Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M306376200 on July 14, 2003

J. Biol. Chem., Vol. 278, Issue 39, 37865-37873, September 26, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/39/37865    most recent
M306376200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chang, D. C.
Right arrow Articles by Luo, K. Q.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chang, D. C.
Right arrow Articles by Luo, K. Q.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Degradation of Cyclin B Is Required for the Onset of Anaphase in Mammalian Cells*

Donald C. Chang {ddagger} § , Naihan Xu {ddagger} and Kathy Q. Luo {ddagger}

From the {ddagger}Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China and the §Marine Biological Laboratory, Woods Hole, Massachusetts 02543

Received for publication, June 17, 2003 , and in revised form, July 14, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Recently, it has been shown that cyclin B1 was degraded mainly before the onset of anaphase in mammalian cells. When a nondegradable form of cyclin B1 was introduced into cells, the metaphase-anaphase transition was blocked. This blockage was not due to a failure in activating anaphase-promoting complex, nor was it due to a failure of degradation of securin. To resolve the question of whether this blockage by overexpressing the nondegradable form of cyclin B1 is physiologically relevant or not, we developed a novel method to estimate the relative protein level of the overexpressed cyclin B1 mutant within an individual cell. We found that a low level of nondegradable cyclin B1 (less than 30% of the endogenous cyclin B1) was sufficient to block the metaphase-anaphase transition, implying that the blockage of anaphase onset by the nondegradable cyclin B1 was not due to an artifact of excessive M-phase-promoting factor activity. This result suggests that, in mammalian cells, the majority of cyclin B1 must be destroyed before the cell can enter anaphase.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
It is well known that cyclin B must be degraded to inactivate M-phase-promoting factor (MPF)1 to exit mitosis (1–8). Yet is inactivation of MPF required for the metaphase to anaphase (M/A) transition? There have been many conflicting reports on this topic (1, 7–19). Up until recently, the majority view was that inactivation of MPF is not required for the onset of anaphase (20, 21). For example, it was demonstrated in an in vitro study using Xenopus egg extracts that the degradation of cyclin B (and thus the inactivation of MPF) was not required for the separation of sister chromatids (9). A similar conclusion was reached in the in vivo study of budding yeast, which reported that the destruction of MPF activity is not required for the M/A transition (10). Recently, Stemmann et al. (15) extended the in vitro study of Holloway et al. (9) and found that when a high concentration of nondestructable cyclin B was added to the Xenopus extract, sister chromatid separation could be blocked. They attributed this blockage to an inhibitory phosphorylation of separase that might be caused by high kinase activity of MPF. Their findings now raise a very important question: In an in vivo metazoan system, is cyclin B required to be degraded before the onset of anaphase?

It is possible that the answer may depend on the cell model. In this study, we try to answer this question in mammalian cells using a living cell imaging technique. To measure the dynamic change of the cyclin B level within a single living cell, we fused the wild type and nondestructible form of cyclin B1 (which is the major form of mitotic cyclin in mammalian cells) with fluorescence proteins such as green fluorescent protein (GFP) and DsRed (22–24). The protein stability of cyclin was then correlated with several key cellular events, including sister chromatid separation and the degradation of securin. To ensure the general applicability of our results, we used both transformed cells (HeLa) and nontransformed cells (Ptk2) as model systems. Our experiments were designed to clarify two major points: 1) In a living mammalian cell, will the presence of nondegradable cyclin B prevent the onset of anaphase? 2) What is the protein level of nondegradable cyclin B required for causing the metaphase arrest? If the amount of nondegradable cyclin B required to block the M/A transition is much higher than the endogenous cyclin B, this blockage could be an artifact due to a hyperactivity of MPF. If a low level of nondegradable cyclin B (which is significantly less than the level of endogenous cyclin B at prometaphase) is sufficient to block the M/A transition, it would indicate that cyclin B must be degraded before the anaphase onset.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids and Transfection—The human cyclin B1 gene was cloned between the HindIII and BamHI sites in a pEGFP-N1 vector and a pDsRed1-N1 vector, respectively (Clontech). Cyclin B1-{Delta}85 was cut out from the full-length cyclin B1 and cloned behind the GFP gene between the KpnI and BamHI sites in a pEGFP-C3 vector (Clontech). Human securin gene (hPTTG) was cloned between the EcoRI and BamHI sites of the pEYFP-N1 vector (Clontech).

The fusion genes were introduced into HeLa or PtK2 cells by electroporation (25). Cells were usually cultured for at least 24 h for expressing the introduced genes. When cell synchronization was required, HeLa cells were allowed to express the transfected genes for 12–16 h and then treated with nocodazole (100 ng/ml) for 10–12 h.

Cell Culture and Immunofluorescence Staining—HeLa cells were cultured as a monolayer at 37 °C in minimum essential medium supplemented with 10% fetal bovine serum and 100 international units/ml penicillin and streptomycin. For immunostaining, cells were fixed with 4% paraformaldehyde plus 0.1% glutaraldehyde (which helped to retain the soluble proteins) and stained with the following primary antibodies: anti-cyclin B1 monoclonal antibody (GNS-11, Pharmingen); anti-kinetochore antibody (human autoantibody control ANA-C-positive, Sigma). They were then stained with one of the following secondary antibodies: rhodamine-conjugated goat anti-mouse IgG (Calbiochem); TRITC-conjugated goat anti-human IgG (Sigma). Autofluorescence of glutaraldehyde was quenched with a freshly prepared solution of 0.1% NaBH4 (in phosphate-buffered saline) for 10 min. Nuclear DNA was stained with Hoechst 33342 (100 ng/ml) at 37 °C for 10 min. The Western blot experiments were performed using the standard procedures. The anti-GFP antibody was from Molecular Probes.

