Cortical ER Research Articles

Coordinated cortical ER remodeling facilitates actomyosin ring assembly

The cortical endoplasmic reticulum (cER) is a reticulated network closely attached to the plasma membrane (PM). In the fission yeast Schizosaccharomyces pombe (S.pombe), ER-PM contacts have been suggested to restrict both the allocation and compaction of large-sized actomyosin assemblies along the lateral cell cortex. However, how cells orchestrate ER-PM contact remodeling in accordance with actomyosin coalescence for contractile ring assembly is unclear. Here, we reveal that actomyosin compaction directs the remodeling of the free tubular cER edges, whereas active exocytosis subsequently promotes the reorganization of the eisosome-bound cER rims by weakening their association or repatterning the eisosome-coated PM furrows. cER-eisosome contacts also act to reserve tubular cER edges and, hence, the ER shaping machinery at the lateral cell cortex. By manipulating or rerouting exocytosis in mutants with compromised actomyosin compaction, due to either the loss of myosin II activity or sheet-like cER morphology, we show that exocytosis facilitates ring formation likely by creating free tubular cER rims allowing robust cER remodeling. We thus propose that coordinated cER remodeling driven by both actomyosin forces and active exocytosis ensures proper contractile ring assembly. Our work also provides mechanistic insights into cER-related modulation in actomyosin ring assembly.

Autophagy of the ER requires actin assembly driven by the interaction of ER with endocytic pits.

Autophagy of the cortical ER in budding yeast was unexpectedly found to require End3, a component of the endocytic machinery that promotes the assembly of actin at endocytic pits on the plasma membrane. The cortical ER transiently interacts with invaginating endocytic pits through a linkage consisting of VAP proteins, oxysterol binding proteins and type I myosins. These proteins are required for actin assembly and for autophagy of the ER. Assembly of actin at these contact sites may direct the movement of ER away from the cortex towards sites of autophagosome assembly.

Zipcode RNA-Binding Proteins and Membrane Trafficking Proteins Cooperate to Transport Glutelin mRNAs in Rice Endosperm.

In rice (Oryza sativa) endosperm cells, mRNAs encoding glutelin and prolamine are translated on distinct cortical-endoplasmic reticulum (ER) subdomains (the cisternal-ER and protein body-ER), a process that facilitates targeting of their proteins to different endomembrane compartments. Although the cis- and trans-factors responsible for mRNA localization have been defined over the years, how these mRNAs are transported to the cortical ER has yet to be resolved. Here, we show that the two interacting glutelin zipcode RNA binding proteins (RBPs), RBP-P and RBP-L, form a quaternary complex with the membrane fusion factors n-ethylmaleimide-sensitive factor (NSF) and the small GTPase Rab5a, enabling mRNA transport on endosomes. Direct interaction of RBP-L with Rab5a, between NSF and RBP-P, and between NSF and Rab5a, were established. Biochemical and microscopic analyses confirmed the co-localization of these RBPs with NSF on Rab5a-positive endosomes that carry glutelin mRNAs. Analysis of a loss-of-function rab5a mutant showed that glutelin mRNA and the quaternary complex were mis-targeted to the extracellular paramural body structure formed by aborted endosomal trafficking, further confirming the involvement of endosomal trafficking in glutelin mRNA transport. Overall, these findings demonstrate that mRNA localization in plants co-opts membrane trafficking via the acquisition of new functional binding properties between RBPs and two essential membrane trafficking factors, thus defining an endosomal anchoring mechanism in mRNA localization.

A Novel Plant Actin-Microtubule Bridging Complex Regulates Cytoskeletal and ER Structure at Endoplasmic Reticulum-Plasma Membrane Contact Sites (EPCS)

