The Ins and Outs of CCR7 in the Thymus

Although it is widely supposed that chemokines play a role in the thymus, most existing evidence is circumstantial. In this issue, two groups provide direct evidence that the chemokine receptor CCR7 is required for normal thymocyte migration (Ueno, T., F. Saito, D. Gray, S. Kuse, K. Hieshima, H. Nakano, T. Kakiuchi, M. Lipp, R. Boyd, and Y. Takahama. 2004. J. Exp. Med. 200:493–505; Misslitz, A., O. Pabst, G. Hintzen, L. Ohl, E. Kremmer, H. T. Petrie, and R. Forster. 2004. J. Exp. Med. 200:481–491). The two papers focus on distinct and opposite migration events, an early outward migration and a later inward migration. Together these papers provide a fascinating picture of the complex role of CCR7 in orchestrating thymocyte migration

The Ubiquitin/Proteasome System Mediates Entry and Endosomal Trafficking of Kaposi's Sarcoma-Associated Herpesvirus in Endothelial Cells

Ubiquitination, a post-translational modification, mediates diverse cellular functions including endocytic transport of molecules. Kaposi's sarcoma-associated herpesvirus (KSHV), an enveloped herpesvirus, enters endothelial cells primarily through clathrin-mediated endocytosis. Whether ubiquitination and proteasome activity regulates KSHV entry and endocytosis remains unknown. We showed that inhibition of proteasome activity reduced KSHV entry into endothelial cells and intracellular trafficking to nuclei, thus preventing KSHV infection of the cells. Three-dimensional (3-D) analyses revealed accumulation of KSHV particles in a cytoplasmic compartment identified as EEA1+ endosomal vesicles upon proteasome inhibition. KSHV particles are colocalized with ubiquitin-binding proteins epsin and eps15. Furthermore, ubiquitination mediates internalization of both KSHV and one of its receptors integrin β1. KSHV particles are colocalized with activated forms of the E3 ligase c-Cbl. Knock-down of c-Cbl or inhibition of its phosphorylation reduced viral entry and intracellular trafficking, resulting in decreased KSHV infectivity. These results demonstrate that ubiquitination mediates internalization of both KSHV and one of its cognate receptors integrin β1, and identify c-Cbl as a potential E3 ligase that facilitates this process

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

Directed migration of positively selected thymocytes visualized in real time.

Development of many vertebrate tissues involves long-range cell migrations. In most cases, these migrations have been inferred from analysis of single time points and the migration process has not been directly observed and quantitated in real time. In the mammalian adult thymus, immature CD4+ CD8+ double-positive (DP) thymocytes are found in the outer cortex, whereas after T cell antigen receptor (TCR) repertoire selection, CD4+ CD8- and CD4- CD8+ single-positive (SP) thymocytes are found in the central medulla. Here we have used two-photon laser-scanning microscopy and quantitative analysis of four-dimensional cell migration data to investigate the movement of thymocytes through the cortex in real time within intact thymic lobes. We show that prior to positive selection, cortical thymocytes exhibit random walk migration. In contrast, positive selection is correlated with the appearance of a thymocyte population displaying rapid, directed migration toward the medulla. These studies provide our first glimpse into the dynamics of developmentally programmed, long-range cell migration in the mammalian thymus

MR^hi Thymocytes Show Preferential Movement Perpendicular to the Thymic Capsule

<div><p>(A) Bar graph showing the average displacement in each direction by wild-type MR^hi cells in a 3-min interval. Data shown were computed from 53 MR^hi cells from four independently imaged thymic lobes. Data from individual runs are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030160#sg003" target="_blank">Figure S3</a>.</p> <p>(B) The upper image is rotated to display the <i>x</i> and <i>z</i> dimensions showing tracks of MR^hi cells. Five of six MR^hi tracks show preferential orientation in the <i>z</i> direction. The lower image shows tracks of MR^lo cells from same dataset.</p> <p>(C) The results of step analysis (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030160#s3" target="_blank">Materials and Methods</a>) on 172 MR^hi cells. Thymocytes are grouped according to their average motility rate (displayed on <i>x</i>-axis) and percentage of cells moving in either the positive or negative direction is displayed on the <i>y</i>-axis. Data are compiled from four independently imaged thymic lobes.</p></div

