FACS has been widely used to determine the DNA content, and hence cell cycle ... chapter, FACS sorting can also be used to analyze a number of other.
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FACS has been widely used to determine the D N A content, and hence cell cycle distribution, of transiently transfected cells. As outlined in this chapter, FACS sorting can also be used to analyze a number of other parameters in transfected populations, for example, apoptosis, direct measurement of S phase through BrdU incorporation, and levels of protein expression and kinase activity.
Acknowledgments We thank Christine Jost, Todd Upton, and Francesco Hofmann for critical reading of the manuscript and many helpful comments; all members of the Dana-Farber Flow Cytometry core facilityfor technical expertise; and Amy Monighetti for expert secretarial assistance.
[6] M i c r o i n j e c t i o n
of Antibodies
into Mammalian
Cells
By NED J. C. LAMB and ANNE FERNANDEZ Introduction Microinjection offers the unique opportunity to manipulate the intracellular environment in a directed and controlled manner. Various techniques have been developed to introduce macromolecules into cells, including osmotic shock, lipid and micelle-mediated fusion, and scrape-loading. These techniques have essentially been favored because they allow largescale injection and subsequent analysis by standard biochemical techniques, they each have a shallow learning curve, and they do not need complicated or delicate equipment. Their principal disadvantages are that they require large volumes of starting material and may cause significant perturbations in cell metabolism (ceils are either shocked or the membranes damaged or fused). Importantly, for cell cycle control-related studies, they do not permit timely controlled studies and analysis of short-term events, nor do they allow the injection of a specific intracellular compartment. In contrast, microneedle microiniection requires extremely small amounts of starting material and allows precise targeting of the microinjection, both temporally and spatially. In addition, it is possible to accurately inject the same cells more than once. This has been particularly important in confirming that a block in cellular growth induced by injection of antibodies or antisense D N A can be reversed through injection of the antigen or complementary protein. In the study of cell cycle control, microneedle microinjection offers the advantage that cells can be injected at precise and
METHODS IN ENZYMOLOGY, VOL. 283
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chosen times in their division cycle. This has been used for the analysis of different phases of the mitotic division processJ Finally, by delineating adjacent areas on a coverslip, it is possible to follow the effect of different solutions (e.g., antibodies against wild-type proteins, mutant proteins, posttranslationally modified proteins) on cells that experience the same growth conditions and environment, thus greatly facilitating the comparative analysis of effects on cell cycle progression or mitotic transit. The principal disadvantages of microneedle microinjection are the requirement for specialized and expensive equipment, the relatively slow learning curve, and difficulties in performing metabolic analysis on injected cells. In this chapter it is hoped that we can minimize the difficulties and pitfalls encountered in applying antibody microinjection to the study of cell cycle regulation. Procedure
Equipment There are a number of simple basic requirements for microneedle microinjection: an inverted microscope with long working distance objectives-10x and 25x are essential, a micromanipulator (manual, pneumatic, or electronic), a means of pulling needles, a source of cells preferably growing on glass coverslips, and a sterile (or sterilizable) room that is not subject to either significant ground or air vibration.
Microscopes The only important point is that the microscope must have a fixed stage. It is impossible to inject on a microscope in which the focal depth is varied by moving the sample and not the objectives. We have used either a Leica Labovert or a Zeiss Axiovert. The Labovert presents the principal advantage over the Axiovert--the eye level of the operator is above the microscope stage. Looking down into the dish significantly facilitates accurate placing of the needle over cells to be injected. All the major inverted microscope manufacturers have now adopted a style similar to the Axiovert with a high stage at or just below the level of the oculars, making it more difficult to look down into the dish to be injected.
Manipulators Essentially three types of manipulators exist: (1) manual manipulators with six controls: coarse and fine X, Y, and Z; (2) pneumatic manipulators A. Fernandez, D. L. Brautigan, and N. J. C. Lamb, J. Cell Biol. 116, 1421 (1992).
