Suborganellar resolution imaging for the localisation of human glycosylation enzymes in tobacco Golgi bodies.

Plant cells are a capable system for producing economically and therapeutically important proteins for a variety of applications, and are considered a safer production system than some existing hosts such as bacteria or yeasts. However, plants do not perform protein modifications in the same manner as mammalian cells do. This can impact on protein functionality for plant-produced human therapeutics. This obstacle can be overcome by creating a plant-based system capable of 'humanising' proteins of interest resulting in a glycosylation profile of synthetic plant-produced proteins as it would occur in mammalian systems. For this, the human glycosylation enzymes (HuGEs) involved in N-linked glycosylation N-acetylglucosaminyltransferase IV and V (GNTIV and GNTV), ß-1,4-galactosyltransferase (B4GALT1), and α-2,6-sialyltransferase (ST6GAL) were expressed in plant cells. For these enzymes to carry out the stepwise glycosylation functions, they need to localise to late Golgi body cisternae. This was achieved by a protein targeting strategy of replacing the mammalian Golgi targeting domains (Cytoplasmic-Transmembrane-Stem (CTS) regions) with plant-specific ones. Using high-resolution and dynamic confocal microscopy, we show that GNTIV and GNTV were successfully targeted to the medial-Golgi cisternae while ST6GAL and B4GALT1 were targeted to trans-Golgi cisternae. Plant cells are a promising system to produce human therapeutics for example proteins used in enzyme replacement therapies. Plants can provide safer and cheaper alternatives to existing expression systems such as mammalian cell culture, bacteria or yeast. An important factor for the functionality of therapeutic proteins though are protein modifications specific to human cells. However, plants do not perform protein modifications in the same manner as human cells do. Therefore, plant cells need to be genetically modified to mimic human protein modifications patterns. The modification of importance here, is called N-linked glycosylation and adds specific sugar molecules onto the proteins. Here we show the expression of four human glycosylation enzymes, which are required for N-linked glycosylation, in plant cells. In addition, as these protein modifications are carried out in cells resembling a factory production line, it is important that the human glycosylation enzymes be placed in the correct cellular compartments and in the correct order. This is carried out in Golgi bodies. Golgi bodies are composed of several defined stacks termed cis-, medial and trans-Golgi body stacks. For correct protein function, two of these human glycosylation enzymes need to be placed in the medial-Golgi attacks and the other two in the trans-Golgi stacks. Using high-resolution laser microscopy in live plant cells, we show here that the human glycosylation enzymes are sent within the cells to the correct Golgi body stacks. These are first steps to modify plant cells in order to produce human therapeutics.

On the nature of the plant ER exit sites.

In plants, the endoplasmic reticulum (ER) and Golgi bodies are not only in close proximity, but are also physically linked. This unique organization raises questions about the nature of the transport vectors carrying cargo between the two organelles. Same as in metazoan and yeast cells, it was suggested that cargo is transported from the ER to Golgi cisternae via COPII-coated vesicles produced at ribosome-free ER exit sites (ERES). Recent developments in mammalian cell research suggest, though, that COPII helps to select secretory cargo, but does not coat the carriers leaving the ER. Furthermore, it was shown that mammalian ERES expand into a tubular network containing secretory cargo, but no COPII components. Because of the close association of the ER and Golgi bodies in plant cells, it was previously proposed that ERES and the Golgi comprise a secretory unit that travels over or with a motile ER membrane. In this study, we aimed to explore the nature of ERES in plant cells and took advantage of high-resolution confocal microscopy and imaged ERES labelled with canonical markers (Sar1a, Sec16, Sec24). We found that ERES are dynamically connected to Golgi bodies and most likely represent pre-cis-Golgi cisternae. Furthermore, we showed fine tubular connections from the ER to Golgi compartments (ERGo tubules) as well as fine protrusions from ERES/Golgi cisternae connecting with the ER. We suggest that these tubules observed between the ER and Golgi as well as between the ER and ERES are involved in stabilizing the physical connection between ER and ERES/Golgi cisternae, but may also be involved in cargo transport from the ER to Golgi bodies.

Insomnia with objective short sleep duration is associated with longer duration of insomnia in the Freiburg Insomnia Cohort compared to insomnia with normal sleep duration, but not with hypertension.

STUDY OBJECTIVES: To replicate the association between insomnia with objective short sleep duration and hypertension, type 2 diabetes and duration of insomnia. DESIGN: Retrospective case-control study. SETTING: Department of Psychiatry and Psychotherapy, Medical Center-University of Freiburg. PARTICIPANTS: 328 patients with primary insomnia classified according to DSM-IV criteria (125 males, 203 females, 44.3 ± 12.2 years). INTERVENTIONS: N/A. MEASUREMENTS: All participants were investigated using polysomnography, blood pressure measurements, and fasting routine laboratory. RESULTS: Insomnia patients with short sleep duration (< 6 hours) in the first night of laboratory sleep presented with a longer duration of insomnia compared to those with normal sleep duration (≥ 6 hours) in the first night of laboratory sleep. Insomnia patients who were categorised as short sleepers in either night were not more likely to suffer from hypertension (systolic blood pressure of ≥ 140 mm Hg, diastolic blood pressure of ≥ 90 mm Hg, or a previously established diagnosis). Data analysis showed that insomnia patients with objective short sleep duration were not more likely to suffer from type 2 diabetes (fasting plasma glucose level of ≥ 126 mg/dl, or a previously established diagnosis). However, the diabetes analysis was only based on a very small number of diabetes cases. As a new finding, insomnia patients who were categorised as short sleepers in either night presented with increases in liver enzyme levels. CONCLUSIONS: The finding on insomnia duration supports the concept of two distinct sub-groups of insomnia, namely insomnia with, and without, objectively determined short sleep duration. However, our data challenges previous findings that insomnia patients with short sleep duration are more likely to suffer from hypertension.