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Synthetic Biology (Oxford, England) 2024Giant unilamellar vesicles (GUVs) provide a powerful model compartment for synthetic cells. However, a key challenge is the incorporation of membrane proteins that allow...
Giant unilamellar vesicles (GUVs) provide a powerful model compartment for synthetic cells. However, a key challenge is the incorporation of membrane proteins that allow for transport, energy transduction, compartment growth and division. Here, we have successfully incorporated the membrane protein complex SecYEG-the key bacterial translocase that is essential for the incorporation of newly synthesized membrane proteins-in GUVs. Our method consists of fusion of small unilamellar vesicles containing reconstituted SecYEG into GUVs, thereby forming SecGUVs. These are suitable for large-scale experiments while maintaining a high protein:lipid ratio. We demonstrate that incorporation of SecYEG into GUVs does not inhibit its translocation efficiency. Robust membrane protein functionalized proteo-GUVs are promising and flexible compartments for use in the formation and growth of synthetic cells.
PubMed: 38807757
DOI: 10.1093/synbio/ysae007 -
BMC Ophthalmology May 2024Macular retinoschisis (MRS) and myopic macular neovascularization (mMNV) are both potentially blinding complications of high myopia. In this case report, we highlight... (Review)
Review
BACKGROUND
Macular retinoschisis (MRS) and myopic macular neovascularization (mMNV) are both potentially blinding complications of high myopia. In this case report, we highlight the progression of MRS after intravitreal anti-vascular endothelial growth factor (anti-VEGF) treatment for mMNV, as well as an extensive review of the literature on this topic.
CASE DESCRIPTION
A 49-year-old woman presented with two weeks of recent onset blurring and metamorphopsia in her right eye. She had high myopia in both eyes (right eye - 20/60 with - 16D, left eye - 20/20 with - 13D). Slit-lamp ophthalmoscopy found a normal anterior segment in both eyes. On fundus examination, features of pathological myopia with posterior staphyloma and peripapillary atrophy were observed in both eyes. An active mMNV, as well as intraretinal fluid, minimal perifoveal inner and outer MRS, and focal posterior vitreous traction along the inferotemporal retinal arcade, were detected on optical coherence tomography (OCT) of the right eye. The patient received an intravitreal injection of Aflibercept (2 mg/0.05 ml).
RESULTS
OCT scans at two- and four-month follow-up visits revealed regressed mMNV with a taut epiretinal membrane, progressive worsening of outer MRS, and the development of multiple perifoveal retinal detachment inferior to the fovea. Pars plana vitrectomy surgery was performed for the progressive MRS with good anatomical (resolved MRS) and functional outcome (maintained visual acuity at 20/60) at the last one-month post-surgery visit.
CONCLUSION
Intravitreal anti-VEGF injections for mMNV can cause vitreoretinal interface changes, exacerbating MRS and causing visual deterioration. Vitrectomy for MRS could be one of several treatment options.
Topics: Humans; Receptors, Vascular Endothelial Growth Factor; Female; Intravitreal Injections; Middle Aged; Retinoschisis; Recombinant Fusion Proteins; Myopia, Degenerative; Tomography, Optical Coherence; Visual Acuity; Angiogenesis Inhibitors; Disease Progression; Retinal Neovascularization; Fluorescein Angiography
PubMed: 38807066
DOI: 10.1186/s12886-024-03497-4 -
Light, Science & Applications May 2024Structured illumination microscopy (SIM) has emerged as a promising super-resolution fluorescence imaging technique, offering diverse configurations and computational...
