Characterisation of organised smooth endoplasmic reticulum suggests a route towards synthetic compartmentalisation

Engineering of subcellular compartmentalisation is one of synthetic biology’s key challenges. Among different approaches, de novo construction of a synthetic compartment is the most coveted but also most difficult option. Restructuring the endoplasmic reticulum (ER), via the introduction of recombinant oligomerising ER-membrane resident proteins, is an alternative starting point for building a new compartment. The presence of such proteins leads to a massive expansion of the ER and the formation of organised smooth endoplasmic reticulum (OSER), a large membranous compartment. However, OSER is poorly characterised and our understanding of its effect on the underlying biology of the plant is limited. Here we characterise a range of OSER compartments and show how the structure of the inducing polyprotein constructs affect the final compartment morphology, with the cytosolic-facing antiparallel oligomerisation domain demonstrated to be an essential component to trigger OSER formation. We show that while OSER retains a connection to the ER, a diffusional barrier exists to both the ER and the cytosol. Using high-resolution quantitative image analysis, we also show that the presence of this large compartment does not disrupt the rest of the ER network. Moreover, transgenic Arabidopsis constitutively expressing the compartment-forming polyproteins grew and developed normally. These properties collectively suggest that OSER could be developed as a plant synthetic biology tool for compartmentalisation, combining the benefits of several existing strategies. Only a single protein construct is necessary to induce its formation, and the compartment retains a delimiting membrane and a diffusional barrier to the rest of the cell

Vesicles versus tubes: is endoplasmic reticulum-Golgi transport in plants fundamentally different from other eukaryotes?

The endoplasmic reticulum (ER) is the gateway to the secretory pathway in all eukaryotic cells. Its products subsequently pass through the Golgi apparatus on the way to the cell surface (true secretion) or to the lytic compartment of the cell (vacuolar protein transport). In animal cells, the Golgi apparatus is present as a stationary larger order complex near the nucleus, and transport between the cortical ER and the Golgi complex occurs via an intermediate compartment which is transported on microtubules. By contrast, higher plant cells have discrete mobile Golgi stacks that move along the cortical ER, and the intermediate compartment is absent. Although many of the major molecular players involved in ER-Golgi trafficking in mammalian and yeast (Saccharomyces cerevisiae) cells have homologs in higher plants, the narrow interface (less than 500 nm) between the Golgi and the ER, together with the motility factor, makes the identification of the transport vectors responsible for bidirectional traffic between these two organelles much more difficult. Over the years, a controversy has arisen over the two major possibilities by which transfer can occur: through vesicles or direct tubular connections. In this article, four leading plant cell biologists attempted to resolve this issue. Unfortunately, their opinions are so divergent and often opposing that it was not possible to reach a consensus. Thus, we decided to let each tell his or her version individually. The review begins with an article by Federica Brandizzi that provides the necessary molecular background on coat protein complexes in relation to the so-called secretory units model for ER-Golgi transport in highly vacuolated plant cells. The second article, written by Chris Hawes, presents the evidence in favor of tubules. It is followed by an article from David Robinson defending the classical notion that transport occurs via vesicles. The last article, by Akihiko Nakano, introduces the reader to possible alternatives to vesicles or tubules, which are now emerging as a result of exciting new developments in high-resolution light microscopy in yeast

Characterisation of intracellular membrane structures derived from a massive expansion of ER membrane due to synthetic ER-membrane-resident polyproteins

The endoplasmic reticulum (ER) is a dynamic organelle that is amenable to major restructuring. Introduction of recombinant ER-membrane-resident proteins that form homo oligomers is a known method of inducing ER-proliferation: interaction of the proteins with each other alters the local structure of the ER network, leading to the formation large aggregations of expanded ER, sometimes leading to the formation organised smooth endoplasmic reticulum (OSER). However, these membrane structures formed by ER proliferation are poorly characterised and this hampers their potential development for plant synthetic biology. Here we characterise a range of ER-derived membranous compartments in tobacco and show how the nature of the polyproteins introduced into the ER membrane affect the final compartment morphology. We show that a cytosol-facing oligomerisation domain is an essential component for compartment formation. Using FRAP, we demonstrate that although the compartment retains a connection to the ER, a diffusional barrier exists to both the ER and the cytosol associated with the compartment. Using quantitative image analysis, we also show that the presence of the compartment does not disrupt the rest of the ER network. Moreover, we demonstrate that it is possible to recruit a heterologous, bacterial enzyme to the compartment and for the enzyme to accumulate to high levels. Finally, transgenic Arabidopsis constitutively expressing the compartment-forming polyproteins grew and developed normally under standard conditions

Interpersonal sensitivity and persistent attenuated psychotic symptoms in adolescence

Interpersonal sensitivity defines feelings of inner-fragility in the presence of others due to the expectation of criticism or rejection. Interpersonal sensitivity was found to be related to attenuated positive psychotic symptom during the prodromal phase of psychosis. The aims of this study were to examine if high level of interpersonal sensitivity at baseline are associated with the persistence of attenuated positive psychotic symptoms and general psychopathology at 18-month follow-up. A sample of 85 help-seeking individuals (mean age = 16.6, SD = 5.05) referred an Italian early detection project, completed the interpersonal sensitivity measure and the structured interview for prodromal symptoms (SIPS) at baseline and were assessed at 18-month follow-up using the SIPS. Results showed that individuals with high level of interpersonal sensitivity at baseline reported high level of attenuated positive psychotic symptoms (i.e., unusual thought content) and general symptoms (i.e., depression, irritability and low tolerance to daily stress) at follow-up. This study suggests that being “hypersensitive” to interpersonal interactions is a psychological feature associated with attenuated positive psychotic symptoms and general symptoms, such as depression and irritability, at 18-month follow-up. Assessing and treating inner-self fragilities may be an important step of early detection program to avoid the persistence of subtle but very distressing long-terms symptom

Is the 6 kDa tobacco etch viral protein a bona fide ERES marker?

