RESUMO
Polarized fluorescence microscopy is a valuable tool for measuring molecular orientations, but techniques for recovering three-dimensional orientations and positions of fluorescent ensembles are limited. We report a polarized dual-view light-sheet system for determining the three-dimensional orientations and diffraction-limited positions of ensembles of fluorescent dipoles that label biological structures, and we share a set of visualization, histogram, and profiling tools for interpreting these positions and orientations. We model our samples, their excitation, and their detection using coarse-grained representations we call orientation distribution functions (ODFs). We apply ODFs to create physics-informed models of image formation with spatio-angular point-spread and transfer functions. We use theory and experiment to conclude that light-sheet tilting is a necessary part of our design for recovering all three-dimensional orientations. We use our system to extend known two-dimensional results to three dimensions in FM1-43-labelled giant unilamellar vesicles, fast-scarlet-labelled cellulose in xylem cells, and phalloidin-labelled actin in U2OS cells. Additionally, we observe phalloidin-labelled actin in mouse fibroblasts grown on grids of labelled nanowires and identify correlations between local actin alignment and global cell-scale orientation, indicating cellular coordination across length scales.
RESUMO
Cancer cell migration during metastasis is mediated by a highly polarized cytoskeleton. MARK2 and its invertebrate homolog Par1B are kinases that regulate the microtubule cytoskeleton to mediate polarization of neurons in mammals and embryos in invertebrates. However, the role of MARK2 in cancer cell migration is unclear. Using osteosarcoma cells, we found that in addition to its known localizations on microtubules and the plasma membrane, MARK2 also associates with the actomyosin cytoskeleton and focal adhesions. Cells depleted of MARK proteins demonstrated that MARK2 promotes phosphorylation of both myosin II and the myosin phosphatase targeting subunit MYPT1 to synergistically drive myosin II contractility and stress fiber formation in cells. Studies with isolated proteins showed that MARK2 directly phosphorylates myosin II regulatory light chain, while its effects on MYPT1 phosphorylation are indirect. Using a mutant lacking the membrane-binding domain, we found that membrane association is required for focal adhesion targeting of MARK2, where it specifically enhances cell protrusion by promoting FAK phosphorylation and formation of focal adhesions oriented in the direction of migration to mediate directionally persistent cell motility. Together, our results define MARK2 as a master regulator of the actomyosin and microtubule cytoskeletal systems and focal adhesions to mediate directional cancer cell migration.
Assuntos
Actomiosina , Adesões Focais , Actomiosina/metabolismo , Animais , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Adesões Focais/metabolismo , Mamíferos , Cadeias Leves de Miosina/metabolismo , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , FosforilaçãoRESUMO
Contact guidance is a powerful topographical cue that induces persistent directional cell migration. Healthy tissue stroma is characterized by a meshwork of wavy extracellular matrix (ECM) fiber bundles, whereas metastasis-prone stroma exhibit less wavy, more linear fibers. The latter topography correlates with poor prognosis, whereas more wavy bundles correlate with benign tumors. We designed nanotopographic ECM-coated substrates that mimic collagen fibril waveforms seen in tumors and healthy tissues to determine how these nanotopographies may regulate cancer cell polarization and migration machineries. Cell polarization and directional migration were inhibited by fibril-like wave substrates above a threshold amplitude. Although polarity signals and actin nucleation factors were required for polarization and migration on low-amplitude wave substrates, they did not localize to cell leading edges. Instead, these factors localized to wave peaks, creating multiple "cryptic leading edges" within cells. On high-amplitude wave substrates, retrograde flow from large cryptic leading edges depolarized stress fibers and focal adhesions and inhibited cell migration. On low-amplitude wave substrates, actomyosin contractility overrode the small cryptic leading edges and drove stress fiber and focal adhesion orientation along the wave axis to mediate directional migration. Cancer cells of different intrinsic contractility depolarized at different wave amplitudes, and cell polarization response to wavy substrates could be tuned by manipulating contractility. We propose that ECM fibril waveforms with sufficiently high amplitude around tumors may serve as "cell polarization barriers," decreasing directional migration of tumor cells, which could be overcome by up-regulation of tumor cell contractility.
