Browse
Submit Data
Databases
API
Help

Dataset Information

24 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

GTP-dependent formation of straight tubulin oligomers leads to microtubule nucleation.

ABSTRACT: Nucleation of microtubules (MTs) is essential for cellular activities, but its mechanism is unknown because of the difficulty involved in capturing rare stochastic events in the early stage of polymerization. Here, combining rapid flush negative stain electron microscopy (EM) and kinetic analysis, we demonstrate that the formation of straight oligomers of critical size is essential for nucleation. Both GDP and GTP tubulin form single-stranded oligomers with a broad range of curvatures, but upon nucleation, the curvature distribution of GTP oligomers is shifted to produce a minor population of straight oligomers. With tubulin having the Y222F mutation in the β subunit, the proportion of straight oligomers increases and nucleation accelerates. Our results support a model in which GTP binding generates a minor population of straight oligomers compatible with lateral association and further growth to MTs. This study suggests that cellular factors involved in nucleation promote it via stabilization of straight oligomers.

SUBMITTER: Ayukawa R

PROVIDER: S-EPMC7871348 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

γ-Tubulin in microtubule nucleation and beyond.

Project description:Microtubules composed of αβ-tubulin dimers are dynamic cytoskeletal polymers that play key roles in essential cellular processes such as cell division, organelle positioning, intracellular transport, and cell migration. γ-Tubulin is a highly conserved member of the tubulin family that is required for microtubule nucleation. γ-Tubulin, together with its associated proteins, forms the γ-tubulin ring complex (γ-TuRC), that templates microtubules. Here we review recent advances in the structure of γ-TuRC, its activation, and centrosomal recruitment. This provides new mechanistic insights into the molecular mechanism of microtubule nucleation. Accumulating data suggest that γ-tubulin also has other, less well understood functions. We discuss emerging evidence that γ-tubulin can form oligomers and filaments, has specific nuclear functions, and might be involved in centrosomal cross-talk between microtubules and microfilaments.

| S-EPMC9503634 | biostudies-literature

Insight into microtubule nucleation from tubulin-capping proteins.

Project description:Nucleation is one of the least understood steps of microtubule dynamics. It is a kinetically unfavorable process that is templated in the cell by the γ-tubulin ring complex or by preexisting microtubules; it also occurs in vitro from pure tubulin. Here we study the nucleation inhibition potency of natural or artificial proteins in connection with their binding mode to the longitudinal surface of α- or β-tubulin. The structure of tubulin-bound CopN, a Chlamydia protein that delays nucleation, suggests that this protein may interfere with two protofilaments at the (+) end of a nucleus. Designed ankyrin repeat proteins that share a binding mode similar to that of CopN also impede nucleation, whereas those that target only one protofilament do not. In addition, an αRep protein predicted to target two protofilaments at the (-) end does not delay nucleation, pointing to different behaviors at both ends of the nucleus. Our results link the interference with protofilaments at the (+) end and the inhibition of nucleation.

| S-EPMC6525497 | biostudies-literature

Microtubule stabilization in vivo by nucleation-incompetent ?-tubulin complex.

Project description:Although the fission yeast Schizosaccharomyces pombe contains many of the ?-tubulin ring complex (?-TuRC)-specific proteins of the ?-tubulin complex (?-TuC), several questions about the organizational state and function of the fission yeast ?-TuC in vivo remain unresolved. Using 3×GFP-tagged ?-TuRC-specific proteins, we show here that ?-TuRC-specific proteins are present at all microtubule organizing centers in fission yeast and that association of ?-TuRC-specific proteins with the ?-tubulin small complex (?-TuSC) does not depend on Mto1, which is a key regulator of the ?-TuC. Through sensitive imaging in mto1? mutants, in which cytoplasmic microtubule nucleation is abolished, we unexpectedly found that ?-TuC incapable of nucleating microtubules can nevertheless associate with microtubule minus-ends in vivo. The presence of ?-TuC at microtubule ends is independent of ?-TuRC-specific proteins and strongly correlates with the stability of microtubule ends. Strikingly, microtubule bundles lacking ?-TuC at microtubule ends undergo extensive treadmilling in vivo, apparently induced by geometrical constraints on plus-end growth. Our results indicate that microtubule stabilization by the ?-TuC, independently of its nucleation function, is important for maintaining the organization and dynamic behavior of microtubule arrays in vivo.

| S-EPMC3065382 | biostudies-literature

Severing enzymes amplify microtubule arrays through lattice GTP-tubulin incorporation.

