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Molecular and Genetic Dissection of the BicD Localization Machinery

Applicant Suter Beat
Number 120635
Funding scheme Project funding
Research institution Institut für Zellbiologie Departement Biologie Universität Bern
Institution of higher education University of Berne - BE
Main discipline Embryology, Developmental Biology
Start/End 01.07.2008 - 30.06.2011
Approved amount 510'000.00
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All Disciplines (5)

Discipline
Embryology, Developmental Biology
Genetics
Biochemistry
Cellular Biology, Cytology
Molecular Biology

Keywords (6)

Drosophila; mRNA localization; Subcellular localization; Oogenesis; Organelle transport; Microtubule transport

Lay Summary (English)

Lead
Lay summary
The human body consists of more than 200 distinct cell types that all arise from one single cell, the fertilized egg. Differentiation into specific cell types is strictly controlled during development. Specification of cellular fate often depends on the localization of cytoplasmic determinants to one part of a developing cell, such that these molecules end up in only one of the two daughter cells after the next division and instruct this cell about its fate. As a result, the two daughter cells then assume different fates. We are interested in how these cytoplasmic determinants get to their specific cellular compartment and will investigate the mechanism and the molecular machinery that achieves this.Localized determinants can be mRNAs, proteins or even organelles. Drosophila Bicaudal-D (BicD) localizes such factors during all stages of development and transports the cargo together with microtubule tracks and engines. This system is not unique to Drosophila, but also active in humans.Our present work addresses the following important questions.1) Characterization of the interaction with vesicles, membranes and the cell cortex5 novel genes and proteins that either genetically or physically interact with BicD are involved with membranes or the cortex. By analyzing their function we want to find out how the BicD transport machinery localizes vesicles and organelles, and how it interacts with the cell membrane and cortex.2) Characterization of the BicD - RNA binding protein interaction BicD complexes also contain RNA binding proteins, which could mediate mRNA localization or translational control. Their functional analysis should show how they contribute to the BicD dependent RNA localization processes during oocyte and embryonic differentiation.3) Analysis of the BicD phosphorylation pathway and its function Protein phosphorylation often acts as a molecular switch that changes the structure and activity of a protein. BicD is phosphorylated at multiple sites and two BicD mutations affect this phosphorylation. We will systematically map BicD phosphosites and test their function.Our work will lead to a better understanding of cellular differentiation and development. Knowledge gained from the analysis of Drosophila BicD has already greatly influenced research on human BicD and has also shown that the same mechanism is important even for human brain development. Furthermore, human BicD1 was recently also linked to aging and cancer. We can therefore expect to make additional significant contributions to the understanding of fundamental cellular mechanisms that are relevant to animal and human development and well-being.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Associated projects

Number Title Start Funding scheme
100629 Localisation of cytoplasmic determinants during development 01.07.2003 Project funding
153280 Composition of different BicD/ mRNA complexes and function of cytoplasmic Cbp80 01.07.2014 Project funding
117446 Fast, accurate, and sensitive mass spectrometry for basic research in life sciences at the university of bern / mass spectrometer 01.10.2007 R'EQUIP
135436 Composition and dynamics of BicD and Cbp transport particles 01.07.2011 Project funding