In plants, the cortical ER network is connected to the plasma membrane through the ER-PM contact sites (EPCS), whose structures are maintained by EPCS resident proteins and the cytoskeleton. Strong co-alignment between EPCS and the cytoskeleton is observed in plants, but little is known of how the cytoskeleton is maintained and regulated at the EPCS. Here we have used a yeast-two-hybrid screen and subsequent in vivo interaction studies in plants by FRET-FLIM analysis, to identify two microtubule binding proteins, KLCR1 (Kinesin Light Chain Related protein 1) and IQD2 (IQ67-Domain 2) that interact with the actin binding protein NET3C and form a component of plant EPCS, that mediates the link between the actin and microtubule networks. The NET3C-KLCR1-IQD2 module, acting as an actin-microtubule bridging complex, has a direct influence on ER morphology. Their loss of function mutants, net3a/NET3C RNAi, 0klcr1 or iqd2, exhibit defects in pavement cell morphology which we suggest is linked to the disorganization of both actin filaments and microtubules. In conclusion, our results reveal a novel cytoskeletal associated complex, which is essential for the maintenance and organization of both cytoskeletal structure and ER morphology at the EPCS, and for normal plant cell morphogenesis.

Influence of modeled microgravity on tobacco mosaic virus

Сlinorotation is an effective method of treating diseases caused by some plant viruses. Therefore, we researched the influence of microgravity (modeled by сlinorotation) on a tobacco mosaic virus (TMV) that infects many agricultural crops. It is known that cells of plants (infected with TMV) contain viral inclusion bodies or viroplasms and amount of viral inclusion bodies correlates with harmfulness of TMV. Therefore, the purpose of this study was to find out the effect of influence of modeled microgravity on inclusion bodies of TMV. In this study, we cultivated Nicotiana tabacum L. and inoculated them with TMV that was isolated from Norway maple (Acer platanoides L.). Then we divided these plants into two groups and cultivated these plants under normal and microgravitation conditions. Microgravity conditions were modeled by сlinorotation at 2 rpm for 4 hours a day. This experiment lasted 36 days. Changes of the amount of TMV inclusion bodies in cells of plants that cultivated under normal and microgravitation conditions was investigated by luminescence microscopy. We found that formation of TMV inclusion bodies under microgravitation conditions is first slowed down compared to formation under normal conditions and then their amount quickly decreasing. These results demonstrate the gravisensitivity of TMV. It was suggested hypothesis that this viroplasm pattern caused by the disorganization of cortical microtubule-associated ER sites (C-MERS) that are nodes of cellular transport pathways and nucleation centers of cortical microtubules and cortical microfilaments. It is known that under microgravity conditions there is a disorganization and disorientation of cortical microtubules, which stabilize C-MERS on which TMV viroplasms are formed. Thus, the disorganization and disorientation of cortical microtubules probably causes the disorganization of C-MERS, which leads to a decrease in the number of TMV viroplasms under the influence of microgravity. In this context, it is worth noting that some plant viruses, such as a Wheat streak mosaic virus (WSMV), a Potato virus M (PVM) and Potato curly dwarf virus (PCDV), are gravisensitive. These viruses belong to different taxa, for example WSMV belong to genus Tritimovirus (family Potyviridae), PVM belong to genus Carlavirus (family Betaflexiviridae) and PCDV belong to plant rhabdoviruses (uncertain taxonomic position), and differ both structurally and functionally. Therefore, the gravisensitivity of these viruses can occur by other mechanisms. Thus, antiviral therapeutic effect of clinorotation based on gravisensitivity of TMV and can be used in the production of virus-free seeds. To confirm this hypothesis, it is necessary to conduct a systematic review, as well as experimentally establish the fact of the disorganization of the C-MERS under microgravity conditions.

Spatially restricted subcellular Ca2+ signaling downstream of store-operated calcium entry encoded by a cortical tunneling mechanism

Agonist-dependent Ca2+ mobilization results in Ca2+ store depletion and Store-Operated Calcium Entry (SOCE), which is spatially restricted to microdomains defined by cortical ER – plasma membrane contact sites (MCS). However, some Ca2+-dependent effectors that localize away from SOCE microdomains, are activated downstream of SOCE by mechanisms that remain obscure. One mechanism proposed initially in acinar cells and termed Ca2+ tunneling, mediates the uptake of Ca2+ flowing through SOCE into the ER followed by release at distal sites through IP3 receptors. Here we show that Ca2+ tunneling encodes exquisite specificity downstream of SOCE signal by dissecting the sensitivity and dependence of multiple effectors in HeLa cells. While mitochondria readily perceive Ca2+ release when stores are full, SOCE shows little effect in raising mitochondrial Ca2+, and Ca2+-tunneling is completely inefficient. In contrast, gKCa displays a similar sensitivity to Ca2+ release and tunneling, while the activation of NFAT1 is selectively responsive to SOCE and not to Ca2+ release. These results show that in contrast to the previously described long-range Ca2+ tunneling, in non-specialized HeLa cells this mechanism mediates spatially restricted Ca2+ rise within the cortical region of the cell to activate a specific subset of effectors.

