Areas Of Plasma Membrane Research Articles

Evaluation of biophysical alterations in the epithelial and endothelial layer of patients with Bullous Keratopathy

The cornea is a fundamental ocular tissue for the sense of sight. Thanks to it, the refraction of two-thirds of light manages to participate in the visual process and protect against mechanical damage. Because it is transparent, avascular, and innervated, the cornea comprises five main layers: Epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. Each layer plays a key role in the functionality and maintenance of ocular tissue, providing unique ultrastructural and biomechanical properties. Bullous Keratopathy (BK) is an endothelial dysfunction that leads to corneal edema, loss of visual acuity, epithelial blisters, and severe pain, among other symptoms. The corneal layers are subject to changes in their biophysical properties promoted by Keratopathy. In this context, the Atomic Force Microscopy (AFM) technique in air was used to investigate the anterior epithelial surface and the posterior endothelial surface, healthy and with BK, using a triangular silicone tip with a nominal spring constant of 0.4 N/m. Six human corneas (n = 6) samples were used for each analyzed group. Roughness data, calculated by third-order polynomial adjustment, adhesion, and Young's modulus, were obtained to serve as a comparison and identification of morphological and biomechanical changes possibly associated with the pathology, such as craters and in the epithelial layer and exposure of a fibrotic layer due to loss of the endothelial cell wall. Endothelial cell membrane area and volume data were calculated, obtaining a relevant comparison between the control and patient. Such results may provide new data on the physical properties of the ocular tissue to understand the physiology of the cornea when it has pathology.

LncNFYB promotes the proliferation of rheumatoid arthritis fibroblast-like synoviocytes via LncNFYB/ANXA2/ERK1/2 axis

Long noncoding RNAs (lncRNAs) are specifically expressed in different diseases and regulate disease progression. To explore the functions of rheumatoid arthritis (RA)- specific lncRNA, we determined the lncRNA expression profile of fibroblast-like synoviocytes (FLS) obtained from patients with RA and osteoarthritis (OA) using an LncRNA microarray and identified up-regulated LncNFYB in RA as a potential therapeutic target. Using gain- and loss-of-function studies, LncNFYB was proved promoting FLS proliferation and cell cycle progress, but not affecting their invasion, migration, and apoptotic abilities. Further investigation discovered that LncRNA could combine with annexin A2 (ANXA2) and enhanced the level of phospho-ANXA2 (Tyr24) in the plasma membrane area, which induced the activation of ERK1/2 to promoting proliferation. These findings provide new insights into the biological functions of LncNFYB on modification of FLS, which may be exploited for the therapy of RA.

Structural characterization and origin of surface vesicles in monocytes: another membranous pathway from cytoplasm to cell surface

ABSTRACT The monocytes in acute monocytic leukemia (AML-M5b) were analyzed by scanning and transmission electron microscopy (SEM and TEM) to understand more fully their structure and origin. By SEM, monocytes exhibited localized expansions of the surface, some of which appeared to bud off as surface vesicles (SVs). Filopodial processes and pseudopodia were also present. TEM demonstrated that the SVs were composed of a double-membrane at the pole away from the cell body, and a single membrane nearer to the cell body. In the peripheral cytoplasm, intracellular vesicles (IVs) had the appearance of vacuoles and were enclosed by single membranes. Most SVs were characterized by a notch as a rER edge and an expanded head. Filopodial processes had the same thickness of 40 nm as the SV walls, which suggested a close developmental relationship between the two. Pseudopodia between SVs were irregular in size. Rod-like rER cisternae were prominent in the peripheral cytoplasm and some showed a close physical juxtaposition as to suggest a transition from rER to IVs to SVs. Ultrastructural cytochemistry demonstrated activity of 5’-nucleotidase over rER, SVs, filopodial processes and pseudopodia, and a patchy reaction over other areas of plasma membrane. Overall, the results indicated that rER transforms into SVs, filopodial processes and pseudopodia, as a way of integrating cytoplasmic membranes into the plasma membrane.

