Imaging Mitosis with Lattice LightSheet.

Mitosis is one of the most fundamental processes of life, allowing organisms to grow, develop, and evolve. Acquiring microscopic images and understanding the detailed mechanism of this process is critical in the fields of cell and developmental biology. Modern fluorescence microscopy is the standard for imaging specific molecules and proteins as they interact during this complicated process. However, researchers must take care to ensure that they are maintaining the basal cell processes during mitosis without disruption by placing the sample on a microscope. In addition, mitosis in itself is an incredibly dynamic process that requires both high-speed and high-resolution imaging (McIntosh and Hays. Biology. 5:55, 2016). The Lattice LightSheet is an advanced system, developed in the lab of Eric Betzig (Chen et al. Science. 346:1257998), that offers imaging speeds in the volumes/second while still resolving fine, intracellular structures. Here we describe how to prepare cell culture samples for ideal mitotic imaging on this cutting-edge light sheet fluorescence microscope.

Neutrophil DREAM promotes neutrophil recruitment in vascular inflammation.

The interaction between neutrophils and endothelial cells is critical for the pathogenesis of vascular inflammation. However, the regulation of neutrophil adhesive function remains not fully understood. Intravital microscopy demonstrates that neutrophil DREAM promotes neutrophil recruitment to sites of inflammation induced by TNF-α but not MIP-2 or fMLP. We observe that neutrophil DREAM represses expression of A20, a negative regulator of NF-κB activity, and enhances expression of pro-inflammatory molecules and phosphorylation of IκB kinase (IKK) after TNF-α stimulation. Studies using genetic and pharmacologic approaches reveal that DREAM deficiency and IKKß inhibition significantly diminish the ligand-binding activity of ß2 integrins in TNF-α-stimulated neutrophils or neutrophil-like HL-60 cells. Neutrophil DREAM promotes degranulation through IKKß-mediated SNAP-23 phosphorylation. Using sickle cell disease mice lacking DREAM, we show that hematopoietic DREAM promotes vaso-occlusive events in microvessels following TNF-α challenge. Our study provides evidence that targeting DREAM might be a novel therapeutic strategy to reduce excessive neutrophil recruitment in inflammatory diseases.

DREAM plays an important role in platelet activation and thrombogenesis.

Downstream regulatory element antagonist modulator (DREAM), a transcriptional repressor, is known to modulate pain responses. However, it is unknown whether DREAM is expressed in anucleate platelets and plays a role in thrombogenesis. By using intravital microscopy with DREAM-null mice and their bone marrow chimeras, we demonstrated that both hematopoietic and nonhematopoietic cell DREAMs are required for platelet thrombus formation following laser-induced arteriolar injury. In a FeCl3-induced thrombosis model, we found that compared with wild-type (WT) control and nonhematopoietic DREAM knockout (KO) mice, DREAM KO control and hematopoietic DREAM KO mice showed a significant delay in time to occlusion. Tail bleeding time was prolonged in DREAM KO control mice, but not in WT or DREAM bone marrow chimeric mice. In vivo adoptive transfer experiments further indicated the importance of platelet DREAM in thrombogenesis. We found that DREAM deletion does not alter the ultrastructural features of platelets but significantly impairs platelet aggregation and adenosine triphosphate secretion induced by numerous agonists (collagen-related peptide, adenosine 5'-diphosphate, A23187, thrombin, or U46619). Biochemical studies revealed that platelet DREAM positively regulates phosphoinositide 3-kinase (PI3K) activity during platelet activation. Using DREAM-null platelets and PI3K isoform-specific inhibitors, we observed that platelet DREAM is important for α-granule secretion, Ca2+ mobilization, and aggregation through PI3K class Iß (PI3K-Iß). Genetic and pharmacological studies in human megakaryoblastic MEG-01 cells showed that DREAM is important for A23187-induced Ca2+ mobilization and its regulatory function requires Ca2+ binding and PI3K-Iß activation. These results suggest that platelet DREAM regulates PI3K-Iß activity and plays an important role during thrombus formation.

