TOG Domains Research Articles

Mechanism of how the universal module XMAP215 γ-TuRC nucleates microtubules.

It has become increasingly evident in recent years that nucleation of microtubules from a diverse set of MTOCs requires both the γ-tubulin ring complex (γ-TuRC) and the microtubule polymerase XMAP215. Despite their essentiality, little is known about how these nucleation factors interact and work together to generate microtubules. Using biochemical domain analysis of XMAP215 and structural approaches, we find that a sixth TOG domain in XMAP215 binds γ-TuRC via γ-tubulin as part of a broader interaction involving the C-terminal region. Moreover, TOG6 is required for XMAP215 to promote nucleation from γ-TuRC to its full extent. Interestingly, we find that XMAP215 also depends strongly on TOG5 for microtubule lattice binding and nucleation. Accordingly, we report a cryo-EM structure of TOG5 bound to the microtubule lattice that reveals promotion of lateral interactions between tubulin dimers. Finally, we find that while XMAP215 constructs' effects on nucleation are generally proportional to their effects on polymerization, formation of a direct complex with γ-TuRC allows cooperative nucleation activity. Thus, we propose that XMAP215's C-terminal TOGs 5 and 6 play key roles in promoting nucleation by promoting formation of longitudinal and lateral bonds in γ-TuRC templated nascent microtubules at cellular MTOCs.

Abstract 1647: Plk4 phosphorylates the distal tip complex to promote centriole growth

Abstract The centrosome is the major microtubule-organizing center of the cell with a key role in forming the mitotic spindle. The centrosome consists of two core duplicating elements, the centrioles, surrounded by pericentriolar material, a cloud of proteins that nucleates microtubules. Centrioles are barrel-like structures composed of microtubule bundles and are essential to maintain centrosome number. Therefore, centriole duplication and growth are tightly regulated to ensure proper centrosome number throughout the cell cycle. Alterations in centrosome number lead to an abnormally shaped mitotic spindle that often mis-segregate chromosomes, thereby causing chromosomal instability, a hallmark of cancer. Defects in centriole growth, such as over-elongation, can increase centrosome numbers (known as ‘centriole amplification’, an overproduction of centrioles frequently observed in cancer cells). For example, over-elongation of the centriolar barrel can cause multiple ectopic daughter centrioles to assemble or simply fragment, producing multiple smaller (but functional) centrioles. Thus, understanding how centrioles grow and define length is a major question in the field. Centrioles elongate at their distal ends and, in Drosophila, elongation is regulated by a group of proteins, Cep97, CP110 and Klp10A. Elongation is negatively regulated by Cep97 and CP110 which cap centrioles at their distal end to maintain centriole length, by counteracting the microtubule depolymerizing kinesin, Klp10A. Recently, we identified a new centriole regulator, Cep104, that also localizes to the centriole distal tip. We discovered that Cep104 promotes centriole elongation through its microtubule-binding TOG domain. Additionally, Cep104 localization to the centriole distal ends is dependent on Cep97. Furthermore, Cep97, CP110, Cep104 and Klp10A form a complex and we have mapped their sites of interaction. Collectively, we refer to this as the Distal Tip Complex (DTC). Although the DTC is first recruited to new daughter centrioles during G1/S-phase, the majority of centriole elongation occurs during mitosis. Here, we explore the mechanisms that regulate the DTC to promote centriole growth during mitosis. We hypothesize that Polo-like kinase 4 (Plk4), the master regulator of centriole duplication, phosphorylates and inhibits the DTC to promote elongation of nascent centrioles. Notably, we found that centrioles are longer in cells overexpressing Plk4. Using tandem mass spectrometry, we identified multiple sites that Plk4 phosphorylates on each DTC protein. Analysis of DTC phosphomutants revealed that the phosphorylation state of Klp10A, Cep97 and CP110 affect centriole number. We are currently investigating how Plk4 phosphorylation regulates the activities of these conserved centriole length factors. Citation Format: Anastasia Amoiroglou, John M. Ryniawec, Daniel W. Buster, Gregory C. Rogers. Plk4 phosphorylates the distal tip complex to promote centriole growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1647.

