Imaging Lipid Dynamics in an Amoeba Isolate Belonging to the

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.
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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.
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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.
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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 SPDiOC18 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
Concentration
5uM DiO
20 min
50 min
No stain
Bacteria well stained
7.5uM DiO
No stain
0.5uM SP-DiOC18
2.5uM SP-DiOC18
5uM SP-DiOC18
8ug/ml FM4-64
5 uM DiOC 6
NucBlue 2 drops/ml
2 hours
Puncta have formed within cell,
unclear if tagging specific or dye
aggregate
Bacteria well stained
Puncta have formed within cell,
unclear if tagging specific or dye
aggregate
Bacteria, maybe some Amoeba puncta
Bacteria well
stained
Bacteria well
Bacteria, maybe some Amoeba puncta
stained
Bacteria stained
Few cells fully stained
Dye forms precipitate after 1 hr
Punctate staining bacteria well stained
Stain showed up after 5 min, washed out extra dye
All nuclei stained, very rapid
Table 1: Concentrations and dyes used during this study
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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.
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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. Laser was able to break the filament but does not
result it much cell damage. Two minutes post laser, new pseudopodia can be seen to rebuilding in region
Figure 7: Laser ablation of larger plasmodium. Laser was not able to break the filament but seem to
damage the filament and induce shrinking. It is possible that laser was not applied for long enough and
eventually breaking would have been induced.
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Future Directions:
There are other membrane staining dyes need to be tried before we can conclude that the
membrane is unstainable. For example, diphenylhexatriene has been used to monitor
membrane dynamics in amoeba [5]. Similarly, vector expressing tags for plasma membrane
such as the BacMam CellLight Plasma Membrane-GFP expression vectors could be useful for
this undertaking.
References:
[1] Tekle, Y.I. et al., A Multigene Analysis of Corallomyxa tenera sp. nov. Suggests its
Membership in a Clade that Includes Gromia, Haplosporidia and Foraminifera. Protist, 2007,
158: p. 457-472
[2] Grell, K.G. Corallomyxa nipponica s. sp. and the phylogeny of plasmodial protists. Arch
Protistenkd, 1991, 140: p. 303-320
[3] Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York:
Garland Science; 2002. The Lipid Bilayer.
[4] Marguet D, Lenne P-F, Rigneault H, He H-T. Dynamics in the plasma membrane: how to
combine fluidity and order. The EMBO Journal. 2006;25(15):3446-3457.
doi:10.1038/sj.emboj.7601204.
[5] Avery SV, Lloyd D, Harwood JL. Temperature-dependent changes in plasma-membrane lipid
order and the phagocytotic activity of the amoeba Acanthamoeba castellanii are closely
correlated. Biochemical Journal. 1995;312(Pt 3):811-816.
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