Regulation Of Protein Trafficking Research Articles

Who's really in control: microbial regulation of protein trafficking in the epithelium

Due to evolutionary pressure, there are many complex interactions at the interface between pathogens and eukaryotic host cells wherein host cells attempt to clear invading microorganisms and pathogens counter these mechanisms to colonize and invade host tissues. One striking observation from studies focused on this interface is that pathogens have multiple mechanisms to modulate and disrupt normal cellular physiology to establish replication niches and avoid clearance. The precision by which pathogens exert their effects on host cells makes them excellent tools to answer questions about cell physiology of eukaryotic cells. Furthermore, an understanding of these mechanisms at the host-pathogen interface will benefit our understanding of how pathogens cause disease. In this review, we describe a few examples of how pathogens disrupt normal cellular physiology and protein trafficking at epithelial cell barriers to underscore how pathogens modulate cellular processes to cause disease and how this knowledge has been utilized to learn about cellular physiology.

Rab GTPases Associate with Isolated Lipid Droplets (LDs) and Show Altered Content After Ethanol Administration: Potential Role in Alcohol-Impaired LD Metabolism

Alcoholic liver disease is manifested by the presence of fatty liver, primarily due to accumulation of hepatocellular lipid droplets (LDs). The presence of membrane-trafficking proteins (e.g., Rab GTPases) with LDs indicates that LDs may be involved in trafficking pathways known to be altered in ethanol (EtOH) damaged hepatocytes. As these Rab GTPases are crucial regulators of protein trafficking, we examined the effect EtOH administration has on hepatic Rab protein content and association with LDs. Male Wistar rats were pair-fed Lieber-DeCarli diets for 5 to 8weeks. Whole liver and isolated LD fractions were analyzed. Identification of LDs and associated Rab proteins was performed in frozen liver or paraffin-embedded sections followed by immunohistochemical analysis. Lipid accumulation was characterized by larger LD vacuoles and increased total triglyceride content in EtOH-fed rats. Rabs 1, 2, 3d, 5, 7, and 18 were analyzed in postnuclear supernatant (PNS) as well as LDs. All of the Rabs were found in the PNS, and Rabs 1, 2, 5, and 7 did not show alcohol-altered content, while Rab 3d content was reduced by over 80%, and Rab 18 also showed EtOH-induced reduction in content. Rab 3d was not found to associate with LDs, while all other Rabs were found in the LD fractions, and several showed an EtOH-related decrease (Rabs 2, 5, 7, 18). Immunohistochemical analysis revealed the enhanced content of a LD-associated protein, perilipin 2 (PLIN2) that was paralleled with an associated decrease of Rab 18 in EtOH-fed rat sections. Chronic EtOH feeding was associated with increased PLIN2 and altered Rab GTPase content in enriched LD fractions. Although mechanisms driving these changes are not established, further studies on intracellular protein trafficking and LD biology after alcohol administration will likely contribute to our understanding of fatty liver disease.

Syntaxin-4 is essential for IgE secretion by plasma cells

The humoral immune system provides a crucial first defense against the invasion of microbial pathogens via the secretion of antigen specific immunoglobulins (Ig). The secretion of Ig is carried out by terminally differentiated B-lymphocytes called plasma cells. Despite the key role of plasma cells in the immune response, the mechanisms by which they constitutively traffic large volumes of Ig out of the cell is poorly understood. The involvement of Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in the regulation of protein trafficking from cells has been well documented. Syntaxin-4, a member of the Qa SNARE syntaxin family has been implicated in fusion events at the plasma membrane in a number of cells in the immune system. In this work we show that knock-down of syntaxin-4 in the multiple myeloma U266 human plasma cell line results in a loss of IgE secretion and accumulation of IgE within the cells. Furthermore, we show that IgE co-localises with syntaxin-4 in U266 plasma cells suggesting direct involvement in secretion at the plasma membrane. This study demonstrates that syntaxin-4 plays a critical role in the secretion of IgE from plasma cells and sheds some light on the mechanisms by which these cells constitutively traffic vesicles to the surface for secretion. An understanding of this machinery may be beneficial in identifying potential therapeutic targets in multiple myeloma and autoimmune disease where over-production of Ig leads to severe pathology in patients.

