Movement of Membrane Domains and Requirement of ... - Cell Press

Developmental Cell, Vol. 9, 781–790, December, 2005, Copyright ª2005 by Elsevier Inc.

DOI 10.1016/j.devcel.2005.11.002

Movement of Membrane Domains and Requirement of Membrane Signaling Molecules for Cytokinesis Michelle M. Ng,1,3 Fred Chang,2,3,4 and David R. Burgess1,3,5,* 1 Department of Biology Boston College 140 Commonwealth Avenue Chestnut Hill, Massachusetts 02467 2 Department of Microbiology Columbia University New York, New York 10032 3 Marine Biological Laboratory Woods Hole, Massachusetts 02543

Summary Plasma membrane subdomains enriched in sphingolipids, cholesterol, and signaling proteins are critical for organization of actin, membrane trafficking, and cell polarity, but the role of such domains in cytokinesis in animal cells is unknown. Here, we show that eggs form a plasma membrane domain enriched in ganglioside GM1 and cholesterol where tyrosine phosphorylated proteins occur at late anaphase at the contractile ring. The equatorial membrane domain forms by movement-specific lipids and proteins and is dependent on anaphase onset, myosin light chain phosphorylation, actin, and microtubules. Isolated detergentresistant membranes contain Src and PLCg, which become tyrosine phosphorylated at cytokinesis, and whose activation is required for furrow progression. These studies suggest that membrane domains at the cleavage furrow possess a signaling pathway that contributes to cytokinesis. Introduction Cytokinesis is the final act of cell division whereby the cytoplasm is pinched apart by constriction of an actinbased contractile ring (Rappaport, 1996). The contractile ring is intimately associated with the plasma membrane to which its assembly and contraction must be anchored and which is the site of new membrane addition during late cytokinesis (Albertson et al., 2005; Strickland and Burgess, 2004). Plasma membrane liquid ordered microdomains floating in the liquid disordered bilayer organize platforms for proteins involved in membrane sorting and trafficking, cell polarization, and signal transduction (Lai, 2003; Foster et al., 2003; Maxfield, 2002; Pike, 2003; Rajendran and Simons, 2005). While there is controversy about the size and mobility of membrane microdomains (Edidin, 2003), FRAP studies indicate that molecules such as the ganglioside GM1 are present in unique domains of the plasma membrane enriched in signaling molecules (Edidin, 2003; Glebov and

*Correspondence: [email protected] 4 Lab address: http://microbiology.columbia.edu/fredchang/. 5 Lab address: http://www.bc.edu/schools/cas/biology/facadmin/ burgess/.

Nichols, 2004a, 2004b; Munro, 2003; Nichols, 2003; Pierce, 2004; Pike, 2003; Rajendran and Simons, 2005). Biochemical preparations of membranes stable to extraction in cold nonionic detergent (detergent-resistant membranes; DRMs) are thought to represent aggregates of these plasma membrane domains (Edidin, 2003; Munro, 2003; Rajendran and Simons, 2005) and possess glycosylphosphatidylinositol (GPI)-anchored proteins, tyrosine kinases of the Src family, flotillins, cholesterol, gangliosides, and sphingolipids. DRMs containing signaling proteins have been shown to be dependent on the actin cytoskeleton for their assembly and maintenance (Holowka et al., 2000; Oliferenko, 1999; Seveau et al., 2001). Recently, FAK-dependent polarization of ganglioside GM1 and stable microtubules have been noted in polarizing migratory cells (Gundersen et al., 2004). Likewise, active Rac in adhering cells binds to low-density, cholesterol-rich membranes identified by GM1 (del Pozo et al., 2004). For activation of signaling to occur, lipid domains may thus cluster to facilitate formation of mobile signaling platforms. There is precedent for membrane domains in the cleavage furrow. Cell surface wheat germ agglutinin receptors in sea urchin eggs (Yoshigaki, 1997) and concanavalin A receptors in mammalian tissue culture cells (Koppel, 1982) appear to accumulate in the cleavage furrow in mid- to late anaphase in a microtubuledependent manner. Late in anaphase, an integral membrane protein, Cps1p, appears at the cell division site in Schizosaccharomyces pombe (Liu et al., 2002). Sterolrich membrane domains have been found at growing tips of S. pombe and at the septum in S. pombe and their integrity is essential for proper cytokinesis (Takeda et al., 2004; Wachtler et al., 2003). Cholesterol depletion results in polyploidy in human tissue culture cells (Fernandez et al., 2004). In hematopoietic cells, the lipid microdomain proteins, flotillins, form large stable domains in the plasma membrane and accumulate at a higher density in the cleavage furrow (Solomon et al., 2002). Ongoing phosphatidylinositol (PtdIns) metabolism, including active PLCg leading to Ca2+ release, is required for cytokinesis (Saul et al., 2004), and Ca2+ release via IP3 receptors is required for furrowing in spermatocytes and sea urchin eggs (Shuster and Burgess, 1999; Wong et al., 2005). PtdIns and its downstream products have been shown, including in sea urchin eggs, to be required for cytokinesis (Becchetti and Whitaker, 1997; Brill et al., 2000). PIP3 and PI3 kinase are localized to the polar membranes of dividing cells, whereas PTEN is found in the furrow (Janetopoulos et al., 2005). Likewise, PI(4,5)P2 elevates in the cleavage furrow of Drosophila spermatocytes and tissue culture cells (Brill et al., 2000; Field et al., 2005; Wong et al., 2005). Maintenance of the cleavage furrow in both yeast and mammalian cells is dependent on a barrier to diffusion in the furrow membrane, with proteins embedded in the inner leaflet showing slower diffusion rates across the furrow membrane than across the polar membrane (Dobbelaere and Barral, 2004; Schmidt and Nichols, 2004). Thus, there is potential for signaling

Developmental Cell 782

platforms in the plasma membrane to play a role in cytokinesis. Here we tested whether the plasma membrane of dividing sea urchin eggs forms signaling platforms and whether an associated signaling complex plays a role in cytokinesis. We find formation of an equatorial membrane domain, with the contractile ring, enriched in ganglioside GM1 and cholesterol and possessing activated tyrosine kinases and PLCg whose activity is essential for cytokinesis. These results suggest that microtubules (MT) and actin filament induce aggregation of unique membrane domains at the future cleavage furrow, bringing together a signaling complex which is required for cytokinesis in embryonic cells. Results Membrane Domains Accumulate in the Equatorial Plasma Membrane at Mid-Anaphase The ganglioside GM1, a marker for cholesterol-rich membrane domains, can be detected in either living cells or in preserved cells with fluorescent cholera toxin subunit B (CTB). CTB evenly labeled the cell surface of live interphase cells (Figure 1A). The spherical eggs elongate during anaphase, and during this period a significant amount of GM1 begins to redistribute and move to the future furrow (Figure 1A; see Movies 1 and 2 in Supplemental Data available with this article online). As the furrow deepens, GM1 concentrates in the furrow by moving away from the polar regions. Quantitation of GM1 in metaphase versus furrowing cells shows that there is a 7-fold increase in GM1 in the furrow region relative to the polar region of dividing cells (Figure 1B), whereas in metaphase, the distribution of GM1 in the pole versus the future furrow was the same. WGA binds primarily to glycoproteins with terminal sialic acid residues, and it has been shown to be concentrated in the cleavage furrow in an MT-dependent manner (Yoshigaki, 1997). Using fluorescent WGA on living cells, we found movement of WGA receptors to the cleavage furrow during anaphase in a similar pattern to the movement of the ganglioside GM1 (Figure 1C; Movie 3). Eggs were fixed prior to staining with filipin to localize cholesterol-rich membrane domains (Figure 1D). Cells from fertilization to mid-anaphase exhibit bright, even fluorescence of cholesterol-containing plasma membrane (Figure 1D). As cells enter mid- to late anaphase, they elongate into an oval shape and while the entire plasma membrane remains fluorescent, cholesterolrich domains accumulate as a fine, even equatorial band centered around the former metaphase plate (Figure 1D). This accumulation of an equatorial cholesterol-rich membrane band occurs prior to furrow formation and remains as a distinct band in the cleavage furrow until late cytokinesis when it disappears, resulting in the two daughter cells showing an even distribution. Costaining fixed cells with filipin and CTB shows the anaphase cholesterol-rich membrane equatorial band colocalizing with GM1 is narrow in forming an equatorial stripe on the cell surface (Figure 2A). The rest of the cell retains weak fluorescence, indicating cholesterol- and GM1-rich domains remain throughout the entire cell surface. Filipin staining of the furrow region occurred in cells regardless of the time after fixation when observations were made.

