N-Linked glycan site occupancy impacts the distribution of a potassium channel in the cell body and outgrowths of neuronal-derived cells

Background

Vacancy of occupied N-glycosylation sites of glycoproteins is quite disruptive to a multicellular organism, as underlined by congenital disorders of glycosylation. Since a neuronal component is typically associated with this disease, we evaluated the impact of N-glycosylation processing of a neuronal voltage gated potassium channel, Kv3.1b, expressed in a neuronal-derived cell line, B35 neuroblastoma cells.

Methods

Total internal reflection fluorescence and differential interference contrast microscopy measurements of live B35 cells expressing wild type and glycosylation mutant Kv3.1b proteins were used to evaluate the distribution of the various forms of the Kv3.1b protein in the cell body and outgrowths. Cell adhesion assays were also employed.

Results

Microscopy images revealed that occupancy of both N-glycosylation sites of Kv3.1b had relatively similar amounts of Kv3.1b in the outgrowth and cell body while vacancy of one or both sites led to increased accumulation of Kv3.1b in the cell body. Further both the fully glycosylated and partially glycosylated N229Q Kv3.1b proteins formed higher density particles in outgrowths compared to cell body. Cellular assays demonstrated that the distinct spatial arrangements altered cell adhesion properties.

Conclusions

Our findings provide direct evidence that occupancy of the N-glycosylation sites of Kv3.1b contributes significantly to its lateral heterogeneity in membranes of neuronal-derived cells, and in turn alters cellular properties.

General significance

Our study demonstrates that N-glycans of Kv3.1b contain information regarding the association, clustering, and distribution of Kv3.1b in the cell membrane, and furthermore that decreased occupancy caused by congenital disorders of glycosylation may alter the biological activity of Kv3.1b.     

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards understanding its mechanism. Protonation and conformational changes affect the vibration pattern of amino acid side chains and of the peptide bond, respectively, both of which can be probed by infrared (IR) difference spectroscopy. For proteins whose function can be repetitively and reproducibly triggered by light, it is possible to obtain infrared difference spectra with (sub)microsecond resolution over a broad spectral range using the step-scan Fourier transform infrared technique. With ~10²-10³ repetitions of the photoreaction, the minimum number to complete a scan at reasonable spectral resolution and bandwidth, the noise level in the absorption difference spectra can be as low as ~10^-4, sufficient to follow the kinetics of protonation changes from a single amino acid. Lower noise levels can be accomplished by more data averaging and/or mathematical processing. The amount of protein required for optimal results is between 5-100 µg, depending on the sampling technique used. Regarding additional requirements, the protein needs to be first concentrated in a low ionic strength buffer and then dried to form a film. The protein film is hydrated prior to the experiment, either with little droplets of water or under controlled atmospheric humidity. The attained hydration level (g of water / g of protein) is gauged from an IR absorption spectrum. To showcase the technique, we studied the photocycle of the light-driven proton-pump bacteriorhodopsin in its native purple membrane environment, and of the light-gated ion channel channelrhodopsin-2 solubilized in detergent.     

Light from below matters: Quantifying the consequences of responses to far‐red light reflected upwards for plant performance in heterogeneous canopies

In vegetation stands, plants receive red to far‐red ratio (R:FR) signals of varying strength from all directions. However, plant responses to variations in R:FR reflected from below have been largely ignored despite their potential consequences for plant performance. Using a heterogeneous rose canopy, which consists of bent shoots down in the canopy and vertically growing upright shoots, we quantified upward far‐red reflection by bent shoots and its consequences for upright shoot architecture. With a three‐dimensional plant model, we assessed consequences of responses to R:FR from below for plant photosynthesis. Bent shoots reflected substantially more far‐red than red light, causing reduced R:FR in light reflected upwards. Leaf inclination angles increased in upright shoots which received low R:FR reflected from below. The increased leaf angle led to an increase in simulated plant photosynthesis only when this low R:FR was reflected off their own bent shoots and not when it reflected off neighbour bent shoots. We conclude that plant response to R:FR from below is an under‐explored phenomenon which may have contrasting consequences for plant performance depending on the type of vegetation or crop system. The responses are beneficial for performance only when R:FR is reflected by lower foliage of the same plants.     

Host cell protein quantification by fourier transform mid infrared spectroscopy (FT‐MIR)

Process development in up‐ and downstream processing requires enhanced, non‐time‐consuming, and non‐expensive monitoring techniques to track product purity, for example, the level of endotoxins, viral particles, and host cell proteins (HCPs). Currently, HCP amounts are measured by laborious and expensive HCP‐enzyme‐linked immunosorbent assay (ELISA) assays best suited for measuring HCP amounts in the low concentration regime. The measurement of higher HCP amounts using this method requires dilution steps, adding dilution errors to the measurement. In this work we evaluated the suitability of attenuated total reflection spectroscopy for HCP quantification in process development, using clarified cell culture fluid from monoclonal antibody producing Chinese hamster ovary‐cells after treatment with different polyelectrolytes for semi‐selective clarification. Forty undiluted samples were chosen for multivariate data analysis in the middle infrared range and predicted HCP‐values were in good agreement with results obtained by an ELISA‐assay, suggesting the suitability of this new method for HCP‐quantification. As this method is able to quantify HCP titers ranging from approximately at least 20,000–200,000 ng mL^?1, it is suitable especially for monitoring of process development steps with higher HCP concentrations, omitting dilution errors associated with ELISA assays. Biotechnol. Bioeng. 2013; 110: 252–259. © 2012 Wiley Periodicals, Inc.     

