A flagellate-to-amoeboid switch in the closest living relatives of animals
- Howard Hughes Medical Institute, United States
- Department of Molecular and Cell Biology, University of California, Berkeley, United States
- Department of Molecular Life Sciences, University of Zürich, Switzerland
- Department of Experimental and Health Sciences, Pompeu Fabra University (UPF), CIBERNED, Spain
Figures
Figure 1 with 4 supplements
Confinement induces an amoeboid phenotype in the choanoflagellate S. rosetta.
(A) Free-swimming cells (bottom left) were confined (bottom right) at a fixed height using confinement slides with micro-spacers (Liu et al., 2015; Le Berre et al., 2014) (top). (B) Confined S. …
Figure 1—figure supplement 1
Flagellar retraction and regeneration during transitions between the flagellate and amoeboid forms.
(A) Most (but not all) S. rosetta cells retracted their flagellum within 500 s of confinement at 2 μm. (B–D) The flagellum regenerated after release from confinement in approximately the same …
Figure 1—figure supplement 2
S. rosetta is competent to undergo the amoeboid switch in rosette and thecate forms.
(A) Schematic drawing of a rosette colony of S. rosetta (from Brunet and King, 2017). (B–E) Cells within rosettes became amoeboid under 2 μm confinement. (A) An unconfined rosette. (C and D) Time …
Figure 1—video 1
Time-lapse of a population of S. rosetta cells before and during 2 μm confinement under a confinement slide controlled by a dynamic cell confiner.
The strain used was SrEpac and the starting cell type was slow swimmer.
Figure 1—video 2
Time-lapse of a population of S. rosetta cells before, during and after 2 μm confinement under a confinement slide controlled by a dynamic cell confiner.
Cells were attached to the substrate by poly-d-lysine to minimize cell movement and help visualizing both conversion of flagellates into amoeboid cells, and reversion of amoeboid cells back into …
Figure 2 with 2 supplements
S. rosetta amoeboid cells generate blebs.
(A) Protrusions in eukaryotic crawling cells can either be F-actin-filled pseudopods that form by polymerization of F-actin (pink) reticulated by the Arp2/3 complex (purple, left) or F-actin-free …
Figure 2—figure supplement 1
Latrunculin B and blebbistatin, but not CK666, inhibited the formation of dynamic cellular protrusions under cell confinement.
Cellular phenotypes observed after pharmacological inhibitor treatments (as shown) under 1 μm confinement. White arrowheads indicate blebs.
Figure 2—figure supplement 2
Flow-through chamber for immunostaining of confined cells.
A flow-through chamber design was used to maintain confinement during immunostaining of amoeboid cells (see Materials and methods).
Figure 3
F-actin dynamics during the lifetime of a bleb.
Time lapse imaging of a LifeAct-mCherry-expressing live cell (Video 5) by DIC (left column) and fluorescence microscopy (middle column) revealed membrane dynamics and actin localization during bleb …
Figure 4 with 4 supplements
Myosin II undergoes intracellular redistribution in response to confinement.
(A) Top: MRLC-mTFP fluorescence in four unconfined flagellate S. rosetta cells from a chain colony (Dayel et al., 2011). The position of the microvilli (black arrowheads) can be inferred from weak …
Figure 4—figure supplement 1
Confinement results in redistribution of MRLC-mTFP from the cytoplasm to the cortex.
All panels are confocal images of MRLC-mTFP-expressing S. rosetta cells, unconfined (A) or confined with 2 μm (B) or 1 μm (C) microbeads. Cells were imaged as early as possible (less than a minute) …
Figure 4—figure supplement 2
Microtubules are present in S. rosetta amoeboid cells.
(A–D) Flagellate S. rosetta cells are characterized by a cage of cortical microtubules that underlie the entire plasma membrane, as previously reported (Karpov and Leadbeater, 1998; Leadbeater, 2015;…
Figure 4—figure supplement 3
The amoeboid switch is independent of calcium signaling.
