Single Particle Fluorescence Burst Analysis of Membrane Fission

The ability to catalyze the fission step of endocytosis is critical to many biological processes including cell communication and synaptic transmission. This activity must be tightly regulated for cellular homeostasis. Limitations of current approaches used for the study of this process, in conjunction with a historical focus on neuronal systems that may not generalize to somatic cells, have left the field torn between several conflicting models. Functional redundancy of several key players complicate interpretation of in vivo studies, stressing the need for a sensitive in vitro approach capable of revealing attenuated activity of tightly regulated machinery. In this document, we introduce Burst Analysis Spectroscopy (BAS) as a simple reagent sparing approach for the investigation of vesicle membrane fission. Using BAS, we accurately map liposome distributions across several orders of magnitude and observe subtle shifts in liposome population as functions of time, temperature, and protein concentration using the fission potent ENTH domain of the protein epsin. We proceed to further uncover an unrecognized fission activity of full-length epsin, consistent with a mechanism in which membrane fission proceeds as a consequence of amphipathic helix insertion into the lipid bilayer

Single Particle Fluorescence Burst Analysis of Epsin Induced Membrane Fission

<div><p>Vital cellular processes, from cell growth to synaptic transmission, rely on membrane-bounded carriers and vesicles to transport molecular cargo to and from specific intracellular compartments throughout the cell. Compartment-specific proteins are required for the final step, membrane fission, which releases the transport carrier from the intracellular compartment. The role of fission proteins, especially at intracellular locations and in non-neuronal cells, while informed by the dynamin-1 paradigm, remains to be resolved. In this study, we introduce a highly sensitive approach for the identification and analysis of membrane fission machinery, called burst analysis spectroscopy (BAS). BAS is a single particle, free-solution approach, well suited for quantitative measurements of membrane dynamics. Here, we use BAS to analyze membrane fission induced by the potent, fission-active ENTH domain of epsin. Using this method, we obtained temperature-dependent, time-resolved measurements of liposome size and concentration changes, even at sub-micromolar concentration of the epsin ENTH domain. We also uncovered, at 37°C, fission activity for the full-length epsin protein, supporting the argument that the membrane-fission activity observed with the ENTH domain represents a native function of the full-length epsin protein.</p></div

Dose dependence of ENTH-mediated vesiculation.

<p>(a) BAS histograms of 200 nm-diameter, TopFluor-labeled, (5%) PtdInsP(4,5)P₂ Folch liposomes before (<i>red</i>) and after incubation at 37°C for 20 min with 500 nM (<i>blue</i>), 1 μM (<i>green</i>), 5μM (<i>purple</i>), and 10 μM (<i>cyan</i>) ENTH. (b) Heat-map representation of the fractional intensity for each reaction shown in (a). The data shown is representative of three experimental replicates.</p

BAS assay distinguishes liposomes of different sizes.

<p>The size distribution of 200 nm, 100 nm and 50 nm fluorescent liposomes was examined by FCS and BAS. (a) Fluorescent burst data of TopFluor-labeled Folch liposomes extruded to 200 nm (<i>green</i>), 100 nm (<i>purple</i>) and 50 nm (<i>cyan</i>). (b) BAS histograms generated from the burst data in (a). Fraction of total events is the concentration of each bin divided by the total concentration, for each sample. Dashed lines show theoretical diameter distributions (35% CV, <i>dash</i>; 50% CV, <i>dot</i>) derived from Monte Carlo simulated intensity data in which fluorescence brightness was set proportional to particle surface area. The resulting simulated intensity distributions were analyzed with BAS analysis code. (c) FCS profiles of 200 nm, 100 nm, and 50 nm liposomes. The data shown is representative of two experimental replicates.</p

BAS analysis of liposomes vesiculated by the ENTH domain of epsin.

<p>Fluorescent burst data for 400 nm-diameter, TopFluor-labeled, (5%) PtdInsP(4,5)P₂ Folch liposomes incubated at 37°C for 40 min before (a) and after addition of 2 μM ENTH (b). Fluorescent burst data for 200 nm-diameter liposomes incubated at 37°C for 40 min before (c) and after addition of 2 μM ENTH (d). (e) BAS histograms generated from starting 200 nm liposomes before (<i>red</i>) and after addition of ENTH (<i>blue</i>; insets indicate resolution of small particles in a 10-fold dilution of the same reaction). (f) FCS profiles of liposomes extruded to 100 nm (<i>green</i>), 50 nm (<i>cyan</i>) and the end products (<i>purple</i>) of the fission reaction of 200 nm liposomes from (d). The data shown is representative of three experimental replicates.</p

Full-length epsin has vesicle fission activity.

<p>(a) BAS histograms of 200 nm-diameter, TopFluor-labeled, (5%) PtdInsP(4,5)P₂ Folch liposomes before (<i>red</i>) and after incubation at 37°C for 40 min with 1 μM (<i>green</i>), 5 μM (<i>purple</i>), and 10 μM (<i>blue</i>) full-length epsin. (b) Heat-map representation of the fractional intensity for each reaction shown in (a). (c) Comparison of ENTH and epsin activity. BAS histograms of starting liposomes (<i>red</i>), and liposomes incubated at 37°C for 90 min with 2 μM ENTH (<i>blue</i>) or full-length epsin (<i>green</i>). The data shown is representative of three experimental replicates.</p

Chemical soil factors influencing plant assemblages along copper-cobalt gradients: implications for conservation and restoration

Aims Define the chemical factors structuring plant communities of three copper-cobalt outcrops (Tenke-Fungurume, Katangan Copperbelt, D. R. Congo) presenting extreme gradients. Methods To discriminate plant communities, 172 vegetation records of all species percentage cover were classified based on NMDS and the Calinski criterion. Soil samples were analyzed for 13 chemical factors and means compared among communities with ANOVA. Partial canonical correspondence analysis (pCCA) was used to determine amount of variation explained individually by each factor and site effect. Results Seven communities were identified. Six of the studied communities were related to distinct sites. Site effect (6.0 % of global inertia) was identified as the most important factor related to plant communities’ variation followed by Cu (5.5 %), pH (3.6 %) and Co (3.5 %). Unique contribution of site effect (3.8 %) was higher than that of Cu (1.1 %) and Co (1.0 %). Conclusions In restoration, not only Cu and Co contents will be important to maintain vegetation diversity, attention should also be given to co-varying factors potentially limiting toxicity of metals: pH, organic matter, Ca and Mn. Physical parameters were also identified as important in the creation of adequate conditions for diverse communities. Further studies should focus on the effect of physical parameters and geology