Direct observation of motor protein stepping in living cells using MINFLUX

Science, Journal Year: 2023, Volume and Issue: 379(6636), P. 1010 - 1015

Published: March 10, 2023

Dynamic measurements of molecular machines can provide invaluable insights into their mechanism, but these have been challenging in living cells. Here, we developed live-cell tracking single fluorophores with nanometer spatial and millisecond temporal resolution two three dimensions using the recently introduced super-resolution technique MINFLUX. Using this approach, resolved precise stepping motion motor protein kinesin-1 as it walked on microtubules Nanoscopic motors walking fixed cells also enabled us to resolve architecture microtubule cytoskeleton protofilament resolution.

Language: Английский

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Optimized Red-Absorbing Dyes for Imaging and Sensing

Journal of the American Chemical Society, Journal Year: 2023, Volume and Issue: 145(42), P. 23000 - 23013

Published: Oct. 16, 2023

Rhodamine dyes are excellent scaffolds for developing a broad range of fluorescent probes. A key property rhodamines is their equilibrium between colorless lactone and zwitterion. Tuning the lactone–zwitterion constant (KL–Z) can optimize dye properties specific biological applications. Here, we use known novel organic chemistry to prepare comprehensive collection rhodamine elucidate structure–activity relationships that govern KL–Z. We discovered auxochrome substituent strongly affects equilibrium, providing roadmap rational design improved dyes. Electron-donating auxochromes, such as julolidine, work in tandem with fluorinated pendant phenyl rings yield bright, red-shifted fluorophores live-cell single-particle tracking (SPT) multicolor imaging. The N-aryl combined fluorination yields Förster resonance energy transfer (FRET) quencher useful creating new semisynthetic indicator sense cAMP using fluorescence lifetime imaging microscopy (FLIM). Together, this expands synthetic methods available synthesis, generates reagents advanced experiments, describes will guide future

Language: Английский

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Recent advances in super-resolution optical imaging based on aggregation-induced emission

Chemical Society Reviews, Journal Year: 2024, Volume and Issue: 53(7), P. 3350 - 3383

Published: Jan. 1, 2024

Super-resolution imaging has rapidly emerged as an optical microscopy technique, offering advantages of high resolution over the past two decades; achieving improved requires significant efforts in developing super-resolution agents characterized by brightness, contrast and sensitivity to fluorescence switching. Apart from technical requirements systems algorithms, relies on fluorescent dyes with special photophysical or photochemical properties. The concept aggregation-induced emission (AIE) was proposed 2001, coinciding unprecedented advancements innovations technology. AIE probes offer many advantages, including brightness aggregated state, low background signal, a larger Stokes shift, ultra-high photostability, excellent biocompatibility, making them highly promising for applications imaging. In this review, we summarize progress implementation methods provide insights into mechanism AIE-based imaging, switching resulting photochemically-converted emission, electrostatically controlled specific binding-regulated emission. Particularly, principle been achieve spontaneous switching, expanding selection application scenarios probes. By combining molecular design, some comprehensive facilitate AIEgens (AIE-active molecules)

Language: Английский

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Live-cell imaging powered by computation

Nature Reviews Molecular Cell Biology, Journal Year: 2024, Volume and Issue: 25(6), P. 443 - 463

Published: Feb. 20, 2024

Language: Английский

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Hybrid Small-Molecule/Protein Fluorescent Probes

Chemical Reviews, Journal Year: 2024, Volume and Issue: 124(10), P. 6198 - 6270

Published: May 8, 2024

Hybrid small-molecule/protein fluorescent probes are powerful tools for visualizing protein localization and function in living cells. These hybrid constructed by diverse site-specific chemical labeling approaches through reactions to exogenous peptide/small tags, enzymatic post-translational modifications, bioorthogonal genetically incorporated unnatural amino acids, ligand-directed reactions. The employed imaging trafficking, conformational changes, bioanalytes surrounding proteins. In addition, facilitate visualization of dynamics at the single-molecule level defined structure with super-resolution imaging. this review, we discuss development bioimaging applications based on hybrids.

