A Spatio-Temporal Analysis of Matrix Protein and Nucleocapsid Trafficking during Vesicular Stomatitis Virus Uncoating

To study VSV entry and the fate of incoming matrix (M) protein during virus uncoating we used recombinant viruses encoding M proteins with a C-terminal tetracysteine tag that could be fluorescently labeled using biarsenical (Lumio) compounds. We found that uncoating occurs early in the endocytic pathway and is inhibited by expression of dominant-negative (DN) Rab5, but is not inhibited by DN-Rab7 or DN-Rab11. Uncoating, as defined by the separation of nucleocapsids from M protein, occurred between 15 and 20 minutes post-entry and did not require microtubules or an intact actin cytoskeleton. Unexpectedly, the bulk of M protein remained associated with endosomal membranes after uncoating and was eventually trafficked to recycling endosomes. Another small, but significant fraction of M distributed to nuclear pore complexes, which was also not dependent on microtubules or polymerized actin. Quantification of fluorescence from high-resolution confocal micrographs indicated that after membrane fusion, M protein diffuses across the endosomal membrane with a concomitant increase in fluorescence from the Lumio label which occurred soon after the release of RNPs into the cytoplasm. These data support a new model for VSV uncoating in which RNPs are released from M which remains bound to the endosomal membrane rather than the dissociation of M protein from RNPs after release of the complex into the cytoplasm following membrane fusion.     

TRIM50 Protein Regulates Vesicular Trafficking for Acid Secretion in Gastric Parietal Cells

Of the TRIM/RBCC family proteins taking part in a variety of cellular processes, TRIM50 is a stomach-specific member with no defined biological function. Our biochemical data demonstrated that TRIM50 is specifically expressed in gastric parietal cells and is predominantly localized in the tubulovesicular and canalicular membranes. In cultured cells ectopically expressing GFP-TRIM50, confocal microscopic imaging revealed dynamic movement of TRIM50-associated vesicles in a phosphoinositide 3-kinase-dependent manner. A protein overlay assay detected preferential binding of the PRY-SPRY domain from the TRIM50 C-terminal region to phosphatidylinositol species, suggesting that TRIM50 is involved in vesicular dynamics by sensing the phosphorylated state of phosphoinositol lipids. Trim50 knock-out mice retained normal histology in the gastric mucosa but exhibited impaired secretion of gastric acid. In response to histamine, Trim50 knock-out parietal cells generated deranged canaliculi, swollen microvilli lacking actin filaments, and excess multilamellar membrane complexes. Therefore, TRIM50 seems to play an essential role in tubulovesicular dynamics, promoting the formation of sophisticated canaliculi and microvilli during acid secretion in parietal cells.     

Astroglial Vesicular Trafficking in Neurodegenerative Diseases

The neocortex represents one of the largest estates of the human brain. This structure comprises ~30–40 billions of neurones and even more of non-neuronal cells. Astrocytes, highly heterogeneous homoeostatic glial cells, are fundamental for housekeeping of the brain and contribute to information processing in neuronal networks. Gray matter astrocytes tightly enwrap synapses, contact blood vessels and, naturally, are also in contact with the extracellular space, where convection of fluid takes place. Thus astrocytes receive signals from several distinct extracellular domains and can get excited by numerous mechanisms, which regulate cytosolic concentration of second messengers, such as Ca²⁺ and cAMP. Excited astrocytes often secrete diverse substances (generally referred to as gliosignalling molecules) that include classical neurotransmitters such as glutamate and ATP or neuromodulators such as d-serine or neuropeptides. Astrocytic secretion occurs through several mechanisms: by diffusion through membrane channels, by translocation via plasmalemmal transporters or by vesicular exocytosis. Vesicular release of gliosignalling molecules appears fundamentally similar to that operating in neurones, since it depends on the SNARE proteins-dependent merger of the vesicle membrane with the plasmalemma. However, the coupling between the stimulus and astroglial vesicular secretion is at least one order of magnitude slower than that in neurones. Here we review mechanisms of astrocytic excitability and the molecular, anatomical and physiological properties of vesicular apparatus mediating the release of gliosignalling molecules in health and in the neurodegenerative pathology.     

