Microinjection Of Antibodies Research Articles

Karyopherin binding interactions and nuclear import mechanism of nuclear pore complex protein Tpr

BackgroundTpr is a large protein with an extended coiled-coil domain that is localized within the nuclear basket of the nuclear pore complex. Previous studies [1] involving antibody microinjection into mammalian cells suggested a role for Tpr in nuclear export of proteins via the CRM1 export receptor. In addition, Tpr was found to co-immunoprecipitate with importins α and β from Xenopus laevis egg extracts [2], although the function of this is unresolved. Yeast Mlp1p and Mlp2p, which are homologous to vertebrate Tpr, have been implicated in mRNA surveillance to retain unspliced mRNAs in the nucleus[3,4]. To augment an understanding of the role of Tpr in nucleocytoplasmic trafficking, we explored the interactions of recombinant Tpr with the karyopherins CRM1, importin β and importin α by solid phase binding assays. We also investigated the conditions required for nuclear import of Tpr using an in vitro assay.ResultsWe found that Tpr binds strongly and specifically to importin α, importin β, and a CRM1 containing trimeric export complex, and that the binding sites for importins α and β are distinct. We also determined that the nuclear import of Tpr is dependent on cytosolic factors and energy and is efficiently mediated by the importin α/β import pathway.ConclusionBased on the binding and nuclear import assays, we propose that Tpr is imported into the nucleus by the importin α/β heterodimer. In addition, we suggest that Tpr can serve as a nucleoporin binding site for importin β during import of importin β cargo complexes and/or importin β recycling. Our finding that Tpr bound preferentially to CRM1 in an export complex strengthens the notion that Tpr is involved in protein export.

Netrin Participates in the Development of Retinotectal Synaptic Connectivity by Modulating Axon Arborization and Synapse Formation in the Developing Brain

Netrin has been implicated in retinal ganglion cell (RGC) axon pathfinding in a number of species. In Xenopus laevis, RGC axons reaching their target in the optic tectum can be repelled by a netrin-1 gradient in vitro, suggesting that netrin may also function in wiring events that follow successful axon pathfinding. Here, we examined the contribution of netrin to RGC axon arborization and synapse formation at the target. Time-lapse confocal microscopy imaging of individual RGC axons coexpressing GFP-synaptobrevin and DsRed in the intact Xenopus brain demonstrated a role for deleted in colorectal cancer (DCC)-mediated netrin signaling. Microinjection of netrin-1 into the tectum induced a rapid and transient increase in presynaptic site addition that resulted in higher presynaptic site density over a 24 h observation period. Moreover, netrin induced dynamic axon branching, increasing branch addition and retraction; a behavior that ultimately increased total branch number. In contrast, microinjection of DCC function-blocking antibodies prevented the increase in presynaptic site number normally observed in control axons as well as the associated increase in branch number and axon arbor growth. Dynamic analysis of axon arbors demonstrated that the effects of anti-DCC on axon morphology and presynaptic connectivity were attributable to a specific decrease in new synapse and branch additions, without affecting the stability of existing synapses and branches. Together, these results indicate that, in the absence of DCC signaling, RGC axons fail to branch and differentiate, and support a novel role for netrin in later phases of retinotectal development.

Nudel Promotes Axonal Lysosome Clearance and Endo‐lysosome Formation via Dynein‐Mediated Transport

Axonal transport is critical for neuronal function and survival. Cytoplasmic dynein and its accessory complex dynactin form a microtubule minus end-directed motor in charge of retrograde transport. In this study, we show that Nudel, a dynein regulator, was highly expressed in dorsal root ganglion (DRG) neurons. Microinjection of anti-Nudel antibody into cultured DRG neurons abolished retrograde transport of membranous organelles in the axon and led to dispersions of Golgi cisternae in the soma. As a result, lysosomes, which are normally enriched in the soma, moved persistently into and thus accumulated in axons. Endo-lysosome formation was also markedly delayed. As anterograde motility of mitochondria was not inhibited, the antibody apparently did not abolish retrograde transport by destructing axonal microtubule tracks. Similar results were obtained by microinjecting N-terminal Nudel, anti-dynein antibody or a p150(Glued) mutant capable of abrogating the dynein-dynactin association. These results indicate a critical role of Nudel in dynein-mediated axonal transport. Moreover, the effects of dynein on endolysosome formation and regional sequestration of lysosomes may contribute to defects in the endocytic pathway seen in neurons of patients or animals with malfunction of dynein.

Kinesin-5–dependent Poleward Flux and Spindle Length Control inDrosophilaEmbryo Mitosis

We used antibody microinjection and genetic manipulations to dissect the various roles of the homotetrameric kinesin-5, KLP61F, in astral, centrosome-controlled Drosophila embryo spindles and to test the hypothesis that it slides apart interpolar (ip) microtubules (MT), thereby controlling poleward flux and spindle length. In wild-type and Ncd null mutant embryos, anti-KLP61F dissociated the motor from spindles, producing a spatial gradient in the KLP61F content of different spindles, which was visible in KLP61F-GFP transgenic embryos. The resulting mitotic defects, supported by gene dosage experiments and time-lapse microscopy of living klp61f mutants, reveal that, after NEB, KLP61F drives persistent MT bundling and the outward sliding of antiparallel MTs, thereby contributing to several processes that all appear insensitive to cortical disruption. KLP61F activity contributes to the poleward flux of both ipMTs and kinetochore MTs and to the length of the metaphase spindle. KLP61F activity maintains the prometaphase spindle by antagonizing Ncd and another unknown force-generator and drives anaphase B, although the rate of spindle elongation is relatively insensitive to the motor's concentration. Finally, KLP61F activity contributes to normal chromosome congression, kinetochore spacing, and anaphase A rates. Thus, a KLP61F-driven sliding filament mechanism contributes to multiple aspects of mitosis in this system.

NuMA is required for proper spindle assembly and chromosome alignment in prometaphase

BackgroundNuMA is a protein that has been previously shown to play a role in focusing microtubules at the mitotic spindle poles. However, most previous work relies on experimental methods that might cause dominant side effects on spindle formation, such as microinjection of antibodies, overexpression of mutant protein, or immunodepletion of NuMA-containing protein complexes.FindingsTo circumvent these technical problems, we performed siRNA experiments in which we depleted the majority of NuMA in human cultured cells. Depleted mitotic cells show a prolonged duration of prometaphase, with spindle pole defects and with unattached, unaligned chromosomes.ConclusionOur data confirm that NuMA is important for spindle pole formation, and for cohesion of centrosome-derived microtubules with the bulk of spindle microtubules. Our findings of NuMA-dependent defects in chromosome alignment suggest that NuMA is involved in stabilizing kinetochore fibres.

Microinjected antibodies interfere with protein nucleocytoplