Glioma cell dispersion is driven by [alfa]5 integrin-mediated cell-matrix and cell-cell interactions

K

Rac1 Dynamics in the Human Opportunistic Fungal Pathogen Candida albicans

The small Rho G-protein Rac1 is highly conserved from fungi to humans, with approximately 65% overall sequence identity in Candida albicans. As observed with human Rac1, we show that C. albicans Rac1 can accumulate in the nucleus, and fluorescence recovery after photobleaching (FRAP) together with fluorescence loss in photobleaching (FLIP) studies indicate that this Rho G-protein undergoes nucleo-cytoplasmic shuttling. Analyses of different chimeras revealed that nuclear accumulation of C. albicans Rac1 requires the NLS-motifs at its carboxyl-terminus, which are blocked by prenylation of the adjacent cysteine residue. Furthermore, we show that C. albicans Rac1 dynamics, both at the plasma membrane and in the nucleus, are dependent on its activation state and in particular that the inactive form accumulates faster in the nucleus. Heterologous expression of human Rac1 in C. albicans also results in nuclear accumulation, yet accumulation is more rapid than that of C. albicans Rac1. Taken together our results indicate that Rac1 nuclear accumulation is an inherent property of this G-protein and suggest that the requirements for its nucleo-cytoplasmic shuttling are conserved from fungi to humans

Imaging and Measuring Vesicular Acidification with a Plasma Membrane-Targeted Ratiometric pH Probe

International audienceTracking the pH variation of intracellular vesicles throughout the endocytosis pathway is of prior importance to better assess the cell trafficking and metabolism of cells. Small molecular fluorescent pH probes are valuable tools in bioimaging but are generally not targeted to intracellular vesicles or are directly targeted to acidic lysosomes, thus not allowing the dynamic observation of the vesicular acidification. Herein, we designed Mem-pH, a fluorogenic ratiometric pH probe based on chromenoquinoline with appealing photophysical properties, which targets the plasma membrane (PM) of cells and further accumulates in the intracellular vesicles by endocytosis. The exposition of Mem-pH toward the vesicle's lumen allowed to monitor the acidification of the vesicles throughout the endocytic pathway and enabled the measurement of their pH via ratiometric imaging

Rdi1 is not necessary for <i>C. albicans</i> Rac1 nuclear accumulation.

<p>DIC and fluorescence images of wild-type cells with <i>PADH1GFPrac1</i> (PY201) and <i>rdi1Δ/rdi1Δ PADH1GFPrac1</i> (PY1598) cells were taken after 75 min without agitation. Bar, 5 µm.</p

Rac1 cycles in and out of the nucleus.

<p>(A) FRAP analysis of nuclear Rac1. Confocal microscopy images of budding <i>rac1</i>Δ<i>/rac1</i>Δ <i>PADH1GFPRAC1</i> (PY205) cells, after 1 h in the absence of agitation, were taken prior to and subsequent to photobleaching of the nucleus. A typical nuclear fluorescence recovery after photobleaching experiment is shown. (B) FLIP analysis of nuclear Rac1. Confocal microscopy images of budding PY205 cells, after 1 h in the absence of agitation, were taken subsequent to photobleaching a region on the plasma membrane (Bleach). For fluorescence loss in photobleaching experiments, images of the nucleus of the photobleached cell (ROI2 nucleus FLIP) together with images of the nucleus of an adjacent cell (ROI3 nucleus control) were captured every 4 sec. Plasma membrane photobleaching was repeated after every 4 images.</p

Rac1 polybasic carboxyl-terminal region is required for membrane localization and function.

<p>(A) Rac1 PBR is required for plasma membrane localization. DIC and fluorescence images of indicated strains <i>rac1</i>Δ<i>/rac1</i>Δ <i>PADH1GFPRAC1</i> (PY205), <i>rac1</i>Δ<i>/rac1</i>Δ <i>PADH1GFPrac1-5Q</i> (PY511), and <i>rac1</i>Δ<i>/rac1</i>Δ <i>PADH1GFPrac1[C233S]</i> (PY415), respectively, are shown. Bar, 5 µm. (B) Rac1 PBR is required for nuclear accumulation. DIC and fluorescence images of <i>rac1</i>Δ<i>/rac1</i>Δ <i>PADH1GFPrac1-5Q</i> (PY511) incubated 75 min without agitation are shown. Bar, 5 µm. (C) In the absence of prenylation, the Rac1 carboxyl-terminal region targets GFP to the nucleus. Fluorescence images of indicated strains (PY357 and PY438, respectively) are shown. Bar, 5 µm. (D) Rac1 PBR is required for function. Cells from <i>rac1</i>Δ<i>/rac1</i>Δ <i>PRAC1RAC1</i> (PY275), <i>rac1</i>Δ<i>/rac1</i>Δ <i>PRAC1rac1-5Q</i> (PY534), and <i>rac1</i>Δ<i>/rac1</i>Δ <i>PRAC1rac1[C233S]</i> (PY406) were embedded in YEPS and images of colonies were taken, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015400#pone-0015400-g003" target="_blank">Figure 3C</a>. Similar results were observed in 3 independent experiments. Bar, 1 mm.</p

