Membrane Relocalization Research Articles

LAMP3 inhibits autophagy and contributes to cell death by lysosomal membrane permeabilization

ABSTRACT Sjögren syndrome (SS) is a chronic and progressive autoimmune disease characterized by dry mouth and dry eyes, and characteristic autoantibodies. Evidence of altered macroautophagy/autophagy and apoptosis has been associated with SS, but a mechanistic understanding of the gene expression changes associated with these abnormal processes has not been realized. Recently, increased LAMP3 (lysosomal associated membrane protein 3) expression was found in a subset of SS patients and was associated with increased apoptosis and autoantigen accumulation and release. To better understand how LAMP3 expression might modulate apoptosis, cell biology, and biochemical studies were used to examine the effect of LAMP3 expression in minor salivary gland cells. LAMP3 expression resulted in degradation of LAMP1 increasing lysosomal membrane permeabilization and relocalization of cathepsins to the cytoplasm, resulting in destabilizing autophagic flux and caspase activation. These findings highlight the central role of LAMP3 expression in the pathogenesis of SS.

Endomembrane-Targeting Plasmodiophora brassicae Effectors Modulate PAMP Triggered Immune Responses in Plants.

Plasmodiophora brassicae is a devastating obligate, intracellular, biotrophic pathogen that causes clubroot disease in crucifer plants. Disease progression is regulated by effector proteins secreted by P. brassicae. Twelve P. brassicae putative effectors (PbPEs), expressed at various stages of disease development [0, 2, 5, 7, 14, 21, and 28 days post inoculation (DPI)] in Arabidopsis and localizing to the plant endomembrane system, were studied for their roles in pathogenesis. Of the 12 PbPEs, seven showed an inhibitory effect on programmed cell death (PCD) as triggered by the PCD inducers, PiINF1 (Phytophthora infestans Infestin 1) and PiNPP1 (P. infestans necrosis causing protein). Showing the strongest level of PCD suppression, PbPE15, a member of the 2-oxoglutarate (2OG) and Fe (II)-dependent oxygenase superfamily and with gene expression during later stages of infection, appears to have a role in tumorigenesis as well as defense signaling in plants. PbPE13 produced an enhanced PiINF1-induced PCD response. Transient expression, in Nicotiana benthamiana leaves of these PbPEs minus the signal peptide (SP) (ΔspPbPEGFPs), showed localization to the endomembrane system, targeting the endoplasmic reticulum (ER), Golgi bodies and nucleo-cytoplasm, suggesting roles in manipulating plant cell secretion and vesicle trafficking. ΔspPbPE13GFP localized to plasma membrane (PM) lipid rafts with an association to plasmodesmata, suggesting a role at the cell-to-cell communication junction. Membrane relocalization of ΔspPbPE13GFP, triggered by flagellin N-terminus of Pseudomonas aeruginosa (flg22 – known to elicit a PAMP triggered immune response in plants), supports its involvement in raft-mediated immune signaling. This study is an important step in deciphering P. brassicae effector roles in the disruption of plant immunity to clubroot disease.

The Role of Diet Related Short-Chain Fatty Acids in Colorectal Cancer Metabolism and Survival: Prevention and Therapeutic Implications.

Colorectal Cancer (CRC) is a major cause of cancer-related death worldwide. CRC increased risk has been associated with alterations in the intestinal microbiota, with decreased production of Short Chain Fatty Acids (SCFAs). SCFAs produced in the human colon are the major products of bacterial fermentation of undigested dietary fiber and starch. While colonocytes use the three major SCFAs, namely acetate, propionate and butyrate, as energy sources, transformed CRC cells primarily undergo aerobic glycolysis. Compared to normal colonocytes, CRC cells exhibit increased sensitivity to SCFAs, thus indicating they play an important role in cell homeostasis. Manipulation of SCFA levels in the intestine, through changes in microbiota, has therefore emerged as a potential preventive/therapeutic strategy for CRC. Interest in understanding SCFAs mechanism of action in CRC cells has increased in the last years. Several SCFA transporters like SMCT-1, MCT-1 and aquaporins have been identified as the main transmembrane transporters in intestinal cells. Recently, it was shown that acetate promotes plasma membrane re-localization of MCT-1 and triggers changes in the glucose metabolism. SCFAs induce apoptotic cell death in CRC cells, and further mechanisms have been discovered, including the involvement of lysosomal membrane permeabilization, associated with mitochondria dysfunction and degradation. In this review, we will discuss the current knowledge on the transport of SCFAs by CRC cells and their effects on CRC metabolism and survival. The impact of increasing SCFA production by manipulation of colon microbiota on the prevention/therapy of CRC will also be addressed.