The claim that the 6 kDa viral protein (VP) of Tobacco Etch Virus is a marker for ER exit sites (ERES) has been investigated. When transiently expressed as a CFP tagged fusion construct in tobacco mesophyll protoplasts, this integral membrane protein co-localizes with both the COPII coat protein YFP-SEC24 and the Golgi marker Man1-RFP. However, when over-expressed the VP locates to larger spherical structures which co-localize with neither ER nor Golgi markers. Nevertheless, deletion of the COPII interactive N-terminal D(X)E motif causes it to be broadly distributed throughout the ER, supporting the notion that this protein could be an ERES marker. Curiously, whereas brefeldin A (BFA) caused a typical Golgi-stack response (redistribution into the ER) of the VP in leaf epidermal cells, in protoplasts it resulted in the formation of structures identical to those formed by over-expression. However, anomalous results were obtained with protoplasts: when co-expressed with the non-cycling cis-Golgi marker Man1-RFP, a BFA-induced redistribution of the VP-CFP signal into the ER was observed, but, in the presence of the cycling Golgi marker ERD2-YFP, this did not occur. High resolution images of side-on views of Golgi stacks in epidermal cells showed that the 6 kDa VP-CFP signal overlapped considerably more with YFP-SEC24 than with Man1-RFP, indicating that the VP is proportionately more associated with ERES. However, based on a consideration of the structure of its cytoplasmic tail, the scenario that the VP collects at ERES and is transported to the cis-Golgi before being recycled back to the ER, is supported

Tubule-Guided Cell-to-Cell Movement of a Plant Virus Requires Class XI Myosin Motors

Cell-to-cell movement of plant viruses occurs via plasmodesmata (PD), organelles that evolved to facilitate intercellular communications. Viral movement proteins (MP) modify PD to allow passage of the virus particles or nucleoproteins. This passage occurs via several distinct mechanisms one of which is MP-dependent formation of the tubules that traverse PD and provide a conduit for virion translocation. The MP of tubule-forming viruses including Grapevine fanleaf virus (GFLV) recruit the plant PD receptors called Plasmodesmata Located Proteins (PDLP) to mediate tubule assembly and virus movement. Here we show that PDLP1 is transported to PD through a specific route within the secretory pathway in a myosin-dependent manner. This transport relies primarily on the class XI myosins XI-K and XI-2. Inactivation of these myosins using dominant negative inhibition results in mislocalization of PDLP and MP and suppression of GFLV movement. We also found that the proper targeting of specific markers of the Golgi apparatus, the plasma membrane, PD, lipid raft subdomains within the plasma membrane, and the tonoplast was not affected by myosin XI-K inhibition. However, the normal tonoplast dynamics required myosin XI-K activity. These results reveal a new pathway of the myosin-dependent protein trafficking to PD that is hijacked by GFLV to promote tubule-guided transport of this virus between plant cells

IRE1/bZIP60-Mediated Unfolded Protein Response Plays Distinct Roles in Plant Immunity and Abiotic Stress Responses

Endoplasmic reticulum (ER)-mediated protein secretion and quality control have been shown to play an important role in immune responses in both animals and plants. In mammals, the ER membrane-located IRE1 kinase/endoribonuclease, a key regulator of unfolded protein response (UPR), is required for plasma cell development to accommodate massive secretion of immunoglobulins. Plant cells can secrete the so-called pathogenesis-related (PR) proteins with antimicrobial activities upon pathogen challenge. However, whether IRE1 plays any role in plant immunity is not known. Arabidopsis thaliana has two copies of IRE1, IRE1a and IRE1b. Here, we show that both IRE1a and IRE1b are transcriptionally induced during chemically-induced ER stress, bacterial pathogen infection and treatment with the immune signal salicylic acid (SA). However, we found that IRE1a plays a predominant role in the secretion of PR proteins upon SA treatment. Consequently, the ire1a mutant plants show enhanced susceptibility to a bacterial pathogen and are deficient in establishing systemic acquired resistance (SAR), whereas ire1b is unaffected in these responses. We further demonstrate that the immune deficiency in ire1a is due to a defect in SA- and pathogen-triggered, IRE1-mediated cytoplasmic splicing of the bZIP60 mRNA, which encodes a transcription factor involved in the expression of UPR-responsive genes. Consistently, IRE1a is preferentially required for bZIP60 splicing upon pathogen infection, while IRE1b plays a major role in bZIP60 processing upon Tunicamycin (Tm)-induced stress. We also show that SA-dependent induction of UPR-responsive genes is altered in the bzip60 mutant resulting in a moderate susceptibility to a bacterial pathogen. These results indicate that the IRE1/bZIP60 branch of UPR is a part of the plant response to pathogens for which the two Arabidopsis IRE1 isoforms play only partially overlapping roles and that IRE1 has both bZIP60-dependent and bZIP60-independent functions in plant immunity