Abstract

Many eukaryotic cells depend on their proper polarization for their development and physiological function. Such cellular asymmetries can be created by the polarized organization of the cytoskeleton and transport of a specific subset of proteins, RNAs and organelles. The questions of how these mRNAs, proteins and organelles are being localized to the appropriate cellular compartment is a fascinating one and we started our molecular dissection of the localization system with the Drosophila BicD gene and protein. We had shown that BicD is part of a localization mechanism that is involved in cell fate determination, oocyte differentiation and growth, and in patterning the oocyte and embryo along the anterior posterior and the dorsal ventral axis. Aside from its function in the female germ line, we also found several processes in the somatic tissue that use this localization machinery. In all these processes, the BicD localization machinery uses microtubules and their motors to localize mRNAs, proteins and sometimes organelles to specific cellular compartments. In the case of early oogenesis it even localizes them from one cell to another cell in a cyst of interconnected cells. While our research focuses on these processes in the Drosophila system, it is interesting to know that the localization system also functions in human cells. After our discovery of this localization machinery, we spent our efforts on characterizing the different BicD functions and on getting additional components using a variety of approaches. On the one hand we used different genetic approaches to screen for contributing genes. On the other hand, we screened for physically interacting proteins by yeast two hybrid system and by immunopurification of BicD complexes. The combined approaches yielded excellent synergies and we have now an interesting pool of good candidates available for further analysis. These include vesicle proteins and 6 RNA binding proteins. We have now entered the most fascinating and rewarding phase and that is to pick out individual genes and groups of genes and find out how they contribute to the localization system. To focus our resources during this harvest phase we selected one group of genes where members were identified in all different approaches, the genetic screen, the genetic interactor screen, and by isolating the physical interactors. These genes and proteins show a connection to vesicle transport and membranes. Our second focus will be on the different mRNA binding proteins, because this large group should reveal us more details about the BicD dependent mRNAs localization. In addition, there is good evidence that phosphorylation of BicD is important for the localization process. Our third research area will therefore focus on the BicD phosphorylation pathway, the identification of the BicD phospho-sites and of their physiological function. Our specific objectives for this granting period will beObjective 1) Characterization of the interaction with vesicles, membranes and the cortexClathrin heavy chain (Chc) co-immunoprecipitate with BicD. We will further ascertain this interaction and analyze the expression of chc and the tagged Chc protein specifically during oogenesis and embryogenesis. This work will be complemented by a genetic analysis of the function of chc in these tissues and during these stages to show whether chc is required for the same processes as BicD. Protein localization studies in various mutants of genes involved in the localization process will show, which genes are required for the localization of Chc. Localization of other machinery components and potential cargos in chc mutants will show, which RNAs and proteins require chc for their localization. We have lethal mutations that show that chc is an essential gene. While these will be used to analyze germ line clones, we also have a female sterile allele that shows oogenesis defects.Based on its phenotypes and its genetic interaction with BicD, sosie functions in (some of) the same processes as BicD. The gene is small, but highly complex in that it encodes two different mRNAs and potentially three different proteins. sosie therefore has a high regulation potential. We will test the usage and function of the different transcripts and ORFs. One of the ORFs encodes a predicted ER protein of unknown function; while the second encodes only the C-term part of it, without the N-terminal signal sequence, and the third ORF encodes an unrelated polypeptide. Because the present allele prevents the expression of only one of the ORFs, we need to create a null allele to study the function of this gene in detail. Work on three genetic interactors encoding the cortical ßH-Spectrin, a seven-pass transmembrane receptor and a multiple transmembrane protein with possibly 10 -12 transmembrane domains may shed light on their involvement with the other BicD interactors sosie, chc, and rab6.Objective 2): Characterization of the BicD - RNA binding protein interactionA declared objective of the BicD complex purification was to identify proteins that could mediate BicD dependent mRNA localization. We are therefore very satisfied having isolated 6 such RNA binding proteins (Rbp) and will focus on the analysis of the first three, Rbp1/PABP -Rbp3. After finishing the verification of the interaction, we will study their expression and function using molecular and classical genetics, immunocytochemistry and embryo injection for localization assays. Objective 3) Analysis of the BicD phosphorylation pathway and its functionThere is good evidence that phosphorylation of BicD dynamically regulates BicD association with either cargo or motor. During this granting period we want to identify the kinases that mediate BicD phosphorylation and to elucidate the function of BicD phosphorylation genetically. We have so far mapped several sites in BicD that are phosphorylated and we are in the process of testing whether their phosphorylation is required for BicD function. We have also designed tests and screens to identify BicD kinases.The discussed experiments will lead to a better understanding of the localization process in Drosophila because they will reveal novel components of this machinery and how they contribute to the localization. Knowledge gained from the analysis of the BicD localization system has already greatly influenced research on the human system. Furthermore, the human disease phenotype caused by a haplo-insufficiency of Lis-1 also bears strong resemblance with the Drosophila Lis-1 phenotype. We can therefore expect to make additional significant contributions to the understanding of a fundamental cellular mechanism that is relevant to animal and human development and well being.
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