Live cell imaging of telomerase RNA dynamics reveals cell cycle-dependent clustering of telomerase at elongating telomeres

The telomerase, which is composed of both protein and RNA, maintains genome stability by replenishing telomeric repeats at the ends of chromosomes. Here, we use live-cell imaging to follow yeast telomerase RNA dynamics and recruitment to telomeres in single cells. Tracking of single telomerase particles revealed a diffusive behavior and transient association with telomeres in G1 and G2 phases of the cell cycle. Interestingly, concurrent with telomere elongation in late S phase, a subset of telomerase enzyme clusters and stably associates with few telomeres. Our data show that this clustering represents elongating telomerase and it depends on regulators of telomerase at telomeres (MRX, Tel1, Rif1/2, and Cdc13). Furthermore, the assay revealed premature telomere elongation in G1 in a rif1/2 strains, suggesting that Rif1/2 act as cell-cycle dependent negative regulators of telomerase. We propose that telomere elongation is organized around a local and transient accumulation of several telomerases on a few telomeres.     

Fluorescent castasterone reveals BRI1 signaling from the plasma membrane

Receptor-mediated endocytosis is an integral part of signal transduction as it mediates signal attenuation and provides spatial and temporal dimensions to signaling events. One of the best-studied leucine-rich repeat receptor-like kinases in plants, BRASSINOSTEROID INSENSITIVE 1 (BRI1), perceives its ligand, the brassinosteroid (BR) hormone, at the cell surface and is constitutively endocytosed. However, the importance of endocytosis for BR signaling remains unclear. Here we developed a bioactive, fluorescent BR analog, Alexa Fluor 647-castasterone (AFCS), and visualized the endocytosis of BRI1-AFCS complexes in living Arabidopsis thaliana cells. Impairment of endocytosis dependent on clathrin and the guanine nucleotide exchange factor for ARF GTPases (ARF-GEF) GNOM enhanced BR signaling by retaining active BRI1-ligand complexes at the plasma membrane. Increasing the trans-Golgi network/early endosome pool of BRI1-BR complexes did not affect BR signaling. Our findings provide what is to our knowledge the first visualization of receptor-ligand complexes in plants and reveal clathrin- and ARF-GEF-dependent endocytic regulation of BR signaling from the plasma membrane.     

Plant pattern recognition receptor complexes at the plasma membrane

A key feature of innate immunity is the ability to recognize and respond to potential pathogens in a highly sensitive and specific manner. In plants, the activation of pattern recognition receptors (PRRs) by pathogen-associated molecular patterns (PAMPs) elicits a defense programme known as PAMP-triggered immunity (PTI). Although only a handful of PAMP-PRR pairs have been defined, all known PRRs are modular transmembrane proteins containing ligand-binding ectodomains. It is becoming clear that PRRs do not act alone but rather function as part of multi-protein complexes at the plasma membrane. Recent studies describing the molecular interactions and protein modifications that occur between PRRs and their regulatory proteins have provided important mechanistic insight into how plants avoid infection and achieve immunity.     

Quantitative proteomics reveals dynamic changes in the plasma membrane during Arabidopsis immune signaling

The plant plasma membrane is a crucial mediator of the interaction between plants and microbes. Understanding how the plasma membrane proteome responds to diverse immune signaling events will lead to a greater understanding of plant immunity and uncover novel targets for crop improvement. Here we report the results from a large scale quantitative proteomics study of plasma membrane-enriched fractions upon activation of the Arabidopsis thaliana immune receptor RPS2. More than 2300 proteins were identified in total, with 1353 proteins reproducibly identified across multiple replications. Label-free spectral counting was employed to quantify the relative protein abundance between different treatment samples. Over 20% of up-regulated proteins have known roles in plant immune responses. Significantly changing proteins include those involved in calcium and lipid signaling, membrane transport, primary and secondary metabolism, protein phosphorylation, redox homeostasis, and vesicle trafficking. A subset of differentially regulated proteins was independently validated during bacterial infection. This study presents the largest quantitative proteomics data set of plant immunity to date and provides a framework for understanding global plasma membrane proteome dynamics during plant immune responses.     

Secretion machinery at the cell plasma membrane

Secretion is a fundamental cellular process involving the regulated release of intracellular products from cells. Physiological functions such as neurotransmission, or the release of hormones and digestive enzymes, are all governed by cell secretion. Three critical activities occur at the cell plasma membrane to ensure secretion. Membrane-bound secretory vesicles dock, fuse, and expel their contents to the outside via specialized and permanent plasma membrane structures, called porosomes or fusion pores. In recent years, significant progress has been made in our understanding of these three key cellular activities required for cell secretion. The molecular machinery and mechanism involving them is summarized in this article.     

Bacterial subversion of host actin dynamics at the plasma membrane

Invasion of non-phagocytic cells by a number of bacterial pathogens involves the subversion of the actin cytoskeletal remodelling machinery to produce actin-rich cell surface projections designed to engulf the bacteria. The signalling that occurs to induce these actin-rich structures has considerable overlap among a diverse group of bacteria. The molecular organization within these structures act in concert to internalize the invading pathogen. This dynamic process could be subdivided into three acts - actin recruitment, engulfment, and finally, actin disassembly/internalization. This review will present the current state of knowledge of the molecular processes involved in each stage of bacterial invasion, and provide a perspective that highlights the temporal and spatial control of actin remodelling that occurs during bacterial invasion.     

Scanning angle interference microscopy reveals cell dynamics at the nanoscale

Emerging questions in cell biology necessitate nanoscale imaging in live cells. Here we present scanning angle interference microscopy, which is capable of localizing fluorescent objects with nanoscale precision along the optical axis in motile cellular structures. We use this approach to resolve nanotopographical features of the cell membrane and cytoskeleton as well as the temporal evolution, three-dimensional architecture and nanoscale dynamics of focal adhesion complexes.     

Analysis of estrogen receptor alpha signaling complex at the plasma membrane

There is accumulating evidence that the estrogen receptor (ER) can transduce specific signals at the plasma membrane. We tried to clarify the biological function of ER as a signaling molecule by identifying proteins that interact with the membrane-localized ER. The activation function 1 and 2 (AF-1 and AF-2) domains of ERalpha with or without the membrane-targeting sequence were stably expressed in the breast cancer cell line, MCF-7. The level of tyrosine phosphorylation of AF-2 was significantly elevated by the membrane localization. By mass-spectrometry analysis, alpha- and beta-tubulins and heat shock protein 70 were identified as the AF-1-associated proteins. Of these, tubulins are associated only with membrane-targeted AF-1.     

Direct cadherin-activated cell signaling: a view from the plasma membrane

Classical cadherin adhesion molecules are key determinants of cell recognition and tissue morphogenesis, with diverse effects on cell behavior. Recent developments indicate that classical cadherins are adhesion-activated signaling receptors. In particular, early-immediate Rac signaling is emerging as a mechanism to coordinate cadherin-actin integration at the plasma membrane.     

Synergistic assembly of linker for activation of T cells signaling protein complexes in T cell plasma membrane domains

Transmembrane adaptor molecule LAT (linker for activation of T cells) forms a central scaffold for signaling protein complexes that accumulate in the vicinity of activated T cell antigen receptors (TCR). Here we used biochemical analysis of immunoisolated plasma membrane domains and fluorescence imaging of green fluorescence protein-tagged signaling proteins to investigate the contributions of different tyrosine-based signaling protein docking sites of LAT to the formation of LAT signaling protein assemblies in TCR membrane domains. We found that the phospholipase C gamma docking site of LAT and different Grb2/Gads docking sites function in an interdependent fashion and synergize to accumulate LAT, Grb2, and phospholipase C gamma in TCR signaling assemblies. Two-dimensional gels showed that Grb2 is a predominant cytoplasmic adaptor in the isolated LAT signaling complexes, whereas Gads, Crk-1, and Grap are present in lower amounts. Taken together our data suggest a synergistic assembly of multimolecular TCR.LAT signal transduction complexes in T cell plasma membrane domains.     

Nerve growth factor signaling in caveolae-like domains at the plasma membrane

Nerve growth factor (NGF) binding to its receptors TrkA and p75(NTR) enhances the survival, differentiation, and maintenance of neurons. Recent studies have suggested that NGF receptor activation may occur in caveolae or caveolae-like membranes (CLM). This is an intriguing possibility because caveolae have been shown to contain many of the signaling intermediates in the TrkA signaling cascade. To examine the membrane localization of TrkA and p75(NTR), we isolated caveolae from 3T3-TrkA-p75 cells and CLM from PC12 cells. Immunoblot analysis showed that TrkA and p75(NTR) were enriched about 13- and 25-fold, respectively, in caveolae and CLM. Binding and cross-linking studies demonstrated that the NGF binding to both TrkA and p75(NTR) was considerably enriched in CLM and that about 90% of high affinity binding to TrkA was present in CLM. When PC12 cells were treated with NGF, virtually all activated (i.e. tyrosine phosphorylated) TrkA was found in the CLM. Remarkably, in NGF-treated cells, it was only in CLM that activated TrkA was coimmunoprecipitated with phosphorylated Shc and PLCgamma. These results document a signaling role for TrkA in CLM and suggest that both TrkA and p75(NTR) signaling are initiated from these membranes.     