Cep104 is a component of the centriole distal tip complex that regulates centriole growth and contributes to Drosophila spermiogenesis

Proper centrosome number and function relies on the accurate assembly of centrioles, barrel-shaped structures that form the core duplicating elements of the organelle. The growth of centrioles is regulated in a cell cycle-dependent manner; while new daughter centrioles elongate during the S/G2/M phase, mature mother centrioles maintain their length throughout the cell cycle. Centriole length is controlled by the synchronized growth of the microtubules that ensheathe the centriole barrel. Although proteins exist that target the growing distal tips of centrioles, such as CP110 and Cep97, these proteins are generally thought to suppress centriolar microtubule growth, suggesting that distal tips may also contain unidentified counteracting factors that facilitate microtubule polymerization. Currently, a mechanistic understanding of how distal tip proteins balance microtubule growth and shrinkage to either promote daughter centriole elongation or maintain centriole length is lacking. Using a proximity-labeling screen in Drosophila cells, we identified Cep104 as a novel component of a group of evolutionarily conserved proteins that we collectively refer to as the distal tip complex (DTC). We found that Cep104 regulates centriole growth and promotes centriole elongation through its microtubule-binding TOG domain. Furthermore, analysis of Cep104 null flies revealed that Cep104 and Cep97 cooperate during spermiogenesis to align spermatids and coordinate individualization. Lastly, we mapped the complete DTC interactome and showed that Cep97 is the central scaffolding unit required to recruit DTC components to the distal tip of centrioles.

Multifaceted modes of γ-tubulin complex recruitment and microtubule nucleation at mitotic centrosomes.

Microtubule nucleation is mediated by γ-tubulin ring complexes (γ-TuRCs). In most eukaryotes, a GCP4/5/4/6 "core" complex promotes γ-tubulin small complex (γ-TuSC) association to generate cytosolic γ-TuRCs. Unlike γ-TuSCs, however, this core complex is non-essential in various species and absent from budding yeasts. In Drosophila, Spindle defective-2 (Spd-2) and Centrosomin (Cnn) redundantly recruit γ-tubulin complexes to mitotic centrosomes. Here, we show that Spd-2 recruits γ-TuRCs formed via the GCP4/5/4/6 core, but Cnn can recruit γ-TuSCs directly via its well-conserved CM1 domain, similar to its homologs in budding yeast. When centrosomes fail to recruit γ-tubulin complexes, they still nucleate microtubules via the TOG domain protein Mini-spindles (Msps), but these microtubules have different dynamic properties. Our data, therefore, help explain the dispensability of the GCP4/5/4/6 core and highlight the robustness of centrosomes as microtubule organizing centers. They also suggest that the dynamic properties of microtubules are influenced by how they are nucleated.

Microtubule nucleation and γTuRC centrosome localization in interphase cells require ch-TOG

Organization of microtubule arrays requires spatio-temporal regulation of the microtubule nucleator γ-tubulin ring complex (γTuRC) at microtubule organizing centers (MTOCs). MTOC-localized adapter proteins are thought to recruit and activate γTuRC, but the molecular underpinnings remain obscure. Here we show that at interphase centrosomes, rather than adapters, the microtubule polymerase ch-TOG (also named chTOG or CKAP5) ultimately controls γTuRC recruitment and activation. ch-TOG co-assembles with γTuRC to stimulate nucleation around centrioles. In the absence of ch-TOG, γTuRC fails to localize to these sites, but not the centriole lumen. However, whereas some ch-TOG is stably bound at subdistal appendages, it only transiently associates with PCM. ch-TOG’s dynamic behavior requires its tubulin-binding TOG domains and a C-terminal region involved in localization. In addition, ch-TOG also promotes nucleation from the Golgi. Thus, at interphase centrosomes stimulation of nucleation and γTuRC attachment are mechanistically coupled through transient recruitment of ch-TOG, and ch-TOG’s nucleation-promoting activity is not restricted to centrosomes.

The short flagella 1 (SHF1) gene in Chlamydomonas encodes a Crescerin TOG-domain protein required for late stages of flagellar growth.

Length control of flagella represents a simple and tractable system to investigate the dynamics of organelle size. Models for flagellar length control in the model organism Chlamydomonas reinhardtii have focused on the length dependence of the intraflagellar transport (IFT) system, which manages the delivery and removal of axonemal subunits at the tip of the flagella. One of these cargoes, tubulin, is the major axonemal subunit, and its frequency of arrival at the tip plays a central role in size control models. However, the mechanisms determining tubulin dynamics at the tip are still poorly understood. We discovered a loss-of-function mutation that leads to shortened flagella and found that this was an allele of a previously described gene, SHF1, whose molecular identity had not been determined. We found that SHF1 encodes a Chlamydomonas orthologue of Crescerin, previously identified as a cilia-specific TOG-domain array protein that can bind tubulin via its TOG domains and increase tubulin polymerization rates. In this mutant, flagellar regeneration occurs with the same initial kinetics as in wild-type cells but plateaus at a shorter length. Using a computational model in which the flagellar microtubules are represented by a differential equation for flagellar length combined with a stochastic model for cytoplasmic microtubule dynamics, we found that our experimental results are best described by a model in which Crescerin/SHF1 binds tubulin dimers in the cytoplasm and transports them into the flagellum. We suggest that this TOG-domain protein is necessary to efficiently and preemptively increase intraflagella tubulin levels to offset decreasing IFT cargo at the tip as flagellar assembly progresses.

