Shape-Based Tracking Allows Functional Discrimination of Two Immune Cell Subsets Expressing the Same Fluorescent Tag in Mouse Lung Explant

Dendritic Cells (DC) represent a key lung immune cell population, which play a critical role in the antigen presenting process and initiation of the adaptive immune response. The study of DCs has largely benefited from the joint development of fluorescence microscopy and knock-in technology, leading to several mouse strains with constitutively labeled DC subsets. However, in the lung most transgenic mice do express fluorescent protein not only in DCs, but also in closely related cell lineages such as monocytes and macrophages. As an example, in the lungs of CX3CR1+/gfp mice the green fluorescent protein is expressed mostly by both CD11b conventional DCs and resident monocytes. Despite this non-specific staining, we show that a shape criterion can discriminate these two particular subsets. Implemented in a cell tracking code, this quantified criterion allows us to analyze the specific behavior of DCs under inflammatory conditions mediated by lipopolysaccharide on lung explants. Compared to monocytes, we show that DCs move slower and are more confined, while both populations do not have any chemotactism-associated movement. We could generalize from these results that DCs can be automatically discriminated from other round-shaped cells expressing the same fluorescent protein in various lung inflammation models

Transmission of West Nile Virus by Culex quinquefasciatus Say Infected with Culex Flavivirus Izabal

Unlike most known flaviviruses (Family, Flaviviridae: Genus, Flavivirus), insect-only flaviviruses are a unique group of flaviviruses that only infect invertebrates. The study of insect-only flaviviruses has increased in recent years due to the discovery and characterization of numerous novel flaviviruses from a diversity of mosquito species around the world. The widespread discovery of these viruses has prompted questions regarding flavivirus evolution and the potential impact of these viruses on the transmission of flaviviruses of public health importance such as WNV. Therefore, we tested the effect of Culex flavivirus Izabal (CxFV Izabal), an insect-only flavivirus isolated from Culex quinquefasciatus mosquitoes in Guatemala, on the growth and transmission of a strain of WNV isolated concurrently from the same mosquito species and location. Prior infection of C6/36 (Aedes albopictus mosquito) cells or Cx. quinquefasciatus with CxFV Izabal did not alter the replication kinetics of WNV, nor did it significantly affect WNV infection, dissemination, or transmission rates in two different colonies of mosquitoes that were fed blood meals containing varying concentrations of WNV. These data demonstrate that CxFV probably does not have a significant effect on WNV transmission efficiency in nature

Podoplanin mediates ECM degradation by squamous carcinoma cells through control of invadopodia stability

This work is licensed under a Creative Commons Attribution 4.0 International License.Invadopodia are actin-rich cell membrane projections used by invasive cells to penetrate the basement membrane. Control of invadopodia stability is critical for efficient degradation of the extracellular matrix (ECM); however, the underlying molecular mechanisms remain poorly understood. Here, we uncover a new role for podoplanin, a transmembrane glycoprotein closely associated with malignant progression of squamous cell carcinomas (SCCs), in the regulation of invadopodia-mediated matrix degradation. Podoplanin downregulation in SCC cells impairs invadopodia stability, thereby reducing the efficiency of ECM degradation. We report podoplanin as a novel component of invadopodia-associated adhesion rings, where it clusters prior to matrix degradation. Early podoplanin recruitment to invadopodia is dependent on lipid rafts, whereas ezrin/moesin proteins mediate podoplanin ring assembly. Finally, we demonstrate that podoplanin regulates invadopodia maturation by acting upstream of the ROCK-LIMK-Cofilin pathway through the control of RhoC GTPase activity. Thus, podoplanin has a key role in the regulation of invadopodia function in SCC cells, controlling the initial steps of cancer cell invasion.This work has been funded by grants from Cancer Research UK (C7125/A9926; A13651) to GEJ and EM-V, the Medical Research Council (G0401026) to GEJ and from the Spanish Ministry of Economy and Competitiveness (SAF 2010-19152/SAF2013-46183R) and Community of Madrid (S2010/BMD-2359, SkinModel-CM) to MQ. EM-V is currently a recipient of a Postdoctoral Research Contract from Fundación Científica Asociación Española Contra el Cáncer (AECC).Peer reviewe

Lamellipodin promotes invasive 3D cancer cell migration via regulated interactions with Ena/VASP and SCAR/WAVE

Cancer invasion is a hallmark of metastasis. The mesenchymal mode of cancer cell invasion is mediated by elongated membrane protrusions driven by the assembly of branched F-actin networks. How deregulation of actin regulators promotes cancer cell invasion is still enigmatic. We report that increased expression and membrane localization of the actin regulator Lamellipodin correlate with reduced metastasis-free survival and poor prognosis in breast cancer patients. In agreement, we find that Lamellipodin depletion reduced lung metastasis in an orthotopic mouse breast cancer model. Invasive 3D cancer cell migration as well as invadopodia formation and matrix degradation was impaired upon Lamellipodin depletion. Mechanistically, we show that Lamellipodin promotes invasive 3D cancer cell migration via both actin-elongating Ena/VASP proteins and the Scar/WAVE complex, which stimulates actin branching. In contrast, Lamellipodin interaction with Scar/WAVE but not with Ena/VASP is required for random 2D cell migration. We identified a phosphorylation-dependent mechanism that regulates selective recruitment of these effectors to Lamellipodin: Abl-mediated Lamellipodin phosphorylation promotes its association with both Scar/WAVE and Ena/VASP, whereas Src-dependent phosphorylation enhances binding to Scar/WAVE but not to Ena/VASP. Through these selective, regulated interactions Lamellipodin mediates directional sensing of epidermal growth factor (EGF) gradients and invasive 3D migration of breast cancer cells. Our findings imply that increased Lamellipodin levels enhance Ena/VASP and Scar/WAVE activities at the plasma membrane to promote 3D invasion and metastasis.Virginia and D.K. Ludwig Fund for Cancer Research (Postdoctoral fellowship)King's College London (Overseas Research PhD Studentship (KORS))National Cancer Institute (U.S.) (U54-CA112967)National Cancer Institute (U.S.) (U54-CA163109)Ludwig Center for Molecular Oncology at MITDavid H. Koch Institute for Integrative Cancer Research at MIT (Support Grant P30-CA14051)National Cancer Institute (U.S.) (Koch Institute Support Grant P30-CA14051)Biotechnology and Biological Sciences Research Council (Great Britain) (BB/F011431/1)Biotechnology and Biological Sciences Research Council (Great Britain) (BB/J000590/1)Biotechnology and Biological Sciences Research Council (Great Britain) (BB/N000226/1)Wellcome Trust (London, England) (082907/Z/07/Z