γ-Tubulin typically forms a ring-shaped complex with 5 related γ-tubulin complex proteins (GCP2 to GCP6), and this γ-tubulin ring complex (γTuRC) serves as a template for microtubule (MT) nucleation in plants and animals. While the γTuRC takes part in MT nucleation in most eukaryotes, in fungi such events take place robustly with just the γ-tubulin small complex (γTuSC) assembled by γ-tubulin plus GCP2 and GCP3. To explore whether the γTuRC is the sole functional γ-tubulin complex in plants, we generated 2 mutants of the
- Award ID(s):
- 1734030
- NSF-PAR ID:
- 10169845
- Date Published:
- Journal Name:
- eLife
- Volume:
- 9
- ISSN:
- 2050-084X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
GCP6 gene encoding the largest subunit of the γTuRC inArabidopsis thaliana . Both mutants showed similar phenotypes of dwarfed vegetative growth and reduced fertility. Thegcp6 mutant assembled the γTuSC, while the wild-type cells had GCP6 join other GCPs to produce the γTuRC. Although thegcp6 cells had greatly diminished γ-tubulin localization on spindle MTs, the protein was still detected there. Thegcp6 cells formed spindles that lacked MT convergence and discernable poles; however, they managed to cope with the challenge of MT disorganization and were able to complete mitosis and cytokinesis. Our results reveal that the γTuRC is not the only functional form of the γ-tubulin complex for MT nucleation in plant cells, and that γ-tubulin-dependent, but γTuRC-independent, mechanisms meet the basal need of MT nucleation. Moreover, we show that the γTuRC function is more critical for the assembly of spindle MT array than for the phragmoplast. Thus, our findings provide insight into acentrosomal MT nucleation and organization. -
To understand how chromosomes are segregated, it is necessary to explain the precise spatiotemporal organization of microtubules (MTs) in the mitotic spindle. We use Xenopus egg extracts to study the nucleation and dynamics of MTs in branched networks, a process that is critical for spindle assembly. Surprisingly, new branched MTs preferentially originate near the minus-ends of pre-existing MTs. A sequential reaction model, consisting of deposition of nucleation sites on an existing MT, followed by rate-limiting nucleation of branches, reproduces the measured spatial profile of nucleation, the distribution of MT plus-ends and tubulin intensity. By regulating the availability of the branching effectors TPX2, augmin and γ-TuRC, combined with single-molecule observations, we show that first TPX2 is deposited on pre-existing MTs, followed by binding of augmin/γ-TuRC to result in the nucleation of branched MTs. In sum, regulating the localization and kinetics of nucleation effectors governs the architecture of branched MT networks.more » « less
-
more » « less
Contents Summary I. Introduction II. MT arrays in plant cells III. γ‐Tubulin and MT nucleation IV. MT nucleation sites or flexible MTOCs in plant cells V. MT‐dependent MT nucleation VI. Generating new MTs for spindle assembly VII. Generation of MTs for phragmoplast expansion during cytokinesis VIII. MT generation for the cortical MT array IX. MT nucleation: looking forward Acknowledgements References Summary Cytoskeletal microtubules (
MT s) have a multitude of functions including intracellular distribution of molecules and organelles, cell morphogenesis, as well as segregation of the genetic material and separation of the cytoplasm during cell division among eukaryotic organisms. In response to internal and external cues, eukaryotic cells remodel theirMT network in a regulated manner in order to assemble physiologically important arrays for cell growth, cell proliferation, or for cells to cope with biotic or abiotic stresses. Nucleation of newMT s is a critical step forMT remodeling. Although many key factors contributing toMT nucleation and organization are well conserved in different kingdoms, the centrosome, representing the most prominent microtubule organizing centers (MTOC s), disappeared during plant evolution as angiosperms lack the structure. Instead, flexibleMTOC s may emerge on the plasma membrane, the nuclear envelope, and even organelles depending on types of cells and organisms and/or physiological conditions.MT ‐dependentMT nucleation is particularly noticeable in plant cells because it accounts for the primary source ofMT generation for assembling spindle, phragmoplast, and cortical arrays when the γ‐tubulin ring complex is anchored and activated by the augmin complex. It is intriguing what proteins are associated with plant‐specificMTOC s and how plant cells activate or inactivateMT nucleation activities in spatiotemporally regulated manners. -
more » « less
Abstract By virtue of their native structures, tubulin dimers are protein building blocks that are naturally preprogrammed to assemble into microtubules (MTs), which are cytoskeletal polymers. Here, polycation‐directed (i.e., electrostatically tunable) assembly of tubulins is demonstrated by conformational changes to the tubulin protofilament in longitudinal and lateral directions, creating tubulin double helices and various tubular architectures. Synchrotron small‐angle X‐ray scattering and transmission electron microscopy reveal a remarkable range of nanoscale assembly structures: single‐ and double‐layered double‐helix tubulin tubules. The phase transitions from MTs to the new assemblies are dependent on the size and concentration of polycations. Two characteristic scales that determine the number of observed phases are the size of polycation compared to the size of tubulin (≈4 nm) and to MT diameter (≈25 nm). This work suggests the feasibility of using polycations that have scissor‐ and glue‐like properties to achieve “programmable breakdown” of protein nanotubes, tearing MTs into double‐stranded tubulins and building up previously undiscovered nanostructures. Importantly, a new role of tubulins is defined as 2D shape‐controllable building blocks for supramolecular architectures. These findings provide insight into the design of protein‐based functional materials, for example, as metallization templates for nanoscale electronic devices, molecular screws, and drug delivery vehicles.
-
The cellular functions of the microtubule (MT) cytoskeleton range from relatively simple to amazingly complex. Assembled from tubulin, a heterodimeric protein with α- and β-tubulin subunits, microtubules are long, hollow cylindrical filaments with inherent polarity. They are intrinsically dynamic polymers that utilize GTP binding by tubulin, and subsequent hydrolysis, to drive spontaneous assembly and disassembly. Early studies indicated that cellular MTs are composed of multiple variants, or isotypes, of α- and β-tubulins, and that these multi-isotype polymers are further diversified by a range of posttranslational modifications (PTMs) to tubulin. These findings support the multi-tubulin hypothesis whereby individual, or combinations of tubulin isotypes possess unique properties needed to support diverse MT structures and/or cellular processes. Beginning 40 years ago researchers have sought to address this hypothesis, and the role of tubulin isotypes, by exploiting experimentally accessible, genetically tractable and functionally conserved model systems. Among these systems, important insights have been gained from eukaryotic microbial models. In this review, we illustrate how using microorganisms yielded among the earliest evidence that tubulin isotypes harbor distinct properties, as well as recent insights as to how they facilitate specific cellular processes. Ongoing and future research in microorganisms will likely continue to reveal basic mechanisms for how tubulin isotypes facilitate MT functions, along with valuable perspectives on how they mediate the range of conserved and diverse processes observed across eukaryotic microbes.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.7554/eLife.54253
