Protocol Dictyostelium Fixation: Two Simple Tricks

Michael Koonce 1,*, Irina Tikhonenko 1 and Ralph Gräf 2

1 Division of Translational Medicine, Wadsworth Center, NYS Department of Health, Albany, NY 12237, USA; [email protected] 2 Department of , University of Potsdam, 14476 Potsdam-Golm, Germany; [email protected] * Correspondence: [email protected]; Tel.: +1-518-486-1490

 Received: 16 June 2020; Accepted: 1 July 2020; Published: 1 July 2020 

Abstract: We share two simple modifications to enhance the fixation and imaging of relatively small, motile, and rounded model cells. These include cell centrifugation and the addition of trace amounts of to existing fixation methods. Though they need to be carefully considered in each context, they have been useful to our studies of the spatial relationships of the microtubule cytoskeletal system.

Keywords: Dictyostelium; cell fixation; ; microtubule; cytoskeleton

1. Introduction The striking mobility of Dictyostelium makes for an interesting and impactful model system to study dynamic cellular events [1]. Directed cell movement, rapid shape changes, and internal reorganization in response to extracellular cues are frequently accentuated here over more sedentary cell types and thus provide novel access to their study and understanding. However, one of the tradeoffs in a dynamic lifestyle is that these cells can be less tightly adherent to their substrate, and thus imaging and downstream structural analyses can present challenges that must be carefully considered. For many years, we have tinkered with fixation and conditions in order to image cytoskeletal arrays and GFP-tagged in D. discoideum [2–6]. Likely not alone, we have been challenged with difficulties in getting cells to sufficiently flatten to facilitate imaging and to remain attached to coverslips for downstream structural processing. In this brief report, we would like to share two insights that make a difference in our work, in the hopes that they benefit others as well.

2. Results

2.1. Cell Flattening

In the early 19800s, Fukui and colleagues developed an agar overlay procedure for D. discoideum that utilizes thin sheets of agarose to flatten cells and significantly improves some aspects of cellular imaging [7]. This is now a widely used method that gently restricts the experimental depth of field to around 1–3 µm. Cells under agarose can undergo division and remain viable for hours in sealed chambers. This overlay remains our preferred method for live cell imaging, enabling us to follow individual microtubule (MT) motions in a minimal number of focal planes [8,9]. However, the procedure is a bit cumbersome to use for routine cell fixation, especially for multiple coverslips. The agar sheet takes some care to set up, only covers a small portion of the coverslip, and takes slightly longer for the fixative to penetrate to the cells, and cell loss (at least in our hands) can be significant upon sheet removal for downstream cell processing. With any perturbation, there are caveats to consider when flattening cells for long periods, and thus multiple strategies can be useful to distinguish between genuine and imposed phenotypes.

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As such an alternative, we have begun to spin cells directly onto coverslips. Five minutes at 400 g in a standard tabletop centrifuge not only promotes attachment but also cell spreading. A small volume of cell suspension is added to buffer overlying a coverslip in a centrifuge tube (Figure 1) and spun for five minutes. The coverslip is then lifted out and gently slid into a dish containing fixative. This approach eliminates the waiting period for cell settling; agarose is not required, and cells labeled with a tubulin antibody [10] appear very similar to those prepared by agar overlay (Figure 2). Methods Protoc. 2020, 3, 47 2 of 6 The take-home message here is that the organization of the MT array in centrifuged cells appears to be very similar if not identical to what we would select as the “best” cells from either direct fix or agar Asoverlay such procedures. an alternative, However, we have a primary begun to benefit spin cells is that directly they are onto significantly coverslips. more Five plentiful minutes on at 400the centrifuged g in a standard coverslip tabletop than centrifuge in the other not onlytwo pr promoteseparations. attachment For example, but also approximately cell spreading. 10% A of small the volumecells on ofa gravity cell suspension settled coverslip is added are to busufficientlyffer overlying flat enough a coverslip to project in a centrifuge a useful tubecharacterization (Figure1) and of spunMT arrays for five for minutes. the types The questions coverslip that is interest then lifted us. On out a andcentrifuged gently slid coverslip, into a dish this containingincreases to fixative. at least This60% approachof the population. eliminates This the percentage waiting period is similar for cell in settling; the agar agarose overlay is notpreparations, required, andbut cellscentrifuged labeled withcells aextend tubulin across antibody the entire [10] appearcoverslip very and similar do no tot suffer those detachment prepared by due agar to overlay agar sheet (Figure removal.2).

Figure 1. CentrifugationCentrifugation devices. devices. Panel Panel (A ()A shows) shows a side a side view, view, panel panel (B) (showsB) shows a top a topdown down view view of a of50 amL 50 Sorvall mL Sorvall centrifugation centrifugation tube fitted tube fittedwith an with EPON an EPON base (yellow base (yellow layer in layer A) and in A a) white and a Delrin white Delrinrod segment rod segment (2.5 cm (2.5 diameter, cm diameter, 3.75 3.75cm cmlength). length). The The notch notch along along the the side side is is helpful helpful to to retrieve coverslips withwith forceps,forceps, allowingallowing accessaccess to to the the underside underside of of the the coverslip coverslip for for lifting. lifting. Panel Panel (C ()C shows) shows a modifieda modified multiwell multiwell holder holder for for processing processing up up to to 12 12 samples samples at at a a time. time. TheThe toptop rowrow containscontains 1212 mm round coverslips;coverslips; thesethese cancan bebe readilyreadily removedremoved withwith curvedcurved forceps.forceps.

The take-home message here is that the organization of the MT array in centrifuged cells appears to be very similar if not identical to what we would select as the “best” cells from either direct fix or agar overlay procedures. However, a primary benefit is that they are significantly more plentiful on the centrifuged coverslip than in the other two preparations. For example, approximately 10% of the cells on a gravity settled coverslip are sufficiently flat enough to project a useful characterization of MT arrays for the types questions that interest us. On a centrifuged coverslip, this increases to at least 60% of the population. This percentage is similar in the agar overlay preparations, but centrifuged cells extend across the entire coverslip and do not suffer detachment due to agar sheet removal.

Methods Protoc. 2020, 3, 47 3 of 6 Methods and Protoc. 2020, 3, 47 3 of 6

FigureFigure 2. ExamplesExamples of of antibody-labeled antibody-labeled microtube microtube (MT) (MT) patterns patterns in cells in (A cells) directly (A) addeddirectly to added coverslips to coverslipsand fixed afterand afixed 10 min after period; a 10 min (B) cellsperiod; fixed (B after) cells overlay fixed withafter agaroverlay sheets; with and agar (C ,sheets;D) cells and fixed (C after,D) cells5 min fixed centrifugation after 5 min onto centrifugation coverslips. onto Panels coverslips. (A–C) show Panels interphase (A)–(C)arrangements show interphase of mononucleated arrangements ofcells mononucleated (left) and binucleates cells (left) (right). and Panelbinucleates (D) shows (right). mitotic Panel cells (D) in shows late anaphase mitotic (left)cells andin late late anaphase telophase (left)(right). and While late telophase the three (right). different While cell the preparations three differe looknt cell similar, preparations it is important look similar, to note it that is important there are totypically note that far there fewer are flattened typically cells far on fewer the direct flattened fix coverslips cells on the and direct substantially fix coverslips fewer and cells substantially in general on fewerthe agarose cells in overlay general coverslip. on the agarose MTs are overlay shown cove in green,rslip. MTs DNA are in blue,shown Scale in green, bar = 5DNAµm. in blue, Scale bar = 5 μm. 2.2. Cell Attachment 2.2. CellThe Attachment second perhaps underappreciated impact derives from a fixation recipe that includes a smallThe amount second of perhaps glutaraldehyde underappreciated (0.05% final). impact This derives concentration from a fixation is not recipe sufficient that includes to preserve a small cell amountstructure of on glutaraldehyde its own, nor does (0.05% it require final). This autofluorescence concentration quenching is not sufficient by sodium to preserve borohydride, cell structure but it substantially improves cell retention on the coverslip (Figure3). Although alone or 20 on its own, nor does it require autofluorescence quenching by sodium borohydride, but− it◦ substantiallymethanol can improves provide structuralcell retention preservation on the coverslip at the light (Figure microscopy 3). Although (LM) formaldehyde level, cell loss alone from theor −coverslip20° is substantial can provide at each structural wash or preservation incubation step. at the This light is particularlymicroscopy a(LM) problem level, for cell mitotic loss from cells, thesince coverslip they are is more substantial rounded at each and lesswash adherent or incubation than during step. This interphase. is particularly The fixation a problem recipe for provided mitotic cells,below since does they not interfereare more with rounded GFP labels, and less and adherent thus expressed than during tags can interphase. be imaged The alongside fixation antibody recipe providedstaining. Comparedbelow does to not the interfere use of higher with concentrationsGFP labels, and of thus glutaraldehyde expressed tags (i.e., can 0.5%) be imaged as the sole alongside fixative, antibody staining. Compared to the use of higher concentrations of glutaraldehyde (i.e., 0.5%) as the sole fixative, this recipe also has the advantage that it improves preservation of MTs without the

