Biology subjects

Roostalu, J.

Publications and source records attributed to Roostalu, J..

4 recordsLinked to original sources

bioRxiv2021

Structural transitions in the GTP cap visualized by cryo-EM of catalytically inactive microtubules

Microtubules (MTs) are polymers of /{beta}-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes, and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here we use cryo-EM and TIRF microscopy of GTP hydrolysis-deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in -tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice, but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wildtype GDP-MTs. End binding proteins of the EB family have the ability to compact both mutant GTP-lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wildtype MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these first high-resolution structures of truly GTP-bound MTs add mechanistic insight to our understanding of MT dynamic instability. Significance StatementMicrotubules (MTs) are non-equilibrium polymers that switch between states of growth and shrinkage. This property is critical for their function and is a consequence of GTP hydrolysis in the MT. The structure of the stable GTP part of the MT (the GTP cap) has previously been inferred from MTs polymerized with non-hydrolyzable GTP analogs. Here, we report the first high-resolution structures of MTs truly containing GTP, polymerized from mutated, hydrolysis-deficient tubulins. We find that GTP-MTs have an "expanded lattice" and a "closed seam", structural characteristics possibly responsible for stabilizing the GTP cap. These results provide new insight into the structural transitions at growing MT ends, furthering our understanding of the bistable nature of MTs.

biophysics↗

bioRxiv2019

Microtubule Nucleation by Single Human γTuRC in a Partly Open Asymmetric Conformation

The {gamma}-tubulin ring complex ({gamma}TuRC) is the major microtubule nucleator in cells. However, the mechanism of its regulation is not understood. Here, we purified human {gamma}TuRC and quantitatively characterized its nucleation properties in a TIRF microscopy-based real-time nucleation assay. We find that microtubule nucleation by {gamma}TuRC is kinetically inhibited compared to microtubule elongation. Determining the cryo-EM structure of {gamma}TuRC at 4 [A] resolution reveals an asymmetric conformation with only part of the complex in a closed conformation matching the microtubule geometry. Several factors stabilise the closed conformation. One is actin in the core of the complex and others, likely MZT1 or MZT2, line the outer perimeter of the closed part of {gamma}TuRC. The opposed side of {gamma}TuRC is in an open, nucleation-incompetent conformation, leading to a structural asymmetry, explaining the kinetic inhibition of nucleation by human {gamma}TuRC. Our data suggest possible regulatory mechanisms for microtubule nucleation by {gamma}TuRC closure.

biochemistry↗

bioRxiv2019

The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability

Microtubules are bistable cytoskeletal polymers whose function depends on their property to switch between states of growth and shrinkage 1. Growing microtubules are thought to be stabilized by a GTP cap at their ends 2-5. The nature of this cap, however, is still poorly understood. How GTP hydrolysis determines the properties of the GTP cap and hence microtubule stability is unclear. End Binding proteins (EBs) recruit a diverse range of regulators of microtubule function to growing microtubule ends 6. Whether these regulatory platforms at growing microtubule ends are identical to the GTP cap is not known. Using mutated human tubulin with blocked GTP hydrolysis, we demonstrate in microscopy-based in vitro reconstitutions that EB proteins bind with high affinity to the GTP conformation of microtubules. Slowing-down GTP hydrolysis leads to extended GTP caps and consequently hyper-stable microtubules. Single molecule experiments reveal that the microtubule conformation gradually changes in the cap as GTP is hydrolyzed. These results demonstrate the critical importance of the kinetics of GTP hydrolysis for microtubule stability; and establish that the GTP cap coincides with the EB-binding regulatory hub that modulates microtubule cytoskeleton function in cells.

biochemistry↗

bioRxiv2017

Combinatorial Regulation Of The Balance Between Dynein Microtubule End Accumulation And Initiation Of Directed Motility

Cytoplasmic dynein is involved in a multitude of essential cellular functions. Dyneins activity is controlled by the combinatorial action of several regulators. The molecular mechanism of this regulation is poorly understood. Using purified proteins, we reconstitute the regulation of the human dynein complex by three prominent regulators on dynamic microtubules in the presence of end binding proteins (EBs). We find that dynein can be in biochemically and functionally distinct pools: either passively tracking dynamic microtubule plus-ends in an EB-dependent manner or moving processively towards minus ends in an adaptor protein-dependent manner. Whereas both dynein pools share the dynactin complex, they have opposite preferences for binding other regulators, either the adaptor protein Bicaudal D2 (BicD2) or the multifunctional regulator Lisencephaly-1 (Lis1). Remarkably, dynactin, but not EBs, strongly biases motility initiation locally from microtubule plus ends by autonomous plus end recognition. BicD2 and Lis1 together control the overall efficiency of motility initiation. Our study provides insight into the mechanism of dynein activity regulation by dissecting the distinct functional contributions of the individual members of a dynein regulatory network.

cell biology↗