Potential future scenarios for Australia's native biodiversity given on-going increases in human population.

Most natural assets, including native biodiversity (our focus), are under increasing threat from direct (loss of habitat, hunting) and indirect (climate change) human actions. Most human impacts arise from increasing human populations coupled with rises in per capita resource use. The rates of change of human actions generally outpace those to which the biota can respond or adapt. If we are to maintain native biodiversity, then we must develop ways to envisage how the biota may be affected over the next several decades to guide management and policy responses. We consider the future for Australia's native biodiversity in the context of two assumptions. First, the human population in Australia will be 40million by 2050, which has been mooted by federal government agencies. Second, greenhouse gas emissions will track the highest rates considered by the Intergovernmental Panel on Climate Change. The scenarios are based on major drivers of change, which were constructed from seven key drivers of change pertinent to native biodiversity. Five scenarios deal with differing distributions of the human population driven by uncertainties in climate change and in the human responses to climate change. Other scenarios are governed largely by global change and explore different rates of resource use, unprecedented rates of technological change, capabilities and societal values. A narrative for each scenario is provided. The set of scenarios spans a wide range of possible future paths for Australia, with different implications for the future of native biodiversity.

A method to identify drivers of societal change likely to affect natural assets in the future, illustrated with Australia's native biodiversity.

Human society has a profound adverse effect on natural assets as human populations increase and as global climate changes. We need to envisage different futures that encompass plausible human responses to threats and change, and become more mindful of their likely impacts on natural assets. We describe a method for developing a set of future scenarios for a natural asset at national scale under ongoing human population growth and climate change. The method involves expansive consideration of potential drivers of societal change, a reduction of these to form a small set of key drivers to which contrasting settings are assigned, which we use to develop a set of different scenarios. We use Australia's native biodiversity as the focus to illustrate the method.

Competition intensity at local versus regional spatial scales.

Studies of competition intensity over natural (i.e. topographic) gradients often contradict the results from studies where artificial (i.e. fertilizer) gradients have been used. Why should the type of gradient matter? To explore the possibilities, we performed experiments to measure competition intensity experienced by tree seedlings from grass competitors across a natural resource gradient, and simultaneously across artificial soil nutrient (fertiliser) gradients. We measured various functional traits (i.e. specific leaf area, leaf area, leaf nitrogen content, delta(15)N, delta(13)C, RGR) to gain mechanistic insight into the nature of competition across these gradients. Competition intensity increased with increasing resource availability, unequivocally at the local scale (i.e. with fertilizer application) but not at the regional scale (i.e. across the natural productivity gradient). Our measurements of plant traits were generally consistent with measurements of competition intensity, and demonstrate that competition occurs even when resource levels are low. Competition mainly acted to reduce the growth of Eucalyptus seedlings. Functional (physiological) traits in the Eucalyptus seedlings were not strongly affected by competitors, with the possible exception of delta(15)N, which may effectively integrate information on soil nutrient, moisture and leaf processes.

Studies of a microtubule-associated protein using a monoclonal antibody elicited against mammalian mitotic spindles.

We have devised a procedure for the identification of individual molecules which are associated with the mitotic spindle apparatus and cytoskeleton in mammalian cells. We prepared monoclonal antibody-producing hybridomas by immunizing mice with mitotic spindles isolated from cultured HeLa cells. Among several antibody-producing clones obtained, one hybridoma (22MA2) produced an antibody that recognizes a putative microtubule-associated protein which exhibits unusual distribution characteristics in cultured cells. Immunofluorescence studies showed that during mitosis the 22MA2 antigen is distributed in parallel with the spindle fibers of the mitotic apparatus, and that during interphase the antigen is always associated to a limited extent with cytoplasmic microtubules. Also, the co-distribution of the antigen with microtubules was found to be Colcemid sensitive. However, the 22MA2 antibody immunofluorescently stained the nuclei of cells in the exponential growth phase, but did not stain the nuclei of cells that had grown to confluence. This nuclear fluorescence appears to be directly related to cell density rather than nutritional (serum) factors in the growth medium. The results suggest that the antigen undergoes some change in structure or distribution in response to changes in the proliferative capacity of the cell. Biochemical analyses of cytoplasmic, nuclear, and mitotic spindle subcellular fractions show that the antigen exhibits a polypeptide molecular weight of 240,000 is found in various mammalian cells ranging from marsupial to human, and is particularly susceptible to proteolysis.

Tubulin assembly sites and the organization of cytoplasmic microtubules in cultured mammalian cells.

The number, distribution, and nucleating capacity of microtubule-organizing centers (MTOCs) has been investigated in a variety of cultured mammalian cells. Most interphase cells contain a single MTOC that is localized at the centrosome region and corresponds to the centriole and pericentriolar material. MTOCs, like centrioles, become duplicated during the S phase of the cell cycle and are equationally distributed to daughter cells in mitosis. Multiple MTOCs were rarely observed in cultured cells except in one cell line (neuroblastoma), which also displayed an equally large number of centrioles in the cytoplasm. The kinetics of microtubule assembly and the tubulin nucleating capacity of MTOCs was assayed by incubating tubulin-depleted, permeabilized 3T3 and simian virus 40-transformed 3T3 cells with phosphocellulose-purified 65 brain tubulin and microtubule assembly buffer. Initiation and assembly of 65 tubulin occurred in association with the cells' endogenous MTOCs, and the length, number, and distribution of microtubules generated about the organizing centers were regulated and cell specific. Our results are consistent with the notion that the specification of microtubule length, number, and spatial arrangement resides largely in the MTOCs and surrounding cytoplasm and not in the tubulin subunits.

Structure of the mitotic apparatus and chromosomes after hypotonic treatment of mammalian cells in vitro.

Cultured mammalian cells were exposed briefly to culture media made hypotonic with various dilutions of distilled water. Electron microscopic observations and tubulin immunofluorescence were carried out on mitotic cells that had been briefly exposed to hypotonic conditions. Similar observations were made on cells that had been exposed to hypotonic medium and then returned to isotonic conditions. These experiments provided new insight into the structure and physical properties of the mitotic apparatus and chromosomes. Spindle microtubules were reversibly depolymerized by hypotonic treatments. Spindle fibers disappeared after a 15-min exposure to hypotonic medium, leaving only two brightly fluorescent spindle poles. When hypotonically swollen cells were returned to isotonic medium for 15 min prior to fixation, the spindle was reassembled. Several types of spindle aberrations were noted in cells recovering from hypotonic treatment. Chromosomes were greatly expanded by hypotonic treatment, and the kinetochores were disrupted. When treated cells were returned to isotonic culture medium, the chromosomes recondensed and the kinetochores reappeared. Brief hypotonic treatments, described herein, had no adverse effect on cell viability and may prove useful in investigations of the structure and physical properties of the mitotic apparatus.

Tubulin nucleation and assembly in mitotic cells: evidence for nucleic acids in kinetochores and centrosomes.

A lysed cell system was used to study the organelle structure and nucleation of exogenous tubulin at kinetochores and centrosomes in mitotic PtK2 cells. We have used this lysed cell system in conjunction with nuclease digestion experiments to determine which specific nucleic acids (DNA or RNA) are involved in either the structure and/or microtubule-initiating capacity of kinetochores and centrosomes. The results indicate that DNase I specifically decondenses the kinetochore plate structure, with the eventual loss in the ability of the chromosomes to nucleate microtubule assembly. DNase I had no effect on either the structure or nucleating capacity of centrosomes. Both RNase T1 and RNase A specifically attacked the amorphous pericentriolar material of the centrosomes, with a concomitant loss in the ability of this material to nucleate microtubule formation. Neither RNase appeared to affect the structure or nucleating capacity of the kinetochore. Therefore, the two types of nucleases appear to exert preferential effects on the different types of microtubule initiation sites in mitotic mammalian cells. The results suggest that DNA is a major component of the kinetochore, while RNA is a major component of the amorphous pericentriolar material. These findings support the concept that microtubule initiation sites in mitotic cells contain nucleic acids which are essential for the structural and functional integrity of the sites.

Microtubule initiation at kinetochores and centrosomes in lysed mitotic cells. Inhibition of site-specific nucleation by tubulin antibody.

A lysed cell system was developed to determine whether tubulin antibody can block the nucleation of exogenous tubulin at kinetochores and centrosomes. Mitotic PtK2 cells were pretreated with colcemid to remove all endogenous microtubules and were lysed with Triton X-100 in PIPES-EGTA-Mg++ buffer. This procedure left centrosomes, chromosomes, and kinetochores intact as determined by electron microscopy of thin-sectioned cells. Exposure of the lysed cells to phorphocellulose-purified tubulin dimers at 37 degrees C in the presence of 1 mM GTP resulted in site-specific nucleation of microtubules at centrosomes and kinetochores. Treatment of the lysed cell preparations with tubulin antibody before subsequent exposure to the exogenous tubulin resulted in almost complete blockage of microtubule nucleation, especially at kinetochores. Pretreatment of the lysed cell preparations with control antibody or buffer without antibody had no effect on the ability of centrosomes and kinetochores to initiate microtubule assembly. The implications of these results with respect to the molecular composition of centrosomes and kinetochores are discussed.

Electron microscopy of spermatocytes previously studied in life: methods and some observations on micromanipulated chromosomes.

A new method is offered for combined living cell and electron-microscopic studies of spermatocytes (or other cells) which normally do not adhere to glass. The key step is micro-injection of glutaraldehyde near the target cell whenever desired during observation in life. Fixation begins and simultaneously the cell is stuck very firmly to the underlying coverslip. The method is easy and reliable: cells are almost never lost and are well preserved, except for membranes. The application of the method is illustrated by studies of micromanipulated grasshopper spermatocytes. A chromosome was detached from the spindle and placed in the cytoplasm. Before or after the beginning of chromosome movement back toward the spindle, the cell was fixed, sectioned and the manipulated chromosome observed in the electron microscope. If the detached chromosome had not moved by the time of fixation, no or only one or two microtubules were seen at its kinetochore, but if movement had occurred, a few microtubules were always present. The arrangement of these microtubules corresponded to the direction of movement, but they commonly were at an unusual angle relative to the kinetochore. The origin and role in chromosome movement of the microtubules seen near moving chromosomes far from the spindle is not yet established, but a speculation is offered. A goal for future work is the detailed analysis of the microtubules associated with individual moving chromosomes. Such an analysis is feasible because the moving chromosome is far removed from the confusing mass of spindle microtubules, and its value is enhanced because the direction of movement at the time of fixation is known.

Localization of tubulin in the mitotic apparatus of mammalian cells by immunofluorescence and immunoelectron microscopy.

Antitubulin antibody was used as an immunofluorescent and immunoelectron microscopic probe to localize tubulin in components of the mitotic apparatus of rat kangaroo (strain PtK1) cells in vitro. In addition to the detection of tubulin in the spindle microtubules and centrioles, other structures were found to display specific staining including kinetochores, amorphous pericentriolar material and small virus-like particles associated with the centrioles. The kinetochores consisted of a densely stained outer layer about 400 A thick which is separated from an inner layer of the same dimension by a lightly staining middle layer. Microtubules were primarily associated with the outermost plate of the kinetochore but tubulin was uniformly distributed in both outer and inner plates. Colcemid treatment prevented the assembly of spindle microtubules and resulted in specific alterations of the kinetochore but failed to diminish the staining of the kinetochores. These observations suggest that tubulin molecules may comprise an important structural component of the kinetochore.