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1.
J Cell Sci ; 136(5)2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-36382597

RESUMO

The endosomal system orchestrates the transport of lipids, proteins and nutrients across the entire cell. Along their journey, endosomes mature, change shape via fusion and fission, and communicate with other organelles. This intriguing endosomal choreography, which includes bidirectional and stop-and-go motions, is coordinated by the microtubule-based motor proteins dynein and kinesin. These motors bridge various endosomal subtypes to the microtubule tracks thanks to their cargo-binding domain interacting with endosome-associated proteins, and their motor domain interacting with microtubules and associated proteins. Together, these interactions determine the mobility of different endosomal structures. In this Review, we provide a comprehensive overview of the factors regulating the different interactions to tune the fascinating dance of endosomes along microtubules.


Assuntos
Dineínas , Cinesinas , Dineínas/metabolismo , Endossomos/metabolismo , Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo
2.
Semin Cell Dev Biol ; 133: 20-31, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-35351373

RESUMO

Ciliogenesis is a complex multistep process used to describe assembly of cilia and flagella. These organelles play essential roles in motility and signaling on the surface of cells. Cilia are built at the distal ends of centrioles through the formation of an axoneme that is surrounded by the ciliary membrane. As is the case in the biogenesis of other cellular organelles, regulators of membrane trafficking play essential roles in ciliogenesis, albeit with a unique feature that membranes are organized around microtubule-based structures. Membrane association with the distal end of the centriole is a critical initiating step for ciliogenesis. Studies of this process in different cell types suggests that a singular mechanism may not be utilized to initiate cilium assembly. In this review, we focus on recent insights into cilium biogenesis and the roles membrane trafficking regulators play in described ciliogenesis mechanisms with relevance to human disease.


Assuntos
Axonema , Centríolos , Humanos , Centríolos/metabolismo , Axonema/metabolismo , Cílios/metabolismo , Microtúbulos/metabolismo , Flagelos
3.
J Cell Biol ; 222(2)2023 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-36416725

RESUMO

Fibronectin (FN) is an essential structural and regulatory component of the extracellular matrix (ECM), and its binding to integrin receptors supports cell adhesion, migration, and signaling. Here, using live-cell microscopy of fibroblasts expressing FN tagged with a pH-sensitive fluorophore, we show that FN is secreted predominantly at the ventral surface of cells in an integrin-independent manner. Locally secreted FN then undergoes ß1 integrin-dependent fibrillogenesis. We find that the site of FN secretion is regulated by cell polarization, which occurs in bursts under stabilized lamellipodia at the leading edge. Moreover, analysis of FN secretion and focal adhesion dynamics suggest that focal adhesion formation precedes FN deposition and that deposition continues during focal adhesion disassembly. Lastly, we show that the polarized FN deposition in spreading and migrating cells requires both intact microtubules and myosin II-mediated contractility. Thus, while FN secretion does not require integrin binding, the site of exocytosis is regulated by membrane and cytoskeletal dynamics with secretion occurring after new adhesion formation.


Assuntos
Fibronectinas , Microtúbulos , Miosina Tipo II , Pseudópodes , Proteínas do Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Integrinas/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Pseudópodes/genética , Pseudópodes/metabolismo , Matriz Extracelular/metabolismo , Exocitose
4.
Semin Cell Dev Biol ; 137: 16-25, 2023 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-34896019

RESUMO

Centrioles are microtubule-based cell organelles present in most eukaryotes. They participate in the control of cell division as part of the centrosome, the major microtubule-organizing center of the cell, and are also essential for the formation of primary and motile cilia. During centriole assembly as well as across its lifetime, centriolar tubulin display marks defined by post-translational modifications (PTMs), such as glutamylation or acetylation. To date, the functions of these PTMs at centrioles are not well understood, although pioneering experiments suggest a role in the stability of this organelle. Here, we review the current knowledge regarding PTMs at centrioles with a particular focus on a possible link between these modifications and centriole's architecture, and propose possible hypothesis regarding centriolar tubulin PTMs's function.