Amyloid beta1–42 (Aβ42) up‐regulates the expression of sortilin via the p75NTR/RhoA signaling pathway

Sortilin, a Golgi sorting protein and a member of the VPS10P family, is the co-receptor for proneurotrophins, regulates protein trafficking, targets proteins to lysosomes, and regulates low density lipoprotein metabolism. The aim of this study was to investigate the expression and regulation of sortilin in Alzheimer's disease (AD). A significantly increased level of sortilin was found in human AD brain and in the brains of 6-month-old swedish-amyloid precursor protein/PS1dE9 transgenic mice. Aβ₄₂ enhanced the protein and mRNA expression levels of sortilin in a dose- and time-dependent manner in SH-SY5Y cells, but had no effect on sorLA. In addition, proBDNF also significantly increased the protein and mRNA expression of sortilin in these cells. The recombinant extracellular domain of p75(NTR) (P75ECD-FC), or the antibody against the extracellular domain of p75(NTR), blocked the up-regulation of sortilin induced by Amyloid-β protein (Aβ), suggesting that Aβ₄₂ increased the expression level of sortilin and mRNA in SH-SY5Y via the p75(NTR) receptor. Inhibition of ROCK, but not Jun N-terminal kinase, suppressed constitutive and Aβ₄₂-induced expression of sortilin. In conclusion, this study shows that sortilin expression is increased in the AD brain in human and mice and that Aβ₄₂ oligomer increases sortilin gene and protein expression through p75(NTR) and RhoA signaling pathways, suggesting a potential physiological interaction of Aβ₄₂ and sortilin in Alzheimer's disease.

Abstract 336: A 14-3-3 Mode-1 Binding Motif Promotes Gap Junction Internalization During Acute Cardiac Ischemia

During each heartbeat, robust cell-cell electrical coupling via connexin 43 (Cx43) gap junctions allows billions of individual cardiomyocytes to contract in synchrony. Cx43 turns over rapidly, and altered Cx43 trafficking during disease contributes to the arrhythmias of sudden cardiac death. The overall phosphorylation status of the Cx43 protein is known to regulate gap junction coupling, but the role of many residue specific phosphorylation events remains unknown. One such residue, Ser373, forms a mode-1 14-3-3 binding motif upon phosphorylation. Given that 14-3-3 proteins are known to regulate protein trafficking, we hypothesized a role for Cx43 Ser373 phosphorylation in regulation of Cx43 gap junction coupling. Using Langendorff-perfused mouse hearts we find robust phosphorylation of Cx43 at Ser373 and Ser368 after 30 min of no-flow ischemia. In human cell lines, a S373A mutation ablated Cx43/14-3-3 complexing and 35 S pulse-chase revealed Cx43 S373A also experiences a longer half-life than wild-type Cx43. Previous reports have implicated phosphorylation of Cx43 Ser368 in PKC mediated Cx43 internalization. We find that upon activation of PKC, the Cx43 S373A mutant undergoes lower and more transient levels of phosphorylation at Ser368 than wild-type Cx43. Consistent with these data, siRNA-mediated ablation of 14-3-3 expression results in enlargement of gap junction plaque formation at cell-cell borders. In conclusion, we propose that phosphorylation of Cx43 Ser373 results in 14-3-3 binding which promotes and maintains phosphorylation of Cx43 Ser368 and the subsequent internalization of gap junction channels. These results identify for the first time a specific role for 14-3-3 proteins in regulation of Cx43 internalization during acute ischemia and contribute to the development of therapies aimed at preserving or enhancing gap junction coupling in the heart.

CD99 suppresses osteosarcoma cell migration through inhibition of ROCK2 activity

CD99, a transmembrane protein encoded by MIC2 gene is involved in multiple cellular events including cell adhesion and migration, apoptosis, cell differentiation and regulation of protein trafficking either in physiological or pathological conditions. In osteosarcoma, CD99 is expressed at low levels and functions as a tumour suppressor. The full-length protein (CD99wt) and the short-form harbouring a deletion in the intracytoplasmic domain (CD99sh) have been associated with distinct functional outcomes with respect to tumour malignancy. In this study, we especially evaluated modulation of cell-cell contacts, reorganisation of the actin cytoskeleton and modulation of signalling pathways by comparing osteosarcoma cells characterised by different metastasis capabilities and CD99 expression, to identify molecular mechanisms responsible for metastasis. Our data indicate that forced expression of CD99wt induces recruitment of N-cadherin and β-catenin to adherens junctions. In addition, transfection of CD99wt inhibits the expression of several molecules crucial to the remodelling of the actin cytoskeleton, such as ACTR2, ARPC1A, Rho-associated, coiled-coil containing protein kinase 2 (ROCK2) as well as ezrin, an ezrin/radixin/moesin family member that has been clearly associated with tumour progression and metastatic spread in osteosarcoma. Functional studies point to ROCK2 as a crucial intracellular mediator regulating osteosarcoma migration. By maintaining c-Src in an inactive conformation, CD99wt inhibits ROCK2 signalling and this leads to ezrin decrease at cell membrane while N-cadherin and β-catenin translocate to the plasma membrane and function as main molecular bridges for actin cytoskeleton. Taken together, we propose that the re-expression of CD99wt, which is generally present in osteoblasts but lost in osteosarcoma, through inhibition of c-Src and ROCK2 activity, manages to increase contact strength and reactivate stop-migration signals that counteract the otherwise dominant promigratory action of ezrin in osteosarcoma cells.