Living Cell Imaging—To study the dynamic changes of cyclin B1-GFP and other GFP-(or DsRed) labeled proteins in living cells, fluorescent images of mitotic cells expressing the fusion gene were recorded continuously for several hours using a digital imaging system. The transfected cells were cultured on a 25-mm-diameter round coverslip, which was placed in a thermally regulated chamber (Medical System Corp.) mounted on the stage of a Zeiss Axiovert 35 inverted microscope. The excitation light was controlled using a Lambda-10 filter wheel shutter (Sutter Instruments). The fluorescent images were observed using appropriate filter sets. The images were recorded using a cooled CCD camera (Micro-Max by Princeton Instruments) or a SPOT camera (Diagnostic Instruments). The imaging system was controlled by a computer using the MetaMorph software (Universal Image).

Chromosome Spreads—HeLa cells (with or without expressing the GFP-cyclin B1-{Delta}85 gene) were arrested in M-phase by nocodazole treatment. GFP-positive cells were sorted out using a fluorescence-activated cell sorter machine. At 0.5 and 3.5 h after releasing from nocodazole arrest, cells were treated with a hypotonic buffer (10 mM Tris, 10 mM NaCl, 5 mM MgCl2) for 15 min before collapsing them on a coverslip by cytospin. The cells were fixed using cold methanol for 20 min and then incubated with primary (ANA-C) and secondary (TRITC-conjugated anti-human IgG) antibody. DNA was stained with Hoechst 33342. The slides were viewed using a Zeiss Axiovert 35 microscope.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Timing of Cyclin B Degradation during Mitosis Is Similar to That of Securin and Is Before the Onset of Anaphase
During mitosis, both cyclin B and securin are known to be degraded by APC through a ubiquitination proteasome system (21, 26). APC can be activated by binding to Cdc20 during metaphase or become activated later through the binding of Cdh1/Hct1 (27–29). It is well known that securin is degraded mainly by the Cdc20-activated APC (30–33). Using living cell imaging techniques, we measured both time courses of cyclin B1 degradation and securin degradation in the same HeLa cell. In this study, we labeled human cyclin B1 with DsRed (red fluorescent protein) and securin with YFP (yellow fluorescent protein). At early metaphase, both DsRed-labeled cyclin B1 and YFP-labeled securin were clearly visible in the cell (Fig. 1A). Their protein levels began to decrease at early metaphase and vanished almost completely before the onset of anaphase. Fig. 1C shows the results of a quantitative analysis of the temporal dependence of the degradation of cyclin B1 and securin. Their time courses were very similar. The same experiment was repeated in the PtK2 cells. Again, we found that both cyclin B1 and securin were degraded before the M/A transition (Fig. 1, B and D). We noticed that cells overexpressing both the cyclin B and securin genes spent a longer time period in the metaphase in comparison with the control cells. This is probably due to the fact that these overexpressed gene products can compete with the endogenous substrate proteins and delay their degradation. Indeed, when we transfected the cells with cyclin B1-GFP alone, the time period for metaphase was shortened. The time-dependent degradation of cyclin B1-GFP in different HeLa cells was also very similar (Fig. 1E). Our results are consistent with the findings of Clute and Pines (14), who also used real time measurements to study the temporal and spatial control of cyclin B1 destruction in mitosis (14).



View larger version (57K):
[in this window]
[in a new window]
  		FIG. 1.
Temporal-dependent proteolysis of cyclin B1-DsRed (red) and securin-YFP (green) in a living HeLa cell (A) and a PtK2 cell (B). The fluorescence image, as well as the phase image of the same cell, was recorded at various time intervals starting from prometaphase. Scale bar, 10 µm. The normalized fluorescence intensities of cyclin B1-DsRed and securin-YFP in a HeLa cell (C) and a PtK2 cell (D) were plotted as a function of time. E, the time-dependent change of normalized fluorescence intensity of GFP-labeled cyclin B1 in three different HeLa cells during mitosis. These results are representative of measurements of 23 cells. Time zero stands for the beginning of metaphase.

 

Based on living cell measurements on 23 HeLa cells, we calculated that 94.2% of cyclin B1 had been destroyed before the onset of anaphase. These results suggest that, in a normal cell cycle, cyclin B1 is largely degraded before the onset of anaphase. The remaining question is whether the cyclin B1 degradation is a necessary condition for the M/A transition.

Onset of Anaphase Is Blocked in the Presence of Nondegradable Cyclin B in Mammalian Cells
From previous studies reported in the literature, there were hints that cyclin B had to be degraded before anaphase onset (5, 14, 15, and see also Ref. 44). For example, Gallant and Nigg (5) reported almost a decade ago that a higher percentage of HeLa cells was found to be arrested in mitosis when a nondegradable form of cyclin B2 was overexpressed. We found similar results also hold for overexpressing cyclin B1. When we transfected HeLa cells with a GFP-labeled nondegradable form of cyclin B1 that had the 85 amino acids removed from its N terminus (cyclin B1-{Delta}85), a large percentage of cells were found in the M-phase, whereas cells transfected with GFP alone were not significantly different from the control (Fig. 2A). Judging from the chromosome distribution pattern, the cyclin B1-{Delta}85-expressing cells appeared to be arrested in metaphase (Fig. 2B). This was true for both PtK2 and HeLa cells (Fig. 2, B and C). Similar findings were also mentioned in a report by Clute and Pines (14), although no data were given there.