Assuntos
Centríolos , Tubulina (Proteína) , Tubulina (Proteína)/genética , Centro Organizador dos Microtúbulos , Microtúbulos , Cílios
5.
Semin Cell Dev Biol ; 137: 46-62, 2023 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-34924330

RESUMO

Among the variety of post-translational modifications to which microtubules are subjected, the detyrosination/re-tyrosination cycle is specific to tubulin. It is conserved by evolution and characterized by the enzymatic removal and re-addition of a gene-encoded tyrosine residue at the C-terminus of α-tubulin. Detyrosinated tubulin can be further converted to Δ2-tubulin by the removal of an additional C-terminal glutamate residue. Detyrosinated and Δ2-tubulin are carried by stable microtubules whereas tyrosinated microtubules are present on dynamic polymers. The cycle regulates trafficking of many cargo transporting molecular motors and is linked to the microtubule dynamics via regulation of microtubule interactions with specific cellular effectors such as kinesin-13. Here, we give an historical overview of the general features discovered for the cycle. We highlight the recent progress toward structure and functioning of the enzymes that keep the levels of tyrosinated and detyrosinated tubulin in cells, the long-known tubulin tyrosine ligase and the recently discovered vasohibin-SVBP complexes. We further describe how the cycle controls microtubule functions in healthy neurons and cardiomyocytes and how deregulations of the cycle are involved in dysfunctions of these highly differentiated cells, leading to neurodegeneration and heart failure in humans.


Assuntos
Miócitos Cardíacos , Tubulina (Proteína) , Humanos , Tubulina (Proteína)/metabolismo , Miócitos Cardíacos/metabolismo , Microtúbulos/metabolismo , Neurônios/metabolismo , Tirosina/química , Tirosina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Transporte/metabolismo
6.
Semin Cell Dev Biol ; 137: 3-15, 2023 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-34922809

RESUMO

Protists are an exceptionally diverse group of mostly single-celled eukaryotes. The organization of the microtubular cytoskeleton in protists from various evolutionary lineages has different levels of sophistication, from a network of microtubules (MTs) supporting intracellular trafficking as in Dictyostelium, to complex structures such as basal bodies and cilia/flagella enabling cell motility, and lineage-specific adaptations such as the ventral disc in Giardia. MTs building these diverse structures have specific properties partly due to the presence of tubulin post-translational modifications (PTMs). Among them there are highly evolutionarily conserved PTMs: acetylation, detyrosination, (poly)glutamylation and (poly)glycylation. In some protists also less common tubulin PTMs were identified, including phosphorylation, methylation, Δ2-, Δ5- of α-tubulin, polyubiquitination, sumoylation, or S-palmitoylation. Not surprisingly, several single-celled organisms become models to study tubulin PTMs, including their effect on MT properties and discovery of the modifying enzymes. Here, we briefly summarize the current knowledge on tubulin PTMs in unicellular eukaryotes and highlight key findings in protists as model organisms.


Assuntos
Dictyostelium , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Dictyostelium/metabolismo , Microtúbulos/metabolismo , Processamento de Proteína Pós-Traducional , Eucariotos/metabolismo
7.
Semin Cell Dev Biol ; 137: 26-37, 2023 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-35067438

RESUMO

Microtubules are cytoskeletal elements that play key roles throughout the different steps of sperm development. As an integral part of the sperm flagellum, the molecular machine that generates sperm motility, microtubules are also essential for the progressive swimming of sperm to the oocyte, which is a prerequisite for fertilisation. Given the central role of microtubules in all steps of spermatogenesis, their functions need to be tightly controlled. Recent work has showcased tubulin posttranslational modifications as key players in sperm development and function, with aberrations often leading to male infertility with a broad spectrum of sperm defects. Posttranslational modifications are part of the tubulin code, a mechanism that can control microtubule functions by modulating the properties of their molecular building blocks, the tubulin proteins. Here we review the current knowledge on the implications of the tubulin code in sperm development and functions and its importance for male fertility.


