The development of complex tissues requires that mitotic progenitor cells integrate information from the environment. The highly varied outcomes of such integration processes undoubtedly depend at least in part upon variations among the gene expression programs of individual progenitor cells. To date, there has not been a comprehensive examination of these differences among progenitor cells of a particular tissue. Here, we used comprehensive gene expression profiling to define these differences among individual progenitor cells of the vertebrate retina. Retinal progenitor cells (RPCs) have been shown by lineage analysis to be multipotent throughout development and to produce distinct types of daughter cells in a temporal, conserved order. A total of 42 single RPCs were profiled on Affymetrix arrays. In situ hybridizations performed on both retinal sections and dissociated retinal cells were used to validate the results of the microarrays. An extensive amount of heterogeneity in gene expression among RPCs, even among cells isolated from the same developmental time point, was observed. While many classes of genes displayed heterogeneity of gene expression, the expression of transcription factors constituted a significant amount of the observed heterogeneity. In contrast to previous findings...
Studies of complex multicellular organisms would benefit from the ability to selectively manipulate the activities of any cell type of interest. Our ability to achieve this is currently limited by technology and available resources. Here, I explore the artificial use of intracellular proteins as signals for conferring cell specificity in gene manipulation.
The Green Fluorescent Protein (GFP) is a useful marker of gene expression and thousands of transgenic GFP reporter lines have been made to label different cell types, particularly in the mouse nervous system. However, the utility of transgenic GFP reporter lines is limited to labeling purposes. I exploited this resource for cell-specific gene manipulation by constructing synthetic systems that become biologically active upon interaction with GFP. Using GFP-binding nanobodies derived from Camelid antibodies, I co-opted GFP as a scaffold protein to bring together complementary split proteins that, when in a complex, can regulate processes such as transcription and DNA recombination. I demonstrate the utility of these systems for selectively manipulating GFP-expressing cells in the mouse nervous system and in zebrafish, for applications such as developmental perturbations, electrophysiology and optogenetic interrogation of neural circuits.
To reduce the complexity of GFP-dependent systems...
Many RNAs show distinct localization patterns in cells with enrichment at particular subcellular sites or organelles. RNA localization is a highly conserved process that both spatially and temporally controls gene expression. A common mechanism to selectively sort RNAs within the cell involves recognition of cis-acting sequences on the RNA by trans-acting RNA-binding proteins. Recently, the ESCRT-II complex was identified as a novel trans-acting factor required for the localization of bicoid mRNA in Drosophila oocytes. ESCRT-II was previously uncharacterized as an RNA-binding complex but has a well-established role in multivesicular body formation and receptor downregulation. Recent studies have revealed links between endosomes and RNA regulatory pathways, and the dual roles of ESCRT-II in both cellular processes suggest that it will be an important factor to better understand as an RNA-binding complex. However, bicoid is the only identified direct RNA target of ESCRT-II, and whether ESCRT-II’s role in RNA localization is conserved in other organisms is unclear.
Here we report that the role of ESCRT-II in RNA regulation is conserved in Xenopus eggs. We found that ESCRT-II interacts with hundreds of RNAs in Xenopus eggs, and we characterized the molecular details of this interaction. Using a UV-crosslinking approach...
Preimplantation genetic diagnosis (PGD) following in vitro fertilization (IVF) offers couples at risk for transmitting genetic disorders the opportunity to identify affected embryos prior to replacement. In particular, embryo gender determination permits screening for X-linked diseases of unknown etiology. Analysis of embryos can be performed by polymerase chain reaction (PCR) amplification of material obtained by micromanipulation. This approach provides an alternative to the termination of an established pregnancy following chorionic villi sampling or amniocentesis. ^ Lately, the focus of preimplantation diagnosis and intervention has been shifting toward an attempt to correct cytoplasmic deficiencies. Accordingly, it is the aim of this investigation to develop methods to permit the examination of single cells or components thereof for clinical evaluation. In an attempt to lay the groundwork for precise therapeutic intervention for age related aneuploidy, transcripts encoding proteins believed to be involved in the proper segregation of chromosomes during human oocyte maturation were examined and quantified. Following fluorescent rapid cycle RT-PCR analysis it was determined that the concentration of cell cycle checkpoint gene transcripts decreases significantly as maternal age increases. Given the well established link between increasing maternal age and the incidence of aneuploidy...
