M. Tlalka

Recruitment of monocytes/macrophages by tissue factor-mediated coagulation is essential for metastatic cell survival and premetastatic niche establishment in mice

Tissue factor (TF) expression by tumor cells correlates with metastasis clinically and supports metastasis in experimental settings. However, the precise pathways coupling TF to malignancy remain incompletely defined. Here, we show that clot formation by TF indirectly enhances tumor cell survival after arrest in the lung, during experimental lung metastasis, by recruiting macrophages characterized by CD11b, CD68, F4/80, and CX(3)CR1 (but not CD11c) expression. Genetic or pharmacologic inhibition of coagulation, by either induction of TF pathway inhibitor ex-pression or by treatment with hirudin, respectively, abrogated macrophage recruitment and tumor cell survival. Furthermore, impairment of macrophage function, in either Mac1-deficient mice or in CD11b-diphtheria toxin receptor mice in which CD11b-positive cells were ablated, decreased tumor cell survival without altering clot formation, demonstrating that the recruitment of functional macrophages was essential for tumor cell survival. This effect was independent of NK cells. Moreover, a similar population of macrophages was also recruited to the lung during the formation of a premetastatic niche. Anticoagulation inhibited their accumulation and prevented the enhanced metastasis associated with the formation of the niche. Our study, for the first time, links TF induced coagulation to macrophage recruitment in the metastatic process.

Fungi in Biogeochemical Cycles: The role of wood decay fungi in the carbon and nitrogen dynamics of the forest floor

decomposed amino acid that tracks the mycelial free amino acid pool. Its movement can be imaged by counting photon emissions from a scintillant screen in contact with the mycelial system. This method allows real-time imaging at high temporal and spatial resolution, for periods of weeks and areas up to 1 m 2 , in microcosms that mimic the mineral/organic soil interface of the forest floor. The results reveal a hitherto unsuspected dynamism and responsiveness in amino acid flows through mycelial networks of cord-forming, wood-decomposing basidiomycetes. We interpret these in the light of current understanding of the pivotal role of fungi in boreal and temperate forest floor nutrient cycling, and attempt to formulate key questions to investigate the effects of mycelial nitrogen translocation on forest floor decomposition and nitrogen absorption.

The vacuole system is a significant intracellular pathway for longitudinal solute transport in basidiomycete fungi.

ABSTRACT Mycelial fungi have a growth form which is unique among multicellular organisms. The data presented here suggest that they have developed a unique solution to internal solute translocation involving a complex, extended vacuole. In all filamentous fungi examined, this extended vacuole forms an interconnected network, dynamically linked by tubules, which has been hypothesized to act as an internal distribution system. We have tested this hypothesis directly by quantifying solute movement within the organelle by photobleaching a fluorescent vacuolar marker. Predictive simulation models were then used to determine the transport characteristics over extended length scales. This modeling showed that the vacuolar organelle forms a functionally important, bidirectional diffusive transport pathway over distances of millimeters to centimeters. Flux through the pathway is regulated by the dynamic tubular connections involving homotypic fusion and fission. There is also a strongly predicted interaction among vacuolar organization, predicted diffusion transport distances, and the architecture of the branching colony margin.

Imaging complex nutrient dynamics in mycelial networks

Transport networks are vital components of multi‐cellular organisms, distributing nutrients and removing waste products. Animal cardiovascular and respiratory systems, and plant vasculature, are branching trees whose architecture is thought to determine universal scaling laws in these organisms. In contrast, the transport systems of many multi‐cellular fungi do not fit into this conceptual framework, as they have evolved to explore a patchy environment in search of new resources, rather than ramify through a three‐dimensional organism. These fungi grow as a foraging mycelium, formed by the branching and fusion of threadlike hyphae, that gives rise to a complex network. To function efficiently, the mycelial network must both transport nutrients between spatially separated source and sink regions and also maintain its integrity in the face of continuous attack by mycophagous insects or random damage. Here we review the development of novel imaging approaches and software tools that we have used to characterise nutrient transport and network formation in foraging mycelia over a range of spatial scales. On a millimetre scale, we have used a combination of time‐lapse confocal imaging and fluorescence recovery after photobleaching to quantify the rate of diffusive transport through the unique vacuole system in individual hyphae. These data then form the basis of a simulation model to predict the impact of such diffusion‐based movement on a scale of several millimetres. On a centimetre scale, we have used novel photon‐counting scintillation imaging techniques to visualize radiolabel movement in small microcosms. This approach has revealed novel N‐transport phenomena, including rapid, preferential N‐resource allocation to C‐rich sinks, induction of simultaneous bi‐directional transport, abrupt switching between different pre‐existing transport routes, and a strong pulsatile component to transport in some species. Analysis of the pulsatile transport component using Fourier techniques shows that as the colony forms, it self‐organizes into well demarcated domains that are identifiable by differences in the phase relationship of the pulses. On the centimetre to metre scale, we have begun to use techniques borrowed from graph theory to characterize the development and dynamics of the network, and used these abstracted network models to predict the transport characteristics, resilience, and cost of the network.

