Biology subjects

Moerner, W. E.

Publications and source records attributed to Moerner, W. E..

At least 19 recordsLinked to original sources

bioRxiv2026

Organization of Myosin H in the Apical Complex of Toxoplasma Gondii Revealed by 3D Single-Molecule Super-Resolution Microscopy

Toxoplasma gondii is a single-celled eukaryotic parasite with prolific invasion capability. The parasite uses an apical complex comprised of proteinaceous structures and secretory organelles to efficiently enter host cells. As a result, the apical complex remains a vital structure of interest, with many studies dedicated to understanding its protein organization. One such protein is the motor Myosin H (MyoH), which is indispensable for parasite motility and host cell invasion. Given the small size of the complex, roughly a diffraction-limited volume in the visible, high-resolution techniques are required to make precise determinations of protein organization. In this work, we use 3D single-molecule localization microscopy in both traditionally fixed and gel-expanded parasites to localize the indispensable motor Myosin H within the apical complex. Labeling of the N- and C-terminus of MyoH in fixed parasites resolved the orientation of the motor protein in the apical complex, showing the motor head radially exterior to the tail. Two-color imaging of MyoH with tubulin in fixed parasites allowed for localization of the MyoH termini relative to the conoid, a barrel of tubulin-based fibers in the apical complex and showed the MyoH tail toward the interior face of the conoid and the head at the conoid exterior. Gel expansion showed improved labeling density for both tubulin and MyoH but altered MyoH localization, highlighting the nuanced effects of gel expansion on protein organization. Statement of SignificanceThis work employs 3D single-molecule super-resolution microscopy to provide quantitative physical analysis of the spatial organization of a vital myosin motor, MyoH, in the model apicomplexan parasite Toxoplasma gondii. While previous studies have provided high-resolution views of the parasites invasion machinery, MyoH has remained elusive at the nanoscale. We resolved differences in radial organization between the N- and C-termini of the motor, thus determining the orientation of the protein in the apical space. Two-color imaging revealed the organization of the motor in the greater context of the parasites invasion complex. 3D single-molecule imaging in gel-expanded samples revealed an increase in labeling efficiency but perturbed localization of only the MyoH C-terminus, highlighting the nuanced effects of gel expansion on protein organization.

biophysics↗

bioRxiv2025

Interferometric Image Scanning Microscopy for label-free imaging at 120 nm resolution inside live cells

Light microscopy remains indispensable in life sciences for visualizing cellular structures and dynamics in live specimens. Yet, conventional fluorescence imaging can suffer from phototoxicity, limited labeling efficiency, or perturbation of biological function. Label-free techniques such as interferometric scattering microscopy (iSCAT) offer a powerful alternative by detecting nanoscale structures based on their light scattering, without the need for dyes or genetic tags. iSCAT has enabled high-sensitivity detection of single proteins and viruses on clean surfaces. More recently, its application to live cells has been extended by using confocal illumination and detection, allowing suppression of out-of-focus light, yielding subcellular structures with high contrast. This development laid the foundation for biologically relevant label-free imaging. Here, we introduce interferometric image scanning microscopy (iISM). This next-generation technique combines interferometric detection with image scanning microscopy to achieve about 120 nm lateral resolution while operating at tenfold lower incident illumination power per diffraction limited spot, significantly reducing photodamage while enhancing signal-to-noise and contrast. Using iISM, we are able to visualize intracellular organelles such as the endoplasmic reticulum, actin cytoskeleton, mitochondria, and vesicles in live cells at essentially unlimited observation times. Importantly, iISM can be readily combined with confocal fluorescence microscopy, enabling correlation of label-free dynamics and structural information with molecular specificity. Our approach opens new avenues for studying dynamic biological processes, such as host-pathogen interactions, intracellular trafficking, or cytoskeletal rearrangements, under label-free, near-native conditions. iISM thus offers a powerful new tool for high-resolution, low-impact imaging of live cells, paving the way for new biological insights.

biophysics↗

bioRxiv2025

Efficient Double Helix Detection with Steerable Filters

We present an efficient detection scheme for localization of Double Helix point-spread functions for 3D single-molecule localization microscopy or tracking. Using steerable filters, we extract both 2D position and lobe orientation (axial position) estimates using just 7 convolutions, orders of magnitude less than used in deep-learning-based approaches. We pair this detection with a fitter and implement both as a plug-in for the open source PYthon Microscopy Environment (PYME), which features percentile-based background subtraction, signal-to-noise-based detection thresholding, and performant parallel analysis. Our complete localization analysis pipeline achieves state-of-the-art performance with minimal user input.

biophysics↗

bioRxiv2025

A Super-Resolution Spatial Atlas of SARS-CoV-2 Infection in Human Cells

Coronaviral replication depends on double-membrane vesicles (DMVs), yet where polyprotein processing occurs and how replication organelles mature remain unresolved. We built a multi{-}color super-resolution atlas of SARS-CoV-2 RNA, non-structural and structural proteins in infected human cells, combining 3D single-molecule localization with radial and angular pair-correlation analysis. The atlas uncovers fundamental spatial principles. First, nsp5 (3CLpro) localizes within the DMV lumen near pores, with nsp7-nsp16 positioned interior to nsp4, supporting a protease-dependent maturation model in which nsp5 action permits membrane closure and subsequently completes intravesicular polyprotein processing to activate replication complexes. Second, we identify dsRNA connectors bridging DMVs and non-canonical, nsp3/4-lacking dsRNA granules decorated with replicase components, consistent with a condensate-mediated route for trafficking replication intermediates. Third, mapping structural proteins reveals M- and S-positive virion assembly intermediates along the secretory route, while premature intracellular S1 shedding captures the baseline instability of the original virus isolate prior to evolutionary adaptation. Finally, the antiviral nirmatrelvir induces multilayered bodies of uncleaved polyproteins (nsp4-5-10-16) that persist after washout and precede rapid rebound, suggesting a drug-induced reservoir state. Beyond mapping the viral architecture, this atlas resolves the spatial context of proteolysis and organelle remodeling, providing a framework for pan-coronaviral mechanisms and antiviral design.

microbiology↗

bioRxiv2024

Exploring transient states of PAmKate to enable improved cryogenic single-molecule imaging

Super-resolved cryogenic correlative light and electron microscopy is a powerful approach which combines the single-molecule specificity and sensitivity of fluorescence imaging with the nano-scale resolution of cryogenic electron tomography. Key to this method is active control over the emissive state of fluorescent labels to ensure sufficient sparsity to localize individual emitters. Recent work has identified fluorescent proteins (FPs) which photoactivate or photoswitch efficiently at cryogenic temperatures, but long on-times due to reduced quantum yield of photobleaching remains a challenge for imaging structures with a high density of localizations. In this work, we explore the photophysical properties of the red photoactivatable FP PAmKate and identify a 2-color process leading to enhanced turn-off of active emitters, improving localization rate. Specifically, after excitation of ground state molecules, we find a transient state forms with a lifetime of [~]2 ms under cryogenic conditions which can be bleached by exposure to a second wavelength. We measure the response of the transient state to different wavelengths, demonstrate how this mechanism can be used to improve imaging, and provide a blueprint for study of other FPs at cryogenic temperatures.

biophysics↗

bioRxiv2023

Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles

The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelle where the replication of viral genomic RNA (vgRNA) occurs. To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain vgRNA clusters along with viral double-stranded RNA (dsRNA) clusters and the replication enzyme, encapsulated by membranes derived from the host endoplasmic reticulum (ER). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of ER labels and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are enclosed by DMVs at early infection stages which then merge into vesicle packets as infection progresses. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.

microbiology↗

bioRxiv2022

Exploring masses and internal mass distributions of single carboxysomes in free solution using fluorescence and interferometric scattering in an anti-Brownian trap

