Biology subjects

Allingham, J. S.

Publications and source records attributed to Allingham, J. S..

3 recordsLinked to original sources

bioRxiv2024

Kinesin-8 motors dimerize by folding their proximal tail domain into a compact helical bundle

Kinesin-8 motor proteins help align and segregate chromosomes during mitosis by regulating the dynamics of kinetochore-attached microtubules and the length and position of the mitotic spindle. Some kinesin-8 isoforms accomplish these roles by operating as multifunctional mechanoenzymes that can traverse microtubules, accumulate at the microtubule plus-ends, and then remove terminal {beta}-tubulin subunits. While these activities are mainly powered by the motor domain, whose unique structure-function relationships have been recently reported, the non-motor tail domain contains integral functional elements that have not been structurally illuminated. Using the Candida albicans Kip3 protein as a kinesin-8 model system, we present an X-ray crystal structure and hydrodynamic data showing how the motor domain-proximal segment of the tail directs the assembly of two kinesin-8 polypeptides into a homodimer that forms the stalk of this motor. Unlike the extended coiled coil-forming helices of most other motile kinesin stalks, the proximal tail of CaKip3 folds into a compact 92 [A]-long four-helix bundle that dimerizes. The first and third helices provide most of the surface area for the dimer interface, while the other two helices brace the folded stalk structure. The upper and lower lobules of the helical bundle are separated by a flexible hinge that gives the exterior faces of the stalk slightly different shapes when bent. We propose that these unique characteristics provide structural rigidity to the kinesin-8 stalk, as well as sites for transient interactions with kinesin-8-associated proteins or other regulatory regions of the motor.

biochemistry↗

bioRxiv2024

Dynein contributes to spindle elongation during anaphase in Candida albicans

AbstractBudding yeast must position the mitotic spindle at the mother-bud junction during anaphase to ensure proper chromosome segregation. Pushing and pulling forces on astral microtubules from dynein motors in the cell cortex are crucial for spindle positioning. In some higher eukaryotes and filamentous fungi, dynein also accelerates spindle elongation during anaphase, but in yeast model systems spindle elongation primarily relies on intra-spindle pushing forces from kinesin-5 motors. The pathogenic yeast Candida albicans is unique because its spindle can elongate without a functional kinesin-5. Our study helps explain C. albicans diminished kinesin-5 requirement by showing that its dynein, Dyn1, collaborates with its kinesin-5, Kip1, to facilitate spindle elongation. Cells lacking Kip1 activity by either KIP1 deletion or Kip1 inhibition have more Dyn1 and astral microtubules, indicating spindle elongation happens through enhanced pulling on spindle pole body-bound astral microtubules by cortical Dyn1. When Dyn1 activity is depleted, spindle elongation speed is normal, but anaphase spindles persist for extended periods, and the number and length of astral microtubules increases. Depletion of both motors prevents spindle elongation and is lethal. The significance of astral microtubules in cells lacking either motor is highlighted by the lethal effects of microtubule-destabilizing drugs and exposure to low temperatures (8{degrees}C) that compromise microtubule stability. These findings demonstrate that C. albicans spindle elongation involves cooperative forces from Kip1 and Dyn1 to ensure chromosome separation during cell division and that C. albicans adapts to changes in abundance of these motors through alterations in their astral microtubule content.

microbiology↗

bioRxiv2022

Kinesin-8-specific loop-2 controls the dual activities of the motor domain according to tubulin protofilament shape

Kinesin-8s are dual-activity motor proteins that can move processively on microtubules and depolymerize microtubule plus-ends, but their mechanism of combining these distinct activities remains unclear. We addressed this by obtaining cryo-EM structures (2.6-3.9 [A]) of Candida albicans Kip3 in different catalytic states on the microtubule lattice and on a curved microtubule end mimic, as well as a microtubule-unbound CaKip3-ADP crystal structure (2.0 [A]). Together with biochemical analyses of CaKip3 and kinesin-1 mutants, we define a model that explains the kinesin-8 mechanism. The microtubule depolymerization activity originates in conformational changes of the kinesin-8 motor core that are amplified by its dynamic loop-2. On curved microtubule ends, loop-1 assists depolymerization by inserting into preceding motor domains, forming head-to-tail arrays of kinesin-8s that complement loop-2 contacts with curved tubulin. On straight tubulin protofilaments in the microtubule lattice, extended loop-2-tubulin contacts inhibit conformational changes in the motor core, but in the ADP-Pi state these contacts are relaxed, allowing neck-linker docking for motility. These tubulin shape-induced alternations between pro-microtubule-depolymerization and pro-motility kinesin states, regulated by loop-2, are the key to the dual activity of kinesin-8 motors.

biochemistry↗