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1.
Proc Natl Acad Sci U S A ; 121(13): e2401625121, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38507449

RESUMO

Molecular motors employ chemical energy to generate unidirectional mechanical output against a track while navigating a chaotic cellular environment, potential disorder on the track, and against Brownian motion. Nevertheless, decades of nanometer-precise optical studies suggest that myosin-5a, one of the prototypical molecular motors, takes uniform steps spanning 13 subunits (36 nm) along its F-actin track. Here, we use high-resolution interferometric scattering microscopy to reveal that myosin takes strides spanning 22 to 34 actin subunits, despite walking straight along the helical actin filament. We show that cumulative angular disorder in F-actin accounts for the observed proportion of each stride length, akin to crossing a river on variably spaced stepping stones. Electron microscopy revealed the structure of the stepping molecule. Our results indicate that both motor and track are soft materials that can adapt to function in complex cellular conditions.


Assuntos
Actinas , Miosina Tipo V , Actinas/química , Miosinas/química , Citoesqueleto de Actina/química , Movimento (Física) , Miosina Tipo V/química
2.
Proc Natl Acad Sci U S A ; 119(39): e2206292119, 2022 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-36122222

RESUMO

Understanding the pathways by which simple RNA viruses self-assemble from their coat proteins and RNA is of practical and fundamental interest. Although RNA-protein interactions are thought to play a critical role in the assembly, our understanding of their effects is limited because the assembly process is difficult to observe directly. We address this problem by using interferometric scattering microscopy, a sensitive optical technique with high dynamic range, to follow the in vitro assembly kinetics of more than 500 individual particles of brome mosaic virus (BMV)-for which RNA-protein interactions can be controlled by varying the ionic strength of the buffer. We find that when RNA-protein interactions are weak, BMV assembles by a nucleation-and-growth pathway in which a small cluster of RNA-bound proteins must exceed a critical size before additional proteins can bind. As the strength of RNA-protein interactions increases, the nucleation time becomes shorter and more narrowly distributed, but the time to grow a capsid after nucleation is largely unaffected. These results suggest that the nucleation rate is controlled by RNA-protein interactions, while the growth process is driven less by RNA-protein interactions and more by protein-protein interactions and intraprotein forces. The nucleated pathway observed with the plant virus BMV is strikingly similar to that previously observed with bacteriophage MS2, a phylogenetically distinct virus with a different host kingdom. These results raise the possibility that nucleated assembly pathways might be common to other RNA viruses.


Assuntos
Bromovirus , Vírus de RNA , Bromovirus/genética , Bromovirus/metabolismo , Capsídeo/metabolismo , Vírus de RNA/genética , RNA Viral/genética , RNA Viral/metabolismo , Vírion/genética , Vírion/metabolismo
3.
Nano Lett ; 24(6): 1874-1881, 2024 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-38295760

RESUMO

Traditional single-nanoparticle sizing using optical microscopy techniques assesses size via the diffusion constant, which requires suspended particles to be in a medium of known viscosity. However, these assumptions are typically not fulfilled in complex natural sample environments. Here, we introduce dual-angle interferometric scattering microscopy (DAISY), enabling optical quantification of both size and polarizability of individual nanoparticles (radius <170 nm) without requiring a priori information regarding the surrounding media or super-resolution imaging. DAISY achieves this by combining the information contained in concurrently measured forward and backward scattering images through twilight off-axis holography and interferometric scattering (iSCAT). Going beyond particle size and polarizability, single-particle morphology can be deduced from the fact that the hydrodynamic radius relates to the outer particle radius, while the scattering-based size estimate depends on the internal mass distribution of the particles. We demonstrate this by differentiating biomolecular fractal aggregates from spherical particles in fetal bovine serum at the single-particle level.

4.
Sensors (Basel) ; 24(2)2024 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-38257678

RESUMO

LiDAR place recognition is a crucial component of autonomous navigation, essential for loop closure in simultaneous localization and mapping (SLAM) systems. Notably, while camera-based methods struggle in fluctuating environments, such as weather or light, LiDAR demonstrates robustness against such challenges. This study introduces the intensity and spatial cross-attention transformer, which is a novel approach that utilizes LiDAR to generate global descriptors by fusing spatial and intensity data for enhanced place recognition. The proposed model leveraged a cross attention to a concatenation mechanism to process and integrate multi-layered LiDAR projections. Consequently, the previously unexplored synergy between spatial and intensity data was addressed. We demonstrated the performance of IS-CAT through extensive validation on the NCLT dataset. Additionally, we performed indoor evaluations on our Sejong indoor-5F dataset and demonstrated successful application to a 3D LiDAR SLAM system. Our findings highlight descriptors that demonstrate superior performance in various environments. This performance enhancement is evident in both indoor and outdoor settings, underscoring the practical effectiveness and advancements of our approach.

