Hook3 is a scaffold for the opposite-polarity microtubule-based motors cytoplasmic dynein-1 and KIF1C.

The unidirectional and opposite-polarity microtubule-based motors, dynein and kinesin, drive long-distance intracellular cargo transport. Cellular observations suggest that opposite-polarity motors may be coupled. We recently identified an interaction between the cytoplasmic dynein-1 activating adaptor Hook3 and the kinesin-3 KIF1C. Here, using in vitro reconstitutions with purified components, we show that KIF1C and dynein/dynactin can exist in a complex scaffolded by Hook3. Full-length Hook3 binds to and activates dynein/dynactin motility. Hook3 also binds to a short region in the "tail" of KIF1C, but unlike dynein/dynactin, this interaction does not activate KIF1C. Hook3 scaffolding allows dynein to transport KIF1C toward the microtubule minus end, and KIF1C to transport dynein toward the microtubule plus end. In cells, KIF1C can recruit Hook3 to the cell periphery, although the cellular role of the complex containing both motors remains unknown. We propose that Hook3's ability to scaffold dynein/dynactin and KIF1C may regulate bidirectional motility, promote motor recycling, or sequester the pool of available dynein/dynactin activating adaptors

Protein aggregation mediates stoichiometry of protein complexes in aneuploid cells

Aneuploidy, a condition characterized by chromosome gains and losses, causes reduced fitness and numerous cellular stresses, including increased protein aggregation. Here, we identify protein complex stoichiometry imbalances as a major cause of protein aggregation in aneuploid cells. Subunits of protein complexes encoded on excess chromosomes aggregate in aneuploid cells, which is suppressed when expression of other subunits is coordinately altered. We further show that excess subunits are either degraded or aggregate and that protein aggregation is nearly as effective as protein degradation at lowering levels of excess proteins. Our study explains why proteotoxic stress is a universal feature of the aneuploid state and reveals protein aggregation as a form of dosage compensation to cope with disproportionate expression of protein complex subunits

Sample multiplexing-based targeted pathway proteomics with real-time analytics reveals the impact of genetic variation on protein expression.

Targeted proteomics enables hypothesis-driven research by measuring the cellular expression of protein cohorts related by function, disease, or class after perturbation. Here, we present a pathway-centric approach and an assay builder resource for targeting entire pathways of up to 200 proteins selected from \u3e10,000 expressed proteins to directly measure their abundances, exploiting sample multiplexing to increase throughput by 16-fold. The strategy, termed GoDig, requires only a single-shot LC-MS analysis, ~1 µg combined peptide material, a list of up to 200 proteins, and real-time analytics to trigger simultaneous quantification of up to 16 samples for hundreds of analytes. We apply GoDig to quantify the impact of genetic variation on protein expression in mice fed a high-fat diet. We create several GoDig assays to quantify the expression of multiple protein families (kinases, lipid metabolism- and lipid droplet-associated proteins) across 480 fully-genotyped Diversity Outbred mice, revealing protein quantitative trait loci and establishing potential linkages between specific proteins and lipid homeostasis

Excessive Cell Growth Causes Cytoplasm Dilution And Contributes to Senescence

Cell size varies greatly between cell types, yet within a specific cell type and growth condition, cell size is narrowly distributed. Why maintenance of a cell-type specific cell size is important remains poorly understood. Here we show that growing budding yeast and primary mammalian cells beyond a certain size impairs gene induction, cell-cycle progression, and cell signaling. These defects are due to the inability of large cells to scale nucleic acid and protein biosynthesis in accordance with cell volume increase, which effectively leads to cytoplasm dilution. We further show that loss of scaling beyond a certain critical size is due to DNA becoming limiting. Based on the observation that senescent cells are large and exhibit many of the phenotypes of large cells, we propose that the range of DNA:cytoplasm ratio that supports optimal cell function is limited and that ratios outside these bounds contribute to aging

Mechanism of cyclin D1-dependent genomic instability and neoplastic transformation

Regulation of cyclin D1-dependent kinase activity is essential for cell cycle progression and DNA replication fidelity. Critically, impaired cyclin D1 phosphorylation and ubiquitin-mediated proteolysis following the G1/S transition drives neoplastic growth, suggesting that posttranslational regulation is required for cell homeostasis. Elucidation of mechanisms facilitating S-phase cyclin D1 accumulation and novel functions of nuclear cyclin D1/CDK4 kinase is critical for understanding the role of cyclin D1 in tumorigenesis. The work presented herein demonstrates that accelerated, Fbx4-dependent cyclin D1 degradation following S-phase DNA damage is essential to maintain genome stability. Furthermore, Fbx4 functions as a bona fide tumor suppressor, as Fbx4-deficient mice develop spontaneous tumors and murine fibroblasts exhibit cyclin D1 stabilization, nuclear accumulation, and associated genomic instability. This work also describes novel regulation of the PRMT5 methyltransferase by nuclear cyclin D1/CDK4, thereby facilitating histone methylation and gene repression during S-phase necessary for neoplastic growth. Finally, current work reveals a synergistic relationship between constitutively nuclear cyclin D1 and impaired DNA damage checkpoint integrity in driving lymphomagenesis in mice. Collectively, these findings define an intricate relationship wherein nuclear cyclin D1/CDK4 activity modulates genetic alterations necessary for perturbed DNA replication, genomic instability, and ultimately neoplasia.

Mechanism of Cyclin D1-Dependent Genomic Instability and Neoplastic Transformation

Regulation of cyclin D1-dependent kinase activity is essential for cell cycle progression and DNA replication fidelity. Critically, impaired cyclin D1 phosphorylation and ubiquitin-mediated proteolysis following the G1/S transition drives neoplastic growth, suggesting that posttranslational regulation is required for cell homeostasis. Elucidation of mechanisms facilitating S-phase cyclin D1 accumulation and novel functions of nuclear cyclin D1/CDK4 kinase is critical for understanding the role of cyclin D1 in tumorigenesis. The work presented herein demonstrates that accelerated, Fbx4-dependent cyclin D1 degradation following S-phase DNA damage is essential to maintain genome stability. Furthermore, Fbx4 functions as a bona fide tumor suppressor, as Fbx4-deficient mice develop spontaneous tumors and murine fibroblasts exhibit cyclin D1 stabilization, nuclear accumulation, and associated genomic instability. This work also describes novel regulation of the PRMT5 methyltransferase by nuclear cyclin D1/CDK4, thereby facilitating histone methylation and gene repression during S-phase necessary for neoplastic growth. Finally, current work reveals a synergistic relationship between constitutively nuclear cyclin D1 and impaired DNA damage checkpoint integrity in driving lymphomagenesis in mice. Collectively, these findings define an intricate relationship wherein nuclear cyclin D1/CDK4 activity modulates genetic alterations necessary for perturbed DNA replication, genomic instability, and ultimately neoplasia

Stock trading pattern

For this Interactive Qualifying Project, basic stock market concepts are discussed along with a six week stock portfolio simulation. The initial investment budget was five-hundred thousand dollars. In addition to the simulation and information on the stock market, a review of investment sites on the World Wide Web has been included

Multi-agent system toolkit

This project concerns the creation of a Multi-Agent System Toolkit designed to ease the creation of Multi-Agent Systems. It includes a number of options for their implementation, is object-oriented and modular, and uses commonly known design patterns. An implementation of the N-Queens problem has been included as an example of a Multi-Agent System using our template