The Search Continues: Determination of a Lipid Raft Targeting Motif in Cytochromes P450

Cytochromes P450 play an essential role in metabolism of both xenobiotics and endogenous compounds. The liver P450s responsible for drug metabolism are predominantly expressed in hepatocytes, where they closely associate with the endoplasmic reticulum (ER). This membranous organelle is mostly composed of phospholipids, which can assemble into disordered regions (ld) or ordered regions (lo, “lipid rafts”).Our group previously showed that different P450 species show strong preference for either ordered or disordered regions. When expressed in human embryonic kidney (HEK) cells, we demonstrated that CYP1A2 is found in ordered (raft) regions, and that CYP1A1 exhibits a strong preference for the disordered region. This pattern was striking, as the two P450 species have over 70% sequence homology. We then showed that a chimeric CYP1A2 construct containing the first 107 amino acids of CYP1A1 no longer localized in the ordered regions, but mobilized to the disordered region. These findings indicate that a P450 raft targeting sequence exists within the first 107 amino acids of CYP1A2.The length and composition of this motif remain unclear. We hypothesize that a 5–9 amino acid sequence within the first 107 amino acids is responsible for the localization of CYP1A2 into the ordered domains, and that substitution of the corresponding region of CYP1A1 can mobilize full‐length CYP1A2 to the disordered region. To test this, we generated 5‐ to 30‐amino acid chimeras using overhang PCR cloning and expressed these constructs in HEK cells. The disordered membrane regions from these cells were solubilized using Brij98, with the ordered regions remaining in the membrane. The ordered membranes were isolated as a pellet after centrifugation at 100,000 x g for 1 h. We found that insertion of the first 28 amino acids of CYP1A1 into CYP1A2 led to its partial relocation into the disordered regions, and that the first 30 amino acids led to more complete relocation of the chimeric protein. We then generated smaller chimeras in an effort to identify the minimum amino acid segment responsible for membrane relocation. Substitution of amino acids 28–30 led to relocation of CYP1A2 from the ordered to disordered regions. These results demonstrate that a raft‐targeting sequence resides within the first 30 N‐terminal amino acids of CYP1A2 and, when eliminated, permits mobilization of the P450.Support or Funding InformationSupported by NIH GM123253 and ES013648 and by a predoctoral fellowship from the PhRMA Foundation.

TTL Proteins Scaffold Brassinosteroid Signaling Components at the Plasma Membrane to Optimize Signal Transduction in Arabidopsis.

Brassinosteroids (BRs) form a group of steroidal hormones essential for plant growth, development, and stress responses. BRs are perceived extracellularly by plasma membrane receptor-like kinases that activate an interconnected signal transduction cascade, leading to the transcriptional regulation of BR-responsive genes. TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) genes are specific for land plants, and their encoded proteins are defined by the presence of protein-protein interaction motives, that is, an intrinsic disordered region at the N terminus, six tetratricopeptide repeat domains, and a C terminus with homology to thioredoxins. TTL proteins thus likely mediate the assembly of multiprotein complexes. Phenotypic, molecular, and genetic analyses show that TTL proteins are positive regulators of BR signaling in Arabidopsis (Arabidopsis thaliana). TTL3 directly interacts with a constitutively active BRASSINOSTEROID INSENSITIVE1 (BRI1) receptor kinase, BRI1-SUPPRESSOR1 phosphatase, and the BRASSINAZOLE RESISTANT1 transcription factor and associates with BR-SIGNALING KINASE1, BRASSINOSTEROID INSENSITIVE2 kinases, but not with BRI1-ASSOCIATED KINASE1. A functional TTL3-green fluorescent protein (GFP) shows dual cytoplasmic plasma membrane localization. Depleting the endogenous BR content reduces plasma membrane localization of TTL3-GFP, while increasing BR content causes its plasma membrane relocalization, where it strengthens the association of BR signaling components. Our results reveal that TTL proteins promote BR responses and suggest that TTL proteins may function as scaffold proteins by bringing together cytoplasmic and plasma membrane BR signaling components.