Glycogen Synthase Kinase 3 (GSK3), a serine/threonine kinase initially characterized in the context of glycogen metabolism, has been repeatedly realized as a multitasking protein that can regulate numerous cellular events in both metazoa and protozoa. I recently found GSK3 plays a role in regulating chemotaxis, a guided cell movement in response to an external chemical gradient, in one of the best studied model systems for chemotaxis - Dictyostelium discoideum. ^ It was initially found that comparing to wild type cells, gsk3 - cells showed aberrant chemotaxis with a significant decrease in both speed and chemotactic indices. In Dictyostelium, phosphatidylinositol 3,4,5-triphosphate (PIP3) signaling is one of the best characterized pathways that regulate chemotaxis. Molecular analysis uncovered that gsk3- cells suffer from high basal level of PIP3, the product of PI3K. Upon chemoattractant cAMP stimulation, wild type cells displayed a transient increase in the level of PIP3. In contrast, gsk3- cells exhibited neither significant increase nor adaptation. On the other hand, no aberrant dynamic of phosphatase and tensin homolog (PTEN), which antagonizes PI3K function, was observed. Upon membrane localization of PI3K, PI3K become activated by Ras...
Aranda, Juan F.; Reglero-Real, Natalia; Kremer, Leonor; Marcos-Ramiro, Beatriz; Ruiz-Sáenz, Ana; Calvo, María; Enrich, Carlos; Correas, Isabel; Millán, Jaime; Alonso, Miguel A.
Fonte: The American Society of Cell BiologyPublicador: The American Society of Cell Biology
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
Copyright (2011) American Institute of Physics. This article may be downloaded for personal use only. Any other use requires prior permission of the author and the American Institute of Physics. The following article appeared in Molecular Biology of the Cell 22.8 (2011): 1252-1262 and may be found at http://www
.molbiolcell.org/cgi/doi/10.1091/mbc.E10-11-0910; Membrane organization into condensed domains or rafts provides molecular platforms for selective recruitment of proteins. Cell migration is a general process that requires spatiotemporal targeting of Rac1 to membrane rafts. The protein machinery responsible for making rafts competent to recruit Rac1 remains elusive. Some members of the MAL family of proteins are involved in specialized processes dependent on this type of membrane. Because condensed membrane domains are a general feature of the plasma membrane of all mammalian cells, we hypothesized that MAL family members with ubiquitous expression and plasma membrane distribution could be involved in the organization of membranes for cell migration. We show that myeloid-associated differentiation marker (MYADM), a protein with unique features within the MAL family, colocalizes with Rac1 in membrane protrusions at the cell surface and distributes in condensed membranes. MYADM knockdown (KD) cells had altered membrane condensation and showed deficient incorporation of Rac1 to membrane raft fractions and...
Endothelin 3 (Edn3) is a ligand important to developing neural crest cells (NCC). Some NCC eventually migrate into the skin and give rise to the pigment-forming melanocytes found in hair follicles. Edn3's effects on NCC have been largely explored through spontaneous mutants and cell culture experiments. These studies have shown the Endothelin receptor B/Edn3 signaling pathway to be important in the proliferation/survival and differentiation of developing melanocytes. To supplement these investigations I have created doxycycline-responsive transgenic mice which conditionally over-express Edn3. These mice will help us clarify Edn3's role during the development of early embryonic melanoblasts, differentiating melanocyte precursors in the skin, and fully differentiated melanocytes in the hair follicle. The transgene mediated expression of Edn3 was predominantly confined to the roof plate of the neural tube and surface ectoderm in embryos and postnatally in the epidermal keratinocytes of the skin. Relative to littermate controls, transgenics develop increased pigmentation on most areas of the skin. My doxycycline-based temporal studies have shown that both embryonic and postnatal events are important for establishing and maintaining pigmented skin. The study of my Edn3 transgenic mice may offer some insight into the genetics behind benign dermal pigmentation and offer clues about the time periods important in establishing these conditions. This apparently abnormal development is echoed in a benign condition of human skin. Cases of dermal melanocytosis...
