Microinjection of p34cdc2 kinase induces marked changes in cell shape, cytoskeletal organization, and chromatin structure in mammalian fibroblasts

Cell, Vol. 60, 151-165,

January

12, 1990, Copyright

0 1990 by Cell Press

Microinjection of p34cdc* Kinase Induces Marked Changes in Cell Shape, Cytoskeletal Organization, and Chromatin Structure in Mammalian Fibroblasts Ned J. C. Lamb,’ Anne Fernandez,’ Annette Watrin, Jean-Claude Labbe, and Jean-Claude Cavadore * Cell Biology CRBM CNRS/INSERM BP. 5051 34033 Montpellier Cedex France

Summary We have examined the effects of elevating the intracellular levels of ~34~~~~ kinase by microinjection into living mammalian cells. These studies reveal rapid and dramatic changes in cell shape with cells becoming round and losing the bulk of their cell-substratum contact. Such effects were induced at all times in the cell cycle except at S phase and were fully reversible at S phase or mitosis. Similar results were obtained with the homogeneous catalytic subunit of ~34~~~ kinase or ~34~~~~ kinase associated with cyclin B. These alterations were accompanied by a marked reduction in interphase microtubules without the spindle formation, actin microfilament redistribution, and premature chromatin condensation. Although these changes closely mimic the events occurring during early phases of mitosis, ~34~~~ kinase-injected cells were not induced to pass further into division. These data provide detailed evidence that ~34~~~~ kinase plays a major prerequisite role in the rearrangement of cellular structures associated with mammalian cell mitosis. Introduction The mechanism that brings about the coordinated control of the eukaryotic cell division cycle remains one of the key problems in cell biology. Although little detail is known of the proteins that bring about the proper timing for the diverse processes that occur during the cell cycle, a number of potential regulators have been identified (reviewed in Lohka, 1989). Probably the most well-documented eukaryotic cell cycle modulator is a 34 kd histone Hl kinase (unresponsive to cyclic nucleotides, calcium calmodulin, diacylglycerol, or polyamines [woodford and Pardee, 1986]), first identified as the product of the cd@ gene of Schizosaccharomyces pombe (Beach et al., 1982; reviewed in Nurse, 1985; Hayles and Nurse, 1986; Lee and Nurse, 1988; Lohka, 1989). Recently, several studies identified that cdc2 kinase was a structural and functional homolog of a 34 kd kinase, component of the maturation promoting factor (MPF), a non-species-specific mitotic inducer that directly triggers the entry of oocytes into mitosis without requirement for protein synthesis (Dunphy et al., 1988; Gautier et al., 1988; Arion et al., 1988). Homologs of the ~34~~~~ kinase have since been identified in all eukary-

otic cell systems so far studied, including human cells (Lee et al., 1988; Draetta et al., 1987, 1988; Draetta and Beach, 1988) suggesting that it forms part of a ubiquitous and conserved cellular regulatory pathway (Lohka, 1989). The inherent role of differential protein phosphorylation has long been implicated in the regulation of the cell cycle with the description of proteins that become specifically phosphorylated during DNA synthesis (Gurley et al., 1974) G2 (Gurley et al., 1974; Domon et al., 1986) or mitosis (Davis et al., 1983; Sahasrabuddha et al., 1984; Vandre et al., 1986; Nishimoto et al., 1987). These include the nuclear lamins (Ottaviano and Gerace, 1984), vimentin (Zieve, 1985) pp60c-src (Chackalaparampil and Shalloway, 1988; Morgan et al., 1989), and the histones (Bradbury et al., 1974; Gurley et al., 1974, 1978; Nishimoto et al., 1987) in addition to many unidentified phosphoproteins (Davis et al., 1983; Sahasrabuddha et al., 1984; Vandre et al., 1986; Capony et al., 1986; Karsenti et al., 1987). ~34~~~~ is homologous to the growth-associated histone Hi kinase or M phase-specific Hi kinase (Arion et al., 1988) one of the protein kinases that becomes transiently activated at the G2-M transition in a wide variety of dividing cells (Lake and Salzman, 1972; Bradbury et al., 1974; Gurley et al., 1974, 1978; reviewed in Wu et al., 1986; Edelman et al., 1987; see Arion et al., 1988, for references). The phosphorylation of mitosis-specific sites on Hl in vitro (Langan, 1978, 1982) and in vivo at the time of chromatin condensation (reviewed in Wu et al., 1986; Nishimoto et al., 1987) suggested that the growth-associated Hl kinase may be directly involved in the condensation of the chromatin (Matsumoto et al., 1980; Mueller et al., 1985; Nishimoto et al., 1987). However, this hypothesis still awaits direct in vivo evidence. Little else is known of the physiological substrates or role of cdc2 kinase in modulating the complex rearrangements of cells that occur at mitosis. Recent evidence has suggested that ~34~~~~ kinase may phosphorylate the ubiquitous cellular tyrosine kinase c-src (Morgan et al., 1989; Shenoy et al., 1989) and in so doing play a role in the activation of src, which takes place at mitosis (Chackalaparampil and Shalloway, 1988). While the level of ~34~~~~ kinase remains constant throughout the cell cycle, its activity oscillates and the cell cycle-dependent phosphorylation/dephosphorylation of the cdc2-MPF complex has been reported (Simanis and Nurse, 1986; Draetta and Beach, 1988; Draetta et al., 1988; Labbe et al., 1989a; Gautier et al., 1989). Alternatively, p34CdC* activity may be regulated by its interaction with associated components, the phosphorylation and/or synthesis of which may also change in a cyclic manner (reviewed in Lohka, 1989). Examples of proteins associated with cdd kinase have been identified in most cdc2 preparations so far reported, including yeast (Brizuela et al., 1987; Hindley et al., 1987; Wittenberg and Reed, 1988), oocytes (Gautier et al., 1988; Dunphy et al., 1988; Arion et al., 1988; Labbe et al., 1989b), starfish eggs (Meijer et al., 1989) and mammalian cells (Draetta et al., 1987; Draetta and Beach, 1988). Although the function of such

Cell 152

G2 cells and second, that the injected cells, although showing all the signs of entry into early prophase, were not induced to pass further through mitosis. These studies provide some of the first insights into the specific function(s) of the ~34~~~~ protein kinase in the major structural arrangements that take place during the mammalian cell division cycle. Results hours post serum-refeeding Figure 1. Analysis of DNA Synthesis Following 36 Hr of Serum Starvation 6% FCS

in Synchronized and Subsequent

REF-52 Cells Refeeding into

REF-52 cells were made quiescent through 36 hr of serum starvation. After refeeding into fresh 6% FCS, the cells were incubated for 2 hr intervals in 0.1 mM 5-Br-dU from 6-24 hr after refeeding. After each 2 hr pulse, the cells were fixed and stained for the incorporation of 5-BrdU into the nucleus as described in Experimental Procedures. The proportion of cells having incorporated the analog is plotted against the time elapsed following refeeding. Also shown are silver-stained one-dimensional gel electrophoretograms of purified ~34~~~’ kinase preparations: the homogeneous catalytic kinase subunit (B) and the ~34~“~~ associated with cyclin B (C).

proteins and their effect on p34 cdc2 kinase activity is still unknown, there is evidence that their binding to the kinase in the formation of multimeric complexes and changes in their phosphorylation state are related to the activity of the kinase (Wittenburg and Reed, 1988) and may change during the cell cycle (Draetta and Beach, 1988). In particular, a group of proteins, the cyclins (Evans et al., 1983; Swenson et al., 1986; Standart et al., 1987; Pines and Hunt, 1987; Booher and Beach, 1988; Solomon et al., 1988; Goebl and Byers, 1988) which show cell cycle-specific synthesis and degradation in oocytes and early embryonic cells (Evans et al., 1983; Swenson et al., 1986; Standart et al., 1987; Solomon et al., 1988; Murray and Kirschner, 1989; Murray et al., 1989), appear to be associated with ~34~~~~ kinase (Draetta et al., 1989; Meijer et al., 1989; Labbe et al., 1989b) and may also be involved in directly or indirectly regulating ~34~~~~ activity (Evans et al., 1983; Swenson et al., 1986; Standart et al., 1987; Draetta et al., 1989; Murray and Kirschner, 1989; Murray et al., 1989). Although a recent study using microinjection of antibodies against ~34~~~~ kinase has documented the requirement for this kinase in proliferating cultured cells (Riabowol et al., 1989), there is a still a paucity of data detailing its potential role and regulation in mammalian cell division. To address these questions, we have investigated the effect of microinjecting into synchronized rat embryo fibroblasts the active protein kinase purified from starfish oocytes, as a catalytic subunit or associated with cyclin B. Elevating intracellular kinase levels using either preparation leads to dramatic changes in cell shape and cytoskeletal and chromatin organization that closely mimic those observed in cells during the initial phases of mitosis. However, the two striking findings are first, that the effects of the injected ~34~~~~ kinase were observed even in cells made quiescent through serum deprivation and in Gl or

Cell Synchrony Initial experiments were performed to assess the effects on cell morphology and structure of microinjecting ~34~~~~ kinase purified from Marthasterias glacialis oocytes in normal rat embryo fibroblast cells. Since cdc2 activity appears to be modulated during the cell cycle (reviewed in Lohka, 1989) we chose to inject cells at different times in their cell division cycle. For synchronization, cells plated on glass coverslips were serum deprived for 36 hr. Under these conditions, the cells have a doubling time of 25-28 hr upon refeeding. To assess more accurately the proportion of cells in a given phase of the cell cycle, we estimated the percentage of cells entering S phase by measuring the incorporation into nuclei of a DNA-specific label, 5-bromodeoxyuridine (5Br-dU), detected by immunofluorescence staining with a monoclonal antibody. Figure 1 shows the proportion of cells that incorporate the label at different times after serum refeeding. Clearly, little or no DNA synthesis occurs during the first 12 hr after refeeding, and less than 15%-20% of cells commence S phase between 12-15 hr. During the period 15-18 hr following refeeding, approximately 500/o-60% of the cells incorporate the label, while over the period 18-20 hr the proportion rises to 75%. Between 20 and 22 hr, less than 25% of the cells incorporate the label. Similar results were obtained when measuring the incorporation of another label, [3H]thymidine, by emulsion autoradiography (data not shown). From these studies we can define the following phases in REF-52 cells under these conditions of synchronization: a quiescent GO phase (when the cells are still serum starved), an early (l-8 hr after refeeding) and late (12-15 hr after refeeding) Gl phase, an S phase between 15 and 20 hr after refeeding, an S-G2 transition between 20 and 22 hr, and a G2 phase from 22 hours till mitosis (G2 was routinely assumed to be 23 hr after refeeding). It is clear that to analyze cells in the Gl phase of the cell cycle we should ensure that they are injected prior to 15 hr, whereas cells in G2 should be injected after 22 hr. Analysis of p34cdc2-lnduced Changes in Cell Morphology at Different Periods of the Cell Cycle To examine specifically the consequences of elevating intracellular levels of p34Cdc2 kinase, we microinjected the purified active ~34~~~~ kinase (or control solutions) into the cytoplasm of synchronized living cells at different times after serum refeeding. The ~34~~~~ kinase was isolated from starfish oocytes by the methods of Labbe et al. (1988a, 1988b, 1989a, 1989b). Two different preparations of active purified ~34~~Cz kinase were used. One con-

Microinjection 153

of ~34~~~~ Kinase

into Mammalian

Cells

tained active ~34~~~~ kinase essentially as a single protein, whereas in the other the ~34~~~~ active kinase had been purified in a 1:l molar ratio with a 45 kd protein identified as cyclin 6 (Labbe et al., 1989b). One-dimensional gel electrophoretograms of the two kinase preparations used throughout this study are shown in Figures 16 (essentially homogeneous ~34~~~~ kinase) and 1C (cyclin B-containing ~34~~~~ kinase). Prior to use, solutions of p34cdc2 were dialyzed into a microinjection buffer (100 mM potassium glutamate, 40 mM potassium citrate, 1.0 mM MgS04 [pH 7.251). Different amounts of the purified ~34~~~~ enzyme were used in initial experiments (up to 0.5 mglml), and we decided to use a concentration of 0.1 mglml (specific activity, 1 ~mollminlmg) in the needle routinely. As discussed in Experimental Procedures, we estimate that the injection of the kinase introduces approximately 6 x 104 molecules into each cell. Figure 2A shows a group of quiescent rat embryo fibroblast (REF-52) cells immediately prior to injection with ~34~~~~ kinase (all the cells in this field, with the exception of the three cells marked with arrows, were subsequently injected with the kinase). Within 30 min, injection of ~34~~~ kinase induces profound changes in cell shape. Initially, the cells become phase darkened around the nucleus and begin to lose cell-substratum contact, particularly at the cell periphery (Figure 28). By 60 min following injection (Figure 2C), the effect has become more pronounced. The original shape of the cells can no longer be discerned, the cells have continued to compact around the nucleus, and have further lost contact with their sub-

stratum. The cells at this phase also show feet-like extensions of the cytoplasm extending from around the nucleus. Moreover, in some cells noticeable changes in the aspect of the nucleus can be observed, with nuclei showing signs of premature DNA condensation. The changes in cell shape induced after kinase injection remained stable for at least 36 hr if the injected cells were kept quiescent, a time after which a majority of the cells (including those uninjected) started to die. However, if the cells were refed with serum, these effects on cell morphology were fully reversed after 15-18 hr as described below. This observation indicates that when cells are maintained in a quiescent state (deprived of serum), they are unable to regulate the additional ~34~~~~ kinase activity brought about by the injection. Microinjection of different amounts (0.01-0.5 mglml) and different preparations (including those from other sources, such as Xenopus oocytes) of the cdc2 kinase always induced such changes in cell morphology as described above (data not shown). Moreover, injection of the cyclin-containing preparation also induced the same extent and rate of cell shape change (data not shown). Similar changes were also observed in cell lines other than FIEF-52, including NIH 3T3, HeLa, CV-1, normal rat kidney cells, bovine fibroblasts, and normal human fibroblasts (data not shown). However, the rate at which these changes in cell shape took place varied markedly with different concentrations, such that injection of higher concentrations (0.3-0.5 mglml) of the ~34~~~~ kinase induced the cells to become round more rapidly (within lo-15 min)

