes s-afadin from l-afadin, and its application to investigate the expression profile of s-afadin in primary cultured neurons and tissue cryosections of adult mouse brain and retina. We describe differential regional and subcellular localization patterns of l- and s-afadin isoforms in the mouse central nervous system.

A short splicing isoform of afadin suppresses the cortical axon branching in a dominant-negative manner.

Precise wiring patterns of axons are among the remarkable features of neuronal circuit formation, and establishment of the proper neuronal network requires control of outgrowth, branching, and guidance of axons. R-Ras is a Ras-family small GTPase that has essential roles in multiple phases of axonal development. We recently identified afadin, an F-actin-binding protein, as an effector of R-Ras mediating axon branching through F-actin reorganization. Afadin comprises two isoforms--l-afadin, having the F-actin-binding domain, and s-afadin, lacking the F-actin-binding domain. Compared with l-afadin, s-afadin, the short splicing variant of l-afadin, contains RA domains but lacks the F-actin-binding domain. Neurons express both isoforms; however, the function of s-afadin in brain remains unknown. Here we identify s-afadin as an endogenous inhibitor of cortical axon branching. In contrast to the abundant and constant expression of l-afadin throughout neuronal development, the expression of s-afadin is relatively low when cortical axons branch actively. Ectopic expression and knockdown of s-afadin suppress and promote branching, respectively. s-Afadin blocks the R-Ras-mediated membrane translocation of l-afadin and axon branching by inhibiting the binding of l-afadin to R-Ras. Thus s-afadin acts as a dominant-negative isoform in R-Ras-afadin-regulated axon branching.

Semaphorin 4D/Plexin-B1-mediated M-Ras GAP activity regulates actin-based dendrite remodeling through Lamellipodin.

Semaphorins have been identified as repulsive guidance molecules in the developing nervous system. We recently reported that the semaphorin 4D (Sema4D) receptor Plexin-B1 induces repulsion in axon and dendrites by functioning as a GTPase-activating protein (GAP) for R-Ras and M-Ras, respectively. In axons, Sema4D stimulation induces growth cone collapse, and downregulation of R-Ras activity by Plexin-B1-mediated GAP activity is required for the action. Axonal R-Ras GAP activity downregulates phosphatidylinositol 3-kinase signaling pathway, and thereby induces inactivation of a microtubule assembly promoter protein, CRMP-2. However, in contrast to the well studied roles of semaphorins and plexins in axonal guidance, signaling molecules linking M-Ras GAP to dendritic cytoskeleton remain obscure. Here we identified an Ena/VASP ligand, Lamellipodin (Lpd), as a novel effector of M-Ras in dendrites. Lpd was expressed in F-actin-rich distal dendritic processes and was required for both basal and M-Ras-mediated dendrite development. Subcellular fractionation showed M-Ras-dependent membrane translocation of Lpd, which was suppressed by Sema4D. Furthermore, the Ena/VASP-binding region within Lpd was required for dendrite development, and its membrane targeting was sufficient to overcome the Sema4D-mediated reduction of dendritic outgrowth and disappearance of F-actin from distal dendrites. Furthermore, in utero electroporation experiments also indicated that regulation of the M-Ras-Lpd system by the GAP activity of Plexin is involved in the normal development of cortical dendrites in vivo. Overall, our study sheds light on how repulsive guidance molecules regulate actin cytoskeleton in dendrites, revealing a novel mechanism that the M-Ras-Lpd system regulates actin-based dendrite remodeling by Sema/Plexin in rats or mice of either sex.

R-Ras controls axon branching through afadin in cortical neurons.