(A–D) Representative micrographs of S. rosetta cells under 2 μm confinement under the following conditions: (A) in control conditions (AKSW 0.1% DMSO), (B) after intracellular calcium depletion …
Figure 4—video 1
Time-lapse of S. rosetta cells under 1 μm confinement expressing MRLC-mTFP (which marks myosin II) and imaged by epifluorescence microscopy.
Note the dynamic intracellular distribution of myosin II foci and fibers.
Figure 5 with 3 supplements
The amoeboid switch allows escape from confinement.
(A–D) Amoeboid cell crawling after flagellar retraction (Figure 1—video 1). White arrow indicates direction of movement. (E and F) Speed and directional persistence of the cells in Figure 1—video 1 …
Figure 5—figure supplement 1
Amoeboid cells elongate during escape from confinement and revert to a rounded shape after escape.
(A) Aspect ratio of a representative escaping cell before, during, and after escape (black line). The aspect ratio increased during escape and decreased after escape. Also depicted is the distance …
Figure 5—video 1
Time-lapse of an mTFP-expressing population of S. rosetta cells trapped in a 0.5 μm space under a circular micropillar.
Some of the most peripheral cells (<10 μm distant of the border) manage to cross the border into the unconfined space around the pillar.
Figure 5—video 2
Close-up of an escaping cell (from Figure 5—video 1) showing DIC channel (top left), mTFP channel (top right), segmented cell shape (bottom left), and segmented cell shape (magenta) overlaid with the DIC channel (gray).
The cell elongates and polarizes in the direction of the border during crossing and resumes a round shape after having crossed.
Figure 6 with 3 supplements
The last common choanozoan ancestor likely had amoeboid and flagellate life-history stages.
Figure 6—figure supplement 1
Phylogenetic distribution of crawling cells, epithelial cells, collar cells, and flagellated sperm cells in animals.
Shown is a phylogenetic tree (modified from Brunet and King, 2017) along with information about the presence or absence (see key) of relevant cell types and cell behaviors in diverse animal …
Figure 6—figure supplement 2
The amoeboid switch is not affected by transcription inhibition.
The fraction of blebbing cells was comparable in DMSO-treated controls (left, N = 42, 60, and 9 cells in three respective biological replicates) and in cells treated with an RNA-polymerase II …
Figure 6—video 1
Time-lapse of a 2 μm-confined choanoflagellate of the species Diaphanoeca grandis.
The cage surrounding the cell is called a ‘lorica’ and is a basket of silicon strips secreted and assembled by the cell. The cell is flattened but does not show blebbing or active deformation.
Videos
Video 1
Time-lapse of an S. rosetta cell undergoing progressive confinement by evaporation and switching to an amoeboid phenotype.
The strain used was SrEpac and the starting cell type was slow swimmer.
Video 2
Time-lapse of a population of S. rosetta cells confined between two glass cover slips using 2 μm microbeads as spacers.
The strain used was SrEpac and the starting cell type was slow swimmer.
Video 3
Time-lapse of a population of S. rosetta cells confined in a thinly spread liquid film under a layer of anti-evaporation oil (see Materials and methods).
The strain used was SrEpac and the starting cell type was slow swimmer.
Video 4
Time-lapse of a population of S. rosetta cells confined together with microbeads on the surface of a 1% agar gel in artificial seawater, under a layer of anti-evaporation oil.
The strain used was SrEpac and the starting cell type was slow swimmer.
Video 5
Time-lapse of two S. rosetta cells under 1 μm confinement expressing LifeAct-mCherry (which marks F-actin).
Blebs first form as cytoplasm-filled, F-actin-free protrusions and are re-invaded by F-actin before retraction.
Video 6
Time-lapse of an S. rosetta cell under 1 μm confinement expressing LifeAct-mCherry (which marks F-actin).