Assuntos
Polaridade Celular , Matriz Extracelular/patologia , Adesões Focais , Metástase Neoplásica , Neoplasias/patologia , Fibras de Estresse/patologia , HumanosRESUMO
Efficient chemotaxis requires rapid coordination between different parts of the cell in response to changing directional cues. Here, we investigate the mechanism of front-rear coordination in chemotactic neutrophils. We find that changes in the protrusion rate at the cell front are instantaneously coupled to changes in retraction at the cell rear, while myosin II accumulation at the rear exhibits a reproducible 9-15-s lag. In turning cells, myosin II exhibits dynamic side-to-side relocalization at the cell rear in response to turning of the leading edge and facilitates efficient turning by rapidly re-orienting the rear. These manifestations of front-rear coupling can be explained by a simple quantitative model incorporating reversible actin-myosin interactions with a rearward-flowing actin network. Finally, the system can be tuned by the degree of myosin regulatory light chain (MRLC) phosphorylation, which appears to be set in an optimal range to balance persistence of movement and turning ability.
Assuntos
Quimiotaxia/fisiologia , Miosina Tipo II/fisiologia , Neutrófilos/fisiologia , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Extensões da Superfície Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Feminino , Humanos , Miosina Tipo II/metabolismo , Miosinas/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Single cell branching during development in vertebrates is typified by neuronal branching to form neurites and vascular branches formed by sprouting angiogenesis. Neurons and endothelial tip cells possess subcellular protrusions that share many common features from the morphological to the molecular level. Both systems utilize filopodia as their cellular protrusion organelles and depend on specific integrin-mediated adhesions to the local extracellular matrix for guidance in their pathfinding. We discuss the similar molecular machineries involved in these two types of cell branch formation and use their analogy to propose a new mechanism for angiogenic filopodia function, namely as adhesion assembly sites. In support of this model we provide primary data of angiogenesis in zebrafish in vivo showing that the actin assembly factor VASP participates in both filopodia formation and adhesion assembly at the base of the filopodia, enabling forward progress of the tip cell. The use of filopodia and their associated adhesions provide a common mechanism for neuronal and endothelial pathfinding during development in response to extracellular matrix cues.
Assuntos
Adesões Focais/metabolismo , Morfogênese/fisiologia , Neovascularização Fisiológica/fisiologia , Pseudópodes/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Orientação de Axônios/fisiologia , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Adesões Focais/genética , Pseudópodes/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The role of nonmuscle myosin 2 (NM2) pulsatile dynamics in generating contractile forces required for developmental morphogenesis has been characterized, but whether these pulsatile contractions are an intrinsic property of all actomyosin networks is not known. Here we used live-cell fluorescence imaging to show that transient, local assembly of NM2A "pulses" occurs in the cortical cytoskeleton of single adherent cells of mesenchymal, epithelial, and sarcoma origin, independent of developmental signaling cues and cell-cell or cell-ECM interactions. We show that pulses in the cortical cytoskeleton require Rho-associated kinase- or myosin light chain kinase (MLCK) activity, increases in cytosolic calcium, and NM2 ATPase activity. Surprisingly, we find that cortical cytoskeleton pulses specifically require the head domain of NM2A, as they do not occur with either NM2B or a 2B-head-2A-tail chimera. Our results thus suggest that pulsatile contractions in the cortical cytoskeleton are an intrinsic property of the NM2A motor that may mediate its role in homeostatic maintenance of tension in the cortical cytoskeleton of adherent cells.
