DOI: 10.5967/sh2f-xt93
title: Dataset for "A close unicellular animal relative and predator of schistosomes exhibits chemokinesis in response to proteins and peptides from its prey"

description: Regulated motility is vital for many cells—both for unicellular microbes and for cells within multicellular bodies. Different conditions require different rates and directions of movement. For the microbial predator Capsaspora owczarzaki, its motility is likely essential for predation. This organism has been shown to prey on diverse organisms, including the schistosome parasites that co-reside with it in Biomphalaria glabrata snails. Capsaspora is also an evolutionary model for the unicellular ancestor of animals. This phylogenic placement makes Capsaspora’s motility an attractive target for understanding the evolution of motility in animal cells. Until now, little was known of how Capsaspora regulates its rate and direction of motility. Here we found that it exhibits chemokinesis (increased movement in response to chemical factors) in response to proteins released from prey cells. Chemokinesis also occurs in response to pure proteins—including bovine serum albumin. We found that this chemokinesis behavior is dependent on Capsaspora cell density, which suggests that the regulated motility is a cooperative behavior (possibly to improve cooperative feeding). We developed a mathematical model of Capsaspora motility and found that chemokinesis alone does not benefit Capsaspora predation. However, when coupled with chemotaxis (directional motility along a chemical gradient toward prey), chemokinesis may improve predation. Finally, we quantitatively analyzed Capsaspora’s previously reported chemotaxis behavior. These findings lay a foundation for characterizing the mechanisms of regulated motility in a predator of a human pathogen and a model for the ancestor of animals.
description: Cell strain and growth conditions Capsaspora owczarzaki cell cultures (strain American Type Culture Collection ATCC®30864) were grown axenically in 25-cm2 culture flasks with 6 ml ATCC media 1034 (modified PYNFH medium) containing 10% v/v heat-inactivated Fetal Bovine Serum (FBS, Corning 35-011-CV), hereafter growth media, in a 23 °C incubator. Cells were obtained during exponential growth phase by passaging ~0.9 – 1.2 ml of adherent cells scraped into suspension within the specified density range, into 5ml of growth media. The culture was incubated at 23 °C for 24 h to achieve desired density range of 3 × 106 to 20 × 106 cells/ml. S. mansoni miracidia isolation and transformation into primary sporocysts S. mansoni miracidia were isolated according to published protocols.47,48 Briefly, 3 infected mice livers were obtained from the Biomedical Research Institute (Rockville, MD) (BRI) Schistosomiasis Resource Center. They were shipped overnight with cold packs and added to ice-cold 1.2% sterile NaCl solution immediately after arrival using a sterile 50 ml centrifuge tube. The tubes with the livers were shaken vigorously, and any floating debris and the saline solution was aspirated. The livers were then poured into the ice-cold cup of a Waring blender with another 30 ml of ice-cold sterile 1.2% NaCl solution and blended for 15–25 seconds non-stop making sure the livers were well-blended. The blended livers were centrifuged at 800 × g for 10 min at 4 °C. The supernatant was discarded, and the pellets were washed with ice-cold sterile 1.25% NaCl solution. The pellets were then resuspended with 40 ml room temperature sterile artificial pond water (recipe from BRI, 0.46 μM FeCl3, 220 μM CaCl2, 100 μM MgSO4, 310 μM KH2PO4, 14 μM (NH4)2SO4 in water adjusted to pH 7.2 with NaOH) and transferred into the aluminum-covered sterile volumetric flask. The flask was carefully filled with sterile pond water to approximately 3 cm above the foil covering the neck. The flask was covered with a small petri dish and a bright light shone across the top of the flask. The eggs started to hatch at room temperature. When miracidia had amassed at the surface after 10 min, around 10 ml of the pondwater with miracidia was transferred to a sterile petri dish. The collected miracidia were centrifuged at 290 × g at 4 °C for 2 minutes. Only 1 ml of pondwater was left with the miracidia pellet and another 1ml of Chernin’s Balanced