Ulipristal Acetate Interferes With Actin Remodeling Induced by 17β-Estradiol and Progesterone in Human Endometrial Stromal Cells.

Ulipristal acetate (UPA) is a selective progesterone receptor modulator (SPRM) used for emergency contraception and for the medical management of symptomatic uterine fibroids (UF). Treatment with UPA turns in amenorrhea and UF volume reduction. Treatment with UPA is associated with the frequent development of benign, transitory endometrial changes known as SPRM-associated endometrial changes (PAECs). Why PAECs develop and their biological or cellular basis is unknown. Sex steroids, including estrogen and progesterone, are established modulators of the actin cytoskeleton in various cells, including endometrial cells. This explains several morphological and functional changes in endometrial cells. We thus hypothesized that UPA may alter the appearance of the endometrium by interfering with the actions of 17β-estradiol (E2) or progesterone (P4) on actin dynamics. We isolated and cultured human endometrial stromal cells (ESC) from endometrial biopsies from healthy fertile women. Treatment with E2 or P4 stimulated visible actin rearrangements with actin remodeling toward the membrane. Activation through phosphorylation of the actin regulatory proteins, Moesin, and focal adhesion kinase (FAK), hacked actin remodeling induced by E2 and P4. Membrane re-localization of Paxillin and Vinculin were also induced by E2 and P4, showing the formation of focal adhesion complexes. All these E2 and P4 actions were inhibited by co-treatment with UPA, which was otherwise inactive if given alone. The cytoskeletal changes induced by E2 and P4 turned into increased motility of ESC, and UPA again blocked the actions E2 and P4. In conclusion, we find that UPA interferes with the cytoskeletal actions of E2 and P4 in ESC. This finding helps understanding the mode of actions of SPRMs in the endometrium and may be relevant for other potential clinical applications of UPA.

Distribution of Endogenous NO Regulates Early Gravitropic Response and PIN2 Localization in Arabidopsis Roots.

High-resolution and automated image analysis of individual roots demonstrated that endogenous nitric oxide (NO) contribute significantly to gravitropism of Arabidopsis roots. Lowering of endogenous NO concentrations strongly reduced and even reversed gravitropism, resulting in upward bending, without affecting root growth rate. Notably, the asymmetric accumulation of NO along the upper and lower sides of roots correlated with a positive gravitropic response. Detection of NO by the specific DAF-FM DA fluorescent probe revealed that NO was higher at the lower side of horizontally-oriented roots returning to initial values 2 h after the onset of gravistimulation. We demonstrate that NO promotes plasma membrane re-localization of PIN2 in epidermal cells, which is required during the early root gravitropic response. The dynamic and asymmetric localization of both auxin and NO is critical to regulate auxin polar transport during gravitropism. Our results collectively suggest that, although auxin and NO crosstalk occurs at different levels of regulation, they converge in the regulation of PIN2 membrane trafficking in gravistimulated roots, supporting the notion that a temporally and spatially coordinated network of signal molecules could participate in the early phases of auxin polar transport during gravitropism.

Role of lysophosphatidic acid in the retinal pigment epithelium and photoreceptors

The human retina is a complex structure of organised layers of specialised cells that support the transmission of light signals to the visual cortex. The outermost layer of the retina, the retinal pigment epithelium (RPE), forms part of the blood retina barrier and is implicated in many retinal diseases. Lysophosphatidic acid (LPA) is a bioactive lipid exerting pleiotropic effects in various cell types, during development, normal physiology and disease. Its producing enzyme, AUTOTAXIN (ATX), is highly expressed by the pigmented epithelia of the human eye, including the RPE. Using human pluripotent stem cell (hPSC)-derived retinal cells, we interrogated the role of LPA in the human RPE and photoreceptors. hPSC-derived RPE cells express and synthesize functional ATX, which is predominantly secreted apically of the RPE, suggesting it acts in a paracrine manner to regulate photoreceptor function. In RPE cells, LPA regulates tight junctions, in a receptor-dependent mechanism, with an increase in OCCLUDIN and ZONULA OCCLUDENS (ZO)-1 expression at the cell membrane, accompanied by an increase in the transepithelial resistance of the epithelium. High concentration of LPA decreases phagocytosis of photoreceptor outer segments by the RPE. In hPSC-derived photoreceptors, LPA induces morphological rearrangements by modulating the actin myosin cytoskeleton, as evidenced by Myosin Light Chain l membrane relocation. Collectively, our data suggests an important role of LPA in the integrity and functionality of the healthy retina and blood retina barrier.

