The role of Arabidopsis 5PTase13 in root gravitropism through modulation of vesicle trafficking

Inositol polyphosphate 5-phosphatases （5PTases） are enzymes of phosphatidylinositol metabolism that affect various aspects of plant growth and development. Arabidopsis 5PTasel3 regulates auxin homeostasis and hormonerelated cotyledon vein development, and here we demonstrate that its knockout mutant 5pt13 has elevated sensitivity to gravistimulation in root gravitropic responses. The altered responses of 5pt13 mutants to 1-N-naphthylphthalamic acid （an auxin transport inhibitor） indicate that 5PTase13 might be involved in the regulation of auxin transport. Indeed, the auxin efflux carrier PIN2 is expressed more broadly under 5PTasel3 deficiency, and observations of the internalization of the membrane-selective dye FM4-64 reveal altered vesicle trafficking in 5pt13 mutants. Compared with wild-type, 5pt13 mutant seedlings are less sensitive to the inhibition by brefeldin A of vesicle cycling, seedling growth, and the intracellular cycling of the PIN1 and PIN2 proteins. Further, auxin redistribution upon gravitropic stimulation is stimulated under 5PTasel3 deficiency. These results suggest that 5PTasel3 may modulate auxin transport by regulating vesicle trafficking and thereby play a role in root gravitropism.

ANP impairs the dose-dependent stimulatory effect of ANG II or AVP on H^(+)-ATPase subcellular vesicle trafficking

The effect of angiotensin II (ANG II) or arginine vasopressin (AVP) alone or plus atrial natriuretic peptide (ANP) on H+-ATPase subcellular vesicle trafficking was investigated in MDCK cells following intracellular pH (pHi) acidification by exposure to20 mMNH4Cl for 2 min in a Na+-free solution containing Schering 28080, conditions under which H+-AT-Pase is the only cell mechanism for pHi recovery. Using the acridine orange fluorescent probe (5mM) and confocal microscopy, the vesicle movement was quantified by determining, for each experimental group, the mean slope of the line indicating the changes in apical/basolateral fluorescence density ratio over time during the first 5.30 min of the pHi recovery period. Under the control conditions, the mean slope was 0.079 ± 0.0033 min-1 (14) and it increased significantly with ANG II [10-12 and 10-7 M, respectively to 0.322 ± 0.038 min-1 (13) and 0.578 ± 0.061 min-1 (12)] or AVP [10-12 and 10-6 M, respectively to 0.301 ± 0.018 min-1 (12) and 0.687 ± 0.049 min-1 (11)]. However, in presence of ANP (10-6 M, decreases cytosolic free calcium), dimethyl-BAPTA/AM (5 × 10-5 M, chelates intracellular calcium) or colchicine (10-5 M, 2-h preincubation;inhibits microtubule-dependent vesicular trafficking) alone or plus ANG II or AVP the mean slopes were similar to the control values, indicating that such agents blocked the stimulatory effect of ANG II or AVP on vesicle trafficking. The results suggest that the pathway responsible for the increase in cytosolic free calcium and the microtu-bule-dependent vesicular trafficking are involved in this hormonal stimulating effect. Whether cytosolic free calcium reduction represents an important direct mechanism for ANP impairs the dose-dependent stimulatory effect of ANG II or AVP on H+-ATPase subcellular vesicle trafficking, or is a side effect of other signaling pathways which will require additional studies.

Membrane Steroid Binding Protein 1 （MSBP1）Stimulates Tropism by Trafficking and Auxin Regulating Vesicle Redistribution

Overexpression of membrane steroid binding protein 1 （MSBP1） stimulates the root gravitropism and antigravitropism of hypocotyl, which is mainly due to the enhanced auxin redistribution in the bending regions of hypocotyls and root tips. The inhibitory effects by 1-N-naphthylphthalamic acid （NPA）, an inhibitor of polar auxin transport, are suppressed under the MSBP1 overexpression, suggesting the positive effects of MSBP1 on polar auxin transport. Interestingly, sub-cellular localization studies showed that MSBP1 is also localized in endosomes and observations of the membraneselective dye FM4-64 revealed the enhanced vesicle trafficking under MSBP1 overexpression. MSBPl-overexpressing seedlings are less sensitive to brefeldin A （BFA） treatment, whereas the vesicle trafficking was evidently reduced by suppressed MSBP1 expression. Enhanced MSBP1 does not affect the polar localization of PIN2, but stimulates the PIN2 cycling and enhances the asymmetric PIN2 redistribution under gravi-stimulation. These results suggest that MSBP1 could enhance the cycling of PIN2-containing vesicles to stimulate the auxin redistribution under gravi-stimulation, providing informative hints on interactions between auxin and steroid binding protein.

Immunohistochemical assessment of NY-ESO-1 expression in esophageal adenocarcinoma resection specimens

AIM: To assess NY-ESO-1 expression in a cohort of esophageal adenocarcinomas.

AtNSF regulates leaf serration by modulating intracellular trafficking of PIN1 in Arabidopsis thaliana

In eukaryotes,N-ethylmaleimide-sensitive factor(NSF)is a conserved AAA+ATPase and a key component of the membrane trafficking machinery that promotes the fusion of secretory vesicles with target membranes.Here,we demonstrate that the Arabidopsis thaliana genome contains a single copy of NSF,At NSF,which plays an essential role in the regulation of leaf serration.The At NSF knock-down mutant,atnsf-1,exhibited more serrations in the leaf margin.Moreover,polar localization of the PINFORMED1(PIN1)auxin efflux transporter was diffuse around the margins of atnsf-1 leaves and root growth was inhibited in the atnsf-1 mutant.More PIN1-GFP accumulated in the intracellular compartments of atnsf-1 plants,suggesting that At NSF is required for intracellular trafficking of PIN between the endosome and plasma membrane.Furthermore,the serration phenotype was suppressed in the atnsf-1 pin1-8 double mutant,suggesting that At NSF is required for PIN1-mediated polar auxin transport to regulate leaf serration.The CUPSHAPED COTYLEDON2(CUC2)transcription factor gene is up-regulated in atnsf-1 plants and the cuc2-3 single mutant exhibits smooth leaf margins,demonstrating that At NSF also functions in the CUC2 pathway.Our results reveal that At NSF regulates the PIN1-generated auxin maxima with a CUC2-mediated feedback loop to control leaf serration.

Protein trafficking during plant innate immunity

Plants have evolved a sophisticated immune system to fight against pathogenic microbes. Upon detection of pathogen invasion by immune receptors, the immune system is turned on, resulting in production of antimicrobial molecules including pathogenesis-related（PR） proteins.Conceivably, an efficient immune response depends on the capacity of the plant cell＇s protein/membrane trafficking network to deploy the right defense-associated molecules in the right place at the right time. Recent research in this area shows that while the abundance of cell surface immune receptors is regulated by endocytosis, many intracellular immune receptors, when activated, are partitioned between the cytoplasm and the nucleus for induction of defense genes and activation of programmed cell death, respectively. Vesicle transport is an essential process for secretion of PR proteins to the apoplastic space and targeting of defense-related proteins to the plasma membrane or other endomembrane compartments. In this review, we discuss the various aspects of protein trafficking during plant immunity, with a focus on the immunity proteins on the move and the major components of the trafficking machineries engaged.

Interplay of transport vesicles during plant-fungal pathogen interaction

Vesicle trafficking is an essential cellular process upon which many physiological processes of eukaryotic cells rely.It is usually the‘language’of communication among the components of the endomembrane system within a cell,between cells and between a cell and its external environment.Generally,cells have the potential to internalize membrane-bound vesicles from external sources by endocytosis.Plants constantly interact with both mutualistic and pathogenic microbes.A large part of this interaction involves the exchange of transport vesicles between the plant cells and the microbes.Usually,in a pathogenic interaction,the pathogen releases vesicles containing bioactive molecules that can modulate the host immunity when absorbed by the host cells.In response to this attack,the host cells similarly mobilize some vesicles containing pathogenesis-related compounds to the pathogen infection site to destroy the pathogen,prevent it from penetrating the host cell or annul its influence.In fact,vesicle trafficking is involved in nearly all the strategies of phytopathogen attack subsequent plant immune responses.However,this field of plant-pathogen interaction is still at its infancy when narrowed down to plant-fungal pathogen interaction in relation to exchange of transport vesicles.Herein,we summarized some recent and novel findings unveiling the involvement of transport vesicles as a crosstalk in plant-fungal phytopathogen interaction,discussed their significance and identified some knowledge gaps to direct future research in the field.The roles of vesicles trafficking in the development of both organisms are also established.