Influence of Joule heating during single-cell electroporation simulation under IRE and H-FIRE pulses

Conventional irreversible electroporation (IRE) and high-frequency irreversible electroporation (H-FIRE) pulses have been widely employed in many biomedical and food applications. To investigate the effect of Joule heating generated by pulsed electric fields on cell electroporation, single-cell electroporation models without (EP only) and with the consideration of Joule heating (EP+T) were subjected to the equivalent energy IRE and H-FIRE pulses. The simulation results showed that with the consideration of Joule heating, the transmembrane voltage of the anodic point (TMPA) reached the electroporation threshold (∼1 V) faster, and a significant increase in the plasma membrane conductivity (σA) and pore number (kp) was found. We further evaluated the effect of the pulse burst repetition frequency (PBRF) of H-FIRE pulses on cell electroporation based on the EP+T model, and discovered kp increased with frequency when PBRF lower than 2 kHz, whereas it decreased with frequency for PBRF higher than 2 kHz. The accumulation of the ratio of the perforation area to the cell membrane area (sum of Ap/A) during the 400th pulse was also reversed when PBRF was 2 kHz. Our results indicated that Joule heating generated by repetitive pulse delivery would facilitate electroporation, and the effect of PBRF on cell electroporation was non-monotonic.

Rapid and reversible optical switching of cell membrane area by an amphiphilic azobenzene

Cellular membrane area is a key parameter for any living cell that is tightly regulated to avoid membrane damage. Changes in area-to-volume ratio are known to be critical for cell shape, but are mostly investigated by changing the cell volume via osmotic shocks. In turn, many important questions relating to cellular shape, membrane tension homeostasis and local membrane area cannot be easily addressed because experimental tools for controlled modulation of cell membrane area are lacking. Here we show that photoswitching an amphiphilic azobenzene can trigger its intercalation into the plasma membrane of various mammalian cells ranging from erythrocytes to myoblasts and cancer cells. The photoisomerization leads to a rapid (250-500 ms) and highly reversible membrane area change (ca 2 % for erythrocytes) that triggers a dramatic shape modulation of living cells.

Coronaviruses Use ACE2 Monomers as Entry‐Receptors

AbstractThe angiotensin‐converting enzyme 2 (ACE2) has been identified as entry receptor on cells enabling binding and infection with the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) via trimeric spike (S) proteins protruding from the viral surface. It has been suggested that trimeric S proteins preferably bind to plasma membrane areas with high concentrations of possibly multimeric ACE2 receptors to achieve a higher binding and infection efficiency. Here we used direct stochastic optical reconstruction microscopy (dSTORM) in combination with different labeling approaches to visualize the distribution and quantify the expression of ACE2 on different cells. Our results reveal that endogenous ACE2 receptors are present as monomers in the plasma membrane with densities of only 1–2 receptors μm−2. In addition, binding of trimeric S proteins does not induce the formation of ACE2 oligomers in the plasma membrane. Supported by infection studies using vesicular stomatitis virus (VSV) particles bearing S proteins our data demonstrate that a single S protein interaction per virus particle with a monomeric ACE2 receptor is sufficient for infection, which provides SARS‐CoV‐2 a high infectivity.

Coronaviruses Use ACE2 Monomers as Entry-Receptors.

The angiotensin-converting enzyme 2 (ACE2) has been identified as entry receptor on cells enabling binding and infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via trimeric spike (S) proteins protruding from the viral surface. It has been suggested that trimeric S proteins preferably bind to plasma membrane areas with high concentrations of possibly multimeric ACE2 receptors to achieve a higher binding and infection efficiency. Here we used direct stochastic optical reconstruction microscopy (dSTORM) in combination with different labeling approaches to visualize the distribution and quantify the expression of ACE2 on different cells. Our results reveal that endogenous ACE2 receptors are present as monomers in the plasma membrane with densities of only 1-2 receptors μm-2 . In addition, binding of trimeric S proteins does not induce the formation of ACE2 oligomers in the plasma membrane. Supported by infection studies using vesicular stomatitis virus (VSV) particles bearing S proteins our data demonstrate that a single S protein interaction per virus particle with a monomeric ACE2 receptor is sufficient for infection, which provides SARS-CoV-2 a high infectivity.