Cleavage activates dispatched for Sonic Hedgehog ligand release.

Hedgehog ligands activate an evolutionarily conserved signaling pathway that provides instructional cues during tissue morphogenesis, and when corrupted, contributes to developmental disorders and cancer. The transmembrane protein Dispatched is an essential component of the machinery that deploys Hedgehog family ligands from producing cells, and is absolutely required for signaling to long-range targets. Despite this crucial role, regulatory mechanisms controlling Dispatched activity remain largely undefined. Herein, we reveal vertebrate Dispatched is activated by proprotein convertase-mediated cleavage at a conserved processing site in its first extracellular loop. Dispatched processing occurs at the cell surface to instruct its membrane re-localization in polarized epithelial cells. Cleavage site mutation alters Dispatched membrane trafficking and reduces ligand release, leading to compromised pathway activity in vivo. As such, convertase-mediated cleavage is required for Dispatched maturation and functional competency in Hedgehog ligand-producing cells.

APT2 Inhibition Restores Scribble Localization and S-Palmitoylation in Snail-Transformed Cells

The multidomain scaffolding protein Scribble (Scrib) organizes key signaling complexes to specify basolateral cell polarity and suppress aberrant growth. In many human cancers, genetically normal Scrib mislocalizes from cell-cell junctions to the cytosol, correlating with enhanced growth signaling and malignancy. Here we confirm that expression of theepithelial-to-mesenchymal transcription factor (EMT-TF) Snail in benign epithelial cells leads to Scribdisplacement from the plasma membrane, mimicking the mislocalization observed in aggressive cancers. Upon further examination, Snail promotes a transcriptional program that targets genes in the palmitoylation cycle, repressing many protein acyl transferases and elevating expression and activity of protein acyl thioesterase 2 (APT2). APT2 isoform-selective inhibition or knockdown rescued Scrib membrane localization and palmitoylation while attenuating MEK activation. Overall, inhibiting APT2 restores balance to the Scrib palmitoylation cycle, promoting membrane re-localization and growth attenuation. These findings emphasize the importance of S-palmitoylation as a post-translational gatekeeper of cell polarity-mediated tumor suppression.

Post-translational myristoylation at the cross roads of cell death, autophagy and neurodegeneration.

In a little more than a decade, post-translational myristoylation (PTMyr) has become an established post-translational modification during cell death. It involves the addition of the fatty acid myristate to newly exposed N-terminal glycines following caspase cleavage. It promotes membrane binding and relocalization of functional protein domains released by caspase cleavage during apoptosis, or programmed cell death. However, as the requirement of caspase cleavage has expanded beyond just cell death, it has become apparent that PTMyr may play a role in cell survival, differentiation and now autophagy. Herein, we describe how myristoylation may play a role in autophagy with an emphasis on PTMyr.

Integration of BMP/Wnt signaling to control clonal growth of limbal epithelial progenitor cells by niche cells

