Report |
Address correspondence to Sergei A. Kuznetsov, Institute of Cell Biology and Biosystems Technology, University of Rostock, Albert-Einstein-Str. 3, D-18051 Rostock, Germany. Tel.: 49-381-498-6311. Fax: 49-381-498-6302. E-mail: Sergei.Kuznetsov{at}biologie.uni-rostock.de
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Abstract |
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Key Words: cell cycle; organelle movement; Xenopus; oocyte; membrane fusion
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Introduction |
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A convenient system to study cell cycledependent motility and network formation of ER is interphase-arrested extract (I extract)* and metaphase-arrested extract (M extract) from Xenopus frog eggs (Allan and Vale, 1991). We use this system here to investigate whether mitotic reorganization affects ER movement on F-actin and found that both myosin Vdriven ER motility and F-actindependent ER network formation, a consequence of membrane movement and fusion, became activated in M extracts. To our knowledge, this is the first study demonstrating an up-regulation of both membrane organelle motility and fusion during mitosis.
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Results and discussion |
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The motion analysis showed that the movement of all membranous organelles on F-actin was ATP dependent and unidirectional. The average velocity of moving globular vesicles was almost identical in both types of extracts (Fig. 1 E, a and b; Student's t test, P = 0.94). However, the velocity of moving ER tubules in M extracts was significantly higher (30%; Student's t test, P = 4.6 x 10-4) than in I extracts (Fig. 1 E, a and b). Greater differences were observed for the distribution of the measured distances of the moving organelles. The average distance moved by globular vesicles in the I extracts was 50% greater (Student's t test, P = 2.2 x 10-4) than in M extracts (Fig. 1 E, c and d). In contrast, the average distance moved by ER tubules in I extracts was 60% shorter (Student's t test, P = 2.0 x 10-5) than in M extracts (Fig. 1 E, c and d). We also performed a quantitative analysis of organelle movement on F-actin. We found that the motile activity (number of organelle movements per field per min) of globular vesicles was down-regulated approximately fivefold (Fig. 2 A). In contrast, the motile activity of the ER tubules was greatly up-regulated (about fourfold) in M extracts (Fig. 2 A). Together, these results demonstrate that ER movement and movement of globular vesicles on F-actin is differentially regulated throughout the cell cycle.
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In M extracts, we detected not only activation of F-actindependent ER motility but also the formation of a reticular network (Fig. 3 A, a). This network formed after incubation of extract for 30 min was labeled with both the fluorescent dye DiOC6 and an antibody to EcaSt/PDI (Fig. 3 A, b and e). Under the conditions used, we did not detect ER network formation in I extracts (Fig. 3 A, c, d, and f). Furthermore, ER network formation in M extracts was dynamic (video 3 available at http://www.jcb.org/cgi/content/full/jcb.200204065/DC1) and strongly dependent on the presence of F-actin, since in the presence cytochalasin D, a drug preventing actin polymerization, no ER network was observed (Fig. 3 B). Collectively, our results argue that both F-actindependent ER network formation and ER tubule movement on F-actin are activated in M extracts. Notably, the microtubule-dependent ER network formation and movement (Allan and Vale, 1991) and the ER network formation by a controlled fusion reaction (Dreier and Rapoport, 2000) are inhibited in these M extracts.
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To test directly whether myosin V is indeed still associated with ER in M extracts, we performed indirect immunofluorescence labeling of ER networks with a DIL2 antibody against mouse myosin Va, which strongly reacted with Xenopus myosin V as shown by immunoblotting (see Fig. 5 B). DIL2 antibody labeling revealed a pattern very similar to the ER network (Fig. 4 A, a). This pattern was not observed when a control antibody against myosin II was used (Fig. 4 A, b). Strikingly, the bulk of myosin V labeling colocalized with the immunosignal for EcaSt/PDI, the ER marker protein (Fig. 4 A, c and d). These data indicate that myosin V is associated with ER in M extracts.
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Because myosin V is more strongly phosphorylated in metaphase compared with interphase (Rogers et al., 1999) and the phosphorylation of myosin V by calcium/calmodulin-dependent kinase II (CaMKII) resulted in the release of the motor from pigment granules (Karcher et al., 2001), we tested the involvement of protein phosphorylation in the movement of membrane organelles on F-actin. We first treated extracts with 2 µM staurosporine, a broad spectrum inhibitor of protein kinases (Ruegg and Burgess, 1989). This treatment blocked strongly both vesicle and ER motility in I and M extracts (Fig. 5 A). Surprisingly, the addition of 2 µM CaMKII (281309) inhibitor peptide, which completely inhibits CaMKII activity in Xenopus egg extracts (Matsumoto and Maller, 2002), also blocked an activation of both ER movement in M extracts and vesicle movement in I extracts (Fig. 5 A).
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Our data provide strong evidence that ER membranes can move along F-actin and fuse during mitosis. The activation of myosin Vdriven motility that we have observed could also explain the increase of ER motility seen during meiotic maturation of Xenopus oocytes (Kume et al., 1997) and the accumulation of ER in the F-actinrich cortex of frog, mouse, and hamster eggs in vivo (Houliston and Elinson, 1991; Mehlmann et al., 1995; Shiraishi et al., 1995). It thus appears that the role of microtubules in moving the ER during interphase is taken over during mitosis by the actin system.
