Cell transitions are characterized by a wide range of cell physiological modifications. As cells progress towards a disease or developmental fate, they change their gene expression, shape, size, motility, polarity, metabolism and signaling, in addition to interactions with their niche. While we know a considerable amount about the molecular processes regulating many cell behaviours, how changes in these dynamic behaviours are coordinated over time is unclear. Superimposed upon many developmental transitions is cell heterogeneity. Cells in clonal populations show considerable heterogeneity in just about any property one can measure. Does this heterogeneity reflect different cell paths towards a common fate, or differences in developmental timing between cells? This generic problem in developmental and disease biology is formalized by two alternative hypotheses: either cells follow a stereotypical linear path from state A to state B; or cells move between A and B via a variety of paths, with different behaviours shown by different cells. Using live imaging of transcriptional bursts in single cells, in combination with measurement of multiple dynamic cell properties, we will generate a multi-parameter description of the dynamic sequence(s) of events occurring during a developmental transition. This will illuminate the implications of heterogeneity on cell behaviour.

Endocytosis plays key roles in various cellular events. However, it remains largely unknown when, where and how endocytosis occurs in each cellular activity, mainly because it is impossible by conventional imaging techniques to visualize the structural dynamics of endocytosis in living cells. Here, I aim to unravel the nature of endocytosis by taking advantage of both two-photon microscopy and high-speed atomic force microscopy (AFM). I will optically identify endocytosis events using two-photon microscopy and visualize the events with nano-meter scale using high-speed AFM. Since endocytosis is involved in a variety of cellular activities and mediated by various molecules, I hypothesize that endocytosis consists of various structural forms. First, I will investigate the location, time-course and shape of endocytosis taking place in cell lines to classify the dynamic structures of endocytosis. Second, I will identify the molecules responsible for each type of endocytosis by gain- and loss-of-function analysis of candidate genes in cell lines. Third, I will reveal the functional relevance of each type of endocytosis by observing when, where and how endocytosis occurs in neurons during synaptic plasticity, in which endocytosis is known to play an essential role. Furthermore, I will clarify the spatio-temporal relationship between molecular activity, endocytosis events and structural changes of dendritic spines during synaptic plasticity by combining two-photon uncaging of caged glutamate, two-photon fluorescent life time imaging microscopy and high-speed AFM. This proposed study may provide new insights into the nature of endocytosis overlooked by conventional techniques.

Movement precision is ultimately determined by the resolution of the motor circuit at the level of single neurons and synapses. However, other factors such as muscle quality/quantity and joint flexibility also affect locomotion. Hence, it is not clear to what extent the fineness of the motor circuit determines physical ability. I propose to tackle this question using Caenorhabditis elegans as a model system. C. elegans moves with a sinusoidal pattern and has no skeletal structures or joints, enabling us to directly test how the synaptic pattern of the motor neurons affects locomotion. In worms locomotion is regulated by 6 types of motor neurons. Each neuron in the same class innervates a non-overlapping segment of the muscle field by restricting its synapses to a distinct sub-axonal region. This type of innervation generates a phenomenon we term “synaptic tiling”. Recently we developed a genetic marker to visualize synaptic tiling between two motor neurons, and showed that Plexin-dependent axon-axon interaction is critical for establishing synaptic tiling. There are, however, many questions that remain unanswered, for example: 1. What are the other components in the Plexin signaling pathway? 2. How is tiling regulated in other neurons? 3. What effects do synaptic tiling defects have on locomotion? To answer these questions, I will attempt to identify the additional molecules involved in synaptic tiling; develop tools to stably distinguish neurons within the same class; and utilize high-sensitivity markers to monitor the effect of synaptic tiling on locomotion. With these studies I hope to use the power of genetics to determine how synaptic patterns affect locomotion.

