Proteomics-Based Monitoring of Pathway Activity Reveals that Blocking Diacylglycerol Biosynthesis Rescues from Alpha-Synuclein Toxicity.

Proteinaceous inclusions containing alpha-synuclein (α-Syn) have been implicated in neuronal toxicity in Parkinson's disease, but the pathways that modulate toxicity remain enigmatic. Here, we used a targeted proteomic assay to simultaneously measure 269 pathway activation markers and proteins deregulated by α-Syn expression across a panel of 33 Saccharomyces cerevisiae strains that genetically modulate α-Syn toxicity. Applying multidimensional linear regression analysis to these data predicted Pah1, a phosphatase that catalyzes conversion of phosphatidic acid to diacylglycerol at the endoplasmic reticulum membrane, as an effector of rescue. Follow-up studies demonstrated that inhibition of Pah1 activity ameliorates the toxic effects of α-Syn, indicate that the diacylglycerol branch of lipid metabolism could enhance α-Syn neuronal cytotoxicity, and suggest a link between α-Syn toxicity and the biology of lipid droplets.

The multi-subunit GID/CTLH E3 ubiquitin ligase promotes cell proliferation and targets the transcription factor Hbp1 for degradation.

In yeast, the glucose-induced degradation-deficient (GID) E3 ligase selectively degrades superfluous gluconeogenic enzymes. Here, we identified all subunits of the mammalian GID/CTLH complex and provide a comprehensive map of its hierarchical organization and step-wise assembly. Biochemical reconstitution demonstrates that the mammalian complex possesses inherent E3 ubiquitin ligase activity, using Ube2H as its cognate E2. Deletions of multiple GID subunits compromise cell proliferation, and this defect is accompanied by deregulation of critical cell cycle markers such as the retinoblastoma (Rb) tumor suppressor, phospho-Histone H3 and Cyclin A. We identify the negative regulator of pro-proliferative genes Hbp1 as a bonafide GID/CTLH proteolytic substrate. Indeed, Hbp1 accumulates in cells lacking GID/CTLH activity, and Hbp1 physically interacts and is ubiquitinated in vitro by reconstituted GID/CTLH complexes. Our biochemical and cellular analysis thus demonstrates that the GID/CTLH complex prevents cell cycle exit in G1, at least in part by degrading Hbp1.

Guard the guardian: A CRL4 ligase stands watch over histone production.

Histones are evolutionarily conserved proteins that together with DNA constitute eukaryotic chromatin in a defined stoichiometry. Core histones are dynamic scaffolding proteins that undergo a myriad of post-translational modifications, which selectively engage chromosome condensation, replication, transcription and DNA damage repair. Cullin4-RING ubiquitin E3 ligases are known to hold pivotal roles in a wide spectrum of chromatin biology ranging from chromatin remodeling and transcriptional repression, to sensing of cytotoxic DNA lesions. Our recent work uncovers an unexpected function of a CRL4 ligase upstream of these processes in promoting histone biogenesis. The CRL4WDR23 ligase directly controls the activity of the stem-loop binding protein (SLBP), which orchestrates elemental steps of canonical histone transcript metabolism. We demonstrate that non-proteolytic ubiquitination of SLBP ensures sufficient histone reservoirs during DNA replication and is vital for genome integrity and cellular fitness.

Cortical dynamics during cell motility are regulated by CRL3(KLHL21) E3 ubiquitin ligase.

Directed cell movement involves spatial and temporal regulation of the cortical microtubule (Mt) and actin networks to allow focal adhesions (FAs) to assemble at the cell front and disassemble at the rear. Mts are known to associate with FAs, but the mechanisms coordinating their dynamic interactions remain unknown. Here we show that the CRL3(KLHL21) E3 ubiquitin ligase promotes cell migration by controlling Mt and FA dynamics at the cell cortex. Indeed, KLHL21 localizes to FA structures preferentially at the leading edge, and in complex with Cul3, ubiquitylates EB1 within its microtubule-interacting CH-domain. Cells lacking CRL3(KLHL21) activity or expressing a non-ubiquitylatable EB1 mutant protein are unable to migrate and exhibit strong defects in FA dynamics, lamellipodia formation and cortical plasticity. Our study thus reveals an important mechanism to regulate cortical dynamics during cell migration that involves ubiquitylation of EB1 at focal adhesions.