Project description:Spastin and katanin sever and destabilize microtubules. Paradoxically, despite their destructive activity they increase microtubule mass in vivo. We combined single-molecule total internal reflection fluorescence microscopy and electron microscopy to show that the elemental step in microtubule severing is the generation of nanoscale damage throughout the microtubule by active extraction of tubulin heterodimers. These damage sites are repaired spontaneously by guanosine triphosphate (GTP)-tubulin incorporation, which rejuvenates and stabilizes the microtubule shaft. Consequently, spastin and katanin increase microtubule rescue rates. Furthermore, newly severed ends emerge with a high density of GTP-tubulin that protects them against depolymerization. The stabilization of the newly severed plus ends and the higher rescue frequency synergize to amplify microtubule number and mass. Thus, severing enzymes regulate microtubule architecture and dynamics by promoting GTP-tubulin incorporation within the microtubule shaft.

| S-EPMC6510489 | biostudies-literature

XMAP215 and γ-tubulin additively promote microtubule nucleation in purified solutions.

Project description:Microtubule nucleation is spatiotemporally regulated in cells by several known molecules, including the template γ-tubulin and the polymerase XMAP215. The role of XMAP215 in nucleation is under debate, specifically whether it acts independently as a polymerase or acts dependently with γ-tubulin. We first confirm XMAP215 as a classically defined nucleator that reduces the nucleation lag seen in bulk tubulin assembly. Secondly, using deletion constructs, we probe the domain requirements for XMAP215 to promote microtubule nucleation. We show that its ability to nucleate microtubules in purified solutions correlates with its ability to elongate existing microtubules and does not depend on the number of tumor overexpressed gene (TOG) domains. Finally, we show that XMAP215 and γ-tubulin promote αβ-tubulin assembly in an additive, not synergistic, manner. Thus, their modes of action during microtubule nucleation are distinct. These findings suggest there are at least two independent processes in nucleation, one promoted by γ-tubulin and one promoted by XMAP215. We propose that XMAP215 accelerates the addition of subunits to existing nucleation intermediates formed either spontaneously or by oligomers of γ-tubulin. [Media: see text].

| S-EPMC7550701 | biostudies-literature

Augmin-dependent microtubule nucleation at microtubule walls in the spindle.

Project description:The formation of a functional spindle requires microtubule (MT) nucleation from within the spindle, which depends on augmin. How augmin contributes to MT formation and organization is not known because augmin-dependent MTs have never been specifically visualized. In this paper, we identify augmin-dependent MTs and their connections to other MTs by electron tomography and 3D modeling. In metaphase spindles of human cells, the minus ends of MTs were located both around the centriole and in the body of the spindle. When augmin was knocked down, the latter population of MTs was significantly reduced. In control cells, we identified connections between the wall of one MT and the minus end of a neighboring MT. Interestingly, the connected MTs were nearly parallel, unlike other examples of end-wall connections between cytoskeletal polymers. Our observations support the concept of augmin-dependent MT nucleation at the walls of existing spindle MTs. Furthermore, they suggest a mechanism for maintaining polarized MT organization, even when noncentrosomal MT initiation is widespread.

| S-EPMC3704994 | biostudies-literature

Structural rearrangements in tubulin following microtubule formation.