Both BMP and Wnt signaling control stem cells in bulge/dermal papilla, intestinal crypt, and bone marrow. To explore their roles in the limbal niche, which govern corneal epithelial homeostasis, we established an in vitro model of sphere growth by reunion between single limbal epithelial progenitor cells (LEPCs) and aggregates of limbal niche cells (LNCs) in 3D Matrigel. Compared to LEPCs alone, spheres formed by LEPC+LNC exhibited higher clonal growth and less corneal epithelial differentiation. Furthermore, pSmad1/5/8 was in the nucleus of LEPCs, but not LNCs, and correlated with upregulation of BMP1, BMP3, BMP4, all three BMP receptors, and BMP target genes. Inactivation of BMP signaling in LNCs was correlated with upregulation of noggin preferentially expressed by LNCs. Additionally, β-catenin was stabilized in the perinuclear cytoplasm in LEPCs and correlated with upregulation of Wnt7A and FZD5 preferentially expressed by LEPCs. Inactivation of Wnt signaling in LNCs was correlated with upregulation of DKK1/2 by LNCs. Addition of XAV939 that expectedly downregulated perinuclear β-catenin in LEPCs led to significant reduction of epithelial clonal growth, but upregulated all three BMP receptors and downregulated LNC-derived noggin, resulting in activation of BMP signaling in LNCs. Addition of noggin that expectedly downregulated nuclear localization of pSmad1/5/8 in LEPCs led to nuclear localization of β-catenin in larger LEPCs but membrane relocation of β-catenin in smaller LEPCs and significant upregulation of DKK1/2. Hence, balancing acts between Wnt signaling and BMP signaling exist not only within LEPCs but also between LEPCs and LNCs to regulate clonal growth of LEPCs.

CAMP signaling regulates platelet myosin light chain (MLC) phosphorylation and shape change through targeting the RhoA-Rho kinase-MLC phosphatase signaling pathway

AbstractCyclic adenosine monophosphate (cAMP)-dependent signaling modulates platelet shape change through unknown mechanisms. We examined the effects of cAMP signaling on platelet contractile machinery. Prostaglandin E1 (PGE1)-mediated inhibition of thrombin-stimulated shape change was accompanied by diminished phosphorylation of myosin light chain (MLC). Since thrombin stimulates phospho-MLC through RhoA/Rho-associated, coiled-coil containing protein kinase (ROCK)-dependent inhibition of MLC phosphatase (MLCP), we examined the effects of cAMP on this pathway. Thrombin stimulated the membrane localization of RhoA and the formation of a signaling complex of RhoA/ROCK2/myosin phosphatase-targeting subunit 1 (MYPT1). This resulted in ROCK-mediated phosphorylation of MYPT1 on threonine 853 (thr853), the disassociation of the catalytic subunit protein phosphatase 1δ (PP1δ) from MYPT1 and inhibition of basal MLCP activity. Treatment of platelets with PGE1 prevented thrombin-induced phospho-MYPT1-thr853 in a protein kinase A (PKA)-dependent manner. Examination of the molecular mechanisms revealed that PGE1 induced the phosphorylation of RhoA on serine188 through a pathway requiring cAMP and PKA. This event inhibited the membrane relocalization of RhoA, prevented the association of RhoA with ROCK2 and MYPT1, attenuated the dissociation of PP1δ from MYPT1, and thereby restored basal MLCP activity leading to a decrease in phospho-MLC. These data reveal a new mechanism by which the cAMP-PKA signaling pathway regulates platelet function.

GDNF restores human blood–nerve barrier function via RET tyrosine kinase-mediated cytoskeletal reorganization

Endoneurial microvessels and the perineurium are responsible for maintaining homeostasis in peripheral nerves. Endoneurial endothelial cells form the blood–nerve barrier (BNB). The molecular pathways responsible for endoneurial microvascular barrier formation in humans are not fully understood. We tested the effect of different mitogens on the transendothelial electrical resistance (TEER) of confluent primary human endoneurial endothelial cell (pHEndEC) cultures following serum withdrawal (mimicking diffuse endothelial injury) in vitro. We show that glial-derived neurotrophic factor (GDNF, 1ng/mL) sufficiently induced a maximal 114.2% recovery in TEER over basal conditions 48h after serum withdrawal. Solute permeability to high molecular weight dextran was reduced by 52.4% following GDNF treatment. GDNF-mediated increase in TEER was dependent on RET tyrosine-kinase signaling pathways and mildly enhanced by cyclic adenosine monophosphate in combination with maximal concentrations of multiple redundant mitogens. There was no significant increase in adherens or tight junction proteins β-catenin, VE-Cadherin, zona occludens-1 and occludin following GDNF treatment. GDNF induced a small increase in total claudin-5 protein expression without significant increase in messenger RNA or modulation in tyrosine phosphorylation following serum withdrawal. Indirect immunocytochemistry revealed membrane relocation of longitudinal F-actin cytoskeletal filaments in pHEndECs following GDNF treatment, resulting in more continuous intercellular contacts that formed adherens and tight junctions. Together, these results demonstrate a sufficient role for GDNF in human BNB recovery following serum withdrawal in vitro, facilitated primarily by endothelial cell cytoskeletal reorganization. These observations provide insights into the regulation of human BNB function during recovery from peripheral nerve injury.

