Bmcc1s, a Novel Brain-Isoform of Bmcc1, Affects Cell Morphology by Regulating MAP6/STOP Functions

The BCH (BNIP2 and Cdc42GAP Homology) domain-containing protein Bmcc1/Prune2 is highly enriched in the brain and is involved in the regulation of cytoskeleton dynamics and cell survival. However, the molecular mechanisms accounting for these functions are poorly defined. Here, we have identified Bmcc1s, a novel isoform of Bmcc1 predominantly expressed in the mouse brain. In primary cultures of astrocytes and neurons, Bmcc1s localized on intermediate filaments and microtubules and interacted directly with MAP6/STOP, a microtubule-binding protein responsible for microtubule cold stability. Bmcc1s overexpression inhibited MAP6-induced microtubule cold stability by displacing MAP6 away from microtubules. It also resulted in the formation of membrane protrusions for which MAP6 was a necessary cofactor of Bmcc1s. This study identifies Bmcc1s as a new MAP6 interacting protein able to modulate MAP6-induced microtubule cold stability. Moreover, it illustrates a novel mechanism by which Bmcc1 regulates cell morphology

The macrophage in HIV-1 infection: From activation to deactivation?

Macrophages play a crucial role in innate and adaptative immunity in response to microorganisms and are an important cellular target during HIV-1 infection. Recently, the heterogeneity of the macrophage population has been highlighted. Classically activated or type 1 macrophages (M1) induced in particular by IFN-γ display a pro-inflammatory profile. The alternatively activated or type 2 macrophages (M2) induced by Th-2 cytokines, such as IL-4 and IL-13 express anti-inflammatory and tissue repair properties. Finally IL-10 has been described as the prototypic cytokine involved in the deactivation of macrophages (dM). Since the capacity of macrophages to support productive HIV-1 infection is known to be modulated by cytokines, this review shows how modulation of macrophage activation by cytokines impacts the capacity to support productive HIV-1 infection. Based on the activation status of macrophages we propose a model starting with M1 classically activated macrophages with accelerated formation of viral reservoirs in a context of Th1 and proinflammatory cytokines. Then IL-4/IL-13 alternatively activated M2 macrophages will enter into the game that will stop the expansion of the HIV-1 reservoir. Finally IL-10 deactivation of macrophages will lead to immune failure observed at the very late stages of the HIV-1 disease

Listeriolysin O promotes the intravacuolar growth of Listeria monocytogenes in epithelial cells

Upon entry into host cells, Listeria monocytogenes (Lm) was described to escape rapidly from internalisation vacuoles and proliferate only after gaining access to the cytosol. Vacuole escape depends upon three secreted virulence factors: the pore-forming toxin listeriolysin O (LLO) and two phospholipases. To quantify the dynamics of vacuolar escape, we used FAST fluorescent tags to monitor bacterial secretion into enclosed compartments. By tracking fluorescently-labelled vacuoles, we quantified the heterogeneity of 20 Lm residence time in primary vacuoles formed in epithelial LoVo cells. Although half of the bacterial population escaped from vacuoles within 13 minutes after internalisation, a fraction of it remained entrapped several hours in Long Residence Vacuoles (LRV), for both wild type and LLO-deficient strains. Unexpectedly, Lm replicated inside LRVs at a rate similar to that in the cytosol. LRVs were decorated with LLO-FAST and LLO was necessary for bacterial proliferation in these compartments, suggesting that 25 permeation of vacuolar membranes sustained growth. LRVs displayed similarities with the spacious Listeria-containing phagosomes described in macrophages, and could constitute an alternative replication niche for Lm in epithelial cells

Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of <i>Listeria monocytogenes</i> in epithelial cells

AbstractReal-time imaging of bacterial virulence factor dynamics is hampered by the limited number of fluorescent tools suitable for tagging secreted effectors. Here, we demonstrated that the fluorogenic reporter FAST could be used to tag secreted proteins, and we implemented it to monitor infection dynamics in epithelial cells exposed to the human pathogen Listeria monocytogenes (Lm). By tracking individual FAST-labelled vacuoles after Lm internalisation into cells, we unveiled the heterogeneity of residence time inside entry vacuoles. Although half of the bacterial population escaped within 13 minutes after entry, 12% of bacteria remained entrapped over an hour inside long term vacuoles, and sometimes much longer, regardless of the secretion of the pore-forming toxin listeriolysin O (LLO). We imaged LLO-FAST in these long-term vacuoles, and showed that LLO enabled Lm to proliferate inside these compartments, reminiscent of what had been previously observed for Spacious Listeria-containing phagosomes (SLAPs). Unexpectedly, inside epithelial SLAP-like vacuoles (eSLAPs), Lm proliferated as fast as in the host cytosol. eSLAPs thus constitute an alternative replication niche in epithelial cells that might promote the colonization of host tissues.Author summaryBacterial pathogens secrete virulence factors to subvert their hosts; however, monitoring bacterial secretion in real-time remains challenging. Here, we developed a convenient method that enabled fluorescent imaging of secreted proteins in live microscopy, and applied it to the human pathogen Listeria monocytogenes. Listeria has been described to invade cells and proliferate in their cytosol; it is first internalized inside vacuoles, from where it escapes thanks to the secretion of virulence factors that disrupt membranes. Our work revealed the existence, in human epithelial cells, of a population of Listeria that failed to escape vacuoles but instead multiplied efficiently therein, despite -and in fact, thanks to— the active secretion of a toxin that permeates membranes. This intravacuolar niche may provide Listeria with an alternative strategy to colonize its host.</jats:sec