Induction of epithelial to mesenchymal transition in HPV16 E6/E7 oncogene transfected C33A cell line

This study focuses on the induction of EMT by HPV16 in the C33A cell line. Expression of β-catenin, EMT-transcription factors (EMT-TFs), and c-myc in the nuclei of HPV16 E6/E7 oncogene transfected and non-transfected C33A cells were investigated through immunofluorescence and RT-PCR. Microphotographs of β-catenin, c-myc, and DAPI-stained nuclei were processed and analyzed by Python and ImageJ respectively. Microphotographs of immunocytochemically stained transfected and control cells were then processed and analyzed with the help of ImageJ and Python programming. The intensity and the integrated density of β-catenin were computed at the cell membrane area as well as the cytoplasmic area along with the integrated density of c-myc and Renyi entropy of DAPI-stained nuclei was quantified by ImageJ software. Python programming was implemented to determine the total percentage of white pixels depicting the presence of β-catenin in the cytoplasmic area of cells. The signal of β-catenin at the cytoplasmic area was found significantly higher in transfected samples which implies the nuclear accumulation of β-catenin. The expression of the c-myc protein was found significantly higher in transfected cells along with significantly higher nuclear entropy. RT-PCR result shows two folds of up-regulation of EMT-TFs Snail1, Twist1, and Zeb2 and down-regulation of Snail2 and Twist2. The study concludes that HPV16 E6/E7 oncogene can induce EMT.

LEMONADE: Lego-like membrane tension analyzer based on self-assembled DNA elastic networks.

Membrane tension regulates and is affected by many cellular processes, including cell motility and membrane trafficking. However, the role of membrane tension in such processes is poorly understood because of difficulties in measuring dynamic membrane tension changes in live cells. Current approaches for membrane tension measurements can be broadly divided into two categories. In the first, fluorescence-based signals from small molecular probes that insert into cellular membranes are used. These allow convenient mapping of the signals throughout the cell membrane, but probe local membrane properties which are not always easy to relate to membrane tension. In addition, they are prone to artefacts due to membrane heterogenicities. The second type of approach relies on pulling a thin membrane tether from the cell surface. The restoring force acting on the tether is related to membrane tension and can be measured using optical tweezers. However, this approach requires specialized equipment and tension is measured at a single location. To overcome these challenges, we are developing a novel approach based on the self-assembly of an elastic network on the cell surface we call LEMONADE (Lego-like membrane tensionanalyzer based on self-assembled DNA elastic networks). The new membrane tension sensor relies on membrane-anchored Lego-like DNA nanostructures that are linked by elastic connectors with a FRET pair modification. Cell membrane area changes will result in transient FRET signals which can be detected throughout the cell surface using standard live fluorescence microscopy. We will report our progress in the design, assembly, and calibration of LEMONADE.

Cell surface detection of vimentin, ACE2 and SARS-CoV-2 Spike proteins reveals selective colocalization at primary cilia

The SARS-CoV-2 Spike protein mediates docking of the virus onto cells prior to viral invasion. Several cellular receptors facilitate SARS-CoV-2 Spike docking at the cell surface, of which ACE2 plays a key role in many cell types. The intermediate filament protein vimentin has been reported to be present at the surface of certain cells and act as a co-receptor for several viruses; furthermore, its potential involvement in interactions with Spike proteins has been proposed. Nevertheless, the potential colocalization of vimentin with Spike and its receptors on the cell surface has not been explored. Here we have assessed the binding of Spike protein constructs to several cell types. Incubation of cells with tagged Spike S or Spike S1 subunit led to discrete dotted patterns at the cell surface, which consistently colocalized with endogenous ACE2, but sparsely with a lipid raft marker. Vimentin immunoreactivity mostly appeared as spots or patches unevenly distributed at the surface of diverse cell types. Of note, vimentin could also be detected in extracellular particles and in the cytoplasm underlying areas of compromised plasma membrane. Interestingly, although overall colocalization of vimentin-positive spots with ACE2 or Spike was moderate, a selective enrichment of the three proteins was detected at elongated structures, positive for acetylated tubulin and ARL13B. These structures, consistent with primary cilia, concentrated Spike binding at the top of the cells. Our results suggest that a vimentin-Spike interaction could occur at selective locations of the cell surface, including ciliated structures, which can act as platforms for SARS-CoV-2 docking.

Folding of the syncytiotrophoblast basal plasma membrane increases the surface area available for exchange in human placenta.

