RESUMO
Synaptic AMPA receptors (AMPARs) on neuronal plasma membranes are correlated with learning and memory. Using a unique labeling and super-resolution imaging, we have visualized the nanoscale synaptic and extra-synaptic organization of native surface AMPARs for the first time in mouse brain slices as a function of brain region and tauopathy. We find that the fraction of surface AMPARs organized in synaptic clusters is two-times smaller in the hippocampus compared to the motor and somatosensory cortex. In 6 months old PS19 model of tauopathy, synaptic and extrasynaptic distributions are disrupted in the hippocampus but not in the cortex. Thus, this optimized super-resolution imaging tool allows us to observe synaptic deterioration at the onset of tauopathy before apparent neurodegeneration.
RESUMO
Dendritic spines are the main sites for synaptic communication in neurons, and alterations in their density, size, and shapes occur in many brain disorders. Current spine segmentation methods perform poorly in conditions with low signal-to-noise and resolution, particularly in the widefield images of thick (10 µm) brain slices. Here, we combined two open-source machine-learning models to achieve automatic 3D spine segmentation in widefield diffraction-limited fluorescence images of neurons in thick brain slices. We validated the performance by comparison with manually segmented super-resolution images of spines reconstructed from direct stochastic optical reconstruction microscopy (dSTORM). Lastly, we show an application of our approach by combining spine segmentation from diffraction-limited images with dSTORM of synaptic protein PSD-95 in the same field-of-view. This allowed us to automatically analyze and quantify the nanoscale distribution of PSD-95 inside the spine. Importantly, we found the numbers, but not the average sizes, of synaptic nanomodules and nanodomains increase with spine size.
RESUMO
Cytoplasmic dynein is a dimeric motor that drives minus-end directed transport on microtubules (MTs). To couple ATP hydrolysis to a mechanical step, a dynein monomer must be released from the MT before undergoing a conformational change that generates a bias towards the minus end. However, the dynamics of dynein stepping have been poorly characterized by tracking flexible regions of the motor with limited resolution. Here, we developed a cysteine-light mutant of yeast dynein and site-specifically labeled its MT-binding domain in vitro. MINFLUX tracking at sub-millisecond resolution revealed that dynein hydrolyzes one ATP per step and takes multitudes of 8 nm steps at physiological ATP. Steps are preceded by the transient movement towards the plus end. We propose that these backward "dips" correspond to MT release and subsequent diffusion of the stepping monomer around its MT-bound partner before taking a minus-end-directed conformational change of its linker. Our results reveal the order of sub-millisecond events that result in a productive step of dynein.
RESUMO
Neurons in the brain communicate with each other at their synapses. It has long been understood that this communication occurs through biochemical processes. Here, we reveal that mechanical tension in neurons is essential for communication. Using in vitro rat hippocampal neurons, we find that 1) neurons become tout/tensed after forming synapses resulting in a contractile neural network, and 2) without this contractility, neurons fail to fire. To measure time evolution of network contractility in 3D (not 2D) extracellular matrix, we developed an ultrasensitive force sensor with 1 nN resolution. We employed Multi-Electrode Array and iGluSnFR, a glutamate sensor, to quantify neuronal firing at the network and at the single synapse scale, respectively. When neuron contractility is relaxed, both techniques show significantly reduced firing. Firing resumes when contractility is restored. This finding highlights the essential contribution of neural contractility in fundamental brain functions and has implications for our understanding of neural physiology.
Assuntos
Neurônios , Sinapses , Ratos , Animais , Neurônios/fisiologia , Sinapses/fisiologia , Hipocampo , Redes Neurais de Computação , Encéfalo/fisiologia , Potenciais de Ação/fisiologia , Modelos NeurológicosRESUMO
DNA-point accumulation for imaging at nanoscale topography (DNA-PAINT) can image fixed biological specimens with nanometer resolution and absolute stoichiometry. In living systems, however, the usage of DNA-PAINT has been limited due to high salt concentration in the buffer required for specific binding of the imager to the docker attached to the target. Here, we used multiple binding motifs of the docker, from 2 to 16, to accelerate the binding speed of the imager under physiological buffer conditions without compromising spatial resolution and maintaining the basal level homeostasis during the measurement. We imaged endogenous α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) in cultured neurons-critical proteins involved in nerve communication-by DNA-PAINT in 3-dimensions using a monovalent single-chain variable fragment (scFv) to the GluA1 subunit of AMPAR. We found a heterogeneous distribution of synaptic AMPARs: ≈60% are immobile, primarily in nanodomains, defined as AMPARs that are within 0.3 µm of the Homer1 protein in the postsynaptic density; the other â¼40% of AMPARs have restricted mobility and trajectory.