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Figure 3. Microinjection of Active p34 cdc* Kinase Leads to Profound Changes in Cell Morphology in Actively Growing Cells during Gl Phase of the Cell Cycle REF-52 cells were synchronized through serum starvation. After refeeding, the cells were microinjected with purified p34cdcZ kinase (without cyclin). Shown are phase-contrast micrographs of living cells 30 min after injection (A) and 60 min after injection (8). The injected cells are marked by arrows. Bar = 10 pm.

than described here, while lower concentrations induced the effects more slowly. At the lowest concentration (0.01 mglml), the induced effects were also less pronounced and could be rapidly reversed in some cases (data not shown). These changes in cell shape are very similar to those observed during early mitosis when cells become round and lose cell surface contact (Aubin et al., 1979). To facilitate a more direct comparison, Figures 2D-2F show the progression of two uninjected REF52 cells (arrows) through mitosis. Figure 2D shows the cells at the earliest phases of prophase when slight differencesin the shape of the cells are apparent. By the next time points (20 and 25 min later), profound changes in cell shape have occurred, as the cell becomes round and pulls away from the surface

of the dish (Figures 2E and 2F). These changes are similar to those observed in ~34~~~~ kinase-injected cells at the peak of the effects of the kinase (Figure 2C). By 60 min a normal mitotic cell has completed mitosis and shows two distinct daughter cells (data not shown). Although the changes in cell morphology shown at the end of prometaphase are very similar to those we observed following injection of the kinase, there is clearly a difference in the speed of these changes in injected cells and mitotic cells; however, this probably reflects the concentrations of the injected kinase. We have previously shown that the injection of the buffer alone does not affect cell shape or morphology (Lamb et al., 1966, 1989). Additionally, supporting the conclusion that the effects on cell shape we observed were specific to the injected kinase, these effects could be abolished in a number of ways, such as incubation of the kinase at 60% for 5 min, repeated freeze-thawing of the kinase (8-10 times), or preincubation of the kinase preparation with either pl3Sepharose beads or an affinity resin containing anti-PSTAIR antibodies, followed by removal of the beads (data not shown). Furthermore, similar effects on cell morphology were observed after injection of a crude inactive oocyte extract, artificially activated in the absence of ATP (data not shown). Since this procedure has previously been shown to result in the specific activation of ~34~~~~ kinase through dephosphorylation (Labbe et al., 19884 1989a), this finding confirms that the effects we have observed are specific for the injection of the active ~34~~~~ kinase preparation. We next assessed the effects of injecting ~34~~~~ kinase in cells that had reentered the cell cycle. Figure 3 shows a group of cells injected 12 hr after serum refeeding (late Gl). Thirty minutes after injection, cells (Figure 3A, marked by arrows) showed changes in morphology similar to those observed in quiescent cells (described above). By 60 min after injection (Figure 38) many of the injected cells (arrows) had undergone extensive changes in shape, compacting around the nucleus and leaving only thin extensions of cytoplasm, in a fashion similar to that observed in quiescent cells. Again, these changes in cell morphology closely mimic those observed in FIEF-52 cells entering mitosis (Figures 2E and 2F), and they are reminiscent of the changes in cell morphology observed in cells after activation of the src tyrosine kinase (Wang and Goldberg, 1976; Edelman and Yahara, 1976). These effects on cell shape (like those in quiescent cells) were induced independently of the site of injection, either in the nucleus or in the cytoplasm. Moreover, immunofluorescence staining of injected cells with anti-p34cdc2 kinase antisera revealed kinase staining also in the cytoplasm of injected cells. Owing to the high background staining for endogenous ~34~~~~ kinase in FIEF-52, specific nuclear localization of the injected kinase could not be discerned (data not shown). Unlike the changes induced in quiescent cells, these changes in cell shape were fully reversible in growing cells. Figure 4 shows the kinetics of reversal in two cells injected with ~34~~~~ kinase 15 hr after refeeding. Initially, as in the cells injected at 12 hr, the cells become round

Microinjection 155

of p34CdQ Kinase

into Mammalian

Ceils

Figure 4. Changes in Cell Shape p34cdcs Kinase Are Fully Reversible Growing Cells

Induced by in Actively

To determine if the changes in cell shape induced by p34cdc2 kinase were reversible, REF52 cells were microinjected with the purified p34cdc* kinase (without cyclin) and followed for the next 2 hr. Cells were synchronized through serum deprivation and injected 14 hr after refeeding. Shown are phasecontrast micrographs of living cells prior to injection (A) and at 30 min (B), 60 min (C), 90 min (D), 120 min (E), and 150 min (F) after injection of the kinase. Two injected cells are marked by arrows. Bar = 10 w.

and lose their contact with the substratum by 30 min (Figure 46). Sixty minutes after injection, both ceils have become round in a way similar to that observed in Figure 2C (Figure 4C). By 90 min after injection (Figure 4D), while one of the cells continues to become round, the other starts to flatten. With further incubation (Figure 4E), both cells have entered the reversal process, the first being almost completely flat, while the other is commencing to flatten. By the last time point (150 min after injection), which is 17 hr after refeeding, both cells have effectively returned to being completely flat (Figure 4F). When we examined the reversibility of the cell shape changes induced by injecting the kinase at times earlier in Gl phase, we found that the majority of cells reversed to a normal flattened morphology only when reaching the period 15-18 hr after refeeding, independent of the time in the cell cycle when they were injected. Furthermore, when injected during this same period 15-19 hr after refeeding, the majority of the cells were refractory to the injection of the kinase with respect to changes in cell morphology and did not become round after this refractory period was over, whereas cells injected after 18-19 hr (in G2) were fully responsive (data not shown). Since this time interval closely corresponds to the phase of DNA synthesis in these cells (see Figure lA), it suggests that there may be features of this process that influence the responsiveness of cells to ~34~~~~ kinase. Further support for this view comes from our recent observations that coinjection of p34Cdc2 kinase and antibodies to phosphatase inhibitor I2 results in restoration of the morphological effects of the kinase (Brautigan et al., submitted). The finding that

