A validation strategy to assess the role of phase separation as a determinant of macromolecular localization.

Liquid-liquid phase separation (LLPS) of putative assembly scaffolds has been proposed to drive the biogenesis of membraneless compartments. LLPS scaffolds are usually identified through in vitro LLPS assays with single macromolecules (homotypic), but the predictive value of these assays remains poorly characterized. Here, we apply a strategy to evaluate the robustness of homotypic LLPS assays. When applied to the chromosomal passenger complex (CPC), which undergoes LLPS in vitro and localizes to centromeres to promote chromosome biorientation, LLPS propensity in vitro emerged as an unreliable predictor of subcellular localization. In vitro CPC LLPS in aqueous buffers was enhanced by commonly used crowding agents. Conversely, diluted cytomimetic media dissolved condensates of the CPC and of several other proteins. We also show that centromeres do not seem to nucleate LLPS, nor do they promote local, spatially restrained LLPS of the CPC. Our strategy can be adapted to purported LLPS scaffolds of other membraneless compartments.

The kinetochore proteins CENP-E and CENP-F directly and specifically interact with distinct BUB mitotic checkpoint Ser/Thr kinases.

The segregation of chromosomes during cell division relies on the function of the kinetochores, protein complexes that physically connect chromosomes with microtubules of the spindle. The metazoan proteins, centromere protein E (CENP-E) and CENP-F, are components of a fibrous layer of mitotic kinetochores named the corona. Several of their features suggest that CENP-E and CENP-F are paralogs: they are very large (comprising ∼2700 and 3200 residues, respectively), contain abundant predicted coiled-coil structures, are C-terminally prenylated, and are endowed with microtubule-binding sites at their termini. Moreover, CENP-E contains an ATP-hydrolyzing motor domain that promotes microtubule plus end-directed motion. Here, we show that both CENP-E and CENP-F are recruited to mitotic kinetochores independently of the main corona constituent, the Rod/Zwilch/ZW10 (RZZ) complex. We identified specific interactions of CENP-F and CENP-E with budding uninhibited by benzimidazole 1 (BUB1) and BUB1-related (BUBR1) mitotic checkpoint Ser/Thr kinases, respectively, paralogous proteins involved in mitotic checkpoint control and chromosome alignment. Whereas BUBR1 was dispensable for kinetochore localization of CENP-E, BUB1 was stringently required for CENP-F localization. Through biochemical reconstitution, we demonstrated that the CENP-E/BUBR1 and CENP-F/BUB1 interactions are direct and require similar determinants, a dimeric coiled-coil in CENP-E or CENP-F and a kinase domain in BUBR1 or BUB1. Our findings are consistent with the existence of structurally similar BUB1/CENP-F and BUBR1/CENP-E complexes, supporting the notion that CENP-E and CENP-F are evolutionarily related.

Structural basis for Arl3-specific release of myristoylated ciliary cargo from UNC119.

Access to the ciliary membrane for trans-membrane or membrane-associated proteins is a regulated process. Previously, we have shown that the closely homologous small G proteins Arl2 and Arl3 allosterically regulate prenylated cargo release from PDEδ. UNC119/HRG4 is responsible for ciliary delivery of myristoylated cargo. Here, we show that although Arl3 and Arl2 bind UNC119 with similar affinities, only Arl3 allosterically displaces cargo by accelerating its release by three orders of magnitude. Crystal structures of Arl3 and Arl2 in complex with UNC119a reveal the molecular basis of specificity. Contrary to previous structures of GTP-bound Arf subfamily proteins, the N-terminal amphipathic helix of Arl3·GppNHp is not displaced by the interswitch toggle but remains bound on the surface of the protein. Opposite to the mechanism of cargo release on PDEδ, this induces a widening of the myristoyl binding pocket. This leads us to propose that ciliary targeting of myristoylated proteins is not only dependent on nucleotide status but also on the cellular localization of Arl3.

Structural analysis of the Ras-like G protein MglA and its cognate GAP MglB and implications for bacterial polarity.