Figure 1. Lipid Raft Marker GM1, Glycoproteins, and Cholesterol Accumulate in the Furrow at Cytokinesis (A) Frames from a confocal time-lapse movie of live cytokinesis stage cells stained with CTB to label lipid raft marker GM1. Frames were taken every 60 s. The scale bar represents 50 mm. (B) Quantitation of CTB fluorescence in the furrow at cytokinesis. Frames were taken from confocal time-lapse movies of live cells stained with CTB. Mean fluorescence intensity (pixel values) readings were taken from equal sized boxes located at equator and pole regions for each cell (n = 14) at metaphase (round) and cytokinesis. Shown on the graph is the ratio of mean fluorescence in the equatorial region versus the pole region. (C) Glycoproteins with terminal sialic or N-acetylglucosamine residues accumulate in the cleavage furrow in cytokinesis. Rhodamine wheat germ agglutinin was visualized in live sea urchin embryos by time-lapse microscopy. The scale bar represents 50 mm. (D) Cholesterol localization through the cell cycle. Sea urchin embryos were fixed at various stages of the cell cycle. Cholesterol was detected by filipin staining and DNA was codetected by addition of Hoechst 33342. Int, interphase; Meta, metaphase; Early Ana, early anaphase; Late Ana, late anaphase; Cyt, cytokinesis. The scale bar represents 20 mm.

In a complementary strategy to image lipids not present in cholesterol-rich domains, we used FAST DiI, which possesses diunsaturated linoleyl (C18:2) tails, to monitor more fluid regions of the bilayer (Hao et al., 2004; Mukherjee et al., 1999). We applied FAST DiI with a micropipette to the cell surface, where it was observed to spread rapidly across the cell membrane.

Membrane Domains and Cytokinesis 783

Table 1. Effects of Inhibitors on GM1- and Cholesterol-Rich Rings

Inhibitor

Target

Blocks CT-B Ring Filipin Ring Cell Division Formation Formation

MG132 Cytochalasin D (high-dose) Cytochalasin D (low-dose) Nocodazole ML-7 Blebbistatin

Proteasome Actin

Yes Yes

No No

No No

Actin

Yes

Yes

Yes

Microtubules MLCK Myosin II ATPase Src family Src, Fyn, Lck

Yes Yes Yes

No No Yes

No No Yes

Yes Yes

No No

No No

Genistein SU6656

Inhibitors of actin, microtubules, and mitosis block formation of GM1- and cholesterol-rich ring formation. Cytokinesis stage cells were fixed and stained with filipin and CTB after exposure to inhibitors to assess the effect of the inhibitors on the formation of the filipin ring.

metaphase, time-lapse imaging by wide-field fluorescence microscopy showed that while the CTB accumulated into the furrow region, the FAST DiI spread up to the furrow evenly but did not accumulate (Figure 2C; Movies 5 and 6). DiI C18, which is able to spread to both liquid ordered and liquid disordered membrane domains, is able to spread into the furrow (Figure 2B). Likewise, we did not observe any relocalization of the lipidanchored Src or Abl proteins to the furrow (Figure 2D).

Figure 2. GM1, Cholesterol, Glycoproteins, and DiI C18 Localize to the Cleavage Furrow in Cytokinesis (A) Colocalization of GM1 and cholesterol rings in cytokinesis stage cells. Fixed cells were costained with filipin and CTB. The scale bar represents 50 mm. (B) DiI C18 enriches in furrow membrane, but FAST DiI is excluded from furrow membrane. Either a FAST DiI- or DiI C18-saturated drop of vegetable oil was applied to the surface of a fertilized egg. Diffusion of the dye through the plasma membrane during cytokinesis was observed by time-lapse fluorescence microscopy (DiI C18) or time-lapse confocal microscopy (FAST DiI). FAST DiI (top panel) did not spread into the cleavage furrow or the interface between the two daughter cells, whereas DiI C18 (bottom panel) moved to the cleavage furrow and was also present in the interface between the two daughter cells. Red arrows indicate location of oil droplet. Time is represented in minutes (0 ) and seconds (0 0 ). The scale bar represents 50 mm. (C) Ganglioside GM1, but not FAST DiI enriches in the furrow at cytokinesis. Still images from time-lapse wide-field fluorescence movies of a live cell labeled with both CTB and FAST DiI. The FAST DiI droplet was added at metaphase. Time is indicated in minutes. The scale bar represents 50 mm. (D) PLCg, c-Src, and Abl do not enrich in the furrow at cytokinesis. Cytokinesis stage cells were fixed and immunostained for PLCg-1, c-Src, or Abl (top panels).

When applied to the surface late in cytokinesis, FAST DiI spread to the furrow membrane but did not migrate into the furrow membrane (Figure 2B; Movie 4), indicating that the furrow membrane is more liquid ordered. In cells which were colabeled with both CTB and FAST DiI at

Formation of the Equatorial GM1- and CholesterolRich Membrane Band Requires Anaphase Onset, Microfilaments, Myosin II, and Microtubules Cleavage furrow formation in embryonic cells requires anaphase onset (Shuster and Burgess, 2002b). In order to determine whether anaphase onset was required for the equatorial membrane domain formation, cells were arrested in metaphase with the proteasome inhibitor MG-132. Metaphase arrested cells do not accumulate GM1- or cholesterol-rich domains into an equatorial band (Table 1). Because the GM1- and cholesterol-rich membrane equatorial band appeared coincident with appearance of the contractile ring, we determined its association with the actin-based contractile ring. The contractile ring in cells early in cytokinesis is a broad band appearing when the cells elongate, as revealed by fluorescent phalloidin staining (Figure 3A) or by electron microscopy (Schroeder, 1972). The equatorial cholesterol band is much narrower than the contractile ring and perfectly bisects the broad actin contractile ring (Figure 3A) but forms coincident with detection of actin in the contractile ring. Cells treated with cytochalasin D (CD) to disrupt actin never form a contractile ring (Schroeder, 1972) nor an equatorial GM1- or cholesterol-rich membrane band (Table 1). A low dose of CD does not inhibit ring assembly but fragments the ring upon contraction (Shuster and Burgess, 2002a). Pools of cholesterol-rich membranes were found to surround fragmented pieces of actin in the early contractile ring in cells after low CD (Figure 3A). Thus, F-actin is necessary to organize and stabilize the equatorial membrane ring.


