Search

Polo-like kinase 4 (Plk4) is the master-regulator of centriole assembly and cell cycle-dependent regulation of its activity maintains proper centrosome number. During most of the cell cycle, Plk4 levels are nearly undetectable due to its ability to autophosphorylate and trigger its own ubiquitin-mediated degradation. However, during mitotic exit, Plk4 forms a single aggregate on the centriole surface to stimulate centriole duplication. Whereas most Polo-like kinase family members are monomeric, Plk4 is unique because it forms homodimers. Notably, Plk4 trans-autophosphorylates a degron near its kinase domain, a critical step in autodestruction. While it is thought that the purpose of homodimerization is to promote trans-autophosphorylation, this has not been tested. Here, we generated separation-of-function Plk4 mutants that fail to dimerize and show that homodimerization creates a binding site for the Plk4 activator, Asterless. Surprisingly however, Plk4 dimer mutants are catalytically-active in cells, promote centriole assembly, and can trans-autophosphorylate through concentration-dependent condensate formation. Moreover, we mapped and then deleted the weak-interacting regions within Plk4 that mediate condensation and conclude that dimerization and condensation are not required for centriole assembly. Our findings suggest that Plk4 dimerization and condensation function simply to downregulate Plk4 and suppress centriole overduplication. [Media: see text]

Epidermal cells of dark-grown plant seedlings reorient their cortical microtubule arrays in response to blue light from a net lateral orientation to a net longitudinal orientation with respect to the long axis of cells. The molecular mechanism underlying this microtubule array reorientation involves katanin, a microtubule severing enzyme, and a plant-specific microtubule associated protein called SPIRAL2. Katanin preferentially severs longitudinal microtubules, generating seeds that amplify the longitudinal array. Upon severing, SPIRAL2 binds nascent microtubule minus ends and limits their dynamics, thereby stabilizing the longitudinal array while the lateral array undergoes net depolymerization. To date, no experimental structural information is available for SPIRAL2 to help inform its mechanism. To gain insight into SPIRAL2 structure and function, we determined a 1.8 Å resolution crystal structure of theArabidopsis thalianaSPIRAL2 C-terminal domain. The domain is composed of seven core α-helices, arranged in an α-solenoid. Amino-acid sequence conservation maps primarily to one face of the domain involving helices α1, α3, α5, and an extended loop, the α6-α7 loop. The domain fold is similar to, yet structurally distinct from the C-terminal domain of Ge-1 (an mRNA decapping complex factor involved in P-body localization) and, surprisingly, the C-terminal domain of the katanin p80 regulatory subunit. The katanin p80 C-terminal domain heterodimerizes with the MIT domain of the katanin p60 catalytic subunit, and in metazoans, binds the microtubule minus-end factors CAMSAP3 and ASPM. Structural analysis predicts that SPIRAL2 does not engage katanin p60 in a mode homologous to katanin p80. The SPIRAL2 structure highlights an interesting evolutionary convergence of domain architecture and microtubule minus-end localization between SPIRAL2 and katanin complexes, and establishes a foundation upon which structure-function analysis can be conducted to elucidate the role of this domain in the regulation of plant microtubule arrays.

BackgroundDysfunction in non-motile cilia is associated with a broad spectrum of developmental disorders characterised by clinical heterogeneity. While over 100 genes have been associated with primary ciliopathies, with wide phenotypic overlap, some patients still lack a molecular diagnosis.ObjectiveTo investigate and functionally characterise the molecular cause of a malformation disorder observed in two sibling fetuses characterised by microphthalmia, cleft lip and palate, and brain anomalies.MethodsA trio-based whole exome sequencing (WES) strategy was used to identify candidate variants in the TOGARAM1 gene. In silico, in vitro and in vivo (Caenorhabditis elegans) studies were carried out to explore the impact of mutations on protein structure and function, and relevant biological processes.ResultsTOGARAM1 encodes a member of the Crescerin1 family of proteins regulating microtubule dynamics. Its orthologue in C. elegans, che-12, is expressed in a subset of sensory neurons and localises in the dendritic cilium where it is required for chemosensation. Nematode lines harbouring the corresponding missense variant in TOGARAM1 were generated by CRISPR/Cas9 technology. Although chemotaxis ability on a NaCl gradient was not affected, che-12 point mutants displayed impaired lipophilic dye uptake, with shorter and altered cilia in sensory neurons. Finally, in vitro analysis of microtubule polymerisation in the presence of wild-type or mutant TOG2 domain revealed a faster polymerisation associated with the mutant protein, suggesting aberrant tubulin binding.ConclusionsOur data are in favour of a causative role of TOGARAM1 variants in the pathogenesis of this novel disorder, connecting this gene with primary ciliopathy.

Summary While the dynamics of microtubule ends are well characterized, the mechanism that repairs breaks in the lattice interior is poorly understood. A new in vitro study finds that the microtubule-associated protein CLASP repairs lattice damage by regulating GTP-tubulin incorporation into the break site.

Polo-like kinase 4 (Plk4) is the master-regulator of centriole assembly and cell cycle-dependent regulation of its activity maintains proper centrosome number. During most of the cell cycle, Plk4 levels are nearly undetectable due to its ability to autophosphorylate and trigger its own ubiquitin-mediated degradation. However, during mitotic exit, Plk4 forms a single aggregate on the centriole surface to stimulate centriole duplication. Whereas most Polo-like kinase family members are monomeric, Plk4 is unique because it forms homodimers. Notably, Plk4 trans-autophosphorylates a degron near its kinase domain, a critical step in autodestruction. While it is thought that the purpose of homodimerization is to promote trans-autophosphorylation, this has not been tested. Here, we generate separation-of-function Plk4 mutants that fail to dimerize, and we show that homodimerization is required to create a binding site for the Plk4 activator, Asterless. Surprisingly, Plk4 dimer mutants are catalytically active in cells, promote centriole assembly, and can trans-autophosphorylate by a process based its on concentration-dependent aggregation. Our findings implicate a concentration-dependent pathway of Plk4 activation that does not require Asterless binding or homodimerization. Lastly, we propose a model of Plk4 cell cycle regulation that utilizes both activation pathways – Asterless-dependent and aggregation-driven – to restrict centriole assembly to mother centrioles.