Assuntos
Neurônios , Receptores de AMPA , Receptores de AMPA/genética , Neurônios/metabolismo , Proteínas de Transporte/metabolismoRESUMO
Point accumulation for imaging in nanoscale topography (PAINT) is a single-molecule technique for super-resolution microscopy, which uses exchangeable single stranded DNA oligos or peptide-pairs to create blinking phenomenon and achieves ≈5-25 nanometer resolution. Here, it is shown that by transfecting the protein-of-interest with a docker-coil, rather than by adding the docker externally-as is the norm when using DNA tethers or antibodies as dockers-similar localization can be achieved, ≈10 nm. However, using a transfected docker has several experimental advances and simplifications. Most importantly, it allows Peptide-PAINT to be applied to transfected live cells for imaging surface proteins in mammalian cells and neurons under physiological conditions. The enhanced resolution of Peptide-PAINT is also shown for organelles in fixed cells to unravel structural details including ≈40-nm and ≈60-nm axial repeats in vimentin filaments in the cytoplasm, and fiber shapes of sub-100-nm histone-rich regions in the nucleus.
Assuntos
DNA , Microscopia , Animais , DNA/genética , DNA/química , Nanotecnologia/métodos , Núcleo Celular , Peptídeos , MamíferosRESUMO
Molecular motors often work in teams to move a cellular cargo. Yet measuring the forces exerted by each motor is challenging. Using a sensor made with denatured ssDNA and multi-color fluorescence, we measured picoNewtons of forces and nanometer distances exerted by individual constrained kinesin-1 motors acting together while driving a common microtubule in vitro. We find that kinesins primarily exerted less than 1 pN force, even while the microtubule is bypassing artificial obstacles of 20-100 nanometer size. Occasionally, individual forces increase upon encountering obstacles, although at other times they do not, with the cargo continuing in a directional manner. Our high-throughput technique, which can measure forces by many motors simultaneously, is expected to be useful for many different types of molecular motors.
Assuntos
Cinesinas , Microtúbulos , Transporte Biológico , Fluorescência , Microtúbulos/metabolismoRESUMO
Mutations of the intracellular estrogen receptor alpha (ERα) is implicated in 70% of breast cancers. Therefore, it is of considerable interest to image various mutants (L536S, Y537S, D538G) in living cancer cell lines, particularly as a function of various anticancer drugs. We therefore developed a small (13 kDa) Affimer, which, after fluorescent labeling, is able to efficiently label ERα by traveling through temporary pores in the cell membrane, created by the toxin streptolysin O. The Affimer, selected by a phage display, predominantly labels the Y537S mutant and can tell the difference between L536S and D538G mutants. The vast majority of Affimer-ERαY537S is in the nucleus and is capable of an efficient, unrestricted navigation to its target DNA sequence, as visualized by single-molecule fluorescence. The Affimer can also differentiate the effect of selective estrogen receptor modulators. More generally, this is an example of a small binding reagent-an Affimer protein-that can be inserted into living cells with minimal perturbation and high efficiency, to image an endogenous protein.