Assuntos
Miosina não Muscular Tipo IIA/metabolismo , Miosina não Muscular Tipo IIA/fisiologia , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Animais , Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Imagem Molecular , Contração Muscular/fisiologia , Quinase de Cadeia Leve de Miosina/metabolismo , Miosinas/metabolismo , Miosina não Muscular Tipo IIA/genética , Miosina não Muscular Tipo IIB/metabolismo , Miosina não Muscular Tipo IIB/fisiologia , Imagem Óptica , Fosforilação , Domínios Proteicos , Quinases Associadas a rho/metabolismoRESUMO
Cell migration in confined 3D tissue microenvironments is critical for both normal physiological functions and dissemination of tumor cells. We discovered a cytoskeletal structure that prevents damage to the nucleus during migration in confined microenvironments. The formin-family actin filament nucleator FMN2 associates with and generates a perinuclear actin/focal adhesion (FA) system that is distinct from previously characterized actin/FA structures. This system controls nuclear shape and positioning in cells migrating on 2D surfaces. In confined 3D microenvironments, FMN2 promotes cell survival by limiting nuclear envelope damage and DNA double-strand breaks. We found that FMN2 is upregulated in human melanomas and showed that disruption of FMN2 in mouse melanoma cells inhibits their extravasation and metastasis to the lung. Our results indicate a critical role for FMN2 in generating a perinuclear actin/FA system that protects the nucleus and DNA from damage to promote cell survival during confined migration and thus promote cancer metastasis.
Assuntos
Núcleo Celular/metabolismo , Adesões Focais , Neoplasias Pulmonares/secundário , Melanoma/patologia , Proteínas dos Microfilamentos/metabolismo , Metástase Neoplásica , Proteínas Nucleares/metabolismo , Actinas/metabolismo , Animais , Quebras de DNA de Cadeia Dupla , Embrião de Mamíferos/citologia , Matriz Extracelular/metabolismo , Feminino , Forminas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido NervosoRESUMO
Cell-cell contact inhibition and the mechanical environment of cells have both been shown to regulate YAP nuclear localization to modulate cell proliferation. Changes in cellular contractility by genetic, pharmacological, and matrix stiffness perturbations regulate YAP nuclear localization. However, because contractility and F-actin organization are interconnected cytoskeletal properties, it remains unclear which of these distinctly regulates YAP localization. Here we show that in the absence of cell-cell contact, actomyosin contractility suppresses YAP phosphorylation at Ser(112), however, neither loss of contractility nor increase in YAP phosphorylation is sufficient for its nuclear exclusion. We find that actin cytoskeletal integrity is essential for YAP nuclear localization, and can override phosphoregulation or contractility-mediated regulation of YAP nuclear localization. This actin-mediated regulation is conserved during mechanotransduction, as substrate compliance increased YAP phosphorylation and reduced cytoskeletal integrity leading to nuclear exclusion of both YAP and Ser(P)(112)-YAP. These data provide evidence for two actin-mediated pathways for YAP regulation; one in which actomyosin contractility regulates YAP phosphorylation, and a second that involves cytoskeletal integrity-mediated regulation of YAP nuclear localization independent of contractility. We suggest that in non-contact inhibited cells, this latter mechanism may be important in low stiffness regimes, such as may be encountered in physiological environments.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Núcleo Celular/metabolismo , Fosfoproteínas/metabolismo , Citoesqueleto de Actina/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Adesão Celular , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Células Cultivadas , Citoesqueleto/fisiologia , Matriz Extracelular , Humanos , Mecanotransdução Celular , Camundongos , Miosina Tipo II/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas de Sinalização YAPRESUMO
The study of cytoskeletal polymers has been an active area of research for more than 70 years. However, despite decades of pioneering work by some of the brightest scientists in biochemistry, cell biology, and physiology, many central questions regarding the polymers themselves are only now starting to be answered. For example, although it has long been appreciated that the actin cytoskeleton provides contractility and couples biochemical responses with mechanical stresses in cells, only recently have we begun to understand how the actin polymer itself responds to mechanical loads. Likewise, although it has long been appreciated that the microtubule cytoskeleton can be post-translationally modified, only recently have the enzymes responsible for these modifications been characterized, so that we can now begin to understand how these modifications alter the polymerization and regulation of microtubule structures. Even the septins in eukaryotes and the cytoskeletal polymers of prokaryotes have yielded new insights due to recent advances in microscopy techniques. In this thematic series of minireviews, these topics are covered by some of the very same scientists who generated these recent insights, thereby providing us with an overview of the State of the Cytoskeleton in 2015.