the effects of the injected ~34~~~~ kinase reverse at specific times implies that the cells can take control of the injected kinase and neutralize its activity, strongly supporting the notion that the effects we described for ~34~~~~ kinase are truly within a physiological range. When cells were injected after the end of S phase, 20-26 hr after refeeding, the changes in cells shape were very similar to those observed in GO quiescent cells or in Gl cells, with cells losing their overall contact with the substratum and becoming round (data not shown). At these late times in the cell cycle, when increasing proportions of the cells on the coverslip were commencing division, injected cells also often proceeded into division. Cells injected at this time (like those injected at Gl) also reversed, but only after passing through division. The observation that injected growing cells can pass through S phase and mitosis also strongly suggests that injection of ~34~~~~ kinase does not interfere with the progression of the cell through the cycle. Reorganization of Cytoskeletal Networks The marked changes in cell morphology induced by the ~34~~~~ kinase closely mimic a prophase cell phenotype, and mitosis is known to be accompanied by dramatic cytoskeletal rearrangements. In light of this, we next investigated if injection of the kinase altered the distribution of the cytoskeletal networks-microtubules, actin microfilaments, and intermediate filaments. Figure 5 shows the changes in microtubule organization in cells injected in Gl. Figures 5A and 58 show a cell injected with injection buffer alone (arrow), and compared

Cell 156

Figure 5. Injection of p34cdc* Kinase Induces Marked Changes in Microtubule Organization Changes in microtubule organization induced by p34CdC* kinase injection were analyzed by immunofluorescence staining with monoclonal anti-tubulin antibody DMA 1A. Subconfluent REF-52 cells growing on glass coverslips were microinjected with ~34~~~~ kinase (without cyclin) and fixed 30 or 60 min afterward before staining for the distribution of tubulin with monoclonal anti-tubulin antibody. The primary antibodies were subsequently visualized for the distribution of the tubulin with fluorescein antimouse antibody. Alternatively, cells were microinjected with ~34~~~’ kinase containing cyclin B and fixed 60 min thereafter in gluteraldehyde with extraction. The cells were subsequentlystained with rat monoclonal antibody YL 112 and visualized with fluorescein-conjugated anti-rat antibodies. Additionally, unsynchronized REF-52 cells were fixed in methanol and stained as described for injected cells. Shown are phase-contrast micrographs (A, C, E, G, and I) and the distribution of the microtubules (B, D, F, H, and J). (A) and (B) show the distribution of microtubules in a cell microinjected with microinjection buffer alone (arrow). (C) and (D) show a cell microinjected with ~34~~~~ kinase 12 hr after refeeding and fixed 30 min afterward. (E) and (F) show the distribution of microtubules in a cell injected with the kinase 12 hr after refeeding and fixed 60 min after. (G) and (H) show the distribution of microtubules in two cells (arrows) injected with the kinase 12 hr after refeeding and fixed 60 min after injection in gluteraldehyde and stained with monoclonal anti-tyrosinated tubulin antibody. The injected cells are marked by arrows in (H). (I) and (J) show the distribution of microtubules in prophase REF-52 cells; the brightly staining asters are marked by arrows. Bar = 5 urn.

with the uninjected cells to its side little or no effect on the microtubules appears to have taken place, and cells show a typically complex distribution of microtubules. In contrast, in a cell fixed 30 min after injection of the kinase, as the first changes in cell shape take place (Figures 5C and 5D), the normal complex organization of the microtubules begins to change such that the majority of the filaments have become disorganized, leaving the cell with a reduced network of microtubules. The cytoplasm of injected cells now contains a bright background of free tubulin staining, possibly suggesting that the microtubules have disassembled into subunits. By 60 min after injection, as the cells have reached the peak of rounding (Figures 5E and 5F), the cytoplasm con-

tains markedly fewer microtubule fibers than control cells, curling throughout the cytoplasm against a bright fluorescent background (Figure 5F). Comparable reorganization of the microtubule networks was observed when Gl cells were injected with the cylin-containing ~34~~~~ kinase (data not shown). Moreover, a similar staining pattern could be obtained when injected cells were stained with two other anti-tubulin antibodies, suggesting these effects do reflect a loss of microtubules and not the loss of the epitope for the anti-tubulin antibody (data not shown). Analogous changes were also observed in injected GO quiescent cells or after fixation under different protocols, for example, -20% methanol with prior extraction (Kreis, 1987) (data not shown).

Microinjection 157

of ~34~~~~ Kinase

Figure 6. Microinjection Prophase

into Mammalian

of p34 cdc2 Kinase

Induces

Cells

Profound

Reorganization

of the Actin

Microfilament

Networks

Similar

to Those

Observed

at

Changes in actin microfilament organization following elevation of intracellular p34 cdc2 kinase levels were followed by indirect immunofluorescence using rhodamine phalloidin (to stain for F-actin). Subconfluent REF-52 cells growing on glass coverslips and synchronized by serum quiescence were microinjected with purified p34 cdc2 kinase associated with cyclin B 12 hr after serum refeeding. Included in the injection buffer was a nonspecific rabbit anti-mouse antibody to act as a marker in identifying injected cells. After incubation for 60 min, cells were fixed and processed for immunofluorescence as described in Experimental Procedures. Alternatively, unsynchronized REF-52 cells were fixed and stained with rhodamine phalloidin. Shown are phase-contrast micrographs (A and D) and immunofluorescence panels for the staining for the microinjected marker antibody (E) and the distribution of the actin microfilaments (C and E). (A-C) Phase-contrast and fluorescence micrographs showing actin distribution in cells injected with p34cdc* kinase and fixed after 60 min. (D and E) Phase and fluorescence staining for actin (E) in a prophase cell. Bar = 5 pm.

Figures 5G and 5H show microtubule staining in two ceils (arrows) injected with ~34~~~~ kinase 12 hr after refeeding and fixed 60 min afterward in gluteraldehyde with extraction (Small et al., 1968). The cells were subsequently stained with a monoclonal anti-tubulin, YL 112, which reacts with carboxy-tyrosylated a-tubulin (Wehland et al., 1983). Again, a similar pattern of reduced and reorganized microtubule staining exists in both cells. Since this antibody reacts with the tyrosylated a-tubulin, this result suggests that the effects of ~34~~~~ kinase on the microtubules do not simply reflect selection for the more stable detyrosylated tubulin. Comparable effects on the microtubule distribution were observed at all times in the cell cycle, except during the period from 15-18 hr when the cells appeared refractory to the injection of the kinase. At no time in the cell cycle (except in late G2, when the majority of cells enter mitosis) did we observe any evidence of the formation of a structure that resembles a spindle in cells injected with the purified kinase solutions. This is a pronounced difference between the effects of injected p34Cdc2 kinase and cells entering mitosis. Figures 5l-5J show a phase-contrast micrograph of a typical rounded prometaphase cell showing similar changes in cell shape to those observed after kinase injection. In contrast, the distribution of the microtubules in this cell reveals two bright asters juxtaposed to the nucleus, each displaying a strong polarized tubulin staining pattern (arrows). Since cell shape appears to be determined by the combined distribution of all three cytoskeletal networks,