Language: Английский

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Understanding the cell: Future views of structural biology

Cell, Journal Year: 2024, Volume and Issue: 187(3), P. 545 - 562

Published: Feb. 1, 2024

Determining the structure and mechanisms of all individual functional modules cells at high molecular detail has often been seen as equal to understanding how work. Recent technical advances have led a flush high-resolution structures various macromolecular machines, but despite this wealth detailed information, our cellular function remains incomplete. Here, we discuss present-day limitations structural biology highlight novel technologies that may enable us analyze functions directly inside cells. We predict progression toward cell will involve shift conceptualizing 4D virtual reality using digital twins. These capture segments in highly enriched detail, include dynamic changes, facilitate simulations processes, leading experimentally testable predictions. Transferring biological questions into algorithms learn from existing data explore solutions ultimately unveil

Language: Английский

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Advancing cell biology with nanoscale fluorescence imaging: essential practical considerations

Trends in Cell Biology, Journal Year: 2024, Volume and Issue: 34(8), P. 671 - 684

Published: Jan. 5, 2024

Here, we provide an overview of the key factors to consider when using fluorescence nanoscopy (FN), including biological questions that can be addressed and aspects might improve reliability effectiveness FN experiments.We cover main related sample preparation, selection appropriate fixation, affinity-based labels, fluorescent dyes.We discuss current limitations possible future developments in field would facilitate a broader application FN.We multiplexing possibilities (allowing simultaneous detection multiple targets single experiment), live cell imaging for study cellular molecular dynamic processes, quantitative workflows. Recently, biologists have gained access several far-field (FN) technologies allow observation components with ~20 nm resolution. is revolutionizing biology by enabling visualization previously inaccessible subcellular details. While technological advances microscopy are critical field, optimal preparation labeling equally important often overlooked experiments. In this review, methodological experimental must considered performing FN. We present concepts dyes, multiplexing, approaches, microscopy. Consideration these greatly enhances FN, making it exquisite tool numerous applications. Conventional microscopy, widefield confocal has been essential studying morphology composition various organelles localization molecules. The resolution (see Glossary) techniques, usually above ~200 nm, limit macromolecular arrangements nanoscale structures. processes (<20 nm) major step forward because bridges world macromolecules. This effort facilitated recent decades use electron (EM), which provides morphological information at level [1.Winey M. et al.Conventional transmission microscopy.Mol. Biol. Cell. 2014; 25: 319-323Crossref PubMed Google Scholar] expenses limited identifications lack applicability. These overcome achieved resolving capabilities progressively reaching those attained EM. Several approaches based on enable researchers address <20 were difficult answer classical Paradigmatic examples applications include characterization periodicity actin rings synaptic sites neurons, structure nuclear pore complexes, maturation viral particles, organization mitochondrial cristae, mechanisms apoptosis, functioning signaling pathways [2.Fornasiero E.F. Opazo F. Super-resolution biologists.BioEssays. 2015; 37: 436-451Crossref Scholar]. Recent studies reached extremely high resolutions [3.Reinhardt S.C.M. al.Ångström-resolution microscopy.Nature. 2023; 617: 711-716Crossref Scopus (21) even enabled follow events such as stepping behavior kinesin vitro cells [4.Wolff J.O. al.MINFLUX dissects unimpeded walking kinesin-1.Science. 379: 1004-1010Crossref (20) Scholar,5.Deguchi T. al.Direct motor protein living MINFLUX.Science. 1010-1015Crossref (22) render individual molecules, GABA receptors, detail almost comparable cryo-EM [6.Shaib A.H. al.Visualizing proteins expansion microscopy.bioRxiv. (Published online March 10, 2023. https://doi.org/10.1101/2022.08.03.502284)Google compendium summarizes practical keep mind harnessing power nanoscopic biology. By bridging theory practice, roadmap researchers, equipping them know-how successfully navigate intricacies implementing, executing, deriving meaningful data from design experiments starts most suitable technique, each its own specific strengths (Figure 1A ; reviews different see [7.Jacquemet G. al.The biologist's guide super-resolution microscopy.J. Cell Sci. 2020; 133jcs240713Crossref (32) Scholar,8.Bond C. al.Technological processes.Mol. 2022; 82: 315-332Abstract Full Text PDF (25) Scholar]). Two strategies currently able reliably <20-nm samples: camera-based single-molecule (SMLM) [9.Rust M.J. al.Sub-diffraction-limit stochastic optical reconstruction (STORM).Nat. Methods. 