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in which the coarse manipulations are still made manually on the manipulator but the fine control is achieved through pneumatic tubing containing either air (de Fonbrune, Paris) or oil (Narashigi, Tokyo, Japan); and (3) electronic manipulators controlled either manually from a joystick or through a computer interface. Microinjection of mammalian cells requires only the gentle lowering of the needle into the cell in a single vertical move. Since neither the plasma membrane nor the nuclear envelope provides any mechanical resistance to the needle, there is no need for complicated maneuvering of the needle in any other direction. We have chosen Leica manual manipulators for a number of reasons. The manipulator is robust and prone to little likelihood of damage with the frequent changing of the needles. The Z (vertical) fine control of the manipulator is separated from the fine X-Y. This physical separation prevents the needle from moving in skew, which rips the membrane and kills cells. Finally, injection is performed by lowering the needle vertically into the cell under the direct control of the thumb and index finger, while the left thumb applies increased pressure on an air-filled syringe. This direct cause-and-effect link between the hand and the needle means no lag is experienced. Pneumatic manipulators that use air are extremely fragile and sensitive to air vibration. Oildriven pneumatic manipulators are less fragile but more sluggish in response. Electronic manipulators in which the coarse and fine X and Y are controlled electronically are very much slower to use. The most robust manipulator in which the needle can be changed and returned to the point of injection in the shortest time scale is the best equipment for performing large-scale microinjection studies.
Pulling Microcapillaries The quality of the needle essentially determines whether injection will be successful. Three essential criteria are the shape of the needle, its size, and the ease of manufacture. Mammalian cells are relatively shallow (3 ~m at the thickest point) and since the user has to accurately place the needle inside the cell volume and at a precise place in the cells (i.e., nucleus or cytoplasm), the better the quality of the image, the more accurate the injection. Since the needle sits in the solution that baths the cells, a needle that widens too rapidly will result in a poor quality phase contrast image. Many commercially available needles widen within the first 0.5 cm, which dramatically reduces the image quality. The needles we use widen out 4-6 cm from the tip, placing the wider region of the capillary out of the media bathing cells. Since a typical fibroblast is 20-30/~m with a nucleus that is 5-7 /.~m in diameter and 1-2 /~m thick, and smaller cells just have less cytoplasm without significant change in the size of the nucleus, needles should range in size from 0.2 to 0.5/~m final inner diameter.
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To produce a long shank and small diameter from a given capillary requires a two-step pulling process in which the bulk of the shank is produced in an initial prepull and the actual needle is formed in a second pull. Double pulling is possible on commercial pullers. The needle shown in Fig. 1A was produced by double pulling, on a Narashige puller. However, for prepulling needles, hand pulling over a small Bunsen flame is the simplest method. Break capillary tubes (1.5 mm o.d., 1.2 mm i.d., Kimble Glass, Toledo, OH) into 25-cm portions. Regulate a Bunsen so that the flame is 1 cm. Holding the tube at each end between the thumb and index finger, introduce the capillary into the flame at the center of the tube and rotate the tube one turn such that it is evenly heated over all sides. As the capillary' begins to melt, withdraw the tube from the flame and gently separate your wrists to produce a constriction of 5-8 cm. The pulled capillary held by one end should bend down evenly at an angle of 70-90 °. The final pull is achieved in a simple horizontal flat-bed puller. The puller comprises two clamps, a central electrode, two springs, and a roller track. The prepulled needle is placed into the clamps with the middle of the constriction passing through the electrode. The electrode is heated to red heat and the needle separated by a constant force generated by the two springs. The natural variation of hand-pulled needles requires an easy means of sizing the product. The simplest method is to place the needle onto a phase-contrast microscope and under a 25 or 32× objective, and look for the presence of defraction rings in the shank of the capillary and the form of the needle, Three or four (ideally) distinct schlieren diffraction patterns should be clearly visible along the wall of the tube (see Fib. 1B). Needles in which the end appears either flattened or as a black point are generally
F:G. 1. A photomicrograph showing micropipettes pulled either by an automated puller (double pull) (A) or by hand prepulled {B). Inset: a higher magnification of the automatically pulled micropipene. Bar: 10 tzm (A); 5 / . m (B).
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too large. The definitive test comes when needles are introduced into the solution to be injected. When observed under the microscope, as the needle enters the solution to be injected, a good needle will immediately draw up liquid and the liquid interface will continue to slowly rise up the capillary. A blocked needle will not draw any liquid into the tube. A needle in which a small amount of liquid enters and the interface rises no more is too small: the capillary action of the opening prevents further liquid from being drawn into the needle.