we also examined the effect of ~34~~~~ kinase injection on the actin microfilaments and vimentin-containing intermediate filaments. Figure 6 shows the reorganized distribution of actin microfilaments (Figure 6C) in a Gl cell injected with ~34~~~~ kinase (stained for injected marker antibodies; Figure 6B) and incubated for 60 min. Concomitant to the changes in cell shape, the number of large actin bundles decreases and the level of diffuse staining increases, possibly resulting from an increase in the number of thin fine actin filaments. The nature and extent of the actin rearrangement is similar to that reported previously in prophase PtK cells (Aubin et al., 1979) and that observed in prophase REF-52 cells, as is clear when comparing the injected cell and the cell shown in Figures 6D and 6E. The vimentin intermediate filaments also show some changes in their distribution, but these appeared to reflect mainly the changes in cell shape (data not shown). Chromatin Condensation Although little is known of the in vivo substrates for ~34~~~~ kinase, there is considerable evidence in vitro and in vivo that this kinase is an important cellular histone Hl kinase (Labbe et al., 1988b; Arion et al., 1988; Langan, 1982; Lohka, 1989). Therefore, we examined if the injection of ~34~~~~ kinase had any significant effect on the organization of the nucleus in cells after injection into synchronized cells at different times in the cell cycle. Following fixation, the cells were concomitantly stained for the distribution of an inert marker antibody, coinjected with

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Figure

7. p3@s

Kinase

Induces

Changes

in Nuclear

Organization

The effect of p3@c2 kinase on the organization of the nucleus was assessed by indirect fluorescence staining ent stages of the cell cycle. To identify the injected cells an inert antibody solution was included in the injection staining for the distribution of the injected marker antibody, i.e., the injected cells (A and C) and the staining of 33258) (B and D). The cells were fixed 60 min after kinase injection. (A) and (B) show the effect of p3@c* cells injected with p34cdc2 kinase while serum starved. (C) and (D) show the DNA staining pattern in a cell after refeeding during Gl phase. Bar = 5 urn

the kinase, and with the DNA-specific stain Hoechst (bisbenzimide 33258). Figure 7 shows a group of serumquiescent cells, two of which have been injected with ~34~~~~ kinase (stained for the distribution of the injected antibody in Figure 7A). In uninjected cells the nucleus forms a smooth ellipsoid structure that shows a uniform even staining when analyzed for Hoechst (Figure 78). In the two cells injected with ~34~~~~ kinase (marked by antibody in Figure 7A), the DNA staining appears brighter than the surrounding uninjected cells, indicative of the more condensed state of the chromatin, and the thin compact form of the nuclei of the two injected cells is clearly different from the normal flat ellipsoid shape characteristic of the nucleus in uninjected cells. Such a distribution is characteristic of cells at the prometaphase of division, when the nuclear material becomes highly condensed and flattened. However, we could not discern any sign of nuclear envelope breakdown on either phase-contrast micrographs or by immunofluorescence using antibodies against the lamins (unpublished data). A similar staining pattern was also observed using another DNA stain, propidium iodide (data not shown). Comparable changes in chromatin staining were observed in cells injected in G2 (data not shown). However, in cell8 injected with ~34~~~~ kinase in Gl phase of the cell cycle (Figures 7C and 7D), a less pronounced change in nuclear structure occurred. Figure 7C shows antibody staining in

of the nuclei of cells injs cted at differbuffer. Shown are double fluorea icence the DNA using Hoechst (t xs-ben zimide kinase on nuclear organ ization in two injected with the ~34~~~s kinast ? 12 hr

a single injected cell (1.8x the magnification of the cells in Figures 7A and 78). Unlike cells injected during GO or G2, cells injected in Gl do not appear to lose the ellipsoid shape of the nucleus. As shown in Figures 6C and 6D, the main difference between the staining pattern in control nuclei and those of Gl cells focuses around the more granular appearance of the nuclear material in the Gl injected cells. To analyze further the nature of these structural changes in chromatin following injection of ~34~~~ kinase, we used thin section electron microscopy. Figure 8A shows a section through the nucleus of a cell in Gi phase of the cell cycle after injection of the injection buffer alone. The nucleus is essentially ellipsoid and shows the fine granular staining characteristic of chromatin. Clearly delineated are the three nucleoli, but they represent essentially the only darkly staining heterochromatic material in these cells. When comparing a similar section from a cell injected with ~34~~~~ kinase and incubated for 30 min after injection (Figure 88) the nucleus now contains a number of areas in which fine points of dense heterochromatin-like staining can be discerned (Figure 8B, arrows) in addition to the fine .granular chromatin. In cells incubated for a longer period after kinase injection (Figure 8C), the nucleus clearly contains considerably more dark heterochromatin-like staining. The nuclear material in such cells is clearly different from that observed in the control cell. The

Microinjection 159

Figure

of ~34~~~s Kinase

8. Changes

in Nuclear

into Mammalian

Organization

Cells

in Gi Cells

Injected

with p34 cdc2 Kinase

Are Similar

to Those

Found

in Early Prophase

cells in Gl phase (12 hr after refeeding) were microinTo obtain a more detailed analysis of the effects of p34 cdc2 kinase on nuclear organization, jetted with the kinase before processing and analyzed by thin section electron microscopy. Cells from the same dish injected with buffer alone were processed at the same time to act as controls. Cells in prophase were obtained by screening an unsynchronized dish of uninjected cells. Shown are comparable sections through the nucleus of a cell injected with buffer 12 hr after refeeding, fixed, and processed 60 min afterward (A), a cell injected with ~34~~’ kinase injected after 12 hr of refeeding and fixed 30 min afterward (B) or 60 min afterward (C), and a section through a prophase cell obtained from an unsynchronized dish (D). The areas of heterochromatin staining are marked by arrows in (B), (C), and (D). Bar = 200 nm

punctate appearance of the areas of heterochromatin staining is highly characteristic of the distribution of chromatin and heterochromatin associated with the early stages of mitosis (Yaniv and Cereghini, 1986). Indeed, a comparison of chromatin distribution in the nucleus of an early prophase REF-52 cell (Figure 80) with that of the ~34~~~~ kinase-injected cells shows a number of close similarities. In both cases the cells still retain an intact nuclear envelope (characteristic of early prophase) and darkly staining nucleoli, and the nuclei contain areas of darkly stained granular heterochromatin. We have consistently observed that cells injected with ~34~~~~ during Gl retain their nuclear envelope and nucleoli and exhibit a varying extent of heterochromatin formation from the state shown in Figure 88 to a state in which as much as 50% of the nuclear staining forms heterochromatin. Discussion Effects of ~34~~~~ Microinjection Mimic Cells in Prophase Although ~34~~~~ kinase is now known to be a ubiquitous component in the induction of mitosis, its specific functions in this process remain obscure. The most striking effect we report following injection of p34CdC2 kinase into