Embryogenesis requires cells to change shape and move without disrupting epithelial integrity. This requires robust, responsive linkage between adherens junctions and the actomyosin cytoskeleton. Using Drosophila morphogenesis we define molecular mechanisms mediating junction-cytoskeletal linkage and explore the role of mechanosensing. We focus on the junction-cytoskeletal linker Canoe, a multidomain protein. We engineered the canoe locus to define how its domains mediate its mechanism of action. To our surprise, the PDZ and FAB domains, which we thought connected junctions and F-actin, are not required for viability or mechanosensitive recruitment to junctions under tension. The FAB domain stabilizes junctions experiencing elevated force, but in its absence most cells recover, suggesting redundant interactions. In contrast, the Rap1-binding RA domains are critical for all Cno functions and for enrichment at junctions under tension. This supports a model in which junctional robustness derives from a large protein network assembled via multivalent interactions, with proteins at network nodes and some node connections more critical than others.

Embryogenesis requires cells to change shape and move without disrupting epithelial integrity. This requires robust, responsive linkage between adherens junctions and the actomyosin cytoskeleton. Using Drosophila morphogenesis, we define molecular mechanisms mediating junction–cytoskeletal linkage and explore the role of mechanosensing. We focus on the junction–cytoskeletal linker Canoe, a multidomain protein. We engineered the canoe locus to define how its domains mediate its mechanism of action. To our surprise, the PDZ and FAB domains, which we thought connected junctions and F-actin, are not required for viability or mechanosensitive recruitment to junctions under tension. The FAB domain stabilizes junctions experiencing elevated force, but in its absence, most cells recover, suggesting redundant interactions. In contrast, the Rap1-binding RA domains are critical for all Cno functions and enrichment at junctions under tension. This supports a model in which junctional robustness derives from a large protein network assembled via multivalent interactions, with proteins at network nodes and some node connections more critical than others.

Cytoplasmic linker protein 170 (CLIP-170) is a microtubule plus-end factor that links vesicles to microtubules and recruits the dynein–dynactin complex to microtubule plus ends. CLIP-170 plus-end localization is end binding 1 (EB1)–dependent. CLIP-170 contains two N-terminal cytoskeleton-associated protein glycine-rich (CAP-Gly) domains flanked by serine-rich regions. The CAP-Gly domains are known EB1-binding domains, and the serine-rich regions have also been implicated in CLIP-170's microtubule plus-end localization mechanism. However, the determinants in these serine-rich regions have not been identified. Here we elucidated multiple EB1-binding modules in the CLIP-170 N-terminal region. Using isothermal titration calorimetry and size-exclusion chromatography, we mapped and biophysically characterized these EB1-binding modules, including the two CAP-Gly domains, a bridging SXIP motif, and a unique array of divergent SXIP-like motifs located N-terminally to the first CAP-Gly domain. We found that, unlike the EB1-binding mode of the CAP-Gly domain in the dynactin-associated protein p150(Glued), which dually engages the EB1 C-terminal EEY motif as well as the EB homology domain and sterically occludes SXIP motif binding, the CLIP-170 CAP-Gly domains engage only the EEY motif, enabling the flanking SXIP and SXIP-like motifs to bind the EB homology domain. These multivalent EB1-binding modules provided avidity to the CLIP-170–EB1 interaction, likely clarifying why CLIP-170 preferentially binds EB1 rather than the α-tubulin C-terminal EEY motif. Our finding that CLIP-170 has multiple non-CAP-Gly EB1-binding modules may explain why autoinhibition of CLIP-170 GAP-Gly domains does not fully abrogate its microtubule plus-end localization. This work expands our understanding of EB1-binding motifs and their multivalent networks.

Centriole assembly initiates when Polo-like kinase 4 (Plk4) interacts with a centriole “targeting-factor.” In Drosophila, Asterless/Asl (Cep152 in humans) fulfills the targeting role. Interestingly, Asl also regulates Plk4 levels. The N-terminus of Asl (Asl-A; amino acids 1-374) binds Plk4 and promotes Plk4 self-destruction, although it is unclear how this is achieved. Moreover, Plk4 phosphorylates the Cep152 N-terminus, but the functional consequence is unknown. Here, we show that Plk4 phosphorylates Asl and mapped 13 phospho-residues in Asl-A. Nonphosphorylatable alanine (13A) and phosphomimetic (13PM) mutants did not alter Asl function, presumably because of the dominant role of the Asl C-terminus in Plk4 stabilization and centriolar targeting. To address how Asl-A phosphorylation specifically affects Plk4 regulation, we generated Asl-A fragment phospho-mutants and expressed them in cultured Drosophila cells. Asl-A-13A stimulated kinase activity by relieving Plk4 autoinhibition. In contrast, Asl-A-13PM inhibited Plk4 activity by a novel mechanism involving autophosphorylation of Plk4’s kinase domain. Thus, Asl-A’s phosphorylation state determines which of Asl-A’s two opposing effects are exerted on Plk4. Initially, nonphosphorylated Asl binds Plk4 and stimulates its kinase activity, but after Asl is phosphorylated, a negative-feedback mechanism suppresses Plk4 activity. This dual regulatory effect by Asl-A may limit Plk4 to bursts of activity that modulate centriole duplication.

Dysfunction in non-motile cilia is associated with a broad spectrum of developmental disorders characterised by clinical heterogeneity. While over 100 genes have been associated with primary ciliopathies, with wide phenotypic overlap, some patients still lack a molecular diagnosis.To investigate and functionally characterise the molecular cause of a malformation disorder observed in two sibling fetuses characterised by microphthalmia, cleft lip and palate, and brain anomalies.A trio-based whole exome sequencing (WES) strategy was used to identify candidate variants in theOur data are in favour of a causative role of