Assuntos
Antineoplásicos , Neoplasias da Mama , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Células MCF-7 , Mutação , Receptores de Estrogênio/genética , Receptores de Estrogênio/uso terapêutico , Moduladores Seletivos de Receptor Estrogênico/uso terapêuticoRESUMO
Macrophages are white blood cells with diverse functions contributing to a healthy immune response as well as the pathogenesis of cancer, osteoarthritis, atherosclerosis, and obesity. Due to their pleiotropic and dynamic nature, tools for imaging and tracking these cells at scales spanning the whole body down to microns could help to understand their role in disease states. Here we report fluorescent and radioisotopic quantum dots (QDs) for multimodal imaging of macrophage cells in vivo, ex vivo, and in situ. Macrophage specificity is imparted by click-conjugation to dextran, a biocompatible polysaccharide that natively targets these cell types. The emission spectral band of the crystalline semiconductor core was tuned to the near-infrared for optical imaging deep in tissue, and probes were covalently conjugated to radioactive iodine for nuclear imaging. The performance of these probes was compared with all-organic dextran probe analogues in terms of their capacity to target macrophages in visceral adipose tissue using in vivo positron emission tomography/computed tomography (PET/CT) imaging, in vivo fluorescence imaging, ex vivo fluorescence, post-mortem isotopic analyses, and optical microscopy. All probe classes exhibited equivalent physicochemical characteristics in aqueous solution and similar in vivo targeting specificity. However, dextran-mimetic QDs provided enhanced signal-to-noise ratio for improved optical quantification, long-term photostability, and resistance to chemical fixation. In addition, the vascular circulation time for the QD-based probes was extended 9-fold compared with dextran, likely due to differences in conformational flexibility. The enhanced photophysical and photochemical properties of dextran-mimetic QDs may accelerate applications in macrophage targeting, tracking, and imaging across broad resolution scales, particularly advancing capabilities in single-cell and single-molecule imaging and quantification.
Assuntos
Pontos Quânticos , Neoplasias da Glândula Tireoide , Dextranos , Humanos , Radioisótopos do Iodo , Macrófagos , Imagem Óptica , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Pontos Quânticos/químicaRESUMO
Quantum dots (QDs) are a class of semiconductor nanocrystal used broadly as fluorescent emitters for analytical studies in the life sciences. These nanomaterials are particularly valuable for single-particle imaging and tracking applications in cells and tissues. An ongoing technological goal is to reduce the hydrodynamic size of QDs to enhance access to sterically hindered biological targets. Multidentate polymer coatings are a focus of these efforts and have resulted in compact and stable QDs with hydrodynamic diameters near 10 nm. New developments are needed to reach smaller sizes to further enhance transport through pores in cells and tissues. Here, we describe how structural characteristics of linear multidentate copolymers determine hydrodynamic size, colloidal stability, and biomolecular interactions of coated QDs. We tune copolymer composition, degree of polymerization, and hydrophilic group length, and coat polymers on CdSe and (core)shell (HgCdSe)CdZnS QDs. We find that a broad range of polymer structures and compositions yield stable colloidal dispersions; however, hydrodynamic size minimization and nonspecific binding resistance can only be simultaneously achieved within a narrow range of properties, requiring short polymers, balanced compositions, and small nanocrystals. In quantitative single-molecule imaging assays in synapses of live neurons, size reduction progressively increases labeling specificity of neurotransmitter receptors. Our findings provide a design roadmap to next-generation QDs with sizes approaching fluorescent protein labels that are the standard of many live-cell biomolecular studies.
RESUMO
Genome editing critically relies on selective recognition of target sites. However, despite recent progress, the underlying search mechanism of genome-editing proteins is not fully understood in the context of cellular chromatin environments. Here, we use single-molecule imaging in live cells to directly study the behavior of CRISPR/Cas9 and TALEN. Our single-molecule imaging of genome-editing proteins reveals that Cas9 is less efficient in heterochromatin than TALEN because Cas9 becomes encumbered by local searches on non-specific sites in these regions. We find up to a fivefold increase in editing efficiency for TALEN compared to Cas9 in heterochromatin regions. Overall, our results show that Cas9 and TALEN use a combination of 3-D and local searches to identify target sites, and the nanoscopic granularity of local search determines the editing outcomes of the genome-editing proteins. Taken together, our results suggest that TALEN is a more efficient gene-editing tool than Cas9 for applications in heterochromatin.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Edição de Genes , Heterocromatina/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Células HeLa , Humanos , Imagem Individual de MoléculaRESUMO
BACKGROUND: Large-scale genome-wide association studies (GWAS) have implicated thousands of germline genetic variants in modulating individuals' risk to various diseases, including cancer. At least 25 risk loci have been identified for low-grade gliomas (LGGs), but their molecular functions remain largely unknown. METHODS: We hypothesized that GWAS loci contain causal single nucleotide polymorphisms (SNPs) that reside in accessible open chromatin regions and modulate the expression of target genes by perturbing the binding affinity of transcription factors (TFs). We performed an integrative analysis of genomic and epigenomic data from The Cancer Genome Atlas and other public repositories to identify candidate causal SNPs within linkage disequilibrium blocks of LGG GWAS loci. We assessed their potential regulatory role via in silico TF binding sequence perturbations, convolutional neural network trained on TF binding data, and simulated annealing-based interpretation methods. RESULTS: We built an interactive website (http://education.knoweng.org/alg3/) summarizing the functional footprinting of 280 variants in 25 LGG GWAS regions, providing rich information for further computational and experimental scrutiny. We identified as case studies PHLDB1 and SLC25A26 as candidate target genes of rs12803321 and rs11706832, respectively, and predicted the GWAS variant rs648044 to be the causal SNP modulating ZBTB16, a known tumor suppressor in multiple cancers. We showed that rs648044 likely perturbed the binding affinity of the TF MAFF, as supported by RNA interference and in vitro MAFF binding experiments. CONCLUSIONS: The identified candidate (causal SNP, target gene, TF) triplets and the accompanying resource will help accelerate our understanding of the molecular mechanisms underlying genetic risk factors for gliomas.