View larger version (50K):
[in this window]
[in a new window]
  		FIG. 2.
A, the percentage of mitotic HeLa cells overexpressing GFP, cyclin B1-GFP, and GFP-cyclin B1-{Delta}85 genes (based on the measurement of 12,203 cells in three experiments). B, time-dependent measurement of GFP-cyclin B1-{Delta}85 in a PtK2 cell. The morphology of its chromosomes and its phase image were recorded at the same time. C, time-dependent changes in the protein distribution of cyclin B1-DsRed and GFP-cyclin B1-{Delta}85 co-expressed in a HeLa cell. The morphology of chromosomes and phase image are also shown. In D, the normalized fluorescence intensities of cyclin B1-DsRed and GFP-cyclin B1-{Delta}85 co-expressed in the same HeLa cell were plotted as a function of time. This result is representative of 16 cells analyzed. E, time-dependent changes in the protein distribution of securin-YFP and CFP-cyclin B1-{Delta}85 co-expressed in a HeLa cell. Morphology of chromosomes and phase image are also shown. In F, the normalized fluorescence intensities of securin-YFP and CFP-cyclin B1-{Delta}85 co-expressed in the same HeLa cell were plotted as a function of time. This result is representative of six cells analyzed. Scale bar, 10 µm. Time zero stands for the beginning of metaphase.

 

Like the full-length cyclin, cyclin B1-{Delta}85 can bind with Cdk1 to form an active MPF complex and maintains its kinase activity (1, 34). Our findings that the onset of anaphase is blocked in the presence of nondegradable cyclin B1 would imply that mammalian cells cannot enter anaphase when the MPF activity remains high.

Blockage of Anaphase Onset Is Due to the Failure of Removing Cohesin Rather than Preventing APC Activation
One important question arises. That is, is the metaphase arrest caused by the active MPF due to its direct effects on the securin/separase/cohesin system, or is it because a high level of MPF may prevent the activation of APC? To answer this question, we co-transfected cells with GFP-cyclin B1-{Delta}85 and cyclin B1-DsRed. Using the green and red fluorescence channels, we measured the temporal-dependent changes of both the wild-type and truncated cyclin B1 in the same HeLa cell during mitosis. We found that cyclin B1-{Delta}85 was not degraded and that the cell cycle was blocked at the M/A transition (Fig. 2C). The full-length cyclin B1, on the other hand, was destroyed when the cell entered metaphase (Fig. 2, C and D), similar to what happened in the control cells (Fig. 1, A and E). Furthermore, by co-transfecting the cell with securin-YFP and CFP-cyclin B1-{Delta}85, we could measure the temporal-dependent changes of both securin and truncated cyclin B1 in the same HeLa cell. We found that like the full-length cyclin B1, securin was also degraded in the presence of nondegradable cyclin B1 (Fig. 2, E and F). Apparently, the presence of the nondegradable cyclin B1 did not prevent the activation of APC.

Thus, the blockage of anaphase onset by the nondegradable cyclin B1 must be due to a direct effect of the active MPF on the securin/separase/cohesin system. We have conducted a chromosome-spread experiment to examine the detailed structure of sister chromatids in cells with or without nondegradable cyclin B1 (see "Experimental Procedures"). We observed that in the majority of cells expressing GFP-cyclin B1-{Delta}85, their paired chromatids remained unseparated at 3.5 h after release from nocodazole treatment, implying that the cohesin proteins holding them together were intact (Fig. 3, A and B). In contrast, the sister chromatids in the control cells (i.e. without GFP-cyclin B1-{Delta}85) had mostly separated (Fig. 3B). These results confirmed that overexpressing cyclin B1-{Delta}85 could indeed arrest cells in metaphase. This finding, in combination with the results shown in Fig. 2, C–F, suggests that in mammalian cells, even after APC is activated and securin is degraded, an active MPF could still prevent the removal of cohesin from the chromosomes.



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 3.
As shown in A, sister chromatid separation was blocked in HeLa cells transfected with GFP-cyclin B1-{Delta}85. Chromosome spreads of HeLa cells were prepared as described under "Experimental Procedures." Panels 1–3, DNA staining of chromatids by Hoechst. In panels 4–6, kinetochors of the same chromatids were revealed by immunostaining using an antibody ANA-C (ANA). Panels 7–9, merged images of panels 1–3 and panels 4–6, respectively. Scale bar,2 µm. B, the percentage of cells showing paired or unpaired chromatids at 0.5 and 3.5 h after nocodazole release (based on the measurement of 7,298 cells). The percentage of cells having decondensed nuclei was counted but not plotted. C, Western blot results of cell extracts obtained from two random populations of cells expressing either cyclin B1-GFP (lane 1) or GFP-cyclin B1-{Delta}85 (lane 2). It is evident that the probing antibody recognized both the endogenous cyclin B1 (band C) as well as the GFP-labeled full-length (band A) or truncated cyclin B1 (band B). The same membrane was reprobed using anti-GFP antibody (lanes 3 and 4).

 

The Relative Protein Level of Cyclin B1-{Delta}85 within an Individual Cell Can Be Determined Using a Combination of GFP Labeling and Immunostaining Techniques
The results of our single living cell measurements shown above are consistent with the findings of an in vitro study by Stemmann et al. (15), who showed that when a high concentration of nondestructible cyclin B was added to the Xenopus extract, sister chromatid separation could be blocked. They attributed this blockage to an inhibitory phosphorylation of separase, which is probably caused by a high kinase activity of MPF (15). Recently, Hagting et al. (33) conducted a preliminary study on living PtK1 cells. They observed that in cells with low levels of nondegradable cyclin B1, separation of sister chromatids was not blocked. However, in a few cells with high levels of nondegradable cyclin B1 (estimated to be 1.5–2-fold of the endogenous cyclin), sister chromatids were observed to remain together for several hours after the disappearance of securin (33). These observations, together with ours as shown in Figs. 2 and 3, raised a very important question: Is the blockage of M/A transition by overexpressing nondegradable cyclin B1 physiologically meaningful? Such a blockage could have two alternative interpretations: 1) The endogenous MPF must be inactivated before the cell can enter anaphase; or 2) A hyperactive MPF may artificially block the M/A transition.

