First Responders to Hyperosmotic Stress in Murine Astrocytes: Connexin 43 Gap Junctions Are Subject to an Immediate Ultrastructural Reorganization

In a short-term model of hyperosmotic stress, primary murine astrocytes were stimulated with a hyperosmolar sucrose solution for five minutes. Astrocytic gap junctions, which are mainly composed of Connexin (Cx) 43, displayed immediate ultrastructural changes, demonstrated by freeze–fracture replica immunogold labeling: their area, perimeter, and distance of intramembrane particles increased, whereas particle numbers per area decreased. Ultrastructural changes were, however, not accompanied by changes in Cx43 mRNA expression. In contrast, transcription of the gap junction regulator zonula occludens (ZO) protein 1 significantly increased, whereas its protein expression was unaffected. Phosphorylation of Serine (S) 368 of the Cx43 C–terminus has previously been associated with gap junction disassembly and reduction in gap junction communication. Hyperosmolar sucrose treatment led to enhanced phosphorylation of Cx43S368 and was accompanied by inhibition of gap junctional intercellular communication, demonstrated by a scrape loading-dye transfer assay. Taken together, Cx43 gap junctions are fast reacting elements in response to hyperosmolar challenges and can therefore be considered as one of the first responders to hyperosmolarity. In this process, phosphorylation of Cx43S368 was associated with disassembly of gap junctions and inhibition of their function. Thus, modulation of the gap junction assembly might represent a target in the treatment of brain edema or trauma.

Rhodopsins build up the birefringent bodies of the dinoflagellate Oxyrrhis marina

The ultrastructure of the birefringent bodies of the dinoflagellate Oxyrrhis marina was investigated by transmission electron microscopy. Ultrathin sectioning revealed that the bodies consist of highly ordered and densely packed lamellae, which show a regular striation along their longitudinal axis. A lattice distance of 6.1 nm was measured for the densely packed lamellae by FFT (Fast Fourier Transformation) analysis. In addition, a rather faint and oblique running striation was registered. Lamellae sectioned rather oblique or almost close to the surface show a honeycombed structure with a periodicity of 7.2–7.8 nm. Freeze-fracture transmission electron microscopy revealed that the lamellae are composed of highly ordered, crystalline arrays of particles. Here, FFT analysis resulted in lattice distances of 7.0–7.6 nm. Freeze-fracture transmission electron microscopy further revealed that the bodies remained intact after cell rupture followed by ascending flotation of the membrane fractions on discontinuous sucrose gradients. The birefringent bodies most likely are formed by evaginations of membranes, which separate the cytoplasm from the food vacuoles. Distinct, slightly reddish-colored areas, which resembled the birefringent bodies with respect to size and morphology, were registered by bright field light microscopy within Oxyrrhis marina cells. An absorbance maximum at 540 nm was registered for these areas, indicating that they are composed of rhodopsins. This was finally proven by immuno-transmission electron microscopy, as antisera directed against the C-terminal amino acid sequences of the rhodopsins AEA49880 and ADY17806 intensely immunolabeled the birefringent bodies of Oxyrrhis marina.

Lipid Polymorphism of the Subchloroplast—Granum and Stroma Thylakoid Membrane–Particles. II. Structure and Functions

In Part I, by using 31P-NMR spectroscopy, we have shown that isolated granum and stroma thylakoid membranes (TMs), in addition to the bilayer, display two isotropic phases and an inverted hexagonal (HII) phase; saturation transfer experiments and selective effects of lipase and thermal treatments have shown that these phases arise from distinct, yet interconnectable structural entities. To obtain information on the functional roles and origin of the different lipid phases, here we performed spectroscopic measurements and inspected the ultrastructure of these TM fragments. Circular dichroism, 77 K fluorescence emission spectroscopy, and variable chlorophyll-a fluorescence measurements revealed only minor lipase- or thermally induced changes in the photosynthetic machinery. Electrochromic absorbance transients showed that the TM fragments were re-sealed, and the vesicles largely retained their impermeabilities after lipase treatments—in line with the low susceptibility of the bilayer against the same treatment, as reflected by our 31P-NMR spectroscopy. Signatures of HII-phase could not be discerned with small-angle X-ray scattering—but traces of HII structures, without long-range order, were found by freeze-fracture electron microscopy (FF-EM) and cryo-electron tomography (CET). EM and CET images also revealed the presence of small vesicles and fusion of membrane particles, which might account for one of the isotropic phases. Interaction of VDE (violaxanthin de-epoxidase, detected by Western blot technique in both membrane fragments) with TM lipids might account for the other isotropic phase. In general, non-bilayer lipids are proposed to play role in the self-assembly of the highly organized yet dynamic TM network in chloroplasts.