Carboxysomes are self-assembled bacterial microcompartments that facilitate carbon assimilation by co-localizing the enzymes of CO2 fixation within a protein shell. These microcompartments can be highly heterogeneous in their composition and filling, so measuring the mass and loading of an individual carboxysome would allow for better characterization of its assembly and function. To enable detailed and extended characterizations of single nanoparticles in solution, we recently demonstrated an improved Interferometric Scattering Anti-Brownian ELectrokinetic (ISABEL) trap, which tracks the position of a single nanoparticle via its scattering of a near-infrared beam and applies feedback to counteract its Brownian motion. Importantly, the scattering signal can be related to the mass of nanoscale proteinaceous objects, whose refractive indices are well-characterized. We calibrate single-particle scattering cross-section measurements in the ISABEL trap and determine individual carboxysome masses in the 50-400 MDa range by analyzing their scattering cross-sections with a core-shell model. We further investigate carboxysome loading by combining mass measurements with simultaneous fluorescence reporting from labeled internal components. This method may be extended to other biological objects, such as viruses or extracellular vesicles, and can be combined with orthogonal fluorescence reporters to achieve precise physical and chemical characterization of individual nanoscale biological objects. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/505029v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@18e9f99org.highwire.dtl.DTLVardef@900912org.highwire.dtl.DTLVardef@4398baorg.highwire.dtl.DTLVardef@1dff103_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

Ratiometric Sensing of Redox Environments Inside Individual Carboxysomes Trapped in Solution

Diffusion of biological nanoparticles in solution impedes our ability to continuously monitor individuals and measure their physical and chemical properties. To overcome this, we previously developed the Interferometric Scattering Anti-Brownian ELectrokinetic (ISABEL) trap, which uses scattering to localize a particle and applies electrokinetic forces which counteract Brownian motion, thus enabling extended observation. Here, we present an improved ISABEL trap that incorporates a near-infrared scatter illumination beam and rapidly interleaves 405 and 488 nm fluorescence excitation reporter beams. With the ISABEL trap, we monitor the internal redox environment of individual carboxysomes labeled with the ratiometric redox reporter roGFP2. Carboxysomes widely vary in scattering contrast (reporting on size) and redox-dependent ratiometric fluorescence. Further, we used redox sensing to explore the chemical kinetics within intact carboxysomes, where bulk measurements may contain unwanted contributions from aggregates or interfering fluorescent proteins. Overall, we demonstrate the ISABEL traps ability to sensitively monitor nanoscale biological objects, enabling new experiments on these systems. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/484789v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1fccfacorg.highwire.dtl.DTLVardef@a639b6org.highwire.dtl.DTLVardef@116fff6org.highwire.dtl.DTLVardef@fe95fd_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Fast and parallel nanoscale 3D tracking of heterogeneous mammalian chromatin dynamics

Chromatin organization and dynamics are critical for gene regulation. In this work we present a methodology for fast and parallel 3D tracking of multiple chromosomal loci of choice over many thousands of frames on various time scales. We achieved this by developing and combining fluorogenic and replenishable nanobody arrays, engineered point spread functions, and light sheet illumination. The result is gentle live-cell 3D tracking with excellent spatiotemporal resolution throughout the mammalian cell nucleus. Correction for both sample drift and nuclear translation facilitated accurate long-term tracking of the chromatin dynamics. We demonstrate tracking of both fast dynamics (50 Hz) and over time scales extending to several hours, and we find both large heterogeneity between cells and apparent anisotropy in the dynamics in the axial direction. We further quantify the effect of inhibiting actin polymerization on the dynamics and find an overall increase in both the apparent diffusion coefficient D* and anomalous diffusion exponent , and a transition to more isotropic dynamics in 3D after such treatment. We think that our methodology in the future will allow researchers to obtain a better fundamental understanding of chromatin dynamics and how it is altered during disease progression and after perturbations of cellular function.

