Membrane trafficking in morphogenesis and planar polarity

Our understanding of how membrane trafficking pathways function to direct morphogenetic movements and the planar polarization of developing tissues is a new and emerging field. While a central focus of developmental biology has been on how protein asymmetries and cytoskeletal force generation direct cell shaping, the role of membrane trafficking in these processes has been less clear. Here, we review recent advances in Drosophila and vertebrate systems in our understanding of how trafficking events are coordinated with planar cytoskeletal function to drive lasting changes in cell and tissue topologies. We additionally explore the function of trafficking pathways in guiding the complex interactions that initiate and maintain core PCP (planar cell polarity) asymmetries and drive the generation of systematically oriented cellular projections during development.      

Expanded and fat regulate growth and differentiation in the Drosophila eye through multiple signaling pathways

Mutations in the expanded gene act as hyperplastic tumor suppressors, interfere with cell competition and elevate Dpp signaling. Unlike Dpp overexpression, ex causes few patterning defects. Our data suggest that patterning effects are partly masked by antagonistic roles of other signaling pathways that are also activated. ex causes proliferation of cells in the posterior eye disc that are normally postmitotic. ex mutations elevate Wg signaling, but Dpp signaling antagonizes patterning effects of Wg. By contrast, if Dpp signaling is blocked in ex mutant cells, the elevated Wg signaling preserves an immature developmental state and prevents retinal differentiation. An effect of ex mutations on vesicle transport is suggested by evidence for altered sterol distribution. Mutations in ft show effects on proliferation, Wg signaling and sterols very similar to those of ex mutations. During disc growth, ex was largely epistatic to ft, and the Warts pathway mutation hippo largely epistatic to ex. Our data suggest that ft and ex act partially through the Warts pathway.     

Rab‐mediated trafficking in the secondary cells of Drosophila male accessory glands and its role in fecundity

The male seminal fluid contains factors that affect female post‐mating behavior and physiology. In Drosophila, most of these factors are secreted by the two epithelial cell types that make up the male accessory gland: the main and secondary cells. Although secondary cells represent only ~4% of the cells of the accessory gland, their contribution to the male seminal fluid is essential for sustaining the female post‐mating response. To better understand the function of the secondary cells, we investigated their molecular organization, particularly with respect to the intracellular membrane transport machinery. We determined that large vacuole‐like structures found in the secondary cells are trafficking hubs labeled by Rab6, 7, 11 and 19. Furthermore, these organelles require Rab6 for their formation and many are essential in the process of creating the long‐term postmating behavior of females. In order to better serve the intracellular membrane and protein trafficking communities, we have created a searchable, online, open‐access imaging resource to display our complete findings regarding Rab localization in the accessory gland.      

MUC1 intra-cellular trafficking is clathrin, dynamin, and rab5 dependent

MUC1, a transmembrane glycoprotein, is abnormally over-expressed in most human adenocarcinomas. MUC1 association with cytoplasmic cell signal regulators and nuclear accumulation are important for its tumor related activities. Little is known about how MUC1 translocates from the cell membrane to the cytoplasm. In this study, live cell imaging was used to study MUC1 intracellular trafficking. The interaction between EGFR and MUC1 was mapped by FRET analysis and EGF stimulated MUC1 endocytosis was observed directly through live cell imaging. MUC1-CT endocytosis was clathrin and dynamin dependent. Rab5 over-expression resulted in decreased cell membrane localization of MUC1, with accumulation of MUC1 endocytic vesicles in the peri-nuclear region. Conversely, over-expression of a Rab5 dominant negative mutant (S34N) resulted in redistribution of MUC1 from the peri-nuclear region to the cytoplasm. Collectively, these results indicated that MUC1 intra-cellular trafficking occurs through a regulated process that was stimulated by direct EGFR and MUC1 interaction, mediated by clathrin coated pits that were dynamin dependent and regulated by Rab5.     