Complex, multicellular organisms are made up of many cell types, each performing different functions. Understanding the evolution of cells' social interactions is hard: multicellularity evolved long ago and dissecting individual interactions is difficult. We therefore evolve social behavior in two microbes, E. coli and budding yeast. Comparing a prokaryote and a eukaryote will suggest hypotheses for how social interactions between cells evolved in plants and animals.
We will use sucrose utilization as a model to study social evolution. Sucrose hydrolysis releases one molecule of glucose and one of fructose, highly preferred carbon sources for many microbes. In yeast, the enzymes that hydrolyze sucrose are extracellular public goods, whereas in E. coli they are plasmid-encoded, private goods.
We will use genetic engineering, experimental evolution, theory, and simulation to investigate three aspects of social evolution: cooperation, the origin of multicellularity, and the division of labor. We have four aims:
1) Engineer and evolve differentiation within multicellular yeast aggregates to test the idea that undifferentiated, multicellular aggregates evolved before the division of labor.
2) Study the role of plasmid transfer in bacterial cooperation. Does the production of secreted public goods by plasmids select for the faster transfer of plasmids to neighboring cells?
3) Ask how prior history and initial conditions determine the trajectory of evolution. We will engineer yeast and bacteria so they can evolve from similar starting points. We will vary whether sucrose-harvesting systems are public or private, whether they are transmissible or chromosomal, and whether cells can differentiate.
4) We will compare our experiments with three different methods to model social evolution: analytical theory, numerical simulation, and evolving digital organisms within computers.
The proposed work will benefit from a) combining engineering and evolution as experimental approaches, b) comparing different approaches to model these problems, c) strong interaction between theory and experiment, d) the interplay between expertise in yeast and bacterial cell biology, and e) regular meetings to exchange results, critiques and ideas that will influence future research.

Toxoplasma gondii is an obligate intracellular parasite infecting an estimated 30% of the human population worldwide. The infection it produces can pose serious health risks in immunocompromised patients or during pregnancy. Because it can infect almost any warm-blooded animal Toxoplasma is considered one of the most successful parasites in nature. My goal is to understand how Toxoplasma has evolved to be such a generalist, what mechanisms confer this ability and what cost this has on transmission in the most relevant hosts. I will do this in light of recent information that the interaction between Toxoplasma and a given host is highly dependent upon allelic differences (both sequence polymorphisms and copy number variants) between effectors secreted by the parasite into the host cell. My working hypothesis is that this genetic diversity is a result of selection for the ability to infect multiple hosts and I will test this by repeatedly passing a recent, natural isolate of Toxoplasma, with and without mutagenesis, through macrophages from different host species and assessing the transcriptomic, genomic and epigenomic changes that occur within the parasite as a result of selection in each host. I expect to see changes in sequence and/or expression of the effectors that reflect the optimal interaction for each host species.

Heart cell differentiation is driven by the sequential action of dedicated transcription factors. These factors orchestrate the activity of thousands of genes at each step of cardiac development by binding to a constellation of regulatory elements. We aim at understanding how the interplay between these transcription factors and cis-regulatory elements underlie proper execution of the mammalian cardiac gene expression programs.
In order to isolate precisely staged cell types we will use a well-controlled ex-vivo system based on the directed differentiation of mouse embryonic stem cells into beating cardiomyocytes, through step-wise commitment to mesoderm and cardiac precursor lineages. DNAseI-sequencing experiments will be performed to create genome-wide maps of cis-regulatory elements and identify transcription factors that bind them. Stage-specific transcription factor regulatory networks will be reconstructed using established computational methods. Overlaying the information from detailed maps of chromatin topology we will obtain from high-throughput Chromosome Conformation Capture experiments will reveal targets of these cis-regulatory elements. Integration with other available datasets, such as RNA-seq, will allow precise understanding of the temporal changes in the structure of the (epi)genome that underlie transcriptional dynamics during cardiac cell differentiation. Finally, analysis of mutant ES cells as well as mouse embryos will provide mechanistic insights as to how disruption of key transcription factors or regulatory elements impairs the execution of cardiac gene expression programs, heart morphogenesis and heart function.

Traditionally, bone has been viewed as a relatively static tissue only fulfilling mechanical and scaffolding functions. In the past decade however, this classical view has considerably evolved towards a more complex picture. Through the secretion of an osteoblast-derived molecule, osteocalcin (Ocn), the skeleton regulates functions as important and diverse as glucose metabolism, energy expenditure and male fertility. The diversity of Ocn’s known functions, together with the powerful inhibition of bone mass accrual exerted by the brain, was a logical incentive to test if the skeleton, through a feedback loop, would signal back to the brain. Consistent with this hypothesis, we uncovered a dialogue of functional relevance between the bone and the brain occurring at several stages during life, and relying on the bone derived hormone Ocn. Post-natally, Ocn crosses the blood-brain barrier and binds to neurons of the brainstem, midbrain and hippocampus. Ocn enhances the synthesis of several neurotransmitters, inhibits GABA synthesis and favors adult neurogenesis. All together, these functions prevent anxiety and depression and favor spatial learning and memory. During embryonic development, maternal Ocn crosses the placenta and mediates the maternal influence on brain development and functions of the offspring. Although we showed that Ocn has an important effect on brain, the mode of action by which this hormone is acting on fetal brain development and adult neurogenesis remain elusive. Indeed, we propose here to identify these biological and molecular mechanism(s) and to enter in a broader search for the Ocn receptor(s) and the downstream cascade triggered by Ocn in the brain. Because of this unexpected bone-brain crosstalk, we believe that this application is novel and has the potential to be truly transformative for the field of bone biology.