biophysics↗

bioRxiv2021

Exploring cell surface-nanopillar interactions with 3D super-resolution microscopy

Plasma membrane topography has been shown to strongly influence the behavior of many cellular processes such as clathrin-mediated endocytosis, actin rearrangements, and others. Recent studies have used 3D nanostructures such as nanopillars to imprint well-defined membrane curvatures (the "nano-bio interface"). In these studies, proteins and their interactions were probed by 2D fluorescence microscopy. However, the low resolution and limited axial detail of such methods are not optimal to determine the relative spatial position and distribution of proteins along a 100 nm-diameter object, which is below the optical diffraction limit. Here, we introduce a general method to explore the nanoscale distribution of proteins at the nano-bio interface with 10-20 nm precision using 3D single-molecule super-resolution (SR) localization microscopy. This is achieved by combining a silicone oil immersion objective and 3D double-helix point-spread function microscopy. We carefully optimize the objective to minimize spherical aberrations between quartz nanopillars and the cell. To validate the 3D SR method, we imaged the 3D shape of surface-labeled nanopillars and compared the results with electron microscopy measurements. Turning to transmembrane-anchored labels in cells, the high quality 3D SR reconstructions reveal the membrane tightly wrapping around the nanopillars. Interestingly, the cytoplasmic protein AP-2 involved in clathrin-mediated endocytosis accumulates along the nanopillar above a specific threshold of 1/R membrane curvature. Finally, we observe that AP-2 and actin preferentially accumulate at positive Gaussian curvature near the pillar caps. Our results establish a general method to investigate the nanoscale distribution of proteins at the nano-bio interface using 3D SR microscopy.

biophysics↗

bioRxiv2021

Multi-color super-resolution imaging to study human coronavirus RNA during cellular infection

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third human coronavirus within 20 years that gave rise to a life-threatening disease and the first to reach pandemic spread. To make therapeutic headway against current and future coronaviruses, the biology of coronavirus RNA during infection must be precisely understood. Here, we present a robust and generalizable framework combining high-throughput confocal and super-resolution microscopy imaging to study coronavirus infection at the nanoscale. Employing the model human coronavirus HCoV-229E, we specifically labeled coronavirus genomic RNA (gRNA) and double-stranded RNA (dsRNA) via multicolor RNA-immunoFISH and visualized their localization patterns within the cell. The exquisite resolution of our approach uncovers a striking spatial organization of gRNA and dsRNA into three distinct structures and enables quantitative characterization of the status of the infection after antiviral drug treatment. Our approach provides a comprehensive framework that supports investigations of coronavirus fundamental biology and therapeutic effects.

cell biology↗

bioRxiv2020

Modulation of kinase activity within a bacterial membraneless organelle

Biomolecular condensates formed via phase separation enable spatial and temporal organization of enzyme activity. The emergent properties of many condensates have been shown to be responsive to intracellular Adenosine triphosphate (ATP) levels, although the consequences of such mechanisms on enzyme activity are unknown. Here, we show that ATP depletion promotes phase separation in condensates composed of a disordered protein, thereby enhancing the activity of a client kinase enabling robust signaling and maintenance of viability under the stress posed by nutrient scarcity. We propose that a diverse repertoire of condensates can serve as control knobs to tune multivalency and reactivity in response to the metabolic state of bacterial cells. One Sentence SummaryBacterial condensates boost kinase activity under ATP depletion

microbiology↗

bioRxiv2020

Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe GalNAzMe that is specific for cancer-relevant Ser/Thr-N-acetylgalactosamine (O-GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn (N)-linked glycans. We equip cells with the capacity to biosynthesize the nucleotide-sugar donor UDP-GalNAzMe from a caged precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR knock-out (KO) screen, and imaging of intestinal organoids. GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation. Significance statementA large portion of all secreted and cell surface proteins in humans are modified by Ser/Thr(O)-linked glycosylation with N-acetylgalactosamine (GalNAc). While of fundamental importance in health and disease, O-GalNAc glycosylation is technically challenging to study because of a lack of specific tools to be used in biological assays. Here, we design an O-GalNAc specific reporter molecule termed GalNAzMe to selectively label O-GalNAc glycoproteins in living human cells. GalNAzMe is compatible with a range of experiments in quantitative biology to broaden our understanding of glycosylation. We further demonstrate that labeling is genetically programmable by expression of a mutant glycosyltransferase, allowing application even to experiments with low inherent sensitivity.

