Formation and three-dimensional architecture of Leishmania adhesion in the sand fly vector
- Department of Biological and Medical Sciences, Oxford Brookes University, United Kingdom
- Department of Parasitology, Charles University, Czech Republic
- Research Center of Mathematics for Social Creativity, Research Institute for Electronic Science, Hokkaido University, Japan
- Laboratory for Spatiotemporal Regulations, National Institute for Basic Biology, Japan
- Spatiotemporal Regulations Group, Exploratory Research Center for Life and Living Systems, Japan
- Department of Basic Biology, School of Life Science, Japan
Figures
Figure 1 with 7 supplements
Haptomonads form a dense, multi-layered complex on the sand fly stomodeal valve.
(A) Cartoon of the sand fly digestive tract highlighting the position of the stomodeal valve, the site of differentiation of free-swimming Leishmania promastigotes into haptomonads, which attach to …
Figure 1—figure supplement 1
Cell cycle analysis of haptomonads at the sand fly stomodeal valve.
(A) Mapping of an infected stomodeal valve showing the position of the nuclei of different attached cells, colour code according to the cell’s division status (Figure 1—video 4). Whole view (left) …
Figure 1—figure supplement 2
Haptomonads detached from the stomodeal valve.
(A) Sequential SBFSEM images (every 100 nm; left) and 3D reconstruction (right) showing a haptomonad flagellum with part of the attachment plaque detached from the stomodeal valve. (B) Sequential …
Figure 1—figure supplement 3
Damage to the stomodeal valve at points of haptomonad attachment.
Top left: lower magnification view showing a SBFSEM cross-section of the stomodeal valve. Bottom left: magnified view of the stomodeal valve surface at the area where no haptomonads were attached …
Figure 1—video 1
SBFSEM imaging of haptomonads on the stomodeal valve in the sand fly (whole view).
3D volume taken in 400 cycles of 100 nm cuttings. Scale bar = 10 µm.
Figure 1—video 2
SBFSEM imaging of haptomonads on the stomodeal valve in the sand fly (enlarged view).
Scale bar = 5 µm.
Figure 1—video 3
3D model generated from the SBFSEM imaging shown in Figure 1—video 2.
Scale bar = 5 µm.
Figure 1—video 4
3D model of mapping of an infected stomodeal valve showing the position of the nuclei of different attached cells.
Different coloured spheres indicate nuclei of cells which have one nucleus and one flagellum (1N1F; blue), nuclei of cells which have one or two nucleus and two flagella (1N2F or 2N2F; magenta), and …
Figure 2 with 3 supplements
The modified haptomonad flagellum has a highly organised ultrastructure, with multiple discrete elements.
Different serial tomograms of haptomonads in the sand fly, focusing on the attachment region (Figure 2—video 1). (A–C) 3D reconstruction (Figure 2—video 2; A) and slices (B, C) from the serial …
Figure 2—figure supplement 1
Tip structure of the in vivo haptomonad and in vitro haptomonad-like cell axoneme.
(A) Sequential tomographic slices (every 10 nm) showing the tip structure of the haptomonad axoneme. The order of the slices is indicated at top left of each image. White arrowheads: ring-shaped …
Figure 2—video 1
Serial section electron tomography of a haptomonad on the stomodeal valve in the sand fly.
3D volume reconstructed by joining tomograms from a series of six 150-nm-thick sections. Scale bar = 500 nm.
Figure 2—video 2
3D model generated from the serial tomogram shown in Figure 2—video 1.
Scale bar = 500 nm.
Figure 3 with 6 supplements
In vitro haptomonad-like cells resemble haptomonads observed in the sand fly.
(A) Scanning electron microscopy images of in vitro haptomonad-like cells on a plastic coverslip. Left: Low-magnification image showing groups of cells (yellow arrowheads) attached to the substrate. …
Figure 3—figure supplement 1
Cell cycle analysis of in vitro haptomonad-like cells.
(A) Mapping of nuclei of in vitro haptomonad-like cells (Figure 3—video 2). Left: whole view. Right: magnified view. Different coloured spheres indicate nuclei of cells which have one nucleus and …
Figure 3—figure supplement 2
Sand fly haptomonads and in vitro haptomonad-like cells are surrounded by an extracellular filamentous gel-like matrix.
SBFSEM slices of sand fly (top) and in vitro (bottom) cells showing attached cells and unattached promastigotes (asterisk) surrounded by a filamentous matrix (arrowheads).
Figure 3—video 1
SBFSEM imaging of in vitro haptomonad-like cells attached to plastic.
3D volume taken in 252 cycles of 100 nm cutting. Scale bar = 10 µm.
Figure 3—video 2
3D model of mapping of nuclei of in vitro haptomonad-like cells.
Different coloured spheres indicate nuclei of cells which have one nucleus and one flagellum (1N1F; blue), nuclei of cells which have one or two nucleus and two flagella (1N2F or 2N2F; magenta), and …
Figure 3—video 3
Serial section electron tomography imaging of an in vitro haptomonad-like cell attached to plastic.
3D volume reconstructed by joining tomograms from a series of six 150-nm-thick sections. Scale bar = 500 nm.
Figure 3—video 4
3D model generated from the serial tomogram shown in Figure 3—video 3.
Scale bar = 500 nm.