Figure 3. Effects of Inhibition of the Actomyosin Ring and Depletion of Cholesterol on Formation of the GM1-Rich Ring and Completion of Cytokinesis (A) Colocalization of equatorial cholesterol ring with actin contractile ring at cytokinesis. Cytokinesis stage cells were fixed and cholesterol in lipid rafts was detected with filipin (green), and F-actin was stained with phalloidin (red). The filipin-stained ring forms a narrow band that colocalizes with the center of the wider actin contractile ring. When a low dose (2 mg/ml) of CD is added to cells following nuclear envelope breakdown, the actin contractile ring is able to form and contract, but later breaks, resulting in binucleate cells. In these low-dose CD-treated cells, the cholesterol ring is absent from the areas where the actin contractile ring is broken. The scale bar represents 50 mm. (B) ML-7 inhibits cytokinesis but not mitosis. Top panel: frames from movie of cell treated with 100 mM ML-7 following nuclear envelope breakdown. The numbers in the upper right corner represent time after fertilization in minutes. The scale bar represents 50 mm. Bottom panel: doseresponse curve of the effects of ML-7 on cell division. (C) Cholesterol depletion blocks completion of cytokinesis. Top panel: Lytechinus pictus eggs were treated with 2.5 mg/ml filipin 5 min prior to the beginning of cell division. Time after fertilization in minutes is indicated. The scale bar represents 50 mm. Bottom panel: dose-response curve of the effects of filipin on cell division. (D) GM1 accumulation in the furrow at cytokinesis is inhibited by cholesterol depletion. GM1 was labeled in live interphase cells with CTB, and at anaphase, either DMSO (control) or filipin (filipin-treated) was added. There is an accumulation of GM1 in the cleavage furrow in control cells (arrow). In the cholesterol-depleted cells, the accumulation of GM1 in the cleavage furrow is inhibited (filipin-treated). (E) Ganglioside GM1 and tyrosine phosphorylated proteins localize to the cell equator at cytokinesis in control cells and cells treated with blebbistatin. Fixed cells were labeled with CTB to visualize GM1 (green) or immunostained for phosphotyrosine (red) and images were acquired using confocal microscopy. The scale bar represents 40 mm.

Astral MTs are required for formation of the cleavage furrow in dividing eggs (Rappaport, 1996). Cells treated with nocodozole to disrupt MTs at the time of nuclear envelope breakdown do not form an equatorial GM1- or cholesterol-rich membrane band at the time control cells enter late anaphase (Table 1). Myosin II light chain phosphorylation, but not its ATPase activity, has been shown to be necessary for actin assembly in the contractile ring, whereas myosin’s ATPase activity is not re-

quired for actin ring assembly but is required for ring contraction (Guha et al., 2005; Murthy and Wadsworth, 2005). Likewise, we find treatment of eggs with ML-7, an inhibitor of MLCK, at nuclear envelope breakdown does not block mitotic progression but blocks formation of a cholesterol- or GM1-rich equatorial band (Table 1; Figure 3B). Cells treated with blebbistatin to inhibit myosin II ATPase activity are able to form a normal actin contractile ring (Guha et al., 2005; Murthy and Wadsworth,


2005) and initiate shallow furrows that later regress to produce binucleate cells. Blebbistatin-treated cells are able to form equatorial GM1- and filipin-staining bands (Figure 3C; Table 1). These results suggest that formation of the cholesterol-rich or GM1-rich equatorial band requires the same components necessary for assembly of the actin contractile ring. Disruption of Unique Equatorial Membrane Domains Blocks Cytokinesis Cells were treated with doses of filipin to deplete cholesterol and associated proteins from the membrane (Liu et al., 1997) at nuclear envelope breakdown, and then fixed at the time when control cells were at the twocell stage. Counts of the percentages of cells divided reveal a dose-dependent inhibitory effect of filipin on cytokinesis (Figure 3C). Nuclear division continues normally in filipin-treated cells at all concentrations used. When filipin was added just prior to cytokinesis onset, cells were able to elongate and began furrowing that failed almost immediately. Filipin addition to cells any time during furrowing stopped furrow advance and caused it to retract (Figure 3C; Movie 7). Live cells were labeled in interphase with CTB to visualize GM1, followed by treatment with filipin early in cleavage to deplete cholesterol. It was found that the ring of GM1 normally seen in the cleavage furrow in control cells was no longer enriched in cholesterol-depleted cells (Figure 3D). DRMs Possess Signaling Molecules Which Are Activated at Cytokinesis Immunofluorescence to localize tyrosine phosphorylated proteins in dividing cells shows that tyrosine phosphorylated proteins accumulate in the furrow membrane during cytokinesis (Figure 3E). Signaling molecules such as Src family members and PLCg, present in cholesterol-rich domains in the plasma membrane, have been isolated in DRMs from eggs (Belton et al., 2001). Because we find tyrosine phosphorylated proteins localized in the furrow membrane, we sought to identify these proteins. However, to date, there is no way to specifically isolate the plasma membrane domains of the furrow, which may include Src family members and PLCg, so we have used a general method of isolating DRMs of the entire plasma membrane containing these signaling molecules. Such a preparation contains the furrow membrane proteins, although they are not enriched, and allows for the analysis of the state of activation of these signaling molecules. Using established methods, DRMs were isolated from eggs at different stages of the cell cycle. A test for the isolation of these membranes is that the preparation should contain ganglioside GM1, tyrosine phosphorylated proteins, Src family members, and PLCg. The protein composition in DRMs isolated from eggs just fertilized, from interphase eggs, or from eggs undergoing cytokinesis was the same (Figure 4A). Analysis of dot blots of these DRMs shows that they possess GM1 and that the amount of GM1 is the same in DRMs isolated from interphase versus dividing cells (Figure 4B). Analysis of the DRMs by Western blotting with a panspecific anti-Src antibody reveals Src family members of 55 kDa, 60 kDa, 115 kDa, and 220 kDa (Figure 4C),

Figure 4. Detergent-Resistant Membranes Possess a Unique Subset of Proteins (A) Coomassie-stained gel of cell fractions. PAGE of whole-cell extracts (whole-cell), cytoplasmic fractions (cytoplasm), and detergent-resistant membrane fractions (DRM) from interphase (Int) cells and dividing (Div) cells. Molecular weight markers are indicated in kilodaltons. (B) GM1 content in DRMs does not change at cytokinesis. One microgram spots of DRM protein isolated from interphase (Int) and cytokinesis (Div) stage cells were spotted onto an Immobilon membrane, which was probed with biotin-CTB and detected using streptavidinHRP (top panel), then stripped and reprobed with a monoclonal antibody against phosphotyrosine (bottom panel). (C) Src family tyrosine kinases are present in DRMs. Western blot of DRM fractions from interphase and cytokinesis stage cells with panspecific polyclonal antibody against Src family kinases. Egg Src at 55 kDa, Fyn at 60 kDa, and Abl at 220 kDa are detected. (D) The Src family member Abl is present in DRMs. Abl is present in the DRM fractions in both interphase and cytokinesis stage samples. (E) Tyrosine phosphorylation of PLCg increases at cytokinesis. PAGE of DRM fractions from interphase stage cells (Int) and cytokinesis stage cells (Div) and then Western blotted with a monoclonal antiPLCg-1 antibody (PLCg-1) or a monoclonal anti-phosphorylatedPLCg antibody (P-PLCg-1). (F) Tyrosine phosphorylation of DRM proteins increases at cytokinesis. Western blot of DRM fractions from interphase (Int) and cytokinesis (Div) stage cells using a monoclonal anti-phosphotyrosine antibody shows increased tyrosine phosphorylation of PLCg-1 and Src in cytokinesis DRMs.