Kv2 potassium channels form endoplasmic reticulum/plasma membrane junctions via interaction with VAPA and VAPB

Kv2.1 exhibits two distinct forms of localization patterns on the neuronal plasma membrane: One population is freely diffusive and regulates electrical activity via voltage-dependent K+ conductance while a second one localizes to micrometer-sized clusters that contain densely packed, but nonconducting, channels. We have previously established that these clusters represent endoplasmic reticulum/plasma membrane (ER/PM) junctions that function as membrane trafficking hubs and that Kv2.1 plays a structural role in forming these membrane contact sites in both primary neuronal cultures and transfected HEK cells. Clustering and the formation of ER/PM contacts are regulated by phosphorylation within the channel C terminus, offering cells fast, dynamic control over the physical relationship between the cortical ER and PM. The present study addresses the mechanisms by which Kv2.1 and the related Kv2.2 channel interact with the ER membrane. Using proximity-based biotinylation techniques in transfected HEK cells we identified ER VAMP-associated proteins (VAPs) as potential Kv2.1 interactors. Confirmation that Kv2.1 and -2.2 bind VAPA and VAPB employed colocalization/redistribution, siRNA knockdown, and Förster resonance energy transfer (FRET)-based assays. CD4 chimeras containing sequence from the Kv2.1 C terminus were used to identify a noncanonical VAP-binding motif. VAPs were first identified as proteins required for neurotransmitter release in Aplysia and are now known to be abundant scaffolding proteins involved in membrane contact site formation throughout the ER. The VAP interactome includes AKAPs, kinases, membrane trafficking machinery, and proteins regulating nonvesicular lipid transport from the ER to the PM. Therefore, the Kv2-induced VAP concentration at ER/PM contact sites is predicted to have wide-ranging effects on neuronal cell biology.

Membrane Trafficking at Kv2.1 Induced Endoplasmic Reticulum/Plasma Membrane Contact Sites

Kv2.1 is the most abundantly expressed and widely distributed voltage-gated K+ channel in mammals. In addition to regulating neuronal electrical activity, we have previously reported that a non-conducting, majority population of Kv2.1 forms micron-sized endoplasmic reticulum/plasma membrane (ER/PM) contact sites in HEK cells, neurons, and cardiac myocytes. As originally shown by the Lotan group, another non-conducting function of Kv2.1 is to enhance exocytosis in neuroendocrine cells via interaction with syntaxin. Interestingly, Kv2.1 remodels the cortical ER by binding ER resident VAPs (VAMP Associated Protein) which were originally identified as being necessary for neurotransmitter release in Aplysia. We have previously demonstrated that ER/PM junctions in non-transfected, Kv2.1-free, HEK cells are trafficking hubs,providing platforms for the delivery and retrieval of transferrin receptors, ion channels and viral coat proteins. Thus an obvious question is how the presence of Kv2.1 alters the functions associated with ER/PM junctions. This presentation will summarize our progress in determining how Kv2.1 at the ER/PM contact site influences both exo and endocytosis. Exocytosis of recycling transferrin receptors becomes sensitive to thapsigargin, i.e. ER-Ca2+, in the presence of Kv2.1. Clathrin-coated pit (CCP) behavior adjacent to the Kv2.1-induced ER/PM contact site is also changed, for in the presence of Kv2.1 both CCP lifetime and mobility are reduced. Kv2.1-induced ER/PM contact sites also remodel the diffusion landscape of the cell surface, concentrating membrane proteins in the immediate vicinity which could in turn enhance CCP/cargo interactions. Perhaps related toboth membrane protein trafficking and diffusion, ER Ca2+ homeostasis is altered when Kv2.1 is present at the ER/PM contact site. Importantly, many of the Kv2.1 influences on membrane biology just described are due to specific Kv2.1 domains and not just close apposition of the ER and PM.