GUV-AP: multifunctional FIJI-based tool for quantitative image analysis of Giant Unilamellar Vesicles

MOTIVATION: Giant Unilamellar Vesicles (GUVs) are widely used synthetic membrane systems that mimic native membranes and cellular processes. Various fluorescence imaging techniques can be employed for their characterization. In order to guarantee a fast and unbiased analysis of imaging data, the development of automated recognition and processing steps is required. RESULTS: We developed a fast and versatile Fiji-based macro for the analysis of digital microscopy images of GUVs. This macro was designed to investigate membrane dye incorporation and protein binding to membranes. Moreover, we propose a fluorescence intensity-based method to quantitatively assess protein binding. AVAILABILITY AND IMPLEMENTATION: The ImageJ distribution package FIJI is freely available online: https://imagej.net/Fiji. The macro file GUV-AP.ijm is available at https://github.com/AG-Roemer/GUV-AP. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online

Yeast strains used in this study.

<p>Yeast strains used in this study.</p

Nuclear accumulation is an inherent property of Rac1.

<p>(A) Human Rac1 (HsRac1) accumulates in the nucleus of <i>C. albicans</i> cells. <i>HsRAC1</i> was corrected for codon usage in <i>C. albicans</i> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015400#s2" target="_blank">Materials and methods</a>). DIC and fluorescence images of <i>rac1</i>Δ<i>/rac1</i>Δ <i>PADH1GFPHsRAC1</i> (PY913) cells were taken after the indicated times without agitation. Bar, 5 µm. (B) Time-course of HsRac1 nuclear accumulation. Cells expressing GFP-HsRac1 (PY913) and GFP-CaRac1 (PY205) with nuclear fluorescent signal were counted after the indicated times in the absence of agitation (<i>n</i> = 100–200 cells for each time point). (C) HsRac1 cannot complement for CaRac1 function. Cells from <i>rac1</i>Δ<i>/rac1</i>Δ (PY191), <i>rac1</i>Δ<i>/rac1</i>Δ <i>PADH1GFPRAC1</i> (PY205), and <i>rac1</i>Δ<i>/rac1</i>Δ <i>PADH1GFPHsRAC1</i> (PY913), were embedded in YEPS as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015400#pone.0015400-Brown1" target="_blank">[25]</a> and images of colonies were taken after 5 d at 25°C. Similar results were observed in 3 independent experiments. Bar, 1 mm. (D) A chimera in which the 14 carboxyl terminal residues of CaRac1 replaced the 12 corresponding residues of HsRac1 (HsRac1CT_CaRac1) localized to the plasma membrane, yet was not functional in embedded filamentous growth. Left two panels: DIC and fluorescence images of <i>rac1</i>Δ<i>/rac1</i>Δ <i>PADH1GFPHsRAC1CT_CaRAC1</i> (PY1222) are shown. Bar, 5 µm. Right panel: PY1222 cells were embedded in YEPS and images of colonies were taken as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015400#pone-0015400-g008" target="_blank">Figure 8C</a>. Bar, 1 mm.</p

Quantitative monitoring of the cytoplasmic release of NCp7 proteins from individual HIV-1 viral cores during the early steps of infection

International audienceFluorescence microscopy imaging of individual HIV-1 viruses necessitates a specific labeling of viral structures that minimally perturbs the infection process. Herein, we used HIV-1 pseudoviruses containing NCp7 fused to a tetracystein (TC) tag, labeled by a biarsenical fluorescein derivative (FlAsH) to quantitatively monitor the NCp7 protein concentration in the viral cores during the early stages of infection. Single particle imaging of individual pseudoviruses with defined ratios of TC-tagged to non tagged NCp7 proteins, together with theoretical modeling of energy transfer between FlAsH dyes, showed that the high packaging of TC-tagged proteins in the viral cores causes a strong fluorescence quenching of FlAsH and that the fluorescence intensity of individual viral complexes is an appropriate parameter to monitor changes in the amount of NCp7 molecules within the viral particles during infection. Interestingly, we observed a dramatic fluorescence increase of individual FlAsH-labeled pseudoviruses containing 100% TC-tagged NCp7 proteins in infected cells at 8 and 16 h post-infection. This effect was significantly lower for pseudoviruses expressing TC-tagged integrase. Therefore, this fluorescence increase is likely related to the cytoplasmic viral transformation and the release of NCp7 molecules from the viral complexes. This loss of quenching effect is largely reduced when reverse transcriptase is inhibited, showing that NCp7 release is connected to viral DNA synthesis. A spatial analysis further revealed that NCp7-TC release is more pronounced in the perinuclear space, where capsid disassembly is thought to be completed. Quantification of NCp7-TC content based on fluorescence quenching presented in this study evidences for the first time the cytoplasmic release of NCp7 during the remodeling of HIV-1 viral particles on their journey toward the nucleus. The developed approach can be applied to quantify dye concentrations in a wide range of nano-objects by fluorescence microscopy techniques