consistent with the known sizes of sea urchin egg Src, Fyn, and Abl tyrosine kinases (Moore and Kinsey, 1994; Walker et al., 1996), although the nature of the


Figure 5. Src, PLCg, and Tyrosine Phosphorylated Proteins Are Present in DRMs, but Are Reduced with Cholesterol Depletion (A) Filipin removes tyrosine phosphorylated proteins from DRM fractions. DRM fractions isolated from unfertilized eggs (UF), embryos collected just after fertilization (JF), interphase stage cells (Int), cytokinesis stage cells (Div), and cytokinesis stage cells treated with 2.5 mg/ml filipin for 5 min (Fil). Equal amounts of total protein were loaded into each lane and then Western blotted with a monoclonal pan-specific anti-Src antibody. Molecular weight markers are indicated in kilodaltons. Arrows mark the locations of PLCg-1 and Src. (B) The Src family kinase inhibitor, genistein, inhibits tyrosine phosphorylation of proteins present in DRMs. Western blots of DRM fractions probed with either monoclonal PLCg-1 antibody (upper set of panels) or polyclonal pan-specific anti-Src antibody (lower set of panels). Interphase cells or cytokinesis stage cells were either untreated (cont) or treated with 100 mM genistein (gen) 5 min prior to collection of DRM fractions. Equal amounts of total protein were loaded into each lane.

115 kDa band is unknown. In addition, Western blots confirm the 220 kDa band present in the DRMs is Abl (Figure 4D). Src is present in DRMs from both interphase and dividing cells, with the amount increasing 2-fold (n = 3) in DRMs from dividing cells when compared with DRMs from interphase cells, whereas the level of Abl does not change. Western blotting also reveals constant levels (n = 3) of the Src effector PLCg in DRMs isolated from eggs at interphase and cytokinesis (Figure 4E). When Western or dot blots of DRMs isolated at different stages of the cell cycle were probed with antibodies against phosphotyrosine, a cell cycle-related change is noted (Figures 4F and 5A). As shown by Belton et al. (2001), we confirm a several-fold increase in both egg 55 kDa Src and PLCg tyrosine phosphorylation at fertilization. However, in DRMs isolated from interphase cells, the level of tyrosine phosphorylation of Src and PLCg is dramatically reduced from levels found in just fertilized DRMs. In DRMs isolated from dividing cells, tyrosine phosphorylation of 55 kDa Src rises 4-fold (n = 3) relative to the levels in interphase DRMs (Figure 5A). No increase in tyrosine phosphorylation of Abl at cytokinesis was detected (Figure 4F). PLCg is found to be activated in DRMs isolated from cells undergoing cytokinesis, as shown by a 2-fold increase in levels of active PLCg or a 2-fold increase in tyrosine phosphorylated PLCg in cytokinesis stage DRMs relative to interphase stage DRMs (Figures 4E and 4F). However, immunolocalization of Src family members and PLCg shows no redistribution to the furrow during cytokinesis (Figure 2E). Treatment of cells beginning cytokinesis with filipin just prior to DRM isolation reveals that Src family members and PLCg are stripped from the DRMs (Figure 5A). DRMs isolated from cells treated with a short pulse of filipin just prior to the time control cells begin cytokinesis do not possess any tyrosine phosphorylated proteins. Blebbistatin-treated cells which just began to furrow, or show weak furrowing on one side of the cell, show phosphotyrosine labeling only in shallow furrow areas (Figure 5B).

members with the broad spectrum inhibitor genistein; Abl with the specific inhibitor Gleevec; and PLCg with the inhibitor U73122, or its inactive isomer, U73343 (Table 1). When U73122 is added at metaphase, cytokinesis, but not mitosis, is blocked in a dose-response manner by inhibition of PLCg (but not by U73343, the inactive isomer). Cells treated with U73122 are able to assemble a contractile ring and initiate a furrow, which does not complete. When genistein is added to cells at metaphase, mitosis proceeds normally but 50% of the cells fail to initiate cytokinesis, whereas the other 50% furrow completely. When the more specific Src, Fyn, and Lyc inhibitor SU6656 is added at metaphase, the initiation of cytokinesis is blocked and the cells do not elongate or form a filipin or GM1 ring, but the cells are able to complete mitosis normally. The Abl inhibitor, Gleevec, had no effect on cytokinesis, mitosis, or formation of cholesterol- or GM1-rich rings. No division-related increase in tyrosine phosphorylation of Src and of PLCg occurred in DRMs from genistein-treated cells isolated at the time control cells were dividing (Figure 5C). Src, Fyn, Abl, and PLCg SH2 Domain Injections Slow Cytokinesis Onset Injection of Src, Fyn, and PLCg SH2 domains inhibits calcium release and egg activation at fertilization (Giusti et al., 2003). To help determine which tyrosine kinases and effectors were involved in cytokinesis, SH2 domains were injected into a single blastomere of two-cell stage embryos at metaphase. The uninjected blastomeres served as timing controls. As shown in Table 2, injection of Src, PLCg-1, Fyn, and Abl SH2 domains either delays or blocks the completion of cytokinesis in the majority of injected cells. Injection of the control phosphatase SHP2-SH2 domain, which has no effect on the fertilization increase in Src activity and PLCg activation, has no effect on cytokinesis or its timing. None of the SH2 domains had an effect on mitotic progression. Discussion

Tyrosine Phosphorylation Is Required for Furrow Progression To test the role of these signaling proteins in cytokinesis, we examined the effects of inhibitors of: Src, Fyn, and Lyc with the specific inhibitor SU6656; general Src family

Here we show that animal cell cytokinesis requires formation of an equatorial membrane ring at the site of cleavage furrow ingression. In sea urchin eggs, formation of the equatorial band enriched in ganglioside


Table 2. Inhibition of Src and PLCg with SH2 Domain Injections Blocks Cytokinesis SH2 Domain

Source

Normal Delayed Blocked

SHP2-SH2 (control) SFK2-SH2 (Src) Fyn-SH2 Abl-SH2 PLCg-SH2

Mouse S. purpuratus Chicken S. purpuratus Bovine

22/23 3/20 2/10 8/29 8/19

1/23 17/20 7/10 21/29 6/19

0 0 1/10 0 5/19

Summary of SH2 injection data. Solutions containing 1–6 mg/ml of specific SH2 domains were injected into single blastomeres of two-cell stage embryos at metaphase. Cells were filmed by timelapse microscopy to monitor effects of the injections. Delayed cells are defined as injected blastomeres that began cytokinesis at least 3 min after uninjected control blastomeres. Mock injections show no effect on cytokinesis onset. SH2 domains show no effect on mitotic progression. Solutions containing 1–6 mg/ml of SH2 domain were injected into single blastomeres of two-cell stage embryos at metaphase. The injection volume was 1%–5% of total cell volume. Cells were filmed by time-lapse microscopy to monitor effects of the injections. Delayed cells are defined as injected blastomeres that divided at least 2 min after uninjected control blastomeres.