Organizational Innovation of Apical Actin Filaments Drives Rapid Pollen Tube Growth and Turning

Polarized tip growth is a fundamental cellular process in many eukaryotes. In this study, we examined the dynamic restructuring of the actin cytoskeleton and its relationship to vesicle transport during pollen tip growth in Arabidopsis. We found that actin filaments originating from the apical membrane form a specialized structure consisting of longitudinally aligned actin bundles at the cortex and inner cytoplasmic fila- ments with a distinct distribution. Using actin-based pharmacological treatments and genetic mutants in combination with FRAP （fluorescence recovery after photobleaching） technology to visualize the transport of vesicles within the growth domain of pollen tubes, we demonstrated that cortical actin filaments facilitate tip-ward vesicle transport. We also discovered that the inner apical actin filaments prevent backward movement of vesicles, thus ensuring that sufficient vesicles accumulate at the pollen tube tip to support the rapid growth of the pollen tube. The combinatorial effect of cortical and internal apical actin filaments perfectly explains the generation of the inverted ＂V＂ cone-shaped vesicle distribution pattern at the pollen tube tip. When pollen tubes turn, apical actin filaments at the facing side undergo depolymerization and repolymerization to reorient the apical actin structure toward the new growth direction. This actin restructuring precedes vesicle accumulation and changes in tube morphology. Thus, our study provides new insights into the functional relationship between actin dynamics and vesicle transport during rapid and directional pollen tube growth.

The Potato Sucrose Transporter StSUT1 Interacts with a DRM-Associated Protein Disulfide Isomerase

Organization of proteins into complexes is crucial for many cellular functions. Recently, the SUT1 protein was shown to form homodimeric complexes, to be associated with lipid raft-like microdomains in yeast as well as in plants and to undergo endocytosis in response to brefeldin A. We therefore aimed to identify SUTl-interacting proteins that might be involved in dimerization, endocytosis, or targeting of SUT1 to raft-like microdomains. Therefore, we identified potato membrane proteins, which are associated with the detergent-resistant membrane （DRM） fraction. Among the proteins identified, we clearly confirmed StSUT1 as part of DRM in potato source leaves. We used the yeast two-hybrid split ubiq- uitin system （SUS） to systematically screen for interaction between the sucrose transporter StSUT1 and other membrane- associated or soluble proteins in vivo. The SUS screen was followed by immunoprecipitation using affinity-purified StSUTl-specific peptide antibodies and mass spectrometric analysis of co-precipitated proteins. A large overlap was ob- served between the StSUTl-interacting proteins identified in the co-immunoprecipitation and the detergent-resistant membrane fraction. One of the SUTl-interacting proteins, a protein disulfide isomerase （PDI）, interacts also with other sucrose transporter proteins. A potential role of the PDI as escort protein is discussed.

The influence, of nanotopography on organelle organization and communication

Cellular differentiation can be affected by the extracellular environment, particularly extracellular substrates. The nanotopography of the substrate may be involved in the mechanisms of cellular differentiation in vivo. Organelles are major players in various cellular functions; however, the influence of nano- topography on organelles has not yet been elucidated. In the present study, a micropit-nanotube topography （MNT） was fabricated on the titanium surface, and organelle-specific fluorescent probes were used to detect the intracellular organelle organization of MG63 cells. Communication between organelles, identified by organelle-specific GTPase expression, was evaluated by quantitative polymerase chain reaction and western blotting. Transmission electron microscopy was performed to evaluate the organelle structure. There were no significant differences in organelle distribution or number between the MNT and flat surface. However, organelle-specific GTPases on the MNT were dramatically downregulated. In addition, obvious endoplasmic reticulum lumen dilation was observed on the MNT surface, and the unfolded protein response （UPR） was also initiated. Regarding the relationships among organelle trafficking, UPR, and osteogenic differentiation, our findings may provide important insights into the signal transduction induced by nanotopography.

FgRab5 and FgRab7 are essential for endosomes biogenesis and nonredundantly recruit the retromer complex to the endosomes in Fusarium graminearum

The retromer complex,composed of the cargo-selective complex(CSC)Vps35-Vps29-Vps26 in complex with the sorting nexin dimer Vps5-Vps17,mediates the sorting and retrograde transport of cargo proteins from the endosomes to the trans-Golgi network in eukaryotic cells.Rab proteins belong to the Ras superfamily of small GTPases and regulate many trafficking events including vesicle formation,budding,transport,tethering,docking and fusion with target membranes.Herein,we investigated the potential functional relationship between the retromer complex and the 11 Rab proteins that exist in Fusarium graminearum using genetic and high-resolution laser confocal microscopic approaches.We found that only FgRab5(FgRab5A and FgRab5B)and FgRab7 associate with the retromer complex.Both FgVps35-GFP and FgVps17-GFP are mis-localized and appear diffused in the cytoplasm ofΔFgrab5A,ΔFgrab5B andΔFgrab7 mutants as compared to their punctate localization within the endosomes of the wild-type.FgRab7 and FgRab5B were found to co-localize with the retromer on endosomal membranes.Most strikingly,we found that these three Rab GTPases are indispensable for endosome biogenesis as both early and late endosomes could not be detected in the cells of the mutants after FM4-64 staining of the cells,while they were very clearly seen in the wild-type PH-1.Furthermore,FgRab7 was found to recruit FgVps35 but not FgVps17 to the endosomal membranes,whereas FgRab5B recruits both FgVps35 and FgVps17 to the membranes.Thus,we conclude that the Rab proteins FgRab5A,FgRab5B and FgRab7 play critical roles in the biogenesis of endosomes and in regulating retromer-mediated trafficking in F.graminearum.