Prolonged EGFR signaling by ERBB2-mediated sequestration at the plasma membrane

We have analyzed the spatial-temporal regulation of epidermal growth factor receptor (EGFR) phosphorylation by the orphan erbB2 receptor. It is shown that EGFR association with erbB2 is sufficient to prolong and enhance the net phosphorylation of EGFR, independent of the kinase activity of erbB2. This enhanced EGFR signaling was rather caused by erbB2-mediated retention of phosphorylated EGFR at the plasma membrane (PM), thereby preventing EGFR dephosphorylation and signal termination by endomembrane-bound protein tyrosine phosphatases (PTPs). EGF-induced EGFR internalization was indeed blocked in the presence of high levels of erbB2 or if cbl binding of EGFR was impaired. This erbB2-mediated blockage of the entry of activated EGFR into clathrin-coated vesicles could be alleviated by antibody-mediated disruption of the interaction between EGFR and erbB2. These results identify erbB2-mediated dominant trapping of phosphorylated EGFR at the PM as a mechanism that prolongs EGFR signaling, by sequestration of activated EGFR away from intracellular sites of high PTP activity.     

Seven transmembrane receptor core signaling complexes are assembled prior to plasma membrane trafficking

Much is known about beta2-adrenergic receptor trafficking and internalization following prolonged agonist stimulation. However, less is known about outward trafficking of the beta2-adrenergic receptor 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. Here, by using a combination of bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and confocal microscopy, we evaluated the steps in the formation of the core receptor-G protein heterotrimer complex. By using dominant negative Rab and Sar GTPase constructs, we demonstrate that receptor dimers and receptor-G betagamma complexes initially associate in the endoplasmic reticulum, whereas G alpha subunits are added to the complex during endoplasmic reticulum-Golgi transit. We also observed that G protein heterotrimers adopt different trafficking itineraries when expressed alone or with stoichiometric co-expression with receptor. Furthermore, deliberate mistargeting of specific components of these complexes leads to diversion of other members from their normal subcellular localization, confirming the role of these early interactions in targeting and formation of specific signaling complexes.     

Trafficking of preassembled opioid mu-delta heterooligomer-Gz signaling complexes to the plasma membrane: coregulation by agonists

The cellular site of formation, Galpha-coupling preference, and agonist regulation of mu-delta opioid receptor (OR) heterooligomers were studied. Bioluminescence resonance energy transfer (BRET) showed that mu-deltaOR heterooligomers, composed of preformed mu and delta homooligomers, interacted constitutively in the endoplasmic reticulum (ER) with Galpha-proteins forming heteromeric signaling complexes before being targeted to the plasma membrane. Compared to muOR homooligomers, the mu-delta heterooligomers showed higher affinity and efficiency of interaction for Gz over Gi, indicating a switch in G-protein preference. Treatment with DAMGO or deltorphin II led to coregulated internalization of both receptors, whereas DPDPE and DSLET had no effect on mu-delta internalization. Staggered expression resulted in non-interacting mu and delta receptors, even though both receptors were colocalized at the cell surface. Agonists failed to induce BRET between staggered receptors, and resulted in internalization solely of the receptor targeted by agonist. Thus, mu-deltaOR heterooligomers form and preferentially associate with Gz to generate a signaling complex in the ER, and have a distinct agonist-internalization profile compared to either mu or delta homooligomers.     

Autophagy modulates dynamics of connexins at the plasma membrane in a ubiquitin-dependent manner

Different pathways contribute to the turnover of connexins, the main structural components of gap junctions (GJs). The cellular pool of connexins targeted to each pathway and the functional consequences of degradation through these degradative pathways are unknown. In this work, we focused on the contribution of macroautophagy to connexin degradation. Using pharmacological and genetic blockage of macroautophagy both in vitro and in vivo, we found that the cellular pool targeted by this autophagic system is primarily the one organized into GJs. Interruption of connexins' macroautophagy resulted in their retention at the plasma membrane in the form of functional GJs and subsequent increased GJ-mediated intercellular diffusion. Up-regulation of macroautophagy alone is not sufficient to induce connexin internalization and degradation. To better understand what factors determine the autophagic degradation of GJ connexins, we analyzed the changes undergone by the fraction of plasma membrane connexin 43 targeted for macroautophagy and the sequence of events that trigger this process. We found that Nedd4-mediated ubiquitinylation of the connexin molecule is required to recruit the adaptor protein Eps15 to the GJ and to initiate the autophagy-dependent internalization and degradation of connexin 43. This study reveals a novel regulatory role for macroautophagy in GJ function that is directly dependent on the ubiquitinylation of plasma membrane connexins.     