The amyloid cascade hypothesis places amyloid-β at the origin of Alzheimer's disease (AD). Amyloid-β (Aβ) is the product of the sequential cleavage of the amyloid precursor protein (APP) by the enzymes β- and γ-secretases. An inflammatory component to AD has been suggested in association with CD40 (a member of the tumor necrosis factor receptor superfamily (TNFRS) and its cognate ligand CD40L. In this study, I hypothesized that the neutralization of pro-inflammatory cytokines produced downstream of CD40/CD40L interaction would reduce APP processing. I also hypothesized that blocking the binding of different adaptor proteins to CD40 by mutating its cytoplasmic tail would result in significant reduction of the APP metabolites: Aβ, sAPPβ, sAPPα, CTFβ and CTFα. ^ Treatment with CD40L of human embryonic kidney cells over-expressing both APP and CD40 (HEK/APPsw/CD40) significantly increased levels of the cytokine granulocyte macrophage colony stimulating factor (GM-CSF). Neutralizing antibodies against GM-CSF mitigated the CD40L-induced production of Aβ in these cells. Treatment of the HEK/APPsw/CD40 cells with recombinant GM-CSF significantly increased Aβ levels. GM-CSF receptor gene silencing with shRNA significantly reduced Aβ levels to below base line in non-stimulated HEK/APPsw/CD40 cells. Silencing of the GM-CSF receptor also decreased APP endocytosis (therefore reducing the availability of APP to be cleaved in the endosomes). ^ Using CD40 mutants...
The nervous system and the immune system are the primary sensory interfaces between the internal and external environment. They are responsible for recognizing, integrating, and responding to stimuli with the appropriate valence and magnitude to optimize host fitness. Furthermore, an alluring parallel concept is that both systems have the capacity to form memories of these encounters leading to optimized and adaptive future responses. Recent work in the fields of neuroscience and immunology has led to a high-resolution map of cell subsets within both systems. Here, we start to leverage these advances to explore the relationship between these two sensory systems at the level of discrete cell subsets in immunological disease and homeostasis with a focus on interactions at barrier tissues. This work stems from the initial hypothesis that sensory neurons for noxious stimuli, nociceptors, regulate inflammation by controlling production of key instructive and effector cytokines derived from tissue-resident immune cells.
In our first investigation focused on the skin, we identified that NaV1.8+ TRPV1+ nociceptors, via interactions with dDCs, are essential in vivo regulators of interleukin-(IL)-23/IL-17 pathway cutaneous immune responses. This set of studies raised several intriguing questions including determining what the inputs and outputs of nociceptors are that regulate IL-23 production from dDCs and whether nociceptors play a role in distinct inflammatory contexts. In further experiments we determined that while TRPV1 itself is dispensable...
Systems Biology has emerged in the last years as a new holistic approach
based on the global understanding of cells instead of only being focused on
their individual parts (genes or proteins), to better understand the complexity
of human cells. Since the Systems Biology still does not provide the most
accurate answers to our questions due to the complexity of cells and the
limited quality of available information to perform a good gene/protein map
analysis, we have created simpler models to ensure easier analysis of the map
that represents the human cell. Therefore, a virtual organism has been designed
according to the main physiological rules for humans in order to replicate the
human organism and its vital functions. This toy model was constructed by
defining the topology of its genes/proteins and the biological functions
associated to it. There are several examples of these toy models that emulate
natural processes to perform analysis of the virtual life in order to design
the best strategy to understand real life. The strategy applied in this study
combines topological and functional analysis integrating the knowledge about
the relative position of a node among the others in the map with the
conclusions generated by mathematical models that reproduce functional data of
the virtual organism. Our results demonstrate that the combination of both
strategies allows better understanding of our virtual organism even with the
lower input of information needed and therefore it can be a potential tool to
better understand the real life.; Comment: KEY WORDS: Systems Biology Functional Analysis Topological Analysis
Algorithm Protein network
Our main tenet argues that the primary role of positive thymic selection and
the resulting T cell population is the maintenance of a homeostatic equilibrium
with self MHC-self peptide complexes. The homeostatic T cell repertoire can
recognize infections non-specifically and this is an indirect (negative)
recognition: the whole homeostatic T cell population together "holds a mirror"
to the whole self, and any MHC-peptide complex that is "not reflected in the
mirror" can be perceived by surrounding homeostatic T cells as a signal of the
presence of a foreign entity. On the other hand, infection-specific T cell
clones arise in a different pathway in the periphery, do not enter the thymus,
and form a functionally different population. Here we summarize the basic
assumptions and consequences of a logic-based new model, which differs from
conventional models in many respects.; Comment: 15 pages, 1 figure, 1 table
Many events in the vertebrate immune system are influenced by some element of
chance. The objective of the present work is to describe affinity maturation of
B lymphocytes (in which random events are perhaps the most characteristic), and
to study a possible network model of immune memory. In our model stochastic
processes govern all events. A major novelty of this approach is that it
permits studying random variations in the immune process. Four basic components
are simulated in the model: non-immune self cells, nonself cells (pathogens), B
lymphocytes, and bone marrow cells that produce naive B lymphocytes. A point in
a generalized shape space plus the size of the corresponding population
represents nonself and non-immune self cells. On the other hand, each
individual B cell is represented by a disc that models its recognition region
in the shape space. Infection is simulated by an "injection" of nonself cells
into the system. Division of pathogens may instigate an attack of naive B
cells, which in turn may induce clonal proliferation and hypermutation in the
attacking B cells, and which eventually may slow down and stop the exponential
growth of pathogens. Affinity maturation of newly produced B cells becomes
expressed as a result of selection when the number of pathogens decreases.
Under favorable conditions...
In the past few years, overwhelming evidence has accrued that a high level of
expression of the protein neuroglobin protects neurons in vitro, in animal
models, and in humans, against cell death associated with hypoxic and amyloid
insult. However, until now, the exact mechanism of neuroglobin's protective
action has not been determined. Using cell biology and biochemical approaches
we demonstrate that neuroglobin inhibits the intrinsic pathway of apoptosis in
vitro and intervenes in activation of pro-caspase 9 by interaction with
cytochrome c. Using systems level information of the apoptotic signalling
reactions we have developed a quantitative model of neuroglobin inhibition of
apoptosis, which simulates neuroglobin blocking of apoptosome formation at a
single cell level. Furthermore, this model allows us to explore the effect of
neuroglobin in conditions not easily accessible to experimental study. We found
that the protection of neurons by neuroglobin is very concentration sensitive.
The impact of neuroglobin may arise from both its binding to cytochrome c and
its subsequent redox reaction, although the binding alone is sufficient to
block pro-caspase 9 activation. These data provides an explanation the action
of neuroglobin in the protection of nerve cells from unwanted apoptosis.; Comment: 11 pages
BH3 mimetics have been proposed as new anticancer therapeutics. They target
anti-apoptotic Bcl-2 proteins, up-regulation of which has been implicated in
the resistance of many cancer cells, particularly leukemia and lymphoma cells,
to apoptosis. Using probabilistic computational modeling of the mitochondrial
pathway of apoptosis, verified by single-cell experimental observations, we
develop a model of Bcl-2 inhibition of apoptosis. Our results clarify how Bcl-2
imparts its anti-apoptotic role by increasing the time-to-death and
cell-to-cell variability. We also show that although the commitment to death is
highly impacted by differences in protein levels at the time of stimulation,
inherent stochastic fluctuations in apoptotic signaling are sufficient to
induce cell-to-cell variability and to allow single cells to escape death. This
study suggests that intrinsic cell-to-cell stochastic variability in apoptotic
signaling is sufficient to cause fractional killing of cancer cells after
exposure to BH3 mimetics. This is an unanticipated facet of cancer
chemoresistance.; Comment: 11 pages, In press
Migrating cells possess intracellular gradients of Rho GTPases, but it is
unknown whether these shallow gradients themselves can induce motility. Here we
describe a new method to present cells with induced linear gradients of active,
endogenous Rac without receptor activation. Gradients as low as 15% were
sufficient to not only trigger cell migration up the synthetic gradient, but
also to induce both cell polarization and repolarization. Response kinetics
were inversely proportional to Rac gradient values, in agreement with a new
mathematical model, suggesting a role for natural input gradient amplification
upstream of Rac. Increases in Rac levels beyond a well-defined threshold
dramatically augmented polarization and decreased sensitivity to the gradient
value. The threshold was governed by initial cell polarity and PI3K activity,
supporting a role for both in defining responsiveness to natural or synthetic
Rac activation. Our methodology suggests a general way to investigate processes
regulated by intracellular signaling gradients.
Apoptosis is an important area of research because of its role in keeping a
mature multicellular organism's number of cells constant hence, ensuring that
the organism does not have cell accumulation that may transform into cancer
with additional hallmarks. Firstly, we have carried out sensitivity analysis on
an existing mitochondria-dependent mathematical apoptosis model to find out
which parameters have a role in causing monostable cell survival i.e.,
malfunction in apoptosis. We have then generated three healthy cell models by
changing these sensitive parameters while preserving bistability i.e., healthy
functioning. For each healthy cell, we varied the proapoptotic production
rates, which were found to be among the most sensitive parameters, to yield
cells that have malfunctioning apoptosis. We simulated caspase-3 activation, by
numerically integrating the governing ordinary differential equations of a
mitochondria-dependent apoptosis model, in a hypothetical malfunctioning cell
which is treated by four potential treatments, namely: (i) proteasome inhibitor
treatment, (ii) Bcl-2 inhibitor treatment, (iii) IAP inhibitor treatment, (iv)
Bid-like synthetic peptides treatment. The simulations of the present model
suggest that proteasome inhibitor treatment is the most effective treatment
though it may have severe side effects. For this treatment...
Tumor Necrosis Factor alpha (TNF) initiates a complex series of biochemical
events in the cell upon binding to its type R1 receptor (TNF-R1). Recent
experimental work has unravelled the molecular regulation of the recruitment of
initial signaling complexes that lead either to cell survival or death.
Survival signals are activated by direct binding of TNF to TNF-R1 at the cell
membrane whereas apoptotic signals by endocytosed TNF/TNF-R1 complexes. Here we
investigate these aspects by developing a quantitative mathematical model of
TNF binding, internalization and intracellular signaling. Model outputs compare
favorably with experimental data and allow to compute TNF-mediated cytotoxicity
as observed in different cell systems. We extensively study the space of
parameters to show that the model is structurally stable and robust over a
broad range of parameter values. Thus, our model is suitable for implementation
in multi-scale simulation programs that are presently under development to
study the behavior of large tumor cell populations.; Comment: 32 pages, 6 figures
Heterochromatin, or condensed chromatin, is a transcriptionally repressive form of chromatin that occurs in many eukaryotic organisms. At its natural locations, heterochromatin is thought to play important roles in genome organization as well as gene expression. Just as important is the restriction of this repressive form of chromatin to appropriate regions of the genome. In the budding yeast Saccaromyces cerevisiae, domains of condensed, transcriptionally silenced chromatin are found at telomeres and at the silent-mating type cassettes, HML and HMR. At these locations, a complex of Silent Information Regulator (SIR) proteins gets recruited to DNA through discrete silencer elements. Once recruited, the Sir protein complex then spreads along chromosomes in a step-wise manner. This process results in the silencing of gene expression. It is unclear whether silenced chromatin is established in the same manner at different genomic locations. Understanding how silenced chromatin is formed is important for determining how these chromatin structures are regulated.
To better understand how silenced chromatin is established in different genomic contexts, I used chromatin immuoprecipitation to follow the rate of silenced chromatin formation at different locations. The rates of Sir protein assembly were compared at two locations...
Pheromones are chemicals from conspecifics that affect innate behavior or hormonal changes. In mammals, the vomeronasal organ (VNO) is thought to play a prominent role in detecting pheromones; the vomeronasal sensory neurons (VSNs) express three families of seven-transmembrane G-protein coupled receptors (GPCRs): the V1Rs, V2Rs, and FPRs, in two molecularly and spatially-distinct regions. In mice, VSNs that express the V2Rs are thought to detect peptide cues, including MHC-presenting peptides, major urinary proteins (MUPs), and exocrine gland-secreting peptides (ESPs). They are thought to be involved in various pheromone-mediated behaviors and physiological changes, such as mating, aggression, and selective pregnancy block. In order to understand how pheromones are detected by the vomeronasal receptors, it is essential to know which receptors are activated by a given chemical. However, identifying cognate ligands for the V2Rs has been challenging, partly because they are poorly localized to the surface of heterologous cells. Here, we show that the calreticulin chaperone family members play a crucial role in trafficking V2Rs. A calreticulin homologue, calreticulin4 is specifically expressed in the VNO, while calreticulin expression level is low. Depleting calreticulin expression in HEK293T cells allows V2Rs to be trafficked to the cell surface...
Reversible acetylation has emerged as an important post-translational modification that rivals phosphorylation in regulating chromatin structure and gene expression. Acetylation of histone is associated with transcriptional activation while deacetylation is linked to transcriptional repression. Moreover, histone deacetylase inhibitors induce growth arrest, differentiation and apoptosis of cancer cells and therefore appear to be promising anti-tumor agents. While transcriptional deregulation is thought to be the main mechanism underlying their therapeutic effects, the exact mechanisms and targets by which HDAC inhibitors, which are mostly non-specific, achieve their anti-tumor effects remain poorly understood. In other words, it is not known which and how each HDAC members are involved in supporting tumor growth.
In this thesis, I have showed that HDAC6, a cytoplasmic localized and cytoskeleton-associated deacetylase, is required for efficient oncogenic transformation and tumor formation. I have found that HDAC6 expression is induced upon oncogenic Ras-induced transformation in both human somatic cells and murine fibroblasts. Conversely, murine fibroblasts deficient in HDAC6 are more resistant to both oncogenic Ras and ErbB2-dependent transformation...