A novel method for monitoring the localization of cytochromes P450 and other endoplasmic reticulum membrane associated proteins: a tool for investigating the formation of metabolons

In plants and possibly other organisms, channelling of the reactive intermediates resulting from P450 oxygenation is thought to require the formation of supramolecular complexes associating membrane-bound and soluble enzymes. This implies a most probably loose membrane association of the soluble proteins. For the assessment of such membrane association in vivo, we propose an imaging strategy based on the accurate evaluation of fluorescent protein repartition and distance around endoplasmic reticulum (ER) tubules. It requires candidate protein fusion constructs with fluorescent reporters and transient expression in leaves of Nicotiana benthamiana. The method was tested with soluble eGFP/mRFP1, with various P450 and P450 reductase fluorescent fusions, and with anchored eGFP/mRFP1. It easily differentiated soluble and anchored proteins and detects subtle changes in ER tubules. The method was further assessed with a soluble protein previously shown to be loosely associated with the ER, the phenylalanine ammonia lyase PAL1 involved in the lignin biosynthetic pathway. This protein was found located in close vicinity to the ER. Taken together, these data indicate that the method proposed herein is suitable to monitor membrane association and relocalization of soluble proteins involved in the formation of metabolons.

Intramyocellular lipid accumulation is associated with permanent relocation ex vivo and in vitro of fatty acid translocase (FAT)/CD36 in obese patients

Intramyocellular lipids (IMCL) accumulation is a classical feature of metabolic diseases. We hypothesised that IMCL accumulate mainly as a consequence of increased adiposity and independently of type 2 diabetes. To test this, we examined IMCL accumulation in two different models and four different populations of participants: muscle biopsies and primary human muscle cells derived from non-obese and obese participants with or without type 2 diabetes. The mechanism regulating IMCL accumulation was also studied. Muscle biopsies were obtained from ten non-obese and seven obese participants without type 2 diabetes, and from eight non-obese and eight obese type 2 diabetic patients. Mitochondrial respiration, citrate synthase activity and both AMP-activated protein kinase and acetyl-CoA carboxylase phosphorylation were measured in muscle tissue. Lipid accumulation in muscle and primary myotubes was estimated by Oil Red O staining and fatty acid translocase (FAT)/CD36 localisation by immunofluorescence. Obesity and type 2 diabetes are independently characterised by skeletal muscle IMCL accumulation and permanent FAT/CD36 relocation. Mitochondrial function is not reduced in type 2 diabetes. IMCL accumulation was independent of type 2 diabetes in cultured myotubes and was correlated with obesity markers of the donor. In obese participants, membrane relocation of FAT/CD36 is a determinant of IMCL accumulation. In skeletal muscle, mitochondrial function is normal in type 2 diabetes, while IMCL accumulation is dependent upon obesity or type 2 diabetes and is related to sarcolemmal FAT/CD36 relocation. In cultured myotubes, IMCL content and FAT/CD36 relocation are independent of type 2 diabetes, suggesting that distinct factors in obesity and type 2 diabetes contribute to permanent FAT/CD36 relocation ex vivo.

