Gcap14 is a microtubule plus-end-tracking protein coordinating microtubule-actin crosstalk during neurodevelopment.

Regulation of microtubule dynamics is required to properly control various steps of neurodevelopment. In this study, we identified granule cell antiserum-positive 14 (Gcap14) as a microtubule plus-end-tracking protein and as a regulator of microtubule dynamics during neurodevelopment. Gcap14 knockout mice exhibited impaired cortical lamination. Gcap14 deficiency resulted in defective neuronal migration. Moreover, nuclear distribution element nudE-like 1 (Ndel1), an interacting partner of Gcap14, effectively corrected the downregulation of microtubule dynamics and the defects in neuronal migration caused by Gcap14 deficiency. Finally, we found that the Gcap14-Ndel1 complex participates in the functional link between microtubule and actin filament, thereby regulating their crosstalks in the growth cones of cortical neurons. Taken together, we propose that the Gcap14-Ndel1 complex is fundamental for cytoskeletal remodeling during neurodevelopmental processes such as neuronal processes elongation and neuronal migration.

GRAMD1B regulates cell migration in breast cancer cells through JAK/STAT and Akt signaling.

Dysregulated JAK/STAT signaling has been implicated in breast cancer metastasis, which is associated with high relapse risks. However, mechanisms underlying JAK/STAT signaling-mediated breast tumorigenesis are poorly understood. Here, we showed that GRAMD1B expression is upregulated on IL-6 but downregulated upon treatment with the JAK2 inhibitor AG490 in the breast cancer MDA-MB-231 cells. Notably, Gramd1b knockdown caused morphological changes of the cells, characterized by the formation of membrane ruffling and protrusions, implicating its role in cell migration. Consistently, GRAMD1B inhibition significantly enhanced cell migration, with an increase in the levels of the Rho family of GTPases. We also found that Gramd1b knockdown-mediated pro-migratory phenotype is associated with JAK2/STAT3 and Akt activation, and that JAK2 or Akt inhibition efficiently suppresses the phenotype. Interestingly, AG490 dose-dependently increased p-Akt levels, and our epistasis analysis suggested that the effect of JAK/STAT inhibition on p-Akt is via the regulation of GRAMD1B expression. Taken together, our results suggest that GRAMD1B is a key signaling molecule that functions to inhibit cell migration in breast cancer by negating both JAK/STAT and Akt signaling, providing the foundation for its development as a novel biomarker in breast cancer.

Regulation of the actin cytoskeleton by the Ndel1-Tara complex is critical for cell migration.

Nuclear distribution element-like 1 (Ndel1) plays pivotal roles in diverse biological processes and is implicated in the pathogenesis of multiple neurodevelopmental disorders. Ndel1 function by regulating microtubules and intermediate filaments; however, its functional link with the actin cytoskeleton is largely unknown. Here, we show that Ndel1 interacts with TRIO-associated repeat on actin (Tara), an actin-bundling protein, to regulate cell movement. In vitro wound healing and Boyden chamber assays revealed that Ndel1- or Tara-deficient cells were defective in cell migration. Moreover, Tara overexpression induced the accumulation of Ndel1 at the cell periphery and resulted in prominent co-localization with F-actin. This redistribution of Ndel1 was abolished by deletion of the Ndel1-interacting domain of Tara, suggesting that the altered peripheral localization of Ndel1 requires a physical interaction with Tara. Furthermore, co-expression of Ndel1 and Tara in SH-SY5Y cells caused a synergistic increase in F-actin levels and filopodia formation, suggesting that Tara facilitates cell movement by sequestering Ndel1 at peripheral structures to regulate actin remodeling. Thus, we demonstrated that Ndel1 interacts with Tara to regulate cell movement. These findings reveal a novel role of the Ndel1-Tara complex in actin reorganization during cell movement.

Disrupted-in-schizophrenia-1 (DISC1) Regulates Endoplasmic Reticulum Calcium Dynamics.