The placental syncytiotrophoblast is the primary barrier between the mother and the fetus. To cross the placenta, nutrients and wastes must be transported across the apical microvillous and basal plasma membranes. While the syncytiotrophoblast basal plasma membrane is typically represented as relatively smooth, it has been shown to have invaginations that may increase its surface area. This study aimed to quantify how folding of the syncytiotrophoblast basal membrane contributes to its surface area and to visualise three-dimensional structures of the basal membrane and cytotrophoblast cell structures. Transmission electron microscope images of human term placenta were analysed using stereological approaches to quantify how folding of the syncytiotrophoblast basal plasma membrane affected surface area. Serial block-face scanning electron microscopy was used to visualise the three-dimensional structure of the syncytiotrophoblast basal membrane and cytotrophoblast cells. Syncytiotrophoblast basal membrane covered 69.1% of the basal lamina, with cytotrophoblast cells covering the remaining 30.9%. In basal lamina adjacent to syncytiotrophoblast, 34% was adjacent to smooth basal membrane and 66% to folded basal membrane. Syncytiotrophoblast basal membrane folds increased the surface area adjacent to basal lamina by 305%. Including regions overlying the cytotrophoblast cells, basal membrane folds increased syncytiotrophoblast basal membrane surface area by 4.4-fold relative to the basal lamina in terminal villi. Terminal and intermediate villi were similar in terms of trophoblast coverage of the basal lamina and basal membrane folding. The three-dimensional structures of the syncytiotrophoblast basal plasma membrane and cytotrophoblast cells were generated from serial block-face scanning electron microscopy image stacks. These findings indicate that the surface area of the syncytiotrophoblast basal plasma membrane is far larger than had been appreciated. We suggest that these folds increase the surface area available for transport to and from the fetus. Changes in the extent of basal membrane folding could affect nutrient transfer capacity and underlie pathological fetal growth, including fetal growth restriction and macrosomia.

Neurons Can Generate Electromagnetic Waves

Based on the potassium channel “origami windmill” model, and the conservation law of cell membrane area and ion inequality equation of based on the potassium channel “origami windmill” model, and Maxwell’s electromagnetic theory, it is theoretically proved that neurons can generate electromagnetic waves. The electromagnetic wave is an energy wave, never disappear. Neurons are equivalent to engineering antennas, and information between neurons can be transmitted through electromagnetic waves. The material basis for neurons to generate electromagnetic waves is the result of the exchange of cations on the inner surface of the cell membrane, especially Na+ and K+; The essence of consciousness should be electromagnetic wave. The conclusion that “neurons can generate electromagnetic waves” provides theoretical support for human beings to finally solve the mystery of the brain. At the same time, the author gives seven falsification schemes. The brain is a huge gold mine, and it is too important to crack the mystery of the brain. It should be a joint operation of “multiple arms”. It should not only be the work of brain scientists, but also the participation of physicists, chemists and mathematicians.

The mesoscale organization of syntaxin 1A and SNAP25 is determined by SNARE-SNARE interactions.

SNARE proteins have been described as the effectors of fusion events in the secretory pathway more than two decades ago. The strong interactions between SNARE domains are clearly important in membrane fusion, but it is unclear whether they are involved in any other cellular processes. Here, we analyzed two classical SNARE proteins, syntaxin 1A and SNAP25. Although they are supposed to be engaged in tight complexes, we surprisingly find them largely segregated in the plasma membrane. Syntaxin 1A only occupies a small fraction of the plasma membrane area. Yet, we find it is able to redistribute the far more abundant SNAP25 on the mesoscale by gathering crowds of SNAP25 molecules onto syntaxin clusters in a SNARE-domain-dependent manner. Our data suggest that SNARE domain interactions are not only involved in driving membrane fusion on the nanoscale, but also play an important role in controlling the general organization of proteins on the mesoscale. Further, we propose these mechanisms preserve active syntaxin 1A-SNAP25 complexes at the plasma membrane.

Par3 cooperates with Sanpodo for the assembly of Notch clusters following asymmetric division of Drosophila sensory organ precursor cells.