5.
Proc Natl Acad Sci U S A ; 117(27): 15632-15641, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571914

RESUMO

KIF3AC is a mammalian neuron-specific kinesin-2 implicated in intracellular cargo transport. It is a heterodimer of KIF3A and KIF3C motor polypeptides which have distinct biochemical and motile properties as engineered homodimers. Single-molecule motility assays show that KIF3AC moves processively along microtubules at a rate faster than expected given the motility rates of the KIF3AA and much slower KIF3CC homodimers. To resolve the stepping kinetics of KIF3A and KIF3C motors in homo- and heterodimeric constructs and determine their transport potential under load, we assayed motor activity using interferometric scattering microscopy and optical trapping. The distribution of stepping durations of KIF3AC molecules is described by a rate (k1 = 11 s-1) without apparent kinetic asymmetry. Asymmetry was also not apparent under hindering or assisting mechanical loads in the optical trap. KIF3AC shows increased force sensitivity relative to KIF3AA yet is more capable of stepping against mechanical load than KIF3CC. Interestingly, the behavior of KIF3C mirrors prior studies of kinesins with increased interhead compliance. Microtubule gliding assays containing 1:1 mixtures of KIF3AA and KIF3CC result in speeds similar to KIF3AC, suggesting the homodimers mechanically impact each other's motility to reproduce the behavior of the heterodimer. Our observations are consistent with a mechanism in which the stepping of KIF3C can be activated by KIF3A in a strain-dependent manner, similar to application of an assisting load. These results suggest that the mechanochemical properties of KIF3AC can be explained by the strain-dependent kinetics of KIF3A and KIF3C.


Assuntos
Cinesinas/metabolismo , Microtúbulos/metabolismo , Multimerização Proteica/fisiologia , Fenômenos Biomecânicos , Cinética , Proteínas Recombinantes/metabolismo
6.
Adv Exp Med Biol ; 1239: 153-181, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32451859

RESUMO

Myosin 5a is a two-headed myosin that functions as a cargo transporter in cells. To accomplish this task it has evolved several unique structural and kinetic features that allow it to move processively as a single molecule along actin filaments. A plethora of biophysical techniques have been used to elucidate the detailed mechanism of its movement along actin filaments in vitro. This chapter describes how this mechanism was deduced.


Assuntos
Movimento , Miosinas , Imagem Individual de Molécula , Citoesqueleto de Actina , Actinas , Biofísica , Humanos , Cinética , Miosinas/metabolismo
7.
Adv Exp Med Biol ; 1239: 85-126, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32451857

RESUMO

Inside the cellular environment, molecular motors can work in concert to conduct a variety of important physiological functions and processes that are vital for the survival of a cell. However, in order to decipher the mechanism of how these molecular motors work, single-molecule microscopy techniques have been popular methods to understand the molecular basis of the emerging ensemble behavior of these motor proteins.In this chapter, we discuss various single-molecule biophysical imaging techniques that have been used to expose the mechanics and kinetics of myosins. The chapter should be taken as a general overview and introductory guide to the many existing techniques; however, since other chapters will discuss some of these techniques more thoroughly, the readership should refer to those chapters for further details and discussions. In particular, we will focus on scattering-based single-molecule microscopy methods, some of which have become more popular in the recent years and around which the work in our laboratories has been centered.