SxIP-iLID is a novel optogenetic tool designed to assess the temporal role of proteins on microtubule dynamics. The authors establish that optogenetic cross-linking of microtubule and actin networks decreases MT growth velocities and increases the cell area void of microtubules., We developed a novel optogenetic tool, SxIP–improved light-inducible dimer (iLID), to facilitate the reversible recruitment of factors to microtubule (MT) plus ends in an end-binding protein–dependent manner using blue light. We show that SxIP-iLID can track MT plus ends and recruit tgRFP-SspB upon blue light activation. We used this system to investigate the effects of cross-linking MT plus ends and F-actin in Drosophila melanogaster S2 cells to gain insight into spectraplakin function and mechanism. We show that SxIP-iLID can be used to temporally recruit an F-actin binding domain to MT plus ends and cross-link the MT and F-actin networks. Cross-linking decreases MT growth velocities and generates a peripheral MT exclusion zone. SxIP-iLID facilitates the general recruitment of specific factors to MT plus ends with temporal control enabling researchers to systematically regulate MT plus end dynamics and probe MT plus end function in many biological processes., Graphical Abstract

The ‘tubulin code’, a set of post-translational modifications to the microtubule cytoskeleton that include removal of the C-terminal Tyr of α-tubulin, regulates the biological function of the polymer. Three studies now report structures of VASH1–SVBP and VASH2–SVBP heterodimers and provide insights into how these proteases recognize and cleave the terminal Tyr of α-tubulin.

Microtubule-associated proteins with arrays of TOG domains differentially regulate microtubule dynamics. Byrnes and Slep show that TOG arrays are polarized containing architecturally distinct TOG domains that bind either free or microtubule lattice-incorporated tubulin, which is essential for microtubule polymerization and mitotic spindle formation., XMAP215, CLASP, and Crescerin use arrayed tubulin-binding tumor overexpressed gene (TOG) domains to modulate microtubule dynamics. We hypothesized that TOGs have distinct architectures and tubulin-binding properties that underlie each family’s ability to promote microtubule polymerization or pause. As a model, we investigated the pentameric TOG array of a Drosophila melanogaster XMAP215 member, Msps. We found that Msps TOGs have distinct architectures that bind either free or polymerized tubulin, and that a polarized array drives microtubule polymerization. An engineered TOG1-2-5 array fully supported Msps-dependent microtubule polymerase activity. Requisite for this activity was a TOG5-specific N-terminal HEAT repeat that engaged microtubule lattice-incorporated tubulin. TOG5–microtubule binding maintained mitotic spindle formation as deleting or mutating TOG5 compromised spindle architecture and increased the mitotic index. Mad2 knockdown released the spindle assembly checkpoint triggered when TOG5–microtubule binding was compromised, indicating that TOG5 is essential for spindle function. Our results reveal a TOG5-specific role in mitotic fidelity and support our hypothesis that architecturally distinct TOGs arranged in a sequence-specific order underlie TOG array microtubule regulator activity.

Like permutating motifs in music, similar protein folds are employed across biology for distinct functions. In this issue of Developmental Cell, Aher et al. (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.

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.

Primary cilia are critical organelles involved in development, sensation, and signaling. Crescerin, a conserved protein family in ciliated and flagellated eukaryotes, uses a TOG domain array with tubulin polymerization activity to regulate cilia microtubules and facilitate proper cilia length, ultrastructure, and function., Eukaryotic cilia are cell-surface projections critical for sensing the extracellular environment. Defects in cilia structure and function result in a broad range of developmental and sensory disorders. However, mechanisms that regulate the microtubule (MT)-based scaffold forming the cilia core are poorly understood. TOG domain array–containing proteins ch-TOG and CLASP are key regulators of cytoplasmic MTs. Whether TOG array proteins also regulate ciliary MTs is unknown. Here we identify the conserved Crescerin protein family as a cilia-specific, TOG array-containing MT regulator. We present the crystal structure of mammalian Crescerin1 TOG2, revealing a canonical TOG fold with conserved tubulin-binding determinants. Crescerin1's TOG domains possess inherent MT-binding activity and promote MT polymerization in vitro. Using Cas9-triggered homologous recombination in Caenorhabditis elegans, we demonstrate that the worm Crescerin family member CHE-12 requires TOG domain–dependent tubulin-binding activity for sensory cilia development. Thus, Crescerin expands the TOG domain array–based MT regulatory paradigm beyond ch-TOG and CLASP, representing a distinct regulator of cilia structure.

Centriole duplication is tightly regulated throughout the cell cycle to ensure one duplication event per centriole. McLamarrah et al. show that a stepwise pattern of Ana2 phosphorylation by Plk4 facilitates proper centriole duplication., Polo-like kinase 4 (Plk4) initiates an early step in centriole assembly by phosphorylating Ana2/STIL, a structural component of the procentriole. Here, we show that Plk4 binding to the central coiled-coil (CC) of Ana2 is a conserved event involving Polo-box 3 and a previously unidentified putative CC located adjacent to the kinase domain. Ana2 is then phosphorylated along its length. Previous studies showed that Plk4 phosphorylates the C-terminal STil/ANa2 (STAN) domain of Ana2/STIL, triggering binding and recruitment of the cartwheel protein Sas6 to the procentriole assembly site. However, the physiological relevance of N-terminal phosphorylation was unknown. We found that Plk4 first phosphorylates the extreme N terminus of Ana2, which is critical for subsequent STAN domain modification. Phosphorylation of the central region then breaks the Plk4–Ana2 interaction. This phosphorylation pattern is important for centriole assembly and integrity because replacement of endogenous Ana2 with phospho-Ana2 mutants disrupts distinct steps in Ana2 function and inhibits centriole duplication.

The Asterless N terminus promotes Plk4 homodimerization and autophosphorylation during interphase, whereas its C terminus stabilizes Plk4 during mitosis., Plk4 (Polo-like kinase 4) and its binding partner Asterless (Asl) are essential, conserved centriole assembly factors that induce centriole amplification when overexpressed. Previous studies found that Asl acts as a scaffolding protein; its N terminus binds Plk4’s tandem Polo box cassette (PB1-PB2) and targets Plk4 to centrioles to initiate centriole duplication. However, how Asl overexpression drives centriole amplification is unknown. In this paper, we investigated the Asl–Plk4 interaction in Drosophila melanogaster cells. Surprisingly, the N-terminal region of Asl is not required for centriole duplication, but a previously unidentified Plk4-binding domain in the C terminus is required. Mechanistic analyses of the different Asl regions revealed that they act uniquely during the cell cycle: the Asl N terminus promotes Plk4 homodimerization and autophosphorylation during interphase, whereas the Asl C terminus stabilizes Plk4 during mitosis. Therefore, Asl affects Plk4 in multiple ways to regulate centriole duplication. Asl not only targets Plk4 to centrioles but also modulates Plk4 stability and activity, explaining the ability of overexpressed Asl to drive centriole amplification.