GM1, tyrosine phosphorylated proteins, and cholesterol occurs in late anaphase coincident with contractile ring formation and, as with contractile ring assembly, depends upon anaphase onset, actin, MTs, and myosin II light chain phosphorylation. Disruption of Src family tyrosine kinase activity, its downstream effector PLCg’s activity, or cholesterol depletion causes defects in cleavage furrow progression leading to a severe cytokinesis defect. Although a similar sterol-rich ring has been detected at the septum in yeast cells (Takeda et al., 2004; Wachtler et al., 2003) and cholesterol has been shown to be essential for cell division in tissue culture cells (Fernandez et al., 2004), this is the first report, to our knowledge, of such a membrane domain at the cleavage furrow in animal cells. Thus, these studies define a new, essential aspect of animal cell cytokinesis. Others have noted unique membrane domains in the cleavage furrow. The redistribution of phosphatidylethanolamine to the outer leaflet of the plasma membrane in the cleavage furrow membrane is required for completion of cytokinesis (Emoto and Umeda, 2000). Sterolrich membrane domains have been found at growing tips of S. pombe and Saccharomyces cerevisiae and at the septum in S. pombe (Bagnat and Simons, 2002; Takeda et al., 2004; Wachtler et al., 2003). Cell surface receptors in sea urchin eggs and in tissue culture cells move to the cleavage furrow after anaphase onset in an actin- and microtubule-dependent manner (Wang et al., 1994; Yoshigaki, 1997). The lipid microdomain marker proteins, flotillins, aggregate at the cleavage furrow in hematopoietic cells (Rajendran and Simons, 2005) and phosphotyrosine has been noted in the ring canals of Drosophila (Hime et al., 1996), suggesting localization or activation of specific proteins in furrows. Our results are also consistent with the report that cholesterol depletion of vertebrate tissue culture cells inhibits cytokinesis (Fernandez et al., 2004) and that PtdIns(4,5)P2 localizes to the cleavage furrow (Field et al., 2005). Actin movement and assembly of the contractile ring is dependent on myosin II light chain phosphorylation, but not myosin II ATPase activity (Guha et al., 2005; Murthy and Wadsworth, 2005) and we find, similarly,

that the membrane equatorial ring accumulation is dependent on myosin II light chain phosphorylation but not its ATPase activity. Further, like the assembly of the contractile ring, formation of the equatorial membrane band requires anaphase onset and microtubules. However, membrane ring formation is not required for assembly of the contractile ring but the signaling molecules activated in this domain are important for furrow progression. Our work and older studies of the egg surface indicate that formation of the equatorial ring is not due to bulk movement of the plasma membrane. We find that not all membrane proteins or lipids migrate to the furrow, as shown by tracing fluorescently labeled phosphatidylethanolamine and phosphatidylcholine (data not shown), Fast DiI, or c-Src. Likewise, Schroeder (1981) monitored the abundant long microvilli, which possess cores of actin overlaying a rich actin cortex (Burgess and Schroeder, 1977), on the surface of eggs through cytokinesis and found that neither their density nor their lengths changed in the poles versus the furrow area during cytokinesis. Thus, the movement of GM1, WGA receptors, and cholesterol in the plasma membrane may reflect movement of part of the membrane to the furrow. Unique membrane domains at the cleavage site may contribute multiple functions, including membrane trafficking, membrane insertion, membrane protein localization, actin assembly, and signaling pathways. The phosphoinositide PI(3,4,5)P3, known to play a role in actin assembly, changes localization via activation and/or localization of PI3K and PTEN in dividing Dictyostelium (Janetopoulos et al., 2005). Tissue culture cells and Drosophila spermatocytes have been shown to accumulate and have essential roles of PtdIns(4,5)P2 in the cleavage furrow and activate PLCg for cytokinesis (Field et al., 2005; Wong et al., 2005). Our results suggest that, as at fertilization (Giusti et al., 2000), Src is activated at cytokinesis. While Src knockout mice are viable, the abundance of related family members suggests that functional redundancy may allow for a Src family member to play a role in cytokinesis. Src family kinases have been implicated in cell division, including in the localization of septins to the ring canal during Drosophila oogenesis (Djagaeva et al., 2005). Inhibition of Src family kinases blocks growth factormediated mitogenesis and they are implicated in mediating the G2-M transition and completion of cytokinesis (Roche et al., 1995a, 1995b; Tominaga et al., 2000). While c-Abl has been shown to interact with PLCg (Plattner et al., 2003) and actin (Woodring et al., 2003), we do not note activation of Abl at cytokinesis nor does Gleevec block cytokinesis in eggs. c-Src may interact with PSTPIP (a protein tyrosine phosphatase-interacting protein homologous to S. pombe Cdc15p), which localizes to the cleavage furrow, interacts with WASP, and plays a role in cytokinesis (Spencer et al., 1997; Cote et al., 2002). S. pombe Cdc15p is essential for multiple aspects of cytokinesis, including formation of both actin and DRM rings, and has been shown to interact with the Arp2/3 complex and the formin Cdc12p (Carnahan and Gould, 2003; Takeda et al., 2004). Further, the tyrosine phosphatase PTP-BL is localized to the contractile ring, and inactivation of its tyrosine phosphatase domain leads to defects in cytokinesis (Herrmann et al., 2003).