The key question here is whether the amount of nondegradable cyclin B1 required for arresting the cell in metaphase is substantially below the level of endogenous cyclin B1 found in prometaphase or not. If the answer is yes, it would suggest that the majority of the endogenous cyclin B1 must be degraded before the M/A transition can occur. Otherwise, the blockage of anaphase onset is just an artifact due to an unphysiologically high level of MPF activity caused by the overexpression of nondegradable cyclin B1.

Thus, we decided to conduct a careful experiment to test this point. Using a combination of GFP imaging and immunofluorescence techniques, we determined the relationship between the probability of metaphase arrest and the relative protein level of GFP-cyclin B1-{Delta}85 within individual cells. The detailed procedures are described in the following.

Step 1—We first synchronized HeLa cells (with or without expressing the GFP-cyclin B1-{Delta}85 gene) at early M-phase using a nocodazole treatment (see "Experimental Procedures"). After nocodazole washout, cells were fixed at 0.5 or 3.5 h. They were then immunostained with anti-cyclin B antibody that recognized both the full-length and truncated cyclin B1 (GNS-11, from Pharmingen). The secondary antibody used for immunostaining was colored red so that it could be distinguished from the GFP signal (Fig. 4A). These cells were also DNA-stained to allow their stage of cell cycle to be determined. The conditions of the four samples are summarized in Table I.



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 4.
A, immunostaining of cyclin B and fluorescence of GFP in HeLa cells with or without expressing the GFP-cyclin B1-{Delta}85 gene. DNA in the same cell was visualized by staining with a Hoechst dye. B, analysis of {Delta}85-positive cells in Sample 4. The number of cells at different cell cycle stage was dependent on the intensity of the GFP-cyclin B1-{Delta}85 signal. Cells were grouped into three categories: metaphase (M), anaphase or telophase (A), and G1-phase (G). C, relative immunofluorescence intensity of individual HeLa cells stained with an antibody against cyclin B1 (analysis of 1,516 cells in three independent experiments). Background due to nonspecific staining was corrected. As shown in D, the percentage of cells arrested before M/A transition was plotted as a function of the protein level of GFP-cyclin B1-{Delta}85 determined at 3.5 h after release from the nocodazole treatment (based on three independent experiments). E, Western blot result showing that the endogenous cyclin B1 protein did not increase during nocodazole treatment. The level of Cdc2 was used as a loading control.

 

View this table:
[in this window]
[in a new window]
  		TABLE I
Conditions of the four sample groups

 

Step 2—Using a fluorescence imaging system (see "Experimental Procedures"), we measured the intensity of the GFP signal in various individual cells in Sample 4. Their cell cycle stages were also determined at the same time. These cells were grouped into three categories: 1) metaphase, 2) anaphase or telophase, and 3) interphase (G1). We then plotted the distribution of cells as a function of the fluorescence intensity of GFP-cyclin B1-{Delta}85 (Fig. 4B). Our results clearly showed that the stage of cell cycle is closely related to the level of expression of the GFP-cyclin B1-{Delta}85 gene. At 3.5 h after the nocodazole release, most cells with a low level of GFP-cyclin B1-{Delta}85 protein had progressed to the G1-phase. In cells with a high level of GFP-cyclin B1-{Delta}85 protein, however, they were mostly arrested in the metaphase (Fig. 4B).

Step 3—To convert the GFP fluorescence into protein level, we used a calibration method based on an immunofluorescence analysis. The relative protein level of GFP-cyclin B1-{Delta}85 (in comparison with the endogenous cyclin B1) was determined based on the following equation.

(Eq. 1)
Here the subscript "cell" refers to the protein concentration of an individual cell, whereas the subscript "average" refers to the average protein level in an ensemble of cells. The first ratio on the right-hand side of Equation 1 was experimentally determined by measuring the fluorescence intensity of GFP in a large number of GFP-positive cells in Sample 4. The second ratio on the right hand side of Equation 2 was determined by measuring the average immunofluorescence intensity of cells in Sample 4 and in Sample 1 (Fig. 4C). (Note: All four samples were processed together and immunostained using identical conditions.) More explicitly, we assumed,

(Eq. 2)
This analysis took advantage of the knowledge that cells in Sample 4 should contain mainly the nondegradable cyclin B1-{Delta}85 since at 3.5 h after nocodazole release, the endogenous cyclin B1 was destroyed. We had shown previously that the presence of nondegradable cyclin B1 did not prevent the destruction of the full-length cyclin B1 (Fig. 2). Cells in Sample 1, on the other hand, were at prometaphase and thus should contain the full amount of endogenous cyclin B1. (Note: Background of immunofluorescence due to nonspecific staining can be determined from Sample 3.)

One key requirement in our immunofluorescence analysis is that the antibody used here (GNS-11, from Pharmingen) must be equally efficient in recognizing the endogenous cyclin B1 and the GFP-cyclin B1-{Delta}85. This was verified in a Western blot study shown in Fig. 3C. Here, we used the GNS-11 antibody to probe protein blots obtained from two random populations of cells expressing either the GFP-cyclin B1 or the GFP-cyclin B1-{Delta}85 genes. It is evident that this antibody was highly specific and recognized both the endogenous cyclin B1 and the GFP-labeled cyclin B1 (or cyclin B1-{Delta}85). To compare the sensitivity of this antibody in recognizing the full-length and truncated cyclin B1, we reprobed the same protein blot membrane with an anti-GFP antibody. As shown in Fig. 3C, we found that the intensity ratios between the GFP-cyclin B1 and GFP-cyclin B1-{Delta}85 bands were similar using either the anti-cyclin B1 or the anti-GFP antibody. (The intensity ratio obtained from GNS-11 and anti-GFP was 2.1 and 1.9, respectively.) These results indicated that this anti-cyclin B1 antibody was almost equally sensitive in recognizing the full-length or truncated form of cyclin B1. Under our experimental conditions, we estimated that the average protein level of cyclin B1-{Delta}85 within the {Delta}85-positive cells in Sample 4 was about 61 ± 21% (analysis of 1516 cells in three independent experiments) of the endogenous cyclin B1 found at the prometaphase (Fig. 4C).

