Saturday, March 25, 2017

Centrioles Have Mothers Too

Penetrating the mysteries of one of our most attractive organelles.

The centriole is one of the more glamorous, yet enigmatic, structures of the eukaryotic cell. Yes, it has beautiful ultrastructure. Yes, it serves as the fabulous astral center of the spindle poles that organize mitosis. And yes, it has been studied for well over a century. But does all that mean it is well understood? Absolutely not.

Electron micrographs of centrioles. The inset is a cross-section of one centriole barrel, showing the 9 X 3 microtubule structure as well as the inner cartwheel that seems to be its construction scaffold. The main image shows a side-ways cross-section of a mother-daughter pair of centrioles, (forming a centrosome), showing their length and relationship. Also shown is the nimbus of microtubules coming out of what is called the peri-centriolar material, or PCM. How those relate functionally to the core structure is still not known.

Centrioles are one of the many radical innovations of eukaryotic cells, forming the core(s) of centrosomes, which organize mitosis, and of cilia, which are the novel eukaryotic solution to cellular locomotion. They are are a barrel-shaped quasi-crystalline arrangement of microtubule proteins, grown on a central wheel of template proteins, called the cartwheel. For cilia, these microtubules clearly serve as the foundation stones of microtubule rods that grow out into the long appendage. But things get far more murky at the mitotic spindle core, where a profusion of microtubules emerge from an amorphous zone around the centrioles, but are not templated directly by them. The nature and history of this structure remains quite mysterious, especially as it turns out that some eukaryotic cells can get by without centrioles at all.

Centrosomes are in red, at what are also called the microtubule organizing centers, organizing the green microtubules that  in turn organize the separation of DNA (blue) during mitosis. 

In addition to these starring, if not obligatory, roles, centrioles have another charismatic property, which is that they reproduce in a clearly parental fashion. Every cell cycle is accompanied by the division of the centriole pair into single centriole mothers, which then give birth to new centriole daughters from their sides. While most of the molecular actors in this centriole mini-drama are known, as are some of their roles, a great deal remains to be found out about how the whole process is put together.

Detailed scheme of centriole (green) and centrosome (the whole green + yellow mess) duplication during the cell cycle. The structures are reasonably well known, the molecules and their roles less so. Note the roles of Plk4, Sas6 and colleagues in generation of the nascent cartwheel scaffold at G1/S. Plk4 itself will be disposed of later on, towards M phase.  

A recent paper described some more about how the first steps of this replication process take place. The cooperation of the centrioles with the larger cell cycle is naturally very deep. Some labs have found that the key kinases (which attach regulatory phosphates to other proteins) that regulate the cell cycle operate out of the centrosome- the structure containing the centriole pair. And the centrioles in turn are subject to key kinase steps that license when they can replicate. Key molecules of this process have the name Sas, after a genetic screen for spindle assembly defective mutants, each of which was given a Sas# name. Sas4, Sas5, and Sas6, for instance, are proteins that make up most of the central cartwheel scaffold upon which the centrosomal microtubules are built, along with another protein, Cep135. But the story starts much earlier. Sas6 is brought to the key location on the mother centriole's side by a kinase, Zyg1, which in turn is brought there by Spd2. How did Spd2 get there, and what does it do? Could it be turtles all the way down?

No, the authors identify Sas7 as the protein that binds to Spd2 and gets centriole pregnancy underway. Sas7, finally, is at the centriole all the time, and is activated not by recruitment, but by the cell cycle. One key finding was that all the other known initiating mutations (of Zyg1 and Spd2) depend on Sas7 for their action and localization, but not the reverse. They also find that the centrioles of mutant cells are significantly diminished, missing quite a bit of the outside structure. This suggests that Sas7 is a structural component at just the right position, around the outsides of the mother centriole, to participate in the construction of daughter centrioles.

Complete deletion of Sas7 renders the organism dead. But a partially inactivating mutation (temperature sensitive) allows its function to be observed. Here, the wild-type cells show flamboyant microtubule (green) organization during mitosis. The mutant at the restrictive temperature shows a mess, where centriole duplication has failed and the DNA (orange) is dispersed around one pole instead of being nicely pulled between two poles.

Mutant Sas7 also causes structural problems for centrioles. The outsides of the lower centrioles are severely depleted of material, whatever it is.

But that leaves one last question- what licenses / initiates the beginning of centriole duplcation, given this need for Sas7 and Spd2 interaction and given the need for tight coupling with the cell cycle, and why does it happen at only one position on the side of the mother, rather than all over? This article does not touch on those issues, and one has to revert to other reviews to gain some insight. Zyg1 is known in other species as Plk4, and seems to be the critical link to the cell cycle. Whether its activation is driven in particular ways is not yet known, but its destruction is known to be driven by the SCF complex that funnels many critical cell cycle proteins into the proteasomal trash bin at the the transition between G2 and M phase (with partner betaTrCP). However, how the localization is restricted to one position on the mother is not known at all. The only relevant fact is that supplying an excess of Plk4 can prompt initiation of multiple daughters. Thus one reviewer is reduced to speculating about a concentration-sensitive positive feedback mechanism that forces all the activity to localize in one place.

So, even after all these years, of both macroscopic and molecular study, this beauty still holds quite a bit of mystery. Will resolving it make us happier?

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