11 Cell transfection, permeabilization and microinjection as means to study Shigella-induced cytoskeletal reorganization

11 Cell Transf ection, Permeabilization and Microinjection as Means to Study Shigella-induced Cytoskeletal Reorganization Guillaume and Guy

Dumknil’**, Laurence Tran Van Nhieu’.*

‘Unite de Pathogenic Cedex 15. France

Microbienne

Moleculaire,

Bougnkes’,

Philippe

Sansonetti’

lnstitut Pasteur, 28 rue du Dr Roux, 75724

Paris,

CONTENTS General considerations on the analysis of Shigella entry effecters Analysis of Shigella-induced cytoskeletal rearrangements during entry into epithelial cells Purification of Shigella type Ill secretion effecters from secretion mutant strains Choice of the strain and growth conditions Microinjection as a means to study the effects of bacterial products on the actin cytoskeleton of eukaryotic cells Semi-permeabilization procedure to study the effects of type Ill secretion effecters on the cell cytoskeleton Cell expression of Shigella effecters by transient transfection Establishing Src stable transfectants of HeLa cells to study bacterial-induced signaling to host cells Investigating the role of a cytoskeletal regulator by transient transfection or microinjection Concluding remarks

List Ab BSA DMEM DTT EDTA FCS FITC HEPES HRP k PFA

of abbreviations Antibody Bovine serum albumin Dulbecco’s modified Eagle’s medium Dithiothreitol Ethylenediamine tetraacetic acid Fetal calf serum Fluorescein isothiocyanate N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic Horse radish peroxidase lmmunoglobulin Paraformaldehyde

acid)

*Present address: Department of Molecular Biology and Microbiology, Medicine, 136, Harrison Avenue, Boston, MA 02111, USA METHODS IN MICROBIOLOGY, ISBN O-12-521531-2

VOLUME 31

Tufts University

School of

Copyright (‘: 2002 Academic Press Ltd All rights of reproduction in any form reserved

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GENERAL ANALYSIS

CONSIDERATIONS ON THE OF SHlGELLA ENTRY EFFECTORS

Various gram-negative pathogens modify the cell cytoskeleton by means of bacterial products injected in the cell cytosol through a type III secretion system (Hueck, 1998). For Shigella, such products determine bacterial invasion in normally non-phagocytic cells. These effecters induce bacterial internalization by inducing cytoskeletal changes resulting in the formation of cell extensions, that dynamically organize to engulf the bacterium in a large vacuole (Tran Van Nhieu et al., 2000). The formation of these cell extensions require actin polymerization, and the activation of the small GTPases Cdc42 and Rat, whereas the later stages of the bacterial entry process require the activation of the GTPase Rho (Dumenil et al., 2000). The Src tyrosine kinase play a dual role during the entry process, by favoring actin polymerization in concert with the activation of Cdc42 and Rat, while down-regulating Rho and actin polymerization during the late stages of entry (Dumenil et al., 2000). This indicates that bacterial entry depends on a finely tuned set of responses, which may be induced by the concerted action of various bacterial determinants. Bacterial genetic studies aiming at identifying such products are limited by the fact that entry defective mutants will not only consist of effector mutants, but also of any mutants that are defective for the type III secretory apparatus. An approach based on analyzing the effect on the cell cytoskeleton of individual bacterial products that translocate through the type III secretion apparatus has proven successful in identifying several effecters of bacterial invasion (Galan and Zhou, 2000; Hayward and Koronakis, 1999; Tran Van Nhieu et al., 2000). This can be achieved by various means, from the expression by transfection, to microinjection or semi-permeabilization of the bacterial effecters in cell lines, In parallel to in vitro assays using single determinants, we have used independent approaches to analyze signaling pathways that regulate cytoskeletal reorganization induced by Shigella. In this chapter, we will discuss experience gained in using these various means to analyze effecters of Shigella entry inside cells.

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ANALYSIS OF SHIGELLA-INDUCED CYTOSKELETAL REARRANGEMENTS DURING ENTRY INTO EPITHELIAL

CELLS

HeLa cells are cells routinely used to analyze entry of Shigella into epithelial cells, Although the reasons for this are mostly historical, the spectacular projections that this bacterium induces during the entry process, as well as the distinct phases of the entry structure that can be distinguished in this type of cell are probably the best rationale for this model. Because Shigella-induced cytoskeletal changes are highly dynamic, it is important to synchronize the infection so as to compare foci at the same stage of development. As Shigella shows little cell-binding activity, it is rendered adhesive to cells by the expression of the E. coli Afa E adhesin 208

11.1. Immunofluorescence analysis of ezrin in a Ski&a focus of internalization. HeLa cells were challenged with Skigella for 15 min at 37°C. Samples were fixed and stained for ezrin (Panel A), actin (Panel B) or Skigeh LPS (Panel C), and analyzed by confocal laser microscopy (LSM510, Zeiss). Images were obtained by reconstruction from sections that do not include the basal cell surface. Panel D shows the superimposition of the triple staining. Ezrin labels the tip of Skigella-induced cell extensions where little F-actin is detected. Scale bar: 5 pm. (This figure is also reproduced in colour between pages 276 and 277.)

Figure

(Labigne-Roussel et al., 1984). Bacterial binding to cells is performed at 22°C and cytoskeletal changes induced by Shigella entry are triggered by placing the samples at 37°C. F-actin staining is used to identify the cytoskeletal rearrangements induced by Shigella. Because the projections that Shigella induces during entry can reach up to 10 microns in length, confocal laser microscopy may be required in double-labeling procedures to precisely characterize the recruitment of a cytoskeletal protein at the level of the entry structure. Figure 11.1 shows an entry structure that has been induced by Shigella on the surface of HeLa cells and that was stained for the cytoskeletal linker ezrin (Panel A) and F-actin (Panel B). Ezrin is recruited at the tip of F-actin rich cell projections, where little F-actin is observed. Bacteria are stained with an anti-LPS antibody (Panel C). Protocol-Day

I l

HeLa cells are plated onto 24 x 24 mm coverslips DMEM containing 10% FCS.

209

in a 35 mm dish, and grown

in

Day

2 l

l

l

l

l

l

Determination

Cells are washed once with DMEM without serum and incubated with bacteria grown in mid-exponential phase and suspended in DMEM containing 50 tnM HEPES pH 7.3. To prepare the bacterial suspension, S&gel/a strains carrying the AfaE encoding plasmid are grown inTCSB containing spectinomycin at 100 ug ml-’ final concentration until mid-exponential phase (OD 600 nm = 0.3). Bacteria are diluted in DMEM-HEPES medium togive an MOI of IO-50 bacteria per cell (0.003 < OD 600 nm < 0.006). I ml of the bacterial suspension is added to the well and samples are incubated at room temperature for I5 min to allow bacterial attachment to the cell surface. Samples are then shifted at 37°C by floating on a water bath.To avoid problems linked to floating samples, a metal plate can be immersed in the 37°C water bath, so as to leave sufficient water above the plate to allow efficient immersion of the samples. After various periods of time, samples are fixed in PBS containing paraformaldehyde at 3.7% final concentration for 20 min at RT Samples are washed three times in PBS. Samples are processed for immunofluorescence staining using standard procedures.

of bacterial

entry

by differential

inside/out

immunostaining

Internalized bacteria can be distinguished from extracellular bacteria because they are not accessible to antibodies unless cells have been permeabilized. This makes it possible to differentially stain the extracellular bacteria prior to sample permeabilization with a given fluorochrome, and stain the total bacteria after permeabilization with a different fluorochrome. To avoid cross-reactivity with the secondary Ab, staining is performed with different antibodies (i.e. rabbit polyclonal and mouse monoclonal Abs, or different subclass of monoclonal Abs, with the corresponding secondary Abs). If only one Ab is available, it is possible to covalently link the fluorochromes to the Ab using commercial reagents (i.e. Molecular probes, Calbiochem). Alternatively, the utilization of a bacterial strain that express the green fluorescent protein (GFI’) (Rathman et al., 2000) can simplify the procedure, as only the labeling of the extracellular bacteria with a fluorochrome that emits in a different spectrum than the GFP (i.e. rhodamine) is necessary.

Protocol l l

l

l l

Fixed samples are blocked in DMEM containing 10% FCS for at least 30 min. Extracellular bacteria are stained with an anti-LPS mAb, followed by an antimouse Ig Ab coupled to FITC. Samples are permeabilized by incubating for4 min in PBS containing O.I%Triton x-too. Samples are washed three times in PBS. Total bacteria are stained with a anti-LPS rabbit polyclonal Ab, followed by an anti-rabbit Ig Ab coupled to rhodamine.

