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

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 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

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

Subcellular localization of Bmcc1s in primary neurons.

<p>(A–C) Confocal section images of primary neurons after 7 days of culture immunostained for endogenous Bmcc1s (green) and α-tubulin or neurofilament subunit M (NF-M) (red). Merge images showed that Bmcc1s colocalizes with α-tubulin (A) and NF-M (C) immunoreactivity signal. Boxed regions in A and C indicate the fields enlarged in each image. B. In nocodazole-treated primary neurons (10 µM, 1 h), Bmcc1s followed the disrupted α-tubulin microtubular staining. (D) Immunogold labeling and electron microscopy analysis of primary neurons showed that Bmcc1s localized on cytoskeleton-type structures compatible with microtubules (left) and intermediate filaments (right). Bars: 10 µm (A–C); 100 nm (D).</p

Immunodetection of Bmcc1s.

<p>(A) Immunoblot of Bmcc1s in lysates of HeLa cells transfected with a plasmid expressing Bmcc1s-V5. Similar profiles were obtained using the Bmcc1 antiserum or anti-V5 antibodies. Note that the Bmcc1 antiserum recognized an endogenous protein around 50 kDa (arrow) of the same size as Bmcc1s in untransfected HeLa cells. Immunostaining of HeLa cells transfected with a plasmid expressing Bmcc1s-V5, using either the Bmcc1 antiserum or anti-V5 antibodies. The antiserum detected only the V5 positive cells, and both signals overlapped. Scale bar: 100 µm (B) Immunoblot of endogenous Bmcc1 isoforms in mouse tissue lysates using Bmcc1 antiserum. GAPDH expression is shown as a loading reference. As in HeLa cells expressing Bmcc1s-V5, the Bmcc1 antiserum detected a band around 50 kDa (arrow) in the brain lysate that appeared specific to this tissue and was the most abundant among the Bmcc1 isoforms. (C) Immunoblot of endogenous Bmcc1 in primary cultures of astrocyte and neuron lysates at DIV7, using Bmcc1 antiserum. As found in brain tissues, a major band around 50 kDa was detected (arrow).</p

Morphological changes induced by Bmcc1s overexpression requires MAP6.

<p>Confocal microscopy image projections of cells transfected with a Bmcc1s-V5 or GFP expressing plasmid, and stained for V5 (green) and F-actin (detected with TRITC-conjugated phalloidin in red). Cells were fixed 24 h after transfection. (A) primary astrocytes; (B) primary neurons. The morphology of GFP-expressing cells (green) was unchanged compared to untransfected cells. In contrast, Bmcc1s-V5-expressing astrocytes and neurons developed numerous membrane protrusions (white arrowheads). Images in B illustrate representative confocal projections of the effect of Bmcc1s-V5 on neuritic growth and number in wild-type neurons. The whole Bmcc1s-V5 transfected neuron is shown in the insert. Histograms present means ± sd of the length of the longest neurite and of the number of neurites. *** p-value<0.0001 ** p-value<0.001. ns, not significant for 3 independent experiments using the two sample independent t-test. In neurons, length of the longest neurite, and number of neurites (or cell extensions starting from the soma) were significantly increased by Bmcc1s-V5 transfection, but not in MAP6-deleted neurons. Bars: 10 µm.</p

Bmcc1s interacts with MAP6.

<p>(A) GST-Bmcc1s or GST immobilized on glutathione sepharose beads were incubated with either a lysis buffer or a mouse brain lysate. After elution, bound proteins were resolved on SDS-PAGE in parallel with the mouse brain lysate, and visualized by Coomassie staining. A unique band (square) was analyzed by MALDI-TOF, where MAP6 was identified. (B) The presence of MAP6 and the specificity of its interaction with Bmcc1s were confirmed by Western blot of the GST eluates with 23N, a polyclonal anti-MAP6 antibody. Several bands corresponding to the neuronal MAP6 isoforms N-STOP (120 kDa) and E-STOP (80 kDa), the astrocyte MAP6 isoform A-STOP (60 KDa) and a 48 kDa isoform described in total brain protein extracts were revealed. (C) MALDI-TOF analysis revealed the presence of 4 peptides (in red) corresponding to MAP6. The microtubule-stabilizing modules Mn1, Mn2 and Mc1 of MAP6 are underlined. (D) Co-immunoprecipitation of MAP6 and Bmcc1s was performed using the 175 monoclonal anti-MAP6 antibody (IP+αMAP6), or no antibody (IP-αMAP6) as control, on mouse brain lysates. Precipitates were analyzed by Western blotting with Bmcc1 antiserum, in parallel with the mouse brain lysate. Bmcc1s was co-immunoprecipitated with MAP6. (E) Pull-down experiments of purified MAP6 isoforms: neuronal, N- and E-STOP and the fibroblast F-STOP, by purified glutathione-S-transferase (GST)-Bmcc1s or GST. Bound proteins were resolved on SDS-PAGE and Coomassie stained. N- and E-STOP were specifically retained by GST-Bmcc1s.</p

Bmcc1s inhibits the MAP6-induced microtubule cold stability.

<p>(<b>A</b>) Inhibition of N-STOP-induced microtubule cold stability by Bmcc1s <i>in vitro</i>. Microtubules polymerized at 37°C and subjected to cold were recovered by sedimentation and analyzed by SDS-PAGE and coomassie staining. The observed 50 kDa band corresponds to polymerized tubulin. At 4°C, almost no microtubules could be recovered. In contrast, they were preserved at 4°C in presence of N-STOP or F-STOP. Adding increasing concentrations of GST-Bmcc1s progressively decreased the level of microtubules in presence of N-STOP, but not of F-STOP. In contrast, GST alone had no effect. Numbers indicate the final concentration of the proteins in micromolar in the depolymerization reaction mix. Concentration of tubulin was 30 µM. (<b>B,C,D</b>) Confocal microscopy image projections of cells transiently transfected with a plasmid expressing Bmcc1s-V5. Twenty-four hours after transfection, cells were exposed to 0°C for 45 minutes. Following free tubulin extraction by cell permeabilization, cells were fixed and double-stained for α-tubulin antibody (red), and V5 (green). Nuclei were stained with DAPI (blue). (B) HeLa cells stably transfected with GFP-N-STOP; (C) Primary culture of astrocytes; (D) Primary culture of neurons. In Bmcc1s-V5 transfected cells (green), α-tubulin staining was almost gone and V5 staining either retracted in a ball shape in the case of GFP-N-STOP HeLa cells and astrocytes, or filled the cell body in neurons. Bars: 10 µm.</p