Biology subjects

Aronova, M. A.

Publications and source records attributed to Aronova, M. A..

5 recordsLinked to original sources

bioRxiv2025

Evolution of a fuzzy ribonucleoprotein complex in viral assembly

Previously we showed that the genetic diversity of SARS-CoV-2 nucleocapsid (N) protein explores a wide range of biophysical properties facilitated by non-local impact of point mutations to its intrinsically disordered regions (Nguyen et al., 2024). This includes modulation of self-association, such as the creation of a de novo binding interface through the P13L mutation characteristic of Omicron variants. In the present work we focus on the key function of N condensing viral RNA into ribonucleoprotein particles (RNPs) for viral assembly. Lacking high-resolution structural information, biochemical and biophysical approaches have revealed architectural principles of RNPs, which involve cooperative interactions of several protein-protein and protein-RNA interfaces, initiated through oligomerization of conserved transient helices in the central disordered linker of N. Here we study the impact of defining N-protein mutations in variants of concern on RNP formation, using biophysical tools, a virus-like particle assay, and reverse genetics experiments. We find convergent evolution in repeated, independent introduction of amino acid substitutions strengthening existing binding interfaces, compensating for other substitutions that promote viral replication but decrease RNP stability. Furthermore, we show that the P13L mutation of Omicron variants enhances RNP assembly and increases viral fitness. Overall, our data reveal RNP complexes to be highly variable not only in sequence and conformations, but also in thermodynamic and kinetic stability, with its pleomorphism affecting basic architectural principles. We hypothesize that the formation of polydisperse, fuzzy N-RNA clusters with multiple distributed weak binding interfaces optimizes reversible RNA condensation, while supporting host adaptation and allowing for a large sequence space to be explored.

biophysics↗

bioRxiv2024

Biological volume EM with focused Ga ion beam depends on formation of radiation-resistant Ga-rich layer at block face

Volume electron microscopy (vEM) enables biologists to visualize nanoscale 3D ultrastructure of entire eukaryotic cells and tissues processed by heavy atom staining and plastic embedding. The vEM technique with the highest resolution is focused ion-beam scanning electron microscopy (FIB-SEM), which provides nearly isotropic ([~]5-10 nm) spatial resolution at fluences up to 10,000 e-/nm2. However, it is still not understood how such resolution is achievable, because serial block-face (SBF) SEM, which incorporates an in-situ ultramicrotome instead of a Ga+ FIB beam, results in radiation-induced collapse of similar specimen blocks at fluences of only [~]20 e-/nm2. Here, we show that FIB-SEM implants a thin concentrated layer of Ga+ ions, which greatly reduces electron beam-damage, reduces the depth from which backscattered electrons are detected, and prevents specimen charging and collapse. Furthermore, we show that the z-resolution (perpendicular to block-face) in FIB-SEM is substantially higher than predicted by Monte Carlo modeling of the backscattered signal when Ga implantation is not included.

biophysics↗

bioRxiv2024

Single-Platelet Mapping of Jugular, Puncture-Wound Thrombi Reveals the Spatial Evolution of Platelet Activation

BackgroundThe contributions of platelet activation to thrombus formation during hemostatic bleeding cessation likely involve multiple activation states. However, the spatial and temporal distribution of platelets in these states has not been defined in a clot. ObjectivesTo use single-platelet mapping of activation states within jugular vein puncture thrombi to determine how the spatial distribution of platelet state evolves during hemostasis. MethodsMontaged, wide-area electron micrographs (EM) were taken at various time points, post-puncture, and annotated for platelet activation state. These classifications were mapped onto the images to identify regions of platelet activation and calculate neighbor associations. The importance of -granule secretion was tested using VAMP8-/- mice. Resultsmapping of platelet activation states at 1 min post-puncture showed extensive spatial intermixing of most platelet activation classes. No high-activation-state, platelet-rich core was observed in 5-min post-puncture thrombi, rather such platelets tended to be localized on the interior surfaces of thrombus vaults open to the circulation. Only at a later stage, 20 min post-puncture, was distinct clustering of high activation, degranulated, cytosol-rich platelets observed. These clusters localized to the central portion of the intravascular platelet-rich crown, and they were now inaccessible to the circulation. Counterintuitively, deletion of the primary platelet v-SNARE, VAMP8, increased the frequency and spatial clustering of highly activated, degranulated platelets in association with intra-thrombus vault surfaces at 5 min post puncture. ConclusionsWe conclude recent multi-activation state models can provide a realistic thrombus formation framework if linked together to encompass the dynamics of puncture wound formation.

systems biology↗

bioRxiv2019

Use of dual electron probes reveals role of ferritin in erythropoiesis

Much is known about the finely regulated process of mammalian erythropoiesis that occurs in the bone marrow, whereby erythropoietic stem cells undergo terminal differentiation accompanied by enormous morphological changes to generate highly functional specialized red blood cells. However, a crucial step in erythropoiesis, the labile iron pool and its transport to mitochondria for heme production, is not well understood1. We apply a dual 3D imaging and spectroscopic technique, based on scanned electron probes, to measure distributions of ferritin iron-storage protein in ex vivo human erythropoietic stem cells, and to determine how those distributions change during terminal differentiation. After seven days of differentiation, the cells display a highly specialized architecture of organelles with anchored clustering of mitochondria and massive accumulation of Fe3+ in loaded ferritin cores localized to lysosomal storage depots, providing an iron source for heme production. Macrophages are not present in our ex vivo cultures, so they cannot be the source of the ferritin2. We suggest that lysosomal iron depots are required by developing reticulocytes while terminally differentiating and continuing to produce heme and globin, which assemble and concentrate to fill the cytoplasm after much of the cellular machinery is expelled.

cell biology↗

bioRxiv2019

Determination of secretory granule maturation times in pancreatic islet beta-cells by serial block face scanning electron microscopy

It is shown how serial block-face electron microscopy (SBEM) of insulin-secreting beta cells in wild-type mouse pancreatic islets of Langerhans can be used to determine maturation times of secretory granules. Although SBEM captures the beta cell structure at a snapshot in time, the observed ultrastructure can be considered representative of a dynamic equilibrium state of the cells since the pancreatic islets are maintained in culture in approximate homeostasis. It is found that 7.2{+/-}1.2% ({+/-}st. dev.) of the beta cell volume is composed of secretory granule dense-cores exhibiting angular shapes surrounded by wide (typically {gtrsim}100 nm) electron-lucent halos. These organelles are identified as mature granules that store insulin for regulated release through the plasma membrane, with a release time of 96{+/-}12 hours, as previously obtained from pulsed 35S-radiolabeling of cysteine and methionine. Analysis of beta cell 3D volumes reveals a subpopulation of secretory organelles without electron-lucent halos, identified as immature secretory granules. Another subpopulation of secretory granules is found with thin (typically [lsim]30 nm) electron-lucent halos, which are attributed to immature granules that are transforming from proinsulin to insulin by action of prohormone convertases. From the volume ratio of proinsulin in the immature granules to insulin in the mature granules, we estimate that the newly formed immature granules remain in morphologically-defined immature states for an average time of 135{+/-}14 minutes, and the immature transforming granules for an average time of 130{+/-}17 minutes.

biophysics↗