Assuntos
Actomiosina/metabolismo , Proteínas Motores Moleculares/metabolismo , Imagem Individual de Molécula , Citoesqueleto de Actina , Humanos , Miosinas
8.
Nano Lett ; 19(8): 4827-4835, 2019 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-31314539

RESUMO

Fluorescence microscopy has been the workhorse for investigating optical phenomena at the nanometer scale but this approach confronts several fundamental limits. As a result, there have been a growing number of activities toward the development of fluorescent-free imaging methods. In this Mini Review, we demonstrate that elastic scattering, the most ubiquitous and oldest optical contrast mechanism, offers excellent opportunities for sensitive detection and imaging of nanoparticles and molecules at very high spatiotemporal resolution. We present interferometric scattering (iSCAT) microscopy as the method of choice, explain its theoretical foundation, discuss its experimental nuances, elaborate on its deep connection to bright-field imaging and other established microscopies, and discuss its promise as well as challenges. A showcase of numerous applications and avenues made possible by iSCAT demonstrates its rapidly growing impact on various disciplines concerned with nanoscopic phenomena.

9.
Nano Lett ; 18(1): 513-519, 2018 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-29227108

RESUMO

Cellular secretion of proteins into the extracellular environment is an essential mediator of critical biological mechanisms, including cell-to-cell communication, immunological response, targeted delivery, and differentiation. Here, we report a novel methodology that allows for the real-time detection and imaging of single unlabeled proteins that are secreted from individual living cells. This is accomplished via interferometric detection of scattered light (iSCAT) and is demonstrated with Laz388 cells, an Epstein-Barr virus (EBV)-transformed B cell line. We find that single Laz388 cells actively secrete IgG antibodies at a rate of the order of 100 molecules per second. Intriguingly, we also find that other proteins and particles spanning ca. 100 kDa-1 MDa are secreted from the Laz388 cells in tandem with IgG antibody release, likely arising from EBV-related viral proteins. The technique is general and, as we show, can also be applied to studying the lysate of a single cell. Our results establish label-free iSCAT imaging as a powerful tool for studying the real-time exchange between cells and their immediate environment with single-protein sensitivity.


Assuntos
Linfócitos B/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Imunoglobulina G/análise , Análise de Célula Única/métodos , Linfócitos B/virologia , Linhagem Celular , Herpesvirus Humano 4/isolamento & purificação , Humanos , Imunoglobulina G/imunologia , Luz , Microscopia de Interferência/métodos , Imagem Óptica/métodos , Espalhamento de Radiação
10.
J Phys D Appl Phys ; 51(23): 235401, 2018 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-29853718

RESUMO

Observation techniques with high spatial and temporal resolution, such as single-particle tracking based on interferometric scattering (iSCAT) microscopy, and fluorescence correlation spectroscopy applied on a super-resolution STED microscope (STED-FCS), have revealed new insights of the molecular organization of membranes. While delivering complementary information, there are still distinct differences between these techniques, most prominently the use of fluorescent dye tagged probes for STED-FCS and a need for larger scattering gold nanoparticle tags for iSCAT. In this work, we have used lipid analogues tagged with a hybrid fluorescent tag-gold nanoparticle construct, to directly compare the results from STED-FCS and iSCAT measurements of phospholipid diffusion on a homogeneous supported lipid bilayer (SLB). These comparative measurements showed that while the mode of diffusion remained free, at least at the spatial (>40 nm) and temporal (50 ⩽ t ⩽ 100 ms) scales probed, the diffussion coefficient was reduced by 20- to 60-fold when tagging with 20 and 40 nm large gold particles as compared to when using dye tagged lipid analogues. These FCS measurements of hybrid fluorescent tag-gold nanoparticle labeled lipids also revealed that commercially supplied streptavidin-coated gold nanoparticles contain large quantities of free streptavidin. Finally, the values of apparent diffusion coefficients obtained by STED-FCS and iSCAT differed by a factor of 2-3 across the techniques, while relative differences in mobility between different species of lipid analogues considered were identical in both approaches. In conclusion, our experiments reveal that large and potentially cross-linking scattering tags introduce a significant slow-down in diffusion on SLBs but no additional bias, and our labeling approach creates a new way of exploiting complementary information from STED-FCS and iSCAT measurements.

11.
Proc Natl Acad Sci U S A ; 112(52): E7186-93, 2015 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-26676576

RESUMO

To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head-bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.


