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Continued mRNA translation and protein production are critical for various neuronal functions. In addition to the precise sorting of proteins from cell soma to distant locations, protein synthesis allows a dynamic remodeling of the local proteome in a spatially variable manner. This spatial heterogeneity of protein synthesis is shaped by several factors such as injury, guidance cues, developmental cues, neuromodulators, and synaptic activity. In matured neurons, thousands of synapses are non-uniformly distributed throughout the dendritic arbor. At any given moment, the activity of individual synapses varies over a wide range, giving rise to the variability in protein synthesis. While past studies have primarily focused on the translation factors or the identity of translated mRNAs to explain the source of this variation, the role of ribosomes in this regard continues to remain unclear. Here, we discuss how several stochastic mechanisms modulate ribosomal functions, contributing to the variability in neuronal protein expression. Also, we point out several underexplored factors such as local ion concentration, availability of tRNA or ATP during translation, and molecular composition and organization of a compartment that can influence protein synthesis and its variability in neurons.

The X family is one of the eight families of DNA polymerases (dPols) and members of this family are known to participate in the later stages of Base Excision Repair. Many prokaryotic members of this family possess a Polymerase and Histidinol Phosphatase (PHP) domain at their C-termini. The PHP domain has been shown to possess 3′–5′ exonuclease activity and may represent the proofreading function in these dPols. PolX from Staphylococcus aureus also possesses the PHP domain at the C-terminus, and we show that this domain has an intrinsic Mn 2+ dependent 3′–5′ exonuclease capable of removing misincorporated dNMPs from the primer. The misincorporation of oxidized nucleotides such as 8oxodGTP and rNTPs are known to be pro-mutagenic and can lead to genomic instability. Here, we show that the PHP domain aids DNA replication by the removal of misincorporated oxidized nucleotides and rNMPs. Overall, our study shows that the proofreading activity of the PHP domain plays a critical role in maintaining genomic integrity and stability. The exonuclease activity of this enzyme can, therefore, be the target of therapeutic intervention to combat infection by methicillin-resistant- Staphylococcus-aureus .

The DNA polymerase module of the Pfprex enzyme (PfpPol) is responsible for duplication of the genome of the apicoplast organelle in the malaria parasite. We show that PfpPol can misincorporate oxidized nucleotides such as 8oxodGTP opposite dA. This event gives rise to transversion mutations that are known to lead to adverse physiological outcomes. The apicoplast genome is particularly vulnerable to the harmful effects of 8oxodGTP due to very high AT content (~ 87%). We show that the proofreading activity of PfpPol has the unique ability to remove the oxidized nucleotide from the primer terminus. Due to this property, the proofreading domain of PfpPol is able to prevent mutagenesis of the AT-rich apicoplast genome and neutralize the deleterious genotoxic effects of ROS generated in the apicoplast due to normal metabolic processes. The proofreading activity of the Pfprex enzyme may, therefore, represent an attractive target for therapeutic intervention. Also, a survey of DNA repair pathways shows that the observed property of Pfprex constitutes a novel form of dynamic error correction wherein the repair of promutagenic damaged nucleotides is concomitant with DNA replication.

Accurate multidimensional localization of isolated fluorescent emitters is a time consuming process in single-molecule based super-resolution microscopy. We demonstrate a functional method for real-time reconstruction with automatic feedback control, without compromising the localization accuracy. Compatible with high frame rates of EM-CCD cameras, it relies on a wavelet segmentation algorithm, together with a mix of CPU/GPU implementation. A combination with Gaussian fitting allows direct access to 3D localization. Automatic feedback control ensures optimal molecule density throughout the acquisition process. With this method, we significantly improve the efficiency and feasibility of localization-based super-resolution microscopy.

A native-SAD (single-wavelength anomalous diffraction) data collection strategy enables phasing using anomalous signal from a single native crystal, facilitating straightforward macromolecular X-ray structure determination. We describe a data collection method that uses a single crystal to solve X-ray structures by native SAD (single-wavelength anomalous diffraction). We solved the structures of 11 real-life examples, including a human membrane protein, a protein-DNA complex and a 266-kDa multiprotein-ligand complex, using this method. The data collection strategy is suitable for routine structure determination and can be implemented at most macromolecular crystallography synchrotron beamlines.

Integrins in focal adhesions (FAs) mediate adhesion and force transmission to extracellular matrices essential for cell motility, proliferation and differentiation. Different fibronectin-binding integrins, simultaneously present in FAs, perform distinct functions. Yet, how integrin dynamics control biochemical and biomechanical processes in FAs is still elusive. Using single-protein tracking and super-resolution imaging we revealed the dynamic nano-organizations of integrins and talin inside FAs. Integrins reside in FAs through free-diffusion and immobilization cycles. Integrin activation promotes immobilization, stabilized in FAs by simultaneous connection to fibronectin and actin-binding proteins. Talin is recruited in FAs directly from the cytosol without membrane free-diffusion, restricting integrin immobilization to FAs. Immobilized β 3 -integrins are enriched and stationary within FAs, whereas immobilized β 1 -integrins are less enriched and exhibit rearward movements. Talin is enriched and mainly stationary, but also exhibited rearward movements in FAs, consistent with stable connections with both β-integrins. Thus, differential transmission of actin motion to fibronectin occurs through specific integrins within FAs. Giannone and colleagues use super-resolution microscopy to analyse the nanoscale dynamic organization of talin and integrins β 1 and β 3 in focal adhesions.