Trafficking of Vacuolar Proteins: The Crucial Role of Arabidopsis Vacuolar Protein Sorting 29 in Recycling Vacuolar Sorting Receptor

The retromer is involved in recycling lysosomal sorting receptors in mammals. A component of the retromer complex in Arabidopsis thaliana, vacuolar protein sorting 29 (VPS29), plays a crucial role in trafficking storage proteins to protein storage vacuoles. However, it is not known whether or how vacuolar sorting receptors (VSRs) are recycled from the prevacuolar compartment (PVC) to the trans-Golgi network (TGN) during trafficking to the lytic vacuole (LV). Here, we report that VPS29 plays an essential role in the trafficking of soluble proteins to the LV from the TGN to the PVC. maigo1-1 (mag1-1) mutants, which harbor a knockdown mutation in VPS29, were defective in trafficking of two soluble proteins, Arabidopsis aleurain-like protein (AALP):green fluorescent protein (GFP) and sporamin:GFP, to the LV but not in trafficking membrane proteins to the LV or plasma membrane or via the secretory pathway. AALP:GFP and sporamin:GFP in mag1-1 protoplasts accumulated in the TGN but were also secreted into the medium. In mag1-1 mutants, VSR1 failed to recycle from the PVC to the TGN; rather, a significant proportion was transported to the LV; VSR1 overexpression rescued this defect. Moreover, endogenous VSRs were expressed at higher levels in mag1-1 plants. Based on these results, we propose that VPS29 plays a crucial role in recycling VSRs from the PVC to the TGN during the trafficking of soluble proteins to the LV.     

Trafficking of Vacuolar Sorting Receptors: New Data and New Problems

Vacuolar sorting receptors bind cargo ligands early in the secretory pathway and show that multivesicular body-vacuole fusion requires a Rab5/Rab7 GTPase conversion with consequences for retromer binding.To serve the purposes of controlled protein turnover, eukaryotic cells compartmentalize the required acid hydrolases in specialized digestive organelles: lysosomes in animals and vacuoles in yeasts and plants. Therefore, a reliable system must be in operation to prevent such proteolytic enzymes being released at the cell surface. Such a mechanism requires that acid hydrolases be identified and diverted away from the secretory flow to the plasma membrane (PM). This process is facilitated by receptors that recognize specific motifs in the hydrolases that are absent in secretory proteins. The most well-known example of this is the mannosyl 6-phosphate receptor (MPR), which is responsible for the sorting of lysosomal enzymes; indeed, it has become a paradigm for protein sorting in most cell biology textbooks. It entails the recognition of phosphomannan cargo ligands by MPRs in the trans-Golgi network (TGN) followed by the sequestration of the MPR-ligand complexes into specific transport vectors (clathrin-coated vesicles [CCVs]). These are then transported to an endosomal compartment (the early endosome [EE]) having a more acidic pH than the TGN, thereby causing the ligands to separate from the MPRs. The MPRs are subsequently recycled back to the TGN via retromer-coated carriers for another round of trafficking (for review, see Braulke and Bonifacino, 2009; Seaman, 2012).Many plant scientists support a scenario for the sorting of soluble vacuolar proteins and the trafficking of their receptors (vacuolar sorting receptors [VSRs]) that closely resembles that of the MPR system of mammalian cells (Hwang, 2008; De Marcos Lousa et al., 2012; Kang et al., 2012; Sauer et al., 2013; Xiang et al., 2013). This working model is based on three key observations: (1) VSRs were first identified in detergent-solubilized CCV fractions isolated from developing pea (Pisum sativum) cotyledons; (2) CCVs are regularly seen budding off the TGN in thin-sectioned plant cells; and (3) depending on the organism, VSRs and VSR-reporter constructs are found concentrated either in the TGN or in multivesicular prevacuolar compartments (PVCs) under steady-state conditions (Robinson and Pimpl, 2014a, 2014b, and refs. therein). Unfortunately, information on VSRs has not been obtained from a single experimental system. Although much work on Arabidopsis (Arabidopsis thaliana) VSR mutants has been published (for review, see De Marcos Lousa et al., 2012) and the majority of immunogold electron microscopic localization experiments have been performed in Arabidopsis, the majority of the fluorescence localizations, particularly with regard to VSR trafficking, have been carried out by transient expression in tobacco (Nicotiana tabacum; agroinfiltration for leaves and electroporation for protoplasts). Nevertheless, it should be stressed that sorting motifs for acid hydrolases and their corresponding receptors in the three major eukaryotic organismal groups differ considerably (Robinson et al., 2012). In addition, the secretory and endocytic pathways of plant cells contrast significantly with mammalian cells, the most important distinctions being (1) the lack of an intermediate compartment between the endoplasmic reticulum (ER) and the Golgi apparatus in plants, (2) that plants have motile Golgi stacks rather than a perinuclear Golgi complex, and (3) the absence of an independent EE in plants, the function of which is assumed by the TGN (Contento and Bassham, 2012). While these differences do not automatically negate the validity of the above working model for VSR trafficking, they at least legitimize a more thorough analysis of the supporting data than has previously been the case (Robinson and Pimpl, 2014a, 2014b).The principal issues at stake are as follows. Where do VSRs bind and release their cargo ligands? What is the actual mechanism resulting in the separation of secretory from vacuolar cargo molecules? What is/are the precise role(s) of TGN-derived CCVs? And where does retromer pick up VSRs and where are they delivered to? The impact of several new publications on these points of dispute is the subject of this article.     