Assuntos
Estudo de Associação Genômica Ampla , Glioma , Sistemas de Transporte de Aminoácidos , Proteínas de Ligação ao Cálcio , Predisposição Genética para Doença , Glioma/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas do Tecido Nervoso , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Quantum dots (QDs) are nanocrystals with bright fluorescence and long-term photostability, attributes particularly beneficial for single-molecule imaging and molecular counting in the life sciences. The size of a QD nanocrystal determines its physicochemical and photophysical properties, both of which dictate the success of imaging applications. Larger nanocrystals typically have better optical properties, with higher brightness, red-shifted emission, reduced blinking, and greater stability. However, larger nanocrystals introduce molecular-labeling biases due to steric hindrance and nonspecific binding. Here, we systematically analyze the impact of nanocrystal size on receptor labeling in live and fixed cells. We designed three (core)shell QDs with red emission (600-700 nm) and crystalline sizes of 3.2, 5.5, and 8.3 nm. After coating with the same multidentate polymer, hydrodynamic sizes were 9.2 nm (QD9.2), 13.3 nm (QD13.3), and 17.4 nm (QD17.4), respectively. The QDs were conjugated to streptavidin and applied as probes for biotinylated neurotransmitter receptors. QD9.2 exhibited the highest labeling specificity for receptors in the narrow synaptic cleft (â¼20-30 nm) in living neurons. However, for dense receptor labeling for molecular counting in live and fixed HeLa cells, QD13.3 yielded the highest counts. Nonspecific binding rose sharply for hydrodynamic sizes larger than 13.3 nm, with QD17.4 exhibiting particularly diminished specificity. Our comparisons further highlight needs to continue engineering the smallest QDs to increase single-molecule intensity, suppress blinking frequency, and inhibit nonspecific labeling in fixed and permeabilized cells. These results lay a foundation for designing QD probes with further reduced sizes to achieve unbiased labeling for quantitative and single-molecule imaging.
Assuntos
Nanopartículas , Pontos Quânticos , Diagnóstico por Imagem , Células HeLa , Humanos , PolímerosRESUMO
The post-synaptic density protein 95 (PSD-95) plays a central role in excitatory synapse development and synaptic plasticity. Phosphorylation of the N-terminus of PSD-95 at threonine 19 (T19) and serine 25 (S25) decreases PSD-95 stability at synapses; however, a molecular mechanism linking PSD-95 phosphorylation to altered synaptic stability is lacking. Here, we show that phosphorylation of T19/S25 recruits the phosphorylation-dependent peptidyl-prolyl cis-trans isomerase (Pin1) and reduces the palmitoylation of Cysteine 3 and Cysteine 5 in PSD-95. This reduction in PSD-95 palmitoylation accounts for the observed loss in the number of dendritic PSD-95 clusters, the increased AMPAR mobility, and the decreased number of functional excitatory synapses. We find the effects of Pin1 overexpression were all rescued by manipulations aimed at increasing the levels of PSD-95 palmitoylation. Therefore, Pin1 is a key signaling molecule that regulates the stability of excitatory synapses and may participate in the destabilization of PSD-95 following the induction of synaptic plasticity.