Disrupted-in-schizophrenia-1 (DISC1) has emerged as a convincing susceptibility gene for multiple mental disorders, but its mechanistic link to the pathogenesis of schizophrenia related psychiatric conditions is yet to be further understood. Here, we showed that DISC1 localizes to the outer surface of the endoplasmic reticulum (ER). EXOC1, a subunit of the exocyst complex, interacted with DISC1 and affected its recruitment to inositol-1,4,5-trisphosphate receptor 1 (IP3R1). Notably, knockdown of DISC1 and EXOC1 elicited an exaggerated ER calcium response upon stimulation of IP3R agonists. Similar abnormal ER calcium responses were observed in hippocampal neurons from DISC1-deficient mutant mice. Moreover, perturbation of ER calcium dynamics upon DISC1 knockdown was effectively reversed by treatment with antipsychotic drugs, such as clozapine and haloperidol. These results collectively indicate that DISC1 is a regulatory factor in ER calcium dynamics, linking a perturbed intracellular calcium signaling and schizophrenia pathogenesis.

The proteins encoded by the Drosophila Planar Polarity Effector genes inturned, fuzzy and fritz interact physically and can re-pattern the accumulation of "upstream" Planar Cell Polarity proteins.

The frizzled/starry night pathway regulates planar cell polarity in a wide variety of tissues in many types of animals. It was discovered and has been most intensively studied in the Drosophila wing where it controls the formation of the array of distally pointing hairs that cover the wing. The pathway does this by restricting the activation of the cytoskeleton to the distal edge of wing cells. This results in hairs initiating at the distal edge and growing in the distal direction. All of the proteins encoded by genes in the pathway accumulate asymmetrically in wing cells. The pathway is a hierarchy with the Planar Cell Polarity (PCP) genes (aka the core genes) functioning as a group upstream of the Planar Polarity Effector (PPE) genes which in turn function as a group upstream of multiple wing hairs. Upstream proteins, such as Frizzled accumulate on either the distal and/or proximal edges of wing cells. Downstream PPE proteins accumulate on the proximal edge under the instruction of the upstream proteins. A variety of types of data support this hierarchy, however, we have found that when over expressed the PPE proteins can alter both the subcellular location and level of accumulation of the upstream proteins. Thus, the epistatic relationship is context dependent. We further show that the PPE proteins interact physically and can modulate the accumulation of each other in wing cells. We also find that over expression of Frtz results in a marked delay in hair initiation suggesting that it has a separate role/activity in regulating the cytoskeleton that is not shared by other members of the group.

Multiparametric analysis of CLASP-interacting protein functions during interphase microtubule dynamics.

The microtubule (MT) plus-end tracking protein (+TIP) CLASP mediates dynamic cellular behaviors and interacts with numerous cytoplasmic proteins. While the influence of some CLASP interactors on MT behavior is known, a comprehensive survey of the proteins in the CLASP interactome as MT regulators is missing. Ultimately, we are interested in understanding how CLASP collaborates with functionally linked proteins to regulate MT dynamics. Here, we utilize multiparametric analysis of time-lapse MT +TIP imaging data acquired in Drosophila melanogaster S2R+ cells to assess the effects on individual microtubule dynamics for RNA interference-mediated depletion of 48 gene products previously identified to be in vivo genetic CLASP interactors. While our analysis corroborates previously described functions of several known CLASP interactors, its multiparametric resolution reveals more detailed functional profiles (fingerprints) that allow us to precisely classify the roles that CLASP-interacting genes play in MT regulation. Using these data, we identify subnetworks of proteins with novel yet overlapping MT-regulatory roles and also uncover subtle distinctions between the functions of proteins previously thought to act via similar mechanisms.

Cdk5 phosphorylates dopamine D2 receptor and attenuates downstream signaling.

The dopamine D2 receptor (DRD2) is a key receptor that mediates dopamine-associated brain functions such as mood, reward, and emotion. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase whose function has been implicated in the brain reward circuit. In this study, we revealed that the serine 321 residue (S321) in the third intracellular loop of DRD2 (D2i3) is a novel regulatory site of Cdk5. Cdk5-dependent phosphorylation of S321 in the D2i3 was observed in in vitro and cell culture systems. We further observed that the phosphorylation of S321 impaired the agonist-stimulated surface expression of DRD2 and decreased G protein coupling to DRD2. Moreover, the downstream cAMP pathway was affected in the heterologous system and in primary neuronal cultures from p35 knockout embryos likely due to the reduced inhibitory activity of DRD2. These results indicate that Cdk5-mediated phosphorylation of S321 inhibits DRD2 function, providing a novel regulatory mechanism for dopamine signaling.

