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Results for “cell biology”

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bioRxiv2022

Effects of microtubule length and crowding on active microtubule network organization

Active filament networks can organize into various dynamic architectures driven by crosslinking motors. Densities and kinetic properties of motors and microtubules have been shown previously to determine active microtubule network self-organization, but the effects of other control parameters are less understood. Using computer simulations, we study here how microtubule lengths and crowding effects determine active network architecture and dynamics. We find that attractive interaction mimicking crowding effects or long microtubules both promote the formation of nematic networks of extensile bundles instead of contractile networks. When microtubules are very long and the network is highly percolated, a new isotropically motile network state resembling a crawling mesh is predicted. Using in vitro reconstitutions, we confirm the existence of this crawling mesh experimentally. These results provide a better understanding of how active microtubule network organization can be controlled, with implications for cell biology and active materials in general.

biophysics↗

bioRxiv2022

Disruptions in cell fate decisions and transformed enteroendocrine cells drive intestinal tumorigenesis in Drosophila.

Most epithelial tissues are maintained by stem cells that produce the different cell lineages required for proper tissue function. Constant communication between different cell types that make up a tissue is essential to ensure that all cell lineages are produced at appropriate numbers and to mount regenerative responses to injury, infection, and other environmental stresses. Cancer-driving alterations change the intrinsic properties of transformed cells and disrupt stem cell regulation, cell fate decisions, and cell-cell communication within transformed tissue. However, mechanisms by which these processes are disrupted and co-opted to support tumorigenesis are not well understood. Here, we report a novel genetic platform, PromoterSwitch, that allows targeting of genetic manipulations to a small subset of cells of any tissue or cell type of interest and all their subsequent progeny. We use this technology to generate large, transformed clones derived from individual stem/progenitor cells in the adult Drosophila intestine. We show that cancer-driving genetic alterations representing common colon tumor genome landscapes drive disruptions in cell fate decisions within transformed clones and changes in the relative abundance of different intestinal cell lineages. We also uncover a critical, context-dependent role for the differentiated, hormone-producing enteroendocrine (EE) cells in the growth and maintenance of transformed clones. Our analysis in different genetic contexts provides insights into how the intrinsic properties of transformed cells --dictated by the genetic alterations they carry-- determine their response to their environment and dependence on niche signals. A better mechanistic understanding of disruptions of cell-cell communication, stem cell regulation, and cell fate decisions within tumors could reveal novel vulnerabilities and druggable regulatory nodes that can be exploited for therapy. Understanding how tissues respond to the emergence of cells with cancer-driving genetic alterations also provides insights into stem cell biology and epithelial homeostasis.

cancer biology↗

bioRxiv2022

Endogenous GFP tagging in the diatom Thalassiosira pseudonana

The regulated abundance and spatial distribution of proteins determines cellular structure and function. The discovery of green fluorescent protein (GFP) and fusing it to a target protein to determine subcellular localization revolutionized cell biology. Most localization studies involve introducing additional copies of a target gene genetically fused to GFP and under the control of a constitutive promoter, resulting in the expression of the GFP-fusion protein at non-native levels. Here we have developed a single vector CRISPR/Cas9 guided GFP knock-in strategy in the diatom Thalassiosira pseudonana. This enables precise and scarless knock-in of GFP at the endogenous genomic location to create GFP fusion proteins under their native cis and trans regulatory elements with knock-in efficiencies of over 50%. We show that a previously uncharacterized bestrophin-like protein localizes to the CO2-fixing pyrenoid and demonstrate that by measuring GFP fluorescence we can track relative protein abundance in response to environmental change. To enable endogenous tagging, we developed a Golden Gate Molecular Cloning system for the rapid assembly of episomes for transformation into Thalassiosira pseudonana via bacterial conjugation. In addition, this versatile toolbox enables CRISPR/Cas9 gene editing, provides a broad range of validated fluorophores and enables future large-scale functional studies in diatoms. Significance statementFluorescent protein (FP) tagging is a widely utilized technique for understanding the spatial distribution of proteins. However, introducing extra gene copies under constitutive promoters that randomly integrate into the genome can result in non-biologically relevant expression levels, unwanted genomic mutations and localization artefacts. To overcome this, we developed a novel single vector system capable of CRISPR/Cas9-guided endogenous GFP tagging in a globally important model diatom. This allows scarless GFP knock-in at precise genomic locations resulting in GFP fusions regulated by native promoters/terminators, which facilitates accurate localization and determination of relative protein abundance. Moreover, the developed modular cloning framework is user-friendly and opens the door for high throughput large-scale studies, including FP tagging, knock-out, and knock-in.

