Three-dimensional electron microscopy reveals the evolution of glomerular barrier injury

Open access articleGlomeruli are highly sophisticated filters and glomerular disease is the leading cause of kidney failure. Morphological change in glomerular podocytes and the underlying basement membrane are frequently observed in disease, irrespective of the underlying molecular etiology. Standard electron microscopy techniques have enabled the identification and classification of glomerular diseases based on two-dimensional information, however complex three-dimensional ultrastructural relationships between cells and their extracellular matrix cannot be easily resolved with this approach. We employed serial block face-scanning electron microscopy to investigate Alport syndrome, the commonest monogenic glomerular disease, and compared findings to other genetic mouse models of glomerular disease (Myo1e−/−, Ptpro−/−). These analyses revealed the evolution of basement membrane and cellular defects through the progression of glomerular injury. Specifically we identified sub-podocyte expansions of the basement membrane with both cellular and matrix gene defects and found a corresponding reduction in podocyte foot process number. Furthermore, we discovered novel podocyte protrusions invading into the glomerular basement membrane in disease and these occurred frequently in expanded regions of basement membrane. These findings provide new insights into mechanisms of glomerular barrier dysfunction and suggest that common cell-matrix-adhesion pathways are involved in the progression of disease regardless of the primary insult

Membrane Tension Orchestrates Rear Retraction in Matrix-Directed Cell Migration

In development, wound healing, and cancer metastasis, vertebrate cells move through 3D interstitial matrix, responding to chemical and physical guidance cues. Protrusion at the cell front has been extensively studied, but the retraction phase of the migration cycle is not well understood. Here, we show that fast-moving cells guided by matrix cues establish positive feedback control of rear retraction by sensing membrane tension. We reveal a mechanism of rear retraction in 3D matrix and durotaxis controlled by caveolae, which form in response to low membrane tension at the cell rear. Caveolae activate RhoA-ROCK1/PKN2 signaling via the RhoA guanidine nucleotide exchange factor (GEF) Ect2 to control local F-actin organization and contractility in this subcellular region and promote translocation of the cell rear. A positive feedback loop between cytoskeletal signaling and membrane tension leads to rapid retraction to complete the migration cycle in fast-moving cells, providing directional memory to drive persistent cell migration in complex matrices. © 2019 The AuthorsCell migration through 3D matrix is critical to developmental and disease processes, but the mechanisms that control rear retraction are poorly understood. Hetmanski et al. show that differential membrane tension allows caveolae to form at the rear of migrating cells and activate the contractile actin cytoskeleton to promote rapid retraction. © 2019 The Author

Membrane Tension Orchestrates Rear Retraction in Matrix-Directed Cell Migration.

In development, wound healing, and cancer metastasis, vertebrate cells move through 3D interstitial matrix, responding to chemical and physical guidance cues. Protrusion at the cell front has been extensively studied, but the retraction phase of the migration cycle is not well understood. Here, we show that fast-moving cells guided by matrix cues establish positive feedback control of rear retraction by sensing membrane tension. We reveal a mechanism of rear retraction in 3D matrix and durotaxis controlled by caveolae, which form in response to low membrane tension at the cell rear. Caveolae activate RhoA-ROCK1/PKN2 signaling via the RhoA guanidine nucleotide exchange factor (GEF) Ect2 to control local F-actin organization and contractility in this subcellular region and promote translocation of the cell rear. A positive feedback loop between cytoskeletal signaling and membrane tension leads to rapid retraction to complete the migration cycle in fast-moving cells, providing directional memory to drive persistent cell migration in complex matrices

Absence of Both IL-7 and IL-15 Severely Impairs the Development of CD8+ T Cell Response against Toxoplasma gondii