Aquaporin-4 Expression In The Human Choroid Plexus

Background: The choroid plexus (CP) consists of specialized ependymal cells and underlying stroma and blood vessels, producing the bulk of the cerebrospinal fluid (CSF). CP epithelial cells are the site of the internal blood-cerebrospinal fluid barrier, show epithelial characteristics (basal lamina, tight junctions), and express aquaporin-1 (AQP1) apically. In contrast, ventricle-lining ependymal cells express aquaporin-4 (AQP4) basolaterallly. The initial purpose of this study was to analyze the expression of aquaporins in the ependyma – CP transition zone in the human brain to gain insights in aquaporin regulation. The results prompted us to investigate aquaporin expression in the mouse CP of different age groups. Methods: We analyzed the CP from eight body donors (age 74-91) applying immunofluorescence, qPCR, and freeze-fracture electron microscopy. We used antibodies against AQP1, AQP4, NKCC1, and Na/K-ATPase. In addition, we compared the CP from young (2 months), adult (12 months) and old (30 months) mice by qPCR and immunofluorescence. Results: Unexpectedly, many cells in the human CP were positive not only for AQP1 but also for AQP4, normally restricted to ependymal cells and astrocytes. Expression of AQP1 and AQP4 was found in the CP of all eight body donors. These results were confirmed by qPCR, and by electron microscopy detecting AQP4-specific orthogonal arrays of particles. To find out whether AQP4 expression correlated with relevant transport-related proteins we investigated expression of NKCC1 and Na/K-ATPase. Immunostaining for NKCC1 was similar to AQP1 and revealed no particular pattern related to AQP4. Co-staining of AQP4 and Na/K-ATPase indicated a trend for an inverse correlation of their expression. To test for the possibility of age-related changes causing AQP4 expression in the CP, we analyzed mouse brains from different age groups and found a significant increase of AQP4 on the mRNA level in old mice compared to young and adult animals. Conclusions: We provide evidence for AQP4 expression in the human and murine CP related to aging which likely contributes to the water flow through the CP epithelium and CSF production. In two alternative hypotheses, we discuss this as a beneficial compensatory, or a detrimental mechanism influencing the previously observed CSF changes during aging.

The molecular basis of Glucose Galactose Malabsorption in a large Swedish Pedigree

Glucose Galactose Malabsorption, GGM, is due to mutations in the gene coding for the intestinal sodium glucose cotransporter SGLT1 (SLC5A1). Here we identify the rare variant Gln457Arg (Q457R) in a large pedigree of patients in the Västerbotten County in Northern Sweden with the clinical phenotype of GGM. The functional effect of the Q457R mutation was determined in protein expressed in Xenopus laevis oocytes using biophysical and biochemical methods. The mutant failed to transport the specific SGLT1 sugar analog α-methyl-D-glucopyranoside (αMDG). Q457R SGLT1 was synthesized in amounts comparable to the wild-type transporter. SGLT1 charge measurements and freeze-fracture electron microscopy demonstrated that the mutant protein was inserted into the plasma membrane. Electrophysiological experiments, both steady-state and presteady-state, demonstrated that the mutant bound sugar with an affinity lower than the wild-type transporter. Together with our previous studies on Q457C and Q457E mutants, we established that the positive charge on Q457R prevented the translocation of sugar from the outward-facing to inward-facing conformation. This is contrary to other GGM cases where missense mutations caused defects in trafficking SGLT1 to the plasma membrane. Thirteen GGM patients are now added to the pedigree traced back to the late 17th Century. The frequency of the Q457R variant in Vasterbotten County genomes, 0.0067, is higher than in the general Swedish population, 0.0015, and higher than the general European population, 0.000067. This explains the high number of GGM cases in this region of Sweden.