Assuntos
Trifosfato de Adenosina/química , Proteínas de Drosophila/química , Cinesinas/química , Trifosfato de Adenosina/metabolismo , Algoritmos , Animais , Proteínas de Drosophila/metabolismo , Hidrólise , Cinesinas/metabolismo , Cinética , Microscopia de Interferência , Modelos Químicos , Modelos Moleculares , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , Estrutura Terciária de Proteína
12.
Proc Natl Acad Sci U S A ; 112(40): 12299-303, 2015 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-26401022

RESUMO

Lipid rafts are submicron proteolipid domains thought to be responsible for membrane trafficking and signaling. Their small size and transient nature put an understanding of their dynamics beyond the reach of existing techniques, leading to much contention as to their exact role. Here, we exploit the differences in light scattering from lipid bilayer phases to achieve dynamic imaging of nanoscopic lipid domains without any labels. Using phase-separated droplet interface bilayers we resolve the diffusion of domains as small as 50 nm in radius and observe nanodomain formation, destruction, and dynamic coalescence with a domain lifetime of 220±60 ms. Domain dynamics on this timescale suggests an important role in modulating membrane protein function.


Assuntos
Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Microdomínios da Membrana/química , Nanoestruturas/química , Difusão , Cinética , Luz , Microscopia de Interferência/métodos , Nanotecnologia/métodos , Espalhamento de Radiação , Solubilidade , Propriedades de Superfície , Temperatura
13.
Nano Lett ; 17(2): 1277-1281, 2017 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-28088861

RESUMO

Label-free detection, analysis, and rapid tracking of nanoparticles is crucial for future ultrasensitive sensing applications, ranging from understanding of biological interactions to the study of size-dependent classical-quantum transitions. Yet optical techniques to distinguish nanoparticles directly among their background remain challenging. Here we present amplified interferometric scattering microscopy (a-iSCAT) as a new all-optical method capable of detecting individual nanoparticles as small as 15 kDa proteins that is equivalent to half a GFP. By balancing scattering and reflection amplitudes the interference contrast of the nanoparticle signal is amplified 1 to 2 orders of magnitude. Beyond high sensitivity, a-iSCAT allows high-speed image acquisition exceeding several hundreds of frames-per-second. We showcase the performance of our approach by detecting single Streptavidin binding events and by tracking single Ferritin proteins at 400 frames-per-second with 12 nm localization precision over seconds. Moreover, due to its extremely simple experimental realization, this advancement finally enables a cheap and routine implementation of label-free all-optical single nanoparticle detection platforms with sensitivity operating at the single protein level.


Assuntos
Técnicas Biossensoriais/métodos , Proteínas de Ligação ao Ferro/análise , Microscopia de Interferência/métodos , Nanopartículas/química , Receptores de Superfície Celular/análise , Estreptavidina/análise , Difusão , Fluorescência , Ouro/química , Humanos , Modelos Teóricos , Peso Molecular , Nanoestruturas/química , Nanotecnologia , Ligação Proteica , Titânio/química
14.
ACS Nano ; 18(29): 19161-19168, 2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-38981021

RESUMO

Quantitative measurements of nanoparticle concentration in liquid suspensions are in high demand, for example, in the medical and food industries. Conventional methods remain unsatisfactory, especially for polydisperse samples with overlapping size ranges. Recently, we introduced interferometric nanoparticle tracking analysis (iNTA) for high-precision measurement of nanoparticle size and refractive index. Here, we show that by counting the number of trajectories that cross the focal plane, iNTA can measure concentrations of subpopulations in a polydisperse mixture in a quantitative manner and without the need for a calibration sample. We evaluate our method on both monodisperse samples and mixtures of known concentrations. Furthermore, we assess the concentration of SARS-CoV-2 in supernatant samples obtained from infected cells.


Assuntos
Interferometria , Nanopartículas , Tamanho da Partícula , SARS-CoV-2 , Interferometria/métodos , Nanopartículas/química , SARS-CoV-2/isolamento & purificação , COVID-19 , Humanos
15.
Methods Mol Biol ; 2623: 177-186, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36602686

RESUMO

The adapter dynactin and the activator BicD2 associate with dynein to form the highly motile dynein-dynactin-BicD2 (DDB) complex. In single-molecule assays, DDB displays processive runs, diffusive episodes, and transient pauses. The switching rates and durations of the different phases can be determined by tracking gold nanoparticle-labeled DDB complexes with interferometric scattering (iSCAT) microscopy and using an algorithm to separate out different motility phases. Here we describe methods for purifying DDB complexes from brain lysate, labeling with gold nanoparticles, imaging by iSCAT, and analyzing the resulting trajectories.


