Biomedical subjects

Anja Habermann

Publications and source records attributed to Anja Habermann.

5 recordsLinked to original sources

PubMed2004

A rapid method for assessing the distribution of gold labeling on thin sections.

Particulate gold labeling on ultrathin sections is in widespread use for antigen localization at the EM level. To extend the usefulness of gold labeling technology, we are evaluating different methods for sampling and estimating quantities of gold labeling. Here we present a simple, rapid, and unbiased method for assessing the relative pool sizes of immunogold labeling distributed over different cell compartments. The method uses a sampling approach developed for stereology in which a regular array of microscopic fields or linear scans is positioned randomly on labeled sections. From these readouts, gold particles are counted and assigned to identifiable cell structures to construct a gold labeling frequency distribution of those labeled compartments. Here we use ultrathin cryosections labeled for a range of different proteins and for a signaling lipid. We show by scanning labeled sections at the electron microscope that counting 100-200 particles on each of two grids is sufficient to obtain a reproducible and rapid assessment of the pattern of labeling proportions over 10-16 compartments. If more precise estimates of labeling proportions over individual compartments are required (e.g., to achieve coefficients of error of 10-20%), then 100-200 particles need to be counted over each compartment of interest.

Animals↗

PubMed2003

Fusion between phagosomes, early and late endosomes: a role for actin in fusion between late, but not early endocytic organelles.

Actin is implicated in membrane fusion, but the precise mechanisms remain unclear. We showed earlier that membrane organelles catalyze the de novo assembly of F-actin that then facilitates the fusion between latex bead phagosomes and a mixture of early and late endocytic organelles. Here, we correlated the polymerization and organization of F-actin with phagosome and endocytic organelle fusion processes in vitro by using biochemistry and light and electron microscopy. When membrane organelles and cytosol were incubated at 37 degrees C with ATP, cytosolic actin polymerized rapidly and became organized into bundles and networks adjacent to membrane organelles. By 30-min incubation, a gel-like state was formed with little further polymerization of actin thereafter. Also during this time, the bulk of in vitro fusion events occurred between phagosomes/endocytic organelles. The fusion between latex bead phagosomes and late endocytic organelles, or between late endocytic organelles themselves was facilitated by actin, but we failed to detect any effect of perturbing F-actin polymerization on early endosome fusion. Consistent with this, late endosomes, like phagosomes, could nucleate F-actin, whereas early endosomes could not. We propose that actin assembled by phagosomes or late endocytic organelles can provide tracks for fusion-partner organelles to move vectorially toward them, via membrane-bound myosins, to facilitate fusion.

Actins↗

PubMed2003

Selected lipids activate phagosome actin assembly and maturation resulting in killing of pathogenic mycobacteria.

Pathogenic mycobacteria such as Mycobacterium tuberculosis and Mycobacterium avium facilitate disease by surviving intracellularly within a potentially hostile environment: the macrophage phagosome. They inhibit phagosome maturation processes, including fusion with lysosomes, acidification and, as shown here, membrane actin assembly. An in vitro assay developed for latex bead phagosomes (LBPs) provided insights into membrane signalling events that regulate phagosome actin assembly, a process linked to membrane fusion. Different lipids were found to stimulate or inhibit actin assembly by LBPs and mycobacterial phagosomes in vitro. In addition, selected lipids activated actin assembly and phagosome maturation in infected macrophages, resulting in a significant killing of M. tuberculosis and M. avium. In contrast, the polyunsaturated sigma-3 lipids behaved differently and stimulated pathogen growth. Thus, lipids can be involved in both stimulatory and inhibitory signalling networks in the phagosomal membrane.

Actins↗

PubMed2003

RanGTP mediates nuclear pore complex assembly.

In metazoa, the nuclear envelope breaks down and reforms during each cell cycle. Nuclear pore complexes (NPCs), which serve as channels for transport between the nucleus and cytoplasm, assemble into the reforming nuclear envelope in a sequential process involving association of a subset of NPC proteins, nucleoporins, with chromatin followed by the formation of a closed nuclear envelope fenestrated by NPCs. How chromatin recruitment of nucleoporins and NPC assembly are regulated is unknown. Here we demonstrate that RanGTP production is required to dissociate nucleoporins Nup107, Nup153 and Nup358 from Importin beta, to target them to chromatin and to induce association between separate NPC subcomplexes. Additionally, either an excess of RanGTP or removal of Importin beta induces formation of NPC-containing membrane structures--annulate lamellae--both in vitro in the absence of chromatin and in vivo. Annulate lamellae formation is strongly and specifically inhibited by an excess of Importin beta. The data demonstrate that RanGTP triggers distinct steps of NPC assembly, and suggest a mechanism for the spatial restriction of NPC assembly to the surface of chromatin.

Amino Acid Substitution↗

PubMed2003

A simpler way of comparing the labelling densities of cellular compartments illustrated using data from VPARP and LAMP-1 immunogold labelling experiments.

Quantitative immunoelectron microscopy of gold label in intracellular compartments often involves calculating labelling densities (LDs). These are related to antigen concentrations and usually refer gold particle counts to the sizes of compartments on sections (for example, golds per microm(2) of organelle profile area or per microm of membrane trace length). Here, we show how LD values can be estimated more simply (without estimating areas or lengths) and also how observed and expected LD values can be used to calculate a relative labelling index (RLI) for each compartment and then test statistically for preferential (non-random) labelling. For random labelling, RLI=1. Compartment size is estimated stereologically by superimposing random test points (which hit organelle profiles in proportion to their area) or test lines (which intersect membrane traces in proportion to their length). By this means, the observed LD of a compartment (LD(obs)) can be expressed simply as golds per test point (organelles) or per intersection (membranes). Furthermore, the LD obtained by dividing total golds (on all compartments) by total points or intersections (on all compartments) is the value to be expected (LD(exp)) when compartments label randomly. For each compartment, RLI=LD(obs)/LD(exp). Statistical analysis is undertaken by comparing observed distributions of golds with predicted random distributions (calculated from point or intersection counts). A compartment is preferentially labelled if two criteria are met: (1) its RLI>1 (i.e. LD(obs) is greater than LD(exp)) and (2) its partial chi-squared value makes a substantial contribution to total chi-squared value. This approach provides a simple and efficient way of comparing LDs in different compartments. Its utility is illustrated using data from VPARP and LAMP-1 labelling experiments.

Animals↗