The bacterium Myxococcus xanthus uses a G protein cycle to dynamically regulate the leading/lagging pole polarity axis. The G protein MglA is regulated by its GTPase-activating protein (GAP) MglB, thus resembling Ras family proteins. Here, we show structurally and biochemically that MglA undergoes a dramatic, GDP-GTP-dependent conformational change involving a screw-type forward movement of the central ß2-strand, never observed in any other G protein. This movement and complex formation with MglB repositions the conserved residues Arg53 and Gln82 into the active site. Residues required for catalysis are thus not provided by the GAP MglB, but by MglA itself. MglB is a Roadblock/LC7 protein and functions as a dimer to stimulate GTP hydrolysis in a 2:1 complex with MglA. In vivo analyses demonstrate that hydrolysis mutants abrogate Myxococcus' ability to regulate its polarity axis changing the reversal behaviour from stochastic to oscillatory and that both MglA GTPase activity and MglB GAP catalysis are essential for maintaining a proper polarity axis.

Structural insights into the small G-protein Arl13B and implications for Joubert syndrome.

Ciliopathies are human diseases arising from defects in primary or motile cilia. The small G-protein Arl13B (ADP-ribosylation factor-like 13B) localizes to microtubule doublets of the ciliary axoneme and is mutated in Joubert syndrome. Its GDP/GTP mechanistic cycle and the effect of its mutations in patients with Joubert syndrome remain elusive. In the present study we applied high resolution structural and biochemical approaches to study Arl13B. The crystal structure of Chlamydomonas rheinhardtii Arl13B, comprising the G-domain and part of its unique C-terminus, revealed an incomplete active site, and together with biochemical data the present study accounts for the absence of intrinsic GTP hydrolysis by this protein. The structure shows that the residues representing patient mutations R79Q and R200C are involved in stabilizing important intramolecular interactions. Our studies suggest that Arg79 is crucial for the GDP/GTP conformational change by stabilizing the large two-residue register shift typical for Arf (ADP-ribosylation factor) and Arl subfamily proteins. A corresponding mutation in Arl3 induces considerable defects in effector and GAP (GTPase-activating protein) binding, suggesting a loss of Arl13B function in patients with Joubert syndrome.

Aurora B SUMOylation Is Restricted to Centromeres in Early Mitosis and Requires RANBP2.

Conjugation with the small ubiquitin-like modifier (SUMO) modulates protein interactions and localisation. The kinase Aurora B, a key regulator of mitosis, was previously identified as a SUMOylation target in vitro and in assays with overexpressed components. However, where and when this modification genuinely occurs in human cells was not ascertained. Here, we have developed intramolecular Proximity Ligation Assays (PLA) to visualise SUMO-conjugated Aurora B in human cells in situ. We visualised Aurora B-SUMO products at centromeres in prometaphase and metaphase, which declined from anaphase onwards and became virtually undetectable at cytokinesis. In the mitotic window in which Aurora B/SUMO products are abundant, Aurora B co-localised and interacted with NUP358/RANBP2, a nucleoporin with SUMO ligase and SUMO-stabilising activity. Indeed, in addition to the requirement for the previously identified PIAS3 SUMO ligase, we found that NUP358/RANBP2 is also implicated in Aurora B-SUMO PLA product formation and centromere localisation. In summary, SUMOylation marks a distinctive window of Aurora B functions at centromeres in prometaphase and metaphase while being dispensable for functions exerted in cytokinesis, and RANBP2 contributes to this control, adding a novel layer to modulation of Aurora B functions during mitosis.

Reconstitution and use of highly active human CDK1:Cyclin-B:CKS1 complexes.

As dividing cells transition into mitosis, hundreds of proteins are phosphorylated by a complex of cyclin-dependent kinase 1 (CDK1) and Cyclin-B, often at multiple sites. CDK1:Cyclin-B phosphorylation patterns alter conformations, interaction partners, and enzymatic activities of target proteins and need to be recapitulated in vitro for the structural and functional characterization of the mitotic protein machinery. This requires a pure and active recombinant kinase complex. The kinase activity of CDK1 critically depends on the phosphorylation of a Threonine residue in its activation loop by a CDK1-activating kinase (CAK). We developed protocols to activate CDK1:Cyclin-B either in vitro with purified CAKs or in insect cells through CDK-CAK co-expression. To boost kinase processivity, we reconstituted a ternary complex consisting of CDK1, Cyclin-B, and CKS1. In this work, we provide and compare detailed protocols to obtain and use highly active CDK1:Cyclin-B (CC) and CDK1:Cyclin-B:CKS1 (CCC).

