TY - JOUR
AU - Forero-Shelton, Manu
AB - Introduction Trypanosoma cruzi, a flagellated protozoa, is the etiological agent of Chagas disease, a condition that afflicts mainly regions in Latin America [1–3]. The life cycle of the pathogen is complex, consisting of three parasitic forms: amastigote, epimastigote, and trypomastigote [1,2,4]; these can be differentiated according to the presence or absence of an external flagellum, in addition to the position of the kinetoplast (extranuclear DNA organelle) relative to the nucleus [4,5]. The flagellum is one of the most characteristic organelles in T. cruzi. It emerges from the basal body, through the flagellar pocket which is an invagination responsible for exocytosis and endocytosis involved in host-parasite interaction [6,7]. The flagellum is involved in morphogenesis, cell division [8,9], evasion of the immune system, by mobilizing surface bound antibodies to the flagellar pocket, where those are transported to the lysosome to be degraded [10], and motility through the different anatomical sites and compartments of both the vector and the mammalian host [11–13]. Adhesion to surfaces is a crucial step for replication and maturation, the process of producing the infective form of the parasite in the vector’s gut [14]. The flagellar membrane plays a fundamental role in the initial process of attachment of the parasites to surfaces [15] and it has been shown that parasites adhere to surfaces by the anterior tip of the flagellum [15]. Most studies of T. cruzi invasion have been focused on the characterization of the molecules involved in the adhesion/invasion process [16], and less attention has been paid to the adaptability of these parasites to variable, hostile and confined microenvironments inside the hosts, which is a challenge for the survival of the pathogen. Fluids present in the host microenvironment can play a defensive role, preventing effective colonization [17,18]. For example, human arterial circulation has shear stresses approximately between 10 and 70 Dyn/cm2 (1–7 Pa); while in venous circulation this magnitude oscillates between 1 and 6 Dyn/cm2 (0.1–0.6 Pa) [19], a hostile environment for hematic parasites. However, studies on the mechanics of adhesion of T. cruzi to surfaces under shear stress, either in the intestine or heart, are absent. Here, in order to understand how T. cruzi epimastigotes attach under flow inside the host, we performed parallel-plate flow chamber experiments with time-varying shear stresses as well as several types of microscopy to study the mechanical response to fluid flow by the membranes of flagella and propose a new role in the adhesion process. Methods Culture and maintenance of T. cruzi epimastigotes T. cruzi epimastigotes from DA isolate (MHOM/CO/01/DA) a TcI genotype were used. Parasite were cultured in LIT (Liver Infusion Tryptose) medium supplemented with 5% bovine fetal serum (Eurobio, Les Ulis, France), and penicillin/streptomycin solution was added to prevent bacterial contamination. From the culture, parasites were diluted with the dye trypan blue (Sigma-Aldrich, Saint Louis, MI, USA) to count them in a Neubauer chamber. Epimastigotes were transferred to 4 mL tubes, in which 3 mL of 0.01 M phosphate buffer saline pH 7,4 (PBS 1X, Sigma-Aldrich) was added. Then, each tube was centrifuged at 1350 g for 5 min to remove excess culture medium. Two washings per sample were carried out, and the whole procedure was repeated twice. Finally, each sample was resuspended in 1 mL of PBS 1X, ensuring a concentration of 1 million epimastigotes per milliliter. Epimastigote fluorescent staining We used Carboxy fluorescein succinimidyl ester (CFSE), a cytoplasmic dye which covalently adheres to intracellular amine residues, and Octadecyl Rhodamine B Chloride (R18), a lipophilic cationic dye derived from rhodamine as a membrane dye. At least 1 μL of CFSE and 1 μL of R18 (250 μg/mL, ethanol) were added per every million epimastigotes contained in a 1.5 mL conic tube. The tube was covered aluminum foil, incubated in dark at 37°C for 15 min and mixed gently every 3 min. Labeled parasites were washed five times in PBS 1x. For CFSE, fluorescence was assessed by flow cytometry with 510/20 PMT comparing with unstained parasite; R18 fluorescence was assessed by fluorescence microscopy, using a TRITC fluorescence cube (excitation filter 528–553 nm, dichroic: 565 nm, and emission filter: 590–650 nm). To check for fluorescence crossover, epimastigotes dyed with only CFSE were tested in the fluorescence microscope using a TRITC fluorescence cube, and no excitation of the dye was observed using the same exposure times, and camera settings as used for taking data for the images. Similarly, epimastigotes dyed only with R18 were tested with a FITC cube (excitation filter 465–495 nm, dichroic: 505 nm, and emission filter: 515–555 nm), and no excitation was observed; thus, fluorescence crossover was discarded. To quantify fluorescence intensity of CFSE and R18, ImageJ [20] software was used. Intensity measurements were taken at the nanotubule, the anchoring pad and the background of each image. The background value was subtracted from the nanotubule and pad measures, and the intensity ratio between the pad and the nanotubule was determined. This procedure was carried out for two different stained parasites and the average measure was used. Microfluidic flow chamber experiments A Glycotech (Gaithersburg, MD, USA) microfluidic chamber (product # 31–001) was used. The silicone gasket used was that corresponding to the channel of 0.25 cm wide and 0.0254 cm high. This system was sealed with either a 35 mm Corning (New York, NY, USA) plastic dish, or a # 1.5 round glass coverslip 45 mm in diameter when short working distance objectives were used, for high resolution or fluorescence. Coverslips were not functionalized for simplicity since good adhesion was observed, although good attachment could also be obtained with poly-l-lysine functionalized coverslips. The chamber was connected to a vacuum pump (Barnant-Company vacuum pump reference 400–3910), to ensure a proper seal of the gasket. A three-way stopcock was used to select epimastigotes or PBS 1X flow. A 1 mL plastic syringe (Nubenco, Paramus, NJ, USA) was used to dispense the epimastigotes. A 60 mL Becton-Dickinson (BD, Queretaro, Mexico) plastic syringe was used to dispense the PBS 1X; its flow was controlled by a syringe pump (Pump 11 Elite Infusion / Withdrawal Programmable Single Syringe, Harvard Apparatus, Inc.) varying from 0.5 to 2.5 mL/min (0.0083–0.0417 cm3/s). Eq 1, which was provided by the manufacturer, was used to calculate shear stress in the parallel flow chamber; results are obtained in Pa, but we use pN/μm2 which are equivalent. Equivalences of PBS 1X flow to shear stress are presented in S1 Table. (1) Eq 1 Shear stress as a function of flow rate and chamber size. Here μ is the fluid viscosity (μ = 0.001 Pa·s for PBS), Q the fluid flow in cm3/s, h the channel height in cm and W the channel width in cm. Optical microscopy Observations with phase contrast and Differential Interference Contrast (DIC) microscopy were made on two systems: one, an inverted Zeiss Axiovert 40 CFL microscope, with Zeiss A-Plan 40X / 0.50 Ph2 and Zeiss A-Plan 100X / 1.25 Oil objectives and Point-Grey GRASSHOPPER 3 camera. Micro-Manager [21] software was used for image acquisition. Images were also obtained with a Nikon Eclipse Ti microscope using the Plan Fluor 40x Oil DIC H N2 and the Apo TIRF 60x Oil DIC N2 objectives with 1.49 NA and DIC. 90x magnification was obtained by using the 60x objective and a magnification changer of 1.5. Videos were taken with an ORCA-R2 (Hamamatsu Photonics K.K., Systems division) camera, while fluorescence images were taken with an Andor ixon 897 (Andor Technology, Oxford Instruments) camera with EM gain set to 0. at 16 bits in order to get the dynamic range necessary to see both the bright and faint features of the epimastigotes, using an exposure time of 300 ms, and a cooling temperature of -60°C. Videos were taken at 10 frames per second, with 100 ms exposure time. Image parameters after video acquisition, such as contrast, were adjusted with ImageJ [20] software for better presentation on computer screens. Flagellated forms of T. cruzi move towards the flagellum, and by following individual parasites on the microscopes it was possible to turn on the shear stress at the moment of initial adhesion to the surface via the anterior tip of flagellum. The structure of flagella did not change, and tracking was achieved by following the position of its anterior tip using ImageJ software with the MTrackJ complement. The position data was obtained as two-dimensional coordinates, in pixels and calibrated using a calibration ruler and the calibration feature of imageJ. Fabrication of microchannels with PDMS Due to the short working distance of fluorescence objectives, we fabricated microchannels with polydimethylsiloxane (PDMS) over ~170 μm thick (#1.5) rectangular glass coverslips. In a plastic container, curing agent and PDMS were mixed, in a 1:10 proportion, with a plastic fork for 5 min. The mixture was left in a degasser for at least two hours and poured over a channel mold 0.25 cm wide, 2 cm long and ~340 μm tall (two cut coverslips bonded with optical glue), and left in the degasser for another two hours, or until the air bubbles disappeared. Then, the cured PDMS was heated to 65°C in an incubator overnight. After solidification, the PDMS was unmolded and cut with a blade in order to fit it on a coverslip, and holes at the longitudinal ends of the channel (approximately 2 mm from the edge) were made with a 3/4 mm perforator. The PDMS was immersed in isopropanol and sonicated for 40 min, dried with filtrated pressure air and then incubated at 65°C overnight. The coverslips were immersed in 1M KOH, sonicated for 30 min, washed 5 times with ultrapure water, dried with filtrated pressure air, and incubated at 65°C overnight. PDMS (channel face up) and coverslips received oxygen plasma treatment for 1 min using a plasma cleaner PDC-32G (Harrick Plasma, Ithaca, NY, USA) in order to activate both surfaces; then, they were assembled and left on a hot plate at 150° C for 5 min, coverslip facing down. Finally, they were incubated overnight at room temperature. Scanning electron microscope (SEM) Epimastigotes were dispensed in the microfluidic chamber and subjected to flow during a period of at least 7 min after the formation of nanotubules, because it was observed that was the time necessary to ensure the nanotubules remained extended (likely due to additional attachments forming as explained in the results section). Once stretched, the parasites were incubated overnight at room temperature with a 2.5% glutaraldehyde solution to fix them to the surface; the chamber was disassembled, maintaining the surface of the coverslip where the epimastigotes were located humidified with glutaraldehyde. Then, epimastigotes were dehydrated by ethanol dilution series and then critical-point dried. A Denton Vacuum Desk IV Standard Sputter Coater was used to cover the samples with a gold layer ~10 nm thick, similar to what was used in [22]. Electron images were obtained in a Lyra3 Tescan, at a 5.0 kV accelerating voltage and working distance of 7.89 mm. Analysis of data and statistics Data was exported to Excel (Microsoft, Redmond, WA, USA). Pythagoras theorem was used to find the length of the nanotubule stretching for each epimastigote. Pixel size was calibrated with a micro ruler in ImageJ. The force threshold required for nanotubule extrusion was estimated from the intercept of the rapid elongation phase presented in Fig 1, by calculating the stokes drag using the formula by Goldman for a sphere [23], with a particle radius R = 1μm, given the diameter of 2 μm for epimastigotes and not taking into account its length. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 1. Filament formation. (a) Surface-bound epimastigote, at 0 shear stress. (b) The same epimastigote under a shear stress of 0.62 pN/μm2. A tether-like structure emerges from the flagellum, the arrows delimitate its length. (c) The flagellum resumes its beating motion while the tether is still attached to the surface. In this case the parasite did not return to its initial position. https://doi.org/10.1371/journal.pone.0283182.g001 The bending modulus was calculated using the formula derived by Hochmuth et al. [24] and Brochard-Wyart et al. [25] (2) Eq 2 Bending modulus (κ) as a function of threshold pulling force f0 and tether radius rt. Surface viscosity ηe was obtained from Eq (16) in Brochard-Wyart et al. [25] using the elongation velocity vs force curve (S1 Fig); data was fit to the following equation using minimum squares and assuming a bond density v between the cytoskeleton and the membrane of 103/μm2, κ as estimated above and rt from the electron microscopy data: (3) Eq 3 Extrusion force as a function of elongation velocity . Culture and maintenance of T. cruzi epimastigotes T. cruzi epimastigotes from DA isolate (MHOM/CO/01/DA) a TcI genotype were used. Parasite were cultured in LIT (Liver Infusion Tryptose) medium supplemented with 5% bovine fetal serum (Eurobio, Les Ulis, France), and penicillin/streptomycin solution was added to prevent bacterial contamination. From the culture, parasites were diluted with the dye trypan blue (Sigma-Aldrich, Saint Louis, MI, USA) to count them in a Neubauer chamber. Epimastigotes were transferred to 4 mL tubes, in which 3 mL of 0.01 M phosphate buffer saline pH 7,4 (PBS 1X, Sigma-Aldrich) was added. Then, each tube was centrifuged at 1350 g for 5 min to remove excess culture medium. Two washings per sample were carried out, and the whole procedure was repeated twice. Finally, each sample was resuspended in 1 mL of PBS 1X, ensuring a concentration of 1 million epimastigotes per milliliter. Epimastigote fluorescent staining We used Carboxy fluorescein succinimidyl ester (CFSE), a cytoplasmic dye which covalently adheres to intracellular amine residues, and Octadecyl Rhodamine B Chloride (R18), a lipophilic cationic dye derived from rhodamine as a membrane dye. At least 1 μL of CFSE and 1 μL of R18 (250 μg/mL, ethanol) were added per every million epimastigotes contained in a 1.5 mL conic tube. The tube was covered aluminum foil, incubated in dark at 37°C for 15 min and mixed gently every 3 min. Labeled parasites were washed five times in PBS 1x. For CFSE, fluorescence was assessed by flow cytometry with 510/20 PMT comparing with unstained parasite; R18 fluorescence was assessed by fluorescence microscopy, using a TRITC fluorescence cube (excitation filter 528–553 nm, dichroic: 565 nm, and emission filter: 590–650 nm). To check for fluorescence crossover, epimastigotes dyed with only CFSE were tested in the fluorescence microscope using a TRITC fluorescence cube, and no excitation of the dye was observed using the same exposure times, and camera settings as used for taking data for the images. Similarly, epimastigotes dyed only with R18 were tested with a FITC cube (excitation filter 465–495 nm, dichroic: 505 nm, and emission filter: 515–555 nm), and no excitation was observed; thus, fluorescence crossover was discarded. To quantify fluorescence intensity of CFSE and R18, ImageJ [20] software was used. Intensity measurements were taken at the nanotubule, the anchoring pad and the background of each image. The background value was subtracted from the nanotubule and pad measures, and the