Like our own bodies, cells have their own skeletons called 'cytoskeletons' and are made of proteins instead of bones.
These network-like structures maintain the cell's shape, provide mechanical support, and are involved in critical processes of the cell's lifecycle. The cytoskeleton is an object of intense scientific and medical research, which often requires being able to observe it directly in cells.
Ideally, this would involve highly-fluorescent molecules that can bind cytoskeletal proteins with high specificity without being toxic to the cell. Publishing in Nature Methods, EPFL scientists have exploited the properties of a new fluorescent molecule, also developed at EPFL, to generate two powerful probes for the imaging of the cytoskeleton with unprecedented resolution. These probes pave the way for the easier and higher quality imaging of cells, offering many scientific and medical advantages.
The cytoskeleton is a large structure inside cells that provides them with mechanical support, keeps their three-dimensional shape and internal organization, and enables them to move and divide. It consists of three major sub-structures inside the cell, which are made up of long, filamentous proteins: tubulin and actin.
Current techniques for observing the cytoskeleton can be difficult to get into living cells, can be toxic, and are usually limited in resolution and duration, since the signal wears off over time. A common technique is fluorescence microscopy, where fluorescent molecules ('probes') are attached to cell structures and then 'lit up' against a dark background.
The team of Kai Johnsson at EPFL has developed novel fluorescent probes that can easily enter live cells, are non-toxic, have long-lasting signals, and most importantly, offer unprecedented image resolution. In 2013, the researchers developed a fluorescent molecule called silicon-rhodamine (SiR), which switches 'on' only when it binds to the charged surface of a protein like the ones found on the cytoskeleton. When SiR switches 'on', it emits light at far-red wavelengths.
The challenge was getting SiR to bind specifically to the cytoskeleton's proteins, actin and tubulin. To achieve this, the scientists fused SiR molecules with compounds that bind tubulin or actin. The resulting hybrid molecules consist of a SiR molecule, which provides the fluorescent signal, and a molecule of a natural compound that can bind the target protein. One such compound was docetaxel, an anticancer drug that binds tubulin, and the other jasplakinolide, which specifically binds the cytoskeletal form of actin. Both compounds, which are used here in very low, non-toxic concentrations, can easily pass through the cell's membrane and into the cell itself.
The probes, named SiR-tubulin and SiR-actin, were used to visualize the dynamics of the cytoskeleton in human skin cells. Because the light signal of the probes is emitted in the far-red spectrum, it is easy to isolate from background noise, which generates images of unprecedented resolution when used with a technique called super-resolution microscopy.
An additional advantage is the practicality of the probes. "You just add them directly into your cell culture, and they are taken up by the cells", says Kai Johnsson. The probes also do not require any washing or preparation of the cells before administration or any subsequent washing steps, which greatly helps in maintaining the stability of their environment and their natural biological functions.
The scientists believe that they can extend their work into other types of proteins and tissues. "Cytoskeletal structures are imaged by biologists all the time", says Johnsson. "Up to now, no probes were available that would allow you to get high quality images of microtubules and microfilaments in living cells without some kind of genetic modification. With this work, we provide the biological community with two high-performing, high-contrast fluorogenic probes that emit in the non-phototoxic part of the light spectrum, and can be even used in tissues like whole-blood samples."
This work represents a collaboration between EPFL's Institute of Chemical Sciences and Engineering (ISIC), Institute of Bioengineering (IBI), and the Bioimaging and Optics Platform (BIOP), with the National Centre of Competence in Research (NCCR) in Chemical Biology; the Max-Planck Institutes for Biophysical Chemistry (Göttingen) and of Molecular Physiology (Dortmund); the Friedrich-Schiller-University's Institute of Organic Chemistry (Jena); and the Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA) (Vienna).
Lukinavičius G, Reymond L, D'Este E, Masharina A, Göttfert F, Ta H, Güther A, Fournier M, Rizzo S, Waldmann H, Blaukopf C, Sommer C, Gerlich DW, Arndt HD, Hell SW, Johnsson K. Fluorogenic probes for live-cell imaging of the cytoskeleton. Nature Methods 25 May 2014. DOI: 10.1038/nmeth.2972
Nik Papageorgiou | Eurek Alert!
Subcutaneous Administration of Multispecific Antibody Makes Tumor Treatment Faster & More Tolerable
01.07.2015 | Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt
Why human egg cells don't age well
01.07.2015 | RIKEN
Wind turbines could be installed under some of the biggest bridges on the road network to produce electricity. So it is confirmed by calculations carried out by a European researchers team, that have taken a viaduct in the Canary Islands as a reference. This concept could be applied in heavily built-up territories or natural areas with new constructions limitations.
The Juncal Viaduct, in Gran Canaria, has served as a reference for Spanish and British researchers to verify that the wind blowing between the pillars on this...
New technique combines electron microscopy and synchrotron X-rays to track chemical reactions under real operating conditions
A new technique pioneered at the U.S. Department of Energy's Brookhaven National Laboratory reveals atomic-scale changes during catalytic reactions in real...
Think of an object made of iron: An I-beam, a car frame, a nail. Now imagine that half of the iron in that object owes its existence to bacteria living two and a half billion years ago.
Think of an object made of iron: An I-beam, a car frame, a nail. Now imagine that half of the iron in that object owes its existence to bacteria living two and...
A team of scientists including PhD student Friedrich Schuler from the Laboratory of MEMS Applications at the Department of Microsystems Engineering (IMTEK) of...
The three-year clinical trial results of the retinal implant popularly known as the "bionic eye," have proven the long-term efficacy, safety and reliability of...
25.06.2015 | Event News
16.06.2015 | Event News
11.06.2015 | Event News
02.07.2015 | Press release
02.07.2015 | Power and Electrical Engineering
02.07.2015 | Earth Sciences