2006; 3: 793-796Crossref (6072) minimal photon fluxes (MINFLUX) [10.Balzarotti al.Nanometer tracking molecules fluxes.Science. 2017; 355: 606-612Crossref (657) Based utilized perform on–off switching fluorophores required obtain super-resolved image, SMLM names; example, (STORM), photoactivated (PALM), DNA-points accumulation topography (DNA-PAINT) [11.Jungmann R. al.Single-molecule kinetics transient binding DNA origami.Nano Lett. 2010; 10: 4756-4761Crossref (576) addition two strategies, depending exact settings, stimulated emission depletion (STED) [12.Hell S.W. Wichmann J. Breaking diffraction emission: stimulated-emission-depletion microscopy.Opt. 1994; 19: 780Crossref achieve ~50 below, although laser not compatible conventional samples. technologies, approach aimed enlarging sample, then imaged either diffraction-limited or [13.Chen al.Optical imaging. Expansion microscopy.Science. 347: 543-548Crossref (858) With exception microcopy, specimens, opening avenue understanding dynamics native conditions (Box 1). Overall, four potential uncover as-yet unexplored detail. At same time, their capability requires protocols introduction artifacts tools linkage errors.Box 1Nanometer-scale cells: there yet?FN visualizes entire structures over time 'whole', allowing changes, recommended endoplasmic reticulum, chemical fixation introduces visible [19.Hoffman D.P. al.Correlative three-dimensional block-face whole vitreously frozen cells.Science. 367eaaz5357Crossref (33) our opinion, least connected implementation detailed herein.PhototoxicityPhototoxicity arises absorbed light generates free radicals reactive oxygen species, ultimately causing genomic damage, stress, degradation Figure 1D text). Light both endogenous additionally increase local temperature sample. To some effects, combinations image adaptive illumination [75.Heine al.Adaptive-illumination STED nanoscopy.Proc. Natl. Acad. U. S. A. 114: 9797-9802Crossref (0) Scholar], event-triggered emerging [76.Alvelid al.Event-triggered imaging.Nat. 1268-1275Crossref (15) Furthermore, novel fluorophore classes self-blinking dyes red photoswitchable exist, do require blue excited lower doses [77.Pennacchietti al.Fast reversibly photoswitching live-cell RESOLFT nanoscopy.Nat. 2018; 15: 601-604Crossref (58) Scholar].Low acquisition frequencyLow frequency resolution, but compromises interpretation fast processes. SMLM, limiting factor sufficient number while, scanning-based steps brightness size view. Reducing view spent pixel (dwell time) speed up imaging, decreased contextual SNR. will strongly benefit parallelization deep learning temporal performances.Availability impact cellsThe availability cannot ignored. Some probes drugs bind target affinity, interfering physiology targeted molecule (e.g., phalloidin). An alternative genetically encoded tags or, better, combined genome-editing [78.Bottanelli al.A physiological role ARF1 formation bidirectional tubules Golgi.Mol. 28: 1676-1687Crossref Table 1 text).Imaging depth large viewImaging many samples monolayers. Although feasibility specialized demonstrated [79.Kim al.Oblique-plane tissues small intact animals.Nat. 2019; 16: 853-857Crossref (54) deeper than 10–50 μm methods challenging. engineered illumination, optics, restoration algorithms, multiphoton excitation.Ultimately, best technology selected setup precise question being addressed, controls phototoxic cause damage considered. should done settings less perturbation troubleshooting conditions. For far-red commonly used compared 405-laser irradiance. herein. Phototoxicity Low performances. Availability Imaging excitation. Ultimately, short 'no', all problems solved. Careful consideration given whether necessary questions. determine interest (POI) localized lysosomes mitochondria, POI outer membrane inner matrix, apart. general, strength increased precision biomolecules 1B,C). A simple rule thumb decide needed understand spatial order tens nanometers allows formulation fundamentally hypotheses process under investigation. serendipitous observations made and, cases, revealed new observable [14.Xu K. al.Actin, spectrin, associated form periodic cytoskeletal axons.Science. 