We recently demonstrated in zebrafish the developmental migration of emerging hematopoietic stem cells (HSCs) that is thought to occur in mammalian embryos, from the aorta-gonad-mesonephros (AGM) area to the successive hematopoietic organs. CD41 is the earliest known molecular marker of nascent HSCs in mammalian development. In this study, we show that in CD41-green fluorescent protein (GFP) transgenic zebrafish embryos, the transgene is expressed by emerging HSCs in the AGM, allowing us for the first time to image their behavior and trace them in real time. We find that the zebrafish AGM contains no intraaortic cell clusters, so far considered a hallmark of HSC emergence. CD41-GFP low HSCs emerge in the subaortic mesenchyme and enter the circulation not through the dorsal aorta but through the axial vein, the peculiar structure of which facilitates their intravasation. The rise in CD41-gfp expression among cmyb ؉ HSC precursors is asynchronous and marks their competence to leave the AGM and immediately seed the caudal hematopoietic tissue (which has a hematopoietic function analogous to that of the mammalian fetal liver). Imaging the later migration of CD41-GFP ؉ precursors to the nascent thymus reveals that although some reach the thymus by extravasating from the nearest vein, most travel for hours through the mesenchyme from surprisingly diverse and remote sites of extravasation. (Blood. 2008;111: 1147-1156)

Leishmania donovani causes human visceral leishmaniasis. The parasite infectious cycle comprises extracellular flagellated promastigotes that proliferate inside the insect vector, and intracellular nonmotile amastigotes that multiply within infected host cells. Using primary macrophages infected with virulent metacyclic promastigotes and high spatiotemporal resolution microscopy, we dissect the dynamics of the early infection process. We find that motile promastigotes enter macrophages in a polarized manner through their flagellar tip and are engulfed into host lysosomal compartments. Persistent intracellular flagellar activity leads to reorientation of the parasite flagellum toward the host cell periphery and results in oscillatory parasite movement. The latter is associated with local lysosomal exocytosis and host cell plasma membrane wounding. These findings implicate lysosome recruitment followed by lysosome exocytosis, consistent with parasite-driven host cell injury, as key cellular events in Leishmania host cell infection. This work highlights the role of promastigote polarity and motility during parasite entry.

The HIV-1 Nef protein is a pathogenic factor modulating the behavior of infected cells. Nef induces actin cytoskeleton changes and impairs cell migration toward chemokines. We further characterized the morphology, cytoskeleton dynamics, and motility of HIV-1-infected lymphocytes. By using scanning electron microscopy, confocal immunofluorescence microscopy, and ImageStream technology, which combines flow cytometry and automated imaging, we report that HIV-1 induces a characteristic remodeling of the actin cytoskeleton. In infected lymphocytes, ruffle formation is inhibited, whereas long, thin filopodium-like protrusions are induced. Cells infected with HIV with nef deleted display a normal phenotype, and Nef expression alone, in the absence of other viral proteins, induces morphological changes. We also used an innovative imaging system to immobilize and visualize living individual cells in suspension. When combined with confocal "axial tomography," this technique greatly enhances three-dimensional optical resolution. With this technique, we confirmed the induction of long filopodium-like structures in unfixed Nef-expressing lymphocytes. The cytoskeleton reorganization induced by Nef is associated with an important impairment of cell movements. The adhesion and spreading of infected cells to fibronectin, their spontaneous motility, and their migration toward chemokines (CXCL12, CCL3, and CCL19) were all significantly decreased. Therefore, Nef induces complex effects on the lymphocyte actin cytoskeleton and cellular morphology, which likely impacts the capacity of infected cells to circulate and to encounter and communicate with bystander cells.

Septins are filament-forming GTPases implicated in several cellular functions, including cytokinesis. We previously showed that SEPT2, SEPT9, and SEPT11 colocalize with several bacteria entering into mammalian non-phagocytic cells, and SEPT2 was identified as essential for this process. Here, we investigated the function of SEPT11, an interacting partner of SEPT9 whose function is still poorly understood. In uninfected HeLa cells, SEPT11 depletion by siRNA increased cell size but surprisingly did not affect actin filament formation or the colocalization of SEPT9 with actin filaments. SEPT11 depletion increased Listeria invasion, and incubating SEPT11-depleted cells with beads coated with the Listeria surface protein InlB also led to increased entry as compared with control cells. Strikingly, as shown by fluorescence resonance energy transfer, the InlB-mediated stimulation of Met signaling remained intact in SEPT11-depleted cells. Taken together, our results show that SEPT11 is not required for the bacterial entry process and rather restricts its efficacy. Because SEPT2 is essential for the InlB-mediated entry of Listeria, but SEPT11 is not, our findings distinguish the roles of different mammalian septins.Septins were discovered in the budding yeast Saccharomyces cerevisiae (1) where they organize into a ring at the mother-bud neck during cell division (2). Septins are GTPases of 30 -65 kDa found in most eukaryotes, except plants, sharing an essential role in cytokinesis (3, 4). Fourteen septins have been identified in humans and classified on the basis of sequence identity into four distinct groups (3, 5). Septins from different groups polymerize into hetero-oligomeric protein complexes and filaments and may associate with cellular membranes, actin filaments, and microtubules (6, 7). Septins are increasingly regarded as novel cytoskeletal elements (8), but their role in post-mitotic events remains poorly understood.The crystal structure of the SEPT2-SEPT6-SEPT7 complex recently highlighted that septins, as opposed to actin and microtubules, form non-polar filaments (9). In the SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7 complex, SEPT2 has a central role in filament formation (9), whereas SEPT6 is thought to be replaceable with other SEPT6 group members, including SEPT11 (3). Widely expressed in mammalian tissues (10), SEPT11 may also be a substitute for SEPT6 in other mammalian septin complexes such as SEPT7-SEPT9-SEPT11 (10) or SEPT5-SEPT7-SEPT11 (11). Because other septins homologous to SEPT11 might compensate for its deficiency (12), the degree to which SEPT11 is required for septin filament structure and function is not yet known. Listeria monocytogenes is an invasive bacterium that enters into most mammalian cells in vitro through the interaction of the bacterial surface protein InlB with its host cellular receptor Met, the hepatocyte growth factor receptor (13). We originally identified SEPT9 associated with phagosomes containing latex beads coated with InlB (14). Given the association of septins with the cytoske...

Conventional flow cytometry (FC) methods report optical signals integrated from individual cells at throughput rates as high as thousands of cells per second. This is further combined with the powerful utility to subsequently sort and/or recover the cells of interest. However, these methods cannot extract spatial information. This limitation has prompted efforts by some commercial manufacturers to produce state-of-the-art commercial flow cytometry systems allowing fluorescence images to be recorded by an imaging detector. Nonetheless, there remains an immediate and growing need for technologies facilitating spatial analysis of fluorescent signals from cells maintained in flow suspension. Here, we report a novel methodological approach to this problem that combines micro-fluidic flow, and microelectrode dielectric-field control to manipulate, immobilize and image individual cells in suspension. The method also offers unique possibilities for imaging studies on cells in suspension. In particular, we report the system's immediate utility for confocal "axial tomography" using micro-rotation imaging and show that it greatly enhances 3-D optical resolution compared with conventional light reconstruction (deconvolution) image data treatment. That the method we present here is relatively rapid and lends itself to full automation suggests its eventual utility for 3-D imaging cytometry.