Sample Preparation Any solution to be microinjected must be in a buffer solution that is physiologically close to the intracellular environment and that alone has little or no strong buffering function in cells. Physiological saline solutions are acceptable. Most antibodies can be injected in 1/2x Dulbecco's phosphate-buffered saline (PBS). Before any microinjection experiment, solutions should be cleared of insoluble debris by centrifugation at 16,000 g for at least 15 min. As soon as the solutions stop spinning, remove a 0.5to 1-tzl aliquot and spot it on the inside surface of a plastic culture dish lid. If the antibody solution to be injected is less than 0.2 mg/ml, add sterile spun immunoglobulins (IgGs) of the same species to act as marker for the injected cells. After aliquoting the spots, cover them with the base of the dish and store on ice. Ceils It is preferable to use cells growing on glass coverslips for two reasons. Subsequent manipulation of the cells after microinjection is easier with cells on glass and the optical quality for immunofluorescence studies is better. Most cells will attach on acid-washed coverslips provided they are seeded as a concentrated drop on top of each coverslip and incubated for a few minutes at 37 °, before dilution by filling the dish with culture medium. A critical point for cell cycle-related studies is to achieve a good synchronization of the cells (over 80-90%) at a given stage of their division cycle. This is easier to do with nontransformed cells than with transformed cell lines because the simplest way to first synchronize cells is via serum withdrawal from the culture medium: normal cells are totally dependent on growth factors to sustain their growth. Therefore, in the absence of growth factors in the culture medium, they will all stop after 36-48 hr in a quiescent stage GO, from which they will synchronously enter G1 on growth factors readdition to the medium. A second level of synchronization is then applied, by blocking cells from entering S phase at the end of G1, which in normal fibroblasts is reached after 16 to 18 hr. To block cells from entering S phase,
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we add 2 mM hydroxyurea (HU) to cells in G1. The drug inhibits DNA synthesis by blocking the upstream synthesis of deoxynucleotides. Reversal from this block is achieved by extensive washing of the HU in serum containing DME, between 20 and 24 hr after cells refeeding from GO. Relief from this second block allows more than 90% cells to enter S phase within 1 hr and pass through mitosis within 8 hr following HU wash. The mitotic index for these cells is more than 30% between 7 and 8 hr after lifting the HU block. This is a good synchronization at that final point of the division cycle, knowing that mitosis lasts 30-40 min in a 22-24 hr division cycle in the fibroblast cell lines we use. Suitably synchronized cells are brought to the microinjection room during the centrifugation of the samples. On the microscope stage, using sterile forceps, areas of cells to be injected are demarked on the cell monolayer by scraping away in a line surrounding cells to delineate a shape (e.g., square, circle, triangle) (Fig. 2). These shapes allow easy subsequent retrieval of the injected cells on the coverslip after immunofluorescence staining, without need to resort to physical demarcation of the coverslip.
F1G, 2. Demarcation of a zone of cells to be microinjected using the cell scraping technique, A phase micrograph of a region of subconfluent HS68 cells in which an area of cells has been demarcated by scraping with fine forceps. This region contains approximately 80 cells. Bar: 40/zm.