Structured illumination microscopy (SIM) has emerged as a promising super-resolution fluorescence imaging technique, offering diverse configurations and computational strategies to mitigate phototoxicity during real-time imaging of biological specimens. Traditional efforts to enhance system frame rates have concentrated on processing algorithms, like rolling reconstruction or reduced frame reconstruction, or on investments in costly sCMOS cameras with accelerated row readout rates. In this article, we introduce an approach to elevate SIM frame rates and region of interest (ROI) coverage at the hardware level, without necessitating an upsurge in camera expenses or intricate algorithms. Here, parallel acquisition-readout SIM (PAR-SIM) achieves the highest imaging speed for fluorescence imaging at currently available detector sensitivity. By using the full frame-width of the detector through synchronizing the pattern generation and image exposure-readout process, we have achieved a fundamentally stupendous information spatial-temporal flux of 132.9 MPixels · s, 9.6-fold that of the latest techniques, with the lowest SNR of -2.11 dB and 100 nm resolution. PAR-SIM demonstrates its proficiency in successfully reconstructing diverse cellular organelles in dual excitations, even under conditions of low signal due to ultra-short exposure times. Notably, mitochondrial dynamic tubulation and ongoing membrane fusion processes have been captured in live COS-7 cell, recorded with PAR-SIM at an impressive 408 Hz. We posit that this novel parallel exposure-readout mode not only augments SIM pattern modulation for superior frame rates but also holds the potential to benefit other complex imaging systems with a strategic controlling approach.
PubMed: 38806501
DOI: 10.1038/s41377-024-01464-8 -
Nature Communications May 2024Membrane fusion, merging two lipid bilayers, is crucial for fabricating artificial membrane structures. Over the past 40 years, in contrast to precise and controllable...
Membrane fusion, merging two lipid bilayers, is crucial for fabricating artificial membrane structures. Over the past 40 years, in contrast to precise and controllable membrane fusion in-vivo through specific molecules such as SNAREs, controlling the fusion in-vitro while fabricating artificial membrane structures in physiological ionic solutions without fusion proteins has been a challenge, becoming a significant obstacle to practical applications. We present an approach consisting of an electric field and a few kPa hydraulic pressure as an additional variable to physically control the fusion, enabling tuning of the shape and size of the 3D freestanding lipid bilayers in physiological ionic solutions. Mechanical model analysis reveals that pressure-induced parallel/normal tensions enhance fusion among membranes in the microwell. In-vitro peptide-membrane assay, mimicking vesicular transport via pressure-assisted fusion, and stability of 38 days with in-chip pressure control via pore size-regulated hydrogel highlight the potential for diverse biological applications.
Topics: Lipid Bilayers; Membrane Fusion; Ions; Membranes, Artificial; Hydrogels; Pressure; Peptides
PubMed: 38806492
DOI: 10.1038/s41467-024-48875-0 -
Frontiers in Cellular Neuroscience 2024The soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) superfamily plays a pivotal role in cellular trafficking by facilitating... (Review)
Review
The soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) superfamily plays a pivotal role in cellular trafficking by facilitating membrane fusion events. These SNARE proteins, including syntaxins, assemble into complexes that actively facilitate specific membrane fusion events. Syntaxins, as integral components of the SNARE complex, play a crucial role in initiating and regulating these fusion activities. While specific syntaxins have been extensively studied in various cellular processes, including neurotransmitter release, autophagy and endoplasmic reticulum (ER)-to-Golgi protein transport, their roles in the retina remain less explored. This review aims to enhance our understanding of syntaxins' functions in the retina by shedding light on how syntaxins mediate membrane fusion events unique to the retina. Additionally, we seek to establish a connection between syntaxin mutations and retinal diseases. By exploring the intricate interplay of syntaxins in retinal function and health, we aim to contribute to the broader comprehension of cellular trafficking in the context of retinal physiology and pathology.