Assuntos
Motilidade Espermática , Tubulina (Proteína) , Animais , Masculino , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Sêmen , Microtúbulos/metabolismo , Processamento de Proteína Pós-Traducional , Espermatozoides/metabolismo , Mamíferos/metabolismo
8.
Semin Cell Dev Biol ; 137: 74-86, 2023 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-35144861

RESUMO

The organ of Corti, located in the cochlea within the inner ear is the receptor organ for hearing. It converts auditory signals into neuronal action potentials that are transmitted to the brain for further processing. The mature organ of Corti consists of a variety of highly differentiated sensory cells that fulfil unique tasks in the processing of auditory signals. The actin and microtubule cytoskeleton play essential function in hearing, however so far, more attention has been paid to the role of actin. Microtubules play important roles in maintaining cellular structure and intracellular transport in virtually all eukaryotic cells. Their functions are controlled by interactions with a large variety of microtubule-associated proteins (MAPs) and molecular motors. Current advances show that tubulin posttranslational modifications, as well as tubulin isotypes could play key roles in modulating microtubule properties and functions in cells. These mechanisms could have various effects on the stability and functions of microtubules in the highly specialised cells of the cochlea. Here, we review the current understanding of the role of microtubule-regulating mechanisms in the function of the cochlea and their implications for hearing, which highlights the importance of microtubules in the field of hearing research.


Assuntos
Actinas , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Actinas/metabolismo , Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos , Processamento de Proteína Pós-Traducional , Audição
9.
Semin Cell Dev Biol ; 137: 63-73, 2023 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-35148939

RESUMO

Blood platelets are small non-nucleated cellular fragments that prevent and stop hemorrhages. They are produced in the bone marrow by megakaryocytes through megakaryopoiesis. This intricate process involves profound microtubule rearrangements culminating in the formation of a unique circular sub-membranous microtubule array, the marginal band, which supports the typical disc-shaped morphology of platelets. Mechanistically, these processes are thought to be controlled by a specific tubulin code. In this review, we summarize the current knowledge on the key isotypes, notably ß1-, α4A- and α8-tubulin, and putative post-translational modifications, involved in platelet and marginal band formation. Additionally, we provide a provisional list of microtubule-associated proteins (MAPs) involved in these processes and a survey of tubulin variants identified in patients presenting defective platelet production. A comprehensive characterization of the platelet tubulin code and the identification of essential MAPs may be expected in the near future to shed new light on a very specialized microtubule assembly process with applications in platelet diseases and transfusion.


Assuntos
Megacariócitos , Tubulina (Proteína) , Humanos , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Megacariócitos/metabolismo , Microtúbulos/metabolismo , Plaquetas/metabolismo , Processamento de Proteína Pós-Traducional
10.
J Cell Sci ; 136(5)2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36250267

RESUMO

Cells are the smallest building blocks of all living eukaryotic organisms, usually ranging from a couple of micrometers (for example, platelets) to hundreds of micrometers (for example, neurons and oocytes) in size. In eukaryotic cells that are more than 100 µm in diameter, very often a self-organized large-scale movement of cytoplasmic contents, known as cytoplasmic streaming, occurs to compensate for the physical constraints of large cells. In this Review, we discuss cytoplasmic streaming in multiple cell types and the mechanisms driving this event. We particularly focus on the molecular motors responsible for cytoplasmic movements and the biological roles of cytoplasmic streaming in cells. Finally, we describe bulk intercellular flow that transports cytoplasmic materials to the oocyte from its sister germline cells to drive rapid oocyte growth.