Affinity purification of tubulin from plant materials.

In the plant cytoskeleton research, mammalian brain tubulin has been widely used to study plant microtubule-interacting proteins in vitro since purification of tubulins from plant sources is generally considered to be challenging and time-consuming. A convenient method for affinity purification of tubulins was devised, which utilized the TOG domains of yeast Stu2 tubulin-binding protein as an affinity ligand (Widlund et al., 2012). We showed that this so-called TOG tubulin affinity chromatography worked efficiently with plant materials, especially actively-dividing cultured cells (Hotta et al., 2016). Plant tubulins purified with the TOG method is highly assembly-competent and thus can be used in various in vitro experiments. Here, we summarize purification strategies of native or tagged plant tubulins as well as an in vitro pull-down assay to monitor their polymerization activity.

Structures of TOG1 and TOG2 from the human microtubule dynamics regulator CLASP1.

Tubulin-binding TOG domains are found arrayed in a number of proteins that regulate microtubule dynamics. While much is known about the structure and function of TOG domains from the XMAP215 microtubule polymerase family, less in known about the TOG domain array found in animal CLASP family members. The animal CLASP TOG array promotes microtubule pause, potentiates rescue, and limits catastrophe. How structurally distinct the TOG domains of animal CLASP are from one another, from XMAP215 family TOG domains, and whether a specific order of structurally distinct TOG domains in the TOG array is conserved across animal CLASP family members is poorly understood. We present the x-ray crystal structures of Homo sapiens (H.s.) CLASP1 TOG1 and TOG2. The structures of H.s. CLASP1 TOG1 and TOG2 are distinct from each other and from the previously determined structure of Mus musculus (M.m.) CLASP2 TOG3. Comparative analyses of CLASP family TOG domain structures determined to date across species and paralogs supports a conserved CLASP TOG array paradigm in which structurally distinct TOG domains are arrayed in a specific order. H.s. CLASP1 TOG1 bears structural similarity to the free-tubulin binding TOG domains of the XMAP215 family but lacks many of the key tubulin-binding determinants found in XMAP215 family TOG domains. This aligns with studies that report that animal CLASP family TOG1 domains cannot bind free tubulin or microtubules. In contrast, animal CLASP family TOG2 and TOG3 domains have reported microtubule-binding activity but are structurally distinct from the free-tubulin binding TOG domains of the XMAP215 family. H.s. CLASP1 TOG2 has a convex architecture, predicted to engage a hyper-curved tubulin state that may underlie its ability to limit microtubule catastrophe and promote rescue. M.m. CLASP2 TOG3 has unique structural elements in the C-terminal half of its α-solenoid domain that our modeling studies implicate in binding to laterally-associated tubulin subunits in the microtubule lattice in a mode similar to, yet distinct from those predicted for the XMAP215 family TOG4 domain. The potential ability of the animal CLASP family TOG3 domain to engage lateral tubulin subunits may underlie the microtubule rescue activity ascribed to the domain. These findings highlight the structural diversity of TOG domains within the CLASP family TOG array and provide a molecular foundation for understanding CLASP-dependent effects on microtubule dynamics.

The microtubule polymerase Stu2 promotes oligomerization of the γ-TuSC for cytoplasmic microtubule nucleation.

Stu2/XMAP215/ZYG-9/Dis1/Alp14/Msps/ch-TOG family members in association with with γ-tubulin complexes nucleate microtubules, but we know little about the interplay of these nucleation factors. Here, we show that the budding yeast Stu2 in complex with the γ-tubulin receptor Spc72 nucleates microtubules in vitro without the small γ-tubulin complex (γ-TuSC). Upon γ-TuSC addition, Stu2 facilitates Spc72-γ-TuSC interaction by binding to Spc72 and γ-TuSC. Stu2 together with Spc72-γ-TuSC increases microtubule nucleation in a process that is dependent on the TOG domains of Stu2. Importantly, these activities are also important for microtubule nucleation in vivo. Stu2 stabilizes Spc72-γ-TuSC at the minus end of cytoplasmic microtubules (cMTs) and an in vivo assay indicates that cMT nucleation requires the TOG domains of Stu2. Upon γ-tubulin depletion, we observed efficient cMT nucleation away from the spindle pole body (SPB), which was dependent on Stu2. Thus, γ-TuSC restricts cMT assembly to the SPB whereas Stu2 nucleates cMTs together with γ-TuSC and stabilizes γ-TuSC at the cMT minus end.

