Mohsen Sabouri-Ghomi

Protein Kinase A Governs a RhoA-RhoGDI Protrusion-Retraction Pacemaker in Migrating Cells

The cyclical protrusion and retraction of the leading edge is a hallmark of many migrating cells involved in processes such as development, inflammation and tumorigenesis. The molecular identity of the signalling mechanisms that control these cycles has remained unknown. Here, we used live-cell imaging of biosensors to monitor spontaneous morphodynamic and signalling activities, and employed correlative image analysis to examine the role of cyclic-AMP-activated protein kinase A (PKA) in protrusion regulation. PKA activity at the leading edge is closely synchronized with rapid protrusion and with the activity of RhoA. Ensuing PKA phosphorylation of RhoA and the resulting increased interaction between RhoA and RhoGDI (Rho GDP-dissociation inhibitor) establish a negative feedback mechanism that controls the cycling of RhoA activity at the leading edge. Thus, cooperation between PKA, RhoA and RhoGDI forms a pacemaker that governs the morphodynamic behaviour of migrating cells.

MicroRNA-17∼92 plays a causative role in lymphomagenesis by coordinating multiple oncogenic pathways

MicroRNAs (miRNAs) have been broadly implicated in cancer, but their exact function and mechanism in carcinogenesis remain poorly understood. Elevated miR‐17∼92 expression is frequently found in human cancers, mainly due to gene amplification and Myc‐mediated transcriptional upregulation. Here we show that B cell‐specific miR‐17∼92 transgenic mice developed lymphomas with high penetrance and that, conversely, Myc‐driven lymphomagenesis stringently requires two intact alleles of miR‐17∼92. We experimentally identified miR‐17∼92 target genes by PAR‐CLIP and validated select target genes in miR‐17∼92 transgenic mice. These analyses demonstrate that miR‐17∼92 drives lymphomagenesis by suppressing the expression of multiple negative regulators of the PI3K and NFκB pathways and by inhibiting the mitochondrial apoptosis pathway. Accordingly, miR‐17∼92‐driven lymphoma cells exhibited constitutive activation of the PI3K and NFκB pathways and chemical inhibition of either pathway reduced tumour size and prolonged the survival of lymphoma‐bearing mice. These findings establish miR‐17∼92 as a powerful cancer driver that coordinates the activation of multiple oncogenic pathways, and demonstrate for the first time that chemical inhibition of miRNA downstream pathways has therapeutic value in treating cancers caused by miRNA dysregulation.

The MicroRNA-183-96-182 Cluster Promotes T Helper 17 Cell Pathogenicity by Negatively Regulating Transcription Factor Foxo1 Expression.

T helper 17 (Th17) cells are key players in autoimmune diseases. However, the roles of non-coding RNAs in Th17 cell development and function are largely unknown. We found that deletion of the endoribonuclease-encoding Dicer1 specifically in Th17 cells protected mice from experimental autoimmune encephalomyelitis. We found that the Dicer1-regulated microRNA (miR)-183-96-182 cluster (miR-183C) was highly expressed in Th17 cells and was induced by cytokine IL-6-STAT3 signaling. miR-183C expression enhanced pathogenic cytokine production from Th17 cells during their development and promoted autoimmunity. Mechanistically, miR-183C in Th17 cells directly repressed expression of the transcription factor Foxo1. Foxo1 negatively regulated the pathogenicity of Th17 cells in part by inhibiting expression of cytokine receptor IL-1R1. These findings indicate that the miR-183C drives Th17 pathogenicity in autoimmune diseases via inhibition of Foxo1 and present promising therapeutic targets.

Modeling of Protrusion Phenotypes Driven by the Actin-Membrane Interaction

We propose a mathematical model for simulating the leading-edge dynamics of a migrating cell from the interplay among elastic properties, architecture of the actin cytoskeleton, and the mechanics of the membrane. Our approach is based on the description of the length and attachment dynamics of actin filaments in the lamellipodium network. It is used to determine the total force exerted on the membrane at each position along the leading edge and at each time step. The model reproduces the marked state switches in protrusion morphodynamics found experimentally between epithelial cells in control conditions and cells expressing constitutively active Rac, a signaling molecule involved in the regulation of lamellipodium network assembly. The model also suggests a mechanistic explanation of experimental distortions in protrusion morphodynamics induced by deregulation of Arp2/3 and cofilin activity.

Protrusion and actin assembly are coupled to the organization of lamellar contractile structures.

Directed cell migration requires continuous cycles of protrusion of the leading edge and contraction to pull up the cell rear. How these spatially distributed processes are coordinated to maintain a state of persistent protrusion remains unknown. During wound healing responses of epithelial sheets, cells along the wound edge display two distinct morphologies: leader cells exhibit persistent edge protrusions, while the greater majority of follower cells randomly cycle between protrusion and retraction. Here, we exploit the heterogeneity in cell morphodynamic behaviors to deduce the requirements in terms of cytoskeleton dynamics for persistent and sporadic protrusion events. We used quantitative Fluorescent Speckle Microscopy (qFSM) to compare rates of F-actin assembly and flow relative to the local protrusion and retraction dynamics of the leading edge. Persistently protruding cells are characterized by contractile actomyosin structures that align with the direction of migration, with converging F-actin flows interpenetrating over a wide band in the lamella. Conversely, non-persistent protruders have their actomyosin structures aligned perpendicular to the axis of migration, and are characterized by prominent F-actin retrograde flows that end into transverse arcs. Analysis of F-actin kinetics in the lamellipodia showed that leader cells have three-fold higher assembly rates when compared to followers. To further investigate a putative relationship between actomyosin contraction and F-actin assembly, myosin II was inhibited by blebbistatin. Treated cells at the wound edge adopted a homogeneously persistent protrusion behavior, with rates matching those of leader cells. Surprisingly, we found that disintegration of actomyosin structures led to a significant decrease in F-actin assembly. Our data suggests that persistent protrusion in these cells is achieved by a reduction in overall F-actin retrograde flow, with lower assembly rates now sufficient to propel forward the leading edge. Based on our data we propose that differences in the protrusion persistence of leaders and followers originate in the distinct actomyosin contraction modules that differentially regulate leading edge protrusion-promoting F-actin assembly, and retraction-promoting retrograde flow.

Visualizing and quantifying adhesive signals

Understanding the structural adaptation and signaling of adhesion sites in response to mechanical stimuli requires in situ characterization of the dynamic activation of a large number of adhesion components. Here, we review high-resolution live cell imaging approaches to measure forces, assembly, and interaction of adhesion components, and the activation of adhesion-mediated signals. We conclude by outlining computational multiplexing as a framework for the integration of these data into comprehensive models of adhesion signaling pathways.

Transfection of microRNA Mimics Should Be Used with Caution

Transient transfection of chemically synthesized microRNA (miRNA) mimics is being used extensively to study the functions and mechanisms of endogenous miRNAs. However, it remains unclear whether transfected miRNAs behave similarly to endogenous miRNAs. Here we show that transient transfection of miRNA mimics into HeLa cells by a commonly used method led to the accumulation of high molecular weight RNA species and a few hundred fold increase in mature miRNA levels. In contrast, expression of the same miRNAs through lentiviral infection or plasmid transfection of HeLa cells, transgenic expression in primary lymphocytes, and endogenous overexpression in lymphoma and leukemia cell lines did not lead to the appearance of high molecular weight RNA species. The increase of mature miRNA levels in these cells was below 10-fold, which was sufficient to suppress target gene expression and to drive lymphoma development in mice. Moreover, transient transfection of miRNA mimics at high concentrations caused non-specific alterations in gene expression, while at low concentrations achieved expression levels comparable to other methods but failed to efficiently suppress target gene expression. Small RNA deep sequencing analysis revealed that the guide strands of miRNA mimics were frequently mutated, while unnatural passenger strands of some miRNA mimics accumulated to high levels. The high molecular weight RNA species were a heterogeneous mixture of several classes of RNA species generated by concatemerization, 5′- and 3′-end tailing of miRNA mimics. We speculate that the supraphysiological levels of mature miRNAs and these artifactual RNA species led to non-specific changes in gene expression. Our results have important implications for the design and interpretation of experiments primarily employing transient transfection of miRNA mimics.

A miR-155–Peli1–c-Rel pathway controls the generation and function of T follicular helper cells

Xiao and collaborators show that miR-155 regulates T follicular helper cell development and function by suppressing the E3 ubiquitin ligase Peli1.

A RhoC Biosensor Reveals Differences in the Activation Kinetics of RhoA and RhoC in Migrating Cells

RhoA and RhoC GTPases share 92% amino acid sequence identity, yet play different roles in regulating cell motility and morphology. To understand these differences, we developed and validated a biosensor of RhoC activation (RhoC FLARE). This was used together with a RhoA biosensor to compare the spatio-temporal dynamics of RhoA and RhoC activity during cell protrusion/retraction and macropinocytosis. Both GTPases were activated similarly at the cell edge, but in regions more distal from the edge RhoC showed higher activation during protrusion. The two isoforms differed markedly in the kinetics of activation. RhoC was activated concomitantly with RhoA at the cell edge, but distally, RhoC activation preceded RhoA activation, occurring before edge protrusion. During macropinocytosis, differences were observed during vesicle closure and in the area surrounding vesicle formation.

Fluid phase biopsy for detection and characterization of circulating endothelial cells in myocardial infarction

Elevated levels of circulating endothelial cells (CECs) occur in response to various pathological conditions including myocardial infarction (MI). Here, we adapted a fluid phase biopsy technology platform that successfully detects circulating tumor cells in the blood of cancer patients (HD-CTC assay), to create a high-definition circulating endothelial cell (HD-CEC) assay for the detection and characterization of CECs. Peripheral blood samples were collected from 79 MI patients, 25 healthy controls and six patients undergoing vascular surgery (VS). CECs were defined by positive staining for DAPI, CD146 and von Willebrand Factor and negative staining for CD45. In addition, CECs exhibited distinct morphological features that enable differentiation from surrounding white blood cells. CECs were found both as individual cells and as aggregates. CEC numbers were higher in MI patients compared with healthy controls. VS patients had lower CEC counts when compared with MI patients but were not different from healthy controls. Both HD-CEC and CellSearch®xa0assays could discriminate MI patients from healthy controls with comparable accuracy but the HD-CEC assay exhibited higher specificity while maintaining high sensitivity. Our HD-CEC assay may be used as a robust diagnostic biomarker in MI patients.