Blebs first form as cytoplasm-filled, F-actin-free protrusions and are re-invaded by F-actin before retraction.
Video 7
Time-lapse of an S. rosetta cell under 1 μm confinement expressing LifeAct-mCherry (which marks F-actin).
Blebs first form as cytoplasm-filled, F-actin-free protrusions and are re-invaded by F-actin before retraction.
Video 8
Time-lapse of S. rosetta cells under 1 μm confinement expressing MRLC-mTFP (which marks myosin II) and imaged by confocal microscopy.
Note the dynamic intracellular distribution of myosin II foci and fibers. Large fluorescent dots in the mTFP channel are autofluorescent food vacuoles previously described in S. rosetta (Wetzel et …
Video 9
Time-lapse of an S. rosetta amoeboflagellate cell under 1 μm confinement expressing MRLC-mTFP (which marks myosin II) and imaged by epifluorescence microscopy.
Note that expanding blebs are devoid of myosin II and are re-invaded by myosin II before retraction, similar to F-actin.
Video 10
Time-lapse of a crawling S. rosetta cell transfected with LifeAct-mCherry and septin2-mTFP.
Note dynamic distribution of F-actin within the leading bleb (Figure 5G–H).
Video 11
Time-lapse of an escaping cell (from a similar experiment to the one shown in Figure 5—video 1) shedding two blebs into the unconfined space prior to escaping.
One of the blebs is then reabsorbed by the cell during escape from confinement.
Video 12
Time-lapse of a population of Monosiga brevicollis confined in a thin liquid film, displaying intense blebbing and crawling (or gliding) motility.
Video 13
Time-lapse of a 2 μm confined Acanthoeca spectabilis showing dynamic bleb extension.
Video 14
Time-lapse of four Salpingoeca helianthica cells, 2 μm confined, displaying long dynamic blebs.
Video 15
Time-lapse of a 2 μm confined S. urceolata cell crawling out of its theca.
The cell crawls over about 40 μm, shedding cellular material at its rear end (possibly similar to the shedding of blebs by S. rosetta; Video 11).
Video 16
Automated recognition of blebs in 2 μm-confined S. rosetta cells.
Left panel: DIC channel. Middle panel: result of the cell segmentation. Right panel: cell protrusions, classified into expanding blebs (orange) and retracting blebs (blue).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Salpingoeca rosetta) | Regulatory myosin light chain short version (PTSG_00375) | NA | NCBI XM_004998867.1 | |
| Strain, strain background (Salpingoeca rosetta) | S. rosetta | PMID:24139741 | ATCC PRA-390; accession number SRX365844 | |
| Strain, strain background (Algoriphagus machipongonensis) | A. machipongonensis | PMID:22368173 | ATCC BAA-2233 | |
| Strain, strain background (Echinicola pacifica) | E. pacifica | PMID:16627637 | DSM 19836 | |
| Transfected construct (S. rosetta) | pEFl5’-Actin3’::pac-P2A-mTFP | Wetzel et al., 2018 | Addgene ID NK676 | |
| Transfected construct (S. rosetta) | pEFL5'-Actin3'::pac, pActin5'-EFL3'::mCherry | This paper | Addgene ID NK802 | |
| Transfected construct (S. rosetta) | pEFL5'-Actin3'::pac, pActin5'-EFL3'::LifeAct-mCherry | This paper | Addgene ID NK803 | |
| Transfected construct (S. rosetta) | pEFL5'-Actin3'::pac, pActin5'-EFL3'::MRLC-mTFP | This paper | Addgene ID NK804 |
Additional files
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Supplementary file 1
Crawling cells across animal diversity.
- https://cdn.elifesciences.org/articles/61037/elife-61037-supp1-v2.docx
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Supplementary file 2
Pharmacological compounds used in inhibitor assays.
- https://cdn.elifesciences.org/articles/61037/elife-61037-supp2-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/61037/elife-61037-transrepform-v2.pdf