2013; 339: 452-456Crossref (888) reason, exploratory Very high-resolution informative, could ignored become challenges Live ultimate goal studies, still due phototoxicity early stage foreseeable relevant near future. fixed already changing field; therefore, concentrate here specimens 1D–F). workflow 'non-live' first optimized avoid artifacts. After antibodies, reveal identities position reporter proximity. relative absolute biologically numbers images (quantitative FN), further measures considerations taken. Box 2, review issues explain why density, stoichiometry, error context. Finally, 3, conditions.Box 2Quantitative FN: versus observed moleculesQuantitative detected matches (or, more precisely, correlates) real Therefore, prerequisites controlled efficiency ability detect fluorophores.A monovalent affinity carrying (or of) reporting 2A Moreover, utilize covalently linked ensure stably labeled preferred. multivalent polyclonal reagents stochastically secondary antibodies approximately one six fluorophores) avoided since correlation between reporters inconsistent.Even ideal case decorated label, possibility exists only decorating label functional. Indeed, inactivated, damaged, during procedure 1E Detecting densely challenging, problem known crowding. When distances single-digit nanometer scale, photophysical interactions occur, resulting undesired alterations properties. happens structure, Förster resonance energy transfer (FRET) H-dimer if separated molecular-scale [80.Ogawa al.H-Type dimer fluorophores: mechanism activatable, vivo imaging.ACS Chem. 2009; 4: 535-546Crossref (153) Notably, reported antibody [81.Helmerich D.A. al.Multiple-labeled behave like emitters buffer.ACS Nano. 14: 12629-12641Crossref (13) serving 'super emitter' while others remain dark state. crowding reversed, physically creating distance Other DNA-PAINT, deal differently modulating concentration probe light-controllable [82.Raymo F.M. Photoactivatable synthetic nanoscale.J. Phys. 2012; 2379-2385Crossref (60) 2D text).While quantification easily achieved, molecule-counting proposed comprehensive recently published challenge [83.Hugelier al.Quantitative microscopy.Annu. Rev. Biophys. 52: 139-160Crossref (1) Importantly, need calibration benchmarked against markers biochemically western blot, liquid chromatography, mass spectrometry).Box 3Fluorophore no one-size-fits-all solutionsA variety developed fulfill requirement technique (Table I). Factors selecting charge, quantum yield, photostability 2C photostable fluorophores, MINFLUX rely switch non-emitting emitting states. mechanisms, refer reader text.The scaffolds cyanines rhodamines. Among cyanines, Alexa Fluor 647 gold standard blinks presence reducing agents UV [84.Berlier J.E. comparison long-wavelength Cy Dyes: bioconjugates.J. Histochem. Cytochem. 2003; 51: 1699-1712Crossref (229) Rhodamines relatively modified tune spectral properties [85.Grimm J.B. general method optimize functionalize red-shifted rhodamine dyes.Nat. 17: 815-821Crossref (85) permeability [86.Lukinavičius near-infrared proteins.Nat. 5: 132-139Crossref (648) equilibrium open closed non-fluorescent forms. regulation latter induces cell-compatible spontaneous blinking Scholar].Other frequently coumarin, oxazine, BODIPY 2E Coumarins among smallest generate variants Stokes shift, advantageous low background [87.Nizamov al.Phosphorylated 3-heteroarylcoumarins nanoscopy.Chem. Eur. 18: 16339-16348Crossref (45) Oxazines [88.Wombacher al.Live-cell trimethoprim conjugates.Nat. 7: 717-719Crossref (282) absorbance, extinction coefficient, 'blink' buffers containing oxidizing agents. valued sharp absorption spectra very yield coefficient [89.Kowada al.BODIPY-based cells.Chem. Soc. 44: 4953-4972Crossref highly hydrophobic nature poor off-switching properties, light-dose photoactivatable make attractive [90.Wijesooriya C.S. localization-based imaging.Angew. Int. Ed. 57 (12685–1268)Crossref (74) Scholar].In after identified, always considering specifications available instrument lasers detectors). choice also driven needs, multicolor autofluorescence reduce background, counting naked may themselves affinities lipophilic stain membranes). evaluated designing experiments, (reporters) without targeting moiety whenever 2B recommend inexperienced users consult expert select dye application.Table IProperties dyesFluorophore classCoumarinsRhodaminesCyaninesBODIPYsOxazinesCommercial examplesAlexa 350, Pacific BlueAlexa 488, silicon-rhodamine, TMRAlexa 647, 555, Cy5BODIPY FL, TMRAtto 655, Atto 680Spectral range (nm)360–700500–750500–1000500–700600–750Extinction (cm–1M–1)15 000–60 00080 000–150 000130 000–250 00060 000–100 000110 000–130 000Quantum yield0.4–0.90.1–0.90.1–0.60.8–0.90.1–0.6Photostability+++++++++++Compatibility methodsSTED, SMLMSTED, MINFLUX, microscopySTED, SMLMSMLM Open table tab Quantitative fluorophores. inconsistent. Even sin