Assuntos
Citoesqueleto/química , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/metabolismo , Animais , Fenômenos Biomecânicos , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Humanos , Microtúbulos/química , Microtúbulos/metabolismoRESUMO
Three-dimensional super-resolution imaging in thick, semi-transparent biological specimens is hindered by light scattering, which increases background and degrades both contrast and optical sectioning. We describe a simple method that mitigates these issues, improving image quality in our recently developed two-photon instant structured illumination microscope without requiring any hardware modifications to the instrument. By exciting the specimen with three laterally-structured, phase-shifted illumination patterns and post-processing the resulting images, we digitally remove both scattered and out-of-focus emissions that would otherwise contaminate our raw data. We demonstrate the improved performance of our approach in biological samples, including pollen grains, primary mouse aortic endothelial cells cultured in a three-dimensional collagen matrix and live tumor-like cell spheroids.
Assuntos
Células Endoteliais/citologia , Aumento da Imagem/instrumentação , Iluminação/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neoplasias Experimentais/patologia , Animais , Desenho de Equipamento , Análise de Falha de Equipamento , Aumento da Imagem/métodos , Iluminação/métodos , Camundongos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
In many cases, cell function is intimately linked to cell shape control. We used endothelial cell branching morphogenesis as a model to understand the role of myosin II in shape control of invasive cells migrating in 3D collagen gels. We applied principles of differential geometry and mathematical morphology to 3D image sets to parameterize cell branch structure and local cell-surface curvature. We find that Rho/ROCK-stimulated myosin II contractility minimizes cell-scale branching by recognizing and minimizing local cell-surface curvature. Using microfabrication to constrain cell shape identifies a positive feedback mechanism in which low curvature stabilizes myosin II cortical association, where it acts to maintain minimal curvature. The feedback between regulation of myosin II by curvature and control of curvature by myosin II drives cycles of localized cortical myosin II assembly and disassembly. These cycles in turn mediate alternating phases of directionally biased branch initiation and retraction to guide 3D cell migration.
Assuntos
Membrana Celular/metabolismo , Movimento Celular , Imageamento Tridimensional , Morfogênese , Miosina Tipo II/metabolismo , Animais , Aorta/citologia , Células Endoteliais/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Proteínas Recombinantes de Fusão/metabolismo , Fatores de TempoRESUMO
BACKGROUND: Cell migration requires coordinated formation of focal adhesions (FAs) and assembly and contraction of the actin cytoskeleton. Nonmuscle myosin II (MII) is a critical mediator of contractility and FA dynamics in cell migration. Signaling downstream of the small GTPase Rac1 also regulates FA and actin dynamics, but its role in regulation of MII during migration is less clear. RESULTS: We found that Rac1 promotes association of MIIA with FA. Live-cell imaging showed that, whereas most MIIA at the leading edge assembled into dorsal contractile arcs, a substantial subset assembled in or was captured within maturing FA, and this behavior was promoted by active Rac1. Protein kinase C (PKC) activation was necessary and sufficient for integrin- and Rac1-dependent phosphorylation of MIIA heavy chain (HC) on serine1916 (S1916) and recruitment to FA. S1916 phosphorylation of MIIA HC and localization in FA was enhanced during cell spreading and ECM stiffness mechanosensing, suggesting upregulation of this pathway during physiological Rac1 activation. Phosphomimic and nonphosphorylatable MIIA HC mutants demonstrated that S1916 phosphorylation was necessary and sufficient for the capture and assembly of MIIA minifilaments in FA. S1916 phosphorylation was also sufficient to promote the rapid assembly of FAs to enhance cell migration and for the modulation of traction force, spreading, and migration by ECM stiffness. CONCLUSIONS: Our study reveals for the first time that Rac1 and integrin activation regulates MIIA HC phosphorylation through a PKC-dependent mechanism that promotes MIIA association with FAs and acts as a critical modulator of cell migration and mechanosensing.