Regulation of axon growth, guidance, and branching is essential for constructing a correct neuronal network. R-Ras, a Ras-family small GTPase, has essential roles in axon formation and guidance. During axon formation, R-Ras activates a series of phosphatidylinositol 3-kinase signaling, inducing activation of a microtubule-assembly promoter-collapsin response mediator protein-2. However, signaling molecules linking R-Ras to actin cytoskeleton-regulating axonal morphology remain obscure. Here we identify afadin, an actin-binding protein harboring Ras association (RA) domains, as an effector of R-Ras inducing axon branching through F-actin reorganization. We observe endogenous interaction of afadin with R-Ras in cortical neurons during the stage of axonal development. Ectopic expression of afadin increases axon branch number, and the RA domains and the carboxyl-terminal F-actin binding domain are required for this action. RNA interference knockdown experiments reveal that knockdown of endogenous afadin suppressed both basal and R-Ras-mediated axon branching in cultured cortical neurons. Subcellular localization analysis shows that active R-Ras-induced translocation of afadin and its RA domains is responsible for afadin localizing to the membrane and inducing neurite development in Neuro2a cells. Overall, our findings demonstrate a novel signaling pathway downstream of R-Ras that controls axon branching.

Rnd1 and Rnd3 targeting to lipid raft is required for p190 RhoGAP activation.

The Rnd proteins Rnd1, Rnd2, and Rnd3/RhoE are well known as key regulators of the actin cytoskeleton in various cell types, but they comprise a distinct subgroup of the Rho family in that they are GTP bound and constitutively active. Functional differences of the Rnd proteins in RhoA inhibition signaling have been reported in various cell types. Rnd1 and Rnd3 antagonize RhoA signaling by activating p190 RhoGAP, whereas Rnd2 does not. However, all the members of the Rnd family have been reported to bind directly to p190 RhoGAP and equally induce activation of p190 RhoGAP in vitro, and there is no evidence that accounts for the functional difference of the Rnd proteins in RhoA inhibition signaling. Here we report the role of the N-terminal region in signaling. Rnd1 and Rnd3, but not Rnd2, have a KERRA (Lys-Glu-Arg-Arg-Ala) sequence of amino acids in their N-terminus, which functions as the lipid raft-targeting determinant. The sequence mediates the lipid raft targeting of p190 RhoGAP correlated with its activation. Overall, our results demonstrate a novel regulatory mechanism by which differential membrane targeting governs activities of Rnd proteins to function as RhoA antagonists.

Semaphorin 4D/Plexin-B1 stimulates PTEN activity through R-Ras GTPase-activating protein activity, inducing growth cone collapse in hippocampal neurons.

Plexins are receptors for axonal guidance molecules semaphorins. We recently reported that the semaphorin 4D (Sema4D) receptor, Plexin-B1, suppresses PI3K signaling through the R-Ras GTPase-activating protein (GAP) activity, inducing growth cone collapse. Phosphatidylinositol 3-phosphate level is critically regulated by PI3K and PTEN (phosphatase and tensin homologue deleted chromosome ten). Here we examined the involvement of PTEN in the Plexin-B1-induced repulsive response. Phosphorylation of PTEN at Ser-380 is known to suppress its phosphatase activity. Sema4D induced the dephosphorylation of PTEN at Ser-380 and stimulated PTEN phosphatase activity in hippocampal neurons. Knockdown of endogenous PTEN suppressed the Sema4D-induced growth cone collapse. Phosphorylation mimic PTEN mutant suppressed the Sema4D-induced growth cone collapse, whereas phosphorylation-resistant PTEN mutant by itself induced growth cone collapse. Plexin-B1-induced PTEN dephosphorylation through R-Ras GAP activity and R-Ras GAP activity was by itself sufficient for PTEN dephosphorylation and activation. We also suggested that the Sema4D-induced PTEN dephosphorylation and growth cone collapse were mediated by the inhibition of casein kinase 2 alpha activity. Thus, we propose that Sema4D/Plexin-B1 promotes the dephosphorylation and activation of PTEN through the R-Ras GAP activity, inducing growth cone collapse.

Plexin-B1 is a GTPase activating protein for M-Ras, remodelling dendrite morphology.