Valproate alters dopamine signaling in association with induction of Par-4 protein expression.

Chromatin remodeling through histone modifications has emerged as a key mechanism in the pathophysiology of psychiatric disorders. Valproate (VPA), a first-line medication for bipolar disorder, is known to have histone deacetylase (HDAC) inhibitor activity, but the relationship between its efficacy as a mood stabilizer and HDAC inhibitory activity is unclear. Here we provide evidence that prostate apoptosis response-4 (Par-4), an intracellular binding partner of dopamine D2 receptors (DRD2), plays a role in mediating the effectiveness of VPA. We found that chronic VPA treatment enhanced the expression of Par-4 in cultured neurons and adult mouse brains. This Par-4 induction phenomenon occurred at the transcriptional level and was correlated with an increase in histone H3 and H4 acetylation of the Par-4 promoter regions. Furthermore, chronic VPA treatment potentiated the suppression of the cAMP signaling cascade upon dopamine stimulation, which was blocked by sulpiride treatment. These results indicate that VPA potentiates DRD2 activity by enhancing Par-4 expression via a chromatin remodeling mechanism.

Disrupted-in-schizophrenia 1 (DISC1) plays essential roles in mitochondria in collaboration with Mitofilin.

Disrupted-in-schizophrenia 1 (DISC1) has emerged as a schizophrenia-susceptibility gene affecting various neuronal functions. In this study, we characterized Mitofilin, a mitochondrial inner membrane protein, as a mediator of the mitochondrial function of DISC1. A fraction of DISC1 was localized to the inside of mitochondria and directly interacts with Mitofilin. A reduction in DISC1 function induced mitochondrial dysfunction, evidenced by decreased mitochondrial NADH dehydrogenase activities, reduced cellular ATP contents, and perturbed mitochondrial Ca(2+) dynamics. In addition, deficiencies in DISC1 and Mitofilin induced a reduction in mitochondrial monoamine oxidase-A activity. The mitochondrial dysfunctions evoked by the deficiency of DISC1 were partially phenocopied by an overexpression of truncated DISC1 that is associated with schizophrenia in human. DISC1 deficiencies induced the ubiquitination of Mitofilin, suggesting that DISC1 is critical for the stability of Mitofilin. Finally, the mitochondrial dysfunction induced by DISC1 deficiency was partially reversed by coexpression of Mitofilin, confirming a functional link between DISC1 and Mitofilin for the normal mitochondrial function. According to these results, we propose that DISC1 plays essential roles for mitochondrial function in collaboration with a mitochondrial interacting partner, Mitofilin.

A small-molecule compound identified through a cell-based screening inhibits JAK/STAT pathway signaling in human cancer cells.

Inappropriate activation of JAK/STAT signaling occurs with high frequency in human cancers and is associated with cancer cell survival and proliferation. Therefore, the development of pharmacologic STAT signaling inhibitors has therapeutic potential in the treatment of human cancers. Here, we report 2-[(3,5-bis-trifluoromethyl-phenyl)-hydroxy-methyl]-1-(4-nitro-phenylamino)-6-phenyl-1,2,4a,7a-tetrahydro-pyrrolo[3,4-b]-pyridine-5,7-dione (AUH-6-96) as a novel small-molecule inhibitor of JAK/STAT signaling that we initially identified through a cell-based high-throughput screening using cultured Drosophila cells. Treatment of Drosophila cells with AUH-6-96 resulted in a reduction of Unpaired-induced transcriptional activity and tyrosine phosphorylation of STAT92E, the sole Drosophila STAT homologue. In human cancer cell lines, AUH-6-96 inhibited both constitutive and interleukin-6-induced STAT3 phosphorylation. Specifically, in Hodgkin lymphoma L540 cells, treatment with AUH-6-96 resulted in reduced levels of tyrosine phosphorylated STAT3 and of the STAT3 downstream target gene SOCS3 in a dose- and time-dependent manner. In addition, AUH-6-96-treated L540 cells showed decreased expression of persistently activated JAK3, suggesting that AUH-6-96 inhibits the JAK/STAT pathway signaling in L540 cells by affecting JAK3 activity and subsequently blocking STAT3 signaling. Importantly, AUH-6-96 selectively affected cell viability only of cancer cells harboring aberrant JAK/STAT signaling. In support of the specificity of AUH-6-96 for inhibition of JAK/STAT signaling, treatment with AUH-6-96 decreased cancer cell survival by inducing programmed cell death by down-regulating the expression of STAT3 downstream target antiapoptotic genes, such as Bcl-xL. In summary, this study shows that AUH-6-96 is a novel small-molecule inhibitor of JAK/STAT signaling and may have therapeutic potential in the treatment of human cancers harboring aberrant JAK/STAT signaling.