New approaches to investigating the function of mycelial networks

Fungi play a key role in ecosystem nutrient cycles by scavenging, concentrating, translocating and redistributing nitrogen. To quantify and predict fungal nitrogen redistribution, and assess the importance of the integrity of fungal networks in soil for ecosystem function, we need better understanding of the structures and processes involved. Until recently nitrogen translocation has been experimentally intractable owing to the lack of a suitable radioisotope tracer for nitrogen, and the impossibility of observing nitrogen translocation in real time under realistic conditions. We have developed an imaging method for recording the magnitude and direction of amino acid flow through the whole mycelial network as it captures, assimilates and channels its carbon and nitrogen resources, while growing in realistically heterogeneous soil microcosms. Computer analysis and modeling, based on these digitized video records, can reveal patterns in transport that suggest experimentally testable hypotheses. Experimental approaches that we are developing include genomics and stable isotope NMR to investigate where in the system nitrogen compounds are being acquired and stored, and where they are mobilized for transport or broken down. The results are elucidating the interplay between environment, metabolism, and the development and function of transport networks as mycelium forages in soil. The highly adapted and selected foraging networks of fungi may illuminate fundamental principles applicable to other supply networks.

Quantifying dynamic resource allocation illuminates foraging strategy in Phanerochaete velutina

Saprotrophic woodland fungi forage for mineral nutrients and woody resources by extension of a mycelial network across the forest floor. Different species explore at different rates and establish networks with qualitatively differing architecture. However, detailed understanding of fungal foraging behaviour has been hampered by the absence of tools to quantify resource allocation and growth accurately and non-invasively. To solve this problem, we have used photon-counting scintillation imaging (PCSI) to map and quantify nutrient allocation and localised growth simultaneously in heterogeneous resource environments. We show that colonies spontaneously shift to an asymmetric growth pattern, even in the absence of added resources, often with a distinct transition between the two growth phases. However, the extent of polarisation was much more pronounced and focussed in the presence of an additional cellulose resource. In this case, there was highly localised growth, often at the expense of growth elsewhere in the colony, and marked accumulation of (14)C-AIB in the sector of the colony with the added resource. The magnitude of the response was greatest when resource was added around the time of the endogenous developmental transition. The focussed response required a metabolisable resource, as only limited changes were seen with glass fibre discs used to mimic the osmotic and thigmotropic stimuli upon resource addition. Overall the behaviour is consistent with an adaptive foraging strategy, both to exploit new resources and also to redirect subsequent foraging effort to this region, presumably with an expectation that the probability of finding additional resources is increased.

Imaging of Long-Distance α-Aminoisobutyric Acid Translocation Dynamics during Resource Capture by Serpula lacrymans

ABSTRACT α-Aminoisobutyric acid (AIB) is a nonmetabolized amino acid analogue of alanine, which at low (μM) concentrations acts as a tracer for amino acid movements. At high concentrations (mM), it competitively inhibits membrane transport and metabolism of protein amino acids and acts as a systemic translocated inhibitor of mycelial extension in fungi. AIB can control mycelial spread of the basidiomycete Serpula lacrymans, the cause of brown rot of wood in buildings. However, it is not known how effectively the inhibitor is distributed throughout the mycelium. Realistically heterogeneous microcosms, in which the fungus grew across nutritionally inert sand to colonize discrete wood resources, were used to investigate patterns of inhibition and translocation following local application of AIB. At a 0.1 M concentration, locally applied AIB caused immediate arrest of extension throughout the whole mycelium, maintained for a 6-week experimental period. The dynamics of translocation of subtoxic amounts of [1-14C]AIB ([14C]AIB) were mapped by photon-counting scintillation imaging in conjunction with destructive harvest to establish the velocity, direction, and rate of translocation and the extent of [14C]AIB reallocation accompanying the invasion of fresh wood. Locally applied [14C]AIB was distributed throughout complex mycelial networks within 2 h of application, becoming localized in growing margins by 12 h. Encounter with a fresh wood resource triggered a widespread response, causing withdrawal of [14C]AIB from throughout the network, accompanied by accumulation in the newly colonized wood and associated mycelium. The results are discussed in the context of nutrient dynamics in wood decomposer fungi and the mechanism of the amino acid reallocation response.