Rop plays conserved roles in the reproductive and digestive processes of spider mites

Ras opposite(Rop)is known to play an essential role in regulating vesicle trafficking,including synaptic transmission and general secretion.The fundamental roles of Rop have been confirmed by the observation that null mutations in many organisms generate lethal phenotypes during embryogenesis.However,the effects of Rop during the postembryonic stages,especially in non-model organisms,remain largely unknown.Here,we provide new data that enhance our understanding of Rop's roles in the adults of multiple species of Tetranychus spider mites(Acari:Tetranychidae),a class of notorious agricultural pests.Our in silico and experimental evidence demonstrated that Rop is under purifying selection and is highly conserved in Tetranychus spp.RNA interference experiments showed that Rop is required for maintaining normal fecundity but has no significant effect on survival.We further demonstrate that knockdown of Rop darkens the body color of spider mites and blocks the excretion of fecal pellets,which is likely to be related to an abnormality in the excretion of food waste in the digestive system.Overall,our findings clarify novel functions of a vesicle trafficking-related gene in the adult stage of multiple Tetranychus species and highlight the need to evaluate the roles of essential genes in various organisms.

An Approach to Quantify Endomembrane Dynamics in Pollen Utilizing Bioactive Chemicals

Tip growth of pollen tubes and root hairs occurs via rapid polar growth. These rapidly elongating cells require tip-focused endomembrane trafficking for the deposition and recycling of proteins, membranes, and cell wall materials. Most of the image-based data published to date are subjective and non-quantified. Quantitative and com- parative descriptors of these highly dynamic processes have been a major challenge, but are highly desirable for genetic and chemical genomics approaches to dissect this biological network. To address this problem, we screened for small molecules that perturbed the localization of a marker for the Golgi Ras-like monomeric G-protein RAB2：GFP expressed in transgenic tobacco pollen. Semi-automated high-throughput imaging and image analysis resulted in the identifica- tion of novel compounds that altered pollen tube development and endomembrane trafficking. Six compounds that caused mislocalization and varying degrees of altered movement of RAB2：GFP-labeled endomembrane bodies were used to generate a training set of image data from which to quantify vesicle dynamics. The area, velocity, straightness, and intensity of each body were quantified using semi-automated image analysis tools revealing quantitative differences in the phenotype caused by each compound. A score was then given to each compound enabling quantitative comparisons between compounds. Our results demonstrate that image analysis can be used to quantitatively evaluate dynamic sub- cellular endomembrane phenotypes induced by bioactive chemicals, mutations, or other perturbing agents as part of a strategy to quantitatively dissect the endomembrane network.

S-acylation of YKT61 modulates its unconventional participation in the formation of SNARE complexes in Arabidopsis

Hetero-tetrameric soluble N-ethylmaleimide-sensitive factor attachment protein receptors(SNAREs)complexes are critical for vesicle-target membrane fusion within the endomembrane system of eukaryotic cells.SNARE assembly involves four different SNARE motifs,Qa,Qb,Qc,and R,providedby three orfour SNARE proteins.YKT6 is an atypical R-SNARE that lacks a transmembrane domain and is involved in multiple vesicle-target membrane fusions.Although YKT6 is evolutionarily conserved and essential,its function and regulation in different phyla seem distinct.Arabidopsis YKT61,the yeast and metazoan YKT6 homologue,is essential for gametophytic development,plays a critical role in sporophytic cells,and me-diates multiple vesicle-target membrane fusion.However,its molecular regulation is unclear.We report here that YKT61 is S-acylated.Abolishing its S-acylation by a C195S mutation dissociates YKT61 from endomembrane structures and causes its functional loss.Although interacting with various SNARE pro-teins,YKT61functions not as a canonical R-SNAREbut coordinates with otherR-SNAREs to participate in theformationof SNAREcomplexes.Phylum-specific molecular regulation of YKT6 may be evolvedto allow more efficient SNARE assembly in different eukaryotic cells.