Aldosterone-induced ENaC and basal Na+/K+-ATPase trafficking via protein kinase D1-phosphatidylinositol 4-kinaseIIIβ trans Golgi signalling in M1 cortical collecting duct cells

Aldosterone regulates Na+ transport in the distal nephron through multiple mechanisms that include the transcriptional control of epithelial sodium channel (ENaC) and Na+/K+-ATPase subunits. Aldosterone also induces the rapid phosphorylation of Protein Kinase D1 (PKD1). PKD isoforms regulate protein trafficking, by the control of vesicle fission from the trans Golgi network (TGN) through activation of phosphatidylinositol 4-kinaseIIIβ (PI4KIIIβ). We report rapid ENaCγ translocation to the plasma membrane after 30min aldosterone treatment in polarized M1 cortical collecting duct cells, which was significantly impaired in PKD1 shRNA-mediated knockdown cells. In PKD1-deficient cells, the ouabain-sensitive current was significantly reduced and Na+/K+-ATPase α and β subunits showed aberrant localization. PKD1 and PI4KIIIβ localize to the TGN, and aldosterone induced an interaction between PKD1 and PI4KIIIβ following aldosterone treatment. This study reveals a novel mechanism for rapid regulation of ENaC and the Na+/K+-ATPase, via directed trafficking through PKD1-PI4KIIIβ signalling at the level of the TGN.

Proteomic analysis of protein palmitoylation in adipocytes

Protein palmitoylation, by modulating the dynamic interaction between protein and cellular membrane, is involved in a wide range of biological processes, including protein trafficking, sorting, sub-membrane partitioning, protein-protein interaction and cell signaling. To explore the role of protein palmitoylation in adipocytes, we have performed proteomic analysis of palmitoylated proteins in adipose tissue and 3T3-L1 adipocytes and identified more than 800 putative palmitoylated proteins. These include various transporters, enzymes required for lipid and glucose metabolism, regulators of protein trafficking and signaling molecules. Of note, key proteins involved in membrane translocation of the glucose-transporter Glut4 including IRAP, Munc18c, AS160 and Glut4, and signaling proteins in the JAK-STAT pathway including JAK1 and 2, STAT1, 3 and 5A and SHP2 in JAK-STAT, were palmitoylated in cultured adipocytes and primary adipose tissue. Further characterization showed that palmitoylation of Glut4 and IRAP was altered in obesity, and palmitoylation of JAK1 played a regulatory role in JAK1 intracellular localization. Overall, our studies provide evidence to suggest a novel and potentially regulatory role for protein palmitoylation in adipocyte function.

Pediatric Acute Myeloid Leukemia with t(8;16)(p11;p13): A Distinct Clinical and Biological Entity. Results of a Collaborative Study by the International Berlin-Frankfurt-Münster AML Study Group.

AbstractAbstract 2516▪▪This icon denotes a clinically relevant abstract Introduction:In pediatric acute myeloid leukemia (AML) cytogenetic abnormalities are important for prognosis and treatment stratification. Some recurring cytogenetic abnormalities occur so rarely that large collaborative studies are required to define their prognostic impact. t(8;16)(p11;p13) is an aberration reported in <1% of pediatric AML patients. Recently, Haferlach et al. (Leukemia, 2009) reported that t(8;16)(p11;p13) represents a distinct clinical and biological subgroup in adult AML, with dismal clinical outcome (median overall survival (OS) 4.7 months). In pediatric AML case reports are available, but studies of larger series are lacking. Methods:We collected data on patients with t(8;16)(p11;p13) (n=39) from 18 collaborative study groups belonging to the International Berlin-Frankfurt-Münster AML-Committee. Data collection included clinical characteristics, karyotype, morphology and immunophenotype, which were centrally reviewed by the co-authors. In addition, the literature was screened for reports on pediatric t(8;16)(p11;p13), which found an additional 23 cases. Results:A total of 62 pediatric AML patients with t(8;16)(p11;p13) were identified, diagnosed between 1978–2010, including 2 secondary AML cases. They had a median age of 1.2 years (range 0–17 years) and 56% of the patients were younger than 2 years old. Median white blood cell count was 21.3 × 109/l (n= 59, range 1.1–173 × 109/l). FAB type was M4 or M5 in 95% of the cases and immunophenotypes showed monocytic differentiation. Erythrophagocytosis (23/33, 70%), leukemia cutis (21/36, 58%) and disseminated intravascular coagulation (DIC, 15/38, 39%) occurred frequently. At initial diagnosis, 35/57 (61%) of the patients showed t(8;16)(p11;p13) as the sole aberration (5 incomplete karyotypes were excluded). In the remaining 22 patients, no recurrent additional cytogenetic aberrations were found. In patients diagnosed after January 1st1993, and treated with chemotherapy at initial diagnosis (n= 34), the CR rate was 85% (29/34), 5-year OS was 59% (±9%), event free survival (EFS) was 57% (±9%) and the cumulative incidence of relapse (CIR) was 28% (±8%). The median follow-up time of survivors was 4 years. Outcome was is comparable to a reference cohort of AML-BFM patients (n= 543) treated in the same era, with OS of 62% (±2%), EFS of 50% (±2%) and CIR of 42% (±2%). Most striking was that in 7 neonates (<1 month of age) diagnosed with AML and t(8;16)(p11;p13), spontaneous complete remission was achieved. Three of these infants survived without recurrence of disease (follow-up 0.9–4.0 years). For those who relapsed (0.7–4 years after initial diagnosis), chemotherapy treatment was successful in 2/4 patients.Gene expression data, using the Affymetrix Human Genome U133 plus 2.0 array, were available from 297 pediatric AML samples, of which 8 were t(8;16)(p11;p13). The gene expression signature of t(8;16)(p11;p13) AML clustered close to but distinct from MLL-rearranged AML using unsupervised analysis. Highly expressed genes included HOXA11, HOXA10, RET, PERP and GGA2, indicating a pathway in common with MLL-rearranged AML with distinct features. PERP is a direct target gene of p53 and acts as an effector of apoptosis, GGA2 codes for a protein that regulates protein trafficking within the cell. PERP and GGA2 knockdown in high expressing cell lines did not result in significant changes in cell proliferation or apoptosis, thus their function in leukemic blasts remains unknown.DNA methylation profiling was performed using Agilent 244K Human CpG Island arrays on 167 pediatric AML samples of which 12 had t(8;16)(p11;p13). Differential methylation of the TRIM59 promoter, an oncogene described in prostate cancer located on chromosome 3, was confirmed by pyrosequencing, with subsequent mRNA overexpression, validated by RT-qPCR. Conclusion:This study shows the distinct nature of t(8;16)(p11;p13) pediatric AML with remarkable clinical features such as high percentage of leukemia cutis and DIC. The 5-year OS of this pediatric cohort was intermediate (59%), and therefore different from the dismal prognosis reported in adult cases. In neonatal t(8;16)(p11;p13) AML, spontaneous remissions were reported. Profiling studies have shed light on potential oncogenic mechanisms involved in AML with t(8;16)(p11;p13), including HOX-gene upregulation similar to MLL-rearranged AML. Disclosures:No relevant conflicts of interest to declare.

Sorting Signals That Mediate Traffic of Chitin Synthase III between the TGN/Endosomes and to the Plasma Membrane in Yeast

Traffic of the integral yeast membrane protein chitin synthase III (Chs3p) from the trans-Golgi network (TGN) to the cell surface and to and from the early endosomes (EE) requires active protein sorting decoded by a number of protein coats. Here we define overlapping signals on Chs3p responsible for sorting in both exocytic and intracellular pathways by the coats exomer and AP-1, respectively. Residues 19DEESLL24, near the N-terminal cytoplasmically-exposed domain, comprise both an exocytic di-acidic signal and an intracellular di-leucine signal. Additionally we show that the AP-3 complex is required for the intracellular retention of Chs3p. Finally, residues R374 and W391, comprise another signal responsible for an exomer-independent alternative pathway that conveys Chs3p to the cell surface. These results establish a role for active protein sorting at the trans-Golgi en route to the plasma membrane (PM) and suggest a possible mechanism to regulate protein trafficking.