plant biology↗

bioRxiv2022

Self-Assembly Coupled to Liquid-Liquid Phase Separation

Liquid condensate droplets with distinct compositions of proteins and nucleic acids are widespread in biological cells. While it is known that such droplets can regulate irreversible protein aggregation, their effect on reversible self-assembly remains largely unexplored. In this article, we use kinetic theory and solution thermodynamics to investigate the effect of liquid-liquid phase separation on the reversible self-assembly of structures with well-defined sizes and architectures. We find that when assembling subunits preferentially partition into liquid domains, robustness against kinetic traps and maximum achievable assembly rates can be significantly increased. In particular, the range of solution conditions over which productive assembly and the corresponding assembly rates can increase by orders of magnitude. We analyze the rate equation predictions using simple scaling estimates to identify effect of liquid-liquid phase separation as a function of relevant control parameters. These results may elucidate self-assembly processes that underlie normal cellular functions or pathogenesis, and suggest strategies for designing efficient bottom-up assembly for nanomaterials applications.

biophysics↗

bioRxiv2022

Ribozyme-phenotype coupling in peptide-based coacervate protocells

Condensed coacervate phases are now understood to be important features of modern cell biology, as well as valuable protocellular models in origin of life studies and synthetic biology. In each of these fields, the development of model systems with varied and tuneable material properties is of great importance for replicating properties of life. Here, we develop a ligase ribozyme system capable of concatenating short RNA fragments into extremely long chains. Our results show that formation of coacervate microdroplets with the ligase ribozyme and poly(L-lysine) enhances ribozyme rate and yield, which in turn increases the length of the anionic polymer component of the system and imparts specific physical properties to the droplets. Droplets containing active ribozyme sequences resist growth, do not wet or spread on unpassivated surfaces, and exhibit reduced transfer of RNA between droplets when compared to controls containing inactive sequences. These altered behaviours, which stem from RNA sequence and catalytic activity, constitute a specific phenotype and potential fitness advantage, opening the door to selection and evolution experiments based on a genotype - phenotype linkage.

synthetic biology↗

bioRxiv2022

Optogenetic manipulation of second messengers in neurons and cardiomyocytes with microbial rhodopsins and adenylyl cyclase

Even though microbial photosensitive proteins have been used for optogenetics, their use should be optimized to precisely control second messengers in vivo. We exploited GtCCR4 and KnChR, cation channelrhodopsins from algae, BeGC1, a guanylyl cyclase rhodopsin from a fungus, and photoactivated adenylyl cyclases (PACs) from cyanobacteria (OPAC) or bacteria (bPAC), to control cell functions in zebrafish. Optical activation of GtCCR4 and KnChR in the hindbrain reticulospinal V2a neurons, which are involved in locomotion, immediately induced swimming behavior, whereas activation of BeGC1 or PACs was achieved at a short latency. KnChR had the highest locomotioninducing activity of all the channelrhodopsins examined. Activation of GtCCR4 and KnChR in cardiomyocytes induced cardiac arrest, whereas activation of bPAC gradually induced bradycardia. KnChR activation led to an increase in intracellular Ca2+ in the heart, suggesting that depolarization caused cardiac arrest. These data suggest that these optogenetic tools can be used to reveal the roles of second messengers in various cell types in vertebrates. Impact statementWe identified efficient and useful microbial channelrhodopsin, guanylyl cyclase rhodopsin, and photoactivated adenylyl cyclase that regulate neural activity and cardiac function in zebrafish. Major subject areasNeuroscience, Cell biology Research organismZebrafish (Danio rerio)

neuroscience↗

bioRxiv2022

Nonlinear microscale mechanics of actin networks governed by coupling of filament crosslinking and stabilization

