analyzing-dendritic-morphology-in-columns-and-layers

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

National Institutes of Health (NIH)

Chromosomes exist in the interphase nucleus as individual chromosome territories. It is unclear to what extent chromosome territories occupy particular positions with respect to each other and how structural rearrangements, such as translocations, affect chromosome organization within the cell nucleus. Here we analyze the relative interphase positioning of chromosomes in mouse lymphoma cells compared to normal splenocytes. We show that in a lymphoma cell line derived from an ATM(-/-) mouse, two translocated chromosomes are preferentially positioned in close proximity to each other. The relative position of the chromosomes involved in these translocations is conserved in normal splenocytes. Relative positioning of chromosomes in normal splenocytes is not due to their random distribution in the interphase nucleus and persists during mitosis. These observations demonstrate that the relative arrangement of chromosomes in the interphase nucleus can be conserved between normal and cancer cells and our data support the notion that physical proximity facilitates rearrangements between chromosomes.

Conservation of relative chromosome positioning in normal and cancer cells.

Cancer cells frequently have disease-specific chromosome rearrangements. It is poorly understood why translocations between chromosomes recur at specific breakpoints in the genome. Here we provide evidence that higher-order spatial genome organization is a contributing factor in the formation of recurrent translocations. We show that MYC, BCL and immunoglobulin loci, which are recurrently translocated in various B-cell lymphomas, are preferentially positioned in close spatial proximity relative to each other in normal B cells. Loci in spatial proximity are non-randomly positioned towards the nuclear interior in normal B cells. This locus proximity is the consequence of higher-order genome structure rather than a property of individual genes. Our results suggest that the formation of specific translocations in human lymphomas, and perhaps other tissues, is determined in part by higher-order spatial organization of the genome.

Spatial proximity of translocation-prone gene loci in human lymphomas.

Malaria parasites and immune responses in an infected human interact on a dynamic landscape, in which a population of replicating parasites depletes a population of replenishing red blood cells (RBCs). These underlying dynamics receive relatively little attention, but they offer unique insights into the processes that control most malaria infections. Here, we focus on the observation that three of the four malaria-parasite species that infect humans are restricted to particular age classes of RBC. We explicitly incorporate this observation in models of infection dynamics to distinguish common from species-specific pressures on host immune responses, and we find that age structuring has profound effects on the course of infection. For all four species conditions exist under which the parasites may persist at low densities, or may clear, even in the absence of an immune response. Catastrophic anemia can occur even with the two species that attack only the youngest RBCs, although only a small fraction of cells are parasitized at any point. Furthermore, with these two, compensatory erythropoetic responses in the host accelerate parasite population growth. A "basic reproduction rate" characterizes these differences in outcomes.

Age-structured red blood cell susceptibility and the dynamics of malaria infections.

Genomes are organized in vivo in the form of chromosomes. Each chromosome occupies a distinct nuclear subvolume in the form of a chromosome territory. The spatial positioning of chromosomes within the interphase nucleus is often nonrandom. It is unclear whether the nonrandom spatial arrangement of chromosomes is conserved among tissues or whether spatial genome organization is tissue-specific.

Tissue-specific spatial organization of genomes.

We assess the consequences of competition for red blood cells (RBCs) in co-infections with the two major agents of human malaria, Plasmodium vivax and Plasmodium falciparum, using differential equations to model the population dynamics of RBCs and parasites. P. vivax parasitizes only the youngest RBCs, but this can reduce the broader RBC population susceptible to P. falciparum. We found that competition for RBCs typically causes one species to suppress the other, depending on their relative reproduction rates and timing of inoculation. However, if the species' reproduction rates are nearly equal, transient increases in RBC production stimulated by the presence of P. falciparum may boost P. vivax parasitemia above its single-species infection level. Conversely, P. falciparum parasitemia is rarely enhanced above its single-species level. Furthermore, transients in RBC production can induce coupled oscillations in the parasitemia of both species. These results are remarkably robust to changes in model parameters.

Competition for red blood cells can enhance Plasmodium vivax parasitemia in mixed-species malaria infections.

