Tubulin cofactors and Arl2 form a cage-like chaperone that catalyzes changes in tubulin state through GTP hydrolysis: Reconstitution of new molecular machine that assembles the a-tubulin dimer

Published in eLife

The building block of microtubules is composed of two proteins called a-tubulin and -tubulin which are assembled into a single polarized a-tubulin subunit. Microtubules are very dynamic structures that can rapidly change length as individual a-tubulin units are either added or removed to their growing or shrinking ends. The assembly of a-tubulin has for long remained a mystery despite extensive genetic and cell biology studies. Several proteins known as tubulin cofactors and an enzyme called Arl2 help to build a vast pool of a-tubulin that are able attach to the microtubules. It has remained unknown how this collection of proteins drives a-tubulin biogenesis and degradation inside cells. And the roles of the conserved tubulin cofactors and Arl2 remain unknown.

Our group used a combination of biochemical reconstitution of all tubulin cofactors and Arl2 GTPase in combination with negative stain electron microscopy and biochemical techniques to study how the tubulin cofactors and Arl2 are organised, and their role in the assembly of a-tubulin and polymerization of dynamic microtubules inside living yeast cells. Our biochemical reconstitution has revealed that Arl2 and two tubulin cofactors TBCD and TBCE associate with each other to form a stable 'complex' that has a cage-like structure. A molecule of a-tubulin binds to the complex, followed by another cofactor called TBCC. The binding of a-tubulin and TBCC activates the enzyme activity of Arl2, which releases the energy needed to alter the shape of the a-tubulin in a larger ternary complex. We also found that inactivating Arl2 GTP hydrolysis inhibits the dissociation of this ternary complex and introducing this mutant form of Arl2 that lacked enzyme activity inside yeast cells had problems forming microtubules characterized by poor polymerization due to pausing and defects in cell division.

Together, our findings show that the tubulin cofactors and Arl2 form a complex that regulates the assembly and maintenance of a-tubulin. The next challenge is to understand how this regulation influences the way that microtubules grow and shrink inside cells.


Structural Basis for the Assembly of the Mitotic motor Kinesin-5 into Bipolar Tetramers

Published in eLife

During cell division, an organized and symmetric intracellular apparatus, named the mitotic spindle, composed of microtubules (and associated molecular machines), coordinate the accurate alignment and equal distribution of newly duplicated chromosomes into two new cells. When the organization of mitotic spindles becomes mis-regulated, a variety of human disease conditions can result, including early onset cancers and human birth defects. Our laboratory studies the molecular mechanisms of microtubule dynamics and organization during mitotic cell division.

Molecular motors in living cells bind and generate motile forces through "step-by-step walking" along microtubules. These motor activities move chromosomes and mechanically orchestrate events during mitotic cell division. We are focused on an evolutionarily well-conserved molecular motor class, Kinesin-5, which are fundamentally responsible for elongating mitotic spindles to form two new cells. The organization of mitotic motor kinesin-5 is unique from all other molecular motors in that they form elongated bipolar molecular assemblies, composed of four subunits, with two sets of active motile motors pointed towards opposite ends of a rigid central rod. The central rod is critical for transmitting forces between the motile ends. The "dual-headed" arrangement of kinesin-5 is critical for its function as it preferentially binds microtubules emanating from opposite poles of the mitotic spindle and mediates their anti-parallel sliding near the end of the cell division cycle.

Mitotic Overview

Our laboratory made a major breakthrough, in collaboration with Jonathan Scholey's group at UC-Davis, in determining the atomic three-dimensional structure four-subunit kinesin-5 central rod region, which specifies the mitotic kinesin-5 organization into "dual headed" bipolar molecular machines. We discovered a precise three-dimensional organization where four kinesin-5 subunits form bipolar assemblies. We observed an alternating pattern of seven symmetrically organized interface elements composed of uniquely interacting amino acids. Four out of seven of these interface elements are ionic in nature and form exposed "molecular pockets" that are accessible to small chemical molecules from the solution. Mutations of the ionic interacting amino acids severely disrupt the kinesin-5 four-subunit organization, leading misassembled kinesin-5 molecules. The kinesin-5 structure suggests a novel strategy in targeting kinesin-5 inactivation that is conceptually more effective in controlling mitotic spindle elongation than the previous strategies as it is specifically focused on disrupting kinesin-5 assembly by disassembling its central bipolar rod filament, the main region that transmits force between the two kinesin-5 motile ends.