CRL4(WDR23)-Mediated SLBP Ubiquitylation Ensures Histone Supply during DNA Replication.

To maintain genome integrity and epigenetic information, mammalian cells must carefully coordinate the supply and deposition of histones during DNA replication. Here we report that the CUL4 E3 ubiquitin ligase complex CRL4(WDR23) directly regulates the stem-loop binding protein (SLBP), which orchestrates the life cycle of histone transcripts including their stability, maturation, and translation. Lack of CRL4(WDR23) activity is characterized by depletion of histones resulting in inhibited DNA replication and a severe slowdown of growth in human cells. Detailed analysis revealed that CRL4(WDR23) is required for efficient histone mRNA 3' end processing to produce mature histone mRNAs for translation. CRL4(WDR23) binds and ubiquitylates SLBP in vitro and in vivo, and this modification activates SLBP function in histone mRNA 3' end processing without affecting its protein levels. Together, these results establish a mechanism by which CUL4 regulates DNA replication and possible additional chromatin transactions by controlling the concerted expression of core histones.

biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes.

Analyses of protein complexes are facilitated by methods that enable the generation of recombinant complexes via coexpression of their subunits from multigene DNA constructs. However, low experimental throughput limits the generation of such constructs in parallel. Here we describe a method that allows up to 25 cDNAs to be assembled into a single baculoviral expression vector in only two steps. This method, called biGBac, uses computationally optimized DNA linker sequences that enable the efficient assembly of linear DNA fragments, using reactions developed by Gibson for the generation of synthetic genomes. The biGBac method uses a flexible and modular "mix and match" approach and enables the generation of baculoviruses from DNA constructs at any assembly stage. Importantly, it is simple, efficient, and fast enough to allow the manual generation of many multigene expression constructs in parallel. We have used this method to generate and characterize recombinant forms of the anaphase-promoting complex/cyclosome, cohesin, and kinetochore complexes.

CRL4(RBBP7) is required for efficient CENP-A deposition at centromeres.

The mitotic spindle drives chromosome movement during mitosis and attaches to chromosomes at dedicated genomic loci named centromeres. Centromeres are epigenetically specified by their histone composition, namely the presence of the histone H3 variant CENP-A, which is regulated during the cell cycle by its dynamic expression and localization. Here, we combined biochemical methods and quantitative imaging approaches to investigate a new function of CUL4-RING E3 ubiquitin ligases (CRL4) in regulating CENP-A dynamics. We found that the core components CUL4 and DDB1 are required for centromeric loading of CENP-A, but do not influence CENP-A maintenance or pre-nucleosomal CENP-A levels. Interestingly, we identified RBBP7 as a substrate-specific CRL4 adaptor required for this process, in addition to its role in binding and stabilizing soluble CENP-A. Our data thus suggest that the CRL4 complex containing RBBP7 (CRL4(RBBP7)) might regulate mitosis by promoting ubiquitin-dependent loading of newly synthesized CENP-A during the G1 phase of the cell cycle.

Parallel feedback loops control the basal activity of the HOG MAPK signaling cascade.