Expansion microscopy (ExM) has revolutionized super-resolution imaging in cell biology due to its simple and inexpensive workflow. The use of ExM has revealed several novel insights into the nanoscale architectures of cellular protein complexes, especially the microtubule cytoskeleton in model and non-model systems. Despite tremendous progress in expansion microscopy protocols that preserve cellular ultrastructure (U-ExM), compatible probes for imaging actin isoforms with U-ExM are still lacking and have hindered the study of diverse actin isoforms and networks across model systems. Here, we use IntAct, an internally tagged actin that incorporates into cellular actin networks, to develop and optimize U-ExM for diverse actin structures in yeast, mammalian cells, and primary neurons. Using ALFA-tagged IntAct variants, we achieve robust visualization of actin patches, cables, and rings in yeast, as well as diverse actin architectures including the cortex, stress fibers, filopodia, and lamellipodia in mammalian cells at improved resolution. In primary hippocampal neurons, IntAct efficiently labels actin throughout the soma and neuronal projections, revealing strong enrichment at dendritic spines and synaptic boutons. Notably, we observe a periodic organization of F-actin along axons consistent with the membrane-associated periodic cytoskeleton, thereby resolving the periodic, sub-diffraction actin ring organization. We also detect transient nuclear actin filaments using IntAct-U-ExM underscoring the advantages offered by our approach to image understudied actin structures. Overall, we demonstrate the effectiveness of IntAct-U-ExM for performing super-resolution imaging of various actin structures in an isoform-specific manner and highlight the potential of IntAct to study the nanoscale organization of diverse actin cytoskeletal networks across species.

Background Asian Indians (AI) are at high risk for both atherosclerotic diseases (ATH) and diabetes mellitus (DM). We analyze the clustering of these two comorbidities as contributing causes of death in AI versus Non-AI populations in the US . Methods Using Mortality Multiple Cause-of-Death Files (2012–2019) from the National Center for Health Statistics, we included deaths at age ≥ 45 years among US residents where AI versus Non-AI status could be ascertained (n = 55,461 AI; n = 20,090,038 Non-AI) and identsified ATH (ICD10: I20-I25, I63, I70) and DM (ICD10: E10-E14) as contributing causes of death. We calculated the tetrachoric correlation (Rho) between these contributing causes and the difference in the fraction of deaths involving DM in those with versus without ATH. Results Among AI decedents, 29.9% of deaths included ATH as a contributing cause, 16.4% included DM as a contributing cause with 8.3% deaths being included in the overlap (Rho = 0.36, SE = 0.007) whereas, among Non-AI, 22.4% of deaths included ATH as a contributing cause, 10.0% included DM as a contributing cause with 4.1% deaths being included in the overlap (Rho = 0.31, SE = 0.001). Thus, DM and ATH as co-occurring causes correlated more strongly in AI versus Non-AI (p < 0.001). Further, this difference in clustering of DM with ATH was highest for younger AI women (age < 60 years) compared to comparable Non-AI women. Conclusions The more frequent co-occurrence of DM and ATH as causes of death among AI compared to Non-AI suggest that the increased burden of these diseases among AI during life has vicious synergistic consequences in terms of mortality. Public health strategies targeted to AI should focus on prevention and clinical treatment of both conditions jointly, in all adults, and especially in women < 60 years.

Synapse associated protein-97/Human Disk Large (SAP97/hDLG) is a conserved, alternatively spliced, modular, scaffolding protein critical in regulating the molecular organization of cell-cell junctions in vertebrates. We confirm that the molecular determinants of first order phase transition of SAP97/hDLG is controlled by morpho-functional changes in its nanoscale organization. Furthermore, the nanoscale molecular signatures of these signalling islands and phase transitions are altered in response to changes in cytosolic Ca 2+ . Additionally, exchange kinetics of alternatively spliced isoforms of the intrinsically disordered region in SAP97/hDLG C-terminus shows differential sensitivities to Ca 2+ bound Calmodulin, affirming that the molecular signatures of local phase transitions of SAP97/hDLG depends on their nanoscale heterogeneity and compositionality of isoforms. SAP97/hDLG is a ubiquitous, alternatively spliced, and conserved modular scaffolding protein involved in the organization cell junctions and excitatory synapses. Here, authors confirm that SAP97/hDLG condenses in to nanosized molecular domains in both heterologous cells and hippocampal pyramidal neurons. Authors demonstrate that in vivo and in vitro condensation, molecular signatures of nanoscale condensates and exchange kinetics of SAP97/hDLG is modulated by the local availability of alternatively spliced isoforms. Additionally, SAP97/hDLG isoforms exhibits a differential sensitivity to Ca 2+ bound Calmodulin, resulting in altered properties of nanocondensates and their real-time regulation

Simultaneous tracking of many thousands of individual particles in live cells is possible now with the advent of high-density superresolution imaging methods. We present an approach to extract local biophysical properties of cell-particle interaction from such newly acquired large collection of data. Because classical methods do not keep the spatial localization of individual trajectories, it is not possible to access localized biophysical parameters. In contrast, by combining the high-density superresolution imaging data with the present analysis, we determine the local properties of protein dynamics. We specifically focus on AMPA receptor (AMPAR) trafficking and estimate the strength of their molecular interaction at the subdiffraction level in hippocampal dendrites. These interactions correspond to attracting potential wells of large size, showing that the high density of AMPARs is generated by physical interactions with an ensemble of cooperative membrane surface binding sites, rather than molecular crowding or aggregation, which is the case for the membrane viral glycoprotein VSVG. We further show that AMPARs can either be pushed in or out of dendritic spines. Finally, we characterize the recurrent step of influenza trajectories. To conclude, the present analysis allows the identification of the molecular organization responsible for the heterogeneities of random trajectories in cells.