A Low Level of Nondegradable Cyclin B Is Sufficient to Block the Metaphase-Anaphase Transition
Using the method discussed above, we were able to determine the relationship between the level of nondegradable cyclin B1 contained in an individual cell and its probability of metaphase arrest. The result is shown in Fig. 4D. We found that a low level of nondegradable cyclin B (equivalent to about 20–30% of the endogenous cyclin B1 at prometaphase) was sufficient to block the M/A transition in a majority of HeLa cells. These results imply that the blockage of M/A transition by expressing cyclin B1-{Delta}85 was not due to an artifact caused by a hyperactivity of MPF. Instead, our findings suggest that the majority of cyclin B1 protein must be degraded before the HeLa cell can enter the anaphase.

One may challenge our findings by questioning that, since we used nocodazole to synchronize the cells, the level of endogenous cyclin B1 in our Sample 1 cells (i.e. cells fixed at 0.5 h after release from nocodazole) could be much higher than that in the normal prometaphase cells. It is well known that the average cyclin B1 concentration in nocodazole-arrested cells is much higher than that of a random cell population. This is mainly due to the fact that cells are arrested at the early M-phase by the nocodazole treatment, and thus, a higher percentage of cells will express the endogenous cyclin B1. Yet once a cell is arrested by nocodazole, will it continue to produce a large amount of cyclin B1 so that the endogenous protein will accumulate to a very high level? To answer this question, we did the following experiment. We first synchronized HeLa cells at the G1/S-boundary using a double thymidine block (35). After this block was released for 8 h, most cells had entered into the late G2-phase. We treated cells with nocodazole for different lengths of time. Then, using a Western blot analysis, we examined whether the cyclin B1 level increases with time in nocodazole-arrested cells. Our result is shown in Fig. 4E. We found that once a cell reached the G2/M-phase, the level of cyclin B1 did not significantly increase further under the nocodazole treatment. This result verified that the level of endogenous cyclin B1 observed in the Sample 1 cells was indeed similar to that found in a typical prometaphase cell.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
A key step in mitosis is the separation of sister chromatids at the metaphase-anaphase transition. This step is known to be regulated by the activation of APC (36), which triggers the removal of cohesin at the chromosome through the degradation of securin and the activation of separase (37–40). Previously, it was not clear whether this step also requires the inactivation of MPF by destroying cyclin B (3–6). Earlier studies on a number of model systems, including Xenopus egg extracts (9), budding yeast and fission yeast (8, 10), sea urchin embryos (16), and Drosophila (17), indicated that the introduction of nondegradable cyclin B arrested the cell cycle mainly at anaphase instead of metaphase, implying that cyclin B degradation is not required for sister chromatid separation. This view has become the standard model in later years (21, 36) (Fig. 5A).



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 5.
Models of signaling control on the metaphase-anaphase transition. A, the standard model that was based on previous studies in yeasts and Xenopus egg extracts. B, an updated model suggested for mammalian cells. MT, microtubule.

 

In reviewing the literature, we noticed that there seems to be a difference in the temporal pattern of cyclin B degradation between eukaryotic cells of different phyla. For example, in mammalian cells, cyclin B appears to degrade before the M/A transition (5, 14, 41, 42), whereas in yeasts, destruction of certain mitotic cyclins (e.g. Clb2) was initiated during anaphase and might persist throughout the G1-phase (10, 43). Thus, it is possible that cyclin B may play a different role in the regulation of M/A transition in different cell models. In this study, we used single cell measurements to investigate such a possibility. Our results suggest that the regulation of M/A transition in mammalian cells is indeed different from the standard model that was based on studies in yeasts and Xenopus egg extracts. First, we observed that in HeLa and PtK2 cells, the time course of cyclin B1 destruction was very similar to that of securin; both of them were degraded before the onset of anaphase. Second, in the presence of nondegradable cyclin B1, the cell cycle was clearly blocked before the M/A transition (Figs. 2 and 3). Such a blockage was independent of the activation of APC since degradation of the full-length cyclin B1 and securin progressed normally during this metaphase arrest (Fig. 2). Third, we found that such a blockage was not an artifact due to the hyperactivity of MPF since a very low level of nondegradable cyclin B1 (about 20–30% of the endogenous cyclin B1 at prometaphase) was sufficient to inhibit the separation of sister chromatids in HeLa cells.

These results suggest that unlike nonvertebrate systems such as yeast or Drosophila, mammalian cells in metaphase must destroy the mitotic cyclin (and thus inactivate MPF) before it can activate the cohesin cleavage system (Fig. 5B). This new model can fit well with the earlier observations indicating that mitotic cyclin in metazoans was destroyed at late metaphase (5, 41, 42, 44). This model is also in qualitative agreement with an in vitro study recently conducted by Stemmann et al. (15), who found that when the amount of nondegradable cyclin B added to the Xenopus extract was increased, sister chromatid separation was blocked (15). Thus, the model depicted in Fig. 5B may be relatively general and could be valid for most vertebrates, although we presented here only supporting evidence in mammalian cells.