Assuntos
Dineínas , Nanopartículas Metálicas , Dineínas/metabolismo , Complexo Dinactina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Ouro , Microtúbulos/metabolismo
16.
bioRxiv ; 2023 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-37503193

RESUMO

Molecular motors employ chemical energy to generate unidirectional mechanical output against a track. By contrast to the majority of macroscopic machines, they need to navigate a chaotic cellular environment, potential disorder in the track and Brownian motion. Nevertheless, decades of nanometer-precise optical studies suggest that myosin-5a, one of the prototypical molecular motors, takes uniform steps spanning 13 subunits (36 nm) along its F-actin track. Here, we use high-resolution interferometric scattering (iSCAT) microscopy to reveal that myosin takes strides spanning 22 to 34 actin subunits, despite walking straight along the helical actin filament. We show that cumulative angular disorder in F-actin accounts for the observed proportion of each stride length, akin to crossing a river on variably-spaced stepping stones. Electron microscopy revealed the structure of the stepping molecule. Our results indicate that both motor and track are soft materials that can adapt to function in complex cellular conditions.

17.
Small Methods ; 5(4): e2000985, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-34927839

RESUMO

Microtubules are cytoskeletal polymers of tubulin dimers assembled into protofilaments that constitute nanotubes undergoing periods of assembly and disassembly. Static electron micrographs suggest a structural transition of straight protofilaments into curved ones occurring at the tips of disassembling microtubules. However, these structural transitions have never been observed and the process of microtubule disassembly thus remains unclear. Here, label-free optical microscopy capable of selective imaging of the transient structural changes of protofilaments at the tip of a disassembling microtubule is introduced. Upon induced disassembly, the transition of ordered protofilaments into a disordered conformation is resolved at the tip of the microtubule. Imaging the unbinding of individual tubulin oligomers from the microtubule tip reveals transient pauses and relapses in the disassembly, concurrent with increased organization of protofilament segments at the microtubule tip. These findings show that microtubule disassembly is a discrete process and suggest a stochastic mechanism of switching from the disassembly to the assembly phase.


Assuntos
Microscopia/métodos , Microtúbulos/química , Polímeros/análise , Conformação Proteica , Tubulina (Proteína)
18.
Essays Biochem ; 65(1): 81-91, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33296454

RESUMO

Single-molecule imaging has mostly been restricted to the use of fluorescence labelling as a contrast mechanism due to its superior ability to visualise molecules of interest on top of an overwhelming background of other molecules. Recently, interferometric scattering (iSCAT) microscopy has demonstrated the detection and imaging of single biomolecules based on light scattering without the need for fluorescent labels. Significant improvements in measurement sensitivity combined with a dependence of scattering signal on object size have led to the development of mass photometry, a technique that measures the mass of individual molecules and thereby determines mass distributions of biomolecule samples in solution. The experimental simplicity of mass photometry makes it a powerful tool to analyse biomolecular equilibria quantitatively with low sample consumption within minutes. When used for label-free imaging of reconstituted or cellular systems, the strict size-dependence of the iSCAT signal enables quantitative measurements of processes at size scales reaching from single-molecule observations during complex assembly up to mesoscopic dynamics of cellular components and extracellular protrusions. In this review, I would like to introduce the principles of this emerging imaging technology and discuss examples that show how mass-sensitive iSCAT can be used as a strong complement to other routine techniques in biochemistry.


Assuntos
Microscopia , Imagem Individual de Molécula , Microscopia/métodos , Nanotecnologia
19.
Front Cell Dev Biol ; 8: 590158, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33224953

RESUMO

The mobility of proteins and lipids within the cell, sculpted oftentimes by the organization of the membrane, reveals a great wealth of information on the function and interaction of these molecules as well as the membrane itself. Single particle tracking has proven to be a vital tool to study the mobility of individual molecules and unravel details of their behavior. Interferometric scattering (iSCAT) microscopy is an emerging technique well-suited for visualizing the diffusion of gold nanoparticle-labeled membrane proteins to a spatial and temporal resolution beyond the means of traditional fluorescent labels. We discuss the applicability of interferometric single particle tracking (iSPT) microscopy to investigate the minutia in the motion of a protein through measurements visualizing the mobility of the epidermal growth factor receptor in various biological scenarios on the live cell.

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