Influenza A causes around 300.000 deaths worldwide each year. Because of a high degree of recombination of its surface epitopes long lasting immunity cannot be achieved. Influenza like many pathogens has developed immune evasion strategies. An important mechanism of antigen delivery for presentation to T cells, autophagy, is targeted by Influenza. The consequence for viral immunogenicity is unknown.
I aim at unraveling the molecular mechanisms underlying the fusion of autophagosomes with antigen loading compartments. To elucidate the cross-talk between viral proteins and the autophagy machinery I will investigate matrix protein 2 (M2) of Influenza A. M2 was shown to cause a block of autophagosome/lysosome fusion, which in turn leads to an accumulation of viral antigens in autophagosomes. I will identify the cellular players interacting with M2 and the mechanism of regulation. Furthermore, the role of autophagy in antigen presentation after Influenza A infection will be addressed applying an inducible mouse model of autophagy deficiency in pneumocytes, the main target of Influenza A infection in mice.
By integrating all findings (molecular mechanism of antigen delivery at steady-state and in viral infection, manipulation of the host machinery by M2, role of autophagy in viral immunogenicity) I will gain a broad overview of the regulation of autophagosome fusion in health and disease. This will help the field to advance in finding new ways to target Influenza infections. However, the implications of these findings are not necessarily limited to infection, as autophagy also plays a major role in many other diseases, such as neurodegenerative diseases.

The methylation of arginine residues is a common post-translational protein modification that plays poorly understood roles in the nervous system. Our work will test the hypothesis that methylation of synaptic proteins is an important regulator nervous system function. Our preliminary findings indicate that numerous synaptic proteins are methylated in vivo, and that at least two members of the PRMT (protein arginine methyltransferase) family, PRMT1 and PRMT8, localize to synaptic sites. We also find that PRMT8 and the postsynaptic scaffolding protein Shank3 (SH3 and multiple ankyrin repeat domains 3) physically interact in vivo, and that PRMT8 can methylate Shank3 in vitro. Further, we find that pharmacological inhibition of arginine methylation or manipulation of specific PRMTs in cultured neurons can alter dendritic spine density and synaptic function. We are proposing to more thoroughly characterize the effects of pharmacological PRMT inhibition in cultured neurons, and then examine the contribution of individual PRMTs to the observed phenotypes. Next, we will characterize the effects of methylation on Shank3 function as well as performing a proteomic screen to identify additional arginine methylated synaptic proteins. Finally, we will explore the importance of arginine methylation in vivo by characterizing the structural, electrophysiological and behavioural phenotypes of PRMT1 and PRMT8 conditional knockout mice. Together, these experiments will allow us to test the importance of this previously un-described modification of synaptic proteins, and if successful will establish arginine methylation as a novel regulator of brain function and plasticity.

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) of eukaryotic cells leads to ER stress eliciting the unfolded protein response (UPR). The UPR is a network of intracellular signaling pathways that adjusts the protein folding capacity of the ER according to the demands of the cell. The transmembrane kinase/RNase Ire1 functions as a signal transducer and represents the most conserved branch. Ire1 oligomerizes in the ER membrane upon stress-induced activation. However, the relationship between different oligomeric states and the activity of Ire1 remains elusive. The goal of the proposed project is to mechanistically dissect how different assembly steps of Ire1 trigger its activation. Using state-of-the-art biochemical and biophysical methods, I will study the assembly steps and structure of higher-order oligomers of Ire1. To this end, I will implement two nanobiological approaches. I will study the structure of full-length Ire1 embedded in artificial lipid bilayers (nanodiscs). In addition, I will exploit the DNA origami nanotechnology to scrutinize the activity of defined oligomeric states assembled on DNA scaffolds. Both approaches will also help to decipher the interaction of Ire1 with RNA substrates. I will further integrate these in vitro results into Ire1 signaling in vivo: By using novel genetically encoded tags, I will determine the localization and ultrastructure of Ire1 signaling platforms in the ER membrane by correlated light and electron microscopy. These analyses hold the promise to significantly enhance our understanding of the molecular activation mechanism of Ire1 and the cellular organization of ER stress signaling.