RESUMO
Materials with short-wave infrared (SWIR) emission are promising contrast agents for in vivo animal imaging, providing high-contrast and high-resolution images of blood vessels in deep tissues. However, SWIR emitters have not been developed as molecular labels for microscopy applications in the life sciences, which require optimized probes that are bright, stable, and small. Here, we design and synthesize semiconductor quantum dots (QDs) with SWIR emission based on HgxCd1-xSe alloy cores red shifted to the SWIR by epitaxial deposition of thin HgxCd1-xS shells with a small band gap. By tuning alloy composition alone, the emission can be shifted across the visible-to-SWIR (VIR) spectra while maintaining a small and equal size, allowing direct comparisons of molecular labeling performance across a broad range of wavelength. After coating with click-functional multidentate polymers, the VIR-QD spectral series has high quantum yield in the SWIR (14-33%), compact size (13 nm hydrodynamic diameter), and long-term stability in aqueous media during continuous excitation. We show that these properties enable diverse applications of SWIR molecular probes for fluorescence microscopy using conjugates of antibodies, growth factors, and nucleic acids. A broadly useful outcome is a 10-55-fold enhancement of the signal-to-background ratio at both the single-molecule level and the ensemble level in the SWIR relative to visible wavelengths, primarily due to drastically reduced autofluorescence. We anticipate that VIR-QDs with SWIR emission will enable ultrasensitive molecular imaging of low-copy number analytes in biospecimens with high autofluorescence.
Assuntos
Microscopia de Fluorescência/métodos , Sondas Moleculares/química , Pontos Quânticos/química , Tecido Adiposo/química , Ligas/química , Animais , Compostos de Cádmio/química , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/análise , Receptores ErbB/metabolismo , Humanos , Camundongos , Tamanho da Partícula , Compostos de Selênio/química , Neoplasias de Mama Triplo Negativas/química , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
How cargoes move within a crowded cell-over long distances and at speeds nearly the same as when moving on unimpeded pathway-has long been mysterious. Through an in vitro force-gliding assay, which involves measuring nanometer displacement and piconewtons of force, we show that multiple mammalian kinesin-1 (from 2 to 8) communicate in a team by inducing tension (up to 4 pN) on the cargo. Kinesins adopt two distinct states, with one-third slowing down the microtubule and two-thirds speeding it up. Resisting kinesins tend to come off more rapidly than, and speed up when pulled by driving kinesins, implying an asymmetric tug-of-war. Furthermore, kinesins dynamically interact to overcome roadblocks, occasionally combining their forces. Consequently, multiple kinesins acting as a team may play a significant role in facilitating smooth cargo motion in a dense environment. This is one of few cases in which single molecule behavior can be connected to ensemble behavior of multiple motors.
The inside of a cell is a crowded space, full of proteins and other molecules. Yet, the molecular motors that transport some of those molecules within the cell move at the same speed as they would in pure water about one micrometer per second. How the molecular motors could achieve such speeds in crowded cells was unclear. Nevertheless, Tjioe et al. suspected that the answer might be related to how multiple motors work together. Molecular motors move by walking along filaments inside the cell and pulling their cargo from one location to another. Other molecules that bind to the filaments should, in theory, act like "roadblocks" and impede the movement of the cargo. Tjioe et al. studied a motor protein called kinesin, which walks on filaments called microtubules. But instead of looking at these motors moving along microtubules inside a cell, Tjioe et al. used a simpler system where the cell was eliminated, and all parts were purified. Specifically, Tjioe et al. tethered purified motors to a piece of glass and then observed them under an extremely accurate microscope as they moved free-floating, fluorescently labelled microtubules. The microtubules, in this scenario, were acting like cargoes, where many kinesins could bind. Each kinesin motor also had a small chemical tag that could emit light. By following the movement of the lights, it was possible to calculate what each kinesin was doing and how the cargo moved. When more than one kinesin molecule was acting, the tension and speed of one kinesin affected the movement of the others. In any group of kinesins, about two-thirds of kinesin pulled the cargo, and unexpectedly, about one-third tended to resist and slow the cargo. These latter kinesins were moved along with the group without actually driving the cargo. These resisting kinesins did come off more rapidly than the driving kinesins, meaning the cargo should be able to quickly bypass roadblocks. This would help to keep the whole group travelling in the right direction at a steady pace.