Summary Centrioles are fundamental and evolutionarily conserved microtubule-based organelles whose assembly is characterized by microtubule growth rates that are orders of magnitude slower than those of cytoplasmic microtubules. Several centriolar proteins can interact with tubulin or microtubules, but how they ensure the exceptionally slow growth of centriolar microtubules has remained mysterious. Here, we bring together crystallographic, biophysical, and reconstitution assays to demonstrate that the human centriolar protein CPAP (SAS-4 in worms and flies) binds and “caps” microtubule plus ends by associating with a site of β-tubulin engaged in longitudinal tubulin-tubulin interactions. Strikingly, we uncover that CPAP activity dampens microtubule growth and stabilizes microtubules by inhibiting catastrophes and promoting rescues. We further establish that the capping function of CPAP is important to limit growth of centriolar microtubules in cells. Our results suggest that CPAP acts as a molecular lid that ensures slow assembly of centriolar microtubules and, thereby, contributes to organelle length control., Highlights • CPAP's PN2-3 domain binds to an exposed site on β-tubulin at microtubule plus ends • CPAP tracks and caps microtubule plus ends in vitro • CPAP dampens microtubule growth in vitro • The capping function of CPAP limits centriolar microtubule growth in human cells, The mechanisms ensuring the extremely slow growth of centriolar microtubules remain elusive. Sharma, Aher, Dynes et al. demonstrate that human CPAP acts as a molecular lid that caps microtubule plus ends and dampens their elongation, thus contributing to centriole length control by ensuring slow processive assembly of centriolar microtubules.

Spectraplakins are large molecules that cross-link F-actin and microtubules (MTs). Mutations in spectraplakins yield defective cell polarization, aberrant focal adhesion dynamics, and dystonia. We present the 2.8 A crystal structure of the hACF7 EF1-EF2-GAR MT-binding module and delineate the GAR residues critical for MT binding. The EF1-EF2 and GAR domains are autonomous domains connected by a flexible linker. The EF1-EF2 domain is an EFβ-scaffold with two bound Ca2+ ions that straddle an N-terminal α helix. The GAR domain has a unique α/β sandwich fold that coordinates Zn2+. While the EF1-EF2 domain is not sufficient for MT binding, the GAR domain is and likely enhances EF1-EF2-MT engagement. Residues in a conserved basic patch, distal to the GAR domain's Zn2+-binding site, mediate MT binding.

The yeast microtubule polymerase Stu2’s C-terminal domain is a 15-nm parallel, homodimeric coiled coil with two spatially distinct conserved regions. Determinants in these conserved regions optimally position Stu2 on the mitotic spindle to drive proper spindle structure and dynamics., XMAP215/Dis1 family proteins are potent microtubule polymerases, critical for mitotic spindle structure and dynamics. While microtubule polymerase activity is driven by an N-terminal tumor overexpressed gene (TOG) domain array, proper cellular localization is a requisite for full activity and is mediated by a C-terminal domain. Structural insight into the C-terminal domain’s architecture and localization mechanism remain outstanding. We present the crystal structure of the Saccharomyces cerevisiae Stu2 C-terminal domain, revealing a 15-nm parallel homodimeric coiled coil. The parallel architecture of the coiled coil has mechanistic implications for the arrangement of the homodimer’s N-terminal TOG domains during microtubule polymerization. The coiled coil has two spatially distinct conserved regions: CRI and CRII. Mutations in CRI and CRII perturb the distribution and localization of Stu2 along the mitotic spindle and yield defects in spindle morphology including increased frequencies of mispositioned and fragmented spindles. Collectively, these data highlight roles for the Stu2 dimerization domain as a scaffold for factor binding that optimally positions Stu2 on the mitotic spindle to promote proper spindle structure and dynamics.

Structures of Drosophila Msps TOG4 and human ch-TOG TOG4 are presented. TOG4 departs from the other TOG structures and predicts novel α-tubulin engagement. Whereas TOG domains across the array have different tubulin-binding properties, cellular studies show that a fully functional TOG array is required for microtubule polymerase activity., XMAP215 family members are potent microtubule (MT) polymerases, with mutants displaying reduced MT growth rates and aberrant spindle morphologies. XMAP215 proteins contain arrayed tumor overexpressed gene (TOG) domains that bind tubulin. Whether these TOG domains are architecturally equivalent is unknown. Here we present crystal structures of TOG4 from Drosophila Msps and human ch-TOG. These TOG4 structures architecturally depart from the structures of TOG domains 1 and 2, revealing a conserved domain bend that predicts a novel engagement with α-tubulin. In vitro assays show differential tubulin-binding affinities across the TOG array, as well as differential effects on MT polymerization. We used Drosophila S2 cells depleted of endogenous Msps to assess the importance of individual TOG domains. Whereas a TOG1-4 array largely rescues MT polymerization rates, mutating tubulin-binding determinants in any single TOG domain dramatically reduces rescue activity. Our work highlights the structurally diverse yet positionally conserved TOG array that drives MT polymerization.

Centrioles play a key role in nucleating polarized microtubule networks. In actively dividing cells, centrioles establish the bipolar mitotic spindle and are essential for genomic stability. Drosophila anastral spindle-2 (Ana2) is a conserved centriole duplication factor. Although recent work has demonstrated that an Ana2-dynein light chain (LC8) centriolar complex is critical for proper spindle positioning in neuroblasts, how Ana2 and LC8 interact is yet to be established. Here we examine the Ana2-LC8 interaction and map two LC8-binding sites within the central region of Ana2, Ana2M (residues 156-251). Ana2 LC8-binding site 1 contains a signature TQT motif and robustly binds LC8 (KD of 1.1 μm), whereas site 2 contains a TQC motif and binds LC8 with lower affinity (KD of 13 μm). Both LC8-binding sites flank a predicted ~34-residue α-helix. We present two independent atomic structures of LC8 dimers in complex with Ana2 LC8-binding site 1 and site 2 peptides. The Ana2 peptides form β-strands that extend a central composite LC8 β-sandwich. LC8 recognizes the signature TQT motif in the first LC8 binding site of Ana2, forming extensive van der Waals contacts and hydrogen bonding with the peptide, whereas the Ana2 site 2 TQC motif forms a uniquely extended β-strand, not observed in other dynein light chain-target complexes. Size exclusion chromatography coupled with multiangle static light scattering demonstrates that LC8 dimers bind Ana2M sites and induce Ana2 tetramerization, yielding an Ana2M4-LC88 complex. LC8-mediated Ana2 oligomerization probably enhances Ana2 avidity for centriole-binding factors and may bridge multiple factors as required during spindle positioning and centriole biogenesis.