Differential requirements of Rab5 and Rab7 for endocytosis of influenza and other enveloped viruses

Enveloped viruses often enter cells via endocytosis; however, specific endocytic trafficking pathway(s) for many viruses have not been determined. Here we demonstrate, through the use of dominant-negative Rab5 and Rab7, that influenza virus (Influenza A/WSN/33 (H1N1) and A/X-31 (H3N2)) requires both early and late endosomes for entry and subsequent infection in HeLa cells. Time-course experiments, monitoring viral ribonucleoprotein colocalization with endosomal markers, indicated that influenza exhibits a conventional endocytic uptake pattern – reaching early endosomes after approximately 10 min, and late endosomes after 40 min. Detection with conformation-specific hemagglutinin antibodies indicated that hemagglutinin did not reach a fusion-competent form until the virus had trafficked beyond early endosomes. We also examined two other enveloped viruses that are also pH-dependent for entry – Semliki Forest virus and vesicular stomatitis virus. In contrast to influenza virus, infection with both Semliki Forest virus and vesicular stomatitis virus was inhibited only by the expression of dominant negative Rab5 and not by dominant negative Rab7, indicating an independence of late endosome function for infection by these viruses. As a whole, these data provide a definitive characterization of influenza virus endocytic trafficking and show differential requirements for endocytic trafficking between pH-dependent enveloped viruses .     

Evolutionary conservancy of the endocytic and trafficking machinery in the unicellular eukaryote Paramecium

Molecular search for the homologues of the mammalian proteins in the unicellular eukaryote Paramecium involved in endocytosis and membrane trafficking is discussed. We cloned and sequenced the gene fragments encoding the following components participating in endosome formation, sorting and maturation of the proprotein precursors, respectively, dynamin 2, Rab7 and furin. There is a proof that all these genes are expressed in this unicellular organism. The function of the identified immunoanalogues of the above described components of Paramecium endocytic machinery as well as a high degree of sequence homology to the respective human counterparts points to the evolutionary conservancy of these pathways.     

FIP5 phosphorylation during mitosis regulates apical trafficking and lumenogenesis

Apical lumen formation is a key step during epithelial morphogenesis. The establishment of the apical lumen is a complex process that involves coordinated changes in plasma membrane composition, endocytic transport, and cytoskeleton organization. These changes are accomplished, at least in part, by the targeting and fusion of Rab11/FIP5‐containing apical endosomes with the apical membrane initiation site (AMIS). Although AMIS formation and polarized transport of Rab11/FIP5‐containing endosomes are crucial for the formation of a single apical lumen, the spatiotemporal regulation of this process remains poorly understood. Here, we demonstrate that the formation of the midbody during cytokinesis is a symmetry‐breaking event that establishes the location of the AMIS. The interaction of FIP5 with SNX18, which is required for the formation of apical endocytic carriers, is inhibited by GSK‐3 phosphorylation at FIP5‐T276. Importantly, we show that FIP5‐T276 phosphorylation occurs specifically during metaphase and anaphase, to ensure the fidelity and timing of FIP5‐endosome targeting to the AMIS during apical lumen formation.     

Dependence on different Rab GTPases for the trafficking of CXCR4 and CCR5 homo or heterodimers between the endoplasmic reticulum and plasma membrane in Jurkat cells

Much is known about G protein coupled receptor trafficking and internalization following agonist stimulation. However, much less is known about outward trafficking of receptors from synthesis in the endoplasmic reticulum to the plasma membrane, or the role that trafficking might play in the assembly of receptor signaling complexes, important for targeting, specificity, and rapidity of subsequent signaling events. Up to now, very little is understood about receptor hetero-oligomers other than the fact that their assembly is done rapidly after biosynthesis. In our study we use bimolecular fluorescence complementation to selectively follow receptor dimers when expressed in Jurkat cells in order to clarify the trafficking itinerary those receptors follow to reach the plasma membrane and the resulting effect on signal transduction. CXCR4 and CCR5, previously shown to form both homo and hetero-oligomers, were used as our model to understand the specificities of trafficking along the anterograde pathway. The CXCR4 homodimer relies on Rabs2, 6 and 8 for anterograde transport regardless of the presence of endogenous CD4. The CCR5 homodimer relies on Rabs1 and 11 when CD4 is absent, but Rabs1 and 8 when CD4 was present. Interestingly, similar to the CCR5 homodimer, the CXCR4-CCR5 heterodimer relied on Rabs1 and 11 but also required Rab2 when CD4 was absent, and only Rab 1 when CD4 was present. Our results demonstrate that, although the receptors composing the heterodimeric complex are the same as in the homodimeric ones, the heterodimer traffics and signals differently than each homodimer. Our study demonstrates the importance of considering the receptor heterodimers as distinct signaling entities that should be carefully and individually characterized.     

