Imaging Lipid Dynamics in an Amoeba Isolate Belonging to the Corallomyxa genus. Irina Mikheyeva Microbial Diversity 2016 Introduction: The Corallomyxa genus is best described by its reticulate plasmodium that forms complex and dynamic net-like structures. Species are multinucleate and can display bidirectional cytoplasmic movement of organelles. Reproduction and dissemination is done through generation of uni- or multinucleate fruiting bodies that are produced from well-established plasmodia [1]. Previously described species have been from marine environments [2], establishing Trunk River as a potential source for isolation of more from this species isolation. Microbial Diversity course 2015 was able to isolate one such species, identified for now as TRL04 which was used for this study. There are four major lipids that make up the mammalian plasma membrane, phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, and sphingomyelin. Their head groups vary in size, shape and charge but their arrangement within a membrane is consistent. The hydrophilic heads face water while the hydrophobic tails are the membrane interior [3]. Different membranes will display different lipid compositions, thicknesses, and symmetry which allows them to perform different functions. Membranes serve as physical barriers, but also play roles in signal transduction, regulation of enzyme activity and interaction with cytoskeleton. It is these interactions with the cytoskeleton that create lipid domains and allow for generation of heterogeneous membranes [4]. The Corallomyxa plasmodium is connected by a network of smaller, dynamic pseudopodia. Scott Dawson has generated evidence that these pseudopodia are generated by microtubule forces. I wanted to build on this foundation and ask what happens to the lipid membrane during the rapid generation and breakdown of these filaments. Specifically, does generation of pseudopodia increase local concentrations of lipid molecules? How do pseudopodia influence membrane heterogeneity? One potential mechanism would be to increase local concentration of lipids prior to extension of membrane, acting as a branching point marker. It is also possible that lipids do not build up at all and are trafficked from larger cells to make up the smaller filaments. 1 Methods and Materials: Cell Culture: A Corallomyxa of unknown species named TRL04 was used for these studies. Cells were grown in 100mmx15mm petri dishes (Fisherbrand) with 1X SW Amoeba media [per liter: 0.1g Tryptone, 0.1g Yeast Extract, 1mL 1M MOPS (pH 7.2), 1X SW Base up to 1L] and fed with Marinobacter. To passage Amoeba, plates were washed with 1X SW Base and a few mL of 1X SW Base added to plate. Disposable sterile cell scrapers (Fisherbrand) were then used to detach cells, which were then pipette to new dishes with fresh media or base. Growth was monitored on a Leitz Diavert scope using 10X objective. Fluorescent staining: Lipophilic dyes (ThermoFisher L7781) were resuspended in an unknown volume by another student. ThermoFisher recommends 1-2mg/ml concentration. For these dilution calculations, assumed the resuspension to be 1mg/ml. Dyes were added to 1X SW Base and allowed to intercalate in a dark drawer. Signal was checked on Scott’s inverted fluorescent scope if in a 6 well dish or using Zeiss Axio Observer.Z1 equipped with Axiocam 702mono using the 63X oil objective. Microscopy: Cells were transferred to 50mm MatTeck glass bottom dishes and allowed to adhere. Several washes with 1X SW Base was used prior to staining and imaging. For extended imaging, dish was filled with 4mL 1X SW Base to prevent sample drying. For time lapse and still images Zeiss Axio Observer.Z1 equipped with Axiocam 702mono camera using the 63X oil objective with phase and/ or fluorescent setting. Laser ablation experiment was performed on Nikon A1 laser scanning microscope under the guidance of Nikon representative. Results and discussion: TLR04 forms intricate reticulate plasmodium. TLR04 was isolated during the course last year and a student began to characterize the species though nuclear imaging and addition of actin and microtubule destabilizing drugs. To begin to address the lipid dynamics, first general growth physiology was observed. Growing TLR04 was transferred to a MatTeck dish, adhered and washed, followed by imaging for 4 hours (Figure 1, time lapse stills). Expansion of the plasmodium seems to be driven by uptake of bacteria but as the feeding stops the network seems to thin and the larger plasmodium seems to be overtaken by smaller, net like structures. The long time lapse fails to highlight the dynamic nature of the pseudopodia that connect the larger stiffer branches of plasmodium. To better understand these dynamics, time lapse was taken of the small pseudopodia (Figure 2). The break down and generation of these filaments is extremely fast. Scott Dawson believes that this movement is microtubule driven. But the rapid generation or movement of lipid particles to generate the surrounding membrane is not well understood. To further understand these dynamics, we proceeded to attempt to stain the membranes and observe their dynamics. 