Salt Solution (CBSS+)20 was added to the petri dish. The miracidia were then transferred to a 24-well plate and incubated at 26 °C overnight to stimulate miracidial transformation into primary sporocysts. Chemotaxis/chemokinesis near schistosomes Capsaspora cells were counted and resuspended such that the final cell density was 60 × 103 cells/ml. 200 µl of the Capsaspora cells were seeded in each well of a TC-treated 96-well plate (Corning #353219) and incubated overnight at 26 °C. In the experiment run on fibronectin-coated plated (Fig S1B–C), a TC-treated 96-well plate (Corning #353219) was pre-coated with 100 µl of 70 µg/ml fibronectin (Sigma #F1141) in 25% FBS-free media before adding Capsaspora. The next day, schistosome sporocysts were centrifuged for 30 seconds at 300 × g at room temperature and the supernatant was aspirated immediately. The pellet was then resuspended with 650 µl 25% FBS-free media. Media was aspirated out of the Capsaspora wells and replaced with 200 µl of the sporocyst cell culture to afford a few schistosomes per well. Negative control wells received media without schistosomes. For imaging each well, a field of view was set such that there is at least one sporocyst surrounded by many Capsaspora cells. Images were collected every one minute for 4 hours using an A1 Nikon inverted microscope. Cells were then analyzed using Imaris image analysis software (Bitplane, version 10.0.1). ImageProcess was used to invert the image, the schistosome was set as the origin reference, and spots were created. Tracking was enabled. Estimated cell diameter was set to 6.43 µm. Background was subtracted, and the automatic threshold was used to quality filter the spots. Cells 70–240 µm from the origin reference frame were selected because cells closer than 70 µm might attach to the schistosome via filopodia, and cells further than 240 µm were near the boundaries of the field of view and may be near other schistosomes.. The autoregressive motion algorithm was used for tracking. To remove spurious readings by floating cells (which appear to migrate abnormally quickly and/or appear/disappear quickly), we excluded cells that migrated &gt; 11.2 µm in adjacent frames or lost tracking for &gt; 3 frames. The ‘fill gap’ function was disabled, and only tracks ≥3 frames were analyzed. For chemotaxis, the distance moved relative to the reference (schistosome) was reported for each cell track. To observe maximal movement, only tracks that had ≥2 hours (120 frames) were included for plotting. For chemokinesis, the distance moved frame-to-frame for each cell in each frame was reported. Only data for the first 15 minutes of the video were plotted, and only for cells 70–90 µm from the schistosome, since this was the timeframe and distance with the most notable chemokinesis. Chemokinesis assay This assay was always performed with the strain ATCC®30864 unless mentioned otherwise. The standard chemokinesis assay was performed on TC-treated 96-well plate (Corning #CLS3997). Either 5 × 103 cells or 10 × 103 cells were seeded in 90 µl of FBS-free growth media per well in the TC-treated 96-well plate and allowed to settle for 2 h. This low cell density was chosen to avoid confounding results from cellular aggregation (which is known to occur with Capsaspora in the presence of FBS at higher cell densities [e.g., 8 × 105 cells per well] in low-attachment [non TC-treated plates]).8-10 Method A After seeding cells and settling for 2 h, 85–90% of the FBS-free media was removed by aspiration and replaced with 85 µl Chernin’s balanced salt solution (CBSS+). Samples were then added at 10 µl volume such that the total volume in each well was 100 µl. At concentrations tested higher than 10%, less CBSS+ was added, and a larger sample volume was added to reach the desired concentration with a final volume of 100 µl. Chemokinesis activity was observed right after the sample addition by taking bright field images every 5 seconds for 3 minutes using an Agilent BioTek Cytation 10 confocal imaging reader. Method B After seeding cells and settling for 2 h, samples were directly added at 10 µl volume such that the total volume in a well is 100 µl. Chemokinesis activity was observed right after the sample addition by taking bright field images every 5 seconds for 3 minutes using an Agilent BioTek