Mechanical stress-induced subcellular re-localization of N-terminally truncated tobacco Nt-4/1 protein

The Nicotiana tabacum 4/1 protein (Nt-4/1) of unknown function expressed in plant vasculature has been shown to localize to cytoplasmic bodies associated with endoplasmic reticulum. Here, we analyzed molecular interactions of an Nt-4/1 mutant with a deletion of 90 N-terminal amino acid residues (Nt-4/1d90) having a diffuse GFP-like localization. Upon transient co-expression with VAP27, a membrane protein known to localize to the ER, ER-plasma membrane contact sites and plasmodesmata, Nt-4/1d90 was concentrated around the cortical ER tubules, forming a network matching the shape of the cortical ER. Additionally, in response to mechanical stress, Nt-4/1d90 was re-localized to small spherical bodies, whereas the subcellular localization of VAP27 remained essentially unaffected. The Nt-4/1d90-containing bodies associated with microtubules, which underwent noticeable bundling under the conditions of mechanical stress. The Nt-4/1d90 re-localization to spherical bodies could also be induced by incubation at an elevated temperature, although under heat shock conditions the re-localization was less efficient and incomplete. An Nt-4/1d90 mutant, which had phosphorylation-mimicking mutations in a predicted cluster of four potentially phosphorylated residues, was found to both inefficiently re-localize to spherical bodies and tend to revert back to the initial diffuse localization. The presented data show that Nt-4/1 has a potential for response to stresses that is manifested by its deletion mutant Nt-4/1d90, and this response can be mediated by protein dephosphorylation.

Genetic analysis of the regulation of the voltage-gated calcium channel homolog Cch1 by the γ subunit homolog Ecm7 and cortical ER protein Scs2 in yeast.

The yeast Cch1/Mid1 Ca2+ channel is equivalent to animal voltage-gated Ca2+ channels and activated in cells incubated in low Ca2+ medium. We herein investigated the third subunit, Ecm7, under the same cell culture conditions. The deletion of ECM7 slightly lowered Ca2+ influx activity in the CNB1+ background, in which calcineurin potentially dephosphorylates Cch1, but markedly lowered this activity in the cnb1Δ background. The deletion of the C-terminal cytoplasmic region of Ecm7 also reduced Ca2+ influx activity. We identified a novel Cch1-interacting protein, Scs2, which is known as a cortical endoplasmic reticulum membrane protein. The deletion of SCS2 did not affect Ca2+ influx activity when calcineurin was inhibited by FK506, but enhanced this activity by 35% when the enzyme was not inhibited. However, this enhancement was canceled by the deletion of ECM7. These results suggest that Cch1/Mid1 is regulated differentially by Ecm7 and Scs2 in a manner that is dependent on the phosphorylation status of Cch1.

Arabidopsis SYT1 maintains stability of cortical endoplasmic reticulum networks and VAP27-1-enriched endoplasmic reticulum-plasma membrane contact sites.

Arabidopsis synaptotagmin 1 (SYT1) is localized on the endoplasmic reticulum-plasma membrane (ER-PM) contact sites in leaf and root cells. The ER-PM localization of Arabidopsis SYT1 resembles that of the extended synaptotagmins (E-SYTs) in animal cells. In mammals, E-SYTs have been shown to regulate calcium signaling, lipid transfer, and endocytosis. Arabidopsis SYT1 was reported to be essential for maintaining cell integrity and virus movement. This study provides detailed insight into the subcellular localization of SYT1 and VAP27-1, another ER-PM-tethering protein. SYT1 and VAP27-1 were shown to be localized on distinct ER-PM contact sites. The VAP27-1-enriched ER-PM contact sites (V-EPCSs) were always in contact with the SYT1-enriched ER-PM contact sites (S-EPCSs). The V-EPCSs still existed in the leaf epidermal cells of the SYT1 null mutant; however, they were less stable than those in the wild type. The polygonal networks of cortical ER disassembled and the mobility of VAP27-1 protein on the ER-PM contact sites increased in leaf cells of the SYT1 null mutant. These results suggest that SYT1 is responsible for stabilizing the ER network and V-EPCSs.