PubMed: 38799985
DOI: 10.3389/fncel.2024.1380064 -
ACS Omega May 2024Cells inhabit a mechanical microenvironment that they continuously sense and adapt to. The plasma membrane (PM), serving as the boundary of the cell, plays a pivotal... (Review)
Review
Cells inhabit a mechanical microenvironment that they continuously sense and adapt to. The plasma membrane (PM), serving as the boundary of the cell, plays a pivotal role in this process of adaptation. In this Review, we begin by examining well-studied processes where mechanoregulation proves significant. Specifically, we highlight examples from the immune system and stem cells, besides discussing processes involving fibroblasts and other cell types. Subsequently, we discuss the common molecular players that facilitate the sensing of the mechanical signal and transform it into a chemical response covering integrins YAP/TAZ and Piezo. We then review how this understanding of molecular elements is leveraged in drug discovery and tissue engineering alongside a discussion of the methodologies used to measure mechanical properties. Focusing on the processes of endocytosis, we discuss how cells may respond to altered membrane mechanics using endo- and exocytosis. Through the process of depleting/adding the membrane area, these could also impact membrane mechanics. We compare pathways from studies illustrating the involvement of endocytosis in mechanoregulation, including clathrin-mediated endocytosis (CME) and the CLIC/GEEC (CG) pathway as central examples. Lastly, we review studies on cell-cell fusion during myogenesis, the mechanical integrity of muscle fibers, and the reported and anticipated roles of various molecular players and processes like endocytosis, thereby emphasizing the significance of mechanoregulation at the PM.
PubMed: 38799362
DOI: 10.1021/acsomega.4c01962 -
BioRxiv : the Preprint Server For... May 2024Bacterial receptors feed into multiple signal transduction pathways that regulate a variety of cellular processes including gene expression, second messenger levels and...
UNLABELLED
Bacterial receptors feed into multiple signal transduction pathways that regulate a variety of cellular processes including gene expression, second messenger levels and motility. Receptors are typically activated by signal binding to ligand binding domains (LBD). Cache domains are omnipresent LBDs found in bacteria, archaea, and eukaryotes, including humans. They form the predominant family of extracytosolic bacterial LBDs and were identified in all major receptor types. Cache domains are composed of either a single (sCache) or a double (dCache) structural module. The functional relevance of bimodular LBDs remains poorly understood. Here, we identify the PacF chemoreceptor in the phytopathogen that recognizes formate at the membrane distal module of its dCache domain, triggering chemoattraction. We further demonstrate that a family of formate-specific sCache domains has evolved from a dCache domain, exemplified by PacF, by losing the membrane proximal module. By solving high-resolution structures of two family members in complex with formate, we show that the molecular basis for formate binding at sCache and dCache domains is highly similar, despite their low sequence identity. The apparent loss of the membrane proximal module may be related to the observation that dCache domains bind ligands typically at the membrane distal module, whereas the membrane proximal module is not involved in signal sensing. This work advances our understanding of signal sensing in bacterial receptors and suggests that evolution by reducing complexity may be a common trend shaping their diversity.
SIGNIFICANCE
Many bacterial receptors contain multi-modular sensing domains indicative of complex sensory processes. The presence of more than one sensing module likely permits the integration of multiple signals, although, the molecular detail and functional relevance for these complex sensors remain poorly understood. Bimodular sensory domains are likely to have arisen from the fusion or duplication of monomodular domains. Evolution by increasing complexity is generally believed to be a dominant force. Here we reveal the opposite - how a monomodular sensing domain has evolved from a bimodular one. Our findings will thus motivate research to establish whether evolution by decreasing complexity is typical of other sensory domains.
PubMed: 38798610
DOI: 10.1101/2024.05.17.594639 -
Research Square May 2024SARS-CoV-2 uses the double-membrane vesicles as replication organelles. However, how virion assembly occurs has not been fully understood. Here we identified a...
SARS-CoV-2 uses the double-membrane vesicles as replication organelles. However, how virion assembly occurs has not been fully understood. Here we identified a SARS-CoV-2-driven membrane structure named the 3a dense body (3DB). 3DBs have unusual electron-dense and dynamic inner structures, and their formation is driven by the accessory protein ORF3a via hijacking a specific subset of the -Golgi network (TGN) and early endosomal membranes. 3DB formation is conserved in related bat and pangolin coronaviruses yet lost during the evolution to SARS-CoV. 3DBs recruit the viral structural proteins spike (S) and membrane (M) and undergo dynamic fusion/fission to facilitate efficient virion assembly. A recombinant SARS-CoV-2 virus with an ORF3a mutant specifically defective in 3DB formation showed dramatically reduced infectivity for both extracellular and cell-associated virions. Our study uncovers the crucial role of 3DB in optimal SARS-CoV-2 infectivity and highlights its potential as a target for COVID-19 prophylactics and therapeutics.