Actin plays a vital role in maintaining the stability and rigidity of biological cells while allowing for cell motility and shape change. The semiflexible nature of actin filaments - along with the myriad actin-binding proteins (ABPs) that serve to crosslink, bundle, and stabilize filaments - are central to this multifunctionality. The effect of ABPs on the structural and mechanical properties of actin network mechanics has been the topic of fervent investigation over the past few decades, revealing diverse structures from isotropic percolated networks to heterogeneous bundles that depend on the crosslinker type and concentration. Yet, the impact of filament stabilization and stiffening via ABPs on the nonlinear response of crosslinked networks has yet to be explored. Here, we perform optical tweezers microheology measurements to characterize the nonlinear force response and relaxation dynamics of actin networks in the presence of varying concentrations of -actinin, which transiently crosslinks actin filaments, and phalloidin, which stabilizes filamentous actin and increases its persistence length. We show that crosslinking and stabilization can act both synergistically and antagonistically to tune the network resistance to nonlinear straining. For example, phalloidin-stabilization leads to enhanced elastic response and reduced dissipation at large strains and timescales, while the initial microscale force response is reduced compared to networks without phalloidin. Moreover, we find that stabilization switches this initial response from that of stress-stiffening to softening despite the increased filament stiffness that phalloidin confers. Finally, we show that both crosslinking and stabilization are necessary to elicit these emergent features, while the effect of stabilization on networks without crosslinkers is much more subdued. We suggest that these intriguing mechanical properties arise from the competition and cooperation between filament connectivity, bundling, and rigidification, shedding light on how ABPs with distinct roles can act in concert to mediate diverse mechanical properties of the cytoskeleton and bio-inspired polymeric materials.

biophysics↗

bioRxiv2022

Growth Mechanics: General principles of optimal cellular resource allocation in balanced growth

The physiology of biological cells evolved under physical and chemical constraints such as mass conservation, nonlinear reaction kinetics, and limits on cell density. For unicellular organisms, the fitness that governs this evolution is mainly determined by the balanced cellular growth rate. We previously introduced Growth Balance Analysis (GBA) as a general framework to model such nonlinear systems, and we presented analytical conditions for optimal balanced growth in the special case that the active reactions are known. Here, we develop Growth Mechanics (GM) as a more general, succinct, and powerful analytical description of the growth optimization of GBA models, which we formulate in terms of a minimal number of dimensionless variables. GM uses Karush-Kuhn-Tucker (KKT) conditions in a Lagrangian formalism. It identifies fundamental principles of optimal resource allocation in GBA models of any size and complexity, including the analytical conditions that determine the set of active reactions at optimal growth. We identify from first principles the economic values of biochemical reactions, expressed as marginal changes in cellular growth rate; these economic values can be related to the costs and benefits of proteome allocation into the reactions catalysts. Our formulation also generalizes the concepts of Metabolic Control Analysis to models of growing cells. GM unifies and extends previous approaches of cellular modeling and analysis, putting forward a program to analyze cellular growth through the stationarity conditions of a Lagrangian function. GM thereby provides a general theoretical toolbox for the study of fundamental mathematical properties of balanced cellular growth.

biophysics↗

bioRxiv2022

The Epithelial Na+ Channel UNC-8 promotes an endocytic mechanism that recycles presynaptic components from old to new boutons in remodeling neurons

SummaryPresynaptic terminals are actively relocated during development to refine circuit function, but the underlying cell biological mechanisms are largely unknown. In C. elegans, the presynaptic boutons of GABAergic DD neurons are moved to new locations during early larval development. We show that developmentally regulated expression of a presynaptic Epithelial Na+ Channel (ENaC), UNC-8, promotes a Ca2+-dependent mechanism, resembling Activity-Dependent Bulk Endocytosis (ADBE), that dismantles presynaptic material for reassembly at nascent DD synapses. ADBE normally functions in highly active neurons to accelerate local recycling of synaptic vesicles. We show that DD presynaptic remodeling depends on canonical features of ADBE including elevated intracellular Ca2+, the phosphatase Calcineurin and its targets, dynamin and the F-BAR protein syndapin, and Arp2/3-driven actin polymerization. Thus, our findings suggest that a native mechanism (ADBE) for maintaining neurotransmitter release at local synapses has been repurposed, in this case, to dismantle presynaptic terminals for reassembly at new locations. HighlightsO_LIDeveloping GABAergic neurons dismantle presynaptic terminals for reassembly at new locations. C_LIO_LIThe DEG/ENaC protein, UNC-8, promotes presynaptic disassembly and recycling C_LIO_LICa2+-dependent endocytosis drives presynaptic disassembly and recycling to new boutons C_LI

neuroscience↗

bioRxiv2022

Microdissection and culturing of adult lateral entorhinal cortex layer II neurons from APP/PS1 Alzheimer model mice