Reticulons Regulate the ER Inheritance Block during ER Stress

Segregation of functional organelles during the cell cycle is crucial to generate healthy daughter cells. In Saccharomyces cerevisiae, ER stress causes an ER inheritance block to ensure cells inherit a functional ER. Here, we report that formation of tubular ER in the mother cell, the first step in ER inheritance,depends on functional symmetry between the cortical ER (cER) and perinuclear ER (pnER). ERstress induces functional asymmetry, blocking tubular ER formation and ER inheritance. Using fluorescence recovery after photobleaching, we show that the ER chaperone Kar2/BiP fused to GFP and an ER membrane reporter, Hmg1-GFP, behave differently in the cER and pnER. The functional asymmetry and tubular ER formation depend on Reticulons/Yop1, which maintain ER structure. LUNAPARK1 deletion in rtn1Δrtn2Δyop1Δ cells restores the pnER/cER functional asymmetry, tubular ER generation, and ER inheritance blocks. Thus, Reticulon/Yop1-dependent changes in ER structure are linked to ER inheritance during the yeast cell cycle.

Tracking Diacylglycerol Pools in Budding Yeast

Many diacylglycerol (DAG) sensing probes have been developed to monitor DAG signalling in mammalian cells. Several C1 domains have been fused to fluorescent proteins that can sense DAG in cellular membranes. The C1 domain of PKCδ is very well characterized and known to bind DAG with high affinity, and has been successfully used as a probe fused to GFP in order to monitor DAG in live cells. We have cloned the C1 domain of PKCδ (C1δ) into a centromeric yeast expression vector under a constitutive promoter for in vivo visualization of DAG pools. Localization of C1δ‐GFP was first monitored in different wild type strains grown under standard conditions. The main localization of this DAG‐binding reporter in actively growing cells was the vacuolar membrane, further confirmed by co‐localization with FM4‐64. This localization is consistent with studies done using similar probes to show that DAG localizes to the membrane of isolated vacuoles from yeast cells. Importantly, no changes in vacuolar morphology of wild type cells due to expression of C1δ‐GFP were detected. In addition to vacuolar membrane localization, the C1δ probe also associated with the plasma membrane of daughter cells and at the bud neck. In contrast to the phosphatidic acid (PA)‐binding reporter GFP‐Spo2051–91, C1δ‐GFP did not show nuclear localization as determined by co‐localization analysis using the nucleolar marker Sik1‐RFP. We then tested if the localization would change in conditions where lipid homeostasis and levels of DAG are known to be altered. For this, we used cells lacking the four enzymes responsible for the final step in the synthesis of triglycerides in yeast, namely DAG acyltransferase Dga1, the transacylase Lro1, and the steryl acyltransferases Are1 and Are2 (4Δ quadruple mutant), which accumulate DAG and have defects in both nuclear and peripheral ER membrane organization. Localization of the C1δ‐GFP probe drastically changed when expressed in 4Δ quadruple mutant cells, clearly revealing its association with the expanded nuclear and cortical ER in addition to the vacuolar membrane. Additionally, the polarized distribution of C1δ‐GFP to the plasma membrane of buds and daughter cells significantly decreased in the quadruple mutant. After validating the use of this probe in yeast, we investigated the localization of DAG pools in response to lysophosphatidylcholine (LysoPC) analogues, as we have determined they induce a 5 times increase in DAG levels. Interestingly, incubation with LysoPC analogues induced a drastic change in the localization of DAG while no differences were observed regarding the distribution of PA. These results further support the use of C1δ‐GFP as a valuable tool to track cytosolic accessible pools of DAG in yeast.Support or Funding InformationThis work was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada to VZ

Dynamic Contacts between the Endoplasmic Reticulum and the Plasma Membrane Regulate Phosphoinositide Metabolism and Control Cellular Excitability