In humans, nucleotide excision repair (NER) is the exclusive mechanism for the removal of DNA damage induced by ultraviolet light and thus protects against the primary cause of skin cancer development. NER must detect minuscule DNA modifications obscured in chromatin, initiate chromatin decondensation and then assemble the NER repairosome. NER employs two damage detection complexes with remarkably contrasting properties. XPE recognizes chromatinized DNA damage and induces ubiquitin dependent chromatin decondensation, but is dispensable for the recruitment of the NER machinery. XPC recognizes major DNA helix distortions but has no affinity for lesions in chromatin, yet it is essential for NER in living cells. Consequently, damage handover must occur in order to initate NER, presumably by the formation of an as yet unidentified bipartite damage detection supercomplex. Here I propose to study the damage handover mechanism and aim to provide experimental and structural evidence in support of the existence of an XPE-XPC-DNA damage handover complex. I will determine the structure of this 0.5MDa nucleoprotein complex by X-ray crystallography and chemical cross-linking followed by mass spectrometry will guide the crystallization of representative subcomplexes. In vitro reconstitution of damage handover and structure-guided mutational analysis will help to dissect the damage handover mechanism in a stepwise manner. Insight into the regulation of damage handover will be validated in living cells. This study will provide a detailed understanding of a process whose malfunction is the cause of a severe genetic disease that condemns its sufferers to life without sunlight.

Nuclear constriction is an important requirement for all cells migrating through narrow pores inside extracellular matrix or between other cells. Upon encountering pathogenic bacteria or viruses, dendritic cells (DCs) internalize antigens and are triggered to then infiltrate lymphatic vessels. Here, the applicant first aims to visualize nuclear constriction of DCs migrating through confined, microfabricated matrices of collagen as well as during their process of entering lymphatic vessels. Nuclear squeezing during DC migration will be observed in mouse ear explants and in the footpad of living mice. The applicant hypothesizes that the layer of lamina just under the nuclear envelope protects the against DNA damage during nuclear squeezing. Intermediate filament polymer Lamin A/C is speculated to contribute to mechanical rigidity of the nucleus, and thus its function during nuclear deformation will be evaluated in this proposal. Lamin A/C will be either mutated at a site found in Muscular Dystrophy, depleted with KO cells, or increased with transient transfection, and the effect this has on DC survival through constrictive matrix will be assessed. The extent of DNA damage caused by nuclear constriction will be assayed for and whether Lamin A/C alleviates the damage will be determined. The final part of the proposal will highlight the importance Lamin A/C has for allowing DCs to reach and enter lymphatic vessels in a mouse model. The proposal will combine the efforts from multiple disciplines by applying what is learned about nuclear squeezing from microfabricated devices to an in vivo context in mice.

Nonmembrane-bound compartments in eukaryotic cells are dynamic intracellular organelles performing multifarious cellular functions. Recent studies suggest that phase separation of proteins with Low Complexity Domains (LCDs) may be a unifying principle of assembly of such compartments. However, regulatory mechanisms and molecular regulators of reversible condensation/dissolution are unknown. Recently, kinase activity of dual-specificity kinase DYRK3 was shown to couple stress-induced RNA granule dynamics with mTOR signalling in a manner dependent on its cycling between the granule and cytosol phase. I propose to investigate the mechanism, based on a working hypothesis, of DYRK3 cycling between liquid-unmixed and –mixed phases via N-terminal LCD auto-phosphorylation. I will also perform an RNA interference (RNAi) and chemical compound screen to identify the phosphatase, which counteracts the phosphorylation event in DYRK3, thereby completing the DYRK cycle of partitioning between two phases. This is followed by an analysis of stress-induced changes in spatio-temporal dynamics of DYRK3 activity by performing FRET/FLIM assays inside cells. Finally, I will test how generalizable is the mechanism of DYRK3 cycling among DYRK family members (DYRKs, HIPKs and PRP4s) and whether there is a single mechanism on how the reversible condensation/dissolution of proteins through LCDs works. This research would reveal a novel mechanistic principle shared by a class of kinases to regulate nonmembrane-bound compartmentalization and the signaling driven by it and will thereby be a major advance for the field of cellular compartmentalization.

