Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

Summary

The social amoeba Dictyostelium discoideum undergoes a developmental transition into a multicellular organism when starved. The evolutionary conserved protein coronin A plays a crucial role in the initiation of development. Using aggregation assays as our main method, we aim to elucidate the role of coronin A in early development.

Abstract

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular development program, during which single cells chemotax towards aggregation centers^1-4. This process is dependent on the release of cyclic adenosine monophosphate (cAMP)⁵. cAMP is produced in waves through the concerted action of adenylate cyclase and phosphodiesterases, and binds to G protein-coupled cAMP receptors^6,7. A widely used assay to analyze the mechanisms involved in the developmental cycle of the lower eukaryote Dictyostelium discoideum is based on the observation of cell aggregation in submerged conditions^8,9. This protocol describes the analysis of the role of coronin A in the developmental cycle by starvation in tissue-culture plates submerged in balanced salt solution (BSS)¹⁰. Coronin A is a member of the widely conserved protein family of coronins that have been implicated in a wide variety of activities^11,12. Dictyostelium cells lacking coronin A are unable to form multicellular aggregates, and this defect can be rescued by supplying pulses of cAMP, suggesting that coronin A acts upstream of the cAMP cascade¹⁰. The techniques described in these studies provide robust tools to investigate functions of proteins during the initial stages of the developmental cycle of Dictyostelium discoideum upstream of the cAMP cascade. Therefore, utilizing this aggregation assay may allow the further study of coronin A function and advance our understanding of coronin biology.

Introduction

The coronin family of proteins is highly conserved throughout eukaryotes. These proteins are characterized by the presence of an amino-terminal tryptophan-aspartate (WD) repeat-containing region followed by a unique region connected to a carboxy-terminal coiled-coil domain^13,14 (Figure 1). Coronins have been implicated in a variety of cellular functions, including cytoskeletal regulation and signal transduction¹². In mammals, up to six short coronin molecules (coronin 1-6) as well as a 'tandem' coronin 7, can be co-expressed^12,15. Coronin 1 is the most extensively studied family member, and was shown to be involved in pathogen destruction, T cell survival and neuronal signaling. How, exactly, coronin 1 carries out these activities remains unclear. While coronin 1 was shown to regulate Ca²⁺ and cAMP-dependent signaling as well as F-actin cytoskeleton modulation ^16-18, the potential co-expression of up to 7 family members in mammals has made it challenging to study the molecular function of coronins in these systems, due to potential redundancies. Unlike mammalian organisms, the lower eukaryote Dictyostelium discoideum expresses only two coronin family members (coronin A, the ortholog of mammalian coronin 1 and coronin B, the ortholog of mammalian coronin 7) with apparently non-redundant functions^15,19,20. This fact makes Dictyostelium discoideum a potent model to study the function of coronins.

To study the role of coronin A in Dictyostelium discoideum, we induced the developmental cycle by starvation in tissue-culture plates containing balanced salt solution (BSS) buffer using either wild type cells or cells lacking coronin A¹⁰. We found that cells lacking coronin A were unable to form multicellular aggregates upon starvation. For an accurate quantitative assessment of this phenotype the automated live cell imaging described in this protocol is a vital tool. The defect in the initiation of the early starvation response in cells lacking coronin A can be rescued by supplying pulses of cAMP, suggesting that coronin A acts upstream of the cAMP cascade. The exogenous application of cAMP pulses to simulate the initiation of development has been utilized by several laboratories in the past^8,9. However, this procedure is also known to be highly dependent on cell densities and timing. Therefore, the protocol described here aims to reduce these variabilities in order to guarantee a high degree of reproducibility. Taken together, the techniques utilized in these studies provide robust tools to investigate functions of proteins during early stages of the developmental cycle of Dictyostelium discoideum and have the potential to identify up- as well as downstream effectors of coronin A function.

Protocol

1. Observe the early starvation response of Dictyostelium discoideum by time-lapse microscopy.
   1. Grow DH1.10 cells or corA-deficient cells in an Erlenmayer flask containing HL-5 medium (for 1 L: 5 g proteose peptone, 5 g thiotone E peptone, 10 g glucose, 5 g yeast extract, 0.35 g Na₂HPO₄*7H₂O, 0.35 g KH₂PO₄, 0.05 g dihydrostreptomycin-sulfate, pH 6.6) at 22 °C in a shaking incubator with a rotation of 160 rpm. Keep cells at a density between 0.01 x 10⁶ cells/ml and 2 x 10⁶ cells/ml.
   2. Examine cell aggregation, by harvesting log-phase-growing DH1.10 cells²¹ or corA-deficient cells¹⁰ generated in the DH1.10 background, grown in shaking culture with HL-5 medium at 22 °C. To do so, take appropriate amount of cells (usually between 10 and 50 ml), centrifuge for 3 min at 400 x g and wash the cells twice in BSS (10 mM NaCl, 10 mM KCl, 2.5 mM CaCl₂, pH 6.5).
   3. Count cells using a hemocytometer. Subsequently, plate cells at a density of (5, 10, 20, or 40) x 10⁴ cells/cm² in a 24-well plate. Allow them to adhere for 1 hr at 22 °C in BSS.
   4. Visualize aggregation by time-lapse microscopy as described before¹⁰, taking images every 135 sec. using a live cell imaging set up equipped with 5X objective and an electron-multiplying charge-coupled device camera automated by the appropriate software (see Materials Table).
2. cAMP pulsing of Dictyostelium discoideum cells during starvation.
   1. Examine the effect of externally applied cAMP pulses on the development of DH1.10 cells²¹ or DH1.10 corA-deficient cells¹⁰, by harvesting cells. To do so, take appropriate amount of cells (usually between 10 and 50 ml), centrifuge for 3 min at 400 x g and wash twice in BSS.
   2. Count cells using a hemocytometer and resuspend the cells to a density of 1 x 10⁷ cells/ml in BSS. Shake the cultures (160 rpm) at 22 °C for 2 hr before applying pulses. Add cAMP pulses using a timer controlled peristaltic pump. Program the pump to deliver a 5 sec pulse every 6.5 min of 15 µl of 50 nM cAMP (final concentration) over a period of 5 hr.
   3. Count cells using a hemocytometer. Subsequently, plate the cells at a density of (5, 10, 20, or 40) x 10⁴ cells/cm² in a 24-well plate. Allow to adhere for 1 hr in BSS.
   4. Visualize aggregation after 16 hr at 22 °C by bright-field microscopy using a 5X objective.
3. Induction of Dictyostelium discoideum development through exposure to conditioned medium.
   1. Prepare fresh conditioned medium as described²². Collect log-phase DH1.10 cells²¹ or DH1.10 corA-deficient cells¹⁰, from shaking cultures with HL-5 medium at 22 °C using a pipette, centrifuge for 3 min at 400 x g and wash cells three times in PBM (0.02 M potassium phosphate, 10 µM CaCl₂, and 1 mM MgCl₂, pH 6.1).
   2. Count cells using a hemocytometer, resuspend these in PBM at a density of 1 x 10⁷ cells/ml and shake for 20 hr at 110 rpm/22 °C.
   3. Collect conditioned medium after centrifugation at 400 × g for 3 min and clarify by centrifugation at 8,000 × g for 15 min at 4°C.
   4. Filter conditioned medium through a 0.45 µm filter (see Materials Table) and dilute threefold in PBM.
   5. Count cells using a hemocytometer and plate at a density of (5, 10, 20, or 40) x 10⁴ cells/cm² in a 24-well plate. Allow them to adhere for 1 hr at 22 °C.
   6. Exchange supernatant of the cells with the previously prepared conditioned medium.
   7. Visualize aggregation after 16 hr by bright-field microscopy using a 5X objective.

Results

Cells deficient in coronin A show a defect in early development (Figure 2). In the absence of coronin A cells are unable to form multicellular aggregates, which is the initial step during the developmental cycle of Dictyostelium discoideum. Therefore, coronin A appears to play a role during the early starvation response and/or cAMP signaling. Indeed, the lack of multicellular aggregate formation in the absence of coronin A is accompanied by reduced cAMP signaling...

Discussion

The coronin proteins are found in most taxa of the eukaryotic clade. Dictyostelium discoideum coronin A, the homologue of mammalian coronin 1, is involved in the early starvation response, since coronin A-deficient cells are not able to form aggregation centers during the early developmental cycle¹⁰. To be able to quantitatively and accurately assess the delay in development between the strains, a microscope live cell imaging set-up with the automated stage controller is an essential tool.

Materials

Name	Company	Catalog Number	Comments
HL-5 media (for 1 L: 5 g proteose peptone, 5 g thiotone E peptone, 10 g glucose, 5 g yeast extract, 0.35 g Na2HPO4*2H2O, 0.35 g KH2PO4, 0.05 g dihydrostreptomycin-sulfate, pH 6.6)
Proteose peptone	BD Bioscience	211693
Thiotone E peptone	BD Bioscience	211684
Yeast extract	BD Bioscience	212750
Glucose	AppliChem	A3666
Na2HPO4*2H2O	Fluka	71643
KH2PO4	AppliChem	A1043
dihydrostreptomycin-sulfate	Sigma-Aldrich	D1954000
PBM (0.02 M potassium phosphate, 10 μM CaCl2, and l mM MgCl2, pH 6.1)			self made
BSS (10 mM NaCl, 10 mM KCl, 2.5 mM CaCl2, pH 6.5)			self made
0.45-μm Filtropure S filter	Sarstedt	83.1826
Falcon 24-well Tissue culture plate	Fisher Scientific	08-772-1H
Cellobserver microscope	Zeiss		custom built
AxioVision software	Zeiss
IPC Microprocessor–controlled dispensing pump	ISMATEC	ISM 931
Axiovert 135M microscope	Zeiss	491237-0001-000
Incubation Shaker	Inforst HT Minitron