Distinct regional and subcellular localization of the actin‐binding protein filamin a in the mature rat brain

Filamin A (FLNa) is an actin-binding protein that regulates cell motility, adhesion, and elasticity by cross-linking filamentous actin. Additional roles of FLNa include regulation of protein trafficking and surface expression. Although the functions of FLNa during brain development are well studied, little is known on its expression, distribution, and function in the adult brain. Here we characterize in detail the neuroanatomical distribution and subcellular localization of FLNa in the mature rat brain, by using two antisera directed against epitopes at either the N' or the C' terminus of the protein, further validated by mRNA expression. FLNa was widely and selectively expressed throughout the brain, and the intensity of immunoreactivity was region dependent. The most intensely FLNa-labeled neurons were found in discrete neuronal systems, including basal forebrain structures, anterior nuclear group of thalamus, and hypothalamic parvocellular neurons. Pyramidal neurons in neocortex and hippocampus and magnocellular cells in basolateral amygdaloid nucleus were also intensely FLNa immunoreactive, and strong FLNa labeling was evident in the pontine and medullary raphe nuclei and in sensory and spinal trigeminal nuclei. The subcellular localization of FLNa was evaluated in situ as well as in primary hippocampal neurons. Punctate expression was found in somata and along the dendritic shaft, but FLNa was not detected in dendritic spines. These subcellular distribution patterns were recapitulated in hippocampal and neocortical pyramidal neurons in vivo. The characterization of the expression and subcellular localization of FLNa may provide new clues to the functional roles of this cytoskeletal protein in the adult brain.

BEX5/RabA1b regulates trans-Golgi network-to-plasma membrane protein trafficking in Arabidopsis.

Constitutive endocytic recycling is a crucial mechanism allowing regulation of the activity of proteins at the plasma membrane and for rapid changes in their localization, as demonstrated in plants for PIN-FORMED (PIN) proteins, the auxin transporters. To identify novel molecular components of endocytic recycling, mainly exocytosis, we designed a PIN1-green fluorescent protein fluorescence imaging-based forward genetic screen for Arabidopsis thaliana mutants that showed increased intracellular accumulation of cargos in response to the trafficking inhibitor brefeldin A (BFA). We identified bex5 (for BFA-visualized exocytic trafficking defective), a novel dominant mutant carrying a missense mutation that disrupts a conserved sequence motif of the small GTPase, RAS GENES FROM RAT BRAINA1b. bex5 displays defects such as enhanced protein accumulation in abnormal BFA compartments, aberrant endosomes, and defective exocytosis and transcytosis. BEX5/RabA1b localizes to trans-Golgi network/early endosomes (TGN/EE) and acts on distinct trafficking processes like those regulated by GTP exchange factors on ADP-ribosylation factors GNOM-LIKE1 and HOPM INTERACTOR7/BFA-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1, which regulate trafficking at the Golgi apparatus and TGN/EE, respectively. All together, this study identifies Arabidopsis BEX5/RabA1b as a novel regulator of protein trafficking from a TGN/EE compartment to the plasma membrane.

Sorting nexin 12 interacts with BACE1 and regulates BACE1-mediated APP processing

Backgroundβ-site APP cleaving enzyme 1 (BACE1) cleaves β-amyloid precursor protein (APP) to initiate the production of β-amyloid (Aβ), the prime culprit in Alzheimer’s disease (AD). Dysregulation of the intracellular trafficking of BACE1 may affect Aβ generation, contributing to AD pathology. In this study, we investigated whether BACE1 trafficking and BACE1-mediated APP processing/Aβ generation are affected by sorting nexin 12 (SNX12), a member of the sorting nexin (SNX) family that is involved in protein trafficking regulation.ResultsHerein, we find that SNX12 is widely expressed in brain tissues and is mainly localized in the early endosomes. Overexpression of SNX12 does not affect the steady-state levels of APP, BACE1 or γ-secretase components, but dramatically reduces the levels of Aβ, soluble APPβ and APP β-carboxyl terminal fragments. Downregulation of SNX12 has the opposite effects. Modulation of SNX12 levels does not affect γ-secretase activity or in vitro β-secretase activity. Further studies reveal that SNX12 interacts with BACE1 and downregulation of SNX12 accelerates BACE1 endocytosis and decreases steady-state level of cell surface BACE1. Finally, we find that the SNX12 protein level is dramatically decreased in the brain of AD patients as compared to that of controls.ConclusionThis study demonstrates that SNX12 can regulate the endocytosis of BACE1 through their interaction, thereby affecting β-processing of APP for Aβ production. The reduced level of SNX12 in AD brains suggests that an alteration of SNX12 may contribute to AD pathology. Therefore, inhibition of BACE1-mediated β-processing of APP by regulating SNX12 might serve as an alternative strategy in developing an AD intervention.