Cholesterol changes in Alzheimer's disease: methods of analysis and impact on the formation of enlarged endosomes

An increasing number of results implicating cholesterol metabolism in the pathophysiology of Alzheimer's disease (AD) suggest cholesterol as a target for treatment. Research in genetics, pathology, epidemiology, biochemistry, and cell biology, as well as in animal models, suggests that cholesterol, its transporter in the brain, apolipoprotein E, amyloid precursor protein, and amyloid-β all interact in AD pathogenesis. Surprisingly, key questions remain unanswered due to the lack of sensitive and specific methods for assessing cholesterol levels in the brain at subcellular resolution. The aims of this review are not only to discuss the various methods for measuring cholesterol and its metabolites and to catalog results obtained from AD patients but also to discuss some new data linking high plasma membrane cholesterol with modifications of the endocytic compartments. These studies are particularly relevant to AD pathology, since enlarged endosomes are believed to be the first morphological change observed in AD brains, in both sporadic cases and Down syndrome.     

The mir‐7 and bag of marbles genes regulate Hedgehog pathway signaling in blood cell progenitors in Drosophila larval lymph glands

Hedgehog (Hh) pathway signaling is crucial for the maintenance of blood cell progenitors in the lymph gland hematopoietic organ present in Drosophila third instar larvae. Previous studies from our lab have likewise shown the importance of the mir‐7 and bag of marbles (bam) genes in maintaining the progenitor state. Thus, we sought to investigate a possible interaction between the Hh pathway and mir‐7/bam in the prohemocyte population within this hematopoietic tissue. Gain of function mir‐7 was able to rescue a blood cell progenitor depletion phenotype caused by Patched (Ptc) inhibition of Hh pathway signaling in these cells. Similarly, expression of a dominant/negative version of Ptc was able to rescue the severe reduction of prohemocytes due to bam loss of function. Furthermore, we demonstrated that Suppressor of fused [Su(fu)], another known inhibitor of Hh signaling, likely serves as a translational repression target of the mir‐7 miRNA. Our results suggest the mir‐7/bam combination regulates the Hh signaling network through repression of Su(fu) to maintain hemocyte progenitors in the larval lymph gland.     

结直肠癌组织RABEX-5、LASS2、HOXB7蛋白的表达及临床意义

摘要 目的：探讨结直肠癌组织Rab鸟嘌呤核苷酸交换因子-5（RABEX-5）、人源长寿保障基因2（LASS2）、同源异型盒基因B7（HOXB7）表达与临床病理特征及预后的关系。方法：选择2013年10月至2015年4月期间在我院接受治疗的105例结直肠癌患者作为研究对象。检测其结直肠癌组织以及癌旁组织中RABEX-5、LASS2、HOXB7表达,分析RABEX-5、LASS2、HOXB7表达与临床病理特征的关系,采用Kaplan-Meier生存曲线分析不同RABEX-5、LASS2、HOXB7表达患者总生存率的差异,Cox比例风险回归分析结直肠癌患者预后的影响因素。结果：与癌旁组织相比,结直肠癌组织中RABEX-5、HOXB7表达阳性率上调,而LASS2阳性率下降（P<0.05）。LASS2表达与TNM分期、浸润深度和淋巴结转移有关（P<0.05）,而RABEX-5、HOXB7表达与TNM分期和淋巴结转移有关（P<0.05）。RABEX-5、HOXB7表达阳性患者的生存率分别低于RABEX-5、HOXB7表达阴性患者,而LASS2表达阳性患者的生存率高于LASS2表达阴性患者（P<0.05）。结直肠癌患者预后的影响因素包括TNM分期Ⅲ期、有淋巴结转移、RABEX-5阳性表达、LASS2阴性表达和HOXB7阳性表达（P<0.05）。结论：在结直肠癌组织中RABEX-5、HOXB7阳性率升高,而LASS2阳性率下降,且与TNM分期以及淋巴结转移密切相关。RABEX-5、LASS2、HOXB7表达与结直肠癌患者的生存和预后联系密切。     