Project description:Microtubules are essential cytoskeletal structures that mediate several dynamic processes in a cell. To shed light on the structural processes relating to microtubule formation and dynamic instability, we investigated microtubules composed of 15 protofilaments using cryo-electron microscopy, helical image reconstruction and computational modelling. Analysis of the configuration of the alpha beta-tubulin heterodimer shows distinct structural differences in both subunits, and illustrates that the tubulin subunits have different roles in the microtubule lattice. Our modelling data suggest that after GTP hydrolysis microtubules, adopt a conformational state somewhere between a straight protofilament conformation--as found in zinc-induced tubulin sheets--and an outward curved conformation--as found in tubulin-stathmin complexes. The tendency towards a curved conformation seems to be mediated mostly by beta-tubulin, whereas alpha-tubulin resembles a state more related to the straight structure. Our data suggest a possible explanation of dynamic instability of microtubules, and for nucleotide-sensitive microtubule-binding properties of microtubule-associated proteins and molecular motors.

| S-EPMC1299270 | biostudies-literature

Crystal structure of ?-tubulin complex protein GCP4 provides insight into microtubule nucleation.

Project description:Microtubule nucleation in all eukaryotes involves ?-tubulin small complexes (?TuSCs) that comprise two molecules of ?-tubulin bound to ?-tubulin complex proteins (GCPs) GCP2 and GCP3. In many eukaryotes, multiple ?TuSCs associate with GCP4, GCP5 and GCP6 into large ?-tubulin ring complexes (?TuRCs). Recent cryo-EM studies indicate that a scaffold similar to ?TuRCs is formed by lateral association of ?TuSCs, with the C-terminal regions of GCP2 and GCP3 binding ?-tubulin molecules. However, the exact role of GCPs in microtubule nucleation remains unknown. Here we report the crystal structure of human GCP4 and show that its C-terminal domain binds directly to ?-tubulin. The human GCP4 structure is the prototype for all GCPs, as it can be precisely positioned within the ?TuSC envelope, revealing the nature of protein-protein interactions and conformational changes regulating nucleation activity.

| S-EPMC3703858 | biostudies-literature

CAMSAP2 organizes a γ-tubulin-independent microtubule nucleation centre through phase separation.

Project description:Microtubules are dynamic polymers consisting of αβ-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αβ-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However, a considerable number of microtubules can polymerize independently of the centrosome in various cell types. Here, we present evidence that the minus-end-binding calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) serves as a strong nucleator for microtubule formation by significantly reducing the nucleation barrier. CAMSAP2 co-condensates with αβ-tubulin via a phase separation process, producing plenty of nucleation intermediates. Microtubules then radiate from the co-condensates, resulting in aster-like structure formation. CAMSAP2 localizes at the co-condensates and decorates the radiating microtubule lattices to some extent. Taken together, these in vitro findings suggest that CAMSAP2 supports microtubule nucleation and growth by organizing a nucleation centre as well as by stabilizing microtubule intermediates and growing microtubules.

| S-EPMC9239687 | biostudies-literature

c-Abl kinase-mediated phosphorylation of γ-tubulin promotes γ-tubulin ring complexes assembly and microtubule nucleation.

Project description:Cytoskeletal microtubules (MTs) are nucleated from γ-tubulin ring complexes (γTuRCs) located at MT organizing centers (MTOCs), such as the centrosome. However, the exact regulatory mechanism of γTuRC assembly is not fully understood. Here, we showed that the nonreceptor tyrosine kinase c-Abl was associated with and phosphorylated γ-tubulin, the essential component of the γTuRC, mainly on the Y443 residue by in vivo (immunofluorescence and immunoprecipitation) or in vitro (surface plasmon resonance) detection. We further demonstrated that phosphorylation deficiency significantly impaired γTuRC assembly, centrosome construction, and MT nucleation. c-Abl/Arg deletion and γ-tubulin Y443F mutation resulted in an abnormal morphology and compromised spindle function during mitosis, eventually causing uneven chromosome segregation. Our findings reveal that γTuRC assembly and nucleation function are regulated by Abl kinase-mediated γ-tubulin phosphorylation, revealing a fundamental mechanism that contributes to the maintenance of MT function.

| S-EPMC8980629 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data