The Unfolded Protein Response as a Guardian of the Secretory Pathway

The endoplasmic reticulum (ER) is the major site of membrane biogenesis in most eukaryotic cells. As the entry point to the secretory pathway, it handles more than 10,000 different secretory and membrane proteins. The insertion of proteins into the membrane, their folding, and ER exit are affected by the lipid composition of the ER membrane and its collective membrane stiffness. The ER is also a hotspot of lipid biosynthesis including sterols, glycerophospholipids, ceramides and neural storage lipids. The unfolded protein response (UPR) bears an evolutionary conserved, dual sensitivity to both protein-folding imbalances in the ER lumen and aberrant compositions of the ER membrane, referred to as lipid bilayer stress (LBS). Through transcriptional and non-transcriptional mechanisms, the UPR upregulates the protein folding capacity of the ER and balances the production of proteins and lipids to maintain a functional secretory pathway. In this review, we discuss how UPR transducers sense unfolded proteins and LBS with a particular focus on their role as guardians of the secretory pathway.

25-Hydroxycholesterol Effect on Membrane Structure and Mechanical Properties

Cholesterol is responsible for the plasticity of plasma membranes and is involved in physiological and pathophysiological responses. Cholesterol homeostasis is regulated by oxysterols, such as 25-hydroxycholesterol. The presence of 25-hydroxycholesterol at the membrane level has been shown to interfere with several viruses’ entry into their target cells. We used atomic force microscopy to assess the effect of 25-hydroxycholesterol on different properties of supported lipid bilayers with controlled lipid compositions. In particular, we showed that 25-hydroxycholesterol inhibits the lipid-condensing effects of cholesterol, rendering the bilayers less rigid. This study indicates that the inclusion of 25-hydroxycholesterol in plasma membranes or the conversion of part of their cholesterol content into 25-hydroxycholesterol leads to morphological alterations of the sphingomyelin (SM)-enriched domains and promotes lipid packing inhomogeneities. These changes culminate in membrane stiffness variations.

Impaired Cytoskeletal and Membrane Biophysical Properties of Acanthocytes in Hypobetalipoproteinemia – A Case Study

Familial hypobetalipoproteinemia is a metabolic disorder mainly caused by mutations in the apolipoprotein B gene. In its homozygous form it can lead without treatment to severe ophthalmological and neurological manifestations. In contrast, the heterozygous form is generally asymptomatic but associated with a low risk of cardiovascular disease. Acanthocytes or thorny red blood cells (RBCs) are described for both forms of the disease. However, those morphological changes are poorly characterized and their potential consequences for RBC functionality are not understood. Thus, in the present study, we asked whether, to what extent and how acanthocytes from a patient with heterozygous familial hypobetalipoproteinemia could exhibit altered RBC functionality. Acanthocytes represented 50% of the total RBC population and contained mitoTracker-positive surface patches, indicating the presence of mitochondrial fragments. While RBC osmotic fragility, calcium content and ATP homeostasis were preserved, a slight decrease of RBC deformability combined with an increase of intracellular free reactive oxygen species were observed. The spectrin cytoskeleton was altered, showing a lower density and an enrichment in patches. At the membrane level, no obvious modification of the RBC membrane fatty acids nor of the cholesterol content were detected but the ceramide species were all increased. Membrane stiffness and curvature were also increased whereas transversal asymmetry was preserved. In contrast, lateral asymmetry was highly impaired showing: (i) increased abundance and decreased functionality of sphingomyelin-enriched domains; (ii) cholesterol enrichment in spicules; and (iii) ceramide enrichment in patches. We propose that oxidative stress induces cytoskeletal alterations, leading to increased membrane stiffness and curvature and impaired lipid lateral distribution in domains and spicules. In addition, ceramide- and spectrin-enriched patches could result from a RBC maturation defect. Altogether, the data indicate that acanthocytes are associated with cytoskeletal and membrane lipid lateral asymmetry alterations, while deformability is only mildly impaired. In addition, familial hypobetalipoproteinemia might also affect RBC precursors leading to disturbed RBC maturation. This study paves the way for the potential use of membrane biophysics and lipid vital imaging as new methods for diagnosis of RBC disorders.

20S proteasomes secreted by the malaria parasite promote its growth

Mature red blood cells (RBCs) lack internal organelles and canonical defense mechanisms, making them both a fascinating host cell, in general, and an intriguing choice for the deadly malaria parasite Plasmodium falciparum (Pf), in particular. Pf, while growing inside its natural host, the human RBC, secretes multipurpose extracellular vesicles (EVs), yet their influence on this essential host cell remains unknown. Here we demonstrate that Pf parasites, cultured in fresh human donor blood, secrete within such EVs assembled and functional 20S proteasome complexes (EV-20S). The EV-20S proteasomes modulate the mechanical properties of naïve human RBCs by remodeling their cytoskeletal network. Furthermore, we identify four degradation targets of the secreted 20S proteasome, the phosphorylated cytoskeletal proteins β-adducin, ankyrin-1, dematin and Epb4.1. Overall, our findings reveal a previously unknown 20S proteasome secretion mechanism employed by the human malaria parasite, which primes RBCs for parasite invasion by altering membrane stiffness, to facilitate malaria parasite growth.