Cytation 10 confocal imaging reader. Chemokinesis quantification When the Cytation 10 was used to record cell movement, analysis was performed using BioTek Gen 5 software. Briefly, images were pre-processed using a dark background with background flattening using a rolling ball diameter of 9 µm and image smoothing strength of 2 cycles. Kinetic frame alignment was employed based on the previous valid image with a maximum offset of 200 µm. The cellular analysis module was employed to track cells. A primary mask was applied using a threshold value of 5000 with a dark background, split touching objects, track objects, and fill holes in masks. Object size was filtered to be 1–100 µm. To remove spurious readings by floating cells (which appear to migrate abnormally quickly and/or appear/disappear quickly), we made a subpopulation of cells that migrated ≤60 µm and were tracked for ≥ 18 frames. The ‘average velocity’ of this subpopulation of cells was reported as the ‘mean velocity’. Cell lysis Biomphalaria glabrata embryonic (Bge) cells49,50 were grown in Bge medium with 10% FBS at 26 °C. The culture was resuspended to 20 × 103 cells/ml and snap-frozen in 20 µl aliquots until further use. The frozen aliquot was freeze-thawed 5 times with a 20 second thawing period between each cycle. An additional 80 µl of FBS-free media was added to make the final volume 100 µl. The lysed culture was centrifuged at 15,000 × g for 5 minutes and the supernatant was collected for further testing. Both animal cell cultures were freshly received and resuspended to appropriate densities using FBS-free media. The cultures were then centrifuged at 15,000 × g for 5 min and the cell pellets were lysed using freeze-thaw method for 5 times with 20 second thawing period between each cycle. The lysed cultures were centrifuged at 15,000 × g for 5 min to collect the lysate supernatant. E. coli MG1655 culture was grown overnight in lysogeny broth (LB) at 37 °C with shaking and sub-cultured to 50 ml with 1:50 dilution of the overnight culture into fresh media. The 50 ml culture was incubated an additional 18 hours with shaking at 37 °C. The culture was centrifuged at 10,000 × g for 15 min. The cell pellet was resuspended in 1 ml of FBS-free media. The culture was lysed using a probe sonication for 10 seconds followed by a 20-second paused, repeated for a total of 5 min. The lysed culture was centrifuged at 10,000 × g for another 15 min and the lysate supernatant was collected for further analysis. B. subtilis 168 culture was grown overnight in lysogeny broth (LB) at 37 °C with shaking and sub-cultured to 50 ml with 1:50 dilution of the overnight culture into fresh media. The 50 ml culture was incubated an additional 18 hours with shaking at 37 °C. The 50 ml culture was centrifuged at 10,000 × g for 15 min. The cell pellet was resuspended in 1 ml of 4 mg/ml lysozyme and incubated for 15 min. The culture was centrifuged again at 15,000 × g for 15 min and the cell pellet was resuspended in 1 ml of FBS-free media. The culture was lysed using sonication for 10 seconds followed by a 20-second paused, repeated for a total of 5 min. The lysed culture was centrifuged at 10,000 × g for another 15 min and the lysate supernatant was collected for further analysis. FBS Heat-treatment 100 µl of FBS in an Eppendorf tube was heated on dry heat block at 75 °C for 30 minutes. The treated and non-treated FBS was tested for chemokinesis assay. The FBS-free growth media was used as negative control. Bioassay-guided fractionation FBS was initially fractionated using either Amicon Ultra 30 kDa or 100 kDa cutoff filters (Sigma, #UFC5030, #UFC5100) according to the manufacturer’s direction. Briefly, the filters were first washed with 500 µl of CBSS by centrifugation at 14,000 × g for 15 min. Then 500 µl of FBS was added to the cutoff filter and centrifuged at 14,000 × g for another 15 min. The small fractions (&lt; 30 kDa, &lt; 100 kDa) were collected for testing, and 20 µl of each large fraction ( &gt; 30 kDa or &gt; 100 kDa) was added to 180 µl of CBSS for testing. 10 µl of each stored fraction was used for testing chemokinesis. Isolation using strong anion exchange column (SAX) Chromatography sample preparation. 