210

l

Samples are washed glycerol and DABCO of the samples.

three times in PBS and mounted onto slides using 50% at a final concentration of IO mg ml-‘to prevent bleaching

If a triple labeling is performed, a UV light excited fluorochrome such as Cascade blue can be used. Because this latter type of fluorescence is more difficult to detect than red or green-emitting fluorochromes, we usually reserve this UV light excited fluorochromes for the labeling giving the strongest and less ambiguous signal, i.e. bacterial LPS labeling. Also, because UV light excitation promotes more sample bleaching than red or green lights, it is preferable to acquire images for quantification purposes. For example, to analyze bacterial internalization in transiently transfected cells, fields containing transfectants will be selected. To limit photobleaching, images corresponding to the UV-excited fluorophore are acquired last. Analysis

of components

of Shigello-induced

entry

foci

Skigella entry structure are identified with F-actin staining. Because these structures are readily distinguishable from other cellular structures, Sk&&-induced foci of actin polymerization can be scored using a computer dedicated program according to the shape and the fluorescence intensity of the entry structure (Dumenil et aI., 1998). When performing kinetics to localize more precisely a cytoskeletal component within the entry structure, bacteria need to be stained. Triple-labeling

Samples are fixed and permeabilized. F-actin is stained with Bodipylinked to phalloidin, the cytoskeletal component to be analyzed is stained with Ab followed by rhodamine-linked secondary Ab. For direct observation, bacteria are labeled with anti-LPS followed by Cascade blue linked Ab. To prepare samples for confocal microscopy analysis, bacteria are labeled with anti-LPS followed by CY-5 linked Ab. Although CY5 cannot easily be distinguished from red-emitting light fluorochrome using commonly used filters, it is readily distinguishable from rhodamine when using the appropriate laser wavelengths, and has the advantage of an excitation light that is less damaging to the sample.

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PURIFICATION OF WIGELLA TYPE Ill SECRETION EFFECTORS FROM SECRETION MUTANT STRAINS It is, in general, not a problem to express Skigella proteins under a recombinant form in E. colt Several proteins that are secreted via the type III secretion apparatus have been obtained after fusion to GST or tagged with a poly-histidine epitope (Chen et al., 1996; Niebuhr et al., 2000). 211

Shigella Ipa proteins that are fused to the GST by their N-terminus, are not secreted by the type III secretion apparatus because the GST moiety probably interferes with the secretion signal. Histidine tagged proteins may be still secreted and have been shown in some instances to functionally complement the invasion defect in the corresponding Shigella mutant (Niebuhr et al., 2000). Because such technologies are now widely used, we will not discuss technical considerations, but there are two types of limitations to recombinant protein technology that may not be specific for Shigella secreted proteins: first, many Shigella recombinant proteins are not soluble and tend to form inclusion bodies when overexpressed in E. coli (De Geyter et al., 1997; Picking et al., 2001). Purification in this case, implies solubilization from inclusion bodies, using agents such as guanidine hydrochloride or urea. Thus, particular care should be taken to ensure that proteins that are purified this way remain functional or do not show altered properties, specially for those proteins whose activity is likely to be regulated. An alternative to recombinant protein technology, is the purification of proteins from Shigella strains, that are expressed under the control of their endogenous promoter. This is feasible for the Shigella Ipa proteins, because they are abundant and because secretion provides a means to fractionate proteins that are secreted via the type III secretion apparatus (BourdetSicard et al., 1999; Tran Van Nhieu et al., 1999). After concentration of secreted proteins from Shigella culture supernatants, proteins are fractionated by FPLC using a combination of ion-exchange chromatography procedures.

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CHOICE OF THE STRAIN CONDITIONS

AND

GROWTH

Shigella mutants ipuB, ipuD or deleted for the ipa operon for which the MxiSpa apparatus shows constitutive activity (Parsot et al., 1995), are used for these purposes. The use of a mutant strain can also simplify the purification steps. For example, IpaB has been shown to form a stable complex with IpaC after secretion (Menard et al., 1994); Shigella ipaD strain may be used for the isolation of the IpaB-C complex, whereas purification of IpaC from a Shigella ipd strain circumvents the problem of dissociating the IpaB-C complex. For proteins other than the Ipa proteins, that are secreted via the Mxi-Spa apparatus, a Shigella strain for which the ipa operon is deleted can be used to avoid contamination by these abundant proteins. Strains are grown in trypticase soya or 2 x YT broth. Although casein products present in this medium tend to ‘stick’ to the chromatography matrix, the yields obtained using TCS broth are significantly higher than those obtained 2 x YT. Purification

procedure-Day l

Inoculate

I a preculture

of the Shigella ipa

212

mutant

strain.