mammalian cells involves a rapid and dramatic alteration in cell shape. In both GO (quiescent) and Gl phases of the cell cycle, cells injected with active cdc2 kinase become round, losing the bulk of their cell surface contact. Control solutions or inactivated kinase do not induce any effects, implying that active ~34~~~~ kinase is integral to these effects on cell morphology. Accompanying alterations in the distribution of all three cytoskeletal networks occur that closely mimic the changes in these cells during entry into prophase. Such changes in cytoskeletal organization differ from those we have previously reported following injection of CAMP-dependent protein kinase (Lamb et al., 1988, 1989). In contrast, there are distinct similarities between the effects of injected ~34~~~~ kinase on cell shape and actin and the consequences of increased levels of the src tyrosine kinase. Transforming mutants of pp60c-Src and pp60v-src particularly are known to show wide-ranging effects on cell morphology and adhesion involving the loss of cell shape and surface contact and alterations in actin microfilament organization (Wang and Goldberg, 1976; Edelman and Yahara, 1976; cf. Morgan et al., 1989). In particular, the disruption of actin bundles we observed in cdcBinjected cells is similar to changes reported foflowing injection of purified src kinase (Maness and Levy,

Cell 160

1983). Many of the ultrastructural alterations reported following src activation mimic the changes that normally occur as cells prepare to divide, and the profound changes in cell shape at mitosis coincide with src kinase activation (Chackalaparampil and Shalloway, 1988). Recently, the site-specific phosphorylation of src kinase by cdc2 kinase at sites that become phosphorylated coincident with mitotic activation of src has been reported (Shenoy et al., 1989), and purification from Hela cells of a kinase that phosphorylates src in vivo at mitosis identified it to be a p34cdc2-associated kinase (Morgan et al., 1989). We also have observed the increased phosphorylation of a protein that migrates identically to an immunoreactive form of pp60c-src within 30 min after injection of cdc2 kinase (unpublished data). Taken together, these data strongly support a mechanism in which cdc2 kinase would modulate the changes in cell morphology we report through activation of the src kinase and/or possibly other related tyrosine kinases. Changes in Microtubule Organization Associated with the changes in cell shape and actin distribution, cdc2 kinase effects a distinct alteration in the organization of the microtubule networks, with injected cells showing a marked reduction in microtubule number. Premitotic cells also reorganize their microtubules, and onset of prophase is accompanied by reassembly of microtubules from one or (more usually) two centrosomes juxtapositioned along the nucleus. One feature of the alterations in microtubule organization induced after ~34~~~~ kinase injection was the apparent absence of a spindlelike structure in either Gl or GO cells, suggesting that factors other than ~34~~~~ are needed to direct this event. Since little is known of the in vivo substrates for ~34~~~~ kinase in mammalian cells other than possibly histone Hl (see Arion et al., 1988) and recently the RNA polymerase II (Cisek and Corden, 1989) the question remains of how ~34~~~~ kinase brings about its acute effect on the microtubule cytoskeleton. Such effects may be related to an activation of src, which has been associated with tubulin phosphorylation in vitro (Mannes and Levy, 1983) and microtubule reorganization in vivo (Edelman and Yahara, 1976). Alternatively, ~34~~~~ kinase may modulate microtubule organization by affecting the centrosome, its assembly promoting activity, or possibly a protein associated with this structure, perhaps even p13 (Booher and Beach, 1988). The recent immunolocalization of cdc2 kinase to these structures in both PC Kl (Riabowol et al., 1989) and HeLa cells (Bailly et al., submitted) may support this hypothesis, although it awaits to be demonstrated that purified ~34~~~~ kinase phosphorylates components of this structure. ~34~~~~ kinase may also act directly on the microtubules by phosphorylating tubulin subunits, the microtubule-associated proteins (MAPs), or a microtubulemodulating protein such as the 62 kd phosphoprotein described by Dinsmore and Sloboda (1988, 1989). In this respect the reported phosphorylation of MAP2 by cdc2 in vitro (cf. the discussion of Morgan et al., 1989, for references) may be relevant because the direct phosphoryla-

tion of MAPS by kinases such as A kinase (MAP2A) (Theurkauf and Vallee, 1982) and casein kinase II (MAPlB and f3-tubulin) (Diaz-Nido et al., 1988; Serrano et al., 1988) and their localization to the microtubule networks (Browne et al., 1980; Serrano et al., 1989) has previously been implicated in microtubule regulation. Finally, the changes in microtubule organization after injection of cdc2 may be related to the activation of another kinase such as casein kinase II or simply to the changes in cell shape and actin, indirectly resulting in a loss of microtubule anchorage. p34cdc2-lnduced Chromatin Condensation Microinjection of ~34~~~~ kinase induced premature chromatin condensation, bringing further in vivo support to the recent reports detailing the functional similarities between ~34~~~~ kinase and the mammalian growth-associated Hl kinase (Arion et al, 1988). The extent of these effects on chromatin structure depended on the phase of the cell cycle when ~34~~~~ kinase was injected, with the most dramatic condensation occurring in quiescent GO cells or cells in G2, while the changes in Gl cells were less prominent. This latter finding is in agreement with the findings of Adlakha et al. (1983) and provides further support for the existence of inhibitors of mitotic factors (IMFs). Such differences in the extent of chromatin reorganization may also reflect differences between the in vivo substrates available for ~34~~~~ kinase within the nucleus during the cell cycle, which could arise from cell cycle-dependent changes in the organization of the DNA itself (Yaniv and Cereghini, 1986) or associated proteins within the nucleus (such as the inhibitory factors localized onto the nuclear scaffold; Adlakha et al., 1982). Histone Hl phosphorylation is known to fluctuate through the cell cycle, culminating in hyperphosphorylation during mitotic chromosome condensation (Lake and Salzman, 1972; Bradbury et al., 1974; Gurley et al., 1974, 1978; reviewed in Wu et al., 1986) and previous experiments involving fusion of Gl and mitotic cells described the induction of premature chromatin condensation (Johnson and Rao, 1970; Rao et al., 1977). Our observations provide direct evidence that ~34~~~~ kinase alone can induce premature chromatin condensation in mammalian cells and strongly suggest that many of the effects of mitotic extracts on chromatin structure (Johnson and Rao, 1970; Rao et al., 1977) reflect an activity similar to ~34~~~~ kinase. Endogenous Regulation of the Injected ~34~~~~ Kinase Supporting the notion that the effects induced following microinjection of ~34~~~~ kinase are physiologically relevant, these effects were fully reversible in growing cells. Interestingly, cells injected at different times returned to their normal flat morphology at specific times either coincident with the onset of DNA synthesis or following cell division. Furthermore, we observed that a majority of cells were apparently refractory to the injection of the ~34~~~~ kinase when injected during S phase. The specific reversibility of the ~34~~~~ effects at the Gl-S and G2-M phase transitions together with the lack of responsiveness of the cells at S phase strongly suggest the involvement of the