Plexins are receptors for axonal guidance molecules known as semaphorins. We recently reported that the semaphorin 4D (Sema4D) receptor, Plexin-B1, induces axonal growth cone collapse by functioning as an R-Ras GTPase activating protein (GAP). Here, we report that Plexin-B1 shows GAP activity for M-Ras, another member of the Ras family of GTPases. In cortical neurons, the expression of M-Ras was upregulated during dendritic development. Knockdown of endogenous M-Ras-but not R-Ras-reduced dendritic outgrowth and branching, whereas overexpression of constitutively active M-Ras, M-Ras(Q71L), enhanced dendritic outgrowth and branching. Sema4D suppressed M-Ras activity and reduced dendritic outgrowth and branching, but this reduction was blocked by M-Ras(Q71L). M-Ras(Q71L) stimulated extracellular signal-regulated kinase (ERK) activation, inducing dendrite growth, whereas Sema4D suppressed ERK activity and down-regulation of ERK was required for a Sema4D-induced reduction of dendrite growth. Thus, we conclude that Plexin-B1 is a dual functional GAP for R-Ras and M-Ras, remodelling axon and dendrite morphology, respectively.

Different requirement for Rnd GTPases of R-Ras GAP activity of Plexin-C1 and Plexin-D1.

Plexins, comprising Plexin-A, -B, -C, and -D subfamilies, are receptors for semaphorins governing cell adhesion, migration, and axon guidance. Among plexin subfamilies, Plexin-A1 and Plexin-B1 have been shown to function as an R-Ras GAP, inducing repulsive responses, and the expression of R-Ras GAP activity requires the binding of Rnd1, a member of Rnd subfamily of Rho GTPases. However, signaling pathways of Plexin-D1 and Plexin-C1 still remain obscure. Here, we found that Plexin-D1 displayed R-Ras GAP activity and inhibited migration of COS-7 cells, and these actions required Rnd2, another Rnd subfamily GTPase. Rnd2 bound to Plexin-D1 in cortical neurons, and Sema3E/Plexin-D1-induced inhibition of axon outgrowth of cortical neurons required Rnd2 and down-regulation of R-Ras activity. On the other hand, Plexin-C1 displayed R-Ras GAP activity and inhibited cell migration of COS-7 cells without Rnd proteins. Therefore, R-Ras GAP activity is a common function of plexin subfamilies but the regulation of R-Ras GAP activity of plexins by Rnd proteins is different among plexin subfamilies.

Plexin-B1 mutations in prostate cancer.

Semaphorins are a large class of secreted or membrane-associated proteins that act as chemotactic cues for cell movement via their transmembrane receptors, plexins. We hypothesized that the function of the semaphorin signaling pathway in the control of cell migration could be harnessed by cancer cells during invasion and metastasis. We now report 13 somatic missense mutations in the cytoplasmic domain of the Plexin-B1 gene. Mutations were found in 89% (8 of 9) of prostate cancer bone metastases, in 41% (7 of 17) of lymph node metastases, and in 46% (41 of 89) of primary cancers. Forty percent of prostate cancers contained the same mutation. Overexpression of the Plexin-B1 protein was found in the majority of primary tumors. The mutations hinder Rac and R-Ras binding and R-RasGAP activity, resulting in an increase in cell motility, invasion, adhesion, and lamellipodia extension. These results identify a key role for Plexin-B1 and the semaphorin signaling pathway it mediates in prostate cancer.

R-Ras controls axon specification upstream of glycogen synthase kinase-3beta through integrin-linked kinase.

The initial event in establishing a polarized neuron is the specification of a single axon. Spatially regulated glycogen synthase kinase-3beta (GSK-3beta) activity is critical for specifying axon-dendrite fate; however, the upstream signaling of GSK-3beta in the determination of neuronal polarity still remains obscure. Here, we found that, in cultured hippocampal neurons, the small GTPase R-Ras selectively localized in a single neurite of stage 2 neurons and that its activity increased after plating and peaked between stages 2 and 3. Ectopic expression of R-Ras induced global inactivation of GSK-3beta and formation of multiple axons, whereas knockdown of endogenous R-Ras by RNA interference blocked GSK-3beta inactivation and axon formation. GSK-3beta inactivation and axon formation by R-Ras required integrin-linked kinase (ILK), and subcellular localization of ILK was strictly regulated by R-Ras-mediated phosphatidylinositol 3-kinase activity. In addition, membrane targeting of ILK was sufficient to inactivate GSK-3beta and to form multiple axons. Our study demonstrates a novel role of R-Ras and ILK upstream of GSK-3beta in the regulation of neuronal polarity.

Sema4D/plexin-B1 activates GSK-3beta through R-Ras GAP activity, inducing growth cone collapse.

Plexins are receptors for the axonal guidance molecules known as semaphorins, and the semaphorin 4D (Sema4D) receptor plexin-B1 induces repulsive responses by functioning as an R-Ras GTPase-activating protein (GAP). Here we characterized the downstream signalling of plexin-B1-mediated R-Ras GAP activity, inducing growth cone collapse. Sema4D suppressed R-Ras activity in hippocampal neurons, in parallel with dephosphorylation of Akt and activation of glycogen synthase kinase (GSK)-3beta. Ectopic expression of the constitutively active mutant of Akt or treatment with GSK-3 inhibitors suppressed the Sema4D-induced growth cone collapse. Constitutive activation of phosphatidylinositol-3-OH kinase (PI(3)K), an upstream kinase of Akt and GSK-3beta, also blocked the growth cone collapse. The R-Ras GAP activity was necessary for plexin-B1-induced dephosphorylation of Akt and activation of GSK-3beta and was also required for phosphorylation of a downstream kinase of GSK-3beta, collapsin response mediator protein-2. Plexin-A1 also induced dephosphorylation of Akt and GSK-3beta through its R-Ras GAP activity. We conclude that plexin-B1 inactivates PI(3)K and dephosphorylates Akt and GSK-3beta through R-Ras GAP activity, inducing growth cone collapse.

Semaphorin 4D/Plexin-B1-mediated R-Ras GAP activity inhibits cell migration by regulating beta(1) integrin activity.

Plexins are cell surface receptors for semaphorins and regulate cell migration in many cell types. We recently reported that the semaphorin 4D (Sema4D) receptor Plexin-B1 functions as a GTPase-activating protein (GAP) for R-Ras, a member of Ras family GTPases implicated in regulation of integrin activity and cell migration. We characterized the role of R-Ras downstream of Sema4D/Plexin-B1 in cell migration. Activation of Plexin-B1 by Sema4D suppressed the ECM-dependent R-Ras activation, R-Ras-mediated phosphatydylinositol 3-kinase activation, and beta(1) integrin activation through its R-Ras GAP domain, leading to inhibition of cell migration. In addition, inactivation of R-Ras by overexpression of the R-Ras-specific GAP or knockdown of R-Ras by RNA interference was sufficient for suppressing beta(1) integrin activation and cell migration in response to the ECM stimulation. Thus, we conclude that R-Ras activity is critical for ECM-mediated beta(1) integrin activation and cell migration and that inactivation of R-Ras by Sema4D/Plexin-B1-mediated R-Ras GAP activity controls cell migration by modulating the activity of beta(1) integrins.

R-ras as a key player for signaling pathway of plexins.

Axon guidance represents an important step in the formation of neuronal networks. Axons are guided by various guidance factors, such as semaphorins, slits, ephrins, and netrins. Plexins are cell surface receptors for the repulsive molecules of the semaphorin family. Cytoplasmic regions of plexins are responsible for initiating cellular signal transduction, resulting in axon repulsion. Recent advances have shed light on the signal transduction mechanism of plexins and the mechanisms by which it leads to a repulsive response. Plexin-B1 possesses an intrinsic guanine triphosphate (GTP)ase activating protein activity for R-Ras, a member of Ras family of small GTPases that has been implicated in promoting cell adhesion and neurite outgrowth through integrin activation. Stimulation of Plexin-B1 by Sema4D induces collapse of the growth cone through downregulation of R-Ras activity. This article summarizes current understanding of the signaling mechanisms of plexins.

Molecular dissection of the semaphorin 4D receptor plexin-B1-stimulated R-Ras GTPase-activating protein activity and neurite remodeling in hippocampal neurons.

Plexins serve as receptors for repulsive axonal guidance molecules semaphorins. The cytoplasmic domain of the semaphorin 4D (Sema4D) receptor, Plexin-B1 has two separated Ras GTPase-activating protein (GAP)-homologous domains, C1 and C2. Recently, we reported that the Rho family small GTPase Rnd1 associates with Plexin-B1, and the Plexin-B1-Rnd1 complex stimulates GTPase activity of R-Ras, inducing growth cone collapse in hippocampal neurons in response to Sema4D. However, the molecular mechanisms by which Plexin-B1 exhibits the GAP activity remain unclear. In this report, critical roles of Rnd1 and Sema4D in Plexin-B1-stimulated R-Ras GAP activity and neurite remodeling were examined. The N-terminal region of the cytoplasmic domain of Plexin-B1 containing the C1 domain interacts with the C-terminal region containing the C2 domain, and Rnd1 disrupts this interaction. On the other hand, Sema4D induces clustering of Rnd1-bound Plexin-B1, in parallel with inactivation of R-Ras in cells. Antibody clustering of the recombinant cytoplasmic domain of Plexin-B1 in the presence of Rnd1 triggers the R-Ras GAP activity. Deletion of the extracellular domain of Plexin-B1 causes ligand-independent clustering of the receptor, rendering the receptor constitutively active in the presence of Rnd1, and induces contraction of COS-7 cells and inhibition of neurite outgrowth in hippocampal neurons. These results indicate that Rnd1 opens the two R-Ras GAP domains of Plexin-B1, and Sema4D-induced receptor clustering stimulates R-Ras GAP activity and neurite remodeling in hippocampal neurons.

The Semaphorin 4D receptor Plexin-B1 is a GTPase activating protein for R-Ras.

Plexins are cell surface receptors for semaphorin molecules, and their interaction governs cell adhesion and migration in a variety of tissues. We report that the Semaphorin 4D (Sema4D) receptor Plexin-B1 directly stimulates the intrinsic guanosine triphosphatase (GTPase) activity of R-Ras, a member of the Ras superfamily of small GTP-binding proteins that has been implicated in promoting cell adhesion and neurite outgrowth. This activity required the interaction of Plexin-B1 with Rnd1, a small GTP-binding protein of the Rho family. Down-regulation of R-Ras activity by the Plexin-B1-Rnd1 complex was essential for the Sema4D-induced growth cone collapse in hippocampal neurons. Thus, Plexin-B1 mediates Sema4D-induced repulsive axon guidance signaling by acting as a GTPase activating protein for R-Ras.

Direct interaction of Rnd1 with Plexin-B1 regulates PDZ-RhoGEF-mediated Rho activation by Plexin-B1 and induces cell contraction in COS-7 cells.

Plexins are receptors for the axon guidance molecule semaphorins, and several lines of evidence suggest that Rho family small GTPases are implicated in the downstream signaling of Plexins. Recent studies have demonstrated that Plexin-B1 activates RhoA and induces growth cone collapse through Rho-specific guanine nucleotide exchange factor PDZ-RhoGEF. Here we show that Rnd1, a member of Rho family GTPases, directly interacted with the cytoplasmic domain of Plexin-B1. In COS-7 cells, coexpression of Rnd1 and Plexin-B1 induced cell contraction in response to semaphorin 4D (Sema4D), a ligand for Plexin-B1, whereas expression of Plexin-B1 alone or coexpression of Rnd1 and a Rnd1 interaction-defective mutant of Plexin-B1 did not. The Sema4D-induced contraction in Plexin-B1/Rnd1-expressing COS-7 cells was suppressed by dominant negative RhoA, a Rho-associated kinase inhibitor, a dominant negative form of PDZ-RhoGEF, or deletion of the carboxyl-terminal PDZ-RhoGEF-binding region of Plexin-B1, indicating that the PDZ-RhoGEF/RhoA/Rho-associated kinase pathway is involved in this morphological effect. We also found that Rnd1 promoted the interaction between Plexin-B1 and PDZ-RhoGEF and thereby dramatically potentiated the Plexin-B1-mediated RhoA activation. We propose that Rnd1 plays an important role in the regulation of Plexin-B1 signaling, leading to Rho activation during axon guidance and cell migration.