Our data show that injection of Src SH2 domain and Src inhibitors delays or blocks cytokinesis. Further work will be needed to characterize mechanisms for how tyrosine kinase pathways operate in cytokinesis. The roles for PLCg in cytokinesis may be part of the signaling pathway that is also used in fertilization. The fertilization signal in sea urchin eggs involves activation of G proteins and Src activation, which in turn tyrosine phosphorylates PLCg leading to PtdIns(4,5)P2-mediated calcium release and a pH change (Runft et al., 2002). These signals lead to actin filament assembly, cortical granule exocytosis, and activation of metabolism, protein synthesis, and the cell cycle. Recent work also supports a role for activation of PLCg in cytokinesis in spermatocytes (Saul et al., 2004). Our findings here that injection of PLCg SH2 domain or inhibition of MLCK blocks furrowing and our previous report that inhibiting the mitotic IP3-mediated Ca2+ release blocks furrowing (Shuster and Burgess, 2002a) are consistent with a role for activating PLCg in cytokinesis. PLCg-mediated Ca2+ release at cytokinesis in eggs may trigger the exocytotic event leading to new membrane addition in the late cleavage furrow (Shuster and Burgess, 2002a) and the activation of Ca2+-calmodulin-dependent myosin II light chain kinase to regulate contractile ring assembly (Chou and Rebhun, 1986; Shuster and Burgess, 1999). Experimental Procedures Sea Urchin Embryo Culture Gamete collection, fertilization, and preparation from the sea urchins Strongylocentrotus drobachiensis (Northeastern University, Nahant, MA) and Lytechinus pictus (Marinus, Long Beach, CA) were as previously described (Shuster and Burgess, 1999, 2002a; Strickland et al., 2005). Filipin (2.5 mg/ml filipin III; Sigma, St. Louis, MO), cytochalasin D (Sigma), nocodazole (1 mM; Sigma), taxol (10 mM paclitaxel; Sigma), genistein (100 mM; Sigma), blebbistatin (30 mM; Calbiochem, La Jolla, CA), PP1 (10 mM; Biomol, Plymouth Meeting, PA), PP2 (10 mM; Biomol), ML-7 (100 mM; Sigma), U73122 (Calbiochem), and U73343 (Calbiochem) were added to the fertilized eggs at metaphase. For MG132 (Sigma) treatments, eggs were treated for 30 min in 50 mM MG132 prior to fertilization cultured in CaFSW containing 50 mM MG132. Live Cell Imaging For GM1 staining, Alexa 488-cholera toxin, subunit B (5 mg/ml; Molecular Probes, Eugene, OR) was added to live cells 30 min postfertilization, and washed out 50 min postfertilization with three rinses of CaFSW. For staining of glycoproteins with terminal sialic or N-acetylglucosamine, live cells were incubated in CaFSW containing 1% BSA (Sigma) and 30 mg/ml rhodamine-WGA (Molecular Probes) for 10 min following nuclear envelope breakdown, and rinsed three times in CaFSW 1% BSA before observation by confocal microscopy. To label plasma membranes of live cells, a DiI C18 or FAST DiI (Molecular Probes)-saturated aliquot of vegetable oil was drawn into a glass microneedle and a small droplet was microinjected against the plasma membrane at metaphase. In some experiments, cells were initially labeled with fluorescent CTB prior to application of a droplet of FAST DiI. Fixation and Microscopy Eggs were fixed in CaFSW containing 3.7% formaldehyde for 1 hr and rinsed three times in PBS. Eggs were permeabilized in PBS containing 0.1% Triton X-100 for 5 min, followed by three washes in PBS. For cholesterol and actin staining, filipin (12.5 mg/ml) and Alexa Fluor 568 phalloidin (1 U/ml; Molecular Probes) were added to fixed cells for 30 min followed by washes in PBS. For immunofluorescence microscopy, fixed cells were incubated overnight at 4ºC in PBS, 0.5% Triton X-100 (Sigma) in primary anti-

bodies: monoclonal anti-phosphotyrosine (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), mixed monoclonal anti-PLCg-1 (1:100; Upstate Biotechnology, Waltham, MA), polyclonal anti-cSrc (1:20; Santa Cruz Biotechnology), or polyclonal anti-mouse Abl SH2 domain (1:100; Upstate Biotechnology). Cells were rinsed in PBS, 0.5% Triton X-100 and incubated in 0.5% Triton X-100 and one of the following secondary antibodies: Alexa Fluor 488 goat anti-mouse (1:200; Molecular Probes) or Alexa Fluor 546 goat antirabbit (1:200; Molecular Probes). Before microscopy, cells were rinsed in PBS, 0.5% Triton X-100. Microscopy For wide-field microscopy, digital images were obtained on a Nikon TE 200 or TE 2000-U inverted microscope (Melville, NY) equipped with a Hamamatsu Orca or ER CCD camera (Hamamatsu City, Japan) driven by Metamorph software (Molecular Devices, Downingtown, PA) using Nikon Plan Fluor 0.60 NA 403 and 0.45 NA 203 air objective lenses. A Brook cooling stage (Lake Villa, IL) was used to maintain temperature for live cell time-lapse experiments. For confocal microscopy, images were obtained on a Leica DM IRBE inverted scope equipped with the Leica TCSSP2 confocal system (Leica Microsystems, Wetzlar, Germany), using N PLAN 203 air, HCX PL APO 403 oil, and HCX PL APO 633 oil objective lenses. Isolation of DRMs DRMs were obtained following protocols previously used in sea urchins (Belton et al., 2001). For filipin- or genistein-treated samples, 25 mg/ml filipin or 100 mM genistein was added 5 min before eggs were homogenized. Immunoblot Analysis Protein concentration was determined using BCA protein assay reagents (Pierce, Rockford, IL), and samples containing equal amounts of protein were run on 10% SDS-PAGE gels. Proteins were transferred to an Immobilon P membrane (Millipore, Billerica, MA) in a Genie apparatus (Idea Scientific, Minneapolis, MN). Blots were blocked with 3% BSA, 0.2% Tween-20, Tris-buffered saline (TBS; 137 mM NaCl [pH 7.4], 10 mM Tris; Fisher Scientific, Pittsburgh, PA) for 1 hr at room temperature, then incubated with either a 1:200 dilution of rabbit anti-c-Src antibody (sc-18; Santa Cruz Biotechnology), a 1:1000 dilution of mouse anti-phosphorylated tyrosine antibody (sc-508; Santa Cruz Biotechnology), a 1:1000 dilution of mouse anti-PLCg (PLCg-1; Upstate Biotechnology), a 1:1000 dilution of rabbit anti-Abl SH2 (06-465; Upstate Biotechnology), or a 1:200 dilution of rabbit anti-p-PLCg-1 (sc-12943-R; Santa Cruz Biotechnology) in 3% BSA, 0.05% Tween-20, TBS for 1 hr at room temperature. Blots were washed three times with TBS containing 0.05% Tween-20 and then incubated in 1:1000 dilutions of horseradish peroxidase (HRP)-conjugated goat anti-mouse or goat anti-rabbit antibodies (Amersham, Buckinghamshire, UK) in 3% BSA, 0.05% Tween-20, TBS for 1 hr at room temperature. Blots were washed three times with TBS containing 0.05% Tween-20, and visualized using ECL (Amersham). For dot blots, 1 ml containing 1 mg total protein was spotted onto an Immobilon P membrane and allowed to air dry. Blots were wetted using methanol, rinsed in Tris-buffered saline, 0.5% (v/v) Tween-20 (Sigma), and then blocked with 3% BSA, 0.2% Tween-20, TBS for 1 hr. Blots were incubated with either a 1:1000 dilution of mouse anti-phosphorylated tyrosine antibody (sc-508; Santa Cruz Biotechnology) or 1 mg/ml biotinXX-cholera toxin subunit B (Molecular Probes) in 3% BSA, 0.05% Tween-20, TBS for 1 hr at room temperature. Blots were washed three times with TBS containing 0.05% Tween-20, then incubated with a 1:1000 dilution of HRP-conjugated goat anti-mouse antibody (Amersham) or with 1 mg/ml streptavidinHRP (Pierce) in 3% BSA, 0.05% Tween-20, TBS for 1 hr at room temperature. Blots were washed three times and visualized using ECL. Films were scanned using a Bio-Rad GS-800 densitometer (Hercules, CA) and the images were quantitated using Bio-Rad Quantity One 1-D Analysis software. Data from three separate experiments were averaged when determining fold increase of band intensity. Microinjections SH2 domain constructs were obtained from Dr. Kathy Foltz and the GST-SH2 domain fusion proteins were purified as previously described (Giusti et al., 2003). Kwik-fil borosilicate glass capillaries


