Spatially distinct functions of Clb2 in the DNA damage response.

In budding yeast four mitotic cyclins (Clb1-4) cooperate in a partially redundant manner to bring about M-phase specific events, including the apical isotropic switch that ends polarized bud growth initiated at bud emergence. How exactly this morphogenetic transition is regulated by mitotic CDKs remains poorly understood. We have taken advantage of the isotropic bud growth that prevails in cells responding to DNA damage to unravel the contribution of mitotic cyclins in this cellular context. We find that clb2∆, in contrast to the other mitotic cyclin mutants, inappropriately respond to the presence of DNA damage. This aberrant response is characterized by a Cdc42- and Bni1-dependent but Cln-independent resumption of polarized bud growth after a brief period of actin depolarization. Biochemical and genetic evidence is presented that formally excludes the possibility of indirect effects due for instance to unrestrained APC activity, untimely mitotic exit or Swe1-mediated CDK inhibition. Importantly, our data demonstrate that in order to maintain the characteristic dumbbell arrest phenotype upon checkpoint activation Clb2 needs to be efficiently exported into the cytoplasm. We propose that the inhibition of mitotic cyclin destruction by the DNA damage checkpoint pathway leads to a buildup of Clb2 in the cytoplasm where this cyclin can stabilize the apical isotropic switch throughout a G 2/M checkpoint arrest. Our study also unveils an essential role of nuclear Clb2 in both survival and adaptation to the DNA damage checkpoint, illustrating a spatially distinct dual function of this mitotic cyclin in the response to DNA damage.

Imaging host cell-Leishmania interaction dynamics implicates parasite motility, lysosome recruitment, and host cell wounding in the infection process.

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.

HIV-1 Nef inhibits ruffles, induces filopodia, and modulates migration of infected lymphocytes.

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.

Septin 11 restricts InlB-mediated invasion by Listeria.

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.

Live imaging of emerging hematopoietic stem cells and early thymus colonization.

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 intra-aortic cell clusters, so far considered a hallmark of HSC emergence. CD41GFP(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 c-myb(+) 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.

High-resolution 3-D imaging of living cells in suspension using confocal axial tomography.

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.