A Cytoskeletal Symphony: Owed to TOG

Like permutating motifs in music, similar protein folds are employed across biology for distinct functions. In this issue of Developmental Cell, Aher etal. (2018) provide insight into how variable TOG domains within an array in the microtubule regulator CLASP are used to prevent microtubule catastrophe and potentiate rescue.

Design principles of a microtubule polymerase.

Stu2/XMAP215 microtubule polymerases use multiple tubulin-binding TOG domains and a lattice-binding basic region to processively promote faster elongation. How the domain composition and organization of these proteins dictate polymerase activity, end localization, and processivity is unknown. We show that polymerase activity does not require different kinds of TOGs, nor are there strict requirements for how the TOGs are linked. We identify an unexpected antagonism between the tubulin-binding TOGs and the lattice-binding basic region: lattice binding by the basic region is weak when at least two TOGs engage tubulins, strong when TOGs are empty. End-localization of Stu2 requires unpolymerized tubulin, at least two TOGs, and polymerase competence. We propose a 'ratcheting' model for processivity: transfer of tubulin from TOGs to the lattice activates the basic region, retaining the polymerase at the end for subsequent rounds of tubulin binding and incorporation. These results clarify design principles of the polymerase.

XMAP215 is a microtubule nucleation factor that functions synergistically with the γ-tubulin ring complex.

How microtubules (MT) 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 cell’s universal MT nucleator. 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 γ-tubulin ring complex (γ-TuRC) possess minimal nucleation activity individually, together these factors synergistically stimulate MT nucleation in vitro. The N-terminal TOG domains 1–5 of XMAP215 bind αβ-tubulin and promote MT polymerization, while the conserved C-terminus is required for efficient MT nucleation and directly binds γ-tubulin. In sum, XMAP215 and γ-TuRC together function as the principal nucleation module that generates MTs in cells.

An isolated CLASP TOG domain suppresses microtubule catastrophe and promotes rescue.

Microtubules are heavily regulated dynamic polymers of αβ-tubulin that are required for proper chromosome segregation and organization of the cytoplasm. Polymerases in the XMAP215 family use arrayed TOG domains to promote faster microtubule elongation. Regulatory factors in the cytoplasmic linker associated protein (CLASP) family that reduce catastrophe and/or increase rescue also contain arrayed TOGs, but how CLASP TOGs contribute to activity is poorly understood. Here, using Saccharomyces cerevisiae Stu1 as a model CLASP, we report structural, biochemical, and reconstitution studies that clarify functional properties of CLASP TOGs. The two TOGs in Stu1 have very different tubulin-binding properties: TOG2 binds to both unpolymerized and polymerized tubulin, and TOG1 binds very weakly to either. The structure of Stu1-TOG2 reveals a CLASP-specific residue that likely confers distinctive tubulin-binding properties. The isolated TOG2 domain strongly suppresses microtubule catastrophe and increases microtubule rescue in vitro, contradicting the expectation that regulatory activity requires an array of TOGs. Single point mutations on the tubulin-binding surface of TOG2 ablate its anti-catastrophe and rescue activity in vitro, and Stu1 function in cells. Revealing that an isolated CLASP TOG can regulate polymerization dynamics without being part of an array provides insight into the mechanism of CLASPs and diversifies the understanding of TOG function.

Solution NMR assignment of the C-terminal domain of human chTOG

The microtubule regulatory protein colonic and hepatic tumor overexpressed gene (chTOG), also known as cytoskeleleton associated protein 5 (CKAP5) plays an important role in organizing the cytoskeleton and in particular in the assembly of k-fibres in mitosis. Recently, we dissected the hitherto poorly understood C-terminus of this protein by discovering two new domains—a cryptic TOG domain (TOG6) and a smaller, helical domain at the very C-terminus. It was shown that the C-terminal domain is important for the interaction with the TACC domain in TACC3 during the assembly of k-fibres in a ternary complex that also includes clathrin. Here we now present the solution NMR assignment of the chTOG C-terminal domain which confirms our earlier prediction that it is mainly made of α-helices. However, the appearance of the 1H–15N HSQC spectrum is indicative of the presence of a considerable amount of unstructured and possibly flexible portions of protein in the domain.

Solution NMR assignment of the 6th TOG domain of minispindles

Solution NMR assignment of the 6th TOG domain of minispindles

A TOGgle for Tension at Kinetochores

Differential stability of kinetochore-microtubule attachments at low versus high tension is critical for accurate chromosome segregation. Miller etal. find that a TOG domain microtubule-binding protein imparts intrinsic tension selectivity to kinetochore-microtubule attachments.

Mutations in Human Tubulin Proximal to the Kinesin-Binding Site Alter Dynamic Instability at Microtubule Plus- and Minus-Ends

The assembly of microtubule-based cellular structures depends on regulated tubulin polymerization and directional transport. Here, we purify and characterize tubulin heterodimers that have human β-tubulin isotype III (TUBB3), as well as heterodimers with one of two β-tubulin mutations (D417H or R262H). Both point mutations are proximal to the kinesin-binding site and have been linked to an ocular motility disorder in humans. Compared to wild-type, microtubules with these mutations have decreased catastrophe frequencies and increased average lifetimes of plus- and minus-end-stabilizing caps. Importantly, the D417H mutation does not alter microtubule lattice structure or Mal3 binding to growing filaments. Instead, this mutation reduces the affinity of tubulin for TOG domains and colchicine, suggesting that the distribution of tubulin heterodimer conformations is changed. Together, our findings reveal how residues on the surface of microtubules, distal from the GTP-hydrolysis site and inter-subunit contacts, can alter polymerization dynamics at the plus- and minus-ends of microtubules.

Identification of microtubule growth deceleration and its regulation by conserved and novel proteins.

Microtubules (MTs) are cytoskeletal polymers that participate in diverse cellular functions, including cell division, intracellular trafficking, and templating of cilia and flagella. MTs undergo dynamic instability, alternating between growth and shortening via catastrophe and rescue events. The rates and frequencies of MT dynamic parameters appear to be characteristic for a given cell type. We recently reported that all MT dynamic parameters vary throughout differentiation of a smooth muscle cell type in intact Caenorhabditis elegans. Here we describe local differences in MT dynamics and a novel MT behavior: an abrupt change in growth rate (deceleration) of single MTs occurring in the cell periphery of these cells. MT deceleration occurs where there is a decrease in local soluble tubulin concentration at the cell periphery. This local regulation of tubulin concentration and MT deceleration are dependent on two novel homologues of human cylicin. These novel ORFs, which we name cylc-1 and -2, share sequence homology with stathmins and encode small, very basic proteins containing several KKD/E repeats. The TOG domain-containing protein ZYG-9(TOGp) is responsible for the faster polymerization rate within the cell body. Thus we have defined two contributors to the molecular regulation for this novel MT behavior.

Solution NMR assignment of the cryptic sixth TOG domain of mini spindles.

TOG domains contribute to the organisation of microtubules through their ability to bind tubulin. They are found in members of the XMAP215 family of proteins, which act as microtubule polymerases and fulfill important roles in the formation of the mitotic spindle and in the assembly of kinetochore fibres. We recently identified a cryptic TOG domain in the XMAP215 family proteins, chTOG and its Drosophila homologue, mini spindles. This domain is not part of the well-established array of TOG domains involved in tubulin polymerisation. Instead it forms part of a binding site for TACC3 family proteins. This interaction is required for the assembly of kinetochore bridges in a trimeric complex with clathrin. Here we present the first NMR assignment of a sixth TOG domain from mini spindles as a first step to elucidate its structure and function.

A tethered delivery mechanism explains the catalytic action of a microtubule polymerase.

Stu2p/XMAP215 proteins are essential microtubule polymerases that use multiple αβ-tubulin-interacting TOG domains to bind microtubule plus ends and catalyze fast microtubule growth. We report here the structure of the TOG2 domain from Stu2p bound to yeast αβ-tubulin. Like TOG1, TOG2 binds selectively to a fully 'curved' conformation of αβ-tubulin, incompatible with a microtubule lattice. We also show that TOG1-TOG2 binds non-cooperatively to two αβ-tubulins. Preferential interactions between TOGs and fully curved αβ-tubulin that cannot exist elsewhere in the microtubule explain how these polymerases localize to the extreme microtubule end. We propose that these polymerases promote elongation because their linked TOG domains concentrate unpolymerized αβ-tubulin near curved subunits already bound at the microtubule end. This tethering model can explain catalyst-like behavior and also predicts that the polymerase action changes the configuration of the microtubule end.