Tight regulation of the MAP kinase Hog1 is crucial for survival under changing osmotic conditions. Interestingly, we found that Hog1 phosphorylates multiple upstream components, implying feedback regulation within the signaling cascade. Taking advantage of an unexpected link between glucose availability and Hog1 activity, we used quantitative single cell measurements and computational modeling to unravel feedback regulation operating in addition to the well-known adaptation feedback triggered by glycerol accumulation. Indeed, we found that Hog1 phosphorylates its activating kinase Ssk2 on several sites, and cells expressing a non-phosphorylatable Ssk2 mutant are partially defective for feedback regulation and proper control of basal Hog1 activity. Together, our data suggest that Hog1 activity is controlled by intertwined regulatory mechanisms operating with varying kinetics, which together tune the Hog1 response to balance basal Hog1 activity and its steady-state level after adaptation to high osmolarity.

A cooperative mechanism drives budding yeast kinetochore assembly downstream of CENP-A.

Kinetochores are megadalton-sized protein complexes that mediate chromosome-microtubule interactions in eukaryotes. How kinetochore assembly is triggered specifically on centromeric chromatin is poorly understood. Here we use biochemical reconstitution experiments alongside genetic and structural analysis to delineate the contributions of centromere-associated proteins to kinetochore assembly in yeast. We show that the conserved kinetochore subunits Ame1(CENP-U) and Okp1(CENP-Q) form a DNA-binding complex that associates with the microtubule-binding KMN network via a short Mtw1 recruitment motif in the N terminus of Ame1. Point mutations in the Ame1 motif disrupt kinetochore function by preventing KMN assembly on chromatin. Ame1-Okp1 directly associates with the centromere protein C (CENP-C) homologue Mif2 to form a cooperative binding platform for outer kinetochore assembly. Our results indicate that the key assembly steps, CENP-A recognition and outer kinetochore recruitment, are executed through different yeast constitutive centromere-associated network subunits. This two-step mechanism may protect against inappropriate kinetochore assembly similar to rate-limiting nucleation steps used by cytoskeletal polymers.

Molecular requirements for the formation of a kinetochore-microtubule interface by Dam1 and Ndc80 complexes.

Kinetochores are large protein complexes that link sister chromatids to the spindle and transduce microtubule dynamics into chromosome movement. In budding yeast, the kinetochore-microtubule interface is formed by the plus end-associated Dam1 complex and the kinetochore-resident Ndc80 complex, but how they work in combination and whether a physical association between them is critical for chromosome segregation is poorly understood. Here, we define structural elements required for the Ndc80-Dam1 interaction and probe their function in vivo. A novel ndc80 allele, selectively impaired in Dam1 binding, displayed growth and chromosome segregation defects. Its combination with an N-terminal truncation resulted in lethality, demonstrating essential but partially redundant roles for the Ndc80 N-tail and Ndc80-Dam1 interface. In contrast, mutations in the calponin homology domain of Ndc80 abrogated kinetochore function and were not compensated by the presence of Dam1. Our experiments shed light on how microtubule couplers cooperate and impose important constraints on structural models for outer kinetochore assembly.

CENP-T proteins are conserved centromere receptors of the Ndc80 complex.

Centromeres direct the assembly of kinetochores, microtubule-attachment sites that allow chromosome segregation on the mitotic spindle. Fundamental differences in size and organization between evolutionarily distant eukaryotic centromeres have in many cases obscured general principles of their function. Here we demonstrate that centromere-binding proteins are highly conserved between budding yeast and humans. We identify the histone-fold protein Cnn1(CENP-T) as a direct centromere receptor of the microtubule-binding Ndc80 complex. The amino terminus of Cnn1 contains a conserved peptide motif that mediates stoichiometric binding to the Spc24-25 domain of the Ndc80 complex. Consistent with the critical role of this interaction, artificial tethering of the Ndc80 complex through Cnn1 allows mini-chromosomes to segregate in the absence of a natural centromere. Our results reveal the molecular function of CENP-T proteins and demonstrate how the Ndc80 complex is anchored to centromeres in a manner that couples chromosome movement to spindle dynamics.

Spatiotemporal regulation of Ipl1/Aurora activity by direct Cdk1 phosphorylation.