(World Precision Instruments, Sarasota, FL) were pulled on a PP-830 micropipette puller to create microinjection needles. Solutions containing 1–5 mg/ml of SH2 domains were microinjected using a Picospritzer II (Parker Instrumentation, Irvine, CA) to inject 1%–5% of total cell volume into a single blastomere of two-cell stage embryos attached to a protamine sulfate-coated coverslip. Injections were done at metaphase, and the uninjected blastomere was used as a timing control. Supplemental Data Supplemental Data include movies and can be found with this article online at http://www.developmentalcell.com/cgi/content/full/9/6/ 781/DC1/. Acknowledgments We thank Dr. Kathy Foltz, UCSB, for SH2 domain constructs and Laila Strickland from our lab for helping with FAST DiI experiments. This work was supported by NIH grant GM58231 to D.R.B. Received: October 20, 2004 Revised: October 31, 2005 Accepted: November 1, 2005 Published: December 5, 2005

Fernandez, C., Lobo, M.V.T., Gomez-Coronado, D., and Lasuncion, M.A. (2004). Cholesterol is essential for mitosis progression and its deficiency induces polyploid cell formation. Exp. Cell Res. 300, 109–120. Field, S., Madson, N., Kerr, M.L., Galbraith, K.A.A., Kennedy, C.E., Tahiliani, M., Wilkins, A., and Cantley, L.C. (2005). PtdIns(4,5)P2 functions at the cleavage furrow during cytokinesis. Curr. Biol. 15, 1407–1412. Foster, L.J., de Hoog, C.L., and Mann, M. (2003). Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors. Proc. Natl. Acad. Sci. USA 100, 5813–5818. Giusti, A.F., Xu, W., Hinkle, B., Teresaki, M., and Jaffe, L. (2000). Evidence that fertilization activates starfish eggs by sequential activation of a src-like kinase and phospholipase Cg. J. Biol. Chem. 275, 16788–16794. Giusti, A.F., O’Neill, F.J., Yamasu, K., Foltz, K.R., and Jaffe, L. (2003). Function of a sea urchin egg Src family kinase in initiating Ca2+ release at fertilization. Dev. Biol. 256, 367–378. Glebov, O.O., and Nichols, B.J. (2004a). Distribution of lipid raft markers in live cells. Biochem. Soc. Trans. 32, 673–675. Glebov, O.O., and Nichols, B.J. (2004b). Lipid raft proteins have a random distribution during localized activation of the T-cell receptor. Nat. Cell Biol. 6, 238–243.

References

Guha, M., Zhou, M., and Wang, Y.L. (2005). Cortical actin turnover during cytokinesis requires myosin II. Curr. Biol. 15, 732–736.

Albertson, R., Riggs, B., and Sullivan, W. (2005). Membrane traffic: a driving force in cytokinesis. Trends Cell Biol. 15, 92–101.

Gundersen, G.G., Gomes, E.R., and Wen, Y. (2004). Cortical control of microtubule stability and polarization. Curr. Opin. Cell Biol. 16, 106–112.

Bagnat, M., and Simons, K. (2002). Cell surface polarization during yeast mating. Proc. Natl. Acad. Sci. USA 99, 14183–14188. Becchetti, A., and Whitaker, M. (1997). Lithium blocks cell cycle transitions in the first cell cycles of sea urchin embryos, an effect rescued by myo-inositol. Development 124, 1099–1107. Belton, R.J., Jr., Adams, N.L., and Foltz, K.R. (2001). Isolation and characterization of sea urchin egg lipid rafts and their possible function during fertilization. Mol. Reprod. Dev. 59, 294–305. Brill, J.A., Hime, G.R., Scharer-Schuksz, M., and Fuller, M.T. (2000). A phospholipid kinase regulates actin organization and intercellular bridge formation during germline cytokinesis. Development 127, 3855–3864. Burgess, D.R., and Schroeder, T.E. (1977). Polarized bundles of actin filaments within microvilli of fertilized sea urchin eggs. J. Cell Biol. 74, 1032–1037. Carnahan, R.H., and Gould, K.L. (2003). The PCH family protein, Cdc15p, recruits two F-actin nucleation pathways to coordinate cytokinetic actin ring formation in Schizosaccharomyces pombe. J. Cell Biol. 162, 851–862. Chou, Y.-H., and Rebhun, L.I. (1986). Purification and characterization of a sea urchin egg Ca2+-calmodulin-dependent kinase with myosin light chain phosphorylating activity. J. Biol. Chem. 261, 5389–5395. Cote, J.F., Chung, P.L., Theberge, J.F., Halle, M., Spencer, S., Lasky, L.A., and Tremblay, M.L. (2002). PSTPIP is a substrate of PTP-PEST and serves as a scaffold guiding PTP-PEST toward a specific dephosphorylation of WASP. J. Biol. Chem. 277, 2973–2986. del Pozo, M.A., Alderson, N.B., Kiosses, W.B., Chiang, H.H., Anderson, R.G., and Schwartz, M.A. (2004). Integrins regulate Rac targeting by internalization of membrane domains. Science 303, 839–842. Djagaeva, I., Doronkin, S., and Beckendorf, S.K. (2005). Src64 is involved in fusome development and karyosome formation during Drosophila oogenesis. Dev. Biol. 284, 143–156. Dobbelaere, J., and Barral, Y. (2004). Spatial coordination of cytokinetic events by compartmentalization of the cell cortex. Science 305, 393–396. Edidin, M. (2003). The state of lipid rafts: from model membranes to cells. Annu. Rev. Biophys. Biomol. Struct. 32, 257–283. Emoto, K., and Umeda, M. (2000). An essential role for a membrane lipid in cytokinesis. Regulation of contractile ring disassembly by redistribution of phosphatidylethanolamine. J. Cell Biol. 149, 1215– 1224.

Hao, M., Mukherjee, S., Sun, Y., and Maxfield, F.R. (2004). Effects of cholesterol depletion and increased lipid unsaturation on the properties of endocytic membranes. J. Biol. Chem. 279, 14171–14178. Herrmann, L., Dittmar, T., and Erdmann, K.S. (2003). The protein tyrosine phosphatase PTP-BL associates with the midbody and is involved in the regulation of cytokinesis. Mol. Biol. Cell 14, 230–240. Hime, G.R., Brill, J.A., and Fuller, M.T. (1996). Assembly of ring canals in the male germ line from structural components of the contractile ring. J. Cell Sci. 109, 2779–2788. Holowka, D., Sheets, E.D., and Baird, B. (2000). Interactions between Fc(3)RI and lipid raft components are regulated by the actin cytoskeleton. J. Cell Sci. 113, 1009–1019. Janetopoulos, C., Borleis, J., Vazquez, F., Iijima, M., and Devreotes, P. (2005). Temporal and spatial regulation of phosphoinositide signaling mediates cytokinesis. Dev. Cell 8, 467–477. Koppel, D.E. (1982). Surface functions during mitosis. III. Quantitative analysis of ligand-receptor movement into the cleavage furrow: diffusion vs. flow. J. Cell Biol. 93, 950–960. Lai, E.C. (2003). Lipid rafts make for slippery platforms. J. Cell Biol. 162, 365–370. Liu, J., Oh, P., Horner, T., Rogers, R.A., and Schnitzer, J.E. (1997). Organized endothelial cell surface signal transduction in calveolae distinct from glycosylphosphatidylinositol-anchored protein microdomains. J. Biol. Chem. 272, 7211–7222. Liu, J., Tang, X., Wang, H., Oliferenko, S., and Balasubramanian, M.K. (2002). The localization of the integral membrane protein Cps1p to the cell division site is dependent on the actomyosin ring and the septation-inducing network in Schizosaccharomyces pombe. Mol. Biol. Cell 13, 989–1000. Maxfield, F.R. (2002). Plasma membrane microdomains. Curr. Opin. Cell Biol. 14, 483–487. Moore, K.L., and Kinsey, W.H. (1994). Identification of an abl-related protein tyrosine kinase in the cortex of the sea urchin egg: possible role at fertilization. Dev. Biol. 164, 444–455. Mukherjee, S., Soe, T.T., and Maxfield, F.R. (1999). Endocytic sorting of lipid analogues differing solely in the chemistry of their hydrophobic tails. J. Cell Biol. 144, 1271–1284. Munro, S. (2003). Lipid rafts: elusive or illusive? Cell 115, 377–388. Murthy, K., and Wadsworth, P. (2005). Myosin II-dependent localization and dynamics of F-actin during cytokinesis. Curr. Biol. 15, 724– 731.