Language: Английский

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Transcription Factor Dynamics: One Molecule at a Time

Annual Review of Cell and Developmental Biology, Journal Year: 2023, Volume and Issue: 39(1), P. 277 - 305

Published: Aug. 4, 2023

Cells must tightly regulate their gene expression programs and yet rapidly respond to acute biochemical biophysical cues within environment. This information is transmitted the nucleus through various signaling cascades, culminating in activation or repression of target genes. Transcription factors (TFs) are key mediators these signals, binding specific regulatory elements chromatin. While live-cell imaging has conclusively proven that TF-chromatin interactions highly dynamic, how such transient can have long-term impacts on developmental trajectories disease progression still largely unclear. In this review, we summarize our current understanding dynamic nature TF functions, starting with a historical overview early experiments. We highlight govern dynamics dynamics, turn, affect downstream transcriptional bursting. Finally, conclude open challenges emerging technologies will further regulation.

Language: Английский

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True-to-scale DNA-density maps correlate with major accessibility differences between active and inactive chromatin

Cell Reports, Journal Year: 2023, Volume and Issue: 42(6), P. 112567 - 112567

Published: May 26, 2023

Chromatin compaction differences may have a strong impact on accessibility of individual macromolecules and macromolecular assemblies to their DNA target sites. Estimates based fluorescence microscopy with conventional resolution, however, suggest only modest (∼2-10×) between the active nuclear compartment (ANC) inactive (INC). Here, we present maps landscapes true-to-scale densities, ranging from <5 >300 Mbp/μm3. Maps are generated human mouse cell nuclei single-molecule localization at ∼20 nm lateral ∼100 axial optical resolution supplemented by electron spectroscopic imaging. Microinjection fluorescent nanobeads sizes corresponding for transcription into living cells demonstrates movements within ANC exclusion INC.

Language: Английский

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Heterogeneous anomalous transport in cellular and molecular biology

Reports on Progress in Physics, Journal Year: 2023, Volume and Issue: 86(12), P. 126601 - 126601

Published: Oct. 20, 2023

It is well established that a wide variety of phenomena in cellular and molecular biology involve anomalous transport e.g. the statistics for motility cells molecules are fractional do not conform to archetypes simple diffusion or ballistic transport. Recent research demonstrates many cases heterogeneous both time space. Thus single exponents generalised coefficients unable satisfactorily describe crucial biology. We consider advances field ofheterogeneous transport(HAT) highlighting: experimental techniques (single molecule methods, microscopy, image analysis, fluorescence correlation spectroscopy, inelastic neutron scattering, nuclear magnetic resonance), theoretical tools data analysis (robust statistical methods such as first passage probabilities, survival different varieties mean square displacements, etc), analytic theory generative models based on simulations. Special emphasis made high throughput machine learning neural networks. Furthermore, we context microrheology viscoelasticity complex fluids. HAT wavefronts reaction-diffusion systems also considered since it plays an important role morphogenesis signalling. In addition, present specific examples from including embryonic cells, leucocytes, cancer bacterial biofilms, eukaryotic microorganisms. Case studies include DNA, membranes, endosomal transport, endoplasmic reticula, mucins, globular proteins, amyloids.

Language: Английский

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