BackgroundPrimary neuronal cultures enable cell-biological studies of Alzheimers disease (AD), albeit typically non-neuron-specific. The first cortical neurons affected in AD reside in layer II of the lateralmost part of the entorhinal cortex, and they undergo early accumulation of intracellular amyloid-{beta}, form subsequent tau pathology, and start degenerating pre-symptomatically. These vulnerable entorhinal neurons uniquely express the glycoprotein reelin and provide selective inputs to the hippocampal memory system. Gaining a more direct access to study these neurons is therefore highly relevant. New methodWe demonstrate a methodological approach for microdissection and long-term culturing of adult lateral entorhinal layer II-neurons from AD-model mice. ResultsWe maintain adult microdissected lateralmost entorhinal layer II-neurons beyond two months in culture. We show that they express neuronal markers, and that they are electrophysiologically active by 15 days in vitro and continuing beyond 2 months. Comparison with existing methodsPrimary neurons are typically harvested from embryonic or early postnatal brains because such neurons are easier to culture compared to adult neurons. Methods to culture adult primary neurons have been reported, however, to our knowledge, culturing of adult entorhinal subregion-specific primary neurons from AD-model animals has not been reported. ConclusionsOur methodological approach offers a window to study initial pathological changes in the AD disease-cascade. This includes the study of proteinopathy, single-neuron changes, and network-level dysfunction. HighlightsO_LIWe microdissect and culture neurons from layer II of the lateralmost part of the entorhinal cortex from adult AD model mice and littermate controls C_LIO_LIThese entorhinal neurons self-organize into networks, express reelin, NeuN and intracellular amyloid-{beta}. C_LIO_LIThe neurons are electrophysiologically active by day 15 in culture and remain viable beyond two months. C_LI

neuroscience↗

bioRxiv2022

Cerebral organoids expressing mutant actin genes reveal cellular mechanism underlying microcephalic cortical malformation

Actins are structural cytoskeletal proteins playing crucial roles in multiple cellular processes. Mutations in the ACTB and ACTG1 genes, encoding the ubiquitous beta- and gamma- cytoskeletal actin isoforms, respectively, cause a broad spectrum of neurodevelopmental disorders, with microcephaly as the most frequent one. Here we used patient-derived cerebral organoids to gain insight into the pathogenesis underlying this cortical malformation. Cerebral organoids from induced pluripotent stem cells (iPSCs) of patients with the Baraitser-Winter- CerebroFrontoFacial syndrome (BWCFF-S), expressing either an ACTB or an ACTG1 missense mutation, are reduced in size, showing a thinner ventricular zone (VZ). This decrease in VZ progenitors is in turn associated with a striking change in the orientation of their cleavage plane from predominantly vertical (control) to predominantly horizontal (BWCFF-S), which is incompatible with increasing VZ progenitor abundance. Various cytoskeletal and morphological irregularities of BWCFF-S VZ progenitors, notably in the apical region of these cells, seemingly contribute to their predominantly horizontal cleavage plane orientation. Our results provide insight into the cell biological basis of the microcephaly associated with BWCFF-S caused by actin mutations.

neuroscience↗

bioRxiv2022

Reanalysis of mtDNA mutations of human primordial germ cells (PGCs) reveals significant contamination with NUMTs, and challenges predominantly purifying selection in late PGCs

The resilience of the mitochondrial genome (mtDNA) to a high mutational pressure depends, in part, on negative purifying selection in the germline. A paradigm in the field has been that such selection, at least in part, takes place in primordial germ cells (PGCs). Specifically, Floros et al. (Nature Cell Biology 20: 144-51) reported an increase in the synonymity of mtDNA mutations (a sign of purifying selection) between pooled early-stage and late-stage PGCs. We re-analyzed Floros et al. pooled PGC data and noticed that their mutational dataset was significantly contaminated with single nucleotide variants (SNVs) derived from a nuclear sequence of mtDNA origin (NUMT) located on chromosome 5. Contamination was caused by co-amplification of the NUMT sequence by cross-specific PCR primers. Importantly, when we removed NUMT-derived SNVs, the evidence of purifying selection was abolished. In addition to pooled PGCs, Floros et al. reported the analysis of single late-stage PGCs, which were amplified with different sets of PCR primers that cannot amplify the NUMT sequence. Accordingly, we found no NUMT-derived SNVs among single PGCs mutations. Interestingly, single PGC mutations show a decrease of synonymity with increased intracellular mutant fraction. This pattern is incompatible with predominantly negative selection. This suggests that germline selection of mtDNA mutations is a complex phenomenon and that the part of this process that takes place in PGCs may be predominantly positive. However counterintuitive, positive germline selection of detrimental mtDNA mutations has been reported previously and potentially may be evolutionarily advantageous.