2 Figure 1: TLR04 growing over 4 hours. The larger hub of plasmodium visibly expands and takes up bacteria found in the dish. Figure 2: TLR04 growth for 1 min. Small pseudopodia connecting stiffer plasmodium show dynamic movement. Branches are built and broken down very rapidly. 3 Cellular Staining: DiO, a long-chain dialkylcarbocyanine green lipophilic dye, was first examined. Stock solution of 1.14mM was diluted to concentration so 1,5, and 10uM in a 6 well dish with overnight growths of TLR04. Initially, cells were stained for 5 min, dye aspirated and 1mL fresh SW base added. This protocol produced no staining. Next using 5uM and 7.5 uM dye concentration staining time course was performed (Table 1). Upon puncta formation, it was unclear if dye was tagging specific lipids inside the cell or aggregating within the cell. Next SP- DiOC18 stain was tried, it is a DiO analogue that has improved solubility in water. Stock concentration was estimated to be 0.9mM and concentrations of 0.5, 2.5 and 5 uM were tried in staining time course assay (Table 1). Imaging of SP-DioC18 lead to intercellular staining of different organelles within the cytoplasm, no dye was retained at the membrane (Figure 3, time lapse stills). Dil DiOC 18 stain was attempted next but showed no signal in the staining time course (images not included). Other stains were used to visualize different compartments of the cells. DiOC6, a dye for endoplasmic reticulum, mitochondria, and vesicles showed differential staining within the larger hubs (Figure 4, concentrations Table 1). While this stain showed some interesting localization, it is unknown which organelles are being highlighted in the differentially stained areas. A few different nuclear dyes were tried including NucRed, Hoechst and NucBlue. NucBlue produced nice nuclear staining that appeared to be brighter than the Hoechst stain. Large plasmodium hubs were filled with a lot of nuclei (Figure 5) and it was possible to observe nuclei being trafficked in and out of the hub (images not shown). It is exciting to speculate about the state of the nuclear environment, whether they are all in synchrony and transcribing the same material or whether they are differentially regulating different processes. This would be a really exciting area of syncytia research. Next, I wanted to determine if the pseudopodia were essential, that is, if one is mechanically broken will it be rebuilt in the same place? Final Dye 20 min 50 min 2 hours Concentration 5uM DiO No stain Bacteria well stained Puncta have formed within cell, unclear if tagging specific or dye aggregate 7.5uM DiO No stain Bacteria well stained Puncta have formed within cell, unclear if tagging specific or dye aggregate 0.5uM SP-DiOC18 Bacteria well Bacteria, maybe some Amoeba puncta stained 2.5uM SP-DiOC18 Bacteria well Bacteria, maybe some Amoeba puncta stained 5uM SP-DiOC18 Bacteria stained Few cells fully stained Dye forms precipitate after 1 hr 8ug/ml FM4-64 Punctate staining bacteria well stained 5 uM DiOC 6 Stain showed up after 5 min, washed out extra dye NucBlue 2 drops/ml All nuclei stained, very rapid Table 1: Concentrations and dyes used during this study 4 Figure 3: Time lapse imaging to SP-DiOC18 stain. Non specific intrecellular organelle staining. Dye was retained in those membranes and movement dynamics were able to be observed. Figure 4: DiOC6 staining. Dye localization is concentrated to plasmodium hubs. Stained organelles were observed to show some movement. Figure 5: NucBlue staining shows multiple nuclei within a plasmodium hub. Some nuclei are seen in the pseudopodia in the process of trafficing. Some of the larger puncta within the plasmodium could represent recently internalized bacteria. 5 Laser Ablation microscopy: Cells were exposed to high level of UV laser for 2 sec, imaging was conducted during the entire experiment. Region of excitation was specified within the software, and covered the center of pseudopodia in image (Figure 6). Laser was able to induce thin filament breaking. Within 2 minutes of laser stress, new pseudopodia were being produced, but not rebuilding of the broken filament was observed. Laser ablation was attempted on the thicker, stiffer plasmodium resulting in shrinking of the width of the filament but no breakage (Figure 7). This observation potentially highlights different roles of the branches. The thicker plasmodium visible traffics a lot of organelles, it is necessary for these structures to be stiffer and more resistant to breaking. The smaller pseudopodia are rarely seen to traffic organelles and potentially play a bigger role in structure maintenance and food searching. Their plasticity is susceptible to laser ablation but it does not lead to damage of the cell since they are not responsible for a high volume of traffic between the plasmodium hubs. These hypothesis would need to be experimentally shown. Figure 6: Laser ablation of small dynamic psedopodia.