Bicaudal-D: Switching motors, cargo and direction

Scope of this thesis Transport of vesicles and organelles is an essential cellular process. Proteins like Rab GTPases, specialized adaptor proteins and motor proteins are involved in targeting and movement of cargos to their destination. This thesis describes the function of the mammalian adaptor protein Bicaudal-D in intracellular transport and its potential role in bidirectional movement of several cargos. Chapter 1 gives an overall introduction of the components involved in transport mechanisms. It starts with a description of the three major types of cytoskeletal filaments, actin, intermediate filaments and microtubules. Subsequently, microtubule associated proteins and motor proteins are discussed in more detail. Chapter 1 concludes with a description of Bicaudal-D, its role in Drosophila development and the function of Bicaudal-D in cargo transport along the microtubules in mammalian cells. Chapter 2 describes the behaviour of vesicles associated with the small GTPase Rab6. It shows that Rab6 is a marker of exocytotic vesicles that are mainly driven by the microtubule plus-end directed motor kinesin-1. Chapter 3 reports a novel interaction partner of mammalian Bicaudal-D, RanBP2. Bicaudal-D binds RanBP2 in the late G2/early prophase and through this interaction targets the microtubule minus end-directed dynein/dynactin motor complex to the nuclear envelope. This interaction contributes to the positioning of the nucleus in close proximity of the microtubule organising centre prior to mitosis. Chapter 4 shows that the N-terminal part of Bicaudal-D acts as a linker between cytoplasmic dynein and its accessory complex dynactin. It explores this finding in the context of dynein motility in vitro. Chapter 5 reviews the role of Rab GTPases in the movement of membrane organelles and discusses several well-studied examples of intracellular transport, such as aggregation and dispersion of melanosomes in pigment cells. The described transport systems are compared with the model of Bicaudal-D function that emerged from the experimental work described in this thesis

X chromosome inactivation in a female carrier of a 1.28 mb deletion encompassing the human X inactivation centre

X chromosome inactivation (XCI) is a mechanism specifically initiated in female cells to silence one X chromosome, thereby equalizing the dose of X-linked gene products between male and female cells. XCI is regulated by a locus on the X chromosome termed the X-inactivation centre (XIC). Located within the XIC is XIST, which acts as a m

Dynamics of gene silencing during X inactivation using allele-specific RNA-seq

Background: During early embryonic development, one of the two X chromosomes in mammalian female cells is inactivated to compensate for a potential imbalance in transcript levels with male cells, which contain a single X chromosome. Here, we use mouse female embryonic stem cells (ESCs) with non-random X chromosome inactivation (XCI) and polymorphic X chromosomes to study the dynamics of gene silencing over the inactive X chromosome by high-resolution allele-specific RNA-seq. Results: Induction of XCI by differentiation of female ESCs shows that genes proximal to the X-inactivation center are silenced earlier than distal genes, while lowly expressed genes show faster XCI dynamics than highly expressed genes. The active X chromosome shows a minor but significant increase in gene activity during differentiation, resulting in complete dosage compensation in differentiated cell types. Genes escaping XCI show little or no silencing during early propagation of XCI. Allele-specific RNA-seq of neural progenitor cells generated from the female ESCs identifies three regions distal to the X-inactivation center that escape XCI. These regions, which stably escape during propagation and maintenance of XCI, coincide with topologically associating domains (TADs) as present in the female ESCs. Also, the previously characterized gene clusters escaping XCI in human fibroblasts correlate with TADs. Conclusions: The gene silencing observed during XCI provides further insight in the establishment of the repressive complex formed by the inactive X chromosome. The association of e

Bicaudal D2, Dynein, and Kinesin-1 Associate with Nuclear Pore Complexes and Regulate Centrosome and Nuclear Positioning during Mitotic Entry

Mammalian Bicaudal D2 is the missing molecular link between cytoplasmic motor proteins and the nucleus during nuclear positioning prior to the onset of mitosis

Bicd2, Dynactin, And Lis1 Cooperate In Regulating Dynein Recruitment To Cellular Structures

Cytoplasmic dynein is the major microtubule minus-end-directed cellular motor. Most dynein activities require dynactin, but the mechanisms regulating cargo-dependent dynein-dynactin interaction are poorly understood. In this study, we focus on dynein-dynactin recruitment to cargo by the conserved motor adaptor Bicaudal D2 (BICD2). We show that dynein and dynactin depend on each other for BICD2-mediated targeting to cargo and that BICD2 N-terminus (BICD2-N) strongly promotes stable interaction between dynein and dynactin both in vitro and in vivo. Direct visualization of dynein in live cells indicates that by itself the triple BICD2-N-dynein-dynactin complex is unable to interact with either cargo or microtubules. However, tethering of BICD2-N to different membranes promotes their microtubule minus-end-directed motility. We further show that LIS1 is required for dynein-mediated transport induced by membrane tethering of BICD2-N and that LIS1 contributes to dynein accumulation at microtubule plus ends and BICD2-positive cellular structures. Our results demonstrate that dynein recruitment to cargo requires concerted action of multiple dynein cofactors. © 2012 Splinter et al