Functional mapping of Ca2+ signaling complexes in plasma membrane microdomains of polarized cells

Many cells cluster signaling complexes in plasma membrane microdomains. Polarized secretory cells cluster all Ca2+ signaling proteins, including GPCRs, at the apical pole. The functional significance of such an arrangement is not known because of a lack of techniques for functional mapping of signaling complexes at plasma membrane patches. In the present work, we developed such a technique based on the use of two patch pipettes, a recording and a stimulating pipette (SP). Including 20% glycerol in the SP solution increased the viscosity and the hydrophobicity to prevent leakage and formation of tight seals on the plasma membrane. This allowed moving the SP between sites to stimulate multiple patches of the same cell and with the same agonist concentrations. Functional mapping of Ca2+ signaling in pancreatic acinar cells revealed that the M3, cholecystokinin, and bombesin signaling complexes at the apical pole are much more sensitive to stimulation than those at the basal pole. Furthermore, at physiological agonist concentrations, Ca2+ signals could be evoked only by stimulation of membrane patches at the apical pole. [Ca2+](i) imaging revealed that Ca2+ waves were invariably initiated at the site of apical membrane patch stimulation, suggesting that long range diffusion of second messengers is not obligatory to initiate and propagate apical-to-basal Ca2+ waves. The present studies reveal a remarkable heterogeneity in responsiveness of Ca2+ signaling complexes at membrane microdomains, with the most responsive complexes confined to the apical pole, probably to restrict the Ca2+ signals to the site of exocytosis and allow the polarized functions of secretory cells.     

Insulin signaling in microdomains of the plasma membrane

Although the effects of insulin on glucose and lipid metabolism are well documented, gaps remain in our understanding of the precise molecular mechanisms of signal transduction. Recent evidence suggests that compartmentalization of signaling molecules and metabolic enzymes may explain the unique cellular effects of the hormone. Signal initiation from the insulin receptor is restricted in part to caveolae microdomains of the plasma membrane. A fraction of the insulin receptor directly interacts with caveolin, thus directing the protein to caveolae. Following its activation by insulin, the receptor recruits a series of adapter proteins, resulting in the activation of the G protein TC10, which also resides in caveolae. TC10 can influence a number of cellular processes, including changes in the actin cytoskeleton, recruitment of effector including the adapter protein CIP4, and assembly of the exocyst complex. These events play crucial roles in the trafficking, docking and fusion of vesicles containing the insulin-responsive glucose transporter Glut4 at the plasma membrane.     

Distribution of sterol-specific complexes in a continually shearing region of a plasma membrane and at procaryotic-eucaryotic cell junctions

A narrow zone of plasma membrane between the head and body of a protozoan from termites undergoes continual in-plane shear because the head rotates continuously in the same direction relative to the cell body (Tamm, S.L., and S. Tamm, 1974, Proc. Natl. Acad. Sci. USA 71:4589- 4593). Using filipin and digitonin as cytochemical probes for cholesterol and related 3-beta-hydroxysterols, we found a high level of sterol-specific complexes, visible as membrane lesions in thin sections, in both shearing and nonshearing regions of the membrane, indicating no difference in sterol content. This confirmed previous observations that any region of the fluid membrane can undergo shear, but that this occurs only at certain locations due to cell geometry and proximity to rotating cytoskeletal structures. Filipin and digitonin did not disrupt the plasma membrane at the junctions with ectosymbiotic rod and fusiform bacteria (i.e., membrane pockets and ridges). However, pepsin degradation of dense material coating the junctional membranes resulted in a positive response of these regions to filipin. Fluorescence microscopy revealed a bright halo around each rod bacterium, due to filipin-sterol binding in the sides of the membrane pockets, but no fluorescence at the bottom of the pockets; the same fluorescence pattern was found in pepsin-treated cells despite the presence of sterols throughout the pocket membrane, as shown by electron microscopy. These findings indicate that (a) regional constraints may restrict the ability of filipin to interact with sterols or form visible membrane lesions, and (b) a negative response to filipin, assayed by either electron or fluorescence microscopy, is not sufficient to demonstrate low membrane sterol concentration, particularly in membrane domains characterized by closely associated proteins.