Figure 4 with 6 supplements
Adhesion of the in vitro haptomonad-like cell has a series of defined sequential steps.
(A) Sequential frames (at 60 min intervals) from a time-lapse video (Figure 4—video 1) of adhesion of a haptomonad-like cell, as viewed by bright field microscopy. The numbers and arrows above the …
Figure 4—figure supplement 1
Leishmania explores the surface while releasing membrane streamers from the flagellum.
(A) Sequential frames from a time-lapse video (Figure 4—video 2) of the adhesion of an in vitro haptomonad-like cell (at 2 min intervals), showing membrane streamers from the flagellum (white …
Figure 4—figure supplement 2
Measurement of mNeonGreen::PFR2 and mCherry::RSP4/6 length in in vitro haptomonad-like cells.
Scatter plot of mCh::RSP4/6 signal length against mNG::PFR2 signal length. The length of the mNG::PFR2 signal of cells whose mNG signal is not visible is indicated as zero, and these points are …
Figure 4—figure supplement 3
In vitro purified metacyclics do not adhere to glass.
(A) Example images of log phase promastigotes and in vitro purified metacyclics. Note: the metacyclics have a flagellum that is longer than the cell body and a short slender cell body. (B) …
Figure 4—video 1
Time-lapse video of adhesion process of an in vitro haptomonad-like cell on a glass coverslip.
Playback of ~9 hr at 1000 x speed. Scale bar = 10 µm.
Figure 4—video 2
Time-lapse video of release of membrane streamers from the flagellum of an in vitro haptomonad-like cell on a glass coverslip.
Playback of ~8 min at 30 x speed. Scale bar = 5 µm.
Figure 4—video 3
Time-lapse video of reversible adhesion of an in vitro haptomonad-like cell on a glass coverslip.
Playback of 1.5 hr at 500 x speed. Scale bar = 10 µm.
Figure 5
Calcium is necessary for adhesion of in vitro haptomonad-like cells.
(A) Growth curve of cells cultured in control M199 growth medium or M199 with 1 or 2 mM EGTA. Data represent mean ± SD (n=3 independent experiments). (B–D) In vitro analysis of adhesion in the …
Figure 6
Models of the haptomonad attachment plaque and adhesion process.
(A) Model of the haptomonad attachment plaque. (B) Model of the haptomonad adhesion process based on the in vitro observations. A promastigote cell explores the surface of the substrate using the …
Author response image 1
Infection rates and intensities of infections on days 6 and 9 post-infection (PI).
Numbers above the bars indicate the number of dissected females. Levels of Leishmania infections were graded into four categories: negative, light (< 100 parasites/gut), moderate (100-1000 …
Author response image 2
Localization of infections on days 6 and 9 post-infection (PI).
Abdominal midgut (AMG), thoracic midgut (TMG) and stomodeal valve (SV). Numbers above the bars indicate the number of evaluated (positive) females.
Author response image 3
L.
mexicana haptomonads attached to the stomodeal valve (SV) Day 9 PI. A, B Light and fluorescence microscopy of infected sand flies fed with sugar. C, D Light and fluorescence microscopy of infected …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Leishmania mexicana) | RSP4/6 | TriTrypDB (http://tritrypdb.org/tritrypdb/) | LmxM.13.0430 | |
| Gene (Leishmania mexicana) | PFR2 | TriTrypDB (http://tritrypdb.org/tritrypdb/) | LmxM.16.1430 | |
| Strain, strain background (Leishmania mexicana) | L. mexicana | Sunter lab stocks | WHO strain MNYC/BZ/1962 /M379 | The identity has been authenticated by genome and mRNA sequencing. The strain was monitored for contamination, including mycoplasma contamination, through DNA staining and microscopy during data capture. |
| Cell line (L. mexicana) | mCherry::RSP4/6, mNeonGreen::PFR2 | Wang et al., 2021 L. mexicana cell line provided by Dr Richard Wheeler (University of Oxford) | NA | The identity has been authenticated by genome sequencing. The cell line was monitored for contamination, including mycoplasma contamination, through DNA staining and microscopy during data capture. |
| Strain, strain background (Lutzomyia longipalpis) | L. longipalpis | Volf and Volfova, 2011 | NA | |
| Software, algorithm | 3dmod | Kremer et al., 1996 | PMID:8742726 | |
| Software, algorithm | IMOD | http://bio3d.colorado.edu/imod | RRID:SCR_003297 | |
| Software, algorithm | SerialEM | Mastronarde, 2005 | PMID:16182563 | |
| Software, algorithm | Fiji | Schindelin et al., 2012 | RRID: SCR_002285 | |
| Software, algorithm | Excel | https://microsoft.com/mac/excel | RRID:SCR_016137 | |
| Software, algorithm | python | https://www.python.org/ | RRID:SCR_008394 | |
| Software, algorithm | matplotlib | Hunter, 2007 | RRID:SCR_008624 | |
| Other | Gridded glass coverslips grid-500 | iBidi | Cat. #: 10816 | In vitro haptomonad-like cell preparation |
| Other | Thermanox plastic coverslips | Nalgene Nunc International | Cat. #: 174950 | In vitro haptomonad-like cell preparation |
| Other | µ-dish 35 mm, high grid-500 glass bottom | iBidi | Cat. #: 81168 | In vitro haptomonad-like cell preparation |