GFP reporters detect the activation of the Drosophila JAK/STAT pathway in vivo.

JAK/STAT signaling is essential for a wide range of developmental processes in Drosophila melanogaster. The mechanism by which the JAK/STAT pathway contributes to these processes has been the subject of recent investigation. However, a reporter that reflects activity of the JAK/STAT pathway in all Drosophila tissues has not yet been developed. By placing a fragment of the Stat92E target gene Socs36E, which contains at least two putative Stat92E binding sites, upstream of GFP, we generated three constructs that can be used to monitor JAK/STAT pathway activity in vivo. These constructs differ by the number of Stat92E binding sites and the stability of GFP. The 2XSTAT92E-GFP and 10XSTAT92E-GFP constructs contain 2 and 10 Stat92E binding sites, respectively, driving expression of enhanced GFP, while 10XSTAT92E-DGFP drives expression of destabilized GFP. We show that these reporters are expressed in the embryo in an overlapping pattern with Stat92E protein and in tissues where JAK/STAT signaling is required. In addition, these reporters accurately reflect JAK/STAT pathway activity at larval stages, as their expression pattern overlaps that of the activating ligand unpaired in imaginal discs. Moreover, the STAT92E-GFP reporters are activated by ectopic JAK/STAT signaling. STAT92E-GFP fluorescence is increased in response to ectopic upd in the larval eye disc and mis-expression of the JAK kinase hopscotch in the adult fat body. Lastly, these reporters are specifically activated by Stat92E, as STAT92E-GFP reporter expression is lost cell-autonomously in stat92E homozygous mutant tissue. In sum, we have generated in vivo GFP reporters that accurately reflect JAK/STAT pathway activation in a variety of tissues. These reporters are valuable tools to further investigate and understand the role of JAK/STAT signaling in Drosophila.

Gene expression during Drosophila wing morphogenesis and differentiation.

The simple cellular composition and array of distally pointing hairs has made the Drosophila wing a favored system for studying planar polarity and the coordination of cellular and tissue level morphogenesis. We carried out a gene expression screen to identify candidate genes that functioned in wing and wing hair morphogenesis. Pupal wing RNA was isolated from tissue prior to, during, and after hair growth and used to probe Affymetrix Drosophila gene chips. We identified 435 genes whose expression changed at least fivefold during this period and 1335 whose expression changed at least twofold. As a functional validation we chose 10 genes where genetic reagents existed but where there was little or no evidence for a wing phenotype. New phenotypes were found for 9 of these genes, providing functional validation for the collection of identified genes. Among the phenotypes seen were a delay in hair initiation, defects in hair maturation, defects in cuticle formation and pigmentation, and abnormal wing hair polarity. The collection of identified genes should be a valuable data set for future studies on hair and bristle morphogenesis, cuticle synthesis, and planar polarity.

The WD40 repeat protein fritz links cytoskeletal planar polarity to frizzled subcellular localization in the Drosophila epidermis.

Much of our understanding of the genetic mechanisms that control planar cell polarity (PCP) in epithelia has derived from studies of the formation of polarized cell hairs during Drosophila wing development. The correct localization of an F-actin prehair to the distal vertex of the pupal wing cell has been shown to be dependent upon the polarized subcellular localization of Frizzled and other core PCP proteins. However, the core PCP proteins do not organize actin cytoskeletal polarity directly but require PCP effector proteins such as Fuzzy and Inturned to mediate this process. Here we describe the characterization of a new PCP effector gene, fritz, that encodes a novel but evolutionarily conserved coiled-coil WD40 protein. We show that the fritz gene product functions cell-autonomously downstream of the core PCP proteins to regulate both the location and the number of wing cell prehair initiation sites.

The microtubule plus end tracking protein Orbit/MAST/CLASP acts downstream of the tyrosine kinase Abl in mediating axon guidance.

Axon guidance requires coordinated remodeling of actin and microtubule polymers. Using a genetic screen, we identified the microtubule-associated protein Orbit/MAST as a partner of the Abelson (Abl) tyrosine kinase. We find identical axon guidance phenotypes in orbit/MAST and Abl mutants at the midline, where the repellent Slit restricts axon crossing. Genetic interaction and epistasis assays indicate that Orbit/MAST mediates the action of Slit and its receptors, acting downstream of Abl. We find that Orbit/MAST protein localizes to Drosophila growth cones. Higher-resolution imaging of the Orbit/MAST ortholog CLASP in Xenopus growth cones suggests that this family of microtubule plus end tracking proteins identifies a subset of microtubules that probe the actin-rich peripheral growth cone domain, where guidance signals exert their initial influence on cytoskeletal organization. These and other data suggest a model where Abl acts as a central signaling node to coordinate actin and microtubule dynamics downstream of guidance receptors.

The grainy head transcription factor is essential for the function of the frizzled pathway in the Drosophila wing.

The Drosophila wing is covered by an array of distally pointing hairs. This tissue planar polarity is regulated by the frizzled pathway. We have found that the function of the grainy head transcription factor is essential for the function of the frizzled pathway. grainy head mutant cells fail to localize planar polarity proteins at either the proximal or distal sides of wing cells and produce multiple hairs of abnormal polarity. Levels of the Starry night protein are strongly reduced in grainy head mutants in both larval wing discs and pupal wings, which is sufficient to account for much of the polarity phenotype. In addition, we found that grh has frizzled pathway independent functions during the development of the adult cuticle.

Neurons take shape.

To construct the intricate network of connections that supports the functions of an adult nervous system, neurons must form highly elaborate processes, extending in the appropriate direction across long distances to form synapses with their partners. As the nervous system takes shape, the process of neuronal morphogenesis is controlled by a broad repertoire of cellular signals. These extracellular cues and cellular interactions are translated by receptors at the cell surface into physical forces that control the dynamic architecture of the neuron as it explores the surrounding terrain. The interpretation of these cues involves a large set of intracellular proteins, whose functional logic we are just beginning to appreciate. We shall consider the basic mechanics of neuronal morphogenesis and some of the emerging pathways that seem to link the outer and inner worlds of the neuron.

The function of the frizzled pathway in the Drosophila wing is dependent on inturned and fuzzy.

The Drosophila epidermis is characterized by a dramatic planar or tissue polarity. The frizzled pathway has been shown to be a key regulator of planar polarity for hairs on the wing, ommatidia in the eye, and sensory bristles on the notum. We have investigated the genetic relationships between putative frizzled pathway downstream genes inturned, fuzzy, and multiple wing hairs (inturned-like genes) and upstream genes such as frizzled, prickle, and starry night (frizzled-like genes). Previous data showed that the inturned-like genes were epistatic to the frizzled-like genes when the entire wing was mutant. We extended those experiments and examined the behavior of frizzled clones in mutant wings. We found the domineering nonautonomy of frizzled clones was not altered when the clone cells were simultaneously mutant for inturned, multiple wing hairs, or dishevelled but it was blocked when the entire wing was mutant for inturned, fuzzy, multiple wing hairs, or dishevelled. Thus, for the domineering nonautonomy phenotype of frizzled, inturned and multiple wing hairs are needed in the responding cells but not in the clone itself. Expressing a number of frizzled pathway genes in a gradient across part of the wing repolarizes wing cells in that region. We found inturned, fuzzy, and multiple wing hairs were required for a gradient of frizzled, starry night, prickle, or spiny-legs expression to repolarize wing cells. These data argue that inturned, fuzzy, and multiple wing hairs are downstream components of the frizzled pathway. To further probe the relationship between the frizzled-like and inturned-like genes we determined the consequences of altering the activity of frizzled-like genes in wings that carried weak alleles of inturned or fuzzy. Interestingly, both increasing and decreasing the activity of frizzled and other upstream genes enhanced the phenotypes of hypomorphic inturned and fuzzy mutants. We also examined the relationship between the frizzled-like and inturned-like genes in other regions of the fly. For some body regions and cell types (e.g., abdomen) the inturned-like genes were epistatic to the frizzled-like genes, but in other body regions (e.g., eye) that was not the case. Thus, the genetic control of tissue polarity is body region specific.