Microinjection 161

of ~34~“~~ Kinase

into Mammalian

Cells

cell cycle-specific synthesis or inactivation of regulatory molecules that modulate the injected ~34~~~~ kinase. The presence of IMFs throughout the early phase (Gl) of the cell cycle, while absent in GO quiescent cells, has been described previously (Adlakha et al., 1983). Such an accumulation of IMFs during Gl in our cells may explain our finding that the effects of injected ~34~~~~ kinase on cell shape and chromatin organization are the most extreme in cells at GO. While little evidence has yet been presented on the nature of such IMFs, similar activities have been proposed in yeast (weel; Russell and Nurse, 1987a) and Aspergillus (bimE7; Osmani et al., 1988). A number of reports have implicated a 13 kd protein homologous to the yeast suc7+ gene product in cdc2 modulation (Brizuela et al., 1987; Hindley et al., 1987) although the potential function of this protein is still poorly understood (Brizuela et al., 1987; Arion et al., 1988). The presence of p13 is sufficient to prevent MPF-induced nuclear envelope breakdown and chromatin condensation (Dunphy et al, 1988) and cause mitotic delay in yeast (Hayles et al., 1986; Hindley et al., 1987), and a recent report implied its activity in regulating tyrosine dephosphorylation of cdc2 (Dunphy and Newport, 1989). One possibility, therefore, is that the reversal of the effect of the injected kinase or its inactivation at S phase could be related to the action of p13 or a similar homolog in rat cells. Indeed, we have observed that the coinjection of p13 (purified from yeast) with ~34~~~~ kinase in REF-52 cells prevents the action of the kinase described here (unpublished data). However, recent immunostaining for p13 did not reveal any evidence of cell cycle changes in p13 distribution (Riabowol et al., 1989). Alternatively, the apparently neutralizing effects on the injected kinase at S phase may result from changes in intracellular phosphorylation brought about by the activation of different kinase/phosphatase pathways since recent reports have identified ~34~~~~ kinase activation with dephosphorylation (Labbe et al., 1989a; Gautier et al., 1989; Dunphy and Newport, 1989). Differential protein phosphatase activity has also been implicated in the modulation of MPF during oocyte maturation (Karsenti et al., 1987; Labbe et al., 1988a; Cyert and Kirschner, 1988). As further support of a role for phosphatase pathways in cdc2 regulation, we have found that the morphological effects induced by injected ~34~~~~ can be restored at S phase by coinjection of neutralizing antibodies directed against the phosphatase inhibitor 12, while both the abundance of this protein and its activity build up rapidly, coincident with the entry of REF-52 cells into S phase (Brautigan et al., submitted). ~34~~~~ Induces Only the Entry of Cells into a Prophase-like Phenotype The marked effects of ~34~~~~ injection we described support the view that ~34~~~~ kinase is essential in the activation of mammalian cells at mitosis. Furthermore, these effects could be induced at all times in the cell cycle, implying that they are brought about specifically by the injected kinase and do not need the cooperation of other regulatory components that may be present at M phase. It is clear, however, that the events induced by ~34~~~~ ki-

nase injection represent only the initial phases of mitosis, and injected cells never enter metaphase (by disassembling the nuclear envelope or reforming the microtubules into a spindle). This finding may appear contradictory because the same kinase, when injected into oocytes, induces the complete entry of cells into mitosis. However, it is conceivable that REF-52 cells lack one or more proteins present in the meiotic oocyte that are prerequisite to completing the progression through metaphase. There is increasing evidence that the assembly of homologs of ~34~~~~ kinase and other proteins into multimerit complexes is implicit in the action of the ~34~~~~ kinase (reviewed in Lohka, 1989). Our studies have examined the effects of one of the best-documented ~34~~~~ kinase-associated proteins, cyclin B. We found that neither the speed nor extent of the induced effects differed when the solution we injected contained only the ~34~~~~ kinase protein (Labbe et al., 1988a, 19884 1989a) or ~34~~~~ kinase stoichiometrically associated with the 45 kd cyclin B (Labbe et al., 1989b). However, since the 45 kd cyclin is highly susceptible to proteolytic degradation, which is prevented by its binding to the sucl column used to purify the heterodimer (Marcel Dome, personal communication), we cannot discount that the preparation of p34 kinase alone may contain proteolytic peptide fragments from cyclin bound to the kinase, which could contribute to its intrinsic activity. The cyclin proteins are known to be involved in the modulation of ~34~~~~ kinase activity (Swenson et al., 1986; Standart et al., 1987; Solomon et al., 1988; see Lohka, 1989, for further references) and in mitotic induction (Murray et al., 1989). In Xenopus oocytes, their synthesis and presence is sufficient to drive the early embryonic cell cycle (Murray and Kirschner, 1989; Murray et al., 1989) and cyclin proteolysis is associated with ~34~~~~ kinase inactivation (Draetta et al., 1989; Murray et al., 1989). However, it is clear from our observations that even with cyclin, the p34cdcz kinase is still insufficient to induce nonembryonic GO or Gi cells to complete mitosis. The existence of other cell cycle-specific mitotic stimulatory proteins has been reported in mammalian cells and a distinct group of nonhistone nuclear-localized phosphoproteins, ranging in molecular mass up to 200 kd (Davis et al., 1983; Sahasrabuddha et al., 1984; Vandre et al., 1986), are synthesized and build up in the nucleus throughout G2 where they are associated with the chromatin (Adlakha et al., 1982). Their mitotic stimulatory activity, which appears to be phosphorylation dependent, coincides with their appearance in the cytoplasm at the onset of mitosis (Adlakha et al., 1982; Davis et al., 1983). While immunological studies have identified these proteins to be common to all eukaryotes and localized to a variety of subcellar structures (Vandre et al., 1986) both their identity and precise intracellular function remain to be determined. Since cdc2 appears to require dephosphorylation for activation (Labbe et al., 1988a; Gautier et al., 1989), whereas cyclin proteins may require phosphorylation (Meijer et al., 1989) the proteins that modulate intracellular phosphorylation are interesting candidates for such positive regulators. Indeed, in yeast genes such as wee7 and niml, which encode protein kinases (Russell