genomics↗

bioRxiv2022

Mirage2's high-quality spliced protein-to-genome mappings produce accurate multiple-sequence alignments of isoforms

The organization of homologous protein sequences into multiple sequence alignments (MSAs) is a cornerstone of modern analysis of proteins. Recent focus on the importance of alternatively-spliced isoforms in disease and cell biology has highlighted the need for MSA software that can appropriately account for isoforms and the exon-length insertions or deletions that isoforms may have relative to each other. We previously developed Mirage, a software package for generating MSAs for isoforms spanning multiple species. Here, we present Mirage2, which retains the fundamental algorithms of the original Mirage implementation while providing substantially improved translated mapping and improving several aspects of usability. We demonstrate that Mirage2 is highly effective at mapping proteins to their encoding exons, and that these protein-genome mappings lead to extremely accurate intron-aware alignments. Additionally, Mirage2 implements a number of engineering improvements that simplify installation and use.

bioinformatics↗

bioRxiv2022

BRCA2 chaperones RAD51 to single molecules of RPA-coated ssDNA

Mutations in the breast cancer susceptibility gene, BRCA2, greatly increase an individuals lifetime risk of developing breast and ovarian cancers. BRCA2 suppresses tumor formation by potentiating DNA repair via homologous recombination. Central to recombination is the assembly of a RAD51 nucleoprotein filament, which forms on single-stranded DNA (ssDNA) generated at or near the site of chromosomal damage. However, Replication Protein-A (RPA) rapidly binds to and continuously sequesters this ssDNA, imposing a kinetic barrier to RAD51 filament assembly that suppresses unregulated recombination. Recombination mediator proteins--of which BRCA2 is the defining member in humans --alleviate this kinetic barrier to catalyze RAD51 filament formation. We combined microfluidics, microscopy, and micromanipulation to directly measure both the binding of full-length BRCA2 to--and the assembly of RAD51 filaments on--a region of RPA-coated ssDNA within individual DNA molecules designed to mimic a resected DNA lesion common in replication-coupled recombinational repair. We demonstrate that a dimer of RAD51 is minimally required for spontaneous nucleation; however, growth self-terminates below the diffraction limit. BRCA2 accelerates nucleation of RAD51 to a rate that approaches the rapid association of RAD51 to naked ssDNA, thereby overcoming the kinetic block imposed by RPA. Furthermore, BRCA2 eliminates the need for the rate-limiting nucleation of RAD51 by chaperoning a short pre-assembled RAD51 filament onto the ssDNA complexed with RPA. Therefore, BRCA2 regulates recombination by initiating RAD51 filament formation. SignificanceDespite decades of genetic and cell biological studies, mechanistic biochemical analyses of human BRCA2 function in recombinational DNA repair have only been possible since the purification of full-length BRCA2. These mechanistic studies crucially inform with respect to the molecular function of BRCA2 in genome maintenance. Here, we use single-molecule methods to visualize the assembly of RAD51 on individual RPA-coated ssDNA molecules and to see how this process is regulated by the tumor suppressor protein, BRCA2. We show that BRCA2 serves as a chaperone to nucleate RAD51 and deliver it to RPA-coated ssDNA. This work advances understanding of the molecular functions of BRCA2 and, consequently, the molecular etiology of breast cancer in an important way.

biochemistry↗

bioRxiv2022

Correlation Single Molecule Localization Microscopy (corrSMLM) Detects Fortunate Molecules for Better Localization Precision and High Signal-to-Noise Ratio

