Geometric constrains for detecting short actin filaments by cryogenic electron tomography

Polymerization of actin into filaments can push membranes forming extensions like filopodia or lamellipodia, which are important during processes such as cell motility and phagocytosis. Similarly, small organelles or pathogens can be moved by actin polymerization. Such actin filaments can be arranged in different patterns and are usually hundreds of nanometers in length as revealed by various electron microscopy approaches. Much shorter actin filaments are involved in the motility of apicomplexan parasites. However, these short filaments have to date not been visualized in intact cells. Here, we investigated Plasmodium sporozoites, the motile forms of the malaria parasite that are transmitted by the mosquito, using cryogenic electron tomography. We detected filopodia-like extensions of the plasma membrane and observed filamentous structures in the supra-alveolar space underneath the plasma membrane. However, these filaments could not be unambiguously assigned as actin filaments. In silico simulations of EM data collection and tomographic reconstruction identify the limits in revealing the filaments due to their length, concentration and orientation

Sozial nachhaltiger Wohnbau und mögliche Perspektiven der Soziokulturellen Animation: Qualitative Forschung zur Umsetzung sozialer Nachhaltigkeit im Wohnbau in Schweizer Städten

Die Anforderungen an den Wohnbau in den Schweizer Städten haben sich in den letzten Jahrzehnten aufgrund der soziodemografischen Entwicklung der Gesellschaft vervielfältigt. Folgen davon sind Pluralisierung, Heterogenisierung, Alterung, Segregation und Individualisierung der Gesellschaft. Ein möglicher Lösungsansatz, um diesen pluralisierten Ansprüchen an den Wohnbau gerecht zu werden, ist das sozial nachhaltige Bauen. Wenn die Realisierungsprozesse von Wohnbauten sozial nachhaltig erfolgen sollen, bedingt dies den partizipativen Einbezug der Bedürfnisse von Betroffenen und Interessierten. Um die Bedürfnisse von Beteiligten zu berücksichtigen, diese in die partizipativen Prozesse zu integrieren und zwischen den verschiedenen Disziplinen, die im Wohnungsbau involviert sind, zu übersetzen und zu vermitteln, braucht es professionelle Unterstützung. Betrachtet man die theoretische Verortung der Soziokulturellen Animation, ist ersichtlich, dass sich der sozial nachhaltige Wohnbau als Handlungsfeld für die Profession anbietet. Die Autorenschaft beschreibt in der vorliegenden Arbeit die soziale Nachhaltigkeit und die soziodemografischen Herausforderungen für den Wohnbau, erforscht Massnahmen zur Umsetzung sozialer Nachhaltigkeit im Wohnbau und geht der Frage nach, welche Rolle der Soziokulturellen Animation dabei zukommt

Positioning of large organelles by a membrane-associated cytoskeleton in Plasmodium sporozoites

Cellular organelles are usually linked to the cytoskeleton, which often provides a scaffold for organelle function. In malaria parasites, no link between the cytoskeleton and the major organelles is known. Here we show that during fast, stop-and-go motion of Plasmodium sporozoites, all organelles stay largely fixed in respect to the moving parasite. Cryogenic electron tomography reveals that the nucleus, mitochondrion, apicoplast and the microtubules of Plasmodium sporozoites are linked to the parasite pellicle via long tethering proteins. These tethers originate from the inner membrane complex and are arranged in a periodic fashion following a 32 nm repeat. The tethers pass through a subpellicular structure that encompasses the entire parasite, probably as a network of membrane-associated filaments. While the spatial organization of the large parasite organelles appears dependent on their linkage to the cortex, the specialized secretory vesicles are mostly not linked to microtubules or other cellular structures that could provide support for movement

Inter-subunit interactions drive divergent dynamics in mammalian and <i>Plasmodium</i> actin filaments

<div><p>Cell motility is essential for protozoan and metazoan organisms and typically relies on the dynamic turnover of actin filaments. In metazoans, monomeric actin polymerises into usually long and stable filaments, while some protozoans form only short and highly dynamic actin filaments. These different dynamics are partly due to the different sets of actin regulatory proteins and partly due to the sequence of actin itself. Here we probe the interactions of actin subunits within divergent actin filaments using a comparative dynamic molecular model and explore their functions using <i>Plasmodium</i>, the protozoan causing malaria, and mouse melanoma derived B16-F1 cells as model systems. Parasite actin tagged to a fluorescent protein (FP) did not incorporate into mammalian actin filaments, and rabbit actin-FP did not incorporate into parasite actin filaments. However, exchanging the most divergent region of actin subdomain 3 allowed such reciprocal incorporation. The exchange of a single amino acid residue in subdomain 2 (N41H) of <i>Plasmodium</i> actin markedly improved incorporation into mammalian filaments. In the parasite, modification of most subunit–subunit interaction sites was lethal, whereas changes in actin subdomains 1 and 4 reduced efficient parasite motility and hence mosquito organ penetration. The strong penetration defects could be rescued by overexpression of the actin filament regulator coronin. Through these comparative approaches we identified an essential and common contributor, subdomain 3, which drives the differential dynamic behaviour of two highly divergent eukaryotic actins in motile cells.</p></div

Mutant coronins reveal distinct binding to membranes and actin filaments.