The two main agents of human malaria, Plasmodium vivax and Plasmodium falciparum, can induce severe anemia and provoke strong, complex immune reactions. Which dynamical behaviors of host immune and erythropoietic responses would foster control of infection, and which would lead to runaway parasitemia and/or severe anemia? To answer these questions, we developed differential equation models of interacting parasite and red blood cell (RBC) populations modulated by host immune and erythropoietic responses. The model immune responses incorporate both a rapidly responding innate component and a slower-responding, long-term antibody component, with several parasite developmental stages considered as targets for each type of immune response. We found that simulated infections with the highest parasitemia tended to be those with ineffective innate immunity even if antibodies were present. We also compared infections with dyserythropoiesis (reduced RBC production during infection) to those with compensatory erythropoiesis (boosted RBC production) or a fixed basal RBC production rate. Dyserythropoiesis tended to reduce parasitemia slightly but at a cost to the host of aggravating anemia. On the other hand, compensatory erythropoiesis tended to reduce the severity of anemia but with enhanced parasitemia if the innate response was ineffective. For both parasite species, sharp transitions between the schizont and the merozoite stages of development (i.e., with standard deviation in intra-RBC development time <or=2.4 h) were associated with lower parasitemia and less severe anemia. Thus tight synchronization in asexual parasite development might help control parasitemia. Finally, our simulations suggest that P. vivax can induce severe anemia as readily as P. falciparum for the same type of immune response, though P. vivax attacks a much smaller subset of RBCs. Since most P. vivax infections are nonlethal (if debilitating) clinically, this suggests that P. falciparum adaptations for countering or evading immune responses are more effective than those of P. vivax.

Host control of malaria infections: constraints on immune and erythropoeitic response kinetics.

Eukaryotic genomes are highly organized within the cell nucleus. Genome organization not only implies the preferential positioning of genetic elements in the interphase nucleus but also the topographic distribution of biological processes. We have investigated the relationship between spatial organization and genome function in single cells. Myc, c-Met, Igf2r, Asb4, and Zac1 genes have the same radial distribution, but they are not positioned in close proximity with respect to each other. Three-dimensional mapping of their transcription sites uncovered a gene-specific pattern of relative positioning with respect to the nucleolus. We found that the Zac1 gene transcription preferentially occurs juxtaposed to the nucleolus, and that its mRNA accumulates at this site of transcription. Nucleoli isolation followed by qRT-PCR provided evidence for a physical interaction between Zac1 mRNA and the nucleolus. Actinomycin-D treatment induced disassembly of the nucleolus, loss of the RNA-FISH signal, and dramatic increase of the ZAC protein level. However, inhibition of RNA polymerase II had no effect over the Zac1 FISH signal and the protein expression. Induction of cell cycle arrest, which involves participation of the ZAC protein, provoked mRNA release from its retention site and protein synthesis. Our data demonstrate that Zac1 mRNA preferentially accumulates in close proximity to nucleoli within the cell nucleus. In addition, our results suggest a functional link between such spatial distribution and protein expression.

Spatial link between nucleoli and expression of the Zac1 gene.

Building a mathematical model of population dynamics of pathogens within their host involves considerations of factors similar to those in ecology, as pathogens can prey on cells in the host. But within the multicellular host, attacked cell types are integrated with other cellular systems, which in turn intervene in the infection. For example, immune responses attempt to sense and then eliminate or contain pathogens, and homeostatic mechanisms try to compensate for cell loss. This review focuses on modeling applied to malarias, diseases caused by single-cell eukaryote parasites that infect red blood cells, with special concern given to vivax malaria, a disease often thought to be benign (if sometimes incapacitating) because the parasite only attacks a small proportion of red blood cells, the very youngest ones. However, I will use mathematical modeling to argue that depletion of this pool of red blood cells can be disastrous to the host if growth of the parasite is not vigorously check by host immune responses. Also, modeling can elucidate aspects of new field observations that indicate that vivax malaria is more dangerous than previously thought. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12551-010-0034-3) contains supplementary material, which is available to authorized users.

Population dynamics of a pathogen: the conundrum of vivax malaria.

Although 80% of malaria occurs in children under five years of age, infants under six months of age are known to have low rates of infection and disease. It is not clear why this youngest age group is protected; possible factors include maternal antibodies, unique nutrition (breast milk), and the presence of foetal haemoglobin (HbF). This work aims to gain insight into possible mechanisms of protection, and suggest pathways for focused empirical work, by modelling a range of possible effects of foetal haemoglobin and other red blood cell (RBC) developmental changes on parasite dynamics in infants.

Foetal haemoglobin and the dynamics of paediatric malaria.

Plasmodium infections trigger complex immune reactions from their hosts against several life stages of the parasite, including gametocytes. These immune responses are highly variable, depending on age, genetics, and exposure history of the host as well as species and strain of parasite. Although the effects of host antibodies that act against gamete stages in the mosquito (due to uptake in the blood meal) are well documented, the effects of host immunity upon within-host gametocytes are not as well understood. This report consists of a theoretical population biology-based analysis to determine constraints that host immunity impose upon gametocyte population growth. The details of the mathematical models used for the analysis were guided by published reports of clinical and animal studies, incorporated plausible modalities of immune reactions to parasites, and were tailored to the life cycl es of the two most widespread human malaria pathogens, Plasmodium falciparum and Plasmodium vivax.

Host immune constraints on malaria transmission: insights from population biology of within-host parasites.

Modeling tumor growth in vitro is essential for cost-effective testing of hypotheses in preclinical cancer research. 3-D cell culture offers an improvement over monolayer culture for studying cellular processes in cancer biology because of the preservation of cell-cell and cell-ECM interactions. Oxygen transport poses a major barrier to mimicking in vivo environments and is not replicated in conventional cell culture systems. We hypothesized that we can better mimic the tumor microenvironment using a bioreactor system for controlling gas exchange in cancer cell cultures with silicone hydrogel synthetic vessels. Soft-lithography techniques were used to fabricate oxygen-permeable silicone hydrogel membranes containing arrays of micropillars. These membranes were inserted into a bioreactor and surrounded by basement membrane extract (BME) within which fluorescent ovarian cancer (OVCAR8) cells were cultured. Cell clusters oxygenated by synthetic vessels showed a ∼100μm drop-off to anoxia, consistent with in vivo studies of tumor nodules fed by the microvasculature. Oxygen transport in the bioreactor system was characterized by experimental testing with a dissolved oxygen probe and finite element modeling of convective flow. Our study demonstrates differing growth patterns associated with controlling gas distributions to better mimic in vivo conditions.

Microfabricated polymeric vessel mimetics for 3-D cancer cell culture.

How neurons form appropriately sized dendritic fields to encounter their presynaptic partners is poorly understood. The Drosophila medulla is organized in layers and columns and innervated by medulla neuron dendrites and photoreceptor axons. Here, we show that three types of medulla projection (Tm) neurons extend their dendrites in stereotyped directions and to distinct layers within a single column for processing retinotopic information. In contrast, the Dm8 amacrine neurons form a wide dendritic field to receive ∼16 R7 photoreceptor inputs. R7- and R8-derived Activin selectively restricts the dendritic fields of their respective postsynaptic partners, Dm8 and Tm20, to the size appropriate for their functions. Canonical Activin signaling promotes dendritic termination without affecting dendritic routing direction or layer. Tm20 neurons lacking Activin signaling expanded their dendritic fields and aberrantly synapsed with neighboring photoreceptors. We suggest that afferent-derived Activin regulates the dendritic field size of their postsynaptic partners to ensure appropriate synaptic partnership.

Photoreceptor-derived activin promotes dendritic termination and restricts the receptive fields of first-order interneurons in Drosophila.

The mechanisms by which α-thalassemia and sickle cell traits confer protection from severe Plasmodium falciparum malaria are not yet fully elucidated. We hypothesized that hemoglobinopathic erythrocytes reduce the intraerythrocytic multiplication of P. falciparum, potentially delaying the development of life-threatening parasite densities until parasite clearing immunity is achieved.

Hemoglobinopathic erythrocytes affect the intraerythrocytic multiplication of Plasmodium falciparum in vitro.

Transposable elements (TE) have both negative and positive impact on the biology of their host. As a result, a balance is struck between the host and the TE that relies on directing integration to specific genome territories. The extraordinary capacity of DNA sequencing can create ultra dense maps of integration that are being used to study the mechanisms that position integration. Unfortunately, the great increase in the numbers of insertion sites detected comes with the cost of not knowing which positions are rare targets and which sustain high numbers of insertions. To address this problem we developed the serial number system, a TE tagging method that measures the frequency of integration at single nucleotide positions. We sequenced 1 million insertions of retrotransposon Tf1 in the genome of Schizosaccharomyces pombe and obtained the first profile of integration with frequencies for each individual position. Integration levels at individual nucleotides varied over two orders of magnitude and revealed that sequence recognition plays a key role in positioning integration. The serial number system is a general method that can be applied to determine precise integration maps for retroviruses and gene therapy vectors.