Membrane Diffusion Occurs by Continuous-Time Random Walk Sustained by Vesicular Trafficking

Diffusion in cellular membranes is regulated by processes that occur over a range of spatial and temporal scales. These processes include membrane fluidity, interprotein and interlipid interactions, interactions with membrane microdomains, interactions with the underlying cytoskeleton, and cellular processes that result in net membrane movement. The complex, non-Brownian diffusion that results from these processes has been difficult to characterize, and moreover, the impact of factors such as membrane recycling on membrane diffusion remains largely unexplored. We have used a careful statistical analysis of single-particle tracking data of the single-pass plasma membrane protein CD93 to show that the diffusion of this protein is well described by a continuous-time random walk in parallel with an aging process mediated by membrane corrals. The overall result is an evolution in the diffusion of CD93: proteins initially diffuse freely on the cell surface but over time become increasingly trapped within diffusion-limiting membrane corrals. Stable populations of freely diffusing and corralled CD93 are maintained by an endocytic/exocytic process in which corralled CD93 is selectively endocytosed, whereas freely diffusing CD93 is replenished by exocytosis of newly synthesized and recycled CD93. This trafficking not only maintained CD93 diffusivity but also maintained the heterogeneous distribution of CD93 in the plasma membrane. These results provide insight into the nature of the biological and biophysical processes that can lead to significantly non-Brownian diffusion of membrane proteins and demonstrate that ongoing membrane recycling is critical to maintaining steady-state diffusion and distribution of proteins in the plasma membrane.     

Specific Sorting and Post-Golgi Trafficking of Dendritic Potassium Channels in Living Neurons

Proper membrane localization of ion channels is essential for the function of neuronal cells. Particularly, the computational ability of dendrites depends on the localization of different ion channels in specific subcompartments. However, the molecular mechanisms that control ion channel localization in distinct dendritic subcompartments are largely unknown. Here, we developed a quantitative live cell imaging method to analyze protein sorting and post-Golgi vesicular trafficking. We focused on two dendritic voltage-gated potassium channels that exhibit distinct localizations: Kv2.1 in proximal dendrites and Kv4.2 in distal dendrites. Our results show that Kv2.1 and Kv4.2 channels are sorted into two distinct populations of vesicles at the Golgi apparatus. The targeting of Kv2.1 and Kv4.2 vesicles occurred by distinct mechanisms as evidenced by their requirement for specific peptide motifs, cytoskeletal elements, and motor proteins. By live cell and super-resolution imaging, we identified a novel trafficking machinery important for the localization of Kv2.1 channels. Particularly, we identified non-muscle myosin II as an important factor in Kv2.1 trafficking. These findings reveal that the sorting of ion channels at the Golgi apparatus and their subsequent trafficking by unique molecular mechanisms are crucial for their specific localizations within dendrites.     