Even for the yeast system, results of some of the recent studies could also fit with this new model. For example, Lim et al. (45) observed that Cdc20 was essential for degradation of Clb2 in budding yeast. Recently, Yeong et al. (46) and Wasch and Cross (47) also suggested that APC-Cdc20 is required for an early phase degradation of Clb2, which is necessary and sufficient for most aspects of mitotic exit. A later phase activation of APC-Cdh1 will cause a further degradation of Clb2, which was thought to be important for regulating cell size and the length of G1. At this point, there is still a question of whether the estimated protein level of nondegradable cyclin B1 required to block the M/A transition in Xenopus egg extracts matched that of an intact cell. According to Hagting et al. (33), a 2-fold excess of nondegradable cyclin B1 (in comparison with the endogenous level) could block sister chromatid separation in Xenopus extracts (Stemmann et al. (15)). In our in vivo study of HeLa cells, we found that a much lower amount of cyclin B1 (less than 30% of endogenous level) was sufficient to block the M/A transition. One possible explanation of this difference could be the variation of the two model systems. In the egg extracts, the volume ratio of cytosol over nuclei was far larger than that of a HeLa cell. It has a significant dilution effect. In the intact cell, cyclin B1 is selectively concentrated in the mitotic spindles (14) (Fig. 1). Since it is mainly the cyclin B/Cdk1 localized near chromosomes that contributes to the phosphorylation of separase, cyclin B/Cdk1 could be more effectively utilized in an intact cell than in egg extracts. In addition, the cyclin B1 added to egg extracts may be less efficient in activating the Cdk1 than a cell due to the less optimal distribution of the required activating kinases and phosphatase (such as Cdc25). Thus, it may not be surprising that a smaller amount of cyclin B1 is sufficient to maintain enough MPF activity to block the M/A transition in an intact cell.

The only major study that appeared to be in conflict with the model presented in Fig. 5B is that by Wheatley et al. (13), who microinjected mRNA of a nondegradable form of cyclin B (cyclin B{Delta}90) into prometaphase normal rat kidney cells and found that expression of cyclin B{Delta}90 did not block chromatid separation but inhibited cytokinesis (13). One possible explanation of their results is that since they injected mRNA instead of mature proteins, it might require a certain period of time for the cyclin B{Delta}90 to be translated and become mature. Even after maturation, it also takes time for the cyclin B{Delta}90 to bind to Cdk1 and go through the proper phosphorylation and dephosphorylation to activate it. Thus, it is possible that in their system, Cdk1 was not yet fully activated by the cyclin B{Delta}90 during the M/A transition. Instead, Cdk1 was activated later and thus was able to block cytokinesis.

At the completion of this work, we noticed that Hagting et al. (33) had just completed a study on securin proteolysis in living cells. Their observations on the kinetics of securin degradation were essentially consistent with ours. They also conducted a preliminary experiment on the effects of nondegradable cyclin B1. They observed that following securin degradation, separation of sister chromatids in PtK1 cells was blocked only when a "moderate to high level" of nondegradable cyclin B1 (estimated to be 1.5–2.0-fold of the endogenous cyclin B1 level) was present. If their estimate was correct, it would imply that the blockage of M/A transition by the nondegradable cyclin B1 was an artifact (due to an unphysiological hyperactivity of MPF). A closer examination of their report, however, indicated that their estimate was very rough. First, they did not actually measure the protein levels of nondegradable cyclin B1 in their cells. Second, their definition of "cells expressing moderate to high level of nondegradable cyclin B1" was arbitrary; they simply classified such cells as those having a "GFP signal >106 pixels." It is well known that it is problematic to use image intensity to define the protein level since the image intensity depends on the experimental conditions and thus is not a reproducible number. Third, their estimate was done very roughly and based on observations of only a few cells. For these reasons, we feel that their estimate on the amount of cyclin B1 required to block the M/A transition was less than reliable. Thus, their result does not necessarily contradict ours.

In summary, we present evidence in this study that, in mammalian cells, mitotic cyclin B1 is required to be destroyed before the anaphase onset (Fig. 5B). Destruction of mitotic cyclin is through the ubiquitination by APC, which can be activated by either binding with Cdc20 or binding with Cdh1 (21). In systems such as yeasts, it is known that the mitotic cyclins can be degraded by either Cdc20- or Cdh1-activated APC (21). Results of this study suggest that in mammalian cells, cyclin B1, like securin, is normally destroyed by the Cdc20-activated APC. Such an action will turn off the MPF activity and allow separase to be activated (through the destruction of securin), which in turns removes cohesin from the sister chromatids and allow the onset of anaphase.


   FOOTNOTES
 
* This work was supported by the Research Grants Council of Hong Kong (HKUST6109/01 M and HKUST6104/02M) and the HIA project of HKUST. 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. Back

To whom correspondence should be addressed. Tel.: 852-2358-7326; Fax: 852-2358-1559; E-mail: bochang{at}ust.hk.