ARQ 092, an orally-available, selective AKT inhibitor, attenuates neutrophil-platelet interactions in sickle cell disease.

Previous studies identified the Ser/Thr protein kinase, AKT, as a therapeutic target in thrombo-inflammatory diseases. Here we report that specific inhibition of AKT with ARQ 092, an orally-available AKT inhibitor currently in phase Ib clinical trials as an anti-cancer drug, attenuates the adhesive function of neutrophils and platelets from sickle cell disease patients in vitro and cell-cell interactions in a mouse model of sickle cell disease. Studies using neutrophils and platelets isolated from sickle cell disease patients revealed that treatment with 50-500 nM ARQ 092 significantly blocks αMß2 integrin function in neutrophils and reduces P-selectin exposure and glycoprotein Ib/IX/V-mediated agglutination in platelets. Treatment of isolated platelets and neutrophils with ARQ 092 inhibited heterotypic cell-cell aggregation under shear conditions. Intravital microscopic studies demonstrated that short-term oral administration of ARQ 092 or hydroxyurea, a major therapy for sickle cell disease, diminishes heterotypic cell-cell interactions in venules of sickle cell disease mice challenged with tumor necrosis factor-α. Co-administration of hydroxyurea and ARQ 092 further reduced the adhesive function of neutrophils in venules and neutrophil transmigration into alveoli, inhibited expression of E-selectin and intercellular adhesion molecule-1 in cremaster vessels, and improved survival in these mice. Ex vivo studies in sickle cell disease mice suggested that co-administration of hydroxyurea and ARQ 092 efficiently blocks neutrophil and platelet activation and that the beneficial effect of hydroxyurea results from nitric oxide production. Our results provide important evidence that ARQ 092 could be a novel drug for the prevention and treatment of acute vaso-occlusive complications in patients with sickle cell disease.

Hydroxyurea with AKT2 inhibition decreases vaso-occlusive events in sickle cell disease mice.

Heterotypic cell-cell adhesion and aggregation mediate vaso-occlusive events in patients with sickle cell disease (SCD). Although hydroxyurea (HU), an inducer of fetal hemoglobin, is the main therapy for treatment of SCD, it is unclear whether it has immediate benefits in acute vaso-occlusive events in SCD patients. Using real-time fluorescence intravital microscopy, we demonstrated that short-term coadministration of HU and Akti XII, an AKT2 inhibitor, efficiently reduced neutrophil adhesion and platelet-neutrophil aggregation in venules of Berkeley (SCD) mice challenged with tumor necrosis factor α (TNF-α) or hypoxia/reoxygenation. Importantly, compared with HU or Akti XII treatment alone, short-term treatment with both agents significantly improved survival in those mice. We found that the level of plasma nitric oxide species was elevated by HU but not Akti XII, AKT2 phosphorylation levels in activated neutrophils and platelets were reduced by Akti XII but not HU, and the expression of endothelial E-selectin and intercellular adhesion molecule 1 was decreased by either agent. Our results suggest that short-term coadministration of HU and Akti XII has immediate benefits for acute vaso-occlusive events and survival in SCD mice exceeding those seen for single therapy.

Platelet-neutrophil interactions under thromboinflammatory conditions.

Platelets primarily mediate hemostasis and thrombosis, whereas leukocytes are responsible for immune responses. Since platelets interact with leukocytes at the site of vascular injury, thrombosis and vascular inflammation are closely intertwined and occur consecutively. Recent studies using real-time imaging technology demonstrated that platelet-neutrophil interactions on the activated endothelium are an important determinant of microvascular occlusion during thromboinflammatory disease in which inflammation is coupled to thrombosis. Although the major receptors and counter receptors have been identified, it remains poorly understood how heterotypic platelet-neutrophil interactions are regulated under disease conditions. This review discusses our current understanding of the regulatory mechanisms of platelet-neutrophil interactions in thromboinflammatory disease.

Real-time imaging of heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus Formation in live mice.

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation. During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion. Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells. It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions. Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions. Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil ß2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells, those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-α-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.