Contacts between the endoplasmic reticulum and the plasma membrane (ER-PM junctions) facilitate intracellular communication and are essential to excitation-contraction coupling and store-operated calcium entry (SOCE). We find that they also regulate membrane phosphoinositides, lipids that control ion channels and electrical excitability. We examined how Sac1, an ER-integral 4-phosphatase, regulates PM phosphoinositides via ER-PM junctions. Through the use of HPLC mass spectrometry to quantitatively monitor phosphoinositides, and super resolution microscopy to determine the localization of Sac1, we have discovered that at steady state the cortical ER tethering protein extended synaptotagmin 2 (E-Syt2) positions the ER and Sac1 in discrete ER-PM junctions. Here, the enzyme's phosphatase activity participates in phosphoinositide homeostasis by limiting PM phosphatidylinositol 4-phosphate (PI(4)P), the precursor of PI(4,5)P2. Activation of G-protein coupled receptors that deplete PM PI(4,5)P2 also dynamically redistributes the ER, and as a consequence Sac1, away from the PM. Conversely, physiological depletion of ER luminal calcium and consequent activation of SOCE leads to increased contact between the ER and the PM and enhanced Sac1 depletion of PM PI(4)P. Further, Sac1 overexpression increases the number of STIM1 puncta at steady-state and during SOCE, resulting in higher cytoplasm calcium levels at rest and during calcium entry. Thus, the dynamic presence of Sac1 at ER-PM junctions allows it to act as a cellular sensor and controller of PM phosphoinositides and thereby influence many PM processes including electrical excitability. Supporting NIH grant R37NS08174.

Synaptotagmin SYTA Forms ER-Plasma Membrane Junctions that Are Recruited to Plasmodesmata for Plant Virus Movement

Metazoan synaptotagmins are Ca(2+) sensors that regulate exocytosis and endocytosis in various cell types, notably in nerve and neuroendocrine cells [1,2]. Recently, the structurally related extended synaptotagmins were shown to tether the cortical ER to the plasma membrane in human and yeast cells to maintain ER morphology and stabilize ER-plasma membrane (ER-PM) contact sites for intracellular lipid and Ca(2+) signaling [3, 4]. The Arabidopsis synaptotagmin SYTA regulates endocytosis and the ability of plant virus movement proteins (MPs) to alter plasmodesmata to promote virus cell-to-cell transport [5,6]. Yet how MPs modify plasmodesmata, the cellular functions of SYTA and how these aid MP activity, and the proteins essential to form plant cell ER-PM contact sites remain unknown. We addressed these questions using an Arabidopsis SYTA knockdown line syta-1 and a Tobamovirus movement protein MP(TVCV) [5, 7]. We report here that SYTA localized to ER-PM contact sites. These sites were depleted and the ER network collapsed in syta-1, and both reformed upon rescue with SYTA. MP(TVCV) accumulation in plasmodesmata, but not secretory trafficking, was also inhibited in syta-1. During infection, MP(TVCV) recruited SYTA to plasmodesmata, and SYTA and the cortical ER were subsequently remodeled to form viral replication sites adjacent to plasmodesmata in which MP(TVCV) and SYTA directly interacted caged within ER membrane. SYTA also accumulated in plasmodesmata active in MP(TVCV) transport. Our findings show that SYTA is essential to form ER-PM contact sites and suggest that MPs interact with SYTA to recruit these sites to alter plasmodesmata for virus cell-to-cell movement.

STIM1L traps and gates Orai1 channels without remodeling the cortical ER.

STIM proteins populate and expand cortical endoplasmic reticulum (ER) sheets to mediate store-operated Ca2+ entry (SOCE) by trapping and gating Orai channels in ER-plasma membrane clusters. A longer splice variant, STIM1L, forms permanent ER-plasma membrane clusters and mediates rapid Ca2+ influx in muscle. Here, we used electron microscopy, total internal reflection fluorescence (TIRF) microscopy and Ca2+ imaging to establish the trafficking and signaling properties of the two STIM1 isoforms in Stim1−/−/Stim2−/− fibroblasts. Unlike STIM1, STIM1L was poorly recruited into ER-plasma membrane clusters and did not mediate store-dependent expansion of cortical ER cisternae. Removal of the STIM1 lysine-rich tail prevented store-dependent cluster enlargement, whereas inhibition of cytosolic Ca2+ elevations or removal of the STIM1L actin-binding domain had no impact on cluster expansion. Finally, STIM1L restored robust but not accelerated SOCE and clustered with Orai1 channels more slowly than STIM1 following store depletion. These results indicate that STIM1L does not mediate rapid SOCE but can trap and gate Orai1 channels efficiently without remodeling cortical ER cisternae. The ability of STIM proteins to induce cortical ER formation is dispensable for SOCE and requires the lysine-rich tail of STIM1 involved in binding to phosphoinositides.