Assuntos
Proteínas de Drosophila , Cinesinas , Transporte Biológico/fisiologia , Corrente Citoplasmática/fisiologia , Proteínas de Drosophila/metabolismo , Dineínas/metabolismo , Microtúbulos/metabolismo , Oogênese
11.
J Cell Biol ; 222(1)2023 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-36269172

RESUMO

Key for accurate chromosome partitioning to the offspring is the ability of mitotic spindle microtubules to respond to different molecular signals and remodel their dynamics accordingly. Spindle microtubules are conventionally divided into three classes: kinetochore, interpolar, and astral microtubules (kMTs, iMTs, and aMTs, respectively). Among all, aMT regulation remains elusive. Here, we show that aMT dynamics are tightly regulated. aMTs remain unstable up to metaphase and are stabilized at anaphase onset. This switch in aMT dynamics, important for proper spindle orientation, specifically requires the degradation of the mitotic cyclin Clb4 by the Anaphase Promoting Complex bound to its activator subunit Cdc20 (APC/CCdc20). These data highlight a unique role for mitotic cyclin Clb4 in controlling aMT regulating factors, of which Kip2 is a prime candidate, provide a framework to understand aMT regulation in vertebrates, and uncover mechanistic principles of how the APC/CCdc20 choreographs the timing of late mitotic events by sequentially impacting on the three classes of spindle microtubules.


Assuntos
Anáfase , Proteínas Cdc20 , Microtúbulos , Fuso Acromático , Animais , Ciclossomo-Complexo Promotor de Anáfase/genética , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclinas/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Fuso Acromático/genética , Fuso Acromático/metabolismo , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Ciclina B
12.
J Cell Sci ; 136(5)2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-36274587

RESUMO

Mitotic cell division requires that kinetochores form microtubule attachments that can segregate chromosomes and control mitotic progression via the spindle assembly checkpoint. During prometaphase, kinetochores shed a domain called the fibrous corona as microtubule attachments form. This shedding is mediated, in part, by the minus-end directed motor dynein, which 'strips' cargoes along K-fibre microtubules. Despite its essentiality, little is known about how dynein stripping is regulated and how it responds to attachment maturation. Lis1 (also known as PAFAH1B1) is a conserved dynein regulator that is mutated in the neurodevelopmental disease lissencephaly. Here, we have combined loss-of-function studies, high-resolution imaging and separation-of-function mutants to define how Lis1 contributes to dynein-mediated corona stripping in HeLa cells. Cells depleted of Lis1 fail to disassemble the corona and show a delay in metaphase as a result of persistent checkpoint activation. Furthermore, we find that although kinetochore-tethered Lis1-dynein is required for error-free microtubule attachment, the contribution of Lis1 to corona disassembly can be mediated by a cytoplasmic pool. These findings support the idea that Lis1 drives dynein function at kinetochores to ensure corona disassembly and prevent chromosome mis-segregation.


Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase , Dineínas , Cinetocoros , Proteínas Associadas aos Microtúbulos , Humanos , Dineínas/metabolismo , Células HeLa , Cinetocoros/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , 1-Alquil-2-acetilglicerofosfocolina Esterase/genética , 1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo
13.
Life Sci Alliance ; 6(1)2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36379670

RESUMO

Membrane organelle function, localization, and proper partitioning upon cell division depend on interactions with the cytoskeleton. Whether membrane organelles also impact the function of cytoskeletal elements remains less clear. Here, we show that acute disruption of the ER around spindle poles affects mitotic spindle size and function in Drosophila syncytial embryos. Acute ER disruption was achieved through the inhibition of ER membrane fusion by the dominant-negative cytoplasmic domain of atlastin. We reveal that when centrosome-proximal ER membranes are disrupted, specifically at metaphase, mitotic spindles become smaller, despite no significant changes in microtubule dynamics. These smaller spindles are still able to mediate sister chromatid separation, yet with decreased velocity. Furthermore, by inducing mitotic exit, we found that nuclear separation and distribution are affected by ER disruption. Our results suggest that ER integrity around spindle poles is crucial for the maintenance of mitotic spindle shape and pulling forces. In addition, ER integrity also ensures nuclear spacing during syncytial divisions.