Intrinsically disordered proteins (IDPs) are widespread in prokaryotes and eukaryotes, and fulfill important functions mainly associated with signaling and regulation. This proposal addresses fundamental questions in a recently discovered subgroup of IDPs, intrinsically disordered chaperones that use the plasticity of intrinsic disorder for their action. My aim in this proposal is to portrait experimentally driven stress response chaperone networks in prokaryotes and eukaryotes, focusing on intrinsically disordered (ID) chaperones. The long-term goal is to map relationships between key proteins maintaining protein homeostasis under diverse stress conditions, and being able to manipulate this network by design of novel chaperones. In this study we will focus on ID chaperones, which undergo conditional unfolding by specific stress conditions (e.g., heat, oxidation, acid and others). The involvement of intrinsic plasticity in molecular recognition is usually explained by such terms as promiscuity and flexibility. Beyond these notions it still remains unclear why specific intrinsically disordered protein were selected to recognize signaling proteins, DNA, small ligands or aggregation-prone proteins. In this proposal, we aim to explore the putative links between intrinsically disordered regions (IDRs) and chaperone activity. In order to establish such links we plan to increase the number of members of this novel protein family. We plan to derive a database of functionally defined IDRs, which will serve to develop a computational toolbox for identification of novel intrinsically disordered chaperones within bacteria and yeast proteomes.

Type-1 interferons (IFNs) play critical roles in antiproliferative, antiviral and immunomodulatory responses. The phenotypic outcome for a cell depends on a complex and largely undetermined interplay between the dose and subtype of IFN it has experienced, the cell type, and any previous signalling events. One hindrance to study has always been an inability to probe the combined long-term response and cellular signalling history of an individual cell. I will design and construct synthetic memory circuits that record multiple aspects of signalling and allow subsequent isolation and profiling. In particular, the pathways activated and degree of activity will be assessed. Using these tools in combination with molecular techniques to quantify and manipulate cell state at the time of signalling, I aim to dissect the factors that direct cell response. A further aspect of signalling modulation occurs through variations in cytokine binding properties. Fundamentally understanding these factors is important for both native and therapeutic cytokines. I therefore aim to use the devices generated to investigate the influence of binding properties on signalling, particularly in the context of targeted IFNs. Together, these systems will provide an unprecedented ability to probe IFN signalling heterogeneity. The project represents a unique approach to understanding cellular outcomes in the context of complex cellular history and includes major advances to mammalian cell synthetic biology. A better fundamental understanding of these type-1 IFN signalling will also have important implications for their use in treating a spectrum of diseases that affect billions worldwide.

Two conditions in humans require allorecognition (the distinction between ‘self’ and ‘non-self’ tissue): pregnancy and transplantation. We will study the role of innate histocompatibility in a model system in Botryllus schlosseri, and in pregnancy immunology and stem cell transplantation in mammals. B. schlosseri is an organism with unusual characteristics that render it a valuable model of naturally accruing allorecognition:(i) a naturally acquired allorecognition where a single highly polymorphic gene determines acceptance or rejection of colonies; this gene (BHF) was recently identified and characterized by our laboratory (ii) genetic/ predicted hierarchies that determine competitive potential among germ line and somatic stem cells respectively;(iii) tissue regeneration with isolatable stem cells mediating formation of body organs de novo every week (iv) known adult stem cell niches;(v) a sequenced genome. The experimental design will take advantage of these natural traits to identify and characterize genes that determine successful chimerism through allorecognition. We will: (a) investigate the B. schlosseri allorecognition pathway and pathways that regulate stem cell transplantation engraftment in B. schlosseri chimeras; (b) identify genes that are associated with human pregnancy disorders and stem cell transplantation using transcriptome analysis; and (c) investigate the roles of homologues genes involved in B. schlosseri and human histocompatibility, in mouse pregnancy disorders models, and (d) stem cell transplantation immunity models.

Although it is clear that during metabolic adaptation and dysfunction information is exchanged between the major organs involved in glucose and lipid homeostasis, the mechanisms that mediate this communication remain in great part elusive. There are inherent difficulties in establishing causality in in vivo systems, and the search for circulating secreted factors is hindered by the challenge of unbiased screening in the complex blood compartment. Our aim is to model the information flow between skeletal muscle, liver, adipose tissue, and mesenchymal stem/stromal cells in a mini-tissue microfluidics system allowing the identification and manipulation of circulating factors affecting tissue function and metabolism. We will develop a system for compartmentalized culture of tissue-specific primary cells with the capability of temporal and spatial control of communication between compartments, allowing us to establish compartment-restricted perturbations (mimicking different physiological and pathophysiological states) and follow their proximal and distal consequences. Tissue-specific function will be monitored by sensors including temperature probes and contractility measurements, and the defined nature of this system will facilitate identification of circulating factors by mass spectrometry.