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Microinjection Technique The principle of mammalian cell microinjection is very simple: a needle filled with the solution to be injected is introduced into the cell either in the nucleus or the cytoplasm by a single vertical move into and out of the cell. If localization is not important, the cell is preferably injected at a point close to the nucleus because this is the thickest part of the cell. The direction of flow from the needle should be toward the nucleus. By eye, injection causes a brief wave of change in phase contrast passing over the entire cell, but otherwise no other externally visible change in cell morphology. Needle Filling. Back-filling needles as recommended with many automatic microinjection apparatuses are not the best option. First, they waste samples, since at least 0.5 /El of solution is required per needle; when the needle blocks this solution is lost. Second, front-filling the needle by aspiration into the needle ensures that the solutions that enter the needle have already passed through the hole via which they will subsequently leave the needle, reducing the time wasted on a blocked needle. METHOD. Place a needle in the needle holder, which is connected via a leakproof air-filled tubing system to a 50-ml plastic syringe. Align the spotted sample to be used over the objective and, by hand, approach the needle to within 0.5 mm of the spot above it (the advantage of the manual manipulator is that this step requires 2 sec). Lower the needle into the spot and focus the needle tip in the microscope, thus checking that liquid freely enters the needle and rises. While still watching in the microscope, pull on the syringe piston to continue to draw in solution for at least 1 min. Microinjection. Once the needle has filled, quickly transfer a dish of cells from the incubator to the microscope and approach the needle so that it is vertically aligned over the coverslip but not in contact with the media. Separate the Tygon tubing from the syringe and fill the syringe with 25-30 ml of air (about half full). Reconnect the Tygon tubing. Moving the needle horizontally locate it as an out-of-focus object above the cells. Change the objective for the 25× (or 32×). With the cells in focus, push lightly on the syringe to provide a low-level positive pressure to prevent capillary action from drawing medium into the needle while lowering it onto the cells. Using the fine X-Y, align the needle to a point juxtaposed to the nucleus of the cell to be injected. Without touching the X-Y, lower the needle into the cell, simultaneously increasing the pressure on the syringe. In the beginning, you can maintain a constant pressure on the syringe if necessary. As the needle touches the cell, a wave of discoloration marks the entering of liquid inside the cell. Quickly lift the needle from the cell by turning the fine Z knob in the opposite direction. With a good needle and some practice, 100 cells can be injected in less than 5 min and, at its maximum,
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1 cell/sec can be injected. It is possible to replace the syringe with an automated injection pump, but we have never found this to be an advantage. Ceils are then immediately returned to the incubator and allowed to recover for 15-30 min before fixation or observation. Do not use a heated stage and enclosed environment because it is preferable that the cells enter a semiresting state during the injection period, which is kept shorter than 15 rain. The typical volume injected per cell depends on the size of the cells and the site of injection. Graessmann and Graessmann 2 estimated that the injection volume into Swiss 3T3 cells was of the order 1-2 × 1014 liters. Using iodinated bovine serum albumin (BSA) and injecting 10 4 cells in the cytoplasm, we also determined that the average injected volume in an REF52 cell is 2-3 × 1014 liters, which represents an average of 5-10% of the cell volume (for REF52 or HS68 fibroblasts). Cell Fixation and Staining We use two methods to fix cells prior to immunofluorescent staining. Formalin/acetone fixation has the advantage that it fixes and crosslinks proteins prior to extraction of membrane components (which are removed by acetone). It has the disadvantage that the fixation is relatively slow and dynamic cellular components may move or be altered during the fixation period. Alternatively, cells are fixed and extracted instantaneously with methanol and acetone. As for all solvent fixations, the methanol and acetone must be below freezing to avoid generation of artifactual intracellular auto fluorescence. Fixation in Formalin. Transfer a coverslip from D M E into 2 ml 3.7% formalin in PBS for 5-10 min. Discard the formalin and extract cells for 30 sec in 2 ml absolute acetone (-20°). Discard the acetone and rehydrate cells in 5 ml PBS supplemented with 0.5 mg/ml BSA (which serves to block reactive sites on the fixed cells). Fixation in Methanol. Transfer a coverslip from D M E into 2 ml absolute methanol at - 2 0 ° (analytical grade); incubate for 3-5 min. Discard the methanol and add 2 ml of acetone ( - 2 0 °) for 30 sec. Rinse cells in 5 ml PBS for 30 sec and block cells as described above in P B S - B S A . The reason for adding acetone after methanol is that acetone is more miscible in water than methanol, thus reducing the risk of damaging cell structures when swapping from solvent to water-based solutions. lmmunostaining. For studies monitoring D N A synthesis, where cells are to be double stained for 5-bromodeoxyuridine (BrdU) incorporation into D N A and anti-cyclin A, we used a technique of double fixation in 2 M. Graessmann and A. Graessmann, Methods Enzymol. 101, 482 (1983).
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which cells are fixed and stained up to the last fluorochrome-conjugated incubation step for the antigen (here cyclin A), and then refixed before processing for BrdU incorporation staining using the acid treatment technique: 10 min in 4 N HC1, before extensive rinsing in water and PBS and staining for BrdU with a monoclonal anti-BrdU. This two-step procedure allows good quality staining for the first antigen, here cyclin A, which otherwise would be lost by the acid treatment required to stain for BrdU. For this two-step technique to be successful, the fluorochrome used with the first step must be Texas Red. Fluorescein is rendered nonfluorescent by acid washing.