1 The abbreviations used are: MPF, M-phase-promoting factor; M/A, metaphase to anaphase; APC, anaphase-promoting complex; GFP, green fluorescent protein; EGFP, enhanced GFP; YFP, yellow fluorescent protein; EYFP, enhanced YFP; CFP, cyan fluorescent protein; TRITC, tetramethylrhodamine isothiocyanate. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Tony Hunter for providing the human cyclin B1 gene and Dr. Francisco Ramos-Morales for providing the human securin (hPTTG) gene.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Murray, A. W., Solomon, M. J., and Kirschner, M. W. (1989) Nature 339, 280–286[CrossRef][Medline] [Order article via Infotrieve]
  2. Nurse, P. (1990) Nature 344, 503–508[CrossRef][Medline] [Order article via Infotrieve]
  3. Ghiara, J. B., Richardson, H. E., Sugimoto, K., Henze, M., Lew, D. J., Wittenberg, C., and Reed, S. I. (1991) Cell 65, 163–174[CrossRef][Medline] [Order article via Infotrieve]
  4. Luca, F. C., Shibuya, E. K., Dohrmann, C. E., and Ruderman, J. V. (1991) EMBO J. 10, 4311–4320[Medline] [Order article via Infotrieve]
  5. Gallant, P., and Nigg, E. A. (1992) J. Cell Biol. 117, 213–224[Abstract/Free Full Text]
  6. Luo, Q., Michaelis, C., and Weeks, G. (1994) J. Cell Sci. 107, 3105–3114[Abstract]
  7. Rimmington, G., Dalby, B., and Glover, D. M. (1994) J. Cell Sci. 107, 2729–2738[Abstract]
  8. Yamano, H., Gannon, J., and Hunt, T. (1996) EMBO J. 15, 5268–5279[Medline] [Order article via Infotrieve]
  9. Holloway, S. L., Glotzer, M., King, R. W., and Murray, A. W. (1993) Cell 73, 1393–1402[CrossRef][Medline] [Order article via Infotrieve]
  10. Surana, U., Amon, A., Dowzer, C., McGrew, J., Byers, B., and Nasmyth, K. (1993) EMBO J. 12, 1969–1978[Medline] [Order article via Infotrieve]
  11. Sigrist, S., Jacobs, H., Stratmann, R., and Lehner, C. F. (1995) EMBO J. 14, 4827–4838[Medline] [Order article via Infotrieve]
  12. Murray, A. W., Desai, A. B., and Salmon, E. D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12327–12332[Abstract/Free Full Text]
  13. Wheatley, S. P., Hinchcliffe, E. H., Glotzer, M., Hyman, A. A., Sluder, G., and Wang, Y. (1997) J. Cell Biol. 138, 385–393[Abstract/Free Full Text]
  14. Clute, P., and Pines, J. (1999) Nat. Cell Biol. 1, 82–87[CrossRef][Medline] [Order article via Infotrieve]
  15. Stemmann, O., Zou, H., Gerber, S. A., Gygi, S. P., and Kirschner, M. W. (2001) Cell 107, 715–726[CrossRef][Medline] [Order article via Infotrieve]
  16. Hinchcliffe, E. H., Cassels, G. O., Rieder, C. L., and Sluder, G. (1998) J. Cell Biol. 140, 1417–1426[Abstract/Free Full Text]
  17. Parry, D. H., and O'Farrell, P. H. (2001) Curr. Biol. 11, 671–683[CrossRef][Medline] [Order article via Infotrieve]
  18. Raff, J. W., Jeffers, K., and Huang, J. Y. (2002) J. Cell Biol. 157, 1139–1149[Abstract/Free Full Text]
  19. Jacobs, H. W., Keidel, E., and Lehner, C. F. (2001) EMBO J. 20, 2376–2386[CrossRef][Medline] [Order article via Infotrieve]
  20. Farr, K. A., and Cohen-Fix, O. (1999) Eur. J. Biochem. 263, 14–19[Medline] [Order article via Infotrieve]
  21. Morgan, D. O. (1999) Nat. Cell Biol. 1, E47–53[CrossRef][Medline] [Order article via Infotrieve]
  22. Brandeis, M., Rosewell, I., Carrington, M., Crompton, T., Jacobs, M. A., Kirk, J., Gannon, J., and Hunt, T. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4344–4349[Abstract/Free Full Text]
  23. Tsien, R. Y. (1998) Annu. Rev. Biochem. 67, 509–544[CrossRef][Medline] [Order article via Infotrieve]
  24. Matz, M. V., Fradkov, A. F., Labas, Y. A., Savitsky, A. P., Zaraisky, A. G., Markelov, M. L., and Lukyanov, S. A. (1999) Nat. Biotechnol. 17, 969–973[CrossRef][Medline] [Order article via Infotrieve]
  25. Chang, D. C. (1997) Methods Mol. Biol. 62, 307–318[Medline] [Order article via Infotrieve]
  26. Zachariae, W. (1999) Curr. Opin. Cell Biol. 11, 708–716[CrossRef][Medline] [Order article via Infotrieve]
  27. Fang, G., Yu, H., and Kirschner, M. W. (1998) Mol. Cell 2, 163–171[CrossRef][Medline] [Order article via Infotrieve]
  28. Kramer, E. R., Gieffers, C., Holzl, G., Hengstschlager, M., and Peters, J. M. (1998) Curr. Biol. 8, 1207–1210[CrossRef][Medline] [Order article via Infotrieve]
  29. Kotani, S., Tanaka, H., Yasuda, H., and Todokoro, K. (1999) J. Cell Biol. 146, 791–800[Abstract/Free Full Text]
  30. Shirayama, M., Zachariae, W., Ciosk, R., and Nasmyth, K. (1998) EMBO J. 17, 1336–1349[CrossRef][Medline] [Order article via Infotrieve]
  31. Visintin, R., Prinz, S., and Amon, A. (1997) Science 278, 460–463[Abstract/Free Full Text]
  32. Zur, A., and Brandeis, M. (2001) EMBO J. 20, 792–801[CrossRef][Medline] [Order article via Infotrieve]
  33. Hagting, A., Den Elzen, N., Vodermaier, H. C., Waizenegger, I. C., Peters, J. M., and Pines, J. (2002) J. Cell Biol. 157, 1125–1137[Abstract/Free Full Text]
  34. Glotzer, M., Murray, A. W., and Kirschner, M. W. (1991) Nature 349, 132–138[CrossRef][Medline] [Order article via Infotrieve]
  35. Fang, G., Yu, H., and Kirschner, M. W. (1998) Genes Dev. 12, 1871–1883[Abstract/Free Full Text]
