citations

                    Choanoflagellate transfection illuminates their cell biology and the ancestry of animal septins.
                

Authors:

Booth DS, Szmidt-Middleton H, King N.

In:

Source: Mol Biol Cell
Publication Date: (2018)
Issue: 29(25): 3026-3038

Research Area:

Parasitology

Gene Expression

Platform:

4D-Nucleofector® 96-well Systems

4D-Nucleofector® X-Unit

Experiment

Prime: After bacteria were washed away, each aliquot of S. rosetta cells was incubated in priming buffer to remove the extracellular material coating the cell. The 100-µl aliquots, which contained 5 ? 106 cells, were centrifuged for 5 min at 800 ? g and at room temperature. The supernatant was removed with a fine-tip micropipette. Cells were resuspended in 100 µl of priming buffer (40 mM HEPES-KOH, pH 7.5; 34 mM lithium citrate; 50 mM l-cysteine; 15% [wt/vol] PEG 8000; and 1 µM papain [Millipore Sigma, St. Louis, MO; Cat. No. P3125-100MG]) and then incubated for 30 min. Priming was quenched by adding 2 µl of 50-mg/ml bovine serum albumin-fraction V (Thermo Fisher Scientific, Waltham, MA; Cat. No. BP1600-100) and then centrifuged for 5 min at 1250 ?g and 22°C with the centrifuge brake set to a “soft” setting. The supernatant was removed with a fine-tip micropipette, and the cells were resuspended in 25 µl of SF Buffer (Lonza, Basel, Switzerland; Cat. No. V4SC-2960).

Transfection: Each transfection reaction was prepared by adding 2 µl of “primed” cells resuspended in SF buffer to a mixture of 14 µl of SF buffer; 2 µl of 20 µg/µl pUC19; 1 µl of 250 mM ATP, pH 7.5; 1 µl of 100 mg/ml sodium heparin; and =7 µl of reporter DNA. (Note that higher volumes of nucleofection lead to lower transfection frequencies; thus, reporter DNA should be as concentrated as possible, not exceeding 7 µl. Also, see Note about titrating reporter plasmids.) The transfection reaction was transferred to one well of a 96-well nucleofection plate (Lonza; Cat. No. V4SC-2960) or a 16-well strip (Lonza; Cat. No. V4XC-2032). The nucleofection plate was placed in a 96-well shuttle device (Lonza; Cat. No. AAM-1001S) or X-unit (Lonza; Cat. No. AAF-1002F) connected to a Nucleofector 4D core unit (Lonza; Cat. No. AAF-1002B), and the CM156 pulse was applied to each well.

Rest and recovery. Immediately after pulsation, 100 µl of ice-cold recovery buffer (10 mM HEPES-KOH, pH 7.5; 0.9 M sorbitol; 8% [wt/vol] PEG 8000) was added to the cells. Recovery buffer was gently mixed with the transfected cells by firmly tapping the side of the plate and then incubating the samples for 5 min. The whole volume of the transfection reaction plus the recovery buffer was transferred to 1 ml of 1× High Nutrient Medium in a 12-well plate

Abstract

As the closest living relatives of animals, choanoflagellates offer unique insights into animal origins and core mechanisms underlying animal cell biology. However, unlike traditional model organisms, such as yeast, flies and worms, choanoflagellates have been refractory to DNA delivery methods for expressing foreign genes. Here we report the establishment of a robust method for expressing transgenes in the choanoflagellate Salpingoeca rosetta, overcoming barriers that have previously hampered DNA delivery and expression. To demonstrate how this method accelerates the study of S. rosetta cell biology, we engineered a panel of fluorescent protein markers that illuminate key features of choanoflagellate cells. We then investigated the localization of choanoflagellate septins, a family of GTP-binding cytoskeletal proteins that are hypothesized to regulate multicellular rosette development in S. rosetta. Fluorescently tagged septins localized to the basal pole of S. rosetta single cells and rosettes in a pattern resembling septin localization in animal epithelia. The establishment of transfection in S. rosetta and its application to the study of septins represent critical advances in the growth of S. rosetta as an experimental model for investigating choanoflagellate cell biology, core mechanisms underlying animal cell biology, and the origin of animals.

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