High-temperature cultivation of recombinant Pichia pastoris increases endoplasmic reticulum stress and decreases production of human interleukin-10.

BackgroundThe yeast Pichia pastoris (P. pastoris) has become a popular ‘cell factory’ for producing heterologous proteins, but production widely varies among proteins. Cultivation temperature is frequently reported to significantly affect protein production; however, the underlying mechanisms of this effect remain unclear.ResultsA P. pastoris strain expressing recombinant human interleukin-10 (rhIL-10) under the control of the AOX1 promoter was used as the model in this study. This system shows high-yield rhIL-10 production with prolonged methanol-induction times when cultured at 20°C but low-yield rhIL-10 production and higher cell death rates when cultured at 30°C. Further investigation showed that G3-pro-rhIL10, an immature form of rhIL-10 that contains the glycosylation-modified signal peptide, remained in the ER for a prolonged period at 30°C. The retention resulted in higher ER stress levels that were accompanied by increased ROS production, Ca2+ leakage, ER-containing autophagosomes, shortened cortical ER length and compromised induction of the unfolded protein response (UPR). In contrast, G3-pro-rhIL10 was quickly processed and eliminated from the ER at 20°C, resulting in a lower level of ER stress and improved rhIL-10 production.ConclusionsHigh-temperature cultivation of an rhIL-10 expression strain leads to prolonged retention of immature G3-pro-rhIL10 in ER, causing higher ER stress levels and thus greater yeast cell death rates and lower production of rhIL-10.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0163-7) contains supplementary material, which is available to authorized users.

Doing the math

The formation of membrane contact sites between cellular organelles is required for proper organelle communication and maintenance in the compartmentalized eukaryotic cell. We recently identified a tether that links peroxisomes to the cortical ER in the yeast, Saccharomyces cerevisiae. The tether is made up of the peroxisome biogenic protein Pex3p and the peroxisome inheritance factor Inp1p, and is formed by Inp1p-mediated linkage of ER-bound Pex3p and peroxisomal Pex3p. Here we discuss how this tether is fine-tuned to ensure that peroxisomes are stably maintained over generations of yeast cells.

Wnt5a Directs Polarized Calcium Gradients by Recruiting Cortical Endoplasmic Reticulum to the Cell Trailing Edge

Wnt5a directs the assembly of the Wnt-receptor-actin-myosin-polarity (WRAMP) structure, which integrates cell-adhesion receptors with F-actin and myosin to form a microfilament array associated with multivesicular bodies (MVBs). The WRAMP structure is polarized to the cell posterior, where it directs tail-end membrane retraction, driving forward translocation of the cell body. Here we define constituents of the WRAMP proteome, including regulators of microfilament and microtubule dynamics, protein interactions, and enzymatic activity. IQGAP1, a scaffold for F-actin nucleation and crosslinking, is necessary for WRAMP structure formation, potentially bridging microfilaments and MVBs. Vesicle coat proteins, including coatomer-I subunits, localize to and are required for the WRAMP structure. Electron microscopy and live imaging demonstrate movement of the ER to the WRAMP structure and plasma membrane, followed by elevation of intracellular Ca2+. Thus, Wnt5a controls directional movement by recruiting cortical ER to mobilize a rear-directed, localized Ca2+ signal, activating actomyosin contraction and adhesion disassembly for membrane retraction.

Give what you can and keep what you need!

EMBO J 32 18, 2439–2453 doi:10.1038/emboj.2013.170; published online July302013 During cell division, peroxisomes are inherited to daughter cells but some are retained in the mother cells. Our knowledge on how peroxisome inheritance and retention is balanced and how this is regulated for each individual organelle remains incompletely understood. The new findings by Knoblach et al (2013) published in this issue of The EMBO Journal demonstrate that Inp1p functions as a bridging protein to connect ER-resident Pex3p and peroxisomal Pex3p, which anchors peroxisomes to the cortical ER for organelle retention in the mother cell. Asymmetric peroxisome division generates peroxisomes, which lack Inp1p but contain Inp2p instead, and only these peroxisomes are primed for myosin-driven transport to daughter cells.