intensity ratio between the pad and the nanotubule was determined. This procedure was carried out for two different stained parasites and the average measure was used. Microfluidic flow chamber experiments A Glycotech (Gaithersburg, MD, USA) microfluidic chamber (product # 31–001) was used. The silicone gasket used was that corresponding to the channel of 0.25 cm wide and 0.0254 cm high. This system was sealed with either a 35 mm Corning (New York, NY, USA) plastic dish, or a # 1.5 round glass coverslip 45 mm in diameter when short working distance objectives were used, for high resolution or fluorescence. Coverslips were not functionalized for simplicity since good adhesion was observed, although good attachment could also be obtained with poly-l-lysine functionalized coverslips. The chamber was connected to a vacuum pump (Barnant-Company vacuum pump reference 400–3910), to ensure a proper seal of the gasket. A three-way stopcock was used to select epimastigotes or PBS 1X flow. A 1 mL plastic syringe (Nubenco, Paramus, NJ, USA) was used to dispense the epimastigotes. A 60 mL Becton-Dickinson (BD, Queretaro, Mexico) plastic syringe was used to dispense the PBS 1X; its flow was controlled by a syringe pump (Pump 11 Elite Infusion / Withdrawal Programmable Single Syringe, Harvard Apparatus, Inc.) varying from 0.5 to 2.5 mL/min (0.0083–0.0417 cm3/s). Eq 1, which was provided by the manufacturer, was used to calculate shear stress in the parallel flow chamber; results are obtained in Pa, but we use pN/μm2 which are equivalent. Equivalences of PBS 1X flow to shear stress are presented in S1 Table. (1) Eq 1 Shear stress as a function of flow rate and chamber size. Here μ is the fluid viscosity (μ = 0.001 Pa·s for PBS), Q the fluid flow in cm3/s, h the channel height in cm and W the channel width in cm. Optical microscopy Observations with phase contrast and Differential Interference Contrast (DIC) microscopy were made on two systems: one, an inverted Zeiss Axiovert 40 CFL microscope, with Zeiss A-Plan 40X / 0.50 Ph2 and Zeiss A-Plan 100X / 1.25 Oil objectives and Point-Grey GRASSHOPPER 3 camera. Micro-Manager [21] software was used for image acquisition. Images were also obtained with a Nikon Eclipse Ti microscope using the Plan Fluor 40x Oil DIC H N2 and the Apo TIRF 60x Oil DIC N2 objectives with 1.49 NA and DIC. 90x magnification was obtained by using the 60x objective and a magnification changer of 1.5. Videos were taken with an ORCA-R2 (Hamamatsu Photonics K.K., Systems division) camera, while fluorescence images were taken with an Andor ixon 897 (Andor Technology, Oxford Instruments) camera with EM gain set to 0. at 16 bits in order to get the dynamic range necessary to see both the bright and faint features of the epimastigotes, using an exposure time of 300 ms, and a cooling temperature of -60°C. Videos were taken at 10 frames per second, with 100 ms exposure time. Image parameters after video acquisition, such as contrast, were adjusted with ImageJ [20] software for better presentation on computer screens. Flagellated forms of T. cruzi move towards the flagellum, and by following individual parasites on the microscopes it was possible to turn on the shear stress at the moment of initial adhesion to the surface via the anterior tip of flagellum. The structure of flagella did not change, and tracking was achieved by following the position of its anterior tip using ImageJ software with the MTrackJ complement. The position data was obtained as two-dimensional coordinates, in pixels and calibrated using a calibration ruler and the calibration feature of imageJ. Fabrication of microchannels with PDMS Due to the short working distance of fluorescence objectives, we fabricated microchannels with polydimethylsiloxane (PDMS) over ~170 μm thick (#1.5) rectangular glass coverslips. In a plastic container, curing agent and PDMS were mixed, in a 1:10 proportion, with a plastic fork for 5 min. The mixture was left in a degasser for at least two hours and poured over a channel mold 0.25 cm wide, 2 cm long and ~340 μm tall (two cut coverslips bonded with optical glue), and left in the degasser for another two hours, or until the air bubbles disappeared. Then, the cured PDMS was heated to 65°C in an incubator overnight. After solidification, the PDMS was unmolded and cut with a blade in order to fit it on a coverslip, and holes at the longitudinal ends of the channel (approximately 2 mm from the edge) were made with a 3/4 mm perforator. The PDMS was immersed in isopropanol and sonicated for 40 min, dried with filtrated pressure air and then incubated at 65°C overnight. The coverslips were immersed in 1M KOH, sonicated for 30 min, washed 5 times with ultrapure water, dried with filtrated pressure air, and incubated at 65°C overnight. PDMS (channel face up) and coverslips received oxygen plasma treatment for 1 min using a plasma cleaner PDC-32G (Harrick Plasma, Ithaca, NY, USA) in order to activate both surfaces; then, they were assembled and left on a hot plate at 150° C for 5 min, coverslip facing down. Finally, they were incubated overnight at room temperature. Scanning electron microscope (SEM) Epimastigotes were dispensed in the microfluidic chamber and subjected to flow during a period of at least 7 min after the formation of nanotubules, because it was observed that was the time necessary to ensure the nanotubules remained extended (likely due to additional attachments forming as explained in the results section). Once stretched, the parasites were incubated overnight at room temperature with a 2.5% glutaraldehyde solution to fix them to the surface; the chamber was disassembled, maintaining the surface of the coverslip where the epimastigotes were located humidified with glutaraldehyde. Then, epimastigotes were dehydrated by ethanol dilution series and then critical-point dried. A Denton Vacuum Desk IV Standard Sputter Coater was used to cover the samples with a gold layer ~10 nm thick, similar to what was used in [22]. Electron images were obtained in a Lyra3 Tescan, at a 5.0 kV accelerating voltage and working distance of 7.89 mm. Analysis of data and statistics Data was exported to Excel (Microsoft, Redmond, WA, USA). Pythagoras theorem was used to find the length of the nanotubule stretching for each epimastigote. Pixel size was calibrated with a micro ruler in ImageJ. The force threshold required for nanotubule extrusion was estimated from the intercept of the rapid elongation phase presented in Fig 1, by calculating the stokes drag using the formula by Goldman for a sphere [23], with a particle radius R = 1μm, given the diameter of 2 μm for epimastigotes and not taking into account its length. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 1. Filament formation. (a) Surface-bound epimastigote, at 0 shear stress. (b) The same epimastigote under a shear stress of 0.62 pN/μm2. A tether-like structure emerges from the flagellum, the arrows delimitate its length. (c) The flagellum resumes its beating motion while the tether is still attached to the surface. In this case the parasite did not return to its initial position. https://doi.org/10.1371/journal.pone.0283182.g001 The bending modulus was calculated using the formula derived by Hochmuth et al. [24] and Brochard-Wyart et al. [25] (2) Eq 2 Bending modulus (κ) as a function of threshold pulling force f0 and tether radius rt. Surface viscosity ηe was obtained from Eq (16) in Brochard-Wyart et al. [25] using the elongation velocity vs force curve (S1 Fig); data was fit to the following equation using minimum squares and assuming a bond density v between the cytoskeleton and the membrane of 103/μm2, κ as estimated above and rt from the electron microscopy data: (3) Eq 3 Extrusion force as a function of elongation velocity . Results Attached epimastigotes extend significantly in response to shear stress Epimastigotes bound nonspecifically to the surface of the flow cell were subjected to different fluid flows of 1X PBS and followed using phase contrast optical microscopy. The parasites responded in one of two manners under flow: some barely shifted their position while others moved significantly from their initial position, up to 10’s of microns, returning near, or to their starting position. We first focus on the group that changed position when shear stress was applied, which were obtained in a systematic manner by following individual parasites under the microscope until they attached and immediately turning on fluid flow. Fluid flow flipped the parasites (which travel flagellum-first) around the tip of the flagellum (Fig 1A), and at higher flows those that remained attached formed thin filaments that generally emerged from the flagellar tip and extended from there to the initial position of the parasite (Fig 1B). Significant extension of the soma of the parasites was not observed, so in this study parasite extension will refer to that of the filaments. Nanotubules connect an anchoring pad to the flagellum To identify the structural changes and the structures involved in epimastigote extension, surface-bound epimastigotes were observed at 100x magnification (Fig 1) starting at no flow (Fig 1A) and subjected to a shear stress of 0.62 pN/μm2. Under the optical microscope, filaments appear to be ~200nm wide, the resolution limit of the transmitted light microscope, and of variable length, up to several times the length of the epimastigote (100 μm). Notably, during flow, parasite movement stopped and resumed when flow stopped (Fig 1C and S1 Movie) suggesting there is a sensing mechanism that stops movement when filaments extend. Scanning electron microscopy (SEM) was used to visualize the filamentous structure at high resolution. Fig 2 shows an epimastigote after being stretched with a shear stress of 1.24 pN/μm2, immediately fixed, then prepared for SEM imaging by dehydration and gold sputtering. In Fig 2A, a nanotubule—as we will refer to these structures from here on, extends ~2 μm from the tip of the flagellum of the epimastigote. The nanotubule is ~60 nm wide (Fig 2B) and connects to a pad at its distal end. Since the gold is ~10 nm thick, the diameter of the nanotubule is estimated at ~40 nm. Near the flagellum, the neck of the nanotubule is funnel shaped, as wide as the flagellum tip (~130 nm) on one side, and as wide as the nanotubule on the other. The main section of the nanotubule is of constant width, and its distal end broadens to form a pad about a micron long and several hundred of nm wide. This structure was not initially observed in transmitted light microscopy, except for the fact that during flagellar movement its distal end remained attached. Thus, the pad is a structure that ensures the adhesion of the nanotubule to the surface, that we will refer to as the “anchoring pad”. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 2. Electron microscopy images of a shear-stress generated nanotubule. (a) An epimastigote after being stretched at 1.24 pN/μm2. The nanotubule is delimited by black arrows. (b) Detailed view of the nanotubule in (a). Note the anchoring pad at the end of the structure and the funnel shape on the flagellum side. https://doi.org/10.1371/journal.pone.0283182.g002 Nanotubules are mainly composed of membrane To determine the composition of the nanotubules, epimastigotes were stained with CFSE, a cytoplasmic dye, and their membrane with R18, a lipophilic cationic dye derived from rhodamine (see methods) and stretched using a continuous shear of 1.24 pN/μm2. Fig 3 shows a representative epimastigote after its extension by shear stress. Using bright field microscopy (Fig 3A) it is possible to see the nanotubule by which the epimastigote is adhered to the surface. At the distal end, the anchoring pad appears as a slightly wider structure than the rest of the extended nanotubule. Fig 3B shows CFSE fluorescence mostly at the soma of the parasite. A faint green signal is visible on the nanotubule and the anchoring pad (inset). The signal is not bleed-through from another channel (see the methods section for the description of the negative controls), suggesting that there are few cytoplasmic components along the nanotubule and the anchoring pad. Fig 3C shows R18 fluorescence, where there is a strong signal at the soma of the parasite as well as the anchoring pad, and a lower signal on the nanotubule. Therefore, the nanotubule as well as the pad are mainly composed of membrane, with some proteins. Fig 3D shows the merged CFSE and R18 images, illustrating the differences in distribution with R18 more on the borders and CSFE on the soma of the parasite. Note that difference of fluorescence intensity between the anchoring pad and the nanotubule (~3:1 for CFSE and 5:1 for R18) can be explained by the diffraction limit of the microscope meaning that intensity differences are not necessarily attributable to differences in protein/membrane content between the structures, but to the fact that the light from an area ~40 nm wide is spread over the width of the point spread function of the microscope, which is over 200 nm wide (5:1). Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 3. Nanotubules dyed with cytoplasmic (CFSE) and membrane (R18) dyes. (a) bright field image of an epimastigote at 90x, after it was extended by shear stress. The anchoring pad is marked by a white arrow at the end of the nanotubule. (b) CFSE fluorescence is mainly at the soma of the parasite, but a faint signal can be seen at both the anchoring pad and the nanotubule (see inset where brightness was increased for visibility) (c) R18 fluorescence levels, are similar to CFSE fluorescence in the soma, but significantly brighter both in the nanotubule and the anchoring pad. (d) composite of CFSE and R18 images. https://doi.org/10.1371/journal.pone.0283182.g003 The membrane dye also made it possible to see additional structures that were not seen in brightfield, such as additional nanotubules and attachment points. Fig 4 shows an epimastigote at 40X, stretched using a shear stress of 1.55 pN/μm2. In bright field (Fig 4A), the presence of nanotubules is not clear, but in fluorescence (Fig 4B), two nanotubules are observed. They have vertices at their distal end, which may correspond to secondary adhesion points formed during the elongation process. Although they appear to be as wide as the nanotubules in the diffraction-limited images, the higher fluorescence intensity in the R18 channel suggests they are larger than the standard nanotubules. This doubly tethered epimastigote was able to resist high flows. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 4. Secondary adhesion points. Epimastigote dyed with R18 and subjected to 1.55 pN/μm2 of shear stress; in bright field microscopy at 40X (a) the adhesion structures are not visible, but in fluorescence (b) two nanotubules by which the parasite is attached are visible, as well as secondary anchoring pads (inset: Adjusted brightness, G = 2.89) marked by white arrows. https://doi.org/10.1371/journal.pone.0283182.g004 Nanotubule extension under shear stresses To determine the dynamics of nanotubules extension as a function of shear stress, parasites were subjected to a cycle of linearly increasing shear stress pulses for 20 s, interspaced by 20 s pauses as shown in Fig 5. When shear stress reached 1.55 pN/μm2 most epimastigotes detached from the surface, so measurements were limited to 1.24 pN/μm2. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 5. (a) Elongation of an epimastigote with increasing shear stress: Starting with 20 secs of no flow, parasites were subjected to a shear stress cycle of 0.31 –– 0.62–0.93–1.24 pN/μm2 pulses; each pulse was 20 s long, followed by a pause lasting 20 s. The curve shows the length of the nanotubules, as measured by changes in position of the flagellum tip. (b) Average extension as a function of shear stress. White circles correspond to the average length reached by each parasite during the fast elongation phase (first 2–4 s) after pulse introduction (n = 12); black triangles represent average data (data available in S1 Dataset). https://doi.org/10.1371/journal.pone.0283182.g005 Filaments require a threshold shear stress to extend, and extension increases with shear stress, until detachment. A similar response was observed in most extending epimastigotes (n > 20) and tracked in detail (n = 12). Fig 5A shows the data for one epimastigote; the filament elongates reversibly after each shear stress pulse. Two elongation regimes appear in the extension curves: the filament first extends rapidly in the first ~2–4 s, essentially the rate of change of the pump and hose system, and then creeps slowly, suggesting two mechanisms of elongation. For the rapid elongation interval, the average extension as a function of shear stress (Fig 5B) increases linearly after a threshold. This response to flow is consistent among epimastigotes and the average data of all analyzed epimastigotes, presented in S2 Fig, illustrates the consistency in these responses. Finally, during these 20 s intervals lengths of a few μm up to several 10’s μm were observed, with longer lengths for epimastigotes exposed for longer times. To evaluate the repeatability and the role of previous elongations on the total extension achieved during the extension-contraction cycles, the parasites were subjected to two increasing shear stress cycles with a 1-min pause with no shear stress between the cycles. In the second cycle, the number of epimastigotes extending was reduced from 12 to 5, with 7 strongly attaching to the surface without extending; there was a small reduction, on average, of the maximum elongation reached, as shown in S3 Fig, but the general shape of the elongation curve remained unchanged. Multiple tethering improves resistance to high flows Although most parasites detached from the surface at high shear stress (≥ 1.55 pN/μm2), some remained attached. The epimastigote in Fig 6 was exposed to a shear stress of 1.55 pN/μm2 and attached to the surface with two filaments: during the elongation of the first filament, another anchoring point was established, and a second filament extended (S2 Movie). Each filament appears to originate from a different point in the flagellum as shown in Fig 6. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 6. Doubly tethered epimastigote at 90x magnification. Inset is a 3X digital magnification of the delimited area. The first filament (on the right) emerged at a shear stress of 1.55 pN/μm2 from an anchoring point established over 100 μm away from the tip of the flagellum (see S4 Fig); During extension, a second anchoring point was established (white arrow), from which the second filament emerged; both extended until the point marked with the black arrowhead for the first filament and the white arrowhead for the second filament, both near the tip of the flagellum. https://doi.org/10.1371/journal.pone.0283182.g006 While for flagellum-attached epimastigotes the flagellum wagged vigorously when fluid flow was stopped, for strongly attached epimastigotes movement stopped or was significantly reduced despite the absence of flow, likely due to additional attachment points on the parasite. We observed that this effect was time-dependent: the longer epimastigotes sat on the surface before fluid flow started, the more likely they were to be strongly attached, with most parasites strongly attached after ~3 min without shear stress. The proximal end of nanotubules can migrate Fig 7 shows the migration of a nanotubule during a cycle of shear stress pulses. The epimastigote was subjected to 0.31 –– 0.62–0.93–1.24 pN/μm2, for 20 s, followed by a 20 s pause. Fig 7A and 7C correspond to the initial state for reference and the pause between the last shear stresses. Flagellar beating in c) induced the rotation of the parasite around its mid-section (S3 Movie). When shear stress was applied again at 1.24 pN/μm2 (Fig 7D), the parasite acquired a “V” shape with a vertex in the middle of the parasite, indicating the migration of the neck of the nanotubule to a location near the flagellar pocket. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 7. Relocation of the parasite-end of a filament during a shear stress cycle with pauses at a) and c). (a) initial adhesion of the parasite to the surface by the anterior part of the flagellum (white arrow) under no flow, (b) after exposure to a shear stress of 0.93 pN/μm2, (c) during the subsequent pause and under 1.24 pN/μm2. Flagellar beating during the pause resulted in the rotation of the parasite around a pivot point marked by the white arrowhead and near the flagellar pocket. At 1.24 pN/μm2, the parasite acquires a “V” shape shown in (d), indicative of the change in the position of the parasite-end of the nanotubule. https://doi.org/10.1371/journal.pone.0283182.g007 Attached epimastigotes extend significantly in response to shear stress Epimastigotes bound nonspecifically to the surface of the flow cell were subjected to different fluid flows of 1X PBS and followed using phase contrast optical microscopy. The parasites responded in one of two manners under flow: some barely shifted their position while others moved significantly from their initial position, up to 10’s of microns, returning near, or to their starting position. We first focus on the group that changed position when shear stress was applied, which were obtained in a systematic manner by following individual parasites under the microscope until they attached and immediately turning on fluid flow. Fluid flow flipped the parasites (which travel flagellum-first) around the tip of the flagellum (Fig 1A), and at higher flows those that remained attached formed thin filaments that generally emerged from the flagellar tip and extended from there to the initial position of the parasite (Fig 1B). Significant extension of the soma of the parasites was not observed, so in this study parasite extension will refer to that of the filaments. Nanotubules connect an anchoring pad to the flagellum To identify the structural changes and the structures involved in epimastigote extension, surface-bound epimastigotes were observed at 100x magnification (Fig 1) starting at no flow (Fig 1A) and subjected to a shear stress of 0.62 pN/μm2. Under the optical microscope, filaments appear to be ~200nm wide, the resolution limit of the transmitted light microscope, and of variable length, up to several times the length of the epimastigote (100 μm). Notably, during flow, parasite movement stopped and resumed when flow stopped (Fig 1C and S1 Movie) suggesting there is a sensing mechanism that stops movement when filaments extend. Scanning electron microscopy (SEM) was used to visualize the filamentous structure at high resolution. Fig 2 shows an epimastigote after being stretched with a shear stress of 1.24 pN/μm2, immediately fixed, then prepared for SEM imaging by dehydration and gold sputtering. In Fig 2A, a nanotubule—as we will refer to these structures from here on, extends ~2 μm from the tip of the flagellum of the epimastigote. The nanotubule is ~60 nm wide (Fig 2B) and connects to a pad at its distal end. Since the gold is ~10 nm thick, the diameter of the nanotubule is estimated at ~40 nm. Near the flagellum, the neck of the nanotubule is funnel shaped, as wide as the flagellum tip (~130 nm) on one side, and as wide as the nanotubule on the other. The main section of the nanotubule is of constant width, and its distal end broadens to form a pad about a micron long and several hundred of nm wide. This structure was not initially observed in transmitted light microscopy, except for the fact that during flagellar movement its distal end remained attached. Thus, the pad is a structure that ensures the adhesion of the nanotubule to the surface, that we will refer to as the “anchoring pad”. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 2. Electron microscopy images of a shear-stress generated nanotubule. (a) An epimastigote after being stretched at 1.24 pN/μm2. The nanotubule is delimited by black arrows. (b) Detailed view of the nanotubule in (a). Note the anchoring pad at the end of the structure and the funnel shape on the flagellum side. https://doi.org/10.1371/journal.pone.0283182.g002 Nanotubules are mainly composed of membrane To determine the composition of the nanotubules, epimastigotes were stained with CFSE, a cytoplasmic dye, and their membrane with R18, a lipophilic cationic dye derived from rhodamine (see methods) and stretched using a continuous shear of 1.24 pN/μm2. Fig 3 shows a representative epimastigote after its extension by shear stress. Using bright field microscopy (Fig 3A) it is possible to see the nanotubule by which the epimastigote is adhered to the surface. At the distal end, the anchoring pad appears as a slightly wider structure than the rest of the extended nanotubule. Fig 3B shows CFSE fluorescence mostly at the soma of the parasite. A faint green signal is visible on the nanotubule and the anchoring pad (inset). The signal is not bleed-through from another channel (see the methods section for the description of the negative controls), suggesting that there are few cytoplasmic components along the nanotubule and the anchoring pad. Fig 3C shows R18 fluorescence, where there is a strong signal at the soma of the parasite as well as the anchoring pad, and a lower signal on the nanotubule. Therefore, the nanotubule as well as the pad are mainly composed of membrane, with some proteins. Fig 3D shows the merged CFSE and R18 images, illustrating the differences in distribution with R18 more on the borders and CSFE on the soma of the parasite. Note that difference of fluorescence intensity between the anchoring pad and the nanotubule (~3:1 for CFSE and 5:1 for R18) can be explained by the diffraction limit of the microscope meaning that intensity differences are not necessarily attributable to differences in protein/membrane content between the structures, but to the fact that the light from an area ~40 nm wide is spread over the width of the point spread function of the microscope, which is over 200 nm wide (5:1). Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 3. Nanotubules dyed with cytoplasmic (CFSE) and membrane (R18) dyes. (a) bright field image of an epimastigote at 90x, after it was extended by shear stress. The anchoring pad is marked by a white arrow at the end of the nanotubule. (b) CFSE fluorescence is mainly at the soma of the parasite, but a faint signal can be seen at both the anchoring pad and the nanotubule (see inset where brightness was increased for visibility) (c) R18 fluorescence levels, are similar to CFSE fluorescence in the soma, but significantly brighter both in the nanotubule and the anchoring pad. (d) composite of CFSE and R18 images. https://doi.org/10.1371/journal.pone.0283182.g003 The membrane dye also made it possible to see additional structures that were not seen in brightfield, such as additional nanotubules and attachment points. Fig 4 shows an epimastigote at 40X, stretched using a shear stress of 1.55 pN/μm2. In bright field (Fig 4A), the presence of nanotubules is not clear, but in fluorescence (Fig 4B), two nanotubules are observed. They have vertices at their distal end, which may correspond to secondary adhesion points formed during the elongation process. Although they appear to be as wide as the nanotubules in the diffraction-limited images, the higher fluorescence intensity in the R18 channel suggests they are larger than the standard nanotubules. This doubly tethered epimastigote was able to resist high flows. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 4. Secondary adhesion points. Epimastigote dyed with R18 and subjected to 1.55 pN/μm2 of shear stress; in bright field microscopy at 40X (a) the adhesion structures are not visible, but in fluorescence (b) two nanotubules by which the parasite is attached are visible, as well as secondary anchoring pads (inset: Adjusted brightness, G = 2.89) marked by white arrows. https://doi.org/10.1371/journal.pone.0283182.g004 Nanotubule extension under shear stresses To determine the dynamics of nanotubules extension as a function of shear stress, parasites were subjected to a cycle of linearly increasing shear stress pulses for 20 s, interspaced by 20 s pauses as shown in Fig 5. When shear stress reached 1.55 pN/μm2 most epimastigotes detached from the surface, so measurements were limited to 1.24 pN/μm2. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 5. (a) Elongation of an epimastigote with increasing shear stress: Starting with 20 secs of no flow, parasites were subjected to a shear stress cycle of 0.31 –– 0.62–0.93–1.24 pN/μm2 pulses; each pulse was 20 s long, followed by a pause lasting 20 s. The curve shows the length of the nanotubules, as measured by changes in position of the flagellum tip. (b) Average extension as a function of shear stress. White circles correspond to the average length reached by each parasite during the fast elongation phase (first 2–4 s) after pulse introduction (n = 12); black triangles represent average data (data available in S1 Dataset). https://doi.org/10.1371/journal.pone.0283182.g005 Filaments require a threshold shear stress to extend, and extension increases with shear stress, until detachment. A similar response was observed in most extending epimastigotes (n > 20) and tracked in detail (n = 12). Fig 5A shows the data for one epimastigote; the filament elongates reversibly after each shear stress pulse. Two elongation regimes appear in the extension curves: the filament first extends rapidly in the first ~2–4 s, essentially the rate of change of the pump and hose system, and then creeps slowly, suggesting two mechanisms of elongation. For the rapid elongation interval, the average extension as a function of shear stress (Fig 5B) increases linearly after a threshold. This response to flow is consistent among epimastigotes and the average data of all analyzed epimastigotes, presented in S2 Fig, illustrates the consistency in these responses. Finally, during these 20 s intervals lengths of a few μm up to several 10’s μm were observed, with longer lengths for epimastigotes exposed for longer times. To evaluate the repeatability and the role of previous elongations on the total extension achieved during the extension-contraction cycles, the parasites were subjected to two increasing shear stress cycles with a 1-min pause with no shear stress between the cycles. In the second cycle, the number of epimastigotes extending was reduced from 12 to 5, with 7 strongly attaching to the surface without extending; there was a small reduction, on average, of the maximum elongation reached, as shown in S3 Fig, but the general shape of the elongation curve remained unchanged. Multiple tethering improves resistance to high flows Although most parasites detached from the surface at high shear stress (≥ 1.55 pN/μm2), some remained attached. The epimastigote in Fig 6 was exposed to a shear stress of 1.55 pN/μm2 and attached to the surface with two filaments: during the elongation of the first filament, another anchoring point was established, and a second filament extended (S2 Movie). Each filament appears to originate from a different point in the flagellum as shown in Fig 6. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 6. Doubly tethered epimastigote at 90x magnification. Inset is a 3X digital magnification of the delimited area. The first filament (on the right) emerged at a shear stress of 1.55 pN/μm2 from an anchoring point established over 100 μm away from the tip of the flagellum (see S4 Fig); During extension, a second anchoring point was established (white arrow), from which the second filament emerged; both extended until the point marked with the black arrowhead for the first filament and the white arrowhead for the second filament, both near the tip of the flagellum. https://doi.org/10.1371/journal.pone.0283182.g006 While for flagellum-attached epimastigotes the flagellum wagged vigorously when fluid flow was stopped, for strongly attached epimastigotes movement stopped or was significantly reduced despite the absence of flow, likely due to additional attachment points on the parasite. We observed that this effect was time-dependent: the longer epimastigotes sat on the surface before fluid flow started, the more likely they were to be strongly attached, with most parasites strongly attached after ~3 min without shear stress. The proximal end of nanotubules can migrate Fig 7 shows the migration of a nanotubule during a cycle of shear stress pulses. The epimastigote was subjected to 0.31 –– 0.62–0.93–1.24 pN/μm2, for 20 s, followed by a 20 s pause. Fig 7A and 7C correspond to the initial state for reference and the pause between the last shear stresses. Flagellar beating in c) induced the rotation of the parasite around its mid-section (S3 Movie). When shear stress was applied again at 1.24 pN/μm2 (Fig 7D), the parasite acquired a “V” shape with a vertex in the middle of the parasite, indicating the migration of the neck of the nanotubule to a location near the flagellar pocket. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 7. Relocation of the parasite-end of a filament during a shear stress cycle with pauses at a) and c). (a) initial adhesion of the parasite to the surface by the anterior part of the flagellum (white arrow) under no flow, (b) after exposure to a shear stress of 0.93 pN/μm2, (c) during the subsequent pause and under 1.24 pN/μm2. Flagellar beating during the pause resulted in the rotation of the parasite around a pivot point marked by the white arrowhead and near the flagellar pocket. At 1.24 pN/μm2, the parasite acquires a “V” shape shown in (d), indicative of the change in the position of the parasite-end of the nanotubule. https://doi.org/10.1371/journal.pone.0283182.g007 Discussion Trypomastigotes travel in the bloodstream of mammals, and in humans they invade the heart. The same occurs for epimastigotes, which must travel along the intestine of the vector until it adheres to an enterocyte to initiate metacyclogenesis; however, little is known about the process of adhesion and invasion under flow. Mechanisms that overcome high fluid flow and make it possible for pathogens to remain attached to the surface have previously described in different pathogens [26–35]. Here, we found a similar mechanism: In epimastigotes attached to the surface, shear stress induces the formation of nanotubules, mainly composed of membrane and a few proteins. The length of nanotubules increases with shear stress and can result in nanotubules up to 100 μm long. At the distal end, attachment is mediated by an anchoring pad (Fig 3), which has a higher protein content in comparison to the filaments, likely proteins associated with surface adhesion. Similar membrane tether formation processes have been described for membrane tethers in erythrocytes [36], neutrophils [37–39], fibroblasts [40], Escherichia coli [41], among others. Characteristically, when fluid flow stops, the nanotubule is reabsorbed and the parasite returns to its original position, implying a reversible process when they are attached by a single tether. A threshold force was necessary to initiate the extraction of membrane tethers, as documented in other systems [42–46], which has been interpreted as the force required to overcome membrane-cytoskeleton interactions [47], and implying the presence of interactions between the membrane and the flagellum. Here, we estimate the force threshold required for nanotubule extrusion at ~7 pN from the intercept of the rapid elongation phase presented in Fig 5B (see methods). This is consistent with the forces required in other types of cells [48]. Additionally, since models for force-extruded tethers or nanotubules relate their radius to the curvature modulus [24,25], using the tether radius obtained in SEM and the force at which the nanotubule was extruded, a curvature modulus of ~60 kbT was estimated for the membrane (see methods), in line with that of other types of cells [25]. It is also possible to estimate the surface viscosity from a viscous drag model [25] using the elongation velocity vs force curve (S1 Fig) at 50 10−6 Pa∙s∙m, also consistent with the properties of the membranes of other types of cells [25]. The dynamics of elongation shows a fast extension (first 2–4 s) followed by creep. Similar elongation processes have been reported in membrane tethers by force experiments, where the initial phase corresponds to the elastic component of the membrane, while the slow regimen can be attributed to a gradual addition of phospholipids to the tether from membrane reservoirs [36,40,41,45,46]. Additionally, the reduction in activity during elongation or multiple tethering (which likely maintains tension as in strongly attached bacteria [30]) is suggestive of a mechanism to sense membrane tension that could establish when the parasite is firmly adhered in order to continue its next step of the invasion process. Epimastigotes that were attached by multiple nanotubules remained attached to the surface at higher shear stress magnitudes than those singly attached. This is consistent with simulations of E. coli attachment via multiple tethers, where increased attachment at high and variable flows is promoted by the distribution of force between tethers with yielding elasticity [30], meaning that force plateaus in the extension curve, as is seen here during the slow extension regime. This is also observed experimentally for membrane tethers extracted mechanically in Chinese hamster ovary and a malignant human brain tumor, and endothelial cells by Sun et al. [42]: individual tethers contribute to increase the total pulling force. Thus, tethers, which have also been related to buffering the force from fluid flow [30,45,49–51], could regulate adhesion under stresses generated in the gut of the triatomine host as can be the case for bacteria in the human gut [52]. Nanotubules can emerge or migrate to parts other than the flagellum tip such as the flagellar pocket. Migration of protein structures on the membrane has been observed in Trypanosoma brucei, the agent of African trypanosomiasis. Engstler et al. described a process involving the clearance of antibodies and immune complexes on the surface of the parasite, where anti-glycoproteins antibodies were mobilized to the flagellar pocket zone for endocytosis and posteriorly eliminated [10]. More experiments are needed to establish the role (if any) of nanotubule migration in these parasites, and a possibility could improve the distribution of force in multiply attached cells. Other membrane/cytoplasmic projections, known as filopodia, have been described in T. cruzi [53], and also in other parasites from the Trypanosomatida order. In T. brucei adhesion and antibody clearance have been proposed [54,55]. Conversely, Szempruch et al. [56] induced the formation of tubular structures from the flagellar membrane of T. brucei. These structures did not result from mechanical stress and were composed of flagellar membrane proteins as well as virulence factors that could be transferred to other cells by direct contact [57] or by vesicles resulting from the subsequent fragmentation of the nanotubules [56]. To our knowledge, the formation of similar structures due to forces from fluid flow have not been previously reported in T. cruzi. The nanotubules described here have characteristics that can facilitate the adhesion process of a flagellated form of the parasite under flow, necessary for replication or maturation inside the host. Membrane-derived trails have been described in T. cruzi amastigotes and epimastigotes [58]. These appeared to emerge spontaneously from the flagellar pocket or the opposite side of the parasites, in a time and temperature dependent manner, in both in-vitro and in-vivo conditions. Although the formation process of the trails was not entirely clear, given that there are similarities with the trails in Fig 4, it is possible that these structures resulted from the flow forces of the washing step to remove unbound parasites in the protocol because the formation was independent of the metabolic conditions of amastigotes. Alternatively, they could be part of a different process because they were beaded in appearance (possibly due to microvesicles as in [59]) and were mainly studied in amastigotes since they were hardly detected in epimastigotes. A significant difference with respect to membrane tethers observed in other cells is that here tethers mediate strong attachment to a surface, whereas in most other systems this role is not observed, and in neutrophils they mediate transient attachment during rolling [39]. Moreover, the anchor points at the distal end of tethers in neutrophils also appear to be smaller than the anchoring pads observed here [39,60] indicating a different role of the nanotubules in neutrophils. Conclusions We showed shear stress can induce nanotubular projections from the flagellar membrane of T. cruzi epimastigotes after surface attachment via an anchoring pad on the surface. Those structures are mainly composed of membrane, as suggested from fluorescence staining, and by their mechanical properties such as extrusion force, curvature modulus, surface viscosity and a yielding force-extension curve. These membrane tethers can extend significantly, up to 100μm, and migrate along the flagellar membrane. Extension is reversible, unless the parasite can form additional attachments, in which case resistance to high fluid forces is greatly enhanced. Although further work is needed to improve our understanding of the biophysical and biochemical properties as well as the biological significance of the nanotubules in the life cycle of epimastigotes, these results suggest a function for membrane nanotubules in T. cruzi: to mediate adhesion under flow inside the microenvironment of the host. In the wider context of cell systems under flow, a novel function for membrane tethers is shown, that of strong adhesion under high shear stresses different from that of transient adhesion, as observed in neutrophils. Supporting information You can find the supporting information at https://doi.org/10.6084/m9.figshare.22178795. S1 Fig. Nanotubule elongation velocity vs force. The line is a fit using the model by Brochard-Wyart et al. [25] and results in an estimate of surface viscosity of the flagellar membrane of Trypanosoma cruzi at ~50 10−6 Pa∙s∙m, consistent with the properties of cell membranes from other types of cells. https://doi.org/10.1371/journal.pone.0283182.s001 (TIF) S2 Fig. Average length reached by 12 epimastigotes along a first cycle. Each epimastigote was subjected to a shear stress cycle of 0.31 –– 0.62–0.93–1.24 pN/um2; each pulse was 20 s long and followed by a 20 s pause. There was an initial pause 20 s long. The curve shows the average elongation of 12 different parasites. Note both elongation regimes: a fast extension and a creep regime. Differences between epimastigotes were mainly related to the maximum length reached at each shear stress. https://doi.org/10.1371/journal.pone.0283182.s002 (TIF) S3 Fig. Average length reached by 5 epimastigotes in two shear stress cycles. Each epimastigote was subjected to two shear stress cycles of 0.31 –– 0.62–0.93–1.24 pN/um2; each pulse was 20 s long and followed by a 20 s pause. There was a pause of 1 min between cycles. The filament length during this pause was averaged and set to 0, assuming this as the initial anchoring point for the second cycle. The curve shows the average elongation of five different parasites. There is a subtle reduction in maximum length reached in the second cycle. https://doi.org/10.1371/journal.pone.0283182.s003 (TIF) S4 Fig. Doubly tethered epimastigote. Illustrative multiply tethered epimastigote, observed at a shear stress of 1.55 pN/μm2. The anchoring point of the first adhered filament is marked with the black arrow. Note how far the filament extends without rupturing or detaching. The second filament emerged during the extension of the first one, its anchoring point is marked by a white arrow. The inset shows the parasite-end of the filaments (black arrow head for the first filament and white arrowheat for the second), note the diferent location of each one. https://doi.org/10.1371/journal.pone.0283182.s004 (TIF) S1 Table. Flow of PBS used, and its shear stress equivalences. https://doi.org/10.1371/journal.pone.0283182.s005 (XLSX) S1 Dataset. Extension data and calculations. https://doi.org/10.6084/m9.figshare.22178789.v1. https://doi.org/10.1371/journal.pone.0283182.s006 (XLSX) S1 Movie. Nanotubule extension affects flagellar beating. While subjected to a shear stress of 0.62 pN/μm2, a tether-like structure emerges from the flagellum of this surface-bound epimastigote and beating is significantly reduced. Then, when flow is stopped, the flagellum beating resumes while the filament is still attached. https://doi.org/10.6084/m9.figshare.22177418.v1. https://doi.org/10.1371/journal.pone.0283182.s007 (MP4) S2 Movie. Emergence of a secondary filament. An epimastigote exposed to a shear stress of 1.55 pN/μm2 is attached to the surface by a single filament. During the elongation of this filament, a new anchoring point is established, and a second filament emerges. https://doi.org/10.6084/m9.figshare.22177583.v1. https://doi.org/10.1371/journal.pone.0283182.s008 (MP4) S3 Movie. Different patterns of extension suggest nanotubule migration. An epimastigote is exposed to a shear stress of 0.93 pN/μm2. Then flow is stopped, and the parasite returns to its initial position where flagellar beating induces the rotation of the parasite. When flow is turned on again to a shear stress of 1.24 pN/μm2, it extends and acquires a "V" shape, with the vertex located near the flagellar pocket. This observation suggests that the nanotubule migrated near the flagellar pocket. https://doi.org/10.6084/m9.figshare.22177586.v1. https://doi.org/10.1371/journal.pone.0283182.s009 (MP4) Acknowledgments We acknowledge the technical assistance of the μ-Core, as well as Elizabeth Suesca for her support in the lab, and Jaime Castro for early experiments.
TI - Extensible membrane nanotubules mediate attachment of Trypanosoma cruzi epimastigotes under flow
JF - PLoS ONE
DO - 10.1371/journal.pone.0283182
DA - 2023-03-22
UR - https://www.deepdyve.com/lp/public-library-of-science-plos-journal/extensible-membrane-nanotubules-mediate-attachment-of-trypanosoma-xX2OlWE5Gb
SP - e0283182
VL - 18
IS - 3
DP - DeepDyve
ER -