Day 2 l

l

l l

l l

Inoculate a culture with a I : 100 dilution of the overnight preculuture. In general, for IpaA or IpaC, it is possible to obtain about 200 pg of protein starting from a liter of culture of the ipaB mutant strain, using the respective endogenous promoters.This yield can be increased by several-folds when using strains transformed with the cloned gene of interest under the control of the Plac promoter. Overexpression, however, appears to alter the solubility of the proteins (not shown). Grow on a rotary shaker at 37”C, 250 rpm until mid-exponential phase (0.6 c OD 600 <: 1.0). The recovery of Ipa proteins from culture supernatant at later stages of growth decreases because they tend to become insoluble. The flasks containing the culture are then chilled on ice, and all the purification steps described below are performed at 4”C, unless otherwise stated.’ Centrifuge the bacterial culture at 7000 rpm for 30 min. Transfer the supernatant in a beaker, and weigh the appropriate amounts of ammonium sulfate to perform a precipitation at 50% final concentration. Ensure the pH of the sample rapidly neutralized after addition of the ammonium sulfate, and stir samples for at least 2 h. Centrifuge samples at 7000 rpm for 30 min and discard supernatant. Resuspend pellet in about I : 20 of the initial volume ofculture in buffer Al containing: 0.1% Nonidet-P40; 25 mM Tris-HCI pH 7.5; 25 mM NaCI; 0.1 mM EDTA; I mM DTT, and a mixture of protease inhibitors. At this stage, the pellet has a dark brown color from the casein precipitates, but it readily comes into solution. Dialyze extensively three times against at least 20 volumes of buffer Al for at least 2 h for each dialysis batch.

Day 3 0 Perform FPLC using a I ml monoQ anion exchange column. Proteins are eluted using a 20 ml s NaCl linear gradient with concentrations ranging from 25 mM to 500 mM. IpaA typically elutes in two peaks; a near homogeneous fraction at around 80 mM NaCI, and another fraction contaminated with proteins that migrate at ca. 60 kDa, which probably correspond to IpaH proteins (Figure ll.2A, arrow). l For IpaC purification, the flow-through of the monoQ column is collected and dialyzed extensively against buffer A2 containing 0.1% Nonidet-P40; 25 mM HEPES pH 7.5; 0.1 mM EDTA; I mM DTT, and a mixture of protease inhibitors (complete TM, Pharmacia). l Proteins are fractionated using a monoS cation exchange column and a 20 ml s NaCl linear gradient with concentrations ranging from 25 mM to 450 mM.Two main peaks are detected that correspond to proteins migrating at 43 kDa, IpaC typically elutes at an NaCl concentration of about 100 mM (Figure I l.2B, arrow). SepA, a secreted protein that shares homology with serine protease, forms a peak that elutes at concentrations slightly inferior to those for IpaC elution, and may contaminate the IpaC containing fractions (Figure 11.28). l Purified samples are dialyzed against microinjection buffer containing 25 mM Tris-HCIpH7.3,100mMKCI,5 mMMgClz,l mMEGTA,O.l mMDTT,andprotease inhibitors, and concentrated using amicon filters C3000 (Millipore Corp.) to a final concentration of about I mgml-‘. Aliquots are flash frozen and stored at -20°C.

213

A.

B.

E

E

1 3 4

8

5

6 7

9 10 I1

8 9

1011

13 15 17

12 13

Figure 11.2. Purification of IpaA and IpaC from Shigella secretion constitutive mutant strains. Concentrated supernatant of a Shigella ipuB mutant strain that shows constitutive secretion, was subjected to FPLC (flow pressure liquid chromatography) on a monoQ anion-exchange column (Panel A). Fractions were eluted with a 20 ml s linear NaCl gradient, with concentrations ranging from 20 mM to 500 mM. The flow-through of the monoQ chromatography was run on a MonoS cation-exchange column (Panel B), and fractions were eluted with a 20 ml s linear NaCl gradient, with concentrations ranging from 0 mM to 450 mM. Panels A and B: E is loaded extract. The numbers above the lanes represent the fraction number from the start of the gradient. The arrow points to IpaA Panel A) and IpaC (Panel B).

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MICROINJECTION AS A MEANS TO STUDY THE EFFECTS OF BACTERIAL PRODUCTS ON THE ACTIN CYTOSKELETON OF EUKARYOTIC CELLS Microinjection has an advantage over the transfection procedure, in the sense that it can allow the analysis of short-term effects linked to the microinjected proteins, by fixing samples within minutes after microinjection and performing immunofluorescence microscopy analysis. Microinjection also allows the use of videomicroscopy to analyze the effects of the injected product on the formation of cell projections. 214

Concerning the choice of the cell line, Swiss 3T3 cells present several advantages over other cell types for the study of cytoskeletal rearrangements (Hall, 1998). These cells show a well-characterized actin cytoskeleton that can be controlled under various cell culture conditions, and there is little heterogeneity in the cell sample (Nobes and Hall, 1995). Furthermore, many studies on the signaling to the actin cytoskeleton have been performed in these cells, and the hierarchy of key regulation events is usually admitted to occur in this cell model. The effects on the cytoskeleton of a particular toxin, or bacterial effector can be visualized best by treating the cells to place them in the optimized responsive state. For example, to study the effects of effecters on depolymerization of actin filaments, cells are usually cultivated in the presence of serum to maximize the presence of stress fibers (Nobes and Hall, 1995). In the case of actin polymerization induced by the IpaC protein, cultivation in the presence of serum appears to interfere with the formation of actin-rich extensions at the cell periphery induced by IpaC (Tran Van Nhieu et al., 1999). Thus, cells need to be cultivated for at least 48 h in the absence of serum. Protocol-Day

Day

I l

Swiss 3T3 cells between passage 7 and 15 are grown in DMEM containing 10% FCS in a 37°C incubator supplemented with 10% COz. Cells are seeded in 24-well plates onto Ilmm-diameter coverslips, that were previously acidwashed and sterilized, at a density of 5 x IO4 cells per well.

l

Cells are washed once in DMEM, resuspended intheabsenceofserum.

2 in DMEM and incubated

for 48 h

Day 4 Cells are processed for microinjection in a chamber supplemented with 10% COz.The microinjection time should not exceed IO min, this period determining the limit in the amounts of cells being microinjected. 0 After microinjection, cells are returned to the incubator in DMEM containing 10% FCS for 5 to 20 min. l Cells are fixed in PFA and processed for staining of the actin cytoskeleton.

l

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SEMI-PERMEABILIZATION PROCEDURE STUDY THE EFFECTS OF TYPE Ill SECRETION EFFECTORS ON THE CELL CYTOSKELETON

TO

As an alternative to microinjection, the effects of IpaC on the cytoskeleton can be visualized by permeabilizing cells with trace amounts of the 215

Figure 11.3. Permeabilization versus microinjection of IpaC. Swiss 3T3 cells were permeabilized in the presence of buffer alone (Panel A) or purified IpaC (Panel B) (see text). Samples were fixed and stained for F-actin. IpaC induces the formation of actin-rich filopodial structures that fill in into leaflets, at the cell periphery. Swiss 3T3 cells were microinjected with purified IpaC (Panel C, arrow). The IpaCmicroinjected cell shows diffuse actin polymerization, and the formation of numerous microspikes over the cell surface.

detergent saponin (Tran Van Nhieu et al., 1999). Although it is not clear to what extent permeabilization applies to all cytoskeletal effecters, it provides a powerful means to visualize cytoskeletal reorganization because the procedure is less cumbersome than microinjection. Also, because the effects are homogenous in cells from within a sample, quantification is rendered easier. In the case of IpaC, permeabilization leads to the formation of actin-rich filopodial extensions that rapidly fill in to form lamellipodial extensions at the cell periphery (Figure 11.3B). This is in contrast to IpaC microinjection, which leads to diffuse actin polymerization and the formation of numerous thin microspikes on the cell surface (Figure 11.3C, arrow). The reasons for this difference are unclear, but could be linked to leakage of cell components regulating cytoskeletal changes during the permeabilization procedure, or alternatively, a difference in the mode of presentation of IpaC in the two techniques. Swiss 3T3 cells are grown in 10% FCS-DMEM 1000 mg ml-’ glucose in a 37°C incubator supplemented with 10% COZ, and used between passage 7 and 15. Serum starvation is in DMEM 1000 mg ml-’ glucose containing 50 mM HEPES pH 7.3. Protocol-Buffers

