Mar 14, 2023
Version 2
Choanoflagellate Ciliogenesis Live Imaging V.2
- 1University of California Berkeley
Protocol Citation: Maxwell C Coyle 2023. Choanoflagellate Ciliogenesis Live Imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7y9n3gwz/v2Version created by Maxwell C Coyle
Manuscript citation:
Coyle, M. C. et al. An RFX transcription factor regulated ciliogenesis in the progenitors of choanoflagellates and animals. bioRxiv 2022.11.11.515474 (2022) doi:10.1101/2022.11.11.515474
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
This protocol works well, although de-ciliation efficiency is in the 80-90% range, not 100. Also you may need to adjust the timing of the -20C step depending on the exact temp and heat exchange of your freezer. The goal is to go as long as possible before the solution freezes.
Created: March 05, 2023
Last Modified: March 14, 2023
Protocol Integer ID: 78159
Funders Acknowledgement:
HHMI
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Abstract
This protocol removes the cilia/flagella from choanoflagellate cells and sets up the cells for live imaging of ciliogenesis. It has been developed for the species Salpingoeca rosetta, but may be portable into other choanoflagellate species. Cells begin to re-generate their cilia/flagella right after removal. The idea of using a cold shock with glycerol for ciliary removal came from Brokaw et al 1960 (doi: 10.1016/0014-4827(60)90027-6).
Materials
High Nutrient Media: 4% AKCGM3 + 4% AKSWC in AKSW - Artificial Known Sea Water (See Booth 2018 Molecular Biology of the Cell for sea water details).
AKSW without addeed supplements
Incubator
Tabletop centrifuge
75cm2 vented flasks
Haemocytomer or automated cell counter
16% Paraformaldehyde
0.1 mg/ml Poly-D-lysine
Forceps
Surface corona treater
Fluorodishes
Circular (22 mm diameter) coverslips
70% ethanol
50% glycerol
-20C freezer for incubation
Widefield microscope
Protocol materials
FluorodishWorld Precision InstrumentsCatalog #FD35-100
Step 4
Poly-D-lysine hydrobromideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6407-5MG
Step 5
Concentrate cells and remove cilia
Concentrate cells and remove cilia
Grow choanoflagellate cells (Salpingoeca rosetta fed with Echinicola pacifica, ATCC PRA-390) in High Nutrient Media to a density of 1-2 x 106 cells/ml. Grow at 22 ºC, 60% humidity
We grow 30 ml of culture in 75 cm2 vented flask. Typically, inoculating this flask with a choanoflagellate cell density of 8,000 cells/ml 48 hours before ciliogenesis works well.
Count cells by haemacytomer or automated cell counter*. Shake culture flask vigorously to homogenize cell populatin and thene mix 99 µl of cell culture with 1 µl of 16% paraformaldehyde to fix cells for counting. Typically 10 µl of fixed cells can be loaded into a haemocytometer or automateed cell counting slide.
*We use LUNA-FL Automated Fluorescence Cell Counter (Logos Biosystems L20001)
5m
Aliquot and pellet 6 x 106 cells 2000 x g, 00:10:00
10m
Corona treat a fluorodish 5-10 secondsFluorodishWorld Precision InstrumentsCatalog #FD35-100
Equipment
BD-20AC Laboratory Corona Treater
NAME
Corona Treateer
TYPE
Electro-Technic Products
BRAND
12051A
SKU
LINK
1m
Rinse fluorodish for 5 seconds with 1 ml of 0.1 mg/ml poly-D-lysine, followed by 3x washes with 1 ml water. Dry by air or by capillary action of kimwipe, being careful to minimize contact with surface of imaging dish.
Poly-D-lysine hydrobromideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6407-5MG
2m
Rinse a coverslip (circular - 22mm diameter) in 70% EtOH followed by water and then lay on kimwipe to dry. Easiest to hold coverslip by forceps and dunk into 50 ml conical tubes with the ethanol or water.
1m
When cells are done pelleting, remove supernatant and resuspend cell pellet in 800 µl of AKSW and transfer to 1.5 ml eppendorf tube.
1m
Add 200 µl of 50% glycerol (final concentration: 10% glycerol) and mix by pipetting
1m
Add cells to a second Fluorodish (i.e. one not treated with poly-D-lysine) and incubate-20 °C 7 mins
Standard laboratory freezer is fine, but depending on exact temperature of your freezer or where in the freezer you place the cells, you may need to adjust the timing.
8m
Set up cells for live imaging of ciliogenesis
Set up cells for live imaging of ciliogenesis
13m
13m
Transfer cells to 1.5 ml eppendorf tube and pellet 4200 x g, 00:08:00
8m
Remove supernatant and resuspend cells in 25 µl of AKSW
1m
Transfer cells to Fluorodish coated with poly-D-lysine and lay clean coverslip slowly on top using forceps
1m
Mount dish on microscope* and find focus. Let cells settle for 1 minute.
*We use a Zeiss Axio Observer.Z1/7 widefield with a 100x NA 1.40 Plan-Apochromatic oil immersion objective and a Hamamatsu Orca-Flash 4.0 LT CMOS digital camera
2m
Float coverslip off of cells by adding AKSW around the side of the coverslip drop by drop with a plastic transfer pipette. If you don't do this, the cells will eventually suffocate.
1m
Image!
On our system we use a short (5 ms exposure) with high light intensity (12.2 V) and a DIC condenser to get the best imaging of ciliogenesis. We use Zeiss Definite Focus and take a 10 µm z-stack with 1 µm between slices every 30 seconds for one hour.
Protocol references
Brokaw, C. J. Decreased adenosine triphosphatase acivity of flagella from a paralyzed mutant of Chlamydomonas moewusii. Exp. Cell Res.19, 430–432 (1960)
Booth, D. S., Szmidt-Middleton, H. & King, N. Transfection of choanoflagellates illuminates their cell biology and the ancestry of animal septins. Mol. Biol. Cell29, 3026–3038 (2018)