Acute Activation of β2-Adrenergic Receptor Regulates Focal Adhesions through βArrestin2- and p115RhoGEF Protein-mediated Activation of RhoA

β(2)-Adrenergic receptors (β(2)ARs) regulate cellular functions through G protein-transduced and βArrestin-transduced signals. β(2)ARs have been shown to regulate cancer cell migration, but the underlying mechanisms are not well understood. Here, we report that β(2)AR regulates formation of focal adhesions, whose dynamic remodeling is critical for directed cell migration. β(2)ARs induce activation of RhoA, which is dependent on βArrestin2 but not G(s). βArrestin2 forms a complex with p115RhoGEF, a guanine nucleotide exchange factor for RhoA that is well known to be activated by G(12/13)-coupled receptors. Our results show that βArrestin2 forms a complex with p115RhoGEF in the cytosol in resting cells. Upon β(2)AR activation, both βArrestin2 and p115RhoGEF translocate to the plasma membrane, with concomitant activation of RhoA and formation of focal adhesions and stress fibers. Activation of RhoA and focal adhesion remodeling may explain, at least in part, the role of β(2)ARs in cell migration. These results suggest that βArrestin2 may serve as a convergence point for non-G(12/13) and non-G(q) protein-coupled receptors to activate RhoA.

Ouabain-stimulated trafficking regulation of the Na/K-ATPase and NHE3 in renal proximal tubule cells

We have demonstrated that ouabain regulates protein trafficking of the Na/K-ATPase α1 subunit and NHE3 (Na/H exchanger, isoform 3) via ouabain-activated Na/K-ATPase signaling in porcine LLC-PK1 cells. To investigate whether this mechanism is species-specific, ouabain-induced regulation of the α1 subunit and NHE3 as well as transcellular (22)Na(+) transport were compared in three renal proximal tubular cell lines (human HK-2, porcine LLC-PK1, and AAC-19 originated from LLC-PK1 in which the pig α1 was replaced by ouabain-resistant rat α1). Ouabain-induced inhibition of transcellular (22)Na(+) transport is due to an ouabain-induced redistribution of the α1 subunit and NHE3. In LLC-PK1 cells, ouabain also inhibited the endocytic recycling of internalized NHE3, but has no significant effect on recycling of endocytosed α1 subunit. These data indicated that the ouabain-induced redistribution of the α1 subunit and NHE3 is not a species-specific phenomenon, and ouabain-activated Na/K-ATPase signaling influences NHE3 regulation.

An antiapoptotic role of sorting nexin 7 is required for liver development in zebrafish

Sorting nexin (SNX) family proteins are best characterized for their abilities to regulate protein trafficking during processes such as endocytosis of membrane receptors, endosomal sorting, and protein degradation, but their in vivo functions remain largely unknown. We started to investigate the biological functions of SNXs using the zebrafish model. In this study, we demonstrated that SNX7 was essential for embryonic liver development. Hepatoblasts were specified normally, and the proliferation of these cells was not affected when SNX7 was knocked down by gene-specific morpholinos; however, they underwent massive apoptosis during the early budding stage. SNX7 mainly regulated the survival of cells in the embryonic liver and did not affect the viability of cells in other endoderm-derived organs. We further demonstrated that down-regulation of SNX7 by short interfering RNAs induced apoptosis in cell culture. At the molecular level, the cellular FLICE-like inhibitory protein (c-FLIP)/caspase 8 pathway was activated when SNX7 was down-regulated. Furthermore, overexpression of c-FLIP(S) was able to rescue the SNX7 knockdown-induced liver defect. SNX7 is a liver-enriched antiapoptotic protein that is indispensable for the survival of hepatoblasts during zebrafish early embryogenesis.