Assuntos
Movimento Celular , Adesões Focais/metabolismo , Proteínas Motores Moleculares/genética , Cadeias Pesadas de Miosina/genética , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/genética , Linhagem Celular , Humanos , Mecanotransdução Celular/fisiologia , Proteínas Motores Moleculares/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Fosforilação , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Polarized epithelial cells create tightly packed arrays of microvilli in their apical membrane, but the fate of these microvilli is relatively unknown when epithelial cell polarity is lost during wound healing. In this issue, Klingner et al. (2014. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201402037) show that, when epithelial cells become subconfluent, actomyosin contractions locally within the apical cortex cause their microvilli to become motile over the dorsal/apical surface. Their unexpected observations may have implications for epithelial responses in wound healing and disease.
Assuntos
Actomiosina/metabolismo , Células Epiteliais/fisiologia , Microvilosidades/fisiologia , Miosina Tipo II/metabolismo , Animais , HumanosRESUMO
How adherent and contractile systems coordinate to promote cell shape changes is unclear. Here, we define a counterbalanced adhesion/contraction model for cell shape control. Live-cell microscopy data showed a crucial role for a contractile meshwork at the top of the cell, which is composed of actin arcs and myosin IIA filaments. The contractile actin meshwork is organized like muscle sarcomeres, with repeating myosin II filaments separated by the actin bundling protein α-actinin, and is mechanically coupled to noncontractile dorsal actin fibers that run from top to bottom in the cell. When the meshwork contracts, it pulls the dorsal fibers away from the substrate. This pulling force is counterbalanced by the dorsal fibers' attachment to focal adhesions, causing the fibers to bend downward and flattening the cell. This model is likely to be relevant for understanding how cells configure themselves to complex surfaces, protrude into tight spaces, and generate three-dimensional forces on the growth substrate under both healthy and diseased conditions.
Assuntos
Citoesqueleto de Actina/metabolismo , Adesão Celular , Movimento Celular , Forma Celular , Adesões Focais/metabolismo , Actinina/metabolismo , Actinas/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Mecanotransdução Celular , Modelos Biológicos , Miosina não Muscular Tipo IIA/genética , Miosina não Muscular Tipo IIA/metabolismo , Pressão , Fatores de Tempo , TransfecçãoRESUMO
Live-cell imaging techniques have been substantially improved due to advances in confocal microscopy instrumentation coupled with ultrasensitive detectors. The spinning disk confocal system is capable of generating images of fluorescent live samples with broad dynamic range and high temporal and spatial resolution. The ability to acquire fluorescent images of living cells in vivo on a millisecond timescale allows the dissection of biological processes that have not previously been visualized in a physiologically relevant context. In vivo imaging of rapidly moving cells such as neutrophils can be technically challenging. In this chapter, we describe the practical aspects of imaging neutrophils in zebrafish embryos using spinning disk confocal microscopy. Similar setups can also be applied to image other motile cell types and signaling processes in translucent animals or tissues.
Assuntos
Doenças do Sistema Imunitário/imunologia , Transtornos Leucocíticos/imunologia , Microscopia Confocal/métodos , Animais , Expressão Gênica , Doenças do Sistema Imunitário/metabolismo , Transtornos Leucocíticos/metabolismo , Especificidade de Órgãos/genética , Transgenes , Peixe-ZebraRESUMO
Existing super-resolution fluorescence microscopes compromise acquisition speed to provide subdiffractive sample information. We report an analog implementation of structured illumination microscopy that enables three-dimensional (3D) super-resolution imaging with a lateral resolution of 145 nm and an axial resolution of 350 nm at acquisition speeds up to 100 Hz. By using optical instead of digital image-processing operations, we removed the need to capture, store and combine multiple camera exposures, increasing data acquisition rates 10- to 100-fold over other super-resolution microscopes and acquiring and displaying super-resolution images in real time. Low excitation intensities allow imaging over hundreds of 2D sections, and combined physical and computational sectioning allow similar depth penetration to spinning-disk confocal microscopy. We demonstrate the capability of our system by imaging fine, rapidly moving structures including motor-driven organelles in human lung fibroblasts and the cytoskeleton of flowing blood cells within developing zebrafish embryos.