HDAC5-mediated Smad7 silencing through MEF2A is critical for fibroblast activation and hypertrophic scar formation

Transforming growth factor-β (TGF-β) signaling plays a key role in excessive fibrosis. As a class IIa family histone deacetylase (HDAC), HDAC5 shows a close relationship with TGF-β signaling and fibrosis. However, the effect and regulatory mechanism of HDAC5 in hypertrophic scar (HS) formation remain elusive. We show that HDAC5 was overexpressed in HS tissues and depletion of HDAC5 attenuated HS formation in vivo and inhibited fibroblast activation in vitro. HDAC5 knockdown (KD) significantly downregulated TGF-β1 induced Smad2/3 phosphorylation and increased Smad7 expression. Meanwhile, Smad7 KD rescued the Smad2/3 phosphorylation downregulation and scar hyperplasia inhibition mediated by HDAC5 deficiency. Luciferase reporter assays and ChIP-qPCR assays revealed that HDAC5 interacts with myocyte enhancer factor 2A (MEF2A) suppressing MEF2A binding to the Smad7 promoter region, which results in Smad7 promoter activity repression. HDAC4/5 inhibitor, LMK235, significantly alleviated hypertrophic scar formation. Our study provides clues for the development of HDAC5 targeting strategies for the therapy or prophylaxis of fibrotic diseases.     

Inhibition of biofilm formation,quorum sensing activity and molecular docking study of isolated 3, 5, 7-Trihydroxyflavone from Alstonia scholaris leaf against P.aeruginosa

The current study is to evaluate the inhibition of biofilm formation and quorum sensing activity of isolated 3, 5, 7-Trihydroxyflavone (TF) from A.scholaris leaf extract against Pseudomonas aeruginosa. The effects of isolated TF on quorum sensing-regulated virulence factors production such as swimming motility, pyocyanin production, proteolytic, EPS, metabolic assay and inhibition of biofilm formation against P.aeruginosa was evaluated by standard protocols. In addition, the interaction between the isolated TF and active sites of QS- gene (LasI/rhlI, LasR/rhlR, and AHLase) in P.aeruginosa was evaluated by molecular docking studies using AutoDock Tools version 1.5.6. Based on the structural elucidation of the isolated compound was identified as 3, 5, 7-Trihydroxyflavone. Consequently, the isolated TF shows a significant reduction of biofilm formation through the inhibition of QS-dependent phenotypes such as pyocyanin production, proteolytic, swimming motility, EPS activities against P.aeruginosa in a dose-dependent manner. Molecular docking analysis of isolated TF can interfere the signaling [N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL)] molecules in P.aeruginosa by QS genes (LasI, LasR, rhlI, and AHLase) regulation. The isolated TF compound from A.scholaris reveals a greater potential to inhibit biofilm and QS dependent virulence factor production in P.aeruginosa. Docking interaction studies of TF-LasR complex express higher binding affinity than the other QS gene in P.aeruginosa.     

Influence of age on the formation of 5alpha-androstanediol and 7alpha-hydroxy-testosterone by incubated rat testes.

Testes from rats of different ages were indubated with or without tritiated testosterone. The exogenously-added or endogenously-produced testosterone is mainly metabolized to 7alpha-hydroxylated testosterone in adult animals, and to 5alpha-reduced metabolites (especially 5alpha-androstanediol) in immature animals.     