Phrenic nerve stimulation in an ovine model with temporary removable pacing leads

International audienceBackground: The objective of this study was to assess the feasibility and safety of a novel, removable, surgically implanted, temporary neurostimulation approach involving the distal portion of the phrenic nerve.Methods: Temporary phrenic nerve pacing electrodes were implanted surgically using an ovine model (4 animals). The primary endpoint was the ability to successfully match the animal's minute-ventilation upon implantation of both phrenic nerve pacers on day 1. Secondary endpoints were successful phrenic neurostimulation by both electrodes 15 and 30 days after initial implantation. We also assessed safe removal of the electrodes at 15 and 30 days after implementation.Results: In 3 of 4 animals, electrodes were successfully implanted in both right and left phrenic nerves. On day 1, median ventilation-minute induced by neurostimulation was not significantly different from baseline ventilation-minute [4.9 L·min-1 (4.4-5.5) vs. 4.4 L·min-1 (4.3-5.2); P=0.4] after 15 minutes. Neurostimulation was still possible 15 and 30 days after implementation in all left side phrenic nerves. On the right side, stimulation was possible at all times in 1 animal but not in the remaining 3 animals for at least one time point, possibly due to lead displacement. Analysis of pathology after percutaneous electrode removal showed integrity of the distal portion of all phrenic nerves.Conclusions: Efficient temporary neurostimulation through the distal portion of the phrenic nerve was possible at baseline. The main complication was the displacement of electrodes on the right phrenic nerve on two occasions, which was due to the anatomy of the ovine model. It compromised diaphragm pacing on day 15 and day 30. The electrodes could be safely removed percutaneously without damage to the phrenic nerves

Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of Listeria monocytogenes in epithelial cells

International audienceReal-time imaging of bacterial virulence factor dynamics is hampered by the limited number of fluorescent tools suitable for tagging secreted effectors. Here, we demonstrated that the fluorogenic reporter FAST could be used to tag secreted proteins, and we implemented it to monitor infection dynamics in epithelial cells exposed to the human pathogen Listeria monocytogenes (Lm). By tracking individual FAST-labelled vacuoles after Lm internalisation into cells, we unveiled the heterogeneity of residence time inside entry vacuoles. Although half of the bacterial population escaped within 13 minutes after entry, 12% of bacteria remained entrapped over an hour inside long term vacuoles, and sometimes much longer, regardless of the secretion of the pore-forming toxin listeriolysin O (LLO). We imaged LLO-FAST in these long-term vacuoles, and showed that LLO enabled Lm to proliferate inside these compartments, reminiscent of what had been previously observed for Spacious Listeria-containing phagosomes (SLAPs). Unexpectedly, inside epithelial SLAP-like vacuoles (eSLAPs), Lm proliferated as fast as in the host cytosol. eSLAPs thus constitute an alternative replication niche in epithelial cells that might promote the colonization of host tissues

Bmcc1s overexpression displaces MAP6 away from the microtubules and induces the formation of membrane protrusions.

<p>(A) HeLa cells stably transfected with GFP-N-STOP (GFP-N-STOP HeLa) transiently transfected with an expression plasmid for Bmcc1s-V5. Twenty-four hours after transfection, cells were fixed and double-stained for N-STOP using the 23N polyclonal MAP6 antibody (green) and for Bmcc1s-V5 using a monoclonal anti V5 antibody (red). In untransfected GFP-N-STOP HeLa, N-STOP staining showed a microtubule-like pattern. The Golgi apparatus was also labeled (asterisks). Insert (a) is an enlargement of the squared region showing N-STOP staining in more detail. In the Bmcc1s-V5 GFP-N-STOP HeLa transfected cell (white arrows), N-STOP labeling became brighter, no longer featuring its typical microtubule-type distribution, and numerous membrane protrusions (white arrowheads) labeled for both V5 and N-STOP were seen. Insert (b) is an enlargement of the Bmcc1s-V5 GFP-N-STOP HeLa transfected cell. (B) Confocal microscopy images of HeLa cells transiently transfected with an expression plasmid for Bmcc1s-V5. Twenty-four hours after transfection, cells were fixed and double-stained for Bmcc1s-V5 using a monoclonal anti V5 antibody (green) and for F-actin using TRITC-conjugated phalloidin (red). (C) Confocal microscopy images of a Bmcc1s-V5 stably transfected HeLa cell (Bmcc1s-V5 HeLa) transiently transfected with an expression plasmid for GFP-N-STOP. Twenty-four hours after transfection, cells were fixed and stained for N-STOP using the 23N polyclonal anti-MAP6 antibody (green), for microtubules using a α-tubulin antibody (blue), and for F-actin using TRITC-conjugated phalloidin (red). Merge images show that N-STOP partially loses its microtubular staining, being more diffuse in the cell, and located in actin-rich membrane protrusions (white arrowheads). Bars: 10 µm.</p