Assuntos
Embrião de Mamíferos/citologia , Animais , Microscopia de FluorescênciaRESUMO
Optimal four-dimensional imaging requires high spatial resolution in all dimensions, high speed and minimal photobleaching and damage. We developed a dual-view, plane illumination microscope with improved spatiotemporal resolution by switching illumination and detection between two perpendicular objectives in an alternating duty cycle. Computationally fusing the resulting volumetric views provides an isotropic resolution of 330 nm. As the sample is stationary and only two views are required, we achieve an imaging speed of 200 images/s (i.e., 0.5 s for a 50-plane volume). Unlike spinning-disk confocal or Bessel beam methods, which illuminate the sample outside the focal plane, we maintain high spatiotemporal resolution over hundreds of volumes with negligible photobleaching. To illustrate the ability of our method to study biological systems that require high-speed volumetric visualization and/or low photobleaching, we describe microtubule tracking in live cells, nuclear imaging over 14 h during nematode embryogenesis and imaging of neural wiring during Caenorhabditis elegans brain development over 5 h.
Assuntos
Rastreamento de Células/métodos , Imageamento Tridimensional/métodos , Microscopia/instrumentação , Microscopia/métodos , Animais , Encéfalo/anatomia & histologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/ultraestrutura , Caenorhabditis elegans/embriologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Iluminação , FotodegradaçãoRESUMO
Regulation of cell functions by the physical properties of the extracellular matrix (ECM) has emerged as a crucial contributor to development and disease. Two specific physical properties of the ECM, stiffness and dimensionality, each influence cell signaling and function. As these ECM physical properties are linked to other properties that also regulate cell behavior, e.g., integrin ligand density, parsing the specific contributions of ECM stiffness and dimensionality has proven difficult. Here we detail a simple protocol, which can be completed in 1-2 d, for combining three-dimensional (3D) ECM engagement with controlled underlying ECM stiffness. In these 'sandwich gels', cells are sandwiched between a 3D fibrillar ECM and an ECM-coupled polyacrylamide gel of defined compliance, allowing the study of the specific effects of ECM compliance on cell function in physiologically relevant 3D ECMs. This type of system enables high-resolution time-lapse imaging and is suitable for a wide range of cell types and molecular perturbations.
Assuntos
Técnicas de Cultura de Células , Matriz Extracelular/química , Resinas Acrílicas , Comunicação Celular , Microambiente Celular , Matriz Extracelular/ultraestrutura , Células Endoteliais da Veia Umbilical Humana , Microscopia de Contraste de FaseRESUMO
We demonstrate three-dimensional (3D) super-resolution in live multicellular organisms using structured illumination microscopy (SIM). Sparse multifocal illumination patterns generated by a digital micromirror device (DMD) allowed us to physically reject out-of-focus light, enabling 3D subdiffractive imaging in samples eightfold thicker than had been previously imaged with SIM. We imaged samples at one 2D image per second, at resolutions as low as 145 nm laterally and 400 nm axially. In addition to dual-labeled, whole fixed cells, we imaged GFP-labeled microtubules in live transgenic zebrafish embryos at depths >45 µm. We captured dynamic changes in the zebrafish lateral line primordium and observed interactions between myosin IIA and F-actin in cells encapsulated in collagen gels, obtaining two-color 4D super-resolution data sets spanning tens of time points and minutes without apparent phototoxicity. Our method uses commercially available parts and open-source software and is simpler than existing SIM implementations, allowing easy integration with wide-field microscopes.