PubMed: 38798602
DOI: 10.21203/rs.3.rs-4292014/v1 -
BioRxiv : the Preprint Server For... May 2024Osteoclasts are multinucleated cells unique in their ability to resorb bone. Osteoclastogenesis involves several steps of actin-driven rearrangements that participate...
Osteoclasts are multinucleated cells unique in their ability to resorb bone. Osteoclastogenesis involves several steps of actin-driven rearrangements that participate not only in the cell-cell fusion process, but also in the formation of the sealing zone, the adhesive structure determining the resorption area. Despite the importance of these actin cytoskeleton-based processes, their precise mechanisms of regulation are still poorly characterized. Here, we found that moesin, a member of the Ezrin/Radixin/Moesin (ERM) protein family, is activated during osteoclast maturation and plays an instrumental role for both osteoclast fusion and function. In mouse and human osteoclast precursors, moesin is negatively regulated to potentiate their ability to fuse and degrade bone. Accordingly, we demonstrated that moesin depletion decreases membrane-to-cortex attachment and enhances formation of tunneling nanotubes (TNTs), F-actin-containing intercellular bridges that we revealed to trigger osteoclast fusion. In addition, via a β3-integrin/RhoA/SLK pathway and independently of its role in fusion, moesin regulates the number and organization of sealing zones in mature osteoclast, and thus participates in the control of bone resorption. Supporting these findings, we found that moesin-deficient mice are osteopenic with a reduced density of trabecular bones and increased osteoclast abundance and activity. These findings provide a better understanding of the regulation of osteoclast biology, and open new opportunities to specifically target osteoclast activity in bone disease therapy.
PubMed: 38798563
DOI: 10.1101/2024.05.13.593799 -
Cell Biology and Toxicology May 2024MYBL1 is a strong transcriptional activator involved in the cell signaling. However, there is no systematic study on the role of MYBL1 in atherosclerosis. The aim of...
MYBL1 is a strong transcriptional activator involved in the cell signaling. However, there is no systematic study on the role of MYBL1 in atherosclerosis. The aim of this study is to elucidate the role and mechanism of MYBL1 in atherosclerosis. GSE28829, GSE43292 and GSE41571 were downloaded from NCBI for differentially expressed analysis. The expression levels of MYBL1 in atherosclerotic plaque tissue and normal vessels were detected by qRT-PCR, Western blot and Immunohistochemistry. Transwell and CCK-8 were used to detect the migration and proliferation of HUVECs after silencing MYBL1. RNA-seq, Western blot, qRT-PCR, Luciferase reporter system, Immunofluorescence, Flow cytometry, ChIP and CO-IP were used to study the role and mechanism of MYBL1 in atherosclerosis. The microarray data of GSE28829, GSE43292, and GSE41571 were analyzed and intersected, and then MYBL1 were verified. MYBL1 was down-regulated in atherosclerotic plaque tissue. After silencing of MYBL1, HUVECs were damaged, and their migration and proliferation abilities were weakened. Overexpression of MYBL1 significantly enhanced the migration and proliferation of HUVECs. MYBL1 knockdown induced abnormal autophagy in HUVEC cells, suggesting that MYBL1 was involved in the regulation of HUVECs through autophagy. Mechanistic studies showed that MYBL1 knockdown inhibited autophagosome and lysosomal fusion in HUVECs by inhibiting PLEKHM1, thereby exacerbating atherosclerosis. Furthermore, MYBL1 was found to repress lipid accumulation in HUVECs after oxLDL treatment. MYBL1 knockdown in HUVECs was involved in atherosclerosis by inhibiting PLEKHM1-induced autophagy, which provided a novel target of therapy for atherosclerosis.
Topics: Animals; Humans; Atherosclerosis; Autophagy; Cell Movement; Cell Proliferation; Down-Regulation; Human Umbilical Vein Endothelial Cells; Membrane Glycoproteins; Plaque, Atherosclerotic; Trans-Activators
PubMed: 38797732
DOI: 10.1007/s10565-024-09873-6