Assuntos
Proteínas de Drosophila , Fuso Acromático , Animais , Fuso Acromático/metabolismo , Centrossomo/metabolismo , Microtúbulos/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Retículo Endoplasmático/metabolismo , Drosophila/metabolismo
14.
J Cell Sci ; 136(5)2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35403186

RESUMO

Primary cilia are microtubule-based sensory organelles whose assembly and function rely on the conserved bidirectional intraflagellar transport (IFT) system, which is powered by anterograde kinesin-2 and retrograde cytoplasmic dynein-2 motors. Nematodes additionally employ a cell-type-specific kinesin-3 motor, KLP-6, which moves within cilia independently of IFT and regulates ciliary content and function. Here, we provide evidence that a KLP-6 homolog, KIF13B, undergoes bursts of bidirectional movement within primary cilia of cultured immortalized human retinal pigment epithelial (hTERT-RPE1) cells. Anterograde and retrograde intraciliary velocities of KIF13B were similar to those of IFT (as assayed using IFT172-eGFP), but intraciliary movement of KIF13B required its own motor domain and appeared to be cell-type specific. Our work provides the first demonstration of motor-driven, intraciliary movement by a vertebrate kinesin other than kinesin-2 motors.


Assuntos
Cílios , Cinesinas , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transporte Biológico , Cílios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Flagelos/metabolismo , Humanos , Cinesinas/genética , Microtúbulos
15.
J Cell Sci ; 136(5)2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35502670

RESUMO

The precise regulation of microtubule length during mitosis is essential to assemble and position the mitotic spindle and segregate chromosomes. The kinesin-13 Kif2C or MCAK acts as a potent microtubule depolymerase that diffuses short distances on microtubules, whereas the kinesin-8 Kif18b is a processive motor with weak depolymerase activity. However, the individual activities of these factors cannot explain the dramatic increase in microtubule dynamics in mitosis. Using in vitro reconstitution and single-molecule imaging, we demonstrate that Kif18b, MCAK and the plus-end tracking protein EB3 (also known as MAPRE3) act in an integrated manner to potently promote microtubule depolymerization at very low concentrations. We find that Kif18b can transport EB3 and MCAK and promotes their accumulation to microtubule plus ends through multivalent weak interactions. Together, our work defines the mechanistic basis for a cooperative Kif18b-MCAK-EB network at microtubule plus ends, that acts to efficiently shorten and regulate microtubules in mitosis, essential for correct chromosome segregation.


Assuntos
Cinesinas , Microtúbulos , Segregação de Cromossomos , Cinesinas/genética , Microtúbulos/metabolismo , Mitose , Fuso Acromático/metabolismo
16.
J Cell Sci ; 136(5)2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35531639

RESUMO

Most motile cilia have a stereotyped structure of nine microtubule outer doublets and a single central pair of microtubules. The central pair of microtubules are surrounded by a set of proteins, termed the central pair apparatus. A specific kinesin, Klp1 projects from the central pair and contributes to ciliary motility in Chlamydomonas. The vertebrate ortholog, Kif9, is required for beating in mouse sperm flagella, but the mechanism of Kif9/Klp1 function remains poorly defined. Here, using Xenopus epidermal multiciliated cells, we show that Kif9 is necessary for ciliary motility and the proper distal localization of not only central pair proteins, but also radial spokes and dynein arms. In addition, single-molecule assays in vitro reveal that Xenopus Kif9 is a long-range processive motor, although it does not mediate long-range movement in ciliary axonemes in vivo. Together, our data suggest that Kif9 is integral for ciliary beating and is necessary for proper axonemal distal end integrity.