In multiple cell lineages, Delta-Notch signalling regulates cell fate decisions owing to unidirectional signalling between daughter cells. In Drosophila pupal sensory organ lineage, Notch regulates the intra-lineage pIIa/pIIb fate decision at cytokinesis. Notch and Delta that localise apically and basally at the pIIa-pIIb interface are expressed at low levels and their residence time at the plasma membrane is in the order of minutes. How Delta can effectively interact with Notch to trigger signalling from a large plasma membrane area remains poorly understood. Here, we report that the signalling interface possesses a unique apico-basal polarity with Par3/Bazooka localising in the form of nano-clusters at the apical and basal level. Notch is preferentially targeted to the pIIa-pIIb interface, where it co-clusters with Bazooka and its cofactor Sanpodo. Clusters whose assembly relies on Bazooka and Sanpodo activities are also positive for Neuralized, the E3 ligase required for Delta activity. We propose that the nano-clusters act as snap buttons at the new pIIa-pIIb interface to allow efficient intra-lineage signalling.

Thermal and Oxygen Flight Sensitivity in Ageing Drosophila melanogaster Flies: Links to Rapamycin-Induced Cell Size Changes.

Simple SummaryCold-blooded organisms can become physiologically challenged when performing highly oxygen-demanding activities (e.g., flight) across different thermal and oxygen environmental conditions. We explored whether this challenge decreases if an organism is built of smaller cells. This is because small cells create a large cell surface, which is costly, but can ease the delivery of oxygen to cells’ power plants, called mitochondria. We developed fruit flies in either standard food or food with rapamycin (a human drug altering the cell cycle and ageing), which produced flies with either large cells (no supplementation) or small cells (rapamycin supplementation). We measured the maximum speed at which flies were flapping their wings in warm and hot conditions, combined with either normal or reduced air oxygen concentrations. Flight intensity increased with temperature, and it was reduced by poor oxygen conditions, indicating limitations of flying insects by oxygen supply. Nevertheless, flies with small cells showed lower limitations, only slowing down their wing flapping in low oxygen in the hot environment. Our study suggests that small cells in a body can help cold-blooded organisms maintain demanding activities (e.g., flight), even in poor oxygen conditions, but this advantage can depend on body temperature.Ectotherms can become physiologically challenged when performing oxygen-demanding activities (e.g., flight) across differing environmental conditions, specifically temperature and oxygen levels. Achieving a balance between oxygen supply and demand can also depend on the cellular composition of organs, which either evolves or changes plastically in nature; however, this hypothesis has rarely been examined, especially in tracheated flying insects. The relatively large cell membrane area of small cells should increase the rates of oxygen and nutrient fluxes in cells; however, it does also increase the costs of cell membrane maintenance. To address the effects of cell size on flying insects, we measured the wing-beat frequency in two cell-size phenotypes of Drosophila melanogaster when flies were exposed to two temperatures (warm/hot) combined with two oxygen conditions (normoxia/hypoxia). The cell-size phenotypes were induced by rearing 15 isolines on either standard food (large cells) or rapamycin-enriched food (small cells). Rapamycin supplementation (downregulation of TOR activity) produced smaller flies with smaller wing epidermal cells. Flies generally flapped their wings at a slower rate in cooler (warm treatment) and less-oxygenated (hypoxia) conditions, but the small-cell-phenotype flies were less prone to oxygen limitation than the large-cell-phenotype flies and did not respond to the different oxygen conditions under the warm treatment. We suggest that ectotherms with small-cell life strategies can maintain physiologically demanding activities (e.g., flight) when challenged by oxygen-poor conditions, but this advantage may depend on the correspondence among body temperatures, acclimation temperatures and physiological thermal limits.

Functional interdependence of the actin nucleator Cobl and Cobl-like in dendritic arbor development

Local actin filament formation is indispensable for development of the dendritic arbor of neurons. We show that, surprisingly, the action of single actin filament-promoting factors was insufficient for powering dendritogenesis. Instead, this required the actin nucleator Cobl and its only evolutionary distant ancestor Cobl-like acting interdependently. This coordination between Cobl-like and Cobl was achieved by physical linkage by syndapins. Syndapin I formed nanodomains at convex plasma membrane areas at the base of protrusive structures and interacted with three motifs in Cobl-like, one of which was Ca2+/calmodulin-regulated. Consistently, syndapin I, Cobl-like's newly identified N terminal calmodulin-binding site and the single Ca2+/calmodulin-responsive syndapin-binding motif all were critical for Cobl-like's functions. In dendritic arbor development, local Ca2+/CaM-controlled actin dynamics thus relies on regulated and physically coordinated interactions of different F-actin formation-promoting factors and only together they have the power to bring about the sophisticated neuronal morphologies required for neuronal network formation in mammals.