Strategies Used in Analysis of Cell Cycle Control through Antibody Microinjection Antibody microinjection has been used to study a wide variety of events within the cell cycle, from the implication of transcriptional regulatory factors to the role of protein kinases and phosphatases in mitotic events. Some of the early examples involved microinjection of antisera directed against transcription factors such as c-fos3 or serum response factor (SRF), 4 to inhibit subsequent G1-S transition. With the discovery of the cyclindependent kinase (cdk) family and their associated regulatory subunits (cyclins), many other studies have subsequently been carried out to determine the particular role and regulation of members of this family of proteins. The key point with respect to microinjection of antibodies relates to their mode of action within the cell. Antibodies can inhibit cellular functions in an immediate manner by one of two means: (1) by preventing the physical interaction between proteins: substrate and enzyme, regulatory and modulator, receptor and activator; and (2) by blocking the activity of the protein by preventing the interaction with its substrates or ligands, for example, for a kinase, with ATP. Despite many studies that have described the use of inhibitory antibodies, very few antibodies act as true inhibitors when microinjected. A third, but less immediate, way of affecting a protein function via antibody microinjection, especially for nuclear proteins, involves binding it in the cytoplasm, as it is synthesized. Here, there are two possibilities: (1) the protein being studied not yet present in the cell, which can be the case for proteins such as c-los or cyclins that are synthesized and degraded at a precise time in the cell cycle. (2) The target protein is constitutively present in the cell. In this case, the effect is comparable to
3 K. R. Riabowol, R. J. Voska, E. B. Ziff, N. J. C. Lamb, and J. R. Feramisco, MoL Cell BioL 8, 1670 (1988). 4 C. Gauthier-Rouvi6re, J.-C. Cavadore, J . M . Blanchard, N. J. C. Lamb, and A. Fernandez, CeHReg. 2, 575 (1991).
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antisense cDNA microinjection in that it requires the existing nuclear protein to turn over before inhibition is effective.
Microinjection of Anti-Cyclin A to Show Requirement .for Cyclin A in S Phase We have demonstrated the requirement for cyclin A in the induction of S phase in mammalian cells initially through the microinjection of affinity purified antibodies. Cyclin A is an example of an ideal substrate for microinjection of antibodies. Cyclin A protein is not synthesized prior to S phase and microinjection of anti-cyclin A was fully inhibitory until just prior to S-phase onset. After this time point, microinjection of anti-cyclin A into the cytoplasm had little or no effect on S-phase transit. During S phase and G2, cells injected in the nucleus with anti-cyclin A block immediately and do not proceed further toward mitosis, whereas cells injected in the cytoplasm continued to mitosis and in most cases divided. In view of this immediate effect of nuclear microinjection of anti-cyclin A, it is difficult to conclude that cyclin A is required for mitotic entry rather than for progression through G2, although some studies have drawn that conclusion. As we demonstrated in the inhibitory effects of anti-cyclin A on S-phase entry, for safe conclusions, the effective knockout of cellular protein function should be achievable in another manner or be possible to rescue. We used antisense cyclin A injection to prevent the synthesis of cyclin A proteins, which also blocked S-phase entry. This event was fully reversible with the injection of purified active cyclin A proteins, s In this particular case, because cyclin A is required in the nucleus and antibody injection acts by sequestering newly synthesized cyclin A in the cytoplasm, reinjection of cyclin A protein in the nucleus could effectively rescue S-phase inhibition, since the injected antibodies are confined in the cytoplasm. Except where inhibition and rescue involve microinjection in two different cellular compartments, we have never seen a convincing rescue of cells blocked by antibody microinjection, probably because to saturate and titrate the injected Igs would involve the injection of very high and, therefore, unphysiological concentrations of antigen.
Probing Role of Phosphatase Type 1 during Mitotic Transit We have also examined the role of protein phosphatases in the regulation of cell division using, in particular, an affinity-purified antibody directed against the catalytic fragment of protein phosphatase type 1 (PP-I). PP-I is involved in a number of different events prior to mitosis, and is particularly 5 F, Girard, U. Strausfeld, A. Fernandez, and N. J. C. Lamb. Cell 67, 1169 (1991).
