Researchers video and measure tubulin transport in cilia for the first time.


Defective cilia can lead to a host of diseases and conditions in the human body, from rare, inherited bone malformations to blindness, male infertility, kidney disease and obesity. It is known that these tiny cell organelles become deformed and cause these diseases because of a problem related to their assembly, which requires the translocation of vast quantities of the vital cell protein tubulin. What they didn’t know was how tubulin and another cell organelle known as flagella fit into the process.

Now, a new study from University of Georgia shows the mechanism behind tubulin transport and its assembly into cilia, including the first video imagery of the process. The study was published in the Journal of Cell Biology.

Cilia are found throughout the body, so defects in cilia formation affect cells that line airways, brain ventricles or the reproductive track.  One of the main causes of male infertility is the cilia won’t function properly.

The team used total internal reflection fluorescence microscopy to analyze moving protein particles inside the cilia of Chlamydomonas reinhardtii, a green alga widely used as a model for cilia analysis.

The team exploited the natural behaviour of the organism, which is to attach by its cilia to a smooth surface, such as a microscope glass cover. This positions the cilia within the 200-nanometer reach of the total internal reflection fluorescence microscope allowing for the imaging of individual proteins as they move inside the cilia.  A video explaining the process was published along with the study.

Tubulin is transported by this process called intraflagellar transport, or IFT.  Though it has long been suspected in the field and there was indirect evidence to support the theory, this is the first time it has been shown directly, through live imaging, that IFT does function as a tubulin pump.  The team observed that about 400,000 tubulin dimers need to be transported within 60 minutes to assemble a single cilium. Being able to see tubulin moving into cilia allowed for first insights into how this transport is regulated to make sure cilia will have the correct size.

The new findings are expected to have wide implications for a variety of diseases and conditions related to cilia defects in the body.  The team state that they are on the very basic side of this research.  But because more and more diseases are being connected to cilia-related conditions, including obesity and even diabetes, the number of people working on cilia has greatly expanded over the last few years.

The team summise that unicellular models are great tools to address the many fundamental questions which remain unanswered such as controlling the size of cilia and how cells determine the proteins to be placed into these sensory organelles. In Chlamydomonas, the researchers were able to initiate cilia formation experimentally, which allowed them to see cilia during construction and analyze protein traffic while they are made.

Source:  UGA Public Affairs Division 

 

Repeated FRAP of growing and nongrowing cilia is followed by similar rates of recovery.  (A and B) Segments of a steady-state (A) and a re-generating (B) cilium were repeatedly bleached (indicated by arrowheads). Kymograms (top) and FRAP quantification (bottom) indicate similar rates (in percentage of pre-bleach GFP– a-tubulin fluorescence) of recovery after each bleaching step. (C and D) Individual frames (C) and kymograms (D) of a long-short cell. Bleached areas are marked by dashed circles. The kymogram (D) is a composite of several recordings, and arrowheads labeled a–e indicate the positions of the frames in C. The time (in seconds) for each recording and the position the bleaching laser (Brackets) is indicated; overexposed frames caused by photobleaching were deleted. Arrows in D: GFP–a-tubulin trajectories. Note fast and strong recovery in subsequent bleachings of the short cilium while the initially bleached area in the long cilium remains visible. The extended observation time will also bleach some of the (axonemal) GFP–a-tubulin outside of the spot bleaching area. This loss of fluorescence in the nonbleached areas results in a higher apparent recovery in the bleached areas.  Tubulin transport by IFT is upregulated during ciliary growth by a cilium-autonomous mechanism.  Lechtreck et al 2015.

Repeated FRAP of growing and nongrowing cilia is followed by similar rates of recovery. (A and B) Segments of a steady-state (A) and a re-generating (B) cilium were repeatedly bleached (indicated by arrowheads). Kymograms (top) and FRAP quantification (bottom) indicate similar rates (in percentage of pre-bleach GFP–
a-tubulin fluorescence) of recovery after each bleaching step. (C and D) Individual frames (C) and kymograms (D) of a long-short cell. Bleached areas are marked by dashed circles. The kymogram (D) is a composite of several recordings, and arrowheads labeled a–e indicate the positions of the frames in C. The time (in seconds) for each recording and the position the bleaching laser (Brackets) is indicated; overexposed frames caused by photobleaching were deleted. Arrows in D: GFP–a-tubulin trajectories. Note fast and strong recovery in subsequent bleachings of the short cilium while the initially bleached area in the long cilium remains visible. The extended observation time will also bleach some of the (axonemal) GFP–a-tubulin outside of the spot bleaching area. This loss of fluorescence in the nonbleached areas results in a higher apparent recovery in the bleached areas. Tubulin transport by IFT is upregulated during ciliary growth by a cilium-autonomous mechanism. Lechtreck et al 2015.

 

 

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