Directed Migration of Positively Selected Thymocytes Visualized in Real Time

<div><p>Development of many vertebrate tissues involves long-range cell migrations. In most cases, these migrations have been inferred from analysis of single time points and the migration process has not been directly observed and quantitated in real time. In the mammalian adult thymus, immature CD4⁺CD8⁺ double-positive (DP) thymocytes are found in the outer cortex, whereas after T cell antigen receptor (TCR) repertoire selection, CD4⁺CD8^– and CD4^–CD8⁺ single-positive (SP) thymocytes are found in the central medulla. Here we have used two-photon laser-scanning microscopy and quantitative analysis of four-dimensional cell migration data to investigate the movement of thymocytes through the cortex in real time within intact thymic lobes. We show that prior to positive selection, cortical thymocytes exhibit random walk migration. In contrast, positive selection is correlated with the appearance of a thymocyte population displaying rapid, directed migration toward the medulla. These studies provide our first glimpse into the dynamics of developmentally programmed, long-range cell migration in the mammalian thymus.</p> </div

Two Distinct Migratory Behaviors within Wild-Type Cortical Thymocytes

<div><p>(A) Histogram showing the frequency distribution of average motility rates (MR) for cortical thymocytes compiled from over 1,250 tracked cells from four independently imaged thymic lobes. The vast majority of cells exhibited speeds ranging from 3 to 8 μm/min (MR^lo). Approximately 7% exhibited speeds of 10 μm/min or greater (MR^hi). Cells migrating between 10–13 μm/min represented approximately 5% of cortical thymocytes, and those with speeds of 14 μm/min or greater represented approximately 2% of cortical thymocytes.</p> <p>(B) Instantaneous velocities versus time for representative MR^hi and MR^lo cells. Data are representative of 53 MR^hi cells and more than 200 MR^lo cells analyzed. No conversions between MR^hi or MR^lo behaviors were observed over a combined imaging time of more than 30 h.</p> <p>(C) Five successive time frames showing the morphology associated with propulsion for an MR^hi and an MR^lo cell (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030160#sv005" target="_blank">Video S5</a>).</p> <p>(D) Graph of displacement versus time for four individual MR^hi and MR^lo cells.</p> <p>(E) Graph of directional index (Traj/D) versus average motility rate. The bars indicate the average values for Traj/D computed from 50 MR^hi and 466 MR^lo cells<b>.</b></p> <p>(F) Graph of MR^lo cells (left), but not MR^hi cells (middle), shows linear relationship between the square of the displacement from origin versus time, indicative of random walk. Right graph shows a linear relationship between displacements from origin (as opposed to their square) with increasing time for MR^hi cells, indicative of ballistic motion (right). Analysis was done on 466 MR^lo cells and 50 MR^hi cells from three independently imaged thymic lobes.</p></div

Positive Selection Leads to an Increased Frequency of MR^hi Thymocytes Migrating away from the Thymic Capsule

<div><p>(A) A histogram showing the frequency distribution of average motility rates for positively selecting (blue, P14) and nonselecting (black, 5CC7) transgenic thymocytes compiled from over 1,200 P14 and 875 5CC7 thymocytes from, respectively, four and three independently imaged thymic lobes. Data (from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030160#pbio-0030160-g002" target="_blank">Figure 2</a>A) from wild-type (WT) thymocytes (red) were overlaid for comparison. P14 cells moving at motility rates greater than 13 μm/min were 34% of total imaged thymocytes (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030160#sv006" target="_blank">Videos S6</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030160#sv007" target="_blank">S7</a>) compared to approximately 1% of wild-type cortical thymocytes. Analysis of 5CC7 thymocytes showed nearly complete absence of cells moving at motility rates greater than 13 μm/min, a value that differed significantly (<i>p</i> = 0.002) from wild-type cortical thymocytes. (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030160#sv008" target="_blank">Video S8</a>)</p> <p>(B) Image showing trajectories of representative P14 MR^hi cells. Note tracks for P14 thymocytes are relatively linear compared to the tracks of wild-type thymocytes (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030160#pbio-0030160-g001" target="_blank">Figure 1</a>).</p> <p>(C) Bar graph showing the average displacement per cell moved in each direction over a 3-min time interval (left). Data was computed from more than 100 P14 MR^hi cortical thymocytes compiled from four independent experiments. Data from individual runs are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030160#sg005" target="_blank">Figure S5</a>.</p> <p>(D) Results of step analysis on 412 P14 thymocytes as a function of motility rate are shown (left). Results of step analysis on 123 5CC7 thymocytes are shown for comparison (right). P14 cells moving at MR greater than 13 μm/min showed strong bias for movement in the –<i>z</i> direction (away from capsule) whereas 5CC7 thymocytes showed random use of both +<i>z</i> and –<i>z</i> directions.</p></div