  36. King, R. W., Deshaies, R. J., Peters, J. M., and Kirschner, M. W. (1996) Science 274, 1652–1659[Abstract/Free Full Text]
  37. Ciosk, R., Zachariae, W., Michaelis, C., Shevchenko, A., Mann, M., and Nasmyth, K. (1998) Cell 93, 1067–1076[CrossRef][Medline] [Order article via Infotrieve]
  38. Uhlmann, F., Lottspeich, F., and Nasmyth, K. (1999) Nature 400, 37–42[CrossRef][Medline] [Order article via Infotrieve]
  39. Uhlmann, F., Wernic, D., Poupart, M. A., Koonin, E. V., and Nasmyth, K. (2000) Cell 103, 375–386[CrossRef][Medline] [Order article via Infotrieve]
  40. Waizenegger, I. C., Hauf, S., Meinke, A., and Peters, J. M. (2000) Cell 103, 399–410[CrossRef][Medline] [Order article via Infotrieve]
  41. Pines, J., and Hunter, T. (1991) J. Cell Biol. 115, 1–17[Abstract/Free Full Text]
  42. Girard, F., Fernandez, A., and Lamb, N. (1995) J. Cell Sci. 108, 2599–2608[Abstract]
  43. Amon, A. (1997) EMBO J. 16, 2693–2702[CrossRef][Medline] [Order article via Infotrieve]
  44. Hunt, T., Luca, F. C., and Ruderman, J. V. (1992) J. Cell Biol. 116, 707–724[Abstract/Free Full Text]
  45. Lim, H. H., Goh, P. Y., and Surana, U. (1998) Curr. Biol. 8, 231–234[CrossRef][Medline] [Order article via Infotrieve]
  46. Yeong, F. M., Lim, H. H., Padmashree, C. G., and Surana, U. (2000) Mol. Cell 5, 501–511[CrossRef][Medline] [Order article via Infotrieve]
  47. Wasch, R., and Cross, F. R. (2002) Nature 418, 556–562[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Clin. Cancer Res.Home page
Y.-M. Wang, L.-X. Hu, Z.-M. Liu, X.-F. You, S.-H. Zhang, J.-R. Qu, Z.-R. Li, Y. Li, W.-J. Kong, H.-W. He, et al.
N-(2,6-Dimethoxypyridine-3-yl)-9-Methylcarbazole-3-Sulfonamide as a Novel Tubulin Ligand against Human Cancer
Clin. Cancer Res., October 1, 2008; 14(19): 6218 - 6227.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
X.-F. Huang, S.-K. Luo, J. Xu, J. Li, D.-R. Xu, L.-H. Wang, M. Yan, X.-R. Wang, X.-B. Wan, F.-M. Zheng, et al.
Aurora kinase inhibitory VX-680 increases Bax/Bcl-2 ratio and induces apoptosis in Aurora-A-high acute myeloid leukemia
Blood, March 1, 2008; 111(5): 2854 - 2865.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
A. G. Scibetta, S. Santangelo, J. Coleman, D. Hall, T. Chaplin, J. Copier, S. Catchpole, J. Burchell, and J. Taylor-Papadimitriou
Functional Analysis of the Transcription Repressor PLU-1/JARID1B
Mol. Cell. Biol., October 15, 2007; 27(20): 7220 - 7235.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
K. E. Strunk, C. Husted, L. C. Miraglia, M. Sandahl, W. A. Rearick, D. M. Hunter, H. S. Earp III, and R. S. Muraoka-Cook
HER4 D-Box Sequences Regulate Mitotic Progression and Degradation of the Nuclear HER4 Cleavage Product s80HER4
Cancer Res., July 15, 2007; 67(14): 6582 - 6590.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
M. Li, J. P. York, and P. Zhang
Loss of Cdc20 Causes a Securin-Dependent Metaphase Arrest in Two-Cell Mouse Embryos
Mol. Cell. Biol., May 1, 2007; 27(9): 3481 - 3488.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
A. J. Holland and S. S. Taylor
Cyclin-B1-mediated inhibition of excess separase is required for timely chromosome disjunction
J. Cell Sci., August 15, 2006; 119(16): 3325 - 3336.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
E. S. Bensen, A. Clemente-Blanco, K. R. Finley, J. Correa-Bordes, and J. Berman
The Mitotic Cyclins Clb2p and Clb4p Affect Morphogenesis in Candida albicans
Mol. Biol. Cell, July 1, 2005; 16(7): 3387 - 3400.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
S. Akhter, C. T. Richie, J. M. Deng, E. Brey, X. Zhang, C. Patrick Jr., R. R. Behringer, and R. J. Legerski
Deficiency in SNM1 Abolishes an Early Mitotic Checkpoint Induced by Spindle Stress
Mol. Cell. Biol., December 1, 2004; 24(23): 10448 - 10455.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. B. Geigl, S. Langer, S. Barwisch, K. Pfleghaar, G. Lederer, and M. R. Speicher
Analysis of Gene Expression Patterns and Chromosomal Changes Associated with Aging
Cancer Res., December 1, 2004; 64(23): 8550 - 8557.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
J. Liu, S. Vasudevan, and E. T. Kipreos
CUL-2 and ZYG-11 promote meiotic anaphase II and the proper placement of the anterior-posterior axis in C. elegans
Development, August 1, 2004; 131(15): 3513 - 3525.
[Abstract] [Full Text] [PDF]

Home page
 Plant CellHome page
M. Weingartner, M.-C. Criqui, T. Meszaros, P. Binarova, A.-C. Schmit, A. Helfer, A. Derevier, M. Erhardt, L. Bogre, and P. Genschik
Expression of a Nondegradable Cyclin B1 Affects Plant Development and Leads to Endomitosis by Inhibiting the Formation of a Phragmoplast
PLANT CELL, March 1, 2004; 16(3): 643 - 657.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/39/37865    most recent
M306376200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chang, D. C.
Right arrow Articles by Luo, K. Q.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chang, D. C.
Right arrow Articles by Luo, K. Q.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement