A cellular railway in motion

By Ruya Houssein

In a dark, windowless lab at VU Amsterdam, a team of scientists in the Peterman lab managed to quietly visualise and quantify the movement of single proteins in the sensory neurons of small, thriving worms. To fully understand their exciting research and how it could help us eventually treat a wide variety of diseases known as ciliopathies, we will first learn more about the oldest known cellular organelles: cilia¹ and how we visualised them.

Cilia are everywhere 

Cilia are highly specialised structures that belong to the list of ancestral eukaryotic compartments together with nuclei and mitochondria. They protrude from the surface of nearly all human cells, acting as cellular antennae, sometimes more than one on each cell. Some cilia are involved in motility where they are found in specialised epithelia to facilitate fluid flow. Other cilia lack motility properties but instead participate in signalling. These sensory cilia, also called primary cilia, are the main characters of this story. 

Sensory cilia are present in every human tissue² and allow us to experience our environment through light, sound, and odorant perception. While you are listening to the sounds coming through your headphones, it is the collective effort of nonstop working cilia. Their ability to sense both internal and external stimuli make cilia crucial to vital pathways for embryonic development, cell migration and even cell proliferation³. 

To accomplish their function, cilia need to concentrate a number of proteins associated with these signal transduction pathways. Cilia have evolved a directed transport mechanism called intraflagellar transport (IFT) because they are not equipped to synthesize their own proteins (Figure 1C). The carriages of this transport system are long polymers called IFT-trains⁴. IFT-trains carry and deliver cargo proteins from the cell body into and out of the cilia. In a pioneering experiment in 1993, this activity of the IFT was first captured in the movement of polystyrene beads in the motile cilium of green algae⁵.

 

The cargo trains of intraflagellar transport

The movement of IFT-trains in cilia occurs at the microtubule-based core called the axoneme (Figure 1C). The core resembles a railway track along which cellular transport takes place. Cargo proteins loaded on these trains include structural components, membrane receptors for sensing and signalling. Motor proteins drive IFT-trains along this axonemal railway. With the activation of motors, IFT-trains and their associated cargo first move from the ciliary base to the tip⁶. At the ciliary tip, cargo is unloaded and trains are recycled back to the rail depot, the ciliary base (Figure 1C). 

The recruitment of cargo molecules into cilia is crucial for building cilia and all ciliary functions⁷,⁸. Recent studies have revealed the structure of the proteins that form the IFT-trains using cryogenic electron microscopy⁹,¹⁰. These beautiful images give insights into the function of IFT structures and their potential binding sites to the ciliary environment. However, the images have been static data until now, not illuminating dynamic process. So, how can scientists study the dynamics of this cellular railway? 

Prof. Erwin Peterman and his team tackled this question using in vivo single-molecule fluorescence microscopy. His laboratory visualises IFTs inside the sensory cilia of living worms, Caenorhabditis elegans (C. elegans) (Figure 1A-B). This method allows us to search for the answers to fundamental biological questions on IFT and study the potential cause of disrupted ciliary function. 

Ciliopathies – Is your heart (literally) in the right place?

While the complete absence of cilia is fatal, malformation or dysfunction of cilia, due to genetic mutations in different ciliary proteins results in a wide variety of diseases. These diseases are collectively known as ciliopathies. Some ciliopathies are related to dysfunctional motile cilia and others to the dysfunction of sensory cilia (Figure 2). Symptoms of dysfunctional motile cilia include infertility, recurrent chest infections, kidney cysts, and reversed internal organs locating the heart to the right chest. Dysfunction of sensory cilia can cause impaired environmental perception and cause neurological disturbance with a whole spectrum of disorders¹¹. 

Nearly 200 genes are known to cause ciliopathies. Interestingly, mutations in the same genes can cause different phenotypes, which often makes it difficult to diagnose patients with ciliopathies. Of these 200 genes, many are related to IFT function¹². Receptors can no longer be transported to the ciliary membrane, thus depleting the sensory function of cilia. Understanding the molecular dynamics of IFT can provide insight into the causes of these ciliopathies and potentially lead to the development of new therapies.

 

Track down those IFT dynamics!

To visualize a particular IFT protein, the researchers tagged the protein with a fluorescent label. The imaging is performed with a laser-illuminated epi-fluorescence microscope¹³. Upon laser excitation, fluorescently-labeled IFT proteins emit light, which is captured on highly-sensitive cameras. This visualises IFT trains moving inside the 8 µm long cilia of living worms. In Figure 3 you see a kymograph, a space-time intensity plot, of an example movie displaying IFT. Here, an IFT-train is labeled with a green-fluorescent protein tag and one can clearly observe bulky IFT trains moving from the ciliary base to the tip and back again. 

 

The Peterman lab has developed advanced image analysis approaches to obtain quantitative insights on the dynamics of IFT-proteins. They can quantify the amount of imaged IFT protein in the cilia and identify turnovers (proteins jumping from one carriage to the other). This method allows visualization of the entry behaviour of the proteins into the cilia, the varying velocities of the proteins as they move along the axoneme and the visualization of turnarounds (movement from tip to base) at the cilia tip. 

In recent years, they have also developed a way to perform single-molecule imaging of IFT. Most IFT-proteins are present in large numbers inside a cilium (in the range of 1000-10000 molecules). Fluorescent imaging provides information regarding the ensemble dynamics of the IFT protein being imaged. Single-molecule imaging is possible when the cilium is illuminated with a high laser-light excitation. This leads to the photo-bleaching of most of the fluorescing molecules, such that only a few molecules are fluorescing at any given time. Such single-molecules can be visualized and tracked with nanometer precision, providing details regarding how they move¹⁴. With quantitative analysis, researchers are slowly enhancing our understanding on how IFT trains are built at the ciliary base, how cargo proteins latch onto these trains, how motor proteins (dis)associate with IFT trains, how the trains remodel at the cilia tip and several other outstanding question¹⁵.  

Beyond intraflagellar transport this single molecule imaging  approach could also be used to study other intracellular transport systems in structures such as axons, dendrites, and microvilli in a variety of species or diseases. Ongoing research aims to gradually unveil the mysteries of the cellular railway system of cilia. The ability to dynamically visualise these systems is an important scientific tool in the kit.

Author information:

Ruya is a second-year biomolecular sciences master student. Interested in the dynamics of the structures that govern molecular neurobiology.

She joined the Peterman laboratory at VU Amsterdam as an intern and later as a research fellow. Here she studies the dynamics of intraflagellar transport using single-molecule fluorescence microscopy.

Acknowledgements:

Ruya would like to thank all members of the lab, especially Dr. Aniruddha Mitra and Elizaveta Loseva M.Sc. for their contributions to the piece.

Cover image credit: Elizaveta Loseva

Further reading: