Mar

Visualize membrane scission under the fluorescence microscope

written by cail • posted in Experiment • 1,583 views • no comments

Nature. 2009 Mar 12;458(7235):172-7. Epub 2009 Feb 22.

Membrane scission by the ESCRT-III complex.

Wollert T, Wunder C, Lippincott-Schwartz J, Hurley JH.

Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, US Department of Health and Human Services, Bethesda, Maryland 20892, USA.

The endosomal sorting complex required for transport (ESCRT) system is essential for multivesicular body biogenesis, in which cargo sorting is coupled to the invagination and scission of intralumenal vesicles. The ESCRTs are also needed for budding of enveloped viruses including human immunodeficiency virus 1, and for membrane abscission in cytokinesis. In Saccharomyces cerevisiae, ESCRT-III consists of Vps20, Snf7, Vps24 and Vps2 (also known as Did4), which assemble in that order and require the ATPase Vps4 for their disassembly. In this study, the ESCRT-III-dependent budding and scission of intralumenal vesicles into giant unilamellar vesicles was reconstituted and visualized by fluorescence microscopy. Here we show that three subunits of ESCRT-III, Vps20, Snf7 and Vps24, are sufficient to detach intralumenal vesicles. Vps2, the ESCRT-III subunit responsible for recruiting Vps4, and the ATPase activity of Vps4 were required for ESCRT-III recycling and supported additional rounds of budding. The minimum set of ESCRT-III and Vps4 proteins capable of multiple cycles of vesicle detachment corresponds to the ancient set of ESCRT proteins conserved from archaea to animals.

PMID: 19234443

Woo! I am completed overwhelmed by the techniques used in this paper.
And the paper: simple, straight forward, and getting to the point.

Fluorescent labelling of proteins

To label Vps20 or Vps20DeltaC, which contain no native Cys residues, the mutant N85C was generated using the QuickChange Mutagenesis Kit (Stratagene). The purified and concentrated protein solution was labelled by incubating Alexa Fluor 488 C5 maleimide (Molecular Probes) at a molar ratio of 1:1 with the protein at 37 °C for 1 h under a N2 atmosphere. The labelling incorporation was evaluated by running samples on an SDS-PAGE gel and carrying out fluorescence scans of the gel with a Typhoon variable mode imager (GE-Healthcare) using 488 nm excitation and 495 nm detection wavelengths. Quantification of labelling efficiency was achieved by separating excess dye from labelled protein using two consecutive HiTrap desalting columns (GE-Healthcare). Molar protein and Alexa Fluor 488 concentrations of the purified protein were determined using absorption at 280 and 495 nm, respectively. Vps4 contains two cysteine residues at positions 317 and 376, which were used for the labelling reaction. The labelling and purification protocol was similar to that of the Vps20 constructs. Labelling efficiencies for all reactions exceeded 90%.

Lipids

The following lipids were purchased from Avanti Polar Lipids: POPS, POPC, cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulphonyl) ( rhodamine-PE). Phosphatidylinositol 3-phosphate and 3,5-bisphosphate ( diC16) were purchased from Echelon. POPS, POPC, cholesterol and rhodamine-PE were dissolved in chloroform. Phosphoinositides were dissolved in 1:2:0.8 chloroform:methanol:water.

Confocal fluorescence microscopy

Images were taken in multi-tracking mode on a Zeiss LSM510 or LSM5 LiveDuo confocal microscope with a times63 Plan Apochromat 1.4 NA objective and a 488/543 dichroic mirror at a resolution of 512 times 512 pixels. The GFP and Alexa 488 dyes were excited using the 488 nm line and the rhodamine was excited with a 543 nm HeNe laser. GFP and Alexa 488 emission was collected with a 505-530 nm bandpass filter. Rhodamine emission was collected with a 560 nm longpass filter. The pinholes for each channel were set for an approximate 1.5–2.5 microm optical slice. Laser power was 9 microW for the 543 nm channel and 24, 400 or 810 microW for GFP bulk phase experiments, Vps20 colocalization or Vps4 colocalization, respectively, in the 488 nm channel. Images were analysed using the LSM Examiner software.

Enumeration of ILV formation

Randomly chosen field of views were evaluated to reveal the number of ILVs per GUV. Reactions were performed as described above and all GUVs in the particular fields of view were scanned in the z direction. All ILV per GUV were counted, and for each experimental condition 100 GUVs were analysed. Results were summarized in histograms.

Three-dimensional fluorescence microscopy of GUVs

Z-stacks over time were taken with an LSM5 LiveDuo (Carl Zeiss MicroImaging Inc.) using times63 1.4 NA Plan-Apochromate oil objective at 90 frames per second, 1,024 times 256 pixels and a piezo focusing motor for fast hyperfine sectioning (0.35 mum). Static and animated three-dimensional visualizations of the data stacks were produced with IMARIS 6.1.0 software package (Bitplane Inc.).