Glycosphingolipid GM3 is localized in both exoplasmic and cytoplasmic leaflets of Plasmodium falciparum malaria parasite plasma membrane

Lipid rafts, sterol-rich and sphingolipid-rich microdomains on the plasma membrane are important in processes like cell signaling, adhesion, and protein and lipid transport. The virulence of many eukaryotic parasites is related to raft microdomains on the cell membrane. In the malaria parasite Plasmodium falciparum, glycosylphosphatidylinositol-anchored proteins, which are important for invasion and are possible targets for vaccine development, are localized in the raft. However, rafts are poorly understood. We used quick-freezing and freeze-fracture immuno-electron microscopy to examine the localization of monosialotetrahexosylganglioside (GM1) and monosialodihexosylganglioside (GM3), putative raft microdomain components in P. falciparum and infected erythrocytes. This method immobilizes molecules in situ, minimizing artifacts. GM3 was localized in the exoplasmic (EF) and cytoplasmic leaflets (PF) of the parasite and the parasitophorous vacuole (PV) membranes, but solely in the EF of the infected erythrocyte membrane, as in the case for uninfected erythrocytes. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) was localized solely in the PF of erythrocyte, parasite, and PV membranes. This is the first time that GM3, the major component of raft microdomains, was found in the PF of a biological membrane. The unique localization of raft microdomains may be due to P. falciparum lipid metabolism and its unique biological processes, like protein transport from the parasite to infected erythrocytes.

Fear Memory Retrieval Is Associated With a Reduction in AMPA Receptor Density at Thalamic to Amygdala Intercalated Cell Synapses

The amygdala plays a crucial role in attaching emotional significance to environmental cues. Its intercalated cell masses (ITC) are tight clusters of GABAergic neurons, which are distributed around the basolateral amygdala complex. Distinct ITC clusters are involved in the acquisition and extinction of conditioned fear responses. Previously, we have shown that fear memory retrieval reduces the AMPA/NMDA ratio at thalamic afferents to ITC neurons within the dorsal medio-paracapsular cluster. Here, we investigate the molecular mechanisms underlying the fear-mediated reduction in the AMPA/NMDA ratio at these synapses and, in particular, whether specific changes in the synaptic density of AMPA receptors underlie the observed change. To this aim, we used a detergent-digested freeze-fracture replica immunolabeling technique (FRIL) approach that enables to visualize the spatial distribution of intrasynaptic AMPA receptors at high resolution. AMPA receptors were detected using an antibody raised against an epitope common to all AMPA subunits. To visualize thalamic inputs, we virally transduced the posterior thalamic complex with Channelrhodopsin 2-YFP, which is anterogradely transported along axons. Using face-matched replica, we confirmed that the postsynaptic elements were ITC neurons due to their prominent expression of μ-opioid receptors. With this approach, we show that, following auditory fear conditioning in mice, the formation and retrieval of fear memory is linked to a significant reduction in the density of AMPA receptors, particularly at spine synapses formed by inputs of the posterior intralaminar thalamic and medial geniculate nuclei onto identified ITC neurons. Our study is one of the few that has directly linked the regulation of AMPA receptor trafficking to memory processes in identified neuronal networks, by showing that fear-memory induced reduction in AMPA/NMDA ratio at thalamic-ITC synapses is associated with a reduced postsynaptic AMPA receptor density.