Cell 162

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Cell Culture and Synchronization Rat embryo fibroblasts (REF-52; McClure et al., 1982) were grown in DMEM supplemented with 10% (w/v) fetal calf serum (FCS) in a humidified atmosphere containing 5% COs as described elsewhere (Lamb et al.. 1988). For use in microinjection studies, cells were subcultured onto 35 mm plastic petri dishes or acid-washed glass coverslips 2-3 days before use (Lamb et al., 1988). For synchronization the cells were placed in serum-free DMEM containing 0.5% (w/v) heat-inactivated serum and allowed to quiesce for 36-48 hr. Quiescent cells were microinjetted without further treatment at 36 hr after serum removal or at various times after refeeding as indicated in the text. To obtain cells in Gl phase of the cell cycle (O-15 hr), cells were refed by the addition of fresh DMEM supplemented with 10% FCS and microinjected routinely 12 hr thereafter. To obtain cells in G2 phase (after DNA synthesis), cells were starved and refed as described above and injected after 22 hr. Purification of Active ~34~~~ Kinase The active catalytic subunit of p34 cdc* kinase was purified from Marthasterias glacialis oocytes essentially as described before (Labbe et al., 1988a, 1989a). The peak of kinase activity, as assayed against histone Hl, corresponded to a fraction containing essentially 95% pure ~34~~~~ kinase (at between 0.7-1.0 mglml) and was taken for use in microinjection experiments. This fraction contained essentially no detectable 47 kd staining or other kinase activity (Labbe et al., 1988a, 1989a). Furthermore, the eventual minor impurities present in different preparations do not immunoprecipitate with anti-p34cdc2 kinase antibodies (against three different peptide epitopes; data not shown). The cylin-containing ~34~~’ kinase preparation was prepared as described elsewhere (Labbe et al., 1989b). We estimate that this preparation contains essentially pure p34 cdc2 kinase (Labbe et al., 1989b) in a stiochiometric association with cylin B. Immediately prior to use for microinjection the kinase preparations were dialyzed into injection buffer with or without antibody as described below. As it is estimated that we are injecting a 20 uM solution of the kinase, which is diluted 20-fold by injection, we assume that the microinjection procedure elevates the intracellular kinase level to approximately 1.0 pM, which reflects 6 x 104 molecules per cell (assuming an injection volume of 5 x lo-j5 liter) (Feramisco et al., 1984). Microinjection Synchronized REF-52 cells were microinjected with a solution of active p34CdC* kinase dialyzed into 100 mM potassium glutamate, 40 mM potassium citrate, 1.0 mM MgS04 (pH 7.24) essentially as described elsewhere (Lamb et al., 1988, 1989). For studies on living cells, the injected cells growing on 35 mm petri dishes were returned to the incubator and subsequent phase-contrast micrographs were recorded on Kodak Technical Pan film (2415) (Kodak Pathe, Avignon, France; developed for 50 ASA in Rodinal, AGFA Geivert, Rueil Mafmaison, France) at 15 min intervals using a Leitz Diavert inverted microscope equipped with a Leicaflex R5 SLR camera.

lmmunofluorescence Techniques For analysis of the changes in microtubule organization, synchronized cells were microinjected with the purified kinase and, after an appropriate incubation period, fixed in absolute methanol at -2O’C. After rehydration in PBS containing 0.1% BSA, the cells were stained for the distribution of microtubules with DMA lA, a mouse monoclonal antitubulin antibody (Blose et al., 1984) or with a rat monoclonal antityrosylated tubulin antibody (Wehland et al., 1983). Microtubules were visualized with fluorescein goat anti-mouse or anti-rat antibodies (Cappel-Organon Technika, Fresnes Cedex, France). Cells were washed in water, mounted in 0.25O/a (w/v) Airvol-205 in PBS (Airvol205 [Air Products Netherlands BV, 3508AB, Utrecht, The Netherlands] is equivalent to Gelvatol) and examined on a Zeiss Axiophot using a 63x planapochromat 1.4 NA lens. Fluorescent images were recorded on Kodak TriX pan film and processed as described before (Lamb et al., 1988). To follow changes in actin microfilament distribution, synchronized cells were injected with p34cdc2 kinase in an injection buffer containing 0.25 mglml inert rabbit anti-mouse antibodies 12 hr after refeeding. After incubation for 60 min the cells were fixed in 3.7% formalin in PBS for 5 min, extracted in -2OOC acetone for 30 s, and hydrated in PBS supplemented with O.l”/, (w/v) BSA. Cells were directly stained for the distribution of F-actin with rhodamine phalloidin (a generous gift from Prof. T. Wheiland) and counter-stained for the marker antibody with fluorescein goat anti-rabbit IgG (Cappel-Organon Technika). Cells were mounted and photographed as described above. Changes in chromatin organization following injection of the kinase were examined by staining cells with Hoechst (bis-benzimide 33258). Essentially, cells at different stages of the division cycle were injected with a solution of ~34~~~” kinase in an injection buffer containing 0.25 mglml rabbit anti-mouse IgG to act as an inert marker for injected cells. After incubation for 60 min, the cells were fixed as described in 3.7% formalin in PBS. Cells were processed for immunofluorescence as described for actin staining, except that the incubation in phalloidin was replaced by a brief incubation in 0.1 pgglml of bis-benzimide in PBS for 60 s before mounting. Cells were photographed using a 40x planapochromat lens. Miscellaneous Methods Assessment of the number of cells entering S phase was made by following the incorporation of 5-Br-dU using a cell proliferation kit (Amersham, Amersham Intl. PLC, Amersham, UK). Refed REF-52 cells growing on glass coverslips were pulse labeled for 2 hr with 5-Br-dU at different times after refeeding and processed as described by the manufacturer, with the exception that cells were initially fixed in absolute methanol at -20°C for 2 min and subsequently extracted in 2.0 M HCI for 15 min. Electron Microscopy Synchronized REF-52 cells growing on plastic petri dishes were injected with the kinase as described above for the changes in cell morphology. Thirty or sixty minutes thereafter the cells were fixed in 2.0% (w/v) gluteraldehyde in PBS for 15 min before processing for thin section electron microscopy as described elsewhere (Lamb et al.. 1989). Alternatively, cells on the same dish were microinjected with injection buffer alone before fixation and processed as described above. Similar sections through the nuclei of selected cells were analyzed. Acknowledgments We would like to thank Prof. Jacques Demaille for his continued and enthusiastic support of this work, Prof. Marcel Doree for initiating these experiments and useful comments and discussions, and Prof. Theodore Weiland for the generous gift of rhodamine phalloidin. This work was supported in part by the Centre National de la Recherche Scientifique (CNRS), lnstitut National de la Sante et de la Recherche Medicale (INSERM), and a grant from the Association Francaise Contre les Myopathies (AFM) to A. F. and N. J. C. L. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

May 9, 1989; revised

November

1, 1989.

Microinjection 163

of ~34~~~’ Kinase

into Mammalian

Cells

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