Teasing out function from morphology: Similarities between primary cilia and immune synapses.

Immune synapses are formed between immune cells to facilitate communication and coordinate the immune response. The reorganization of receptors involved in recognition and signaling creates a transient area of plasma membrane specialized in signaling and polarized secretion. Studies on the formation of the immune synapse between cytotoxic T lymphocytes (CTLs) and their targets uncovered a critical role for centrosome polarization in CTL function and suggested a striking parallel between the synapse and primary cilium. Since these initial observations, a plethora of further morphological, functional, and molecular similarities have been identified between these two fascinating structures. In this review, we describe how advances in imaging and molecular techniques have revealed additional parallels as well as functionally significant differences and discuss how comparative studies continue to shed light on the molecular mechanisms underlying the functions of both the immune synapse and primary cilium.

Integrin adhesion complexes

Tissue architecture and function are orchestrated by an intricate repertoire of cellular adhesion and signalling receptors, and by the surrounding extracellular matrix (ECM). The essential role of cell-tissue interactions in guiding organogenesis was identified in experimental embryology studies over a century ago, and in 1954 Grobstein laid down the fundamental concept of ECM being the ultimate integrator of cellular systems. Long before the main cell adhesion receptors were identified, Abercrombie and colleagues proposed in 1971 that cell attachment to the ECM substratum was mediated through electron-dense plaques containing longitudinal cytoplasmic filaments that localise to areas of the ventral cell membrane that lie close to the substratum. In 1982, Bissell and co-workers proposed "the minimum required unit for expression of tissue specific functions", a model depicting a structure in which the nucleus links to the ECM via cytoskeletal filament bundles that connect to a hypothetical transmembrane ECM adhesion receptor.

FIB-SEM as a Volume Electron Microscopy Approach to Study Cellular Architectures in SARS-CoV-2 and Other Viral Infections: A Practical Primer for a Virologist.

The visualization of cellular ultrastructure over a wide range of volumes is becoming possible by increasingly powerful techniques grouped under the rubric “volume electron microscopy” or volume EM (vEM). Focused ion beam scanning electron microscopy (FIB-SEM) occupies a “Goldilocks zone” in vEM: iterative and automated cycles of milling and imaging allow the interrogation of microns-thick specimens in 3-D at resolutions of tens of nanometers or less. This bestows on FIB-SEM the unique ability to aid the accurate and precise study of architectures of virus-cell interactions. Here we give the virologist or cell biologist a primer on FIB-SEM imaging in the context of vEM and discuss practical aspects of a room temperature FIB-SEM experiment. In an in vitro study of SARS-CoV-2 infection, we show that accurate quantitation of viral densities and surface curvatures enabled by FIB-SEM imaging reveals SARS-CoV-2 viruses preferentially located at areas of plasma membrane that have positive mean curvatures.

Evidence for CAT gene being functionally involved in the susceptibility of COVID-19.

Novel coronary pneumonia (COVID‐19) is a respiratory distress syndrome caused by a new type of coronavirus. Understanding the genetic basis of susceptibility and prognosis to COVID‐19 is of great significance to disease prevention, molecular typing, prognosis, and treatment. However, so far, there have been only two genome‐wide association studies (GWASs) on the susceptibility of COVID‐19. Starting with these reported DNA variants, we found the genes regulated by these variants through cis‐eQTL and cis‐meQTL acting. We further did a series of bioinformatics analysis on these potential risk genes. The analysis shows that the genetic variants on EHF regulate the expression of its neighbor CAT gene via cis‐eQTL. There was significant evidence that CAT and the SARS‐CoV‐2‐related S protein binding protein ACE2 interact with each other. Intracellular localization results showed that CAT and ACE2 proteins both exists in the cell membrane and extracellular area and their interaction could have an impact on the cell invasion ability of S protein. In addition, the expression of these three genes showed a significant positive correlation in the lungs. Based on these results, we propose that CAT plays a crucial intermediary role in binding effectiveness of ACE2, thereby affecting the susceptibility to COVID‐19.