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Materials and methods |
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For some experiments, the transition from I to mitotic M extracts was induced by 1:0.75 (vol:vol) dilution of I extract in glycine buffer (300 mM glycine, 120 mM D-gluconic acid, 100 mM taurine, 20 mM KCl, 5 mM MgCl2, 5 mM EGTA, 30 µM nocodazole, pH 7.2) supplemented with 0.1 mg/ml bacterially expressed CYC (Glotzer et al., 1991) and after incubation at RT in the presence of 0.5 µM rhodamine-phalloidin and ATP-regenerating system (1 mM ATP, 0.5 mg/ml phosphocreatine kinase, 10 mM creatine phosphate). The morphology of added sperm chromatin (Murray, 1991) and the histone H1 kinase assays were used to monitor the status of the cell cycle and the activity of p34cdc2kinase (Felix et al., 1993).
In vitro motility assay and ER network formation
I or M extracts were diluted by 1:0.75 (vol:vol) in glycine buffer. Diluted extracts were supplemented with an ATP-regenerating system and 0.5 µM rhodamine-phalloidin and incubated for 15, 30, 45, and 60 min at RT. At each time point, the extracts were perfused into a microscope chamber. Organelle motility was monitored by video-enhanced contrast differential interference (DIC) microscopy, and the presence of rhodamine-phalloidinlabeled F-actin was confirmed by fluorescence microscopy. At each time point of incubation, three random fields (23 µm x 22 µm) were recorded for 3 min. The myosin-based motile activity was determined by counting moving organelles on tracks invisible with DIC microscopy and averaging the numbers obtained from 12 random fields after all time points of extract incubation. For organelle motility during transition from interphase to metaphase, the extract was examined by microscopy immediately after dilution, and continuous recording of 30 random fields for 3 min during 90 min of observation was performed.
ER network formation was monitored by both DIC microscopy and fluorescence microscopy after staining with 1 µg/ml DiOC6 (Molecular Probes). To stain the Golgi apparatus, the extracts were incubated with 5 µM C6-NBD-Cer (N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproyl D-erythro-sphingosine) (Molecular Probes) and examined by fluorescence microscopy. For immunofluorescence labeling of ER, 510 µl of extracts prepared as for DIC microscopy were placed on a poly-L-lysinecoated coverslip followed by incubation for 30 min at RT and then fixed by adding carefully 200 µl of 4% PFA with 4% sucrose in PBS. After fixation for 30 min, immunofluorescence labeling of the ER was performed as previously described (Kaether et al., 1997) using an mAb against rat ECaSt/PDI (StressGen Biotechnologies) and DIL2 polyclonal rabbit antibody against a GST fusion protein containing heavy chain residues 9101,106 of mouse myosin-Va (Wu et al., 1997).
The effect of the following reagents on organelle motile activity and ER network formation was tested: a DIL2 antibody at 1:25 dilution, a polyclonal rabbit antibody raised against skeletal myosin II (M7648; Sigma-Aldrich) at 1:25 dilution, and 5 µM cytochalasin D (Sigma-Aldrich), 2 µM staurosporine (Calbiochem), and 2 µM CaMKII (281309) inhibitor peptide (Calbiochem). For this, the diluted extracts were preincubated with the agents mentioned above for 30 min at RT.
Fractionation of extracts, 32P labeling of membranes, and myosin V immunoprecipitation
Membrane fractions were prepared by flotation as described in Lane and Allan (1999) which was slightly modified. 300 µl of I extracts were diluted 1:0.75 (vol:vol) in glycine buffer containing an ATP-regenerating system. For obtaining the membrane fraction from mitotic M extracts, diluted I extracts were supplemented with CYC. To study the effect of CaMKII activity on membrane protein phosphorylation, 2 µM CaMKII inhibitor and 50 µCi [
-32P]ATP (Amersham Biosciences) were added to diluted extracts. All samples were incubated for 60 min at RT and then were diluted once again with 2.75 ml of ice-cold glycine buffer containing 65% sucrose and 10 mM ß-glycerophosphate. After centrifugation, the membrane fractions floated through 0.8 ml of glycine buffer with 50% sucrose were subjected to SDS-PAGE followed by Western blot and phosphoimaging. The immunoprecipitation of myosin V from the membrane fractions using affinity-purified DIL2 antibody was performed as described (Kromer et al., 1998).
Online supplemental material
Video 1 shows globular organelle movement on F-actin in I extract. The video illustrates the movement of globular organelles along F-actin observed with DIC microscopy and described in Fig. 1 B. Video 2 shows movement of tubular membrane structures (ER) on F-actin in meiosis II M extract. The video illustrates the movement of tubular membrane structures (ER) along F-actin observed with DIC microscopy and described in Fig. 1 C. Video 3 shows F-actindependent dynamics of ER network in meiosis II
M extract. The video illustrates the movement along F-actin of tubular extensions of ER network observed with DIC microscopy and shown in Fig. 3 A. Videos 13 are available online at http://www.jcb.org/cgi/content/full/jcb.200204065/DC1.
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Footnotes |
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* Abbreviations used in this paper: CaMKII, calcium/calmodulim-dependent kinase II; CYC
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Acknowledgments |
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This work was supported by grants from the Deutsche Forschungsgemeinschaft (WE 790/19-1; GE 550/3-1) and the Deutsche Forschungsgemeinschaft Innovationskolleg "Komplexe und Zelluläre Sensorsysteme."
Submitted: 15 April 2002
Revised: 16 October 2002
Accepted: 17 October 2002
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