Serial number tagging reveals a prominent sequence preference of retrotransposon integration.

Transposable elements (TEs) constitute a substantial fraction of the eukaryotic genome and, as a result, have a complex relationship with their host that is both adversarial and dependent. To minimize damage to cellular genes, TEs possess mechanisms that target integration to sequences of low importance. However, the retrotransposon Tf1 of Schizosaccharomyces pombe integrates with a surprising bias for promoter sequences of stress-response genes. The clustering of integration in specific promoters suggests that Tf1 possesses a targeting mechanism that is important for evolutionary adaptation to changes in environment. We report here that Sap1, an essential DNA-binding protein, plays an important role in Tf1 integration. A mutation in Sap1 resulted in a 10-fold drop in Tf1 transposition, and measures of transposon intermediates support the argument that the defect occurred in the process of integration. Published ChIP-Seq data on Sap1 binding combined with high-density maps of Tf1 integration that measure independent insertions at single-nucleotide positions show that 73.4% of all integration occurs at genomic sequences bound by Sap1. This represents high selectivity because Sap1 binds just 6.8% of the genome. A genome-wide analysis of promoter sequences revealed that Sap1 binding and amounts of integration correlate strongly. More important, an alignment of the DNA-binding motif of Sap1 revealed integration clustered on both sides of the motif and showed high levels specifically at positions +19 and -9. These data indicate that Sap1 contributes to the efficiency and position of Tf1 integration.

Single-Nucleotide-Specific Targeting of the Tf1 Retrotransposon Promoted by the DNA-Binding Protein Sap1 of Schizosaccharomyces pombe.

The most striking structure in the nervous system is the complex yet stereotyped morphology of the neuronal dendritic tree. Dendritic morphologies and the connections they make govern information flow and integration in the brain. The fundamental mechanisms that regulate dendritic outgrowth and branching are subjects of extensive study. In this review, we summarize recent advances in the molecular and cellular mechanisms for routing dendrites in layers and columns, prevalent organizational structures in the brain. We highlight how dendritic patterning influences the formation of synaptic circuits.

Article	Year
Conservation of relative chromosome positioning in normal and cancer cells. Current biology : CB\| PubMed ID: 12361574	2002
Spatial proximity of translocation-prone gene loci in human lymphomas. Nature genetics\| PubMed ID: 12808455	2003
Age-structured red blood cell susceptibility and the dynamics of malaria infections. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 15178766	2004
Tissue-specific spatial organization of genomes. Genome biology\| PubMed ID: 15239829	2004
Competition for red blood cells can enhance Plasmodium vivax parasitemia in mixed-species malaria infections. The American journal of tropical medicine and hygiene\| PubMed ID: 16837717	2006
Host control of malaria infections: constraints on immune and erythropoeitic response kinetics. PLoS computational biology\| PubMed ID: 18725923	2008
Spatial link between nucleoli and expression of the Zac1 gene. Chromosoma\| PubMed ID: 19649645	2009
Population dynamics of a pathogen: the conundrum of vivax malaria. Biophysical reviews\| PubMed ID: 20730124	2010
Foetal haemoglobin and the dynamics of paediatric malaria. Malaria journal\| PubMed ID: 23190739	2012
Host immune constraints on malaria transmission: insights from population biology of within-host parasites. Malaria journal\| PubMed ID: 23767770	2013
Microfabricated polymeric vessel mimetics for 3-D cancer cell culture. Biomaterials\| PubMed ID: 23911071	2013
Photoreceptor-derived activin promotes dendritic termination and restricts the receptive fields of first-order interneurons in Drosophila. Neuron\| PubMed ID: 24462039	2014
Hemoglobinopathic erythrocytes affect the intraerythrocytic multiplication of Plasmodium falciparum in vitro. The Journal of infectious diseases\| PubMed ID: 24688070	2014
Serial number tagging reveals a prominent sequence preference of retrotransposon integration. Nucleic acids research\| PubMed ID: 24948612	2014
Single-Nucleotide-Specific Targeting of the Tf1 Retrotransposon Promoted by the DNA-Binding Protein Sap1 of Schizosaccharomyces pombe. Genetics\| PubMed ID: 26358720	2015
Wiring dendrites in layers and columns. Journal of neurogenetics\| PubMed ID: 27315108	2016

Philip G. McQueen

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