used l

UB buffer: 50 mM HEPES pH 7.3, 100 mM Kcl, 3 mM 0.2% BSA. 216

MgCl2, 0.1 mM

DTT,

Day

Day

l

Permeabilization buffer: 0.003% saponin, 50 mM HEPES pH 7.3, 100 mM KCI, 3 mM MgClz, 0.1 mM DTT, 0.2% BSA, I mM ATP, 100 pM GTP, 100 pM UTP.

l

Semi-confluent Swiss 3T3 cells (use between passages 7 and 12) are split and plated at 4 x IO4 cells/l3 mm diameter coverslip or 2 x IO5 cells/24 mm x 24 mmcoverslip.

I

2 Serum starved

for 30-48

h.

Wash I x PBS, 2 x UB Dilute protein (I mg ml-‘) onto parafrlm, at least I : IO in permeabilization bufferon parafrlm parafilm piece. Use: 40 pl for I3 mm diametercoverslip or 150 pl for 24 x 24 mm coverslip. Incubate cells with samples by putting the coverslip face down onto the droplet on parafilm. Incubate at 37°C in humid chamber. 20 min on prewarmed metal plate. Transfer cells to 3.7% PFA for 30 min at 22°C. Process for fluorescence staining of the actin cytoskeleton.

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CELL EXPRESSION OF WIGELLA BY TRANSIENT TRANSFECTION

EFFECTORS

The approach consists of cloning the bacterial gene of interest on an eukaryotic expression vector, and introducing the recombinant vector by transfection inside epithelial cells. Although straightforward because it does not require purification of the bacterial product, this approach may be limited by a few considerations that are discussed below. First, any visible effects have to correspond to those occurring at concentrations of protein that allow immunodetection, without warranty that these relate to effects occurring at physiological concentrations. Furthermore, transfection requires incubation, usually from a few hours to a few days, to allow expression and immunodetection of the transfected protein. Thus, the changes observed on the cytoskeleton result from an equilibrium between direct effects of transfected protein and secondary effects that this protein induces. If the direct effects are predominant over secondary effects, transient transfection may be an adequate technique to analyze the effects of a protein on the cytoskeleton. If the direct effects are subtle , or very transient, as one may suspect for the bacterial effecters of entry, the changes observed on the cytoskeleton may correlate only remotely with the real effects of the protein. This is well exemplified by activated Cdc42, which induces filopodia and microspike formation when microinjected

217

(Nobes and Hall, 1995), whereas it induces stress fiber formation after transient transfection. In this latter situation, the formation of stress fibers probably results from the activation of Rat and Rho GTPases, downstream of Cdc42 (Hall, 1998). For these reasons, transfection should be used as a means to detect obvious effects on the cytoskeleton, to confirm a phenotype, or in conjunction with other pieces of evidences to assess a precise function of a protein. Choice

of the

eukaryotic l

Transfection

Day

expression

vector

It is convenient to use vectors that also carry a prokaryotic for in-frame cloning and expression of the protein to be many commercially available vectors allow insertion of an extremity of the protein coding sequence that will facilitate tion of the protein. procedure-Day

promoter to check transfected. Also, epitope tag at one the immunodetec-

I

l

Cells are seeded at a density of 5 x IO4 cells on a I3 mm diameter incubated overnight in DMEM containing 10% FCS.

l

Cells are transfected using the Fugene reagent according turer’s instructions, and incubated from 6 h to 3 days.

coverslip

and

2

Analysis

of the

to the manufac-

transfectants

Cells are fixed with PFA and processed for staining of F-actin and to detect expression of the transfected protein. In the case of IpaC cloned into pCDNA.3, immunoanalysis of the transfectants indicate the formation of filopodial structures at the cell periphery, as well as of a reorganization of actin fibers into a branched meshwork of thinner actin cables (Tran Van Nhieu et al., 1999). The changes that are observed on the organization of actin cables of the transfectants are likely to be secondary effects due to the expression of IpaC, as those are not visible during short-term kinetics of cells that have been microinjected with the purified IpaC protein (Figure 11.2B).

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ESTABLISHING SRC STABLE TRANSFECTANTS OF HELA CELLS TO STUDY BACTERIAL-INDUCED SIGNALING TO HOST CELLS For biochemical purposes, microinjection or transient transfection techniques are not suitable. To analyze association of proteins, or regulation of cytoskeletal organization during bacterial entry, the use of stable 218

transfectants may be required. Although obtaining stable transfectants may not always be possible for proteins that interfere with cell growth, and although it requires significant investment in terms of effort and time, the establishment of stable cell lines clearly opens the way for analysis that cannot be achieved with transient transfection. It allows the obtention of cells that express relatively homogeneous levels of the transfected proteins. Also, it then becomes possible to study a combination of effects, for example, by microinjection or super transfection. The procedure to obtain stable transfectant is similar to the transient transfection protocol, except that selective medium is added to the cells, usually 24 to 48 h after transfection. In the case of HeLa cells, clones growing as individual clusters of about 200 cells are detectable after 10 days. These clusters should be subcloned at least twice by the limiting dilution or cloning ring technique, to limit the mixture of cell clones. Because not all clones that are resistant to the selective marker will express the protein of interest, it is necessary to analyze several clones growing in the selective medium. In the case of Src constructs, the screening of expressing clones was performed by Western blot on cell lysates corresponding to 5 x lo5 to 5 x lo6 cells, using the ECL chemiluminescent substrate (Dumenil et al., 1998). Although cumbersome, this was necessary because the levels of expression of the transfected Src were too low to detect by immunofluorescence microscopy. Clones expressing levels of the kinase deficient Src could be readily obtained with levels above ten-fold those of endogenous Src, whereas clones expressing constitutively active Src showed only moderate expression of the kinase, with at best a two-fold expression over the endogenous kinase. These latter clones showed a strongly altered morphology, with a disappearance of stress fibers and a decrease in focal adhesions (Dumenil et al., 2000). The various Src transfectants are stable and can be transiently transfected with Rho GTPases constructs to study the hierarchy between Src and these GTPases during Shigella entry (Dumenil et al., 2000).

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INVESTIGATING THE ROLE OF A CYTOSKELETAL REGULATOR BY TRANSIENT TRANSFECTION OR MICROINJECTION Considerable progress has been made in the knowledge of signaling pathways that govern actin dynamics in recent years, and many key regulators have been identified and characterized in terms of molecular structure and partners. This is opening up the possibility to directly interfere with the function of defined molecules by the introduction of dominant-negative forms that will specifically inhibit or disconnect a given pathway. With the reserves of technical considerations (i.e. difficulties to transfect or to microinject), it is logical to use the same cell model as is commonly used for the bacterial entry process, although one

219

has to be careful about drawing conclusions from what has been established for the implication of a given molecule in another cell type. We have used microinjection and transfection of dominant-negative forms of Rho GTPases to study the role of these GTPases in Shigella entry. Both techniques gave similar results in that inhibition of Cdc42 or Racinhibited actin polymerization induced by Shigella at the site of entry, whereas inhibition of Rho did not inhibit actin polymerization but the recruitment of ezrin and the Src tyrosine kinase at entry foci (Dumenil et al., 2000). Transfection is a straightforward approach because it does not involve protein purification and large-scale microinjection. When expressing a dominant interfering by transfection, however, there are constraints linked to the period of time allowed to visualize expression of the construct that ranges from a minimum of 6 h to several days. This incubation time may lead to the accumulation of pleiotropic effects, and may lead to inhibitory results that are not directly linked to the protein analyzed. Although more cumbersome, microinjection allows one to control the concentration of the inhibitor used, and also to study the effects of the inhibitor a few min after its introduction into the cell cytosol. Analysis Shigella

Day

Day

of the expression entry by transient

of dominant-interfering transfection-Day

forms

of Rho

GTPases

on

I

l

HeLa cells are seeded at a density of 2 x IO5 cells per 24 mm x 24 mm coverslip in a 35 mm dish, in Dubellco Modifred Earl’s Medium (Gibco BRL) containing lO%fetal calf serum, in a 37°C incubater supplemented with 10% CO*.

l

Cells are transfected with the corresponding construct using the Fugene reagent (Boerhinger Mannheim) according to the manufacturer’s instructions. When performing immunofluorescence analysis, transfection using CazP04 procedures needs to be avoided because of the interference of CazP04 precipitates with the immunodetection procedure.

l

Cells are challenged with Shigella and the formation of foci of actin polymerization is analyzed by immunofluorescence as described in earlier.

