Jens Lüders

Microtubule-organizing centres: a re-evaluation

The number, length, distribution and polarity of microtubules are largely controlled by microtubule-organizing centres, which nucleate and anchor microtubule minus ends in a process that requires γ-tubulin. Here we discuss recent evidence indicating that γ-tubulin-dependent formation of new microtubules is not restricted to conventional microtubule-organizing centres. These findings suggest that the spatio-temporal control of microtubule nucleation is more complex than previously thought, leading us to a re-evaluation of the concept of the microtubule-organizing center.

GCP-WD is a gamma-tubulin targeting factor required for centrosomal and chromatin-mediated microtubule nucleation

The γ-tubulin ring complex (γTuRC) is a large multi-protein complex that is required for microtubule nucleation from the centrosome. Here, we show that the GCP-WD protein (originally named NEDD1) is the orthologue of the Drosophila Dgrip71WD protein, and is a subunit of the human γTuRC. GCP-WD has the properties of an attachment factor for the γTuRC: depletion or inhibition of GCP-WD results in loss of the γTuRC from the centrosome, abolishing centrosomal microtubule nucleation, although the γTuRC is intact and able to bind to microtubules. GCP-WD depletion also blocks mitotic chromatin-mediated microtubule nucleation, resulting in failure of spindle assembly. Mitotic phosphorylation of GCP-WD is required for association of γ-tubulin with the spindle, separately from association with the centrosome. Our results indicate that GCP-WD broadly mediates targeting of the γTuRC to sites of microtubule nucleation and to the mitotic spindle, which is essential for spindle formation.

The Carboxy-Terminal Domain of Hsc70 Provides Binding Sites for a Distinct Set of Chaperone Cofactors

ABSTRACT The modulation of the chaperone activity of the heat shock cognate Hsc70 protein in mammalian cells involves cooperation with chaperone cofactors, such as Hsp40; BAG-1; the Hsc70-interacting protein, Hip; and the Hsc70-Hsp90-organizing protein, Hop. By employing the yeast two-hybrid system and in vitro interaction assays, we have provided insight into the structural basis that underlies Hsc70’s cooperation with different cofactors. The carboxy-terminal domain of Hsc70, previously shown to form a lid over the peptide binding pocket of the chaperone protein, mediates the interaction of Hsc70 with Hsp40 and Hop. Remarkably, the two cofactors bind to the carboxy terminus of Hsc70 in a noncompetitive manner, revealing the existence of distinct binding sites for Hsp40 and Hop within this domain. In contrast, Hip interacts exclusively with the amino-terminal ATPase domain of Hsc70. Hence, Hsc70 possesses separate nonoverlapping binding sites for Hsp40, Hip, and Hop. This appears to enable the chaperone protein to cooperate simultaneously with multiple cofactors. On the other hand, BAG-1 and Hip have recently been shown to compete in binding to the ATPase domain. Our data thus establish the existence of a network of cooperating and competing cofactors regulating the chaperone activity of Hsc70 in the mammalian cell.

Plk1-Dependent Recruitment of γ-Tubulin Complexes to Mitotic Centrosomes Involves Multiple PCM Components

The nucleation of microtubules requires protein complexes containing γ-tubulin, which are present in the cytoplasm and associate with the centrosome and with the mitotic spindle. We have previously shown that these interactions require the γ-tubulin targeting factor GCP-WD/NEDD1, which has an essential role in spindle formation. The recruitment of additional γ-tubulin to the centrosomes occurs during centrosome maturation at the G2/M transition and is regulated by the mitotic kinase Plk1. However, the molecular details of this important pathway are unknown and a Plk1 substrate that controls γ-tubulin recruitment has not been identified. Here we show that Plk1 associates with GCP-WD in mitosis and Plk1 activity contributes to phosphorylation of GCP-WD. Plk1 depletion or inhibition prevents accumulation of GCP-WD at mitotic centrosomes, but GCP-WD mutants that are defective in Plk1-binding and -phosphorylation still accumulate at mitotic centrosomes and recruit γ-tubulin. Moreover, Plk1 also controls the recruitment of other PCM proteins implicated in centrosomal γ-tubulin attachment (Cep192/hSPD2, pericentrin, Cep215/Cdk5Rap2). Our results support a model in which Plk1-dependent recruitment of γ-tubulin to mitotic centrosomes is regulated upstream of GCP-WD, involves multiple PCM proteins and therefore potentially multiple Plk1 substrates.

The where, when and how of microtubule nucleation – one ring to rule them all

Summary The function of microtubules depends on their arrangement into highly ordered arrays. Spatio-temporal control over the formation of new microtubules and regulation of their properties are central to the organization of these arrays. The nucleation of new microtubules requires &ggr;-tubulin, an essential protein that assembles into multi-subunit complexes and is found in all eukaryotic organisms. However, the way in which &ggr;-tubulin complexes are regulated and how this affects nucleation and, potentially, microtubule behavior, is poorly understood. &ggr;-tubulin has been found in complexes of various sizes but several lines of evidence suggest that only large, ring-shaped complexes function as efficient microtubule nucleators. Human &ggr;-tubulin ring complexes (&ggr;TuRCs) are composed of &ggr;-tubulin and the &ggr;-tubulin complex components (GCPs) 2, 3, 4, 5 and 6, which are members of a conserved protein family. Recent work has identified additional unrelated &ggr;TuRC subunits, as well as a large number of more transient &ggr;TuRC interactors. In this Commentary, we discuss the regulation of &ggr;TuRC-dependent microtubule nucleation as a key mechanism of microtubule organization. Specifically, we focus on the regulatory roles of the &ggr;TuRC subunits and interactors and present an overview of other mechanisms that regulate &ggr;TuRC-dependent microtubule nucleation and organization.

