Song J-G, Petry S.
Dissecting Protein Complexes in Branching Microtubule Nucleation Using Meiotic Egg Extracts. Cold Spring Harb Protoc 2018;2018(9):pdb.prot100958.
AbstractThe mitotic spindle is the microtubule-based apparatus that reliably segregates chromosomes during cell division. Recently, it was discovered that microtubules originate within the mitotic spindle by nucleating off of existing spindle microtubules. This mechanism, termed branching microtubule nucleation, allows the efficient amplification of microtubules while preserving their original polarity as required in the spindle. Three molecular players are known to be involved in this process, namely, the protein TPX2, the protein complex augmin, and the gamma-tubulin ring complex; however, little is known about the assembly of the protein complexes. Here, we use the eight-subunit augmin complex as an example of how to dissect the function and assembly of a protein complex using meiotic egg extracts. Specifically, immunodepletion combined with total internal reflection fluorescence (TIRF) microscopy is used to identify the role of the protein complex. In parallel, immunoprecipitation (IP) and tandem mass spectrometry (MS/MS) are used to infer how it is assembled. This approach can be applied to investigate the assembly of other multisubunit protein complexes that function in branching microtubule nucleation and mitotic spindle assembly.
Dixit R, Petry S.
The life of a microtubule. Mol Biol Cell 2018;29(6):689.
Rale MJ, Kadzik RS, Petry S.
Phase Transitioning the Centrosome into a Microtubule Nucleator. Biochemistry 2018;57(1):30-37.
AbstractCentrosomes are self-assembling, micron-scale, nonmembrane bound organelles that nucleate microtubules (MTs) and organize the microtubule cytoskeleton of the cell. They orchestrate critical cellular processes such as ciliary-based motility, vesicle trafficking, and cell division. Much is known about the role of the centrosome in these contexts, but we have a less comprehensive understanding of how the centrosome assembles and generates microtubules. Studies over the past 10 years have fundamentally shifted our view of these processes. Subdiffraction imaging has probed the amorphous haze of material surrounding the core of the centrosome revealing a complex, hierarchically organized structure whose composition and size changes profoundly during the transition from interphase to mitosis. New biophysical insights into protein phase transitions, where a diffuse protein spontaneously separates into a locally concentrated, nonmembrane bounded compartment, have provided a fresh perspective into how the centrosome might rapidly condense from diffuse cytoplasmic components. In this Perspective, we focus on recent findings that identify several centrosomal proteins that undergo phase transitions. We discuss how to reconcile these results with the current model of the underlying organization of proteins in the centrosome. Furthermore, we reflect on how these findings impact our understanding of how the centrosome undergoes self-assembly and promotes MT nucleation.
Thawani A, Kadzik RS, Petry S.
XMAP215 is a microtubule nucleation factor that functions synergistically with the γ-tubulin ring complex. Nat Cell Biol 2018;20(5):575-585.
AbstractHow microtubules (MTs) are generated in the cell is a major question in understanding how the cytoskeleton is assembled. For several decades, γ-tubulin has been accepted as the universal MT nucleator of the cell. Although there is evidence that γ-tubulin complexes are not the sole MT nucleators, identification of other nucleation factors has proven difficult. Here, we report that the well-characterized MT polymerase XMAP215 (chTOG/Msps/Stu2p/Alp14/Dis1 homologue) is essential for MT nucleation in Xenopus egg extracts. The concentration of XMAP215 determines the extent of MT nucleation. Even though XMAP215 and the γ-tubulin ring complex (γ-TuRC) possess minimal nucleation activity individually, together, these factors synergistically stimulate MT nucleation in vitro. The amino-terminal TOG domains 1-5 of XMAP215 bind to αβ-tubulin and promote MT polymerization, whereas the conserved carboxy terminus is required for efficient MT nucleation and directly binds to γ-tubulin. In summary, XMAP215 and γ-TuRC together function as the principal nucleation module that generates MTs in cells.
Song J-G, King MR, Zhang R, Kadzik RS, Thawani A, Petry S.
Mechanism of how augmin directly targets the γ-tubulin ring complex to microtubules. J Cell Biol 2018;217(7):2417-2428.
AbstractMicrotubules (MTs) must be generated from precise locations to form the structural frameworks required for cell shape and function. MTs are nucleated by the γ-tubulin ring complex (γ-TuRC), but it remains unclear how γ-TuRC gets to the right location. Augmin has been suggested to be a γ-TuRC targeting factor and is required for MT nucleation from preexisting MTs. To determine augmin's architecture and function, we purified augmin from insect cells. We demonstrate that augmin is sufficient to target γ-TuRC to MTs by in vitro reconstitution. Augmin is composed of two functional parts. One module (tetramer-II) is necessary for MT binding, whereas the other (tetramer-III) interacts with γ-TuRC. Negative-stain electron microscopy reveals that both tetramers fit into the Y-shape of augmin, and MT branching assays reveal that both are necessary for MT nucleation. The finding that augmin can directly bridge MTs with γ-TuRC via these two tetramers adds to our mechanistic understanding of how MTs can be nucleated from preexisting MTs.