Severe influenza infection represents a leading cause of global morbidity and mortality. Although influenza is primarily considered a viral infection that results in pathology limited to the ...respiratory system, clinical reports suggest that influenza infection is frequently associated with a number of clinical syndromes that involve organ systems outside the respiratory tract. A comprehensive MEDLINE literature review of articles pertaining to extra‐pulmonary complications of influenza infection, using organ‐specific search terms, yielded 218 articles including case reports, epidemiologic investigations, and autopsy studies that were reviewed to determine the clinical involvement of other organs. The most frequently described clinical entities were viral myocarditis and viral encephalitis. Recognition of these extra‐pulmonary complications is critical to determining the true burden of influenza infection and initiating organ‐specific supportive care.
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.
A key feature of angiogenesis is directional control of endothelial cell (EC) morphogenesis and movement
1. During angiogenic sprouting, endothelial “tip cells” directionally branch from existing ...vessels in response to biochemical cues such as VEGF or hypoxia and migrate and invade the surrounding extracellular matrix (ECM) in a process that requires ECM remodeling by matrix metalloproteases (MMPs)
2–4. Tip EC branching is mediated by directional protrusion of subcellular pseudopodial branches
5, 6. Here, we seek to understand how EC pseudopodial branching is locally regulated to directionally guide angiogenesis. We develop an in vitro 3D EC model system in which migrating ECs display branched pseudopodia morphodynamics similar to those in living zebrafish. Using this system, we find that ECM stiffness and ROCK-mediated myosin II activity inhibit EC pseudopodial branch initiation. Myosin II is dynamically localized to the EC cortex and is partially released under conditions that promote branching. Local depletion of cortical myosin II precedes branch initiation, and initiation can be induced by local inhibition of myosin II activity. Thus, local downregulation of myosin II cortical contraction allows pseudopodium initiation to mediate EC branching and hence guide directional migration and angiogenesis.
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.
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.
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.
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.
•Rac1 and integrin activation promotes myosin IIA localization at focal adhesions (FAs)•PKC-mediated Myo IIA HC phosphorylation is required for its localization to FAs•Phosphomutants of Myo IIA alter FA dynamics, cell migration, and mechanosensation
Pasapera et al. find that Rac1 and integrin activation regulates MIIA heavy-chain phosphorylation through a PKC-dependent mechanism that promotes MIIA association with focal adhesions, which acts as a critical modulator of cell migration and mechanosensing.
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.
Significance
Tumor progression to enable metastasis includes remodeling the wavy bundles of collagen making up the tissue stromal extracellular matrix (ECM) into straight bundles within the tumor ...microenvironment. While wavy collagen bundles are thought to be inhibitory to cell polarization and migration in tissue, straight ECM fibers are thought to be conducive, thereby mediating metastasis. We used nanofabricated cell culture substrates that mimic the ECM fiber waveforms seen in both benign- and metastases-promoting tumor ECMs. Large amplitude ECM waves depolarized tumor cells and decreased directional migration via cell contractility-mediated organization of the cytoskeleton and adhesions. Thus, ECM architecture of normal tissue and benign tumors may generally inhibit tumor cell exit, but this may be overcome by increasing tumor cell contractility.
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.
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.
•Neurons and endothelial cells share many molecular cues in pathfinding, including cell-matrix adhesion signals.•This review suggests that neurons and endothelial both use filopodia as adhesion “factories” during pathfinding, similar to how other cell types use lamellipodia.•Adhesion complexes created at the tips of filopodia are matured into larger focal adhesions in the lamella area near the base of filopodia.•Demonstrating this, VASP decorates growing filopodia tips and accumulates in adhesion sites at or near the base of filopodia in endothelial tip cells in vivo.
Abstract
Paragonimiasis is a zoonotic, food-borne trematode infection that affects 21 million people globally. Trematodes interact with their hosts via extracellular vesicles (EV) that carry protein ...and RNA cargo. We analyzed EV in excretory-secretory products (ESP) released by
Paragonimus kellicotti
adult worms cultured in vitro (EV ESP) and EV isolated from lung cyst fluid (EV CFP) recovered from infected gerbils. The majority of EV were approximately 30–50 nm in diameter. We identified 548
P. kellicotti
-derived proteins in EV ESP by mass spectrometry and 8 proteins in EV CFP of which 7 were also present in EV ESP. No parasite-derived proteins were reliably detected in EV isolated from plasma samples. A cysteine protease (MK050848, CP-6) was the most abundant protein found in EV CFP in all technical and biological replicates. Immunolocalization of CP-6 showed strong labeling in the tegument of
P. kellicotti
and in the adjacent cyst and lung tissue that contained worm eggs. It is likely that CP-6 present in EV is involved in parasite-host interactions. These results provide new insights into interactions between
Paragonimus
and their mammalian hosts, and they provide potential clues for development of novel diagnostic tools and treatments.
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.
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