JNK and Ceramide Kinase Govern the Biogenesis of Lipid Droplets through Activation of Group IVA Phospholipase A2

The biogenesis of lipid droplets (LD) induced by serum depends on group IVA phospholipase A(2) (cPLA(2)alpha). This work dissects the pathway leading to cPLA(2)alpha activation and LD biogenesis. Both processes were Ca(2+)-independent, as they took place after pharmacological blockade of Ca(2+) transients elicited by serum or chelation with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester). The single mutation D43N in cPLA(2)alpha, which abrogates its Ca(2+) binding capacity and translocation to membranes, did not affect enzyme activation and formation of LD. In contrast, the mutation S505A did not affect membrane relocation of the enzyme in response to Ca(2+) but prevented its phosphorylation, activation, and the appearance of LD. Expression of specific activators of different mitogen-activated protein kinases showed that phosphorylation of cPLA(2)alpha at Ser-505 is due to JNK. This was confirmed by pharmacological inhibition and expression of a dominant-negative form of the upstream activator MEKK1. LD biogenesis was accompanied by increased synthesis of ceramide 1-phosphate. Overexpression of its synthesizing enzyme ceramide kinase increased phosphorylation of cPLA(2)alpha at Ser-505 and formation of LD, and its down-regulation blocked the phosphorylation of cPLA(2)alpha and LD biogenesis. These results demonstrate that LD biogenesis induced by serum is regulated by JNK and ceramide kinase.

Dual FGF-2 and Intergrin α5β1 Signaling Mediate GRAF-Induced RhoA Inactivation in a Model of Breast Cancer Dormancy

Interactions with the bone marrow stroma regulate dormancy and survival of breast cancer micrometastases. In an in vitro model of dormancy in the bone marrow, we previously demonstrated that estrogen-dependent breast cancer cells are partially re-differentiated by FGF-2, re-express integrin α5β1 lost with malignant transformation and acquire an activated PI3K/Akt pathway. Ligation of integrin α5β1 by fibronectin and activation of the PI3K pathway both contribute to survival of these dormant cells. Here, we investigated mechanisms responsible for the dormant phenotype. Experiments demonstrate that integrin α5β1 controls de novo cytoskeletal rearrangements, cell spreading, focal adhesion kinase rearrangement to the cell perimeter and recruitment of a RhoA GAP known as GRAF. This results in the inactivation of RhoA, an effect which is necessary for the stabilization of cortical actin. Experiments also demonstrate that activation of the PI3K pathway by FGF-2 is independent of integrin α5β1 and is also required for cortical actin reorganization, GRAF membrane relocalization and RhoA inactivation. These data suggest that GRAF-mediated RhoA inactivation and consequent phenotypic changes of dormancy depend on dual signaling by FGF-2-initiated PI3K activation and through ligation of integrin α5β1 by fibronectin.

Single-Molecule Photoactivation and Localization of Signaling Complexes in T cells

In T cells, membrane receptor and ligand engagement initiates a signaling cascade. Ligand binding is relayed through specific membrane receptors, protein tyrosine kinases, and critical adaptors to regulate downstream activation of transcription factors, cytokine production and cell proliferation. The membrane segregation and relocalization of these signaling components display highly dynamic protein networks in response to distinct stimulations. However, the membrane-protein and protein-protein interactions involved in the formation of signaling complexes are still poorly understood. We are currently using a high-resolution imaging technique to visualize the early stage of signal transduction events at the single-molecule level with photoactivatable or photoconvertable fluorescent markers. The photoactivation and localization of T-cell signaling components allow measurements of molecular spatial and temporal correlation in response to different stimulations. With dual-color application, molecular correlation functions will enhance the understanding of the protein-protein interactions in signaling complexes.

Cell migration by a FRS2‐adaptor dependent membrane relocation of ret receptors

During development neural progenitor cells migrate with extraordinary precision to inhabit tissues and organs far from their initial position. Little is known about the cellular basis for directional guidance by tyrosine kinase receptors (RTKs). RET is a RTK with important functions in guiding the migration of neuronal cells, and RET dysregulation leads to clinical disease such as agangliosis of the colon. We show here that RET migration in neuroepitheliomal and non-neuronal cells is elicited by the activation of specific signaling pathways initiated by the competitive recruitment of the FRS2 adaptor molecule to tyrosine 1062 (Y1062) in RET. FRS2 selectively recruited RET to focal complexes and led to activation of SRC family kinases and focal adhesion kinase (FAK). Activation of SRC depended on its direct interaction with RET at a different intracellular tyrosine (Y981) and activation of molecular signaling from these two separate sites in concert regulated migration. Our data suggest that an important function for FRS2 is to concentrate RET in membrane foci, leading to an engagement of specific signaling complexes localized in these membrane domains.