Nichols, B. (2003). Caveosomes and endocytosis of lipid rafts. J. Cell Sci. 116, 4707–4714. Oliferenko, S. (1999). Analysis of CD44-containing lipid rafts: recruitment of annexin II and stabilization by the actin cytoskeleton. J. Cell Biol. 146, 843–854. Pierce, S.K. (2004). To cluster or not to cluster: FRETting over rafts. Nat. Cell Biol. 6, 180–181.

Wachtler, V., Rajagopalan, S., and Balasubramanian, M. (2003). Sterol-rich plasma membrane domains in the fission yeast Schizosaccharomyces pombe. J. Cell Sci. 116, 867–874. Walker, G., Burgess, D., and Kinsey, W.H. (1996). Fertilization promotes selective association of the Abl [correction of AbI] kinase with the egg cytoskeleton. Eur. J. Cell Biol. 70, 165–171.

Pike, L.J. (2003). Lipid rafts: bringing order to chaos. J. Lipid Res. 44, 655–667.

Wang, Y.L., Silverman, J.D., and Cao, L.G. (1994). Single particle tracking of surface receptor movement during cell division. J. Cell Biol. 127, 963–971.

Plattner, R., Irvin, B.J., Guo, S., Blackburn, K., Kazlauskas, A., Abraham, R.T., York, J.D., and Pendergast, A.M. (2003). A new link between the c-Abl tyrosine kinase and phosphoinositide signalling through PLC-g1. Nat. Cell Biol. 5, 309–319.

Wong, R., Hadjiyanni, I., Wei, H.-C., Polevoy, G., McBride, R., Sem, K.-P., and Brill, J.A. (2005). PIP2 hydrolysis and calcium release are required for cytokinesis in Drosophila spermatocytes. Curr. Biol. 15, 1401–1406.

Rajendran, L., and Simons, K. (2005). Lipid rafts and membrane dynamics. J. Cell Sci. 118, 1099–1102.

Woodring, P.J., Hunter, T., and Wang, J.Y. (2003). Regulation of F-actin-dependent processes by the Abl family of tyrosine kinases. J. Cell Sci. 116, 2613–2626.

Rappaport, R. (1996). Cytokinesis in Animal Cells (Cambridge, UK: Cambridge University Press). Roche, S., Fumagalli, S., and Courtneidge, S.A. (1995a). Requirement for Src family protein tyrosine kinases in G2 for fibroblast cell division. Science 269, 1567–1569. Roche, S., Koegl, M., Barone, M.V., Roussel, M.F., and Courtneidge, S.A. (1995b). DNA synthesis induced by some but not all growth factors requires Src family protein tyrosine kinases. Mol. Cell. Biol. 15, 1102–1109. Runft, L.L., Jaffe, L.A., and Mehlmann, L.M. (2002). Egg activation at fertilization: where it all begins. Dev. Biol. 245, 237–254. Saul, D., Fabian, L., Forer, A., and Brill, J.A. (2004). Continuous phosphatidylinositol metabolism is required for cleavage of crane fly spermatocytes. J. Cell Sci. 117, 3887–3896. Schmidt, K., and Nichols, B.J. (2004). A barrier to lateral diffusion in the cleavage furrow of dividing mammalian cells. Curr. Biol. 14, 1002–1006. Schroeder, T.E. (1972). The contractile ring. II. Determining its brief existence, volumetric changes, and vital role in cleaving Arbacia eggs. J. Cell Biol. 53, 419–434. Schroeder, T.E. (1981). Interrelations between the cell surface and the cytoskeleton in cleaving sea urchin eggs. In Cytoskeletal Elements and Plasma Membrane Organization, G.L. Nicolson, ed. (Amsterdam: Elsevier/North Holland), pp. 169–216. Seveau, S., Eddy, R.J., Maxfield, F.R., and Pierini, L.M. (2001). Cytoskeleton-dependent membrane domain segregation during neutrophil polarization. Mol. Biol. Cell 12, 3550–3562. Shuster, C.B., and Burgess, D.R. (1999). Parameters that specify the timing of cytokinesis. J. Cell Biol. 146, 981–992. Shuster, C.B., and Burgess, D.R. (2002a). Targeted new membrane addition in the cleavage furrow is a late, separate event in cytokinesis. Proc. Natl. Acad. Sci. USA 99, 3633–3638. Shuster, C.B., and Burgess, D.R. (2002b). Transitions regulating the timing of cytokinesis in embryonic cells. Curr. Biol. 12, 1–20. Solomon, S., Masilamani, M., Rajendran, L., Bastmeyer, M., Stuermer, C.A., and Illges, H. (2002). The lipid raft microdomainassociated protein reggie-1/flotillin-2 is expressed in human B cells and localized at the plasma membrane and centrosome in PBMCs. Immunobiology 205, 108–119. Spencer, S., Dowbenko, D., Chemg, C., Li, W., Brush, J., Utzig, S., Simanis, V., and Lasky, L.A. (1997). PSTPIP: a tyrosine phosphorylated cleavage furrow-associated protein that is a substrate for a PEST tyrosine phosphatase. J. Cell Biol. 138, 845–860. Strickland, L.I., and Burgess, D.R. (2004). Pathways for membrane trafficking during cytokinesis. Trends Cell Biol. 14, 115–118. Strickland, L.I., Donnelly, E.J., and Burgess, D.R. (2005). Induction of cytokinesis is independent of precisely regulated microtubule dynamics. Mol. Biol. Cell 16, 4485–4494. Takeda, T., Kawate, T., and Chang, F. (2004). Organization of a sterol-rich membrane domain by cdc15p during cytokinesis in fission yeast. Nat. Cell Biol. 6, 1142–1144. Tominaga, T., Sahai, E., Chardin, P., McCormick, F., Courtneidge, S.A., and Alberts, A.S. (2000). Diaphanous-related formins bridge Rho GTPase and Src tyrosine kinase signaling. Mol. Cell 5, 13–25.

Yoshigaki, T. (1997). Accumulation of WGA receptors in the cleavage furrow during cytokinesis of sea urchin eggs. Exp. Cell Res. 236, 463–471.