CEP120 and SPICE1 Cooperate with CPAP in Centriole Elongation

Centrosomes organize microtubule (MT) arrays and are comprised of centrioles surrounded by ordered pericentriolar proteins. Centrioles are barrel-shaped structures composed of MTs, and as basal bodies they template the formation of cilia/flagella. Defects in centriole assembly can lead to ciliopathies and genome instability. The assembly of procentrioles requires a set of conserved proteins. It is initiated at the G1-to-S transition by PLK4 and CEP152, which help recruit SASS6 and STIL to the vicinity of the mother centriole to organize the cartwheel. Subsequently, CPAP promotes centriolar MT assembly and elongation in G2. While centriole integrity is maintained by CEP135 and POC1 through MT stabilization, centriole elongation requires POC5 and is restricted by CP110 and CEP97. How strict control of centriole length is achieved remains unclear. Here, we show that CEP120 and SPICE1 are required to localize CEP135 (but not SASS6, STIL, or CPAP) to procentrioles. CEP120 associates with SPICE1 and CPAP, and depletion of any of these proteins results in short procentrioles. Furthermore, CEP120 or CPAP overexpression results in excessive centriole elongation, a process dependent on CEP120, SPICE1, and CPAP. Our findings identify a shared function for these proteins in centriole length control.

The γTuRC Revisited: A Comparative Analysis of Interphase and Mitotic Human γTuRC Redefines the Set of Core Components and Identifies the Novel Subunit GCP8

We compared the composition of γ-tubulin ring complexes tandem-affinity purified from asynchronous and mitotic human cells by mass spectrometry. We identified various interactors including the novel core subunit GCP8. GCP8 is the first subunit with an interphase-specific role in centrosomal γ-tubulin recruitment and microtubule nucleation.

The ubiquitin‐like protein HUB1 forms SDS‐resistant complexes with cellular proteins in the absence of ATP

Ubiquitin and ubiquitin‐like modifiers (UBLs) form covalent complexes with other proteins by isopeptide formation between their carboxyl (C)‐termini and ε‐amino groups of lysine residues of acceptor proteins. A hallmark of UBLs is a protruding C‐terminal tail with a terminal glycine residue, which is required for ATP‐dependent conjugation. Recently, the highly conserved protein HUB1 (homologous to ubiquitin 1) has been reported to function as a UBL following C‐terminal processing. HUB1 exhibits sequence similarity with ubiquitin but lacks a C‐terminal tail bearing a glycine residue. Here we show that HUB1 can form SDS‐resistant complexes with cellular proteins, but provide evidence that these adducts are not formed through covalent C‐terminal conjugation of HUB1 to substrates. The adducts are still formed when the C‐terminus of HUB1 was altered by epitope tagging, amino‐acid exchange or deletion, or when cells were depleted of ATP. We propose that HUB1 may act as a novel protein modulator through the formation of tight, possibly noncovalent interactions with target proteins.

The dynamics of microtubule minus ends in the human mitotic spindle

During mitotic spindle assembly, γ-tubulin ring complexes (γTuRCs) nucleate microtubules at centrosomes, around chromosomes, and, by interaction with augmin, from pre-existing microtubules. How different populations of microtubules are organized to form a bipolar spindle is poorly understood, in part because we lack information on the dynamics of microtubule minus ends. Here we show that γTuRC is associated with minus ends of non-centrosomal spindle microtubules. Recruitment of γTuRC to spindles occurs preferentially at pole-distal regions, requires nucleation and/or interaction with minus ends, and is followed by sorting of minus-end-bound γTuRC towards the poles. Poleward movement of γTuRC exceeds k-fibre flux, involves the motors dynein, HSET (also known as KIFC1; a kinesin-14 family member) and Eg5 (also known as KIF11; a kinesin-5 family member), and slows down in pole-proximal regions, resulting in the accumulation of minus ends. Thus, in addition to nucleation, γTuRC actively contributes to spindle architecture by organizing microtubule minus ends.

Cep63 and Cep152 Cooperate to Ensure Centriole Duplication

Centrosomes consist of two centrioles embedded in pericentriolar material and function as the main microtubule organising centres in dividing animal cells. They ensure proper formation and orientation of the mitotic spindle and are therefore essential for the maintenance of genome stability. Centrosome function is crucial during embryonic development, highlighted by the discovery of mutations in genes encoding centrosome or spindle pole proteins that cause autosomal recessive primary microcephaly, including Cep63 and Cep152. In this study we show that Cep63 functions to ensure that centriole duplication occurs reliably in dividing mammalian cells. We show that the interaction between Cep63 and Cep152 can occur independently of centrosome localisation and that the two proteins are dependent on one another for centrosomal localisation. Further, both mouse and human Cep63 and Cep152 cooperate to ensure efficient centriole duplication by promoting the accumulation of essential centriole duplication factors upstream of SAS-6 recruitment and procentriole formation. These observations describe the requirement for Cep63 in maintaining centriole number in dividing mammalian cells and further establish the order of events in centriole formation.