Assuntos
Axonema , Cílios , Cinesinas , Animais , Axonema/metabolismo , Cílios/metabolismo , Dineínas/metabolismo , Flagelos/metabolismo , Cinesinas/genética , Microtúbulos/metabolismo , Xenopus
17.
J Cell Sci ; 136(5)2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36074043

RESUMO

Microtubules are dynamic cytoskeletal filaments that can generate forces when polymerizing and depolymerizing. Proteins that follow growing or shortening microtubule ends and couple forces to cargo movement are important for a wide range of cellular processes. Quantifying these forces and the composition of protein complexes at dynamic microtubule ends is challenging and requires sophisticated instrumentation. Here, we present an experimental approach to estimate microtubule-generated forces through the extension of a fluorescent spring-shaped DNA origami molecule. Optical readout of the spring extension enables recording of force production simultaneously with single-molecule fluorescence of proteins getting recruited to the site of force generation. DNA nanosprings enable multiplexing of force measurements and only require a fluorescence microscope and basic laboratory equipment. We validate the performance of DNA nanosprings against results obtained using optical trapping. Finally, we demonstrate the use of the nanospring to study proteins that couple microtubule growth and shortening to force generation.


Assuntos
Citoesqueleto , Microtúbulos , Citoesqueleto/metabolismo , Fenômenos Mecânicos , Microscopia de Fluorescência , Microtúbulos/metabolismo
18.
J Cell Sci ; 136(5)2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36093836

RESUMO

Intracellular mature viruses (IMVs) are the first and most abundant infectious form of vaccinia virus to assemble during its replication cycle. IMVs can undergo microtubule-based motility, but their directionality and the motor involved in their transport remain unknown. Here, we demonstrate that IMVs, like intracellular enveloped viruses (IEVs), the second form of vaccinia that are wrapped in Golgi-derived membranes, recruit kinesin-1 and undergo anterograde transport. In vitro reconstitution of virion transport in infected cell extracts revealed that IMVs and IEVs move toward microtubule plus ends with respective velocities of 0.66 and 0.56 µm/s. Quantitative imaging established that IMVs and IEVs recruit an average of 139 and 320 kinesin-1 motor complexes, respectively. In the absence of kinesin-1, there was a near-complete loss of in vitro motility and reduction in the intracellular spread of both types of virions. Our observations demonstrate that kinesin-1 transports two morphologically distinct forms of vaccinia. Reconstitution of vaccinia-based microtubule motility in vitro provides a new model to elucidate how motor number and regulation impacts transport of a bona fide kinesin-1 cargo.


Assuntos
Cinesinas , Vaccinia , Extratos Celulares , Humanos , Microtúbulos/metabolismo , Vaccinia/metabolismo , Vírus Vaccinia , Vírion/fisiologia
20.
Curr Protoc ; 2(11): e618, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36426921

RESUMO

Recent advances in super resolution microscopy have enabled imaging at the 10-20 nm scale on a light microscope, providing unprecedented details of native biological structures and processes in intact and hydrated samples. Of the existing strategies, DNA points accumulation in imaging nanoscale topography (DNA-PAINT) affords convenient multiplexing, an important feature in interrogating complex biological systems. A practical limitation of DNA-PAINT, however, has been the slow imaging speed. In its original form, DNA-PAINT imaging of each target takes tens of minutes to hours to complete. To address this challenge, several improved implementations have been introduced. These include DNA-PAINT-ERS (where E = ethylene carbonate; R = repeat sequence; S = spacer), a set of strategies that leads to both accelerated DNA-PAINT imaging speed and improved image quality. With DNA-PAINT-ERS, imaging of typical cellular targets such as microtubules takes only 5-10 min. Importantly, DNA-PAINT-ERS also facilitates multiplexing and can be easily integrated into current workflows for fluorescence staining of biological samples. Here, we provide a detailed, step-by-step guide for fast and multiplexed DNA-PAINT-ERS imaging of fixed and immunostained cells grown on glass substrates as adherent monolayers. The protocol should be readily extended to biological samples of a different format (for example tissue sections) or staining mechanisms (for example using nanobodies). © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of probes for DNA-PAINT-ERS Basic Protocol 2: Sample preparation for imaging membrane targets with DNA-PAINT-ERS in fixed cells Alternate Protocol: Immunostaining of extracted U2OS cells Basic Protocol 3: Super resolution image acquisition and analysis.


Assuntos
DNA , Microtúbulos , Microscopia de Fluorescência/métodos , DNA/química , Coloração e Rotulagem
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