Variations in strain affect friction and microstructure evolution in copper under a reciprocating tribological load

The microstructure of the materials constituting a metallic frictional contact strongly influences tribological performance. Being able to tailor friction and wear is challenging due to the complex microstructure evolution associated with tribological loading. Here, we investigate the effect of the strain distribution on these processes. High-purity copper plates were morphologically surface textured with two parallel rectangles—referred to as membranes—over the entire sample length by micro-milling. By keeping the width of these membranes constant and only varying their height, reciprocating tribological loading against sapphire discs resulted in different elastic and plastic strains. Finite element simulations were carried out to evaluate the strain distribution in the membranes. It was found that the maximum elastic strain increases with decreasing membrane stiffness. The coefficient of friction decreases with increasing membrane aspect ratio. By analyzing the microstructure and local crystallographic orientation, we found that both show less change with decreasing membrane stiffness. Graphic abstract

ASSESSMENT OF THERAPY EFFICACY BY SYNOVIAL ELASTOMETRY DATA IN PATIENTS WITH RHEUMATOID ARTHRITIS

Rheumatoid arthritis is a widespread disease in general population, which can progress to the permanent disability as in a short-time as in long-time periods. It was considered that estimated changes of the joints' morphology in patients suffering on rheumatoid arthritis may depend on the basic treatment received and, eventually, on the control over rheumatoid arthritis progression achieved. The aim of our work is to study the accuracy of using elastometry method in evaluating the severity of knee joints' synovitis in patients with rheumatoid arthritis and its dependence on received basic treatment and achieved control over rheumatoid arthritis progression. By using a correlation analysis, we found a close relationship between the stiffness of the synovial membrane of the knee joints and the level of C-reactive protein (r = 0.63, p = 0.022), as well as with the level of erythrocyte sedimentation rate (r = 0.66, p = 0.019) in all groups of patients at the time of inclusion to the study. This relationship was present even after 6 months of therapy (r = 0.61, p = 0.043) and (r = 0.59, p = 0.023), respectively. Performed correlation analysis also showed the dependence of the morning joints' stiffness severity with the synovial membrane stiffness indicators (r = 0.57, p = 0.018), with the severity of pain in the knee joints (calculated by using The Visual Analogue Scale) and with the severity of knee joint synovial membrane stiffness (r = 0.56, p = 0,024) at the time of inclusion. So, it was considered a presence of relationship between synovial stiffness and clinical manifestations of rheumatoid arthritis. This relationship was present even after 6 months of therapy (r = 0.57, p = 0.038) and (r = 0.63, p = 0.024), respectively. The obtained results clearly demonstrate the accuracy of evaluating the knee joints' synovial stiffness by using elastometric indicators in assessing the effectiveness of received basic treatment in patients with rheumatoid arthritis.

Abstract 13664: Membrane Stiffness as a Function of Plakophilin-2 Expression: Implications to Arrhythmogenic Right Ventricular Cardiomyopathy

Mutations in PKP2 , the gene coding for the desmosomal protein Plakophilin-2 (PKP2), can lead to an inheritable cardiac disease called Arrhythmogenic Right Ventricle Cardiomyopathy (ARVC). Various studies investigated the molecular and electrical properties of cardiomyocytes (CMs) with PKP2 deficiency. However, systematic studies on the mechanical properties of PKP2-deficient CMs, and their relation to the cardiomyopathic phenotype, are lacking. We studied the relation between PKP2 expression, membrane transverse Young’s modulus (tYM; a surrogate measure of membrane stiffness), and their correlation with tubulin expression and with substrate stiffness. Left and right ventricular (LV, RV) CMs were isolated from murine hearts control (ctrl), or with a cardiomyocyte-specific, tamoxifen-activated knockout of PKP2 (PKP2cKO). Three time points were studied: 14, 15 and 21 days post-tamoxifen injection (dpi). The phenotypes at these time points correspond to three disease stages: concealed, arrhythmogenic and cardiomyopathy of RV predominance, respectively. tYM was evaluated by mechanoSICM. Tubulin was visualized by confocal microscopy. tYM in LV myocytes from control hearts showed a tendency toward higher values when compared to RV myocytes (LV 2.9±0.2 kPa vs RV 2.6±0.2 kPa, n=53-59). At 14 dpi, LV and RV PKP2cKO CMs were softer than ctrl (LV 2.1±0.1 kPa, RV 2.1±0.1 kPa vs ctrl, n=30-59). The trend reversed a day later and by 21 dpi there was a clear increase in tYM, particularly in PKP2cKO RV myocytes (LV 4.5±0.3 kPa, RV 4.8±0.6 kPa vs ctrl, n=44-59). Z-groove index, a parameter of membrane organization, decreased in PKP2cKO CMs. Increased tYM at 21 dpi corresponded to upregulation of α-tubulin and particularly, acetylated tubulin, which was reduced at 14dpi but went up in 21dpi. We also observed an inverse correlation between membrane tYM, and substrate stiffness. We conclude that loss of PKP2 expression affects, in a disease stage-specific manner, CM mechanical properties and the MT network organization. Whether these effects are correlative or one is consequence of the other, remains to be determined. We speculate that changes in membrane mechanics at the single cell level are a component of mechanical dysfunction in hearts affected with ARVC.