Localisation of endothelin B receptor variants to plasma membrane microdomains and its effects on downstream signalling

The endothelin B (ET_B) receptor can undergo a proteolytic cleavage resulting in an unglycosylated N-terminally truncated receptor. We investigated whether ET_B receptor processing affects caveolar localisation and mitogenic signalling. Distinct subcellular localisations of ET_B receptor constructs and epidermal growth factor (EGF) receptor ligands were analysed performing detergent-free caveolae preparations and total internal reflection fluorescence microscopy. ET_B receptor-induced transactivation of the EGF receptor and its downstream signalling was investigated performing shedding assays and ERK1/2 phosphorylation analyses. In COS7 cells, the N-terminally truncated but not the full-length or glycosylation-deficient ET_B receptor localised to caveolae. In caveolae-free HEK293 cells, only ET_B receptor constructs fused to caveolin-2 localised to membrane microdomains. A caveolar accumulation of the ET_B receptor disfavoured EGF receptor ligand shedding. Nonetheless, the activation of ERK1/2 was efficient and long-lasting. In HEK293 cells, the shedding activity was also impaired by N-terminal truncation. The subsequent ERK1/2 phosphorylation was long-lasting only for the full-length ET_B receptor. We conclude that the ET_B receptor localisation might depend on the presence of caveolae within the cell investigated. The data further suggest that caveolar enrichment of ET_B receptors does not facilitate the release of EGF receptor ligands. However, independent of their localisation, ET_B receptors are able to induce an ERK1/2 phosphorylation.     

Protein adsorption and displacement at lipid layers determined by total internal reflection fluorescence (TIRF)

In many drug delivery systems such as liposomes, the adsorption of interstitial proteins upon administration can have a huge effect on the elimination, release, and stability of the delivery system. For example, it is assumed that PEGylated liposomes prevent the adsorption of opsonins and thereby prolong the circulation time in vivo, and EMEA guidelines recommend that more than 80% of the protein antigen is adsorbed in the formulation of adjuvant systems. However, few methods exist to elucidate this protein adsorption. The present study indicates that total internal reflection fluorescence (TIRF) is a possible method to examine the adsorption and exchange of proteins at lipid surfaces. In the TIRF set-up, a lipid layer can be formed [exemplified with dimethyldioctadecylammonium bromide (DDA) and D-(+)-trehalose 6,6’-dibehenate (TDB)] whereafter protein (i.e., ovalbumin or an antigen, Ag85B-ESAT-6) is adsorbed, and these proteins can subsequently be displaced by the abundant interstitial protein (i.e., serum albumin).     

Make an Art Slide Library

Self-assessment can play an important role in teachers’ personal and professional development and is encouraged by educational programs worldwide. This article reports on a Greek study that aimed to investigate generalist preservice kindergarten teachers’ self-assessment of their music teaching ability. One hundred participants were asked to design and deliver three music sessions for the kindergarten and then prepare a short reflective portfolio. Qualitative analysis of the portfolios led to the identification of different issues related to student practices, including preservice teacher training, teaching ability, the effect of different school environments and policies, and personal thoughts and feelings. Finally, the article discusses the role of self-assessment in understanding students’ academic needs and suggests initiatives and policies that might promote better teaching practices in music education, both at kindergarten and university level.     

Results of human evolution research in Czechoslovakia

The results of anthropological research into human evolution have been discussed. Only two research places in CSSR have been engaged with the human evolution: the Department Anthropos in the Moravian Museum in Brno and the Laboratory of Evolutionary Biology of the Czechoslovak Academy of Sciences in Praha. The first of them has been mainly concerned with the paleontological and archeological research reported elsewhere. In the second, the following main questions of human evolution have been discussed: the evolution of bipedal locomotion, the morphological aspects, both qualitative and quantitative ones, the locomotion in other primates and various aspects of their behaviour have been studied. In this connection also various questions of the evolution the of human mind and social consciousness were studied. Special attention has been paid to role of neoteny in the evolution of man and to the import of synergism of the main evolutionary factors. As one of the main results is the principle of sociogenesis viewed and its various aspects. Its top product is human consciousness integrating the most important results of human thinking. Much attention has been paid to its evolution on the basis of the principle of reflection. Also the philosophical and ideological consequences of the sociogenesis as the main trend in the evolution of organisms have been elaborated in detail.     

In vitro reconstitution of a transport complex containing Rab27a, melanophilin and myosin Va

Rab27a and melanophilin (Mlph) are required in vivo to form a melanosome receptor for myosin Va in which Rab27a anchored in the melanosome membrane recruits Mlph, which in turn recruits myosin Va. Here, we show by reconstitution using purified proteins that Rab27a and Mlph are sufficient to form a transport complex with myosin Va in vitro. These results suggest that additional proteins are not required in vivo for assembly of the myosin Va receptor, although other proteins may associate with this tripartite complex to regulate its activity and/or to assist Rab27a in anchoring the complex to the melanosome membrane.