Mo1809 Enteropathogenic E. coli Non-Lee Encoded Effectors NleH1 and NleH2 Attenuate Activation of Mitogen-Activated Protein Kinases

BACKGROUND: Regulation of the brush border (BB) Na+/H+ exchanger, NHE3, accounts for most of the recognized digestion-related changes in neutral Na+ absorption, as well as most of the inhibition of Na+ absorption that occurs in diarrheal diseases. NHE3 regulation usually occurs by changes in the rates of endocytosis and/or exocytosis. NHE3 traffics between the plasma membrane and recycling endosomes under basal and regulated conditions. Constitutive NHE3 endocytosis occurs through a clathrin-dependent process. In intestinal epithelial cells, carbachol (CCH) elevation of intracellular Ca2+ ([Ca]i) decreases NHE3 activity and stimulates endocytosis as early as one minute; however, the mechanism involved in calcium-mediated endocytosis of NHE3 remains to be determined. Furthermore, a pool of BB NHE3 resides in lipid rafts which contributes to basal, but not cAMP-mediated, NHE3 trafficking, suggesting an alternative mechanism exists for NHE3 endocytosis. Recently, the Rho GTPase, Cdc42, was demonstrated to play an integral role in cholesterol-sensitive, clathrin-independent endocytosis. However, it has not been determined whether [Ca]imediated NHE3 endocytosis occurs through a clathrin-independent, lipid raft-dependent pathway involving Cdc42. PURPOSE: Therefore, the current study was designed to test the hypothesis that (1) inhibition of clathrin-mediated endocytosis (CME) prevents constitutive, but not CCH-mediated, endocytosis of NHE3, and (2) CCH-mediated endocytosis of NHE3 occurs through a lipid raft, activated Cdc42-dependent pathway that does not involve clathrin. METHODS: 12 day post-confluent WT, Rab5 and Cdc42 shRNA knockdown (KD) Caco-2/BBe cells grown on filters were transiently infected with a 3HA-NHE3 adenovirus construct and studied 48 hours later. NHE3 activity was measured by fluorimetry using the pH-sensitive dye, BCECF, in cells treated with vehicle or 10μM CCH to elevate [Ca]i as well as 10mM methyl-β-cyclodextrin (MβCD) to disrupt lipid rafts. CME was blocked by chlorpromazine (CPZ) or K+ depletion. Cdc42 activity was determined in total cell lysates obtained (with 0.1% Triton X-100) from cells treated with vehicle or CCH at various time points. RESULTS: Basal NHE3 activity, but not CCH-inhibition, was increased in WT cells treated with CPZ or K+ depletion as well as in Rab5 KD cells supporting that constitutive NHE3 endocytosis is regulated by CME. CCH rapidly stimulated Cdc42 activity as early as 1min. CCH-mediated inhibition of NHE3 activity was abolished in WT cells treated with MβCD as well as in Cdc42 KD cells. CONCLUSIONS: CCH-mediated inhibition of NHE3 activity is not dependent on clathrin or Rab5 and is regulated by lipid rafts and requires Cdc42. Elevation of [Ca]i may specifically stimulate a novel clathrin-independent endocytic process to regulate protein trafficking in intestinal epithelial cells.

Mo1810 Modulation of the Expression of Sar1b, a Key Regulator of COPII Transport Vesicles, in Intestinal Caco-2/15 Cells

BACKGROUND: Regulation of the brush border (BB) Na+/H+ exchanger, NHE3, accounts for most of the recognized digestion-related changes in neutral Na+ absorption, as well as most of the inhibition of Na+ absorption that occurs in diarrheal diseases. NHE3 regulation usually occurs by changes in the rates of endocytosis and/or exocytosis. NHE3 traffics between the plasma membrane and recycling endosomes under basal and regulated conditions. Constitutive NHE3 endocytosis occurs through a clathrin-dependent process. In intestinal epithelial cells, carbachol (CCH) elevation of intracellular Ca2+ ([Ca]i) decreases NHE3 activity and stimulates endocytosis as early as one minute; however, the mechanism involved in calcium-mediated endocytosis of NHE3 remains to be determined. Furthermore, a pool of BB NHE3 resides in lipid rafts which contributes to basal, but not cAMP-mediated, NHE3 trafficking, suggesting an alternative mechanism exists for NHE3 endocytosis. Recently, the Rho GTPase, Cdc42, was demonstrated to play an integral role in cholesterol-sensitive, clathrin-independent endocytosis. However, it has not been determined whether [Ca]imediated NHE3 endocytosis occurs through a clathrin-independent, lipid raft-dependent pathway involving Cdc42. PURPOSE: Therefore, the current study was designed to test the hypothesis that (1) inhibition of clathrin-mediated endocytosis (CME) prevents constitutive, but not CCH-mediated, endocytosis of NHE3, and (2) CCH-mediated endocytosis of NHE3 occurs through a lipid raft, activated Cdc42-dependent pathway that does not involve clathrin. METHODS: 12 day post-confluent WT, Rab5 and Cdc42 shRNA knockdown (KD) Caco-2/BBe cells grown on filters were transiently infected with a 3HA-NHE3 adenovirus construct and studied 48 hours later. NHE3 activity was measured by fluorimetry using the pH-sensitive dye, BCECF, in cells treated with vehicle or 10μM CCH to elevate [Ca]i as well as 10mM methyl-β-cyclodextrin (MβCD) to disrupt lipid rafts. CME was blocked by chlorpromazine (CPZ) or K+ depletion. Cdc42 activity was determined in total cell lysates obtained (with 0.1% Triton X-100) from cells treated with vehicle or CCH at various time points. RESULTS: Basal NHE3 activity, but not CCH-inhibition, was increased in WT cells treated with CPZ or K+ depletion as well as in Rab5 KD cells supporting that constitutive NHE3 endocytosis is regulated by CME. CCH rapidly stimulated Cdc42 activity as early as 1min. CCH-mediated inhibition of NHE3 activity was abolished in WT cells treated with MβCD as well as in Cdc42 KD cells. CONCLUSIONS: CCH-mediated inhibition of NHE3 activity is not dependent on clathrin or Rab5 and is regulated by lipid rafts and requires Cdc42. Elevation of [Ca]i may specifically stimulate a novel clathrin-independent endocytic process to regulate protein trafficking in intestinal epithelial cells.