The ATF4/ATF3 stress pathway is strongly upregulated in both neurons and microglia during neuronal injury, ER stress and in response to neuroinflammatory stimuli. However, the role of this pathway in mediating microglia cell function and in the pathogenesis of neurodegenerative diseases such as Alzheimer's Disease (AD) and Amyotrophic Lateral Sclerosis (ALS) remains to be elucidated. Here, I hypothesize that the ATF4/ATF3 stress response has a crucial role in maintaining CNS homeostasis and preventing aberrant neurodegeneration by repressing damaging pro-inflammatory cytokines in microglia and promoting neuronal cell proliferation. I will first assess how the ATF4/ATF3 pathway affects microglia function and gene expression ex vivo by isolating primary microglia cells from C57Bl6 wild-type mice (WT), ATF3-deficient mice (ATF3-/-) and ATF-deficient mice (ATF4-/-) and assessing their proliferation, microglia activation markers, cytokine production, gene and microRNA profiling and phagocytic capacity. Next, I will assess the role of the ATF3 stress response in mouse models of chronic neurodegeneration by generating ATF3-/-APP/PS1 mice to model AD and ATF3-/-SOD1G93A mice to model ALS. The survival, disease severity, microglia activation and CNS pathology will be assessed in the ATF3-/-APP/PS1 and ATF3-/-SOD1G93A mice compared to their respective controls. Finally, I will determine the impact of specifically deleting ATF4 or ATF3 in microglia in vivo by crossing ATF3flox/flox and ATF4flox/flox mice to the microglia-specific CX3CR1(cre/ER-EYFP) mouse, which will generate mice deficient for ATF3 and ATF4 in CX3CR1+ cells, including all microglial cells.

A particularly well-understood model system for studying the mechanisms of morphogen-mediated patterning is the vertebrate neural tube. Here, Sonic Hedgehog (Shh) induces discrete neuronal progenitor domains through regulation of a combinatorial code of transcription factors. The observed precision and reproducibility of gene expression appears greater than can be accounted for by the precision of Shh signaling. Thus, mechanisms must exist that filter signaling noise. We hypothesize that these involve properties of the downstream transcriptional network regulated by Shh signaling and/or active cell sorting.
I propose an interdisciplinary project to quantify the amount of noise in the pattern of gene expression in the neural tube and investigate how noise is transmitted and buffered. To achieve this, I will generate fluorescent reporters for the expression of three key Shh target genes, Nkx2.2, Olig2 and Pax6, and spatiotemporally correlate their activity with the activity of the Shh pathway. In parallel, a quantitative analysis of the behavior of cells will be performed to elucidate the contribution of differential cell adhesion and active cell sorting. The combined results will be analyzed using dynamical systems, theoretical frameworks, and computational tissue level simulations. For this I will take advantage of an existing collaboration with mathematical physicists and computer scientists.
Despite the importance of precision in development, relatively little is known about the underlying cellular and molecular mechanisms, thus the mechanisms deduced from our quantitative studies are likely to be relevant in other tissues patterned by morphogens.

Epithelial cells have an apical-basal polarity, which is regulated by polarity proteins. Lgl is an important polarity protein that localizes to basolateral membrane. The fly genetic studies suggest that Lgl functions not only as a polarity protein but also as a tumor suppressor.
In the breast cancer cells, the downregulation of Lgl is frequently observed but the pathophysiological significance of Lgl is not understood. Thus, this research project aims to reveal the molecular mechanisms on how Lgl contributes to the development of mammary carcinoma. Especially, this project will focus on the effects of Lgl on cell proliferation and try to reveal the molecular mechanisms on how Lgl regulates cell proliferation in mammary epithelium. To achieve the goal of this project, I designed several experiments.
First, to examine the effects of Lgl in mammary epithelial cells, Lgl will be knockdowned in mammary cells using RNAi techniques. The effects of Lgl-downregulation on cell proliferation will be examined in the culture condition of 3D space, which enable the mammary cells to form acinus structure in vitro. In addition, the effects of Lgl-downregulation on tumorigenicity will be examined by xenograft assay. Secondly, to confirm the results obtained from cultured cells, the phenotype of Lgl null mice will be examined. Finally, the molecular mechanisms on how Lgl-downregulation contributes to carcinogenesis will be addressed. Briefly, by using global analyses such as expression analyses with microarray and mass spectrometry, the downstream molecules of Lgl, which relates to cell proliferation, will be searched to understand the Lgl-mediated mammary carcinogenesis.