CD8+ T cells play an essential role in the protection against both acute as well as chronic Toxoplasma gondii infection. Although the role of IL-15 has been reported to be important for the development of long-term CD8+ T cell immunity against the pathogen, the simultaneous roles played by both IL-15 and related γ-chain family cytokine IL-7 in the generation of this response during acute phase of infection has not been described. We demonstrate that while lack of IL-7 or IL-15 alone has minimal impact on splenic CD8+ T cell maturation or effector function development during acute Toxoplasmosis, absence of both IL-7 and IL-15 only in the context of infection severely down-regulates the development of a potent CD8+ T cell response. This impairment is characterized by reduction in CD44 expression, IFN-γ production, proliferation and cytotoxicity. However, attenuated maturation and decreased effector functions in these mice are essentially downstream consequences of reduced number of antigen-specific CD8+ T cells. Interestingly, the absence of both cytokines did not impair initial CD8+ T cell generation but affected their survival and differentiation into memory phenotype IL-7Rαhi cells. Significantly lack of both cytokines severely affected expression of Bcl-2, an anti-apoptotic protein, but minimally affected proliferation. The overarching role played by these cytokines in eliciting a potent CD8+ T cell immunity against T. gondii infection is further evidenced by poor survival and high parasite burden in anti IL-7 treated IL-15−/− mice. These studies demonstrate that the two cytokines, IL-7 and IL-15, are exclusively important for the development of protective CD8+ T cell immune response against T. gondii. To the best of our knowledge this synergism between IL-7 and IL-15 in generating an optimal CD8+ T cell immunity against intracellular parasite or any other infectious disease model has not been previously reported

Remodeling of the Purkinje Network in Congestive Heart Failure in the Rabbit

BACKGROUND: Purkinje fibers (PFs) control timing of ventricular conduction and play a key role in arrhythmogenesis in heart failure (HF) patients. We investigated the effects of HF on PFs. METHODS: Echocardiography, electrocardiography, micro-computed tomography, quantitative polymerase chain reaction, immunohistochemistry, volume electron microscopy, and sharp microelectrode electrophysiology were used. RESULTS: Congestive HF was induced in rabbits by left ventricular volume- and pressure-overload producing left ventricular hypertrophy, diminished fractional shortening and ejection fraction, and increased left ventricular dimensions. HF baseline QRS and corrected QT interval were prolonged by 17% and 21% (mean±SEMs: 303±6 ms HF, 249±11 ms control; n=8/7; P=0.0002), suggesting PF dysfunction and impaired ventricular repolarization. Micro-computed tomography imaging showed increased free-running left PF network volume and length in HF. mRNA levels for 40 ion channels, Ca2+-handling proteins, connexins, and proinflammatory and fibrosis markers were assessed: 50% and 35% were dysregulated in left and right PFs respectively, whereas only 12.5% and 7.5% changed in left and right ventricular muscle. Funny channels, Ca2+-channels, and K+-channels were significantly reduced in left PFs. Microelectrode recordings from left PFs revealed more negative resting membrane potential, reduced action potential upstroke velocity, prolonged duration (action potential duration at 90% repolarization: 378±24 ms HF, 249±5 ms control; n=23/38; P<0.0001), and arrhythmic events in HF. Similar electrical remodeling was seen at the left PF-ventricular junction. In the failing left ventricle, upstroke velocity and amplitude were increased, but action potential duration at 90% repolarization was unaffected. CONCLUSIONS: Severe volume- followed by pressure-overload causes rapidly progressing HF with extensive remodeling of PFs. The PF network is central to both arrhythmogenesis and contractile dysfunction and the pathological remodeling may increase the risk of fatal arrhythmias in HF patients

A cell-body groove housing the new flagellum tip suggests an adaptation of cellular morphogenesis for parasitism in the bloodstream form of Trypanosoma brucei.

Flagella are highly conserved organelles present in a wide variety of species. In Trypanosoma brucei the single flagellum is necessary for morphogenesis, cell motility and pathogenesis, and is attached along the cell body. A new flagellum is formed alongside the old during the cell division cycle. In the (insect) procyclic form, the flagella connector (FC) attaches the tip of the new flagellum to the side of the old flagellum, ensuring faithful replication of cell architecture. The FC is not present in the bloodstream form of the parasite. We show here, using new imaging techniques including serial block-face scanning electron microscopy (SBF-SEM), that the distal tip of the new flagellum in the bloodstream form is embedded within an invagination in the cell body plasma membrane, named the groove. We suggest that the groove has a similar function to the flagella connector. The groove is a mobile junction located alongside the microtubule quartet (MtQ) and occurred within a gap in the subpellicular microtubule corset, causing significant modification of microtubules during elongation of the new flagellum. It appears likely that this novel form of morphogenetic structure has evolved to withstand the hostile immune response in the mammalian blood