Moriwaki and Goshima identify the five molecular components that are necessary for recapitulation of all three phases of microtubule dynamics in vitro and show that Plk1 acts to shift microtubules to the mitotic mode., In vitro reconstitution is the fundamental test for identification of the core components of a biological process. In this issue, Moriwaki and Goshima (2016. J. Cell Biol. https://doi.org/10.1083/jcb.201604118) reconstitute all phases of microtubule dynamics through the inclusion of five key regulators and demonstrate that Polo kinase activity shifts the system from an interphase mode into an enhanced mitotic mode.

The centrosome is the major microtubule-organizing centre of many cells, best known for its role in mitotic spindle organization. How the proteins of the centrosome are accurately assembled to carry out its many functions remains poorly understood. The non-membrane-bound nature of the centrosome dictates that protein–protein interactions drive its assembly and functions. To investigate this massive macromolecular organelle, we generated a ‘domain-level' centrosome interactome using direct protein–protein interaction data from a focused yeast two-hybrid screen. We then used biochemistry, cell biology and the model organism Drosophila to provide insight into the protein organization and kinase regulatory machinery required for centrosome assembly. Finally, we identified a novel role for Plk4, the master regulator of centriole duplication. We show that Plk4 phosphorylates Cep135 to properly position the essential centriole component Asterless. This interaction landscape affords a critical framework for research of normal and aberrant centrosomes., The centrosome is a large intracellular structure that serves as the microtubule-organising center, but how it is accurately assembled is not known. Here the authors generate a ‘domain-level' centrosome interactome and show that Plk4 positions the essential centriole component Asterless by phosphorylating Cep135.

The dynamics of actin and microtubules are coordinated in many cellular processes, but little is known about molecules mediating cross-talk. We describe intracellular dynamics of Shot in a structure-function analysis of its role as a cross-linker. Shot interacts with microtubules two ways through EB1 and along microtubule lattices by the GAS2 domain., The dynamics of actin and microtubules are coordinated in a variety of cellular and morphogenetic processes; however, little is known about the molecules mediating this cytoskeletal cross-talk. We are studying Short stop (Shot), the sole Drosophila spectraplakin, as a model actin–microtubule cross-linking protein. Spectraplakins are an ancient family of giant cytoskeletal proteins that are essential for a diverse set of cellular functions; yet, we know little about the dynamics of spectraplakins and how they bridge actin filaments and microtubules. In this study we describe the intracellular dynamics of Shot and a structure–function analysis of its role as a cytoskeletal cross-linker. We find that Shot interacts with microtubules using two different mechanisms. In the cell interior, Shot binds growing plus ends through an interaction with EB1. In the cell periphery, Shot associates with the microtubule lattice via its GAS2 domain, and this pool of Shot is actively engaged as a cross-linker via its NH2-terminal actin-binding calponin homology domains. This cross-linking maintains microtubule organization by resisting forces that produce lateral microtubule movements in the cytoplasm. Our results provide the first description of the dynamics of these important proteins and provide key insight about how they function during cytoskeletal cross-talk.

The XMAP215 (Xenopus microtubule-associated protein 215) and CLASP [CLIP-170 (cytoskeletal linker protein 170) associated protein] microtubule plus end tracking families play central roles in the regulation of interphase microtubule dynamics and the proper formation of mitotic spindle architecture and flux. XMAP215 members comprise N-terminally-arrayed hexa-HEAT (huntingtin, elongation factor 3, the PR65/A subunit of protein phosphatase 2A and the lipid kinase Tor) repeats known as TOG (tumour overexpressed gene) domains. Higher eukaryotic XMAP215 members are monomeric and have five TOG domains. Yeast counterparts are dimeric and have two TOG domains. Structure determination of the TOG domain reveals that the six HEAT repeats are aligned to form an oblong scaffold. The TOG domain face composed of intra-HEAT loops forms a contiguous, conserved tubulin-binding surface. Nested within the conserved intra-HEAT loop 1 is an invariant, signature, surface-exposed tryptophan residue that is a prime determinant in the TOG domain–tubulin interaction. The arrayed organization of TOG domains is critical for the processive mechanism of XMAP215, indicative that multiple tubulin/microtubule-binding sites are required for plus end tracking activity. The CLASP family has been annotated as containing a single N-terminal TOG domain. Using XMAP215 TOG domain structure determinants as a metric to analyse CLASP sequence, it is anticipated that CLASP contains two additional cryptic TOGL (TOG-like) domains. The presence of additional TOGL domains implicates CLASP as an ancient XMAP215 relative that uses a similar, multi-TOG-based mechanism to processively track microtubule ends.

Microtubule plus end binding proteins (+TIPs) localize to the dynamic plus ends of microtubules, where they stimulate microtubule growth and recruit signaling molecules. Three main +TIP classes have been identified (XMAP215, EB1, and CLIP-170), but whether they act upon microtubule plus ends through a similar mechanism has not been resolved. Here, we report crystal structures of the tubulin binding domains of XMAP215 (yeast Stu2p and Drosophila Msps), EB1 (yeast Bim1p and human EB1), and CLIP-170 (human), which reveal diverse tubulin binding interfaces. Functional studies, however, reveal a common property that native or artificial dimerization of tubulin binding domains (including chemically induced heterodimers of EB1 and CLIP-170) induces tubulin nucleation/assembly in vitro and, in most cases, plus end tracking in living cells. We propose that +TIPs, although diverse in structure, share a common property of multimerizing tubulin, thus acting as polymerization chaperones that aid in subunit addition to the microtubule plus end.