GNOM-Mediated Vesicular Trafficking Plays an Essential Role in Hydrotropism of Arabidopsis Roots

Roots respond not only to gravity but also to moisture gradient by displaying gravitropism and hydrotropism, respectively, to control their growth orientation, which helps plants obtain water and become established in the terrestrial environment. As gravitropism often interferes with hydrotropism, however, the mechanisms of how roots display hydrotropism and differentiate it from gravitropism are not understood. We previously reported MIZU-KUSSEI1 (MIZ1) as a gene required for hydrotropism but not for gravitropism, although the function of its protein was not known. Here, we found that a mutation of GNOM encoding guanine-nucleotide exchange factor for ADP-ribosylation factor-type G proteins was responsible for the ahydrotropism of Arabidopsis (Arabidopsis thaliana), miz2. Unlike other gnom alleles, miz2 showed no apparent morphological defects or reduced gravitropism. Instead, brefeldin A (BFA) treatment inhibited both hydrotropism and gravitropism in Arabidopsis roots. In addition, a BFA-resistant GNOM variant, GN^M696L, showed normal hydrotropic response in the presence of BFA. Furthermore, a weak gnom allele, gnom^B/E, showed defect in hydrotropic response. These results indicate that GNOM-mediated vesicular trafficking plays an essential role in hydrotropism of seedling roots.Stationary growth is a distinct feature of plants and distinguishes them from other organisms. Plants have evolved a variety of mechanisms for responding to environmental cues, which enables them to survive in the presence of limited resources or environmental stresses. One of the most important growth adaptations plants have acquired is tropism, growth response that involves bending or curving of plant organs toward or away from a stimulus. For example, roots display tropisms in response to environmental cues such as gravity, light, touch, and moisture (Darwin and Darwin, 1880; Takahashi, 1997; Correll and Kiss, 2002; Monshausen et al., 2008). Gravitropism has been the subject of intense study, while other tropic responses of roots have been less well characterized. There is some evidence of hydrotropism in roots, but this response has proven difficult to differentiate from gravitropism, as the latter always interferes with hydrotropism (Jaffe et al., 1985; Takahashi, 1994; Takahashi, 1997). The demonstration of true hydrotropism in roots has facilitated the identification of some of the physiological aspects of hydrotropism and its existence in a wide range of plant species. However, the underlying mechanisms that regulate hydrotropism remain unknown. The limited supply of water and precipitation in many parts of the world greatly affects agriculture and ecosystems. Elucidating the molecular mechanism of hydrotropism in roots is therefore important not only for understanding how terrestrial plants adapt to changes in moisture, but also for improving crop yields and biomass production.The isolation and analysis of hydrotropism-deficient mutants using the model plant species Arabidopsis (Arabidopsis thaliana) represents a potent tool for dissecting the molecular mechanism of hydrotropism. Previously, we isolated an ahydrotropic mutant of Arabidopsis, mizu-kussei1 (miz1), and showed that MIZ1 encodes a protein of unknown function (Kobayashi et al., 2007). In light of both the physiological features of hydrotropism, as well as what we have learned from genetic studies of other tropisms, it is unlikely that miz1 alone governs the hydrotropic response. In support of this, we have identified a second ahydrotropic mutant, miz2, a unique allele of gnom that confers ahydrotropic but not agravitropic growth, which implies distinct roles of vesicular trafficking between hydrotropism and gravitropism in roots.     

A Computationally Guided Protein-Interaction Screen Uncovers Coiled-Coil Interactions Involved in Vesicular Trafficking

Mapping protein-protein interactions at a domain or motif level can provide structural annotation of the interactome. The α-helical coiled coil is among the most common protein-interaction motifs, and proteins predicted to contain coiled coils participate in diverse biological processes. Here, we introduce a combined computational/experimental screening strategy that we used to uncover coiled-coil interactions among proteins involved in vesicular trafficking in Saccharomyces cerevisiae. A number of coiled-coil complexes have already been identified and reported to play important roles in this important biological process. We identify additional examples of coiled coils that can form physical associations. The computational strategy used to prioritize coiled-coil candidates for testing dramatically improved the efficiency of discovery in a large experimental screen. As assessed by comprehensive yeast two-hybrid assays, computational prefiltering retained 90% of positive interacting pairs and eliminated > 60% of negatives from a set of interaction candidates. The coiled-coil-mediated interaction network elucidated using the combined computational/experimental approach comprises 80 coiled-coil associations between 58 protein pairs, among which 21 protein interactions have not been previously reported in interaction databases and 26 interactions were previously known at the protein level but have now been localized to the coiled-coil motif. The coiled-coil-mediated interactions were specific rather than promiscuous, and many interactions could be recapitulated in a green fluorescent protein complementation assay. Our method provides an efficient route to discovering new coiled-coil interactions and uncovers a number of associations that may have functional significance for vesicular trafficking.     

Mechanisms of Protein Sorting in Mitochondria

A protein’s function is intimately linked to its correct subcellular location, yet the machinery required for protein synthesis is predominately cytosolic. How proteins are trafficked through the confines of the cell and integrated into the appropriate cellular compartments has puzzled and intrigued researchers for decades. Indeed, studies exploring this premise revealed elaborate cellular protein translocation and sorting systems, which ensure that all proteins are shuttled to the appropriate cellular destination, where they fulfill their specific functions. This holds true for mitochondria, where sophisticated molecular machines serve to recognize incoming precursor proteins and integrate them into the functional framework of the organelle. We summarize the recent progress in our understanding of mitochondrial protein sorting and the machineries and mechanisms that mediate and regulate this highly dynamic cellular process essential for survival of virtually all eukaryotic cells.Mitochondria are multifunctional double-membrane-bound organelles that arose from a bacterial endosymbiont during the evolution of eukaryotic cells. Known as the powerhouses of the cell, mitochondria harbor the oxidative phosphorylation machinery for ATP synthesis, but also a large number of biosynthetic pathways. Moreover, they are intimately involved in complex cellular processes, like calcium homeostasis and programmed cell death. As a relic of their evolutionary origin, mitochondria contain their own genetic material and machineries to manufacture their own RNAs and proteins. However, the small circular mitochondrial genome encodes only a few proteins (8 and 13 polypeptides in yeast and humans, respectively). All remaining mitochondrial proteins (approximately 99%) are encoded by the nuclear genome and synthesized on cytosolic ribosomes in their precursor forms. To acquire their mature, functional state these precursor proteins need to be efficiently targeted and imported into mitochondria and sorted to the correct submitochondrial compartment: outer membrane, intermembrane space (IMS), inner membrane, and matrix. The inner mitochondrial membrane is further subdivided into the inner boundary membrane, which is closely opposed to the outer membrane, and large tubular invaginations, termed cristae membranes. Within the four mitochondrial compartments, sophisticated translocation, sorting, and assembly machineries serve to establish incoming precursors in a functional state within the context of their new environment. Advances in the last decade, particularly because of the application of proteomic approaches, have significantly extended the number of components and machineries known to be involved in mitochondrial protein import (Sickmann et al. 2003; Prokisch et al. 2004; Reinders et al. 2006; Pagliarini et al. 2008). These and previous discoveries have provided us with the current framework, which suggests the presence of at least six distinct translocation and assembly machineries within mitochondria (Fig. 1). In this article, we will summarize our current understanding of the machineries for mitochondrial protein import and describe the different molecular mechanisms that execute this essential task.Open in a separate windowFigure 1.Overview of mitochondrial protein sorting pathways. Cytosolic chaperones deliver precursor proteins to the organelle in a translocation-competent state. Some α-helical proteins are inserted into the outer membrane with the help of Mim1. Virtually all other precursors initially traverse the outer membrane via the TOM complex and are subsequently routed to downstream sorting pathways. Biogenesis of outer membrane β-barrel proteins requires the small TIM chaperones of the IMS and the SAM complex. Cysteine-containing IMS proteins are imported via the MIA pathway. Metabolite carriers of the inner mitochondrial membrane are transferred by the small TIM chaperones to the TIM22 complex, which mediates their membrane integration. Presequence-containing precursors are directly taken over from the TOM complex by the TIM23 machinery that either inserts these proteins into the membrane or translocates them into the matrix in cooperation with the import motor PAM. OM, outer membrane; IMS, intermembrane space; IM, inner membrane, Δψ, membrane potential across the inner mitochondrial membrane.     

Viral Bimolecular Fluorescence Complementation: A Novel Tool to Study Intracellular Vesicular Trafficking Pathways

The Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Nef interacts with a multitude of cellular proteins, manipulating the host membrane trafficking machinery to evade immune surveillance. Nef interactions have been analyzed using various in vitro assays, co-immunoprecipitation studies, and more recently mass spectrometry. However, these methods do not evaluate Nef interactions in the context of viral infection nor do they define the sub-cellular location of these interactions. In this report, we describe a novel bimolecular fluorescence complementation (BiFC) lentiviral expression tool, termed viral BiFC, to study Nef interactions with host cellular proteins in the context of viral infection. Using the F2A cleavage site from the foot and mouth disease virus we generated a viral BiFC expression vector capable of concurrent expression of Nef and host cellular proteins; PACS-1, MHC-I and SNX18. Our studies confirmed the interaction between Nef and PACS-1, a host membrane trafficking protein involved in Nef-mediated immune evasion, and demonstrated co-localization of this complex with LAMP-1 positive endolysosomal vesicles. Furthermore, we utilized viral BiFC to localize the Nef/MHC-I interaction to an AP-1 positive endosomal compartment. Finally, viral BiFC was observed between Nef and the membrane trafficking regulator SNX18. This novel demonstration of an association between Nef and SNX18 was localized to AP-1 positive vesicles. In summary, viral BiFC is a unique tool designed to analyze the interaction between Nef and host cellular proteins by mapping the sub-cellular locations of their interactions during viral infection.     

Impairment of Protein Trafficking upon Overexpression and Mutation of Optineurin

Background

Glaucoma is a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and axons. Optineurin is one of the candidate genes identified so far. A mutation of Glu⁵⁰ to Lys (E50K) has been reported to be associated with a more progressive and severe disease. Optineurin, known to interact with Rab8, myosin VI and transferrin receptor (TfR), was speculated to have a role in protein trafficking. Here we determined whether, and how optineurin overexpression and E50K mutation affect the internalization of transferrin (Tf), widely used as a marker for receptor-mediated endocytosis.

Methodology/Principal Findings

Human retinal pigment epithelial (RPE) and rat RGC5 cells transfected to overexpress wild type optineurin were incubated with Texas Red-Tf to evaluate Tf uptake. Granular structures or dots referred to as foci formed in perinuclear regions after transfection. An impairment of the Tf uptake was in addition observed in transfected cells. Compared to overexpression of the wild type, E50K mutation yielded an increased foci formation and a more pronounced defect in Tf uptake. Co-transfection with TfR, but not Rab8 or myosin VI, construct rescued the optineurin inhibitory effect, suggesting that TfR was the factor involved in the trafficking phenotype. Forced expression of both wild type and E50K optineurin rendered TfR to colocalize with the foci. Surface biotinylation experiments showed that the surface level of TfR was also reduced, leading presumably to an impeded Tf uptake. A non-consequential Leu¹⁵⁷ to Ala (L157A) mutation that displayed much reduced foci formation and TfR binding had normal TfR distribution, normal surface TfR level and normal Tf internalization.

Conclusions/Significance

The present study demonstrates that overexpression of wild type optineurin results in impairment of the Tf uptake in RPE and RGC5 cells. The phenotype is related to the optineurin interaction with TfR. Our results further indicate that E50K induces more dramatic effects than the wild type optineurin, and is thus a gain-of-function mutation. The defective protein trafficking may be one of the underlying bases why glaucoma pathology develops in patients with E50K mutation.     

Coronin-1C Protein and Caveolin Protein Provide Constitutive and Inducible Mechanisms of Rac1 Protein Trafficking

Sustained directional fibroblast migration requires both polarized activation of the protrusive signal, Rac1, and redistribution of inactive Rac1 from the rear of the cell so that it can be redistributed or degraded. In this work, we determine how alternative endocytic mechanisms dictate the fate of Rac1 in response to the extracellular matrix environment. We discover that both coronin-1C and caveolin retrieve Rac1 from similar locations at the rear and sides of the cell. We find that coronin-1C-mediated extraction, which is responsible for Rac1 recycling, is a constitutive process that maintains Rac1 protein levels within the cell. In the absence of coronin-1C, the effect of caveolin-mediated endocytosis, which targets Rac1 for proteasomal degradation, becomes apparent. Unlike constitutive coronin-1C-mediated trafficking, caveolin-mediated Rac1 endocytosis is induced by engagement of the fibronectin receptor syndecan-4. Such an inducible endocytic/degradation mechanism would predict that, in the presence of fibronectin, caveolin defines regions of the cell that are resistant to Rac1 activation but, in the absence of fibronectin leaves more of the membrane susceptible to Rac1 activation and protrusion. Indeed, we demonstrate that fibronectin-stimulated activation of Rac1 is accelerated in the absence of caveolin and that, when caveolin is knocked down, polarization of active Rac1 is lost in FRET experiments and culminates in shunting migration in a fibrous fibronectin matrix. Although the concept of polarized Rac1 activity in response to chemoattractants has always been apparent, our understanding of the balance between recycling and degradation explains how polarity can be maintained when the chemotactic gradient has faded.     

Populus euphratica APYRASE2 Enhances Cold Tolerance by Modulating Vesicular Trafficking and Extracellular ATP in Arabidopsis Plants

Apyrase and extracellular ATP play crucial roles in mediating plant growth and defense responses. In the cold-tolerant poplar, Populus euphratica, low temperatures up-regulate APYRASE2 (PeAPY2) expression in callus cells. We investigated the biochemical characteristics of PeAPY2 and its role in cold tolerance. We found that PeAPY2 predominantly localized to the plasma membrane, but punctate signals also appeared in the endoplasmic reticulum and Golgi apparatus. PeAPY2 exhibited broad substrate specificity, but it most efficiently hydrolyzed purine nucleotides, particularly ATP. PeAPY2 preferred Mg²⁺ as a cofactor, and it was insensitive to various, specific ATPase inhibitors. When PeAPY2 was ectopically expressed in Arabidopsis (Arabidopsis thaliana), cold tolerance was enhanced, based on root growth measurements and survival rates. Moreover, under cold stress, PeAPY2-transgenic plants maintained plasma membrane integrity and showed reduced cold-elicited electrolyte leakage compared with wild-type plants. These responses probably resulted from efficient plasma membrane repair via vesicular trafficking. Indeed, transgenic plants showed accelerated endocytosis and exocytosis during cold stress and recovery. We found that low doses of extracellular ATP accelerated vesicular trafficking, but high extracellular ATP inhibited trafficking and reduced cell viability. Cold stress caused significant increases in root medium extracellular ATP. However, under these conditions, PeAPY2-transgenic lines showed greater control of extracellular ATP levels than wild-type plants. We conclude that Arabidopsis plants that overexpressed PeAPY2 could increase membrane repair by accelerating vesicular trafficking and hydrolyzing extracellular ATP to avoid excessive, cold-elicited ATP accumulation in the root medium and, thus, reduced ATP-induced inhibition of vesicular trafficking.Low temperature is a major environmental factor that restrains plant growth and crop productivity (Yamaguchi-Shinozaki and Shinozaki, 2006). When temperatures fall below 0°C, plant cells experience dehydration and mechanical injury caused by ice crystallization (Webb and Steponkus, 1993; Yamazaki et al., 2008). Cold stress reduces plasma membrane (PM) integrity, which leads to the leakage of intracellular solutes. ATP can be an important signaling molecule when released into the extracellular matrix (ECM). Extracellular ATP (eATP) was shown to regulate a wide range of cellular processes (Roux and Steinebrunner, 2007; Clark and Roux, 2009, 2011; Tanaka et al., 2010; Clark et al., 2014), but its functions are dose dependent. For example, in Arabidopsis (Arabidopsis thaliana), low concentrations of eATP triggered stomatal opening, but high concentrations caused stomatal closure (Clark et al., 2011). Application of eATP at 100 to 200 μm increased hypocotyl elongation in etiolated seedlings, but higher doses led to a reduction of hypocotyl elongation (Roux et al., 2006). In fibers of cotton (Gossypium hirsutum), application of ATPγS and ADPβS at 30 μm each induced an increase in average fiber length, whereas 150 μm ATPγS or ADPβS inhibited the growth of fibers (Clark et al., 2010a). In addition, low doses of eATP (less than 100 μm) induced an obvious Ca²⁺ influx into the elongation zone of Arabidopsis roots, and higher doses of eATP (1,000 μm) caused a significant increase in Ca²⁺ efflux (Demidchik et al., 2011). In Populus euphratica, exposure to high levels of ATP (more than 500 μm) was shown to trigger programmed cell death in callus cells, but at low doses (less than 200 μm), callus cells did not die over the observation period (Sun et al., 2012a). The cold-elicited release of ATP through disrupted membranes and the buildup of ATP in the ECM may cause a reduction in cell viability. eATP also plays a fundamental role in mediating plant responses to environmental stresses, such as pathogens (Chivasa et al., 2009), wounds (Cao et al., 2014), high salt (Sun et al., 2012b), and osmotic stress (Jeter et al., 2004; Kim et al., 2009); however, the link between eATP and cold tolerance has not been fully established.Extracellular apyrases (or ectoapyrases) are the principal enzymes that limit eATP accumulation in both animals and plants (Todorov et al., 1997; Marcus et al., 2003; Wu et al., 2007). In Arabidopsis, suppression of apyrases (AtAPY1 and AtAPY2) led to a slight increase in eATP levels, indicating that APY1 and APY2 controlled the concentration of eATP (Lim et al., 2014). Apyrases are suggested to be involved in some of the signaling steps in plant growth. Apyrases play a crucial role in pollen germination (Steinebrunner et al., 2003; Wu et al., 2007), cotton fiber elongation (Clark et al., 2010a), and root hair growth (Liu et al., 2012). In addition, ectoapyrases contribute to regulating stomatal functions; chemical and immunological inhibition of apyrase activity induced stomatal closure (Clark et al., 2011). Recently, apyrases were shown to play important roles in the signaling steps in plant defense responses (Lim et al., 2014). Suppression of apyrase significantly altered the expression of genes involved in biotic stress responses (Lim et al., 2014). Additionally, our previous finding suggested that apyrase contributed to salt tolerance in P. euphratica (Sun et al., 2012b). NaCl shock elicited a significant rise in ATP in the ECM, but the eATP levels returned to basal levels after 20 min of salt treatment (Sun et al., 2012b). This was presumably due to ATP hydrolysis by ectoapyrase, which enabled P. euphratica to maintain low levels of eATP in a prolonged duration of salinity and, thus, prevent eATP-induced cell death (Sun et al., 2012a). Apyrase was also postulated to serve as a signal in stress responses. However, no studies have investigated in higher order plants whether apyrase promotes the hydrolysis of ATP at low temperatures and whether this activity is correlated to cold tolerance.In general, in higher order plants, low temperature causes a reduction in PM integrity. It is necessary for plant cells to reseal the PM disruption to prevent a decrease in cell viability (Yamazaki et al., 2008, 2010). PM resealing requires vesicular trafficking that includes both endocytosis and exocytosis (Togo et al., 1999; McNeil et al., 2003; Tam et al., 2010; Los et al., 2011). Ca²⁺-dependent exocytosis provides a membrane patch to the wound site, which relieves PM tension for resealing (Togo et al., 2000; Sonnemann and Bement, 2011). In animals, lysosomes are the major organelles that contribute to exocytosis-mediated membrane repair (Gerasimenko et al., 2001; Reddy et al., 2001; McNeil, 2002). Endocytosis also contributes to membrane repair by retrieving the wound site from the PM in a Ca²⁺-dependent manner (Idone et al., 2008). Shibasaki et al. (2009) suggested that low temperature inhibited the intracellular trafficking of auxin efflux carriers after the initiation of cold stress (9–12 h). However, it remains unclear whether vesicular trafficking is mediated by apyrase and eATP and contributes to cold tolerance during long-term cold stress and the subsequent recovery period.This study evaluated the roles of apyrase and eATP in cold stress signaling in woody plants. We focused on P. euphratica, because this species plays very important roles in stabilizing sand dunes and in sheltering agricultural regions in northwest China (Wei, 1993). In addition, P. euphratica trees can adapt to harsh temperature conditions in saline and alkaline desert sites (Wei, 1993). In this study, we showed that cold stress up-regulated APY2 expression in P. euphratica callus cells, but it did not induce the expression of APY1, another apyrase (Supplemental Fig. S1). Thus, APY2 may contribute to cold adaptation in P. euphratica. We tested this hypothesis by cloning the PeAPY2 gene from P. euphratica callus cells and transferring it into a model species, Arabidopsis. We then investigated the roles of PeAPY2 in eATP control and cold tolerance. Our data showed that PeAPY2 overexpression increased root membrane integrity and cold tolerance. This was likely due to effective PM repair, because endocytosis and exocytosis were up-regulated in transgenic plants. We concluded that PeAPY2 modulated eATP levels and enhanced vesicular trafficking and that these activities may have contributed to membrane resealing in cold-stressed PeAPY2-transgenic plants.