Giant vesicles, minibeads, and molecular motors : An original system to emulate intracellular transport

Communication, clearly essential to humans, is also essential to cells, their elemental building blocks. In order to preserve organic cohesion, cells need to communicate with their environment, but they also need to ensure adequate communication between their various compartments.

These forms of intracellular exchange are essential and require the setting up of actual networks. Membrane transport tubes were evidenced some years ago, but their formation has up till now remained a mystery.

A team of CNRS (1) biologists and physicists working at the Institut Curie has now, for the first time, managed to produce in vitro a minimal system which emulates this form of intracellular transport.

This system should help better to understand intracellular protein transport. Furthermore the tubes it generates may lend themselves to a number of nanotechnological applications as well as to the study of antigens expressed on the surface of tumor cells.

The paper presenting this work is published in the Proceedings of the National Academy of Sciences of the USA on April 16, 2002.

Exchanges between cell compartments occur constantly, and are indispensable to the preservation of all main organic functions. In order properly to communicate, cells use molecules on which information is inscribed (2). But as this information cannot be deposited randomly within cells, it needs to be ferried, or transported.

Transportation was long ascribed to small bead-like structures known as vesicles. We now know that other, more elongated membrane structures are also involved in this process: these larger tubes carry molecules towards their destination. In order to study the transportation of intracellular information, the effectiveness of which is vital in vivo, teams headed by Bruno Goud (“Compartimentation et dynamique cellulaires” UMR 144 CNRS/Institut Curie) and Patricia Bassereau (“Physico-chimie Curie” UMR 168 CNRS/Institut Curie) have for the first time developed a minimal system which generates tubes in vitro on the basis of artificial membranes.

Microtubules used as railway tracks…

The system was developed using natural cell constituents.
The first phase involved emulating a microtubule-based support structure. These long strands are distributed homogeneously within cells and serve as railway tracks along which molecules are ferried to their destination.

…and molecular motors to drive the train forward

In order to transport molecules, you need engines, or motors that will pull them in the right direction. This is what kinesins do. Kinesins are made up of two chains tipped with mechanisms onto which the fuel needed for transportation (ie, ATP) can lock. This is how molecular motors can ?move down the track?, traveling in a given direction along the microtubules.

An original system based on giant vesicles and minibeads

Giant vesicles (diameter>10 microns) were prepared: basically, these are large pockets made up of a single lipid membrane and filled with fluid. Constitutionally, these vesicles resemble the membrane-surrounded cell compartments from which information-inscribed molecules travel. The vesicles? largeness makes them easy to visualize with microscopes and furthermore provides a sufficient store of membrane – as the experiment does not provide for membrane renewal, contrary to what happens in vivo.
The research team then decided to use small polysterene beads (100 nm) coated with molecules devised to lock on to the giant vesicle at one end, and to the kinesins at the other.

The two bead locking links (to the vesicle and to the kinesins) are biotin ’handles’ (a vitamin used here as a fixation molecule)
Once the polysterene bead has locked on to the giant lipid vesicle membrane, it starts stretching it, as the kinesin ’arms’ pull it out, their ’feet’ meanwhile rolling along the network of in vitro replicated microtubules.

The tube formation mechanism is very tricky as it involves applying just the right amount of traction to the vesicle membrane, while protecting it against possible tears.
This artificial and sensitive system uses beads as a sort of ?resistor? to help avoid tube rupture. The cellular equivalent to this mechanism is not yet fully understood but may well correspond to a protein complex surfaced with a number of different motors.

A network of tubes emulating tubes in live cells

A number of very fine tubes (with diameters of a few dozen nanometers) were thus produced by stretching the membrane from a number of different bead anchoring sites on the surface of the vesicle.

Once the process was initiated, the tubes proceeded to grow and generate a complex microtubule-aligned network, as expected. This network is similar to the one which forms in vivo in the endoplasmic reticulum or the Golgi apparatus.

This minimal and original system thus provides for the generation of membrane tubes with a very limited number of inputs: lipid vesicle membranes, kinesins, microtubules, ATP.

Possible applications?

• In cell biology : This minimal system is a significant measure of progess in terms of cell transport studies, a broad area of research of great relevance to a number of different fields. Hence the significance of tool optimization. However, inter-compartmental information transmission involves many different players both for direction selection and for actual transmission. Which is why this minimal system, which is easy to replicate in vitro, should help speed up experiments in cell transport. It will in particular make it easy to add ?extraneous? elements to base preparations so as to observe their direct impact, simple comparison to the reference system thus allowing for easy assessment of these elements? possible role in cell transport. Until now, visualizing given molecular functions involved de-activating other molecules, a task both complex and fastidious.
• In nanotechnology : In the future, nanotechnologies are going to make very many new applications possible. If tubes within cells transport molecules, why couldn?t they transport pre-selected objects in vitro ? One possible application involves using nanotubes to transport fluids and thus create nanoreactors. Another, making these tubes solid so as to generate fibers which can then be used, inter alia, as nanooscillators. And these are but a few of the new investigative possibilities being considered…
• In oncology : Analyzing membrane proteins in cancer cells is hard work. Scientists are thus contemplating systems which would allow them to ?pull out? artificial tubes onto which they would slide the proteins they wish to study. These systems would be made up of beads onto which would be placed antibodies specific to given tumoral antigens expressed on the surface membranes of cancer cells, so as to allow for the sorting and typing of these antigens.