Abstract P-42: Structure of Hydrogels of an Anionic Polysaccharide Studied by Freeze-Fracture Transmission Electron Microscopy

Background: Polysaccharide hydrogels draw attention due to the ability to form mechanically tough gels at low concentrations (typically 1 wt% or lower), combined with biocompatibility and biodegradability. Biopolymer hydrogels can be used as a matrix for cell growth, in order to obtain materials for the replacement of damaged tissues. “Physical” gels with macromolecules cross-linked by dynamic reversible cross-links are of great interest due to their self-healing ability. However, investigation of the native un-perturbed structure of such hydrogels presents a challenge, since they collapse upon drying, and present a difficulty for preparing a thin specimen for cryo-TEM experiments due to very high viscosity. The aim of this work is to study the native structure of hydrogels of an anionic polysaccharide – carboxymethyl hydroxypropyl guar (CMHPG) – cross-linked by borax. Methods: Freeze-fracture transmission electron microscopy (FF-TEM) was conducted on a Phillips EM-301 microscope. A small volume of the sample (100 μl) was put into the copper cell and cooled down by liquid nitrogen, put under vacuum (10−5 torr) at continuous cooling with liquid nitrogen, and fractured. The surface was etched for 10–20 min at 10−5 torr and then replicated by spraying platinum and carbon. Results: The gels have a microphase-separated microstructure – a rather thick (several nm) polymer backbone is seen, which is presumably formed by multiple aggregated macromolecules, and meshes between the backbone do not contain polymer and are filled with solvent. Mesh size determined from the micrographs qualitatively coincides with the value determined from the elastic modulus of the gels. Upon increasing the concentrations of cross-linker, the network becomes denser: the mesh size becomes lower, and the thickness of the backbone increases. Thus, the addition of cross-linker favors the aggregation of polymer chains forming the backbone. Conclusion: It was shown by FF-TEM that cross-linked CMHPG gels have a microphase-separated structure with a dense backbone formed by polymer chains and rather large meshes between them.

Extracellular and intracellular lanthanide accumulation in the methylotroph Beijerinckiaceae bacterium RH AL1

Recent work in Methylorubrum extorquens AM1 identified intracellular, cytoplasmic lanthanide storage in an organism that harnesses these metals for its metabolism. Here, we describe the extracellular and intracellular accumulation of lanthanides in Beijerinckiaceae bacterium RH AL1, a newly isolated and recently characterized methylotroph. Using ultrathin-section transmission electron microscopy (TEM), freeze-fracture TEM (FFTEM), and energy-dispersive X-ray spectroscopy, we demonstrated that strain RH AL1 accumulates lanthanides extracellularly at outer membrane vesicles (OMVs) and stores them in the periplasm. High-resolution elemental analyses of biomass samples revealed that strain RH AL1 can accumulate ions of different lanthanide species with a preference for heavier lanthanides. Its methanol oxidation machinery is supposably adapted to light lanthanides, and their selective uptake is mediated by dedicated uptake mechanisms. Based on RNAseq analysis, these presumably include the previously characterized TonB-ABC transport system encoded by the lut-cluster, but eventually also a type VI secretion system. A high constitutive expression of genes coding for lanthanide-dependent enzymes suggested that strain RH AL1 maintains a stable transcript pool to flexibly respond to changing lanthanide availability. Genes coding for lanthanide-dependent enzymes are broadly distributed taxonomically. Our results support the hypothesis that central aspects of lanthanide-dependent metabolism partially differ between the various taxa. Importance: Although multiple pieces of evidence have been added to the puzzle of lanthanide-dependent metabolism, we are still far from understanding the physiological role of lanthanides. Given how widespread lanthanide-dependent enzymes are, only limited information is available with respect to how lanthanides are taken up and stored in an organism. Our research complements work in commonly studied model organisms and showed the localized storage of lanthanides in the periplasm. This storage occurred at comparably low concentrations. Strain RH AL1 is able to accumulate lanthanide ions extracellularly and to selectively utilize lighter lanthanides. Beijerinckiaceae bacterium RH AL1 might be an attractive target for developing biorecovery strategies to win these economically highly demanded metals in more environmentally friendly ways.