The crystal structure of the Ran-Nup153ZnF2 complex: a general Ran docking site at the nuclear pore complex.

Nucleoporin (Nup) 153 is a highly mobile, multifunctional, and essential nuclear pore protein. It contains four zinc finger motifs that are thought to be crucial for the regulation of transport-receptor/cargo interactions via their binding to the small guanine nucleotide binding protein, Ran. We found this interaction to be independent of the phoshorylation state of the nucleotide. Ran binds with the highest affinity to the second zinc finger motif of Nup153 (Nup153ZnF2). Here we present the crystal structure of this complex, revealing a new type of Ran-Ran interaction partner interface together with the solution structure of Nup153ZnF2. According to our complex structure, Nup153ZnF2 binding to Ran excludes the formation of a Ran-importin-beta complex. This finding suggests a local Nup153-mediated Ran reservoir at the nucleoplasmic distal ring of the nuclear pore, where nucleotide exchange may take place in a ternary Nup153-Ran-RCC1 complex, so that import complexes are efficiently terminated.

A G-protein activation cascade from Arl13B to Arl3 and implications for ciliary targeting of lipidated proteins.

Small G-proteins of the ADP-ribosylation-factor-like (Arl) subfamily have been shown to be crucial to ciliogenesis and cilia maintenance. Active Arl3 is involved in targeting and releasing lipidated cargo proteins from their carriers PDE6δ and UNC119a/b to the cilium. However, the guanine nucleotide exchange factor (GEF) which activates Arl3 is unknown. Here we show that the ciliary G-protein Arl13B mutated in Joubert syndrome is the GEF for Arl3, and its function is conserved in evolution. The GEF activity of Arl13B is mediated by the G-domain plus an additional C-terminal helix. The switch regions of Arl13B are involved in the interaction with Arl3. Overexpression of Arl13B in mammalian cell lines leads to an increased Arl3·GTP level, whereas Arl13B Joubert-Syndrome patient mutations impair GEF activity and thus Arl3 activation. We anticipate that through Arl13B's exclusive ciliary localization, Arl3 activation is spatially restricted and thereby an Arl3·GTP compartment generated where ciliary cargo is specifically released.

Combination of BMP2 and MSCs significantly increases bone formation in the rat arterio-venous loop model.

INTRODUCTION: In this study the induction of bone formation in an axially vascularized bone matrix using mesenchymal stem cells (MSCs) and application of bone morphogenetic protein 2 (BMP2) was analyzed in the arteriovenous loop (AVL) model. MATERIALS AND METHODS: An AVL was created in the medial thigh of 42 rats and placed in a porous titanium chamber filled with a particulated porous hydroxyapatite and beta-tricalcium phosphate matrix and fibrin. In group A the fibrin was loaded with 5×10(6) DiI-stained fibrin gel-immobilized primary MSCs from syngenic Lewis rats, in group B the matrix was loaded with 60 µg/mL BMP2 and in group C both, BMP2 and MSCs were applied at implantation time point. After 6 and 12 weeks, specimens were investigated by means of histological, morphometrical, and micro-computed tomography analysis. RESULTS: After implantation of an AVL a dense vascular network was visible in all groups. In group A, newly generated bone islands were detected in the periphery of the main vascular axis. Using BMP2 alone (group B), small islands of newly formed bone were visible evenly distributed in all parts of the constructs. In group C nearly the whole matrix was interspersed with bone formations. In all groups there was an increase of bone formation between the 6 and 12 weeks explantation time points. CONCLUSIONS: This study demonstrates for the first time the successful generation of axially vascularized bone substitutes using MSCs and BMP2 in the AVL rat model using a one step procedure. Using the combination of BMP2 and MSCs there was a significant increase of bone formations detectable compared to the BMP2 or MSCs alone groups.