Assuntos
Cinesinas/metabolismo , Animais , Transporte Biológico , Fenômenos Biomecânicos , Camundongos , Microtúbulos/metabolismoRESUMO
Stimulated emission depletion microscopy (STED) is one of the pivotal super-resolution techniques. It overcomes the spatial resolution limit imposed by the diffraction by using an additional laser beam, the STED beam, intensity of which is directly related to the achievable resolution. Despite reaching nanometer resolution, much effort in recent years has been devoted to reducing the STED beam intensity because it may lead to photo-damaging effects. Accessing the spatial information encoded in the temporal dynamics of the detected fluorescent photons has been proved to be a powerful strategy and has contributed to the separation by lifetime tuning (SPLIT) technique. The SPLIT method uses the phasor analysis to efficiently distinguish photons emitted from the center and the periphery of the excitation spot. It thus improves the resolution without increasing the STED beam intensity. This method was proposed for architectures based on the STED beam running in continuous waves (CW-STED microscopy). Here, we extend it to pulsed STED beam implementations (pSTED microscopy). We show, through simulated and experimental data, that the pSTED-SPLIT method reduces the detection volume of the pSTED microscope without significantly decreasing the signal-to-noise ratio of the final image, thus effectively improving the resolution without increasing the STED beam intensity.
RESUMO
Calcium dynamics in presynaptic terminals regulate the response dynamics of most central excitatory synapses. However, this dogma has been challenged by the hypothesis that mobility of the postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype glutamate receptors (AMPAR) plays a role in tuning fast excitatory synaptic transmission. In this review, we reevaluate the factors regulating postsynaptic AMPAR mobility, reassess the modeling parameters, analyze the experimental tools, and end by providing alternative ideas stemming from recent results. In particular, newer methods of labeling AMPARs with small fluorophores in live neurons, combined with super-resolution microscopy and sub-second dynamics, lends support to the idea that AMPARs are primarily within the synapse, are greatly constrained, and have much slower mobility than previously thought. We discuss new experiments which may be necessary to readdress the role of postsynaptic AMPAR mobility in tuning fast excitatory synaptic transmission.
RESUMO
Methods to efficiently deliver fluorophores across the cell membrane are crucial for imaging the dynamics of intracellular proteins using fluorescence. Here we describe a simple protocol for permeabilizing living cells using streptolysin O, a bacterial toxin, which allows transient uptake of fluorescent probes for labeling specific intracellular proteins. The technique is applicable for delivering different classes of fluorescent probes with a molecular weight of <150 kDa, and it is also applicable to a variety of different cell lines. The technique enables the utilization of a broad range of fluorophores for live cell imaging of intracellular proteins. Extended observation of intracellular fluorescence bound to specific proteins is now possible through super-resolution microscopy by using fluorophores that are photostable in "cell-friendly" deoxygenating and reducing conditions. © 2018 by John Wiley & Sons, Inc.
Assuntos
Corantes Fluorescentes/química , Coloração e Rotulagem/métodos , Estreptolisinas/química , Proteínas de Bactérias/química , Linhagem Celular , Microscopia de Fluorescência/métodosRESUMO
We develop magnetic cytoskeleton affinity (MiCA) purification, which allows for rapid isolation of molecular motors conjugated to large multivalent quantum dots, in miniscule quantities, which is especially useful for single-molecule applications. When purifying labeled molecular motors, an excess of fluorophores or labels is usually used. However, large labels tend to sediment during the centrifugation step of microtubule affinity purification, a traditionally powerful technique for motor purification. This is solved with MiCA, and purification time is cut from 2 h to 20 min, a significant time-savings when it needs to be done daily. For kinesin, MiCA works with as little as 0.6 µg protein, with yield of â¼27%, compared to 41% with traditional purification. We show the utility of MiCA purification in a force-gliding assay with kinesin, allowing, for the first time, simultaneous determination of whether the force from each motor in a multiple-motor system drives or hinders microtubule movement. Furthermore, we demonstrate rapid purification of just 30 ng dynein-dynactin-BICD2N-QD (DDB-QD), ordinarily a difficult protein-complex to purify.