Localization of pectins and arabinogalactan-proteins in lily (Lilium longiflorum L.) pollen tube and style, and their possible roles in pollination

In lily, adhesion of the pollen tube to the transmitting-tract epidermal cells (TTEs) is purported to facilitate the effective movement of the tube cell to the ovary. In this study, we examine the components of the extracellular matrices (ECMs) of the lily pollen tubes and TTEs that may be involved in this adhesion event. Several monoclonal antibodies to plant cell wall components such as esterified pectins, unesterified pectins, and arabinogalactan-proteins (AGPs) were used to localize these molecules in the lily pollen tube and style at both light microscope (LM) and transmission electron microscope (TEM) levels. In addition, (-d-Glc)₃ Yariv reagent which binds to AGPs was used to detect AGPs in the pollen tube and style. At the LM level, unesterified pectins were localized to the entire wall in in-vivo- and in-vitro-grown pollen tubes as well as to the surface of the stylar TTEs. Esterified pectins occurred at the tube tip region (with some differences in extent in in-vivo versus in-vitro tubes) and were evenly distributed in the entire style. At the TEM level, esterified pectins were detected inside pollen tube cell vesicles and unesterified pectins were localized to the pollen tube wall. The in-vivo pollen tubes adhere to each other and can be separated by pectinase treatment. At the LM level, AGP localization occurred in the tube tip of both in-vivo- and in-vitro-grown pollen tubes and, in the case of one AGP probe, on the surface of the TTEs. Another AGP probe localized to every cell of the style except the surface of the TTE. At the TEM level, AGPs were mainly found on the plasma membrane and vesicle membranes of in-vivo-grown pollen tubes as well as on the TTE surface, with some localization to the adhesion zone between pollen tubes and style. (-d-Glc)₃ Yariv reagent bound to the in-vitro-grown pollen tube tip and significantly reduced the growth of both in-vitro- and in-vivo-grown pollen tubes. This led to abnormal expansion of the tube tip and random deposition of callose. These effects could be overcome by removal of (-d-Glc)₃ Yariv reagent which resulted in new tube tip growth zones emerging from the flanks of the arrested tube tip. The possible roles of pectins and AGPs in adhesion during pollination and pollen tube growth are discussed.Abbreviations AGP arabinogalactan-protein - ECM extracellular matrix - Glc glucose - MAbs monoclonal antibodies - LM light microscope - Man mannose - TEM transmission electron microscope - TTE transmitting tract epidermal cell The authors thank Michael Georgiady for assistance with the preparation of material for the TEM immunolocalization, Diana Dang for her help with the pectinase experiment, and Kathleen Eckard for assistance in all aspects of this study. The MAbs were the generous gifts of Dr. J.P. Knox. G.Y. Jauh thanks Dr. E.A. Nothnagel for assistance in making the Yariv reagent and for the gift of the control (-d-Man)₃ Yariv reagent. This work is in partial fulfilment of the dissertation requirements for a PhD degree in Botany and Plant Sciences for G.Y. Jauh at the University of California, Riverside. This work was supported by National Science Foundation grant 91-18554 and an R.E.U. grant to E.M.L.     

Patterning, prestress, and peeling dynamics of myocytes

As typical anchorage-dependent cells myocytes must balance contractility against adequate adhesion. Skeletal myotubes grown as isolated strips from myoblasts on micropatterned glass exhibited spontaneous peeling after one end of the myotube was mechanically detached. Such results indicate the development of a prestress in the cells. To assess this prestress and study the dynamic adhesion strength of single myocytes, the shear stress of fluid aspirated into a large-bore micropipette was then used to forcibly peel myotubes. The velocity at which cells peeled from the surface, V(peel), was measured as a continuously increasing function of the imposed tension, T(peel), which ranges from approximately 0 to 50 nN/ micro m. For each cell, peeling proved highly heterogeneous, with V(peel) fluctuating between 0 micro m/s ( approximately 80% of time) and approximately 10 micro m/s. Parallel studies of smooth muscle cells expressing GFP-paxillin also exhibited a discontinuous peeling in which focal adhesions fractured above sites of strong attachment (when pressure peeled using a small-bore pipette). The peeling approaches described here lend insight into the contractile-adhesion balance and can be used to study the real-time dynamics of stressed adhesions through both physical detection and the use of GFP markers; the methods should prove useful in comparing normal versus dystrophic muscle cells.