Oscillating cyclin-dependent kinase 1 (Cdk1) activity is the major regulator of cell-cycle progression, whereas the Aurora B kinase, as part of the chromosome passenger complex (CPC), controls critical aspects of mitosis such as chromosome condensation and biorientation on the spindle. How these kinases mechanistically coordinate their important functions is only partially understood. Here, using budding yeast, we identify a regulatory mechanism by which the Cdk1 kinase Cdc28 directly controls the Aurora kinase Ipl1. We show that Cdk1 phosphorylates Ipl1 on two serine residues in the N-terminal domain, thereby suppressing its association with the microtubule plus-end tracking protein Bim1 until the onset of anaphase. Failure to phosphorylate Ipl1 leads to its premature targeting to the metaphase spindle and results in constitutive Bim1 phosphorylation, which is normally restricted to anaphase. Cells expressing an Ipl1-Sli15 complex that cannot be phosphorylated by Cdk1 display a severe growth defect. Our work shows that Ipl1/Aurora is not only the catalytic subunit of the CPC but also an important regulatory target that allows Cdk1 to coordinate chromosome biorientation with spindle morphogenesis.

A blueprint for kinetochores - new insights into the molecular mechanics of cell division.

Kinetochores are large proteinaceous complexes that physically link centromeric DNA to the plus ends of spindle microtubules. Stable kinetochore-microtubule attachments are a prerequisite for the accurate and efficient distribution of genetic material over multiple generations. In the past decade, concerted research has resulted in the identification of the individual kinetochore building blocks, the characterization of critical microtubule-interacting components, such as the NDC80 complex, and the development of an approximate model of the architecture of this sophisticated biological machine.

The Dam1 complex confers microtubule plus end-tracking activity to the Ndc80 kinetochore complex.

Kinetochores must remain associated with microtubule ends, as they undergo rapid transitions between growth and shrinkage. The molecular basis for this essential activity that ensures correct chromosome segregation is unclear. In this study, we have used reconstitution of dynamic microtubules and total internal reflection fluorescence microscopy to define the functional relationship between two important budding yeast kinetochore complexes. We find that the Dam1 complex is an autonomous plus end-tracking complex. The Ndc80 complex, despite being structurally related to the general tip tracker EB1, fails to recognize growing ends efficiently. Dam1 oligomers are necessary and sufficient to recruit Ndc80 to dynamic microtubule ends, where both complexes remain continuously associated. The interaction occurs specifically in the presence of microtubules and is subject to regulation by Ipl1 phosphorylation. These findings can explain how the force harvested by Dam1 is transmitted to the rest of the kinetochore via the Ndc80 complex.

Ca2+/Calmodulin-dependent kinase II signaling causes skeletal overgrowth and premature chondrocyte maturation.

The long bones of vertebrate limbs originate from cartilage templates and are formed by the process of endochondral ossification. This process requires that chondrocytes undergo a progressive maturation from proliferating to postmitotic prehypertrophic to mature, hypertrophic chondrocytes. Coordinated control of proliferation and maturation regulates growth of the skeletal elements. Various signals and pathways have been implicated in orchestrating these processes, but the underlying intracellular molecular mechanisms are often not entirely known. Here we demonstrated in the chick using replication-competent retroviruses that constitutive activation of Calcium/Calmodulin-dependent kinase II (CaMKII) in the developing wing resulted in elongation of skeletal elements associated with premature differentiation of chondrocytes. The premature maturation of chondrocytes was a cell-autonomous effect of constitutive CaMKII signaling associated with down-regulation of cell-cycle regulators and up-regulation of chondrocyte maturation markers. In contrast, the elongation of the skeletal elements resulted from a non-cell autonomous up-regulation of the Indian hedgehog responsive gene encoding Parathyroid-hormone-related peptide. Reduction of endogenous CaMKII activity by overexpressing an inhibitory peptide resulted in shortening of the skeletal elements associated with a delay in chondrocyte maturation. Thus, CaMKII is an essential component of intracellular signaling pathways regulating chondrocyte maturation.