Spitzenkörper, vacuoles, ring-like structures, and mitochondria of Phanerochaete velutina hyphal tips visualized with carboxy-DFFDA, CMAC and DiOC6(3)

Growth and organelle morphology in the wood rotting basidiomycete fungus Phanerochaete velutina were examined in Petri dishes, on agar-coated slides, and in submerged cultures, using DIC, fluorescence and four-dimensional (4-D; x,y,z,t) confocal microscopy, with several fluorescent probes. Phanerochaete is ideal for this work because of its fast growth, robustness, and use in a wide range of other studies. The probe carboxy-DFFDA, widely used for labelling vacuoles, has no effect either on hyphal tip extension or colony growth at the concentrations usually applied in labelling experiments. Carboxy-DFFDA labels the vacuoles and these form a tubular reticulum in hyphal tip cells. The probe also labels extremely small vesicles (punctate fluorescence) in the apex of tip cells, the Spitzenkörper, and short tubules that undergo sequences of characteristic movements and transformations to produce various morphologies, including ring-like structures. Their location and behaviour suggest that they are a distinct group of structures, possibly a subset of vacuoles, but as yet to be fully identified. Regular incursions of tubules extending from these structures and from the vacuolar reticulum into the apical dome indicate the potential for delivery of material to the apex via tubules as well as vesicles. Such structures are potential candidates for delivering chitin synthases to the apex. Spitzenkörper behaviour has been followed as hyphal tips with linear growth encounter obstacle hyphae and, as the hydrolysis product of carboxy-DFFDA only accumulates in membrane-enclosed compartments, it can be inferred that the labelled structures represent the Spitzenkörper vesicle cloud. Mitochondria also form a reticular continuum of branched tubules in growing hyphal tips, and dual localisation with DiOC6(3) and CMAC allows this to be distinguished from the vacuolar reticulum. Like vacuolar tubules, mitochondrial tubules also span the septa, indicating that they may also be a conduit for intercellular transport.

Quantitative Confocal Fluorescence Measurements in Living Tissue

Fluorescent probes offer unparalleled opportunities to visualize and quantify dynamic events within single living cells with a minimum of perturbation. Cells or monolayers maintained in culture can be readily imaged; however, measurements are often also needed from cells within intact tissues that are operating in their correct physiological context to include the effects of cell-cell interactions and the mechanical, ionic and physiological effects of the extracellular matrix (e.g., Errington et al. 1997). A range of different measurement techniques are now available to quantify fluorescence signals from reporter molecules within biological specimens, including fluorimetry, flow cytometry, microscope photometry, camera and confocal microscopy. Each system performs well for a specific range of sampling conditions and specimens; thus, several techniques in combination may be needed to provide a sufficiently flexible balance between the spatial, temporal and spectral resolution required. Several recent volumes cover many of the technical details and practical applications of these techniques (e.g., Wang and Taylor 1989; Taylor and Wang 1990; Mason 1993; Matsumoto 1993; Nuccitelli 1994; Pawley 1995a). Here we focus on the practical advantages and disadvantages of quantitative fluorescence measurements using confocal microscopy as a tool to study living cells in intact animal, plant and fungal tissues (Errington et al. 1997; Fricker et al. 1994; White et al. 1996).

Chapter 3 Mycelial networks: Nutrient uptake, translocation and role in ecosystems

Sequestration and release of carbon in the decomposer subsystem of the forest floor are key ecosystem functions of saprotrophic basidiomycetes. Both are the result of fungal metabolic processes commonly regulated by nitrogen availability. Saprotrophic basidiomycetes are the primary wood decomposer organisms in N-limited boreal and temperate forests. To predict the ecosystem effects of atmospheric nitrogen deposition in forests, we need better understanding of the fungal adaptive responses that link carbon conversions to nitrogen dynamics. Some Basidiomycota clades have evolved the capacity to develop mass flow nutrient channels-cords-in response to nutrient context. Rapid bidirectional nutrient transport in cords enables these fungi to operate extensive and persistent resource supply networks, and to exploit the spatiotemporally uncoupled carbon and nitrogen resources of the upper soil horizons of the forest floor. Both the initiation of cord development and the velocity, direction and magnitude of amino acid flows within the corded network are regulated in response to the amounts and geometry of its carbon and nitrogen supply. Predictive models of fungal metabolic, physiological and developmental responses to environmental nitrogen, at cell and organism scale, can be realistically parameterized with data from experimentally manipulated saprotrophic mycelia in microcosms and ecosystems. In future, the whole-genome sequence of the basidiomycete cord-forming wood decay fungus Serpula lacrymans will provide a model for -omics technologies to dissect the extracellular and intracellular nutrient responses that underlie the functions of basidiomycete networks in ecosystems.