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important in the regulation of cytoskeletal dynamics and glycogen metabolism. It is therefore difficult to envisage the use of an antibody that will deplete the cell of the protein phosphatase. First, this approach would require a large excess of antisera, and second, the phosphatase is present and active in both the nuclear and cytoplasmic compartments. The study therefore requires the use of an inhibitory antisera. Very few antisera are directly active against the in vitro activity of a protein kinase or phosphatase. We were fortunate to obtain from Dr. David Brautigan (Markey Center for Cell Signalling, Charlottesville, VA) one such antisera directed against the active catalytic site of PP-I. The combination of direct inhibitory antibodies and microneedle microinjection offers the unique opportunity to precisely inhibit the action of a protein in a spatial and temporal manner, which is completely impossible to achieve by other microinjection methods. Using microinjection of this affinity-purified antibody, we chose to examine the in vivo role of PP-1 during different phases of mitosis. In REF52 cells, mitotic figures can be identified as early as prophase. To study the role of PP-1 during early mitosis, cells were synchronized by serum starvation and hydroxyurea (HU). Five hours after release from HU, cells were microinjected either in the cytoplasmic or nuclear compartments and allowed to continue into mitosis. Cells were left sufficient time to have completed mitosis (a further 8 hr) before fixation and observation. Using this technique, we observed that greater than 90% of injected cells successfully complete the progression from prophase to metaphase, but became blocked with an intact microtubule spindle unable to enter anaphase. This state was stable for between 16 and 20 hr, which reflects the half-life of this affinity-purified antibody. Cells were injected with a 1.0 mg/ml solution of affinity-purified antibodies in the needle, which reflects introducing about 0.05 mg/ml of proteins per cell. Assuming the antibodies to be IgG, this would reflect introducing 30,000 molecules per cells, an intracellular concentration of -0.5 txM. The true intracellular concentration of PP-1 has not been determined in these cells, but considering that the level of an abundant kinase is in the order of 1.0/zM, this concentration of injected antibodies is unlikely to be excessive. Indeed, reducing the concentration of antibodies from 0.5 to 0.1 to 0.05 caused a progressive increase in the numbers of cells that would successfully break through the mitotic inhibition. To determine if PP-1 inhibition was still inhibitory at a later time in mitotic progression, we chose to inject cells at different phases after metaphase. When cells were injected in anaphase A, the antibody freely distributes throughout the anaphase figure. These cells were immediately blocked. If, however, cells were allowed to proceed to the end of anaphase A (as judged by phase contrast visually) before microinjection, liquid from the needle could be accurately guided into one of the reforming pair of daughter cells.
[7]
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83
Under these circumstances we could determine that anti-PP-1 injection was preventing one of the daughter cells from completing its mitotic exit-respreading and chromatin decondensation. Staining for the injected IgGs after incubation and fixing not only allowed us to unambiguously identify the injected cells, but also to show the location of injection, since the IgGs remain accumulated at their site of injection. Although these two examples reveal some of the possibilities of the antibody microinjection technique, the new user should always consider the effective controls that are required for antibody microinjection. A simple set of rules should include the following: the inhibitory activity should be present in the antisera from the outset (i.e., in the serum). Antibodies that show good binding by blotting may be of no use for microinjection since the first case involves recognition of denatured epitope sequences and the second requires binding to sites present in the native conformation.
Acknowledgments Work and technical developments described here were supported by grants from Liguc Nationale contre le Cancer (to N.L.), A.R.C. (1306 to N.L.), and Association Francaise contre les Myopathies ( A F M to A.F.).
[7] S m a l l P o o l E x p r e s s i o n S c r e e n i n g : I d e n t i f i c a t i o n of Genes Involved in Cell Cycle Control, Apoptosis, and Early Development
By K E V I N D.
LUST1G, P. T O D D S T U K E N B E R G , T H O M A S J. M C G A R R Y , RANDALL W. KING, VINCENT t . CRYNS, PAUL E. MEAD, LEONARD I. ZON, JUNY1NGYUAN, and MARC W. KIRSCHNER
Introduction Traditional genetic and biochemical methods have been quite successful in identifying genes that are essential for cell cycle progression and early embryonic development, among other diverse biological processes. Nevertheless, only a small fraction of the genes in the vertebrate genome has been functionally characterized. In this chapter, we describe a systematic and broadly applicable approach to cloning genes based solely on the biological activities or biochemical properties of the gene products. This approach does not depend on knowledge of the D N A sequence of the
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