20 ml of FBS was fractionated using an Amicon Ultra 100 kDa cutoff filter (Sigma, #UFC8100) according to the manufacturer’s direction. Briefly, the filters were first washed with 5 ml CBSS+ by centrifuging at 4,000 × g for 25 min. Then 10 ml of FBS was added to each cutoff filter and centrifuged at 4,000 × g for 50 min. The &gt; 100 kDa was washed by adding 5 ml of buffer A (50 mM Tris Ethanolamine, pH 9.5), centrifuged at 4,000 × g for 25 min, and the concentrated sample was collected. The &gt; 100 kDa fraction was then sterile-filtered using Costar Spin-X Centrifuge filters (Corning, #29442-752). Chromatography Method. A GE HealthCare HiPrepTM Q XL 16/10 column (Fisher, #45-002-068) was used. For the mobile phase, 50 mM Tris Ethanolamine, pH 9.5 was used as buffer A and 50 mM Tris Ethanolamine supplemented with 1 M NaCl, pH 9.5 was used as buffer B with a flowrate of 5 ml/min. The column was first washed with buffer B for 5 min and equilibrated with buffer A using 5 column volumes (CV). The 5 ml sample loop was then washed with buffer A followed by injecting 1.2 ml of the sample (&gt; 100 kDa). Unbound material was washed from the column using 10 CV buffer A. The fractions were then eluted by using a linear gradient from 0–55% B using 30 CV and 55–100% B using 20 CV followed by holding 100% B for 10 CV. All fractions including the flow through were collected using a fixed volume of 5 ml per fraction. Chemokinesis sample preparation. The unbound fraction was pooled and labelled as fraction 1. To simplify, a systematic pooling strategy was employed. Initially, a primary pool was made by combining 50 µl from each of the 30 consecutive eluted fractions and labelled them sequentially from fraction 2 through fraction 9. Each of these pooled fractions were concentrated using the 100 kDa cut off filters, centrifuged at 14,000 × g for 15 min at 10 °C. &gt; 100 kDa fractions were then washed by adding 300 µl of CBSS and centrifuged again at 14,000 × g for 15 min at 10 °C and collected the &gt; 100 kDa fractions. 20 µl of each of these fractions were added to another 70 µl of CBSS to such that the total volume was 90 µl and stored at 4 °C until testing. Once the active pooled fractions were identified (pools 3 and 4), secondary pools were made by combining 100 µl from 10 consecutive initial fractions and labeled them sequentially. For example, fraction 31 contained original fractions 31–40, while fraction 33 contained fractions from 51–60. The same approach was applied to the other active initial pool 4. Pool 4 was subdivided into fractions 41, 42, and 43, where fraction 42 contained original fractions 71–80. The combined fractions were concentrated and washed as above. After collecting the &gt; 100 kDa fractions, 20 µl of each of these fractions were added to 180 µl of CBSS to such that the total volume was 200 µl. These were stored at 4 °C until testing. Upon identifying activity in specific secondary pools (pools 33 and 41), a tertiary pooling step was performed. Each active secondary pool was further divided by combining 50 µl from 5 consecutive initial fractions into a new pool and labeled accordingly. For example, fraction 33 was divided into 331 and 332, where fraction 332 contained original fractions 56–60 and fraction 421 contained original fractions 71–75. This iterative process allowed for the stepwise narrowing of active fractions. 20 µl of each of these fractions were added to another 80 µl of CBSS such that the total volume was 100 µl. These were stored at 4 °C until testing. 10 µl from each of these pools were added to the chemokinesis assay and CBSS was used as negative control Isolation using gel filtration (size exclusion chromatography [SEC]) Chromatography sample preparation. 2 ml of each fraction contributing to the 331 pool (i.e. 51–55) was combined to achieve a final volume of 10 ml to prepare for SEC. First, the 100 kDa cutoff filter was washed with 5 ml CBSS+ by centrifuging at 4,000 × g for 25 min. The sample was then centrifuged at 4,000 × g for 35 min using 100 kDa cutoff filter. To the &gt; 100 kDa fraction, another 3 ml of CBSS was added to the sample and centrifuged for another 30 min using 100 kDa cutoff filter and collected the final &gt; 100 kDa for SEC fractionation. Chromatography Method. A HiLoad 16/600 Superdex 200 pg (GE healthcare #28989335) column was used. For the mobile phase, 50 mM Tris Ethanolamine supplemented with 1 M NaCl, pH 9.5 was used with a flowrate of 1 ml/min. The column was first washed with the buffer for 5 min and equilibrated with the buffer using 0.2 column volumes (CV). The 1 ml sample loop was then washed with the buffer followed by injecting 1 ml of the sample (&gt; 100 kDa). The fractions were then eluted by using isocratic condition using 5 CV. All fractions were collected using a fixed volume of 3 ml per fraction. Chemokinesis sample preparation. 