The scientific merits are very well summarized by the F1000 reviews:

David Stephens
University of Bristol, United Kingdom
Cell Biology

This work provides a clear, elegant, and yet conceptually very simple data set that demonstrates the role of ESCRT-III subunits during membrane vesicle formation and scission. Using a fluorescence-based imaging approach, based on incubation of ESCRT-III subunits with giant unilamellar vesicles (GUVs), the Hurley lab demonstrates that the Vps20, Vps24, and Snf7 subunits mediate the formation and detachment of intralumenal vesicles, while Vps2 recruits Vps4 to recycle subunits in an ATPase-dependent manner for further rounds of budding. This work has key implications for multivesicular body biogenesis but also viral budding and membrane resolution during cytokinesis where ESCRT-III has also been implicated.
The parallels with other processes are intriguing, such as SNARE-mediated membrane fusion; here, fusion is driven largely by the thermodynamics of SNARE complex assembly, with NSF utilising ATP to disassemble SNARE complexes for further rounds of fusion. While the role of ESCRT-III in multivesicular body formation is clear, the precise role of individual subunits within this process have remained unclear. This study resolves these questions and provides a great example of an elegant and yet simple study, in a very hot area, which is precisely the kind of work that merits a high profile.

Tom Rapoport with Ann Marie Stanley
Harvard Medical School & Howard Hughes Medical Institute, United States of America
Cell Biology

Wollert at al. use an in vitro reconstitution assay in giant unilamellar vesicles to investigate the role of the ESCRT-III complex in intralumenal vesicle formation (ILVs), a process imitating multi-vesicular body formation and virus budding. The generation of ILVs is visualized by fluorescence microscopy, and the assay leads to new and exciting insight into the mechanism of ILV formation.
In this paper, Wollert et al. examine the role of the ESCRT-III complex in the formation of intralumenal vesicles (ILVs). The authors use giant unilamellar vesicles, whose membranes are labeled with a fluorescent dye, and visualize the reaction by fluorescence microscopy. By using a truncated version of Vps20, the authors are able get Vps20 bound to membranes and bypass the requirement for the ESCRT-II complex. In the presence of the ESCRT-III components Snf7, Vps24, and Vps2, a significant number of ILV’s are formed, and the in vitro assay recapitulates the ordered assembly observed in vivo. The straightforward assay leads to novel and significant insight into the mechanism of ILV scission. First, the authors are able to demonstrate that the ESCRT-III complex alone is sufficient for ILV formation. In fact, of the ESCRT-III components, Vps20, Snf7, Vps24 are sufficient for ILV formation, with Snf7 being the most important. A model is proposed in which Snf7 oligomerization constricts the neck of the vesicle to the point of scission, a model initially suggested by the formation of spirals observed when Snf7 is overexpressed {1}. Interestingly, the results from Wollert et al. demonstrate that the ATPase Vps4 and the ESCRT-III subunit that recruits it, Vps2, are not required for membrane scission. Instead, Vps4 acts after the scission step to recycle the ESCRT-III components for subsequent rounds of vesicle formation. In a related paper, Saksena et al. also find that the Vps4 ATPase is required for disassembly of the Snf7 oligomers in vitro, but the Saksena et al. model differs in that they propose that in vivo disassembly would be concurrent with membrane scission {2}.

References: {1} Hanson et al. J Cell Biol 2008, 180:389-402 [PMID: 18209100]. {2} Saksena et al. Cell 2009, 136:97-109 [PMID: 19135892].

Lois Weisman
University of Michigan, United States of America
Cell Biology

Wollert et al. present an elegant set of studies that provide both new insights and tools to study how multivesicular bodies form. Using giant unilamellar vesicles, the authors show that only three ESCRT (endosomal sorting complex required for transport) III subunits were sufficient to achieve scission of intralumenal vesicles. These findings provide strong support to the hypothesis that control of inward budding occurs primarily (and possibly exclusively) on the cytoplasmic side of the membrane. Moreover, these findings provide a new critical tool to determine the mechanism of inward vesicle budding.
The best studied mechanisms of vesicle budding and scission are those that utilize protein coats that produce the membrane curvature required to form vesicles. In this case, vesicles bud towards the cytoplasmic side of the membrane, so it is intuitively obvious how cytoplasmic proteins are recruited to achieve membrane curvature and fission. It is significantly more difficult to conceive a mechanism for multivesicular body formation, and other membrane events that are catalyzed by the ESCRT proteins. In these cases, the proteins that catalyze these events are also on the cytoplasmic side of the membrane, but the vesicles are budding away from the cytoplasm. Wollert et al. propose a mechanism whereby the ESCRT III subunit Vps20 initiates a spiral of Snf7, which then terminates with Vps2 followed by recruitment of the triple ATPase, Vps4. The authors show that Vps4 dissembles Vps20/Snf7/Vps2 and enables new rounds of vesicle formation. This model fits with the in vitro studies presented, and with earlier in vivo studies. However, these elegant studies also underscore how far we are from understanding the earliest steps that initiate the formation of inward-budding vesicles.

Bruno Goud with Winfried Römer
Mecanismes moleculaires du transport intracellulaire/ Institut Curie, France
Cell Biology

The authors point out that the ESCRT-III complex, required for multivesicular body biogenesis, is, alone, capable of detaching intralumenal vesicles from the limiting membrane and that Vps4 acts after the membrane scission step to recycle the ESCRT-III proteins.
ESCRT-III is composed of four protein subunits (Vps20, Snf7, Vps24 and Vps2) that are monomeric in the cytosol and oligomerize into a protein lattice only upon binding to endosomal membrane. Using an in vitro ESCRT scission assay, ESCRT-III-dependent budding and scission of intralumenal vesicles into giant unilamellar vesicles was reconstituted with a minimal set of purified proteins and lipids, and visualized by fluorescence microscopy. Giant unilamellar liposomes emerge more and more as a tool to reconstitute cellular processes with the motto: make it as simple as possible and as complicated as necessary!