The Mitotic Arrest Deficient Protein MAD2B Interacts with the Clathrin Light Chain A during Mitosis

Contains fulltext : 87811.pdf (publisher's version ) (Open Access)BACKGROUND: Although the mitotic arrest deficient protein MAD2B (MAD2L2) is thought to inhibit the anaphase promoting complex (APC) by binding to CDC20 and/or CDH1 (FZR1), its exact role in cell cycle control still remains to be established. METHODOLOGY/PRINCIPAL FINDINGS: Using a yeast two-hybrid interaction trap we identified the human clathrin light chain A (CLTA) as a novel MAD2B binding protein. A direct interaction was established in mammalian cells via GST pull-down and endogenous co-immunoprecipitation during the G2/M phase of the cell cycle. Through subsequent confocal laser scanning microscopy we found that MAD2B and CLTA co-localize at the mitotic spindle. Clathrin forms a trimeric structure, i.e., the clathrin triskelion, consisting of three heavy chains (CLTC), each with an associated light chain. This clathrin structure has previously been shown to be required for the function of the mitotic spindle through stabilization of kinetochore fibers. Upon siRNA-mediated MAD2B depletion, we found that CLTA was no longer concentrated at the mitotic spindle but, instead, diffusely distributed throughout the cell. In addition, we found a marked increase in the percentage of misaligned chromosomes. CONCLUSIONS/SIGNIFICANCE: Previously, we identified MAD2B as an interactor of the renal cell carcinoma (RCC)-associated protein PRCC. In addition, we found that fusion of PRCC with the transcription factor TFE3 in t(X;1)(p11;q21)-positive RCCs results in an impairment of this interaction and a concomitant failure to shuttle MAD2B to the nucleus. Our current data show that MAD2B interacts with CLTA during the G2/M phase of the cell cycle and that depletion of MAD2B leads to a marked increase in the percentage of misaligned chromosomes and a redistribution of CLTA during mitosis

Asymmetry in skeletal distribution of mouse hematopoietic stem cell clones and their equilibration by mobilizing cytokines

Hematopoietic stem cells (HSCs) are able to migrate through the blood stream and engraft bone marrow (BM) niches. These features are key factors for successful stem cell transplantations that are used in cancer patients and in gene therapy protocols. It is unknown to what extent transplanted HSCs distribute throughout different anatomical niches in the BM and whether this changes with age. Here we determine the degree of hematopoietic migration at a clonal level by transplanting individual young and aged mouse HSCs labeled with barcoded viral vector, followed by assessing the skeletal distribution of hundreds of HSC clones. We detected highly skewed representation of individual clones in different bones at least 11 mo after transplantation. Importantly, a single challenge with the clinically relevant mobilizing agent granulocyte colony-stimulating factor (G-CSF) caused rapid redistribution of HSCs across the skeletal compartments. Old and young HSC clones showed a similar level of migratory behavior. Clonal make- up of blood of secondary recipients recapitulates the barcode composition of HSCs in the bone of origin. These data demonstrate a previously unanticipated high skeletal disequilibrium of the clonal composition of HSC pool long- term after transplantation. Our findings have important implications for experimental and clinical and stem cell transplantation protocols

Towards understanding the catalytic properties of lead-based ballistic modifiers in double-base propellants

Double-base propellants, derived from nitrocellulose and nitroglycerin, are a combined solid fuel and oxidiser system. They present smokeless combustion, and are typically utilised for small rocket motor applications. In order to provide stability in combustion performance, ballistic modifiers, which modify the burn-rate properties in three distinct ways, are added to the formulation. However, these additives are lead-based, which poses personal and environmental safety concerns. Moreover, impending European legislation will soon ban their use. Despite years of experimental research, no viable alternative currently exists, and so the impending ban presents a considerable challenge for our defence industries. For this reason, the main aim of this thesis is to develop a better understanding of the fundamental role played by lead as a ballistic modifier. Catalysis with the lead-based ballistic modifier is known to occur at the solid/gas-phase boundary of the propellant, which is known as the burning surface. It is assumed throughout this work that the lead additives, presented as metal salts in the propellant formulation, will decompose to lead oxide in the high temperatures (> 300 C) of the propellant flame. This is taken as the baseline catalytic model. As such, in this work two computational models have been employed; one to investigate catalysis in the solid-state, the other in the gas-phase. The lead additives are known to generate (i) super-rate, (ii) plateau-burn and (iii) mesa-burning effects, and it is known that carbon-soot, which builds up and is subsequently lost from the burning surface, plays an essential (but unknown) role in these burn-rate phenomena. Thus the interaction of carbon with lead oxide is a recurring theme throughout this work. Looking first to the solid-state, Chapter 3 documents a comparative study of the properties of several metal oxides, namely lead, tin and bismuth oxide. Tin and bismuth oxide are ballistic modifiers which demonstrate super-rate burning, but fail to produce plateau- and mesa-rate burning. This chapter examines the chemical reactivities of each metal oxide through computation of their electronic band gaps, surface energies and surface work functions, to deduce any unique properties that separates the behaviour of lead oxide from the other metal oxides. A layer of amorphous carbon is also bound to the stable surfaces of each metal oxide to ascertain whether any significant differences in bond strength and surface integrity arise. Chapter 4 turns its attention to investigate the formulation of industry-standard ballistic modifiers, which are derived from a blend of lead and copper salts. Here the structures of stable small metal oxide clusters which could form in the gas-phase above the burning surface are investigated with respect to their interaction with carbon. The individual roles of each metal in terms of the burn-rate effects are accounted for, and a phenomenological model is proposed that accounts for the three burn-rate effects. Finally, Chapter 5 presents a continuation of the narrative from Chapter 4, and widens the gas-phase discussion to include tin and bismuth oxide. The results obtained further validate the catalytic model presented in Chapter 4. Thus overall, the work reported in this thesis provides an atomistic interpretation of ballistic modifiers in double-base propellants, routed in first principles simulation, that provides a new platform for the continued search for lead-free additives

Illuminating trajectories of adolescent thriving and contribution through the words of youth: qualitative findings from the 4-H Study of Positive Youth Development

Theory and research in adolescent development have emphasized that contributing to self, others, and community is important to the success of society and predictive of positive youth and later adult development. Despite this emphasis, there is a lack of qualitative and youth-centered research exploring whether adolescents themselves value contribution as part of their daily lives or future goals. Understandings of contribution are, thus, limited in their generalizability. To lessen this gap, we implemented qualitative analyses of open-ended responses from youth in the 4-H Study of Positive Youth Development. We addressed questions about what is meaningful to youth and about their future goals through descriptive and thematic analyses of responses from 56 youth (66% female) who participated in the 4-H Study in each of three grades (6, 9, and 12). Findings indicated that most youth in this study valued acts and/or ideologies of contribution at some point in their adolescence, and several were committed to facets of contribution across grades. The analyses also identified other aspects of these youth experiences (e.g., athletics, family relationships, and academic competencies) that were described as meaningful across adolescence. Findings are discussed in relationship to youth programming aimed at encouraging well-being and contribution in adolescence