synthetic biology↗

bioRxiv2019

Continuous, Topologically Guided Protein Crystallization Controls Bacterial Surface Layer Self-Assembly

Bacteria assemble the cell envelope using localized enzymes to account for growth and division of a topologically complicated surface1-3. However, a regulatory pathway has not been identified for assembly and maintenance of the surface layer (S-layer), a 2D crystalline protein coat surrounding the curved 3D surface of a variety of bacteria4,5. By specifically labeling, imaging, and tracking native and purified RsaA, the S-layer protein (SLP) from C. crescentus, we show that protein self-assembly alone is sufficient to assemble and maintain the S-layer in vivo. By monitoring the location of newly produced S-layer on the surface of living bacteria, we find that S-layer assembly occurs independently of the site of RsaA secretion and that localized production of new cell wall surface area alone is insufficient to explain S-layer assembly patterns. When the cell surface is devoid of a pre-existing S-layer, the location of S-layer assembly depends on the nucleation characteristics of SLP crystals, which grow by capturing RsaA molecules freely diffusing on the outer bacterial surface. Based on these observations, we propose a model of S-layer assembly whereby RsaA monomers are secreted randomly and diffuse on the lipopolysaccharide (LPS) outer membrane until incorporated into growing 2D S-layer crystals. The complicated topology of the cell surface enables formation of defects, gaps, and grain boundaries within the S-layer lattice, thereby guiding the location of S-layer assembly without enzymatic assistance. This unsupervised mechanism poses unique challenges and advantages for designing treatments targeting cell surface structures or utilizing S-layers as self-assembling macromolecular nanomaterials. As an evolutionary driver, 2D protein self-assembly rationalizes the exceptional S-layer subunit sequence and species diversity6.

biophysics↗

bioRxiv2018

Revealing the nanoscale morphology of the primary cilium using super-resolution fluorescence microscopy

Super-resolution (SR) microscopy has been used to observe structural details beyond the diffraction limit of ~250 nm in a variety of biological and materials systems. By combining this imaging technique with both computer-vision algorithms and topological methods, we reveal and quantify the nanoscale morphology of the primary cilium, a tiny tubular cellular structure (~2-6 m long and 200-300 nm diameter). The cilium in mammalian cells protrudes out of the plasma membrane and is important in many signaling processes related to cellular differentiation and disease. After tagging individual ciliary transmembrane proteins, specifically Smoothened (SMO), with single fluorescent labels in fixed cells, we use three-dimensional (3D) single-molecule SR microscopy to determine their positions with a precision of 10-25 nm. We gain a dense, pointillistic reconstruction of the surfaces of many cilia, revealing large heterogeneity in membrane shape. A Poisson surface reconstruction (PSR) algorithm generates a fine surface mesh, allowing us to characterize the presence of deformations by quantifying the surface curvature. Upon impairment of intracellular cargo transport machinery by genetic knockout or small-molecule treatment of cells, our quantitative curvature analysis shows significant morphological differences not visible by conventional fluorescence microscopy techniques. Furthermore, using a complementary SR technique, 2-color, 2D STimulated Emission Depletion (STED) microscopy, we find that the cytoskeleton in the cilium, the axoneme, also exhibits abnormal morphology in the mutant cells, similar to our 3D results on the SMO-measured ciliary surface. Our work combines 3D SR microscopy and computational tools to quantitatively characterize morphological changes of the primary cilium under different treatments and uses STED to discover correlated changes in the underlying structure. This approach can be useful for studying other biological or nanoscale structures of interest.