Single-molecule localization microscopy can decipher fine details that are otherwise not possible using diffraction-limited microscopy. Often the reconstructed super-resolved image contains unwanted noise, random background and is prone to false detections. This cause spurious data that necessitates several trials, multiple experimentations, and repeated preparation of specimens. Moreover, this is not suitable for experiments that require time-lapse imaging and real-time microscopy. To overcome these limitations, we propose a technique (corrSMLM) that can recognize and detect fortunate molecules (molecules with long fluorescence cycles) from the recorded data. The technique uses correlation between two or more consecutive frames to extract fortunate molecules that blink for longer than the standard blinking time. Accordingly, strongly-correlated spots (single molecule signatures) are compared in consecutive frames, followed by data integration (mean centroid position and the total number of photons) and estimation of critical parameters (position and localization precision). The technique addresses two major problems that plague SMLM : (1) random noise due to false detection that contributes to strong background, and (2) poor localization precision offered by standard SMLM techniques. On the brighter side, corrSMLM allows only fortunate molecules contribute to the super-resolved image, thereby suppressing the background and improving localization precision by a factor of 2-4 times as compared to standard SMLM. To substantiate, corrSMLM is used for imaging fixed cell samples (Dendra2-Actin and Dendra2-Tubulin transfected NIH3T3 cells). Results show multi-fold reduction in noise and localization precision with a marked improvement in overall resolution and SBR. We anticipate corrSMLM to improve overall image quality and offer a better understanding of single molecule dynamics in cell biology.

biophysics↗

bioRxiv2023

Wetting by biomolecular condensates increases membrane lipid packing and dehydration

Membrane wetting by biomolecular condensates recently emerged as a key phenomenon in cell biology, playing an important role in a diverse range of processes across different organisms. However, an understanding of the molecular mechanisms behind condensate formation and interaction with lipid membranes is still missing. To study this, we exploited the properties of the dyes ACDAN and LAURDAN as nano-environmental sensors in combination with phasor analysis of hyperspectral and lifetime imaging microscopy. Using glycinin as a model condensate-forming protein and giant vesicles as model membranes, we obtained vital information on the process of condensate formation and membrane wetting. Our results reveal that glycinin condensates display differences in water dynamics when changing the salinity of the medium as a consequence of rearrangements in the secondary structure of the protein. Remarkably, analysis of membrane-condensates interaction with protein as well as polymer condensates indicated a correlation between increased wetting affinity and enhanced lipid packing. This is demonstrated by a decrease in the dipolar relaxation of water across all membrane-condensate systems, suggesting a general mechanism to tune membrane packing by condensate wetting.

biophysics↗

bioRxiv2023

Limits on Inferring Gene Regulatory Networks Subjected to Different Noise Mechanisms

One of the most difficult and pressing problems in computational cell biology is the inference of gene regulatory network structure from transcriptomic data. Benchmarking network inference methods on model organism datasets has yielded mixed results, in which the methods sometimes perform reasonably well and other times fail to outperform random guessing. In this paper, we analyze the feasibility of network inference under different noise conditions using stochastic simulations. We show that gene regulatory interactions with extrinsic noise appear to be more amenable to inference than those with only intrinsic noise, especially when the extrinsic noise causes the system to switch between distinct expression states. Furthermore, we analyze the problem of false positives between genes that have no direct interaction but share a common upstream regulator, and explore a strategy for distinguishing between these false positives and true interactions based on noise profiles of mRNA expression levels. Lastly, we derive mathematical formulas for the mRNA noise levels and correlation using moment analysis techniques, and show how these levels change as the mean mRNA expression level changes.

systems biology↗

bioRxiv2023

Artificial channel separation of monochrome multi-structure microscopy image

Fluorescence microscopy is a powerful tool for life sciences, which employs fluorescent tags to label and observe cellular structures and their dynamics. However, due to the spectral overlap between different dyes, a limited number of structures can be separately labeled and imaged for live cell applications. Here we propose a novel double-structure network (DBSN) that consists of multiple connected models, which can extract six subcellular structures from three images with only two separate fluorescent labels. DBSN combines the intensity-balance models to compensate for uneven fluorescent labels for different structures and the structure-separation models to extract multiple different structures with the same fluorescent labels. Therefore, DBSN permits the imaging of multiple structures with only one fluorescent label. It significantly reduces photobleaching, breaks the bottleneck of the existing technologies, and would have vast applications in cell biology.

biophysics↗