CP190 is a large, multi-domain protein, first identified as a centrosome protein with oscillatory localization over the course of the cell cycle. During interphase it has a well-established role within the nucleus as a chromatin insulator. Upon nuclear envelope breakdown, there is a striking redistribution of CP190 to centrosomes and the mitotic spindle, in addition to the population at chromosomes. Here, we investigate CP190 in detail by performing domain analysis in cultured Drosophila S2 cells combined with protein structure determination by X-ray crystallography, in vitro biochemical characterization, and in vivo fixed and live imaging of cp190 mutant flies. Our analysis of CP190 identifies a novel N-terminal centrosome and microtubule (MT) targeting region, sufficient for spindle localization. This region consists of a highly conserved BTB domain and a linker region that serves as the MT binding domain. We present the 2.5 Å resolution structure of the CP190 N-terminal 126 amino acids, which adopts a canonical BTB domain fold and exists as a stable dimer in solution. The ability of the linker region to robustly localize to MTs requires BTB domain-mediated dimerization. Deletion of the linker region using CRISPR significantly alters spindle morphology and leads to DNA segregation errors in the developing Drosophila brain neuroblasts. Collectively, we highlight a multivalent MT-binding architecture in CP190, which confers distinct subcellular cytoskeletal localization and function during mitosis.

Microtubule-associated proteins regulate microtubule (MT) dynamics spatially and temporally, which is essential for proper formation of the bipolar mitotic spindle. The XMAP215 family is comprised of conserved microtubule-associated proteins that use an array of tubulin-binding tumor overexpressed gene (TOG) domains, consisting of six (A-F) Huntingtin, elongation factor 3, protein phosphatase 2A, target of rapamycin (HEAT) repeats, to robustly increase MT plus-end polymerization rates. Recent work showed that TOG domains have differentially conserved architectures across the array, with implications for position-dependent TOG domain tubulin binding activities and function within the XMAP215 MT polymerization mechanism. Although TOG domains 1, 2, and 4 are well described, structural and mechanistic information characterizing TOG domains 3 and 5 is outstanding. Here, we present the structure and characterization of Drosophila melanogaster Mini spindles (Msps) TOG3. Msps TOG3 has two unique features as follows: the first is a C-terminal tail that stabilizes the ultimate four HEAT repeats (HRs), and the second is a unique architecture in HR B. Structural alignments of TOG3 with other TOG domain structures show that the architecture of TOG3 is most similar to TOG domains 1 and 2 and diverges from TOG4. Docking TOG3 onto recently solved Stu2 TOG1· and TOG2·tubulin complex structures suggests that TOG3 uses similarly conserved tubulin-binding intra-HEAT loop residues to engage α- and β-tubulin. This indicates that TOG3 has maintained a TOG1- and TOG2-like TOG-tubulin binding mode despite structural divergence. The similarity of TOG domains 1-3 and the divergence of TOG4 suggest that a TOG domain array with polarized structural diversity may play a key mechanistic role in XMAP215-dependent MT polymerization activity.

A critical challenge of structural genomics is to extract functional information from protein structures. We present an example of how this may be accomplished using the Evolutionary Trace (ET) method in the context of the regulators of G protein signaling (RGS) family. We have previously applied ET to the RGS family and identified a novel, evolutionarily privileged site on the RGS domain as important for regulating RGS activity. Here we confirm through targeted mutagenesis of RGS7 that these ET-identified residues are critical for RGS domain regulation and are likely to function as global determinants of RGS function. We also discuss how the recent structure of the complex of RGS9, Gt/i1α–GDP–AlF4− and the effector subunit PDEγ confirms their contact with the effector–G protein interface, forming a structural pathway that communicates from the effector-contacting surface of the G protein and RGS catalytic core domain to the catalytic interface between Gα and RGS. These results demonstrate the effectiveness of ET for identifying binding sites and efficiently focusing mutational studies on their key residues, thereby linking raw sequence and structure data to functional information.

Wnt signaling has key roles in embryogenesis and oncogenesis, yet several aspects of Wnt signal transduction remain mysterious. Schwarz-Romond et al. demonstrate that the Wnt signaling protein Dishevelled has the surprising ability to form dynamic polymers and present evidence that polymerization is crucial in signaling, perhaps forming an inducible scaffold for further signal transduction.

Genetic studies in the yeast Saccharomyces cerevisiae have shown that SST2 promotes pheromone desensitization in vivo. Sst2 is the founding member of the RGS (regulators of G protein signaling) family of proteins, which in mammals act as GAPs (GTPase activating proteins) for several subfamilies of Galpha proteins in vitro. A similar activity for Sst2 has not been demonstrated, and it is not self-evident from sequence homology arguments alone. Here we describe the purification of Sst2 and its cognate Galpha protein (Gpa1) in yeast, and demonstrate Sst2-stimulated Gpa1 GTPase activity. His-tagged versions of Sst2 and Gpa1 were expressed in E. coli, and purified using Ni2+-agarose and ion exchange chromatography. Time-course binding experiments reveal that Sst2 does not affect the binding or release of guanine nucleotides. Similarly, steady-state GTPase assays reveal that Sst2 does not alter the overall rate of hydrolysis, including the rate-limiting nucleotide exchange step. Single-turnover GTPase assays reveal, however, that Sst2 is a potent stimulator of GTP hydrolysis. Sst2 also exhibits GAP activity for mammalian Goalpha, and the mammalian RGS protein GAIP exhibits GAP activity for Gpa1. Finally, we show that Sst2 binds with highest affinity to the transition state of Gpa1 (GDP-AlF4--bound), and with much lower affinity to the inactive (GDP-bound) and active (GTPgammaS-bound) conformations. These experiments represent the first biochemical characterization of Gpa1 and Sst2, and provide a molecular basis for their well-established biological roles in signaling and desensitization.

Studying the role of accessory chains in dynein single-molecule motility shows that the dynein light chain (LC) and intermediate chain (IC) promote motor dimerization, increase velocity, and potentiate processivity. The crystal structure of the yeast LC–IC complex is determined, and the interaction is biochemically characterized., Cytoplasmic dynein is the major microtubule minus end–directed motor. Although studies have probed the mechanism of the C-terminal motor domain, if and how dynein's N-terminal tail and the accessory chains it binds regulate motor activity remain to be determined. Here, we investigate the structure and function of the Saccharomyces cerevisiae dynein light (Dyn2) and intermediate (Pac11) chains in dynein heavy chain (Dyn1) movement. We present the crystal structure of a Dyn2-Pac11 complex, showing Dyn2-mediated Pac11 dimerization. To determine the molecular effects of Dyn2 and Pac11 on Dyn1 function, we generated dyn2Δ and dyn2Δpac11Δ strains and analyzed Dyn1 single-molecule motor activity. We find that the Dyn2-Pac11 complex promotes Dyn1 homodimerization and potentiates processivity. The absence of Dyn2 and Pac11 yields motors with decreased velocity, dramatically reduced processivity, increased monomerization, aggregation, and immobility as determined by single-molecule measurements. Deleting dyn2 significantly reduces Pac11-Dyn1 complex formation, yielding Dyn1 motors with activity similar to Dyn1 from the dyn2Δpac11Δ strain. Of interest, motor phenotypes resulting from Dyn2-Pac11 complex depletion bear similarity to a point mutation in the mammalian dynein N-terminal tail (Loa), highlighting this region as a conserved, regulatory motor element.

CLASP is a key regulator of microtubule (MT) dynamics and bipolar mitotic spindle structure with CLASP mutants displaying a distinctive monopolar spindle phenotype. It has been postulated that cryptic TOG domains underlie CLASP’s ability regulate MT dynamics. Here, we report the crystal structure of the first cryptic TOG domain (TOG2) from human CLASP1, revealing the existence of a bona fide TOG array in the CLASP family. Strikingly, CLASP1 TOG2 exhibits a unique, convex architecture across the tubulin-binding surface that contrasts with the flat tubulin-binding surface of XMAP215 family TOG domains. Mutations in key, conserved TOG2 determinants abrogate the ability of CLASP mutants to rescue bipolar spindle formation in Drosophila cells depleted of endogenous CLASP. These findings highlight the common mechanistic use of TOG domains in XMAP215 and CLASP families to regulate MT dynamics, and suggest that differential TOG domain architecture may confer distinct functions to these critical cytoskeletal regulators.

The nuclear pore complex gates nucleocytoplasmic transport through a massive, eight-fold symmetric channel capped by a nucleoplasmic basket and structurally unique, cytoplasmic fibrils whose tentacles bind and regulate asymmetric traffic. The conserved Nup82 complex, composed of Nsp1, Nup82, and Nup159, forms the unique cytoplasmic fibrils that regulate mRNA nuclear export. Although the nuclear pore complex plays a fundamental, conserved role in nuclear trafficking, structural information about the cytoplasmic fibrils is limited. Here, we investigate the structural and biochemical interactions between Saccharomyces cerevisiae Nup159 and the nucleoporin, Dyn2. We find that Dyn2 is predominantly a homodimer and binds arrayed sites on Nup159, promoting the Nup159 parallel homodimerization. We present the first structure of Dyn2, determined at 1.85 A resolution, complexed with a Nup159 target peptide. Dyn2 resembles homologous metazoan dynein light chains, forming homodimeric composite substrate binding sites that engage two independent 10-residue target motifs, imparting a β-strand structure to each peptide via antiparallel extension of the Dyn2 core β-sandwich. Dyn2 recognizes a highly conserved QT motif while allowing sequence plasticity in the flanking residues of the peptide. Isothermal titration calorimetric analysis of the comparative binding of Dyn2 to two Nup159 target sites shows similar affinities (18 and 13 μm), but divergent thermal binding modes. Dyn2 homodimers are arrayed in the crystal lattice, likely mimicking the arrayed architecture of Dyn2 on the Nup159 multivalent binding sites. Crystallographic interdimer interactions potentially reflect a cooperative basis for Dyn2-Nup159 complex formation. Our data highlight the determinants that mediate oligomerization of the Nup82 complex and promote a directed, elongated cytoplasmic fibril architecture.

Dynamic instability is a hallmark of the microtubule cytoskeleton. In order to regulate microtubule dynamics in vivo, a varied host of microtubule-associated proteins are mobilized to promote microtubule nucleation, growth, stabilization, catastrophe, depolymerization, rescue, and severing. To confer these various functions, cytoskeletal regulators have highly tuned affinities for tubulin, recognizing the unpolymerized αβ-tubulin heterodimer, dynamic microtubule lattice, stabilized microtubule lattice, or a combination therein. The protocols presented here probe αβ-tubulin and microtubule binding using gel filtration and cosedimentation, respectively.

Dynamic instability is a hallmark of the microtubule cytoskeleton. In order to regulate microtubule dynamics in vivo, a varied host of microtubule-associated proteins are mobilized to promote microtubule nucleation, growth, stabilization, catastrophe, depolymerization, rescue, and severing. To confer these various functions, cytoskeletal regulators have highly tuned affinities for tubulin, recognizing the unpolymerized αβ-tubulin heterodimer, dynamic microtubule lattice, stabilized microtubule lattice, or a combination therein. The protocols presented here probe αβ-tubulin and microtubule binding using gel filtration and cosedimentation, respectively.

Recent experiments reconstituting microtubule plus end tracking activity coupled with structural determination of microtubule plus end domains and plus end complexes are revealing the hierarchy, regulatory features, and potential mechanisms of plus end tracking proteins. Primary plus end tracking proteins include EB1 and XMAP215, while a host of secondary, EB1-dependent plus end proteins have been identified and characterized, including CLIP-170 and SKIP-motif proteins. Single molecule in vitro reconstitution assays show that XMAP215 is a processive polymerases that drives tubulin polymerization. Analysis of the EB1–microtubule interaction indicates EB1 actively promotes A-form microtubule lattice growth and rapidly exchanges with subsecond dwell times.

Cadherin-based adherens junctions (AJs) mediate cell adhesion and regulate cell shape change. The nectin–afadin complex also localizes to AJs and links to the cytoskeleton. Mammalian afadin has been suggested to be essential for adhesion and polarity establishment, but its mechanism of action is unclear. In contrast, Drosophila melanogaster’s afadin homologue Canoe (Cno) has suggested roles in signal transduction during morphogenesis. We completely removed Cno from embryos, testing these hypotheses. Surprisingly, Cno is not essential for AJ assembly or for AJ maintenance in many tissues. However, morphogenesis is impaired from the start. Apical constriction of mesodermal cells initiates but is not completed. The actomyosin cytoskeleton disconnects from AJs, uncoupling actomyosin constriction and cell shape change. Cno has multiple direct interactions with AJ proteins, but is not a core part of the cadherin–catenin complex. Instead, Cno localizes to AJs by a Rap1- and actin-dependent mechanism. These data suggest that Cno regulates linkage between AJs and the actin cytoskeleton during morphogenesis.

Heterotrimeric G proteins relay extracellular cues from heptahelical transmembrane receptors to downstream effector molecules. Composed of an α subunit with intrinsic GTPase activity and a βγ heterodimer, the trimeric complex dissociates upon receptor-mediated nucleotide exchange on the α subunit, enabling each component to engage downstream effector targets for either activation or inhibition as dictated in a particular pathway. To mitigate excessive effector engagement and concomitant signal transmission, the Gα subunit's intrinsic activation timer (the rate of GTP hydrolysis) is regulated spatially and temporally by a class of GTPase accelerating proteins (GAPs) known as the regulator of G protein signaling (RGS) family. The array of G protein-coupled receptors, Gα subunits, RGS proteins and downstream effectors in mammalian systems is vast. Understanding the molecular determinants of specificity is critical for a comprehensive mapping of the G protein system. Here, we present the 2.9 Å crystal structure of the enigmatic, neuronal G protein Gα o in the GTP hydrolytic transition state, complexed with RGS16. Comparison with the 1.89 Å structure of apo-RGS16, also presented here, reveals plasticity upon Gα o binding, the determinants for GAP activity, and the structurally unique features of Gα o that likely distinguish it physiologically from other members of the larger Gα i family, affording insight to receptor, GAP and effector specificity.

EB1 is a member of a conserved protein family that localizes to growing microtubule plus ends. EB1 proteins also recruit cell polarity and signaling molecules to microtubule tips. However, the mechanism by which EB1 recognizes cargo is unknown. Here, we have defined a repeat sequence in adenomatous polyposis coli (APC) that binds to EB1's COOH-terminal domain and identified a similar sequence in members of the microtubule actin cross-linking factor (MACF) family of spectraplakins. We show that MACFs directly bind EB1 and exhibit EB1-dependent plus end tracking in vivo. To understand how EB1 recognizes APC and MACFs, we solved the crystal structure of the EB1 COOH-terminal domain. The structure reveals a novel homodimeric fold comprised of a coiled coil and four-helix bundle motif. Mutational analysis reveals that the cargo binding site for MACFs maps to a cluster of conserved residues at the junction between the coiled coil and four-helix bundle. These results provide a structural understanding of how EB1 binds two regulators of microtubule-based cell polarity.

A multitude of heptahelical receptors use heterotrimeric G proteins to transduce signals to specific effector target molecules. The G protein transducin, Gt, couples photon-activated rhodopsin with the effector cyclic GMP phosophodiesterase (PDE) in the vertebrate phototransduction cascade. The interactions of the Gt alpha-subunit (alpha(t)) with the inhibitory PDE gamma-subunit (PDEgamma) are central to effector activation, and also enhance visual recovery in cooperation with the GTPase-activating protein regulator of G-protein signalling (RGS)-9 (refs 1-3). Here we describe the crystal structure at 2.0 A of rod transducin alpha x GDP x AlF4- in complex with the effector molecule PDEgamma and the GTPase-activating protein RGS9. In addition, we present the independently solved crystal structures of the RGS9 RGS domain both alone and in complex with alpha(t/i1) x GDP x AlF4-. These structures reveal insights into effector activation, synergistic GTPase acceleration, RGS9 specificity and RGS activity. Effector binding to a nucleotide-dependent site on alpha(t) sequesters PDEgamma residues implicated in PDE inhibition, and potentiates recruitment of RGS9 for hydrolytic transition state stabilization and concomitant signal termination.

RGS (regulators of G protein signaling) proteins regulate G protein signaling by accelerating GTP hydrolysis, but little is known about regulation of GTPase-accelerating protein (GAP) activities or roles of domains and subunits outside the catalytic cores. RGS9-1 is the GAP required for rapid recovery of light responses in vertebrate photoreceptors and the only mammalian RGS protein with a defined physiological function. It belongs to an RGS subfamily whose members have multiple domains, including G(gamma)-like domains that bind G(beta)(5) proteins. Members of this subfamily play important roles in neuronal signaling. Within the GAP complex organized around the RGS domain of RGS9-1, we have identified a functional role for the G(gamma)-like-G(beta)(5L) complex in regulation of GAP activity by an effector subunit, cGMP phosphodiesterase gamma and in protein folding and stability of RGS9-1. The C-terminal domain of RGS9-1 also plays a major role in conferring effector stimulation. The sequence of the RGS domain determines whether the sign of the effector effect will be positive or negative. These roles were observed in vitro using full-length proteins or fragments for RGS9-1, RGS7, G(beta)(5S), and G(beta)(5L). The dependence of RGS9-1 on G(beta)(5) co-expression for folding, stability, and function has been confirmed in vivo using transgenic Xenopus laevis. These results reveal how multiple domains and regulatory polypeptides work together to fine tune G(talpha) inactivation.

Summary Centrioles are key microtubule polarity determinants. Centriole duplication is tightly controlled to prevent cells from developing multipolar spindles, a situation that promotes chromosomal instability. A conserved component in the duplication pathway is Plk4, a polo kinase family member that localizes to centrioles in M/G1. To limit centriole duplication, Plk4 levels are controlled through trans -autophosphorylation that primes ubiquitination. In contrast to Plks 1-3, Plk4 possesses a unique central region called the "cryptic polo box." Here, we present the crystal structure of this region at 2.3 A resolution. Surprisingly, the structure reveals two tandem homodimerized polo boxes, PB1-PB2, that form a unique winged architecture. The full PB1-PB2 cassette is required for binding the centriolar protein Asterless as well as robust centriole targeting. Thus, with its C-terminal polo box (PB3), Plk4 has a triple polo box architecture that facilitates oligomerization, targeting, and promotes trans -autophosphorylation, limiting centriole duplication to once per cell cycle.