The Interaction of CCDC104/BARTL1 with Arl3 and Implications for Ciliary Function.

Cilia are small antenna-like cellular protrusions critical for many developmental signaling pathways. The ciliary protein Arl3 has been shown to act as a specific release factor for myristoylated and farnesylated ciliary cargo molecules by binding to the effectors Unc119 and PDE6δ. Here we describe a newly identified Arl3 binding partner, CCDC104/CFAP36. Biochemical and structural analyses reveal that the protein contains a BART-like domain and is called BARTL1. It recognizes an LLxILxxL motif at the N-terminal amphipathic helix of Arl3, which is crucial for the interaction with the BART-like domain but also for the ciliary localization of Arl3 itself. These results seem to suggest a ciliary role of BARTL1, and possibly link it to the Arl3 transport network. We thus speculate on a regulatory mechanism whereby BARTL1 aids the presentation of active Arl3 to its GTPase-activating protein RP2 or hinders Arl3 membrane binding in the area of the transition zone.

RasGEF1A and RasGEF1B are guanine nucleotide exchange factors that discriminate between Rap GTP-binding proteins and mediate Rap2-specific nucleotide exchange.

The highly conserved RasGEF1 family of proteins contain a C-terminal CDC25-Ras exchange motif domain and an N-terminal RasGEF-N domain, and are of unknown function and specificity. Using purified RasGEF1A and RasGEF1B proteins, as well as Ras family proteins, we established that RasGEF1A and RasGEF1B function as very specific exchange factors for Rap2, a member of the Rap subfamily of Ras-like G-proteins. They do not act on Rap1 or other members of the Ras subfamily. Although Rap2 was implicated in the regulation of cell adhesion, the establishment of cell morphology, and the modulation of synapses in neurons, no specific guanine nucleotide exchange factor for Rap2 was previously identified. Using reciprocal site-directed mutagenesis, we analyzed residues that allow RasGEF1 proteins to discriminate between Rap1 and Rap2, and we were able to identify Phe39 in the switch I region of Rap2 as a specificity residue. Mutation of the corresponding Ser39 in Rap1 changed the specificity and allowed the nucleotide exchange of Rap1(S39F) to be stimulated by RasGEF1B.

An acylation cycle regulates localization and activity of palmitoylated Ras isoforms.

We show that the specific subcellular distribution of H- and Nras guanosine triphosphate-binding proteins is generated by a constitutive de/reacylation cycle that operates on palmitoylated proteins, driving their rapid exchange between the plasma membrane (PM) and the Golgi apparatus. Depalmitoylation redistributes farnesylated Ras in all membranes, followed by repalmitoylation and trapping of Ras at the Golgi, from where it is redirected to the PM via the secretory pathway. This continuous cycle prevents Ras from nonspecific residence on endomembranes, thereby maintaining the specific intracellular compartmentalization. The de/reacylation cycle also initiates Ras activation at the Golgi by transport of PM-localized Ras guanosine triphosphate. Different de/repalmitoylation kinetics account for isoform-specific activation responses to growth factors.

Synergy of silent and hot spot mutations in importin beta reveals a dynamic mechanism for recognition of a nuclear localization signal.

Molecular recognition of the importin beta-binding (IBB) domain of importin alpha by importin beta is critical for the nuclear import of protein cargoes containing a classical nuclear localization signal. We have studied the function of four conserved tryptophans of importin beta (Trp-342, Trp-430, Trp-472, and Trp-864) located at the binding interface with the IBB domain by systematic alanine substitution mutagenesis. We found that Trp-864 is a mutational hot spot that significantly affects IBB-binding and import activity, whereas residues Trp-342, Trp-430, and Trp-472 are mutationally silent when analyzed individually. Interestingly, the combination of the hot spot at residue Trp-864 with mutations in the other three tryptophans gives rise to a striking synergy that diminishes IBB domain binding by up to approximately 1000-fold and, in turn, abolishes import activity. We propose that importin beta uses the tryptophans to select and stabilize a helical conformation of the IBB domain, which, in turn, conveys specific, high affinity binding.