500 µl from each fraction containing proteins (5–12) was individually centrifuged at 4,000 × g for 15 min at 10 °C using 100 kDa cutoff filters. The &gt;100 kDa fraction was washed once with 200 µl CBSS and the remained 20 µl of &gt;100 kDa fraction was directly tested for chemokinesis assay and stored at 4 °C for further analysis. Different cell density seeding After measuring the cell density in a culture flask, the culture was appropriately diluted such that seeding with 90 µl of each diluted culture achieves the desired density. Desensitization experiments Chemokinesis assay was performed using the method B and Gen5 for imaging analysis. 10 µl of 30 mg/ml BSA was added to induce chemokinesis as mentioned at t =0 and 6h. Adhesion assay 5 × 103 cells were seeded in 90 µl of FBS-free growth media per well in the TC-treated 96-well plate and allowed to settle for 2 h. After seeding cells, 85–90% of FBS-free media was removed by aspiration and replaced with Chernin’s balanced salt solution (CBSS+). A Cytation 10 imaging reader was set to capture ~80% of the well’s area to count this initial cell number. After imaging, 10 µl of either BSA or water were added to each well and allowed to sit for 2 min (to mimic the chemokinesis conditions). 85–90% of FBS-free media was removed by aspiration and replaced with 85 µl CBSS+ solution. The Cytation 10 imaging reader was again used to capture ~80% of the well’s area to count the remaining cell number. The remaining cell number was divided by the initial cell number to determine the % cells remaining for each well. Calcium signaling assay 7 × 104 cells were seeded in 200 µl of FBS-free growth media per well in the TC-treated black-walled 96-well plate (Corning 353219) and allowed to settle for 2 h at room temperature. After seeding cells, 85–90% of FBS-free media was removed by aspiration and replaced with 100 µl Chernin’s balanced salt solution (CBSS+) containing 5 µM Fluo-4 AM dye with 0.1% DMSO and 0.02% pluruonic F-127 surfactant. The cells were incubated in the dark for 45–60 minutes before aspirating the solution and replacing with 100 µl of fresh CBSS+. Cells were imaged using a Olympus IX83 P2ZF inverted microscope fitted with a Yokogawa CSU-W1 spinning disk confocal system, a Hamamatsu ORCA-Flash4.0 camera, and a UPlanSApo 20x/0.75 objective. Fluorescence excitation was performed with a 488 nm laser, and emission was detected at 519 nm with 2x2 binning. Images were taken every 5 seconds for 3 minutes. After 25 seconds of baseline readings, 10 µl of test sample was added (i.e., FBS, CBSS, &gt;30 kDa FBS, &lt; 30 kDa FBS). Images were analyzed using Imaris image analysis software (Bitplane, version 10.0.1). ImageProcess was used to invert the image, and spots were created. Tracking was enabled. Estimated cell diameter was set to 3.00 µm. Background was subtracted, and the automatic threshold was used to quality filter the spots. The Brownian motion algorithm was used for tracking. To remove spurious readings by floating cells (which appear to migrate abnormally quickly and/or appear/disappear quickly), we excluded cells that migrated &gt; 2.30 µm in adjacent frames or lost tracking for &gt; 3 frames. The ‘fill gap’ function was disabled. The center intensity for each spot (i.e., each cell) was exported for each frame along the entire track. In Microsoft Excel, cell tracks without all frames included were removed. For each cell track, the initial spot intensity was subtracted from all spot intensity readings, thereby normalizing the initial intensity for every cell to zero. For each well at each time point, the normalized spot intensities for each cell were averaged (n~200 cells). Three replicate wells were analyzed, and their mean ± standard error were plotted (n=3).
description: Capsaspora cell migration was measured primarily with a Cytation C10 imager (Agilent) and reported here.