Abstract 1199: Sorting nexin 2 (SNX2) controls drug sensitivity to molecular targeted anticancer agents through membrane trafficking of c-Met protein in cancer cell

Abstract Various molecular targeted anticancer drugs are often developed by targeting activated growth factor receptors that are expressed on plasma membrane of cancer cells. Expression of such active receptors on the cell surface is essential to limit the drug sensitivity to molecular targeted drugs in lung cancer cells and others. The sorting nexin (SNX) family is a diverse group of cytoplasmic and membrane-associated proteins that are involved in membrane-trafficking steps within the endocytotic network. Human SNX family proteins comprise about 34 proteins with wide range of biological functions, which play specialized and/or generalized roles in the regulation of protein trafficking, including endosomal trafficking of membrane receptors and transporters. SNX1 and SNX2 are components of the mammalian retromer complex. In this study, we show that the small-interfering RNA (siRNA)-mediated knockdown of SNX2 decreased the cell-surface localization of c-Met, but not that of EGFR, resulting in degradation of the c-Met protein. Knockdown of SNX2 markedly altered sensitivity to anticancer drugs targeting c-Met and EGFR through promotion of compensatory activation of the EGFR pathway in lung cancer cells. Furthermore, SNX2 knockdown also specifically decreased c-Met expression, and altered drug sensitivity to c-Met-targeted drug in gastric cancer cells. These findings suggest that SNX2 is a key protein that regulates c-Met-driven growth-signalling pathways, affecting the therapeutic efficacy of growth factor receptor-targeted drugs in lung and gastric cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1199. doi:1538-7445.AM2012-1199

The GTPase ARFRP1 controls assembly of apoA1 to and lipidation of chylomicron in the Golgi of intestinal enterocyte

Intestinal lipid metabolism is comprised of multi‐steps of vesicle trafficking. ADP‐ribosylation factor‐related protein 1 (ARFRP1) regulates protein trafficking between intracellular organelles. To study the role of ARFRP1 in intestinal lipid metabolism, Arfrp1 was deleted in mouse intestine (Arfrp1vil−/−), or depleted by siRNA transfection in Caco‐2 cells. Lipid absorption and lipoprotein production were examined. Decreased lipid absorption was detected in oral fat tolerance test in Arfrp1vil−/− mice. However, the apolipoprotein (Apo) B48 and ApoA4 in the plasma were higher, whereas the level of ApoA1 was markedly lower. Fractionation of the plasma showed that the chylomicron fractions of Arfrp1vil−/− mice contain lower triglyceride level, indicating that the Arfrp1vil−/− enterocytes produce a large number of chylomicron particles that are smaller in size containing less fat. Caco‐2 cells demonstrated that the defect in Arfrp1 expression caused decreased lipid release, but normal lipid uptake. In conclusion, ARFRP1 plays important roles in chylomicron lipidation in the Golgi and the secretion of ApoA1 in the intestine.Grant Funding Source : Deutsche Forschungsgemeinschaft (Schu 750/5‐2; Schu 750/5‐3; GRK 1459; SFB958), Alexander von Humboldt Foundation