Saturday, November 4, 2017

Travels up the Cilium, and Back Down Again

Cilia are far more than wavy arms.  They are key sensory organs for most cells and have complex internal dynamics.

Cilia are the fuzz of Paramecia, and the sweepers of our respiratory system. They can be flagella, but are structurally unrelated to bacterial flagella. They are yet another eukaryotic innovation, bound by a membrane and composed of a microtubule bundle with complex boundary and transport mechanisms to construct themselves and provide unique functions. While some cells have lots of cilia, virtually all eukaryotic cells have at least one, and that one is called the primary cilium. It arises from the microtubule organizing center left over after cell division, which then migrates to the cell surface (via a vesicle intermediate) and grows a new bundle of microtubules pointed outside to create a key sensory organ. Indeed, primary cilia are the eyes, ears and noses of many eukaryotic cells.

Comb jellies move using cilia.

The cilium membrane is kept distinct from the bulk membrane of the cell via a collar or "necklace" of proteins around the base, implying special transport mechanisms for membrane components as well as for the many internal components needed to build and maintain cilia. This allows various signaling molecules to specialize to the cilium, forming a concentrated and specialized sensory platform.

For example, the photoreceptors in our retinas sit inside modified primary cilia, elaborated and evolved from the more primitive sensory system common to all eukaryotic cells. Fat cells have a primary cilium that displays G-protein coupled signal receptors that receive status updates from the body, and are defective in at least one genetic syndrome of obesity. Other genetic syndromes of ciliary function result in developmental defects, kidney disease, diabetes, and cancer.
The primary cilium is a product of the centriole or MTOC previously used in cell division.

A recent paper (review) investigated the intgernal dynamics that makes all this possible, a train system of cargo carriers that travel up and down the microtubule bundles of cilia. The microtubules are oriented in a single direction, minus at the base to plus at the tip. Two classes of motor proteins go in opposite directions, kinesins going up towards the plus end, or anterograde, and dyneins going in opposite fashion, towards the base, or retrograde. How cargoes attach themselves to these motors, and how their transport is regulated, makes up a very interesting field of study.

This paper observed the transit time of transport carrier complexes (IFT) at the tip, before heading back down to the cilium base.

These researchers used a fluorescent technique to look at individual cargoes as they were making this trip. If cargoes are made fluorescent, and then most of them are bleached out by overexposure, then a few remaining ones can be tracked. Their questions were mainly about how long these materials spent at the tip, and how the decision was made to reverse course. The operative acronym here is IFT, for intra-flagellar transport. Multiple cargoes seem to gang up into literal trains, which seems to depend a special coupling mechanism. For example, the authors labeled one of these dedicated transport proteins, called intraflagellar transport 27, or IFT27. They found that it takes about three seconds for this protein to hold up at the tip before being re-organized into a retrograde train. One of the cargoes being carried up the cilium is dynein, the motor required for all the return trips.

Experimental image set, showing time slices (going down). The fluorescent train comes up the cilium (towards the right) a first, then waits around for a couple of seconds at the tip, before dissociated fluorescent components start their return trips (on two different train sets, here) going back towards the left.

On the other hand, kinesin, which powers the trip up the cilium, does not get actively carried back down, but diffuses back, which is ten times slower. The authors suggest that this may be the rate limiting step restricting cilium length, since as the cilium grows, more of the kinesin is clogging its length, and less is available at the base to serve new anterograde trains. but since kinesin is expressed cytoplasm-wide, this is not an entirely compelling speculation.
In contrast to the train protein tracked above, fluorescently labeled kinesin powers its way up the cilium, but then dilly-dallies about, diffusing slowly back down without joining any powered trains.

What regulates all this? The motors and train proteins are nucleotide binders of various sorts, powered by ATP or GTP, so there is ample precedent for fine regulation using such systems. Perhaps just coming to the (+) end of a microtubule fiber sets switches in the kinesin and train proteins, priming them for the return conformation. What turns the dyneins on at the right time, and what regulates cargo attachment/detachment is still a mystery, however. As for cargo proteins like signaling receptors, some are known to have special targeting sequences which let them bind to the vicinity of the ciliary base, where they are transferred to the IFT train system. Others are sent to the primary cilium via the golgi protein sorting and vesicle generation system. If the targeting is high enough in affinity, such receptors can be essentially vacuumed from the rest of the cell and localized entirely to the cilium, thereby explaining the extreme localization/specialization of some signaling pathways and receptors to the primary cilium.


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