<p>(A) Multiple sequence alignment shows that amino acids found to be important for actin binding in yeast are conserved between mouse, yeast and apicomplexans and are marked in red. (B) Fluorescence microscopy of a parasite line overexpressing coronin-mCherry from the uis3 promoter. Note the peripheral fluorescence in non-motile (RPMI) and motile (RPMI+BSA) salivary gland derived sporozoites. Numbers indicate time in seconds. Scale bar: 5 μm. (C) Localization of a number of overexpressed mutant coronin-mCherry fusion proteins. Note the three different types of localizations: cytoplasmic (coronin-8mut; K283A/E, D285A/R), peripheral (R24E, R28E; R349A/E, K350A/E) and polarized (WT, R24A, R28A). Scale bar: 5 μm. (D) Graph showing a quantitative assessment of the front versus rear ratio from 20 images of the various parasite lines overexpressing mutated coronin-mCherry as indicated on the x-axis. (E) The peripheral localization of R349A/E, K350A/E mutants is not altered when sporozoites are incubated with cytochalasin (100 nM), jasplakinolide (100 nM), ionomycin (1000 nM) or cytochalasin D (100 nM) + ionomycin (500 nM) Scale bar: 5 μm.</p

Comparison of the different parasite strains for their growth within cultured liver cells and their infectivity for mice.

<p>Note that all parasites with a lowered number of sporozoites in their salivary glands (all but WT and coronin-mCherry) failed to infect all mice following mosquito bites. While 100% (24 out of 24) of the mice bitten by mosquitoes infected with either wild type or coronin-mCherry expressing parasites were infected, only 78% (53 out of 68) of the mice bitten by mosquitoes infected with the various mutants developed a blood stage infection. As the motility was similar between WT, coronin-mCherry and the R24A/R28A mutant on one hand and between all the other mutants on the other hand (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005710#ppat.1005710.t001" target="_blank">Table 1</a>), we further analyzed the time to infection of mice successfully infected by mosquitoes. Hence, the first group showed an average time to blood stage infection of 3.4 days and the second group of 3.9 days for i.v. injected parasites (p<0.001, one way ANOVA) and 3.5 versus 4.1 days (p<0.01, one way ANOVA) for mice receiving parasites by mosquito bites.</p

Comparison of the different parasite strains in the infectivity of mosquitoes and the <i>in vitro</i> speed of salivary gland sporozoites.

<p>Significant differences are indicated with an asterisk. Note that sporozoites from the R24A, R28A mutant, despite lower numbers in the salivary gland moved with comparable speed to the coronin-mCherry parasites. Note that coronin-mCherry parasites are significantly slower than WT parasites.</p

Speculative working model on coronin function at the interface between calcium and cAMP signaling during motility (red Arab numbers) and invasion (orange Roman numbers).

<p>Upon activation of a trans-membrane receptor by extracellular ligands on the salivary gland or in the skin (1), phospholipase C is activated and converts PIP2 into IP3 (2). This leads to the release of calcium from intracellular stores (3) and subsequent exocytosis of micronemes, which bring more receptors to the plasma membrane (5). These reinforce signaling and activate actin polymerization (6). Actin filaments are organized by surface receptors and relocalization of coronin from peripheral membranes (either PM or IMC) to actin filaments (7). This leads to efficient adhesion and force production essential for motility in 2D. Coronin (crn) relocalizes from actin filaments as these are disassembled through the action of PKA, which is possibly activated by cytosolic adenylate cyclase (AC). Upon stimulation of membrane bound adenylate cyclase more cAMP is produced leading to higher PKA activity and further calcium release from intracellular stores. The additionally released receptors then mediate invasion.</p

Mutant coronins exhibit defects in sporozoite motility.

<p>Time-lapse images and randomly selected tracks of different parasite lines expressing endogenous coronin fused to mCherry with the indicated mutations in their putative actin-binding sites. Note that R24A, R28A moves in a similar manner to WT, while the other mutants move differently and exhibit bending movements so far not seen in any wild type or mutated sporozoite, most strikingly seen in the time lapse of a sporozoites expressing the R349E, K350E mutation. A small but discernable fraction of sporozoite expressing the K283A, D285A mutation in coronin can move in persistent circles. Scale bars: 5 μm (images), 10 μm (tracks).</p