biophysics↗

bioRxiv2017

Phospho-signal flow from a pole-localized microdomain spatially patterns transcription factor activity

Selective recruitment and concentration of signaling proteins within membrane-less compartments is a ubiquitous mechanism for subcellular organization. However, little is known about how such dynamic recruitment patterns intracellular signaling and cellular development. Here, we combined transcriptional profiling, reaction-diffusion modeling, and single-molecule tracking to study signal exchange in and out of a microdomain at the cell pole of the asymmetrically dividing bacterium Caulobacter crescentus. Our study revealed that the microdomain is selectively permeable, and that each protein in the signaling pathway that activates the cell fate transcription factor CtrA is sequestered and uniformly concentrated within the microdomain or its proximal membrane. Restricted rates of entry into and escape from the microdomain enhance phospho-signaling, leading to a sublinear gradient of CtrA~P along the long axis of the cell. The spatial patterning of CtrA~P creates a gradient of transcriptional activation that serves to prime asymmetric development of the two daughter cells.

cell biology↗

bioRxiv2017

Spatial organization and dynamics of RNase E and ribosomes in Caulobacter crescentus

We report the dynamic spatial organization of Caulobacter crescentus RNase E (RNA degradosome) and ribosomal protein L1 (ribosome) using 3D single particle tracking and super-resolution microscopy. RNase E formed clusters along the central axis of the cell, while weak clusters of ribosomal protein L1 were deployed throughout the cytoplasm. These results contrast with RNase E and ribosome distribution in E. coli, where RNase E co-localizes with the cytoplasmic membrane and ribosomes accumulate in polar nucleoid-free zones. For both RNase E and ribosomes in Caulobacter, we observed a decrease in confinement and clustering upon transcription inhibition and subsequent depletion of nascent RNA, suggesting that RNA substrate availability for processing, degradation, and translation facilitates confinement and clustering. Moreover, RNase E cluster positions correlate with the subcellular location of chromosomal loci of two highly transcribed ribosomal RNA genes, suggesting that RNase Es function in ribosomal RNA processing occurs at the site of rRNA synthesis. Thus, components of the RNA degradosome and ribosome assembly are spatiotemporally organized in Caulobacter, with chromosomal readout serving as the template for this organization.

biophysics↗

bioRxiv2017

Single-molecule diffusometry reveals the nucleotide-dependent oligomerization pathways of Nicotiana tabacum Rubisco activase

Oligomerization plays an important role in the function of many proteins, but a quantitative picture of the oligomer distribution has been difficult to obtain using existing techniques. Here we describe a method that combines sub-stoichiometric labeling and recently-developed single-molecule diffusometry to measure the size distribution of oligomers under equilibrium conditions in solution, one molecule at a time. We use this technique to characterize the oligomerization behavior of Nicotiana tabacum (Nt) rubisco activase (Nt-Rca), a chaperone-like, AAA-plus ATPase essential in regulating carbon fixation during photosynthesis. We directly observed monomers, dimers and a tetramer/hexamer mixture, and extracted their fractional abundance as a function of protein concentration. We show that the oligomerization pathway of Nt-Rca is nucleotide dependent: ATP{gamma}S binding strongly promotes tetramer/hexamer formation from dimers and results in a preferred tetramer/hexamer population for concentrations in the 1-10M range. Furthermore, we directly observed dynamic assembly and disassembly processes of single complexes in real time, and from there estimated the rate of subunit exchange to be ~0.1s-1 with ATP{gamma}S. On the other hand, ADP binding destabilizes Rca complexes by enhancing the rate of subunit exchange by >2 fold. These observations provide a quantitative starting point to elucidate the structure-function relations of Nt-Rca complexes. We envision the method to fill a critical gap in defining and quantifying protein assembly pathways in the small-oligomer regime.

biophysics↗