Methods Protoc. 2020, 3, 47 4 of 6 Methods and Protoc. 2020, 3, 47 4 of 6 thisoften-encountered recipe also has thenegative advantage effects that that it improvesglutaraldehy preservationde has on of antigenicity MTs without for the antibodies often-encountered directed negativeagainst other effects structures. that glutaraldehyde has on antigenicity for antibodies directed against other structures.

Figure 3. Phase contrast images of identical aliquots of D. discoideum cells spun onto coverslips and fixedfixed (A) with or ( B) without the the addition addition of of 0.05% 0.05% glutaralde glutaraldehydehyde and and rinsed rinsed four four times times with with buffer. buffer. Scale bar = 5050 μµm.

3. Discussion As with anyany cellcell perturbation,perturbation, centrifugation centrifugation may may not not universally universally be be a gooda good idea idea and and needs needs to beto approachedbe approached with with caution. caution. However, However, side-by-side side-by-side comparisons comparisons of the of MT the array MT (interphasearray (interphase or mitotic or cells)mitotic prepared cells) prepared by agar by overlay agar overlay or centrifugation or centrifugation are indistinguishable, are indistinguishable, and thus and in ourthus case, in our we case, feel thewe feel method the method affords aaffords useful a strategy useful strategy for our imaging.for our imaging. An important benefit benefit is is that that this this method method can can be be applied applied to tocells cells that that do donot not readily readily attach attach to the to µ thecoverslip. coverslip. For example, For example, treatment treatment of D. discoideum of D. discoideum with latrunculinwith latrunculin A (5 μM) A results (5 M) in results actin filament in actin filamentdisruption disruption and cell rounding and cell rounding[11]. Centrifugation [11]. Centrifugation of Lat-A-treated of Lat-A-treated cells onto coverslips cells onto followed coverslips by followedimmediate by fixation immediate preserves fixation a significant preserves percentage a significant of percentage the population of the for population downstream for imaging. downstream We imaging.have used We this have strategy used this to strategy examine to examinehow the how MT the array MT arraymay maybe beintegrated integrated into into the the cortical actin meshwork. Laboratories already comfortable with their stai stainingning procedures are not likely to do anything didifferent,fferent, but for those frustrated that D. discoideum cells (or other cell types)types) are less adherent than mammalian culture cells, these hints provideprovide alternativealternative strategiesstrategies toto consider.consider. 4. Experimental Design 4. Experimental Design 4.1. Centrifugation 4.1. Centrifugation For a limited number of samples, we used 50 mL polycarbonate Sorvall centrifuge tubes, which wereFor roughly a limited the number same diameter of samples, as the we ubiquitousused 50 mL 50 polycarbonate mL disposable Sorvall screw centrifuge cap tubes tubes, and which could accommodatewere roughly 18the mm same2 coverslips. diameter as We the partially ubiquitous filled 50 a set mL with disposable EPON resin screw (a commoncap tubes epoxy and could resin 2 foundaccommodate in electron 18 mm microscopy coverslips. labs) We to partially serve as filled a base, a set then with inserted EPON 3.75 resin cm (a long common Delrin epoxy spacers resin of almostfound in the electron same diameter microscopy as the labs) centrifuge to serve tube, as a forbase, the then coverslip inserted to rest 3.75 on. cm We long added Delrin a small spacers notch of inalmost the topthe ofsame the diameter spacer to as facilitate the centrifuge forceps tube, removal for the of coverslip the coverslip. to rest In on. order We foradded the a spacer small notch alone orin the multiple top of otherthe spacer configurations to facilitate to forceps work, removal one only of needs the coverslip. a smooth In surface order for that the will spacer support alone the or coverslip;multiple other remain configurations perpendicular to to work, the centrifugal one only force; needs and, a importantly,smooth surface provide that thewill ability support to reach the thecoverslip; coverslip remain with perpendicular a pair of forceps to the for centrifugal retrieval. force; and, importantly, provide the ability to reach the coverslipFor processing with a more pair of than forceps a few for coverslips, retrieval. we used custom 12-(flat) well titer plates (about half the size ofFor a standardprocessing 24-well more plate)than a that few fit coverslips, into the buckets we used of acustom swing-out 12-(flat) rotor. well Round titer coverslips plates (about (12 mm)half werethe size placed of a standard into the wells 24-well prior plate) toadding that fit cellsinto the and buckets centrifugation. of a swing-out There wererotor.also Round commercially coverslips available(12 mm) 24-wellwere placed 0.17 mm into glass the bottom wells platesprior thatto adding fit into manycells and swing-out centrifugation. buckets. UsingThere these were plates, also thecommercially centrifugation available method 24-well may more0.17 mm easily glass be applicablebottom plates for live that cell fit imaging.into many swing-out buckets. Using these plates, the centrifugation method may more easily be applicable for live cell imaging. A clean coverslip was added to the tube followed by sufficient phosphate buffer to cover the coverslip by about 5 mm. Using a micropipette, ~100 μL of D. discoideum cells were scraped from the bottom of a nearly confluent culture dish and gently added into the buffer volume directly over the

Methods Protoc. 2020, 3, 47 5 of 6

A clean coverslip was added to the tube followed by sufficient phosphate buffer to cover the coverslip by about 5 mm. Using a micropipette, ~100 µL of D. discoideum cells were scraped from the bottom of a nearly confluent culture dish and gently added into the buffer volume directly over the coverslip. Depending on the desired cell density, this volume of cells can be adjusted. Tubes can be centrifuged as soon as convenient after adding cells; no settling period is required. While fixing D. discoideum cells directly in culture media should be avoided due to auto fluorescent components in some recipes, the dilution into buffer (~1:50) and time over the brief spin period is sufficient to substantially reduce the fluorescent background. The tubes or dishes were placed in a table top clinical centrifuge with a swinging bucket rotor and spun for five minutes (400 g setting). Coverslips were then quickly removed and placed cell side up in 35 mm petri dishes prefilled with fixative, and incubated for 15–30 min. All downstream washing and incubation steps were performed as usual for antibody or reporter labeling. The centrifugation time and force settings were not rigorously optimized; we tinkered with them, and these settings worked for our purpose.

4.2. D. discoideum Fixative 50% PHEM buffer (30 mM PIPES, 12.5 mM HEPES, pH 6.9, 4 mM EGTA, 1 mM MgCl ). pH 6.9 • 2 3.7% formaldehyde • 0.05% glutaraldehyde • 0.1% Triton X-100 • Coverslips are acid washed by soaking in 1N HCl for one hour, followed by rinsing with milli Q H2O, and a brief 100% ETOH wash, then air dried in a glass petri dish.

4.3. Phosphate Development Buffer 20 mM KCL • 2.5 mM MgCl 6H 0 • 2· 2 27 mM NaH PO4 H 0 • 2 · 2 24 mM Na HPO • 2 4 pH 6.4 (see reference [12] for detail) • 4.4. Light Microscopy Imaging was performed on a DeltaVision microscope workstation using the softWoRx 2.50 imaging package. Z-series stacks were collected at 0.5 µm step intervals and deconvolved. Maximum intensity projections of the entire stack are presented here, performed using FIJI [13]. DNA is labeled with Hoechst 33342. The panels were assembled using Adobe Photoshop.

Author Contributions: Conceptualization, M.K., R.G.; methodology and investigation, M.K., I.T., and R.G.; resources, M.K., I.T., and R.G.; writing, M.K., R.G. All authors have read and agreed to the published version of the manuscript. Funding: Our work is supported in part by the National Science Foundation (MCB-1510511 to M.P.K.). Acknowledgments: We are grateful to Alexey Khodjakov for his long-term assistance with cellular imaging. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Methods Protoc. 2020, 3, 47 6 of 6

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