2

3-5

The expression of the construct is monitored by immunofluorescence analysis. Because most recombinant proteins are tagged with an exogenous epitope, the antibodies and the conditions used for detection of the recombinant proteins are in general well characterized. In theory, it is also possible to interpolate the levels of molecule expressed in the transfectants, by performing the proper calibrations. In practice, however, the levels of inhibitor that should be analyzed are those which lead to inhibition of specific pathways, without causing gross alterations of the cytoskeleton. In this respect, it is important to define the antibody

220

probing conditions so as to observe a range of expression levels related to the fluorescence intensity in the various transfectants. For incubation periods that exceed 24 h after transfection, transfectants are usually observed that expressed weak, intermediate, or high levels of the transfected construct. In the case of dominant interfering N17 Cdc42, N17 Rat or N19 Rho GTPases, transfectants that express high levels of the construct should be omitted from the analysis because of their profoundly altered cytoskeleton. The analysis is performed on cells that express low or intermediate levels of the transfected construct on a statistically significant number of cells from at least three independent experiments. Analysis Shigelfa

Day

Day

of the expression of dominant-interfering entry by microinjection

forms

of Rho

GTPases

on

l

Purification of dominant-interfering forms of Rho GTPases using recombinant GST fusions in E. co/i has been described elsewhere (Self and Hall, 1995). After purification, protein samples are dialyzed against microinjection buffer (25 mM Tris-HCI pH 7.3, 100 mM KCI, 5 mM MgClz, I mM EGTA, 0.1 mM DTTand protease inhibitors) and concentrated by centrifugation using Amicon microconcentraters to obtain a protein concentration in the order of I mgml-‘. Samples are stored aliquoted at -20°C. The day of microinjection, samples are thawed and mixed with FITC-Dextran (70 kDa, Molecular Probes) previously dialyzed in microinjection buffer, to give a final concentration of 0.2 mg ml-‘. Samples are centrifuged for 5 min at I3 K just prior to injection to pellet potential aggregates and the supernatant is used to load microinjection capillaries.

l

Cells are seeded the day before on I3 mm diameter at a density of 5 x IO4 cells per well.

I coverslips

in a24-well

plate

2 Coverslips are kept in DMEM containing 10% FCS in the 37°C incubator supplemented with 10% COz. Just prior to microinjection, samples are transferred to a 60 mm diameter dish in DMEM containing 25 mM HEPES pH 7.3. l Microinjection is performed on a maximal number of cells for IO min. With practice, it is usually possible to microinject as many as 50 to 100 cells per sample with a high recovery rate. Samples are returned to the incubator in DMEM containing 10% FCS for 30 min. l Samples are challenged with Shigella and fixed in PFA as described above. 0 For IF analysis, microinjected cells are distinguished by the FITC fluorescence. To analyze entry foci induced by Shigella, it is convenient to perform staining of bacteria as well as of the component of the foci to be analyzed (i.e. actin) in a manner that is compatible with direct microscopy observation. This can be performed by using red-light emitting fluorophores such as rhodamine, combined with UV light such as Cascade blue-linked antibodies.

l

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++++++

CONCLUDING

REMARKS

Because the action of the various Shigellu effecters, as well as the responses they induce, are finely regulated during the entry process, it is clear that none of the in vitro approaches described in this chapter can reproduce what the bacterium achieves during entry. This is well-illustrated in the case of the IpaA protein, which favors the transformation of cell extensions into a structure that is proficient for bacterial uptake. IpaA carries two activities. IpaA binds to the focal adhesion protein vinculin and stimulates its binding to F-actin; this may account for the formation of a focal adhesion-like structure at the intimate contact site between the bacterium and the host cell membrane. The IpaA-vinculin complex also carries an Factin depolymerizing activity that may account for the transition from filopodial to leaflet-like extensions (Bourdet-Sicard et al., 1999). It is likely that these activities are dynamically regulated during Shigellu entry, either by the cellular environment, or by the concerted action of other bacterial effecters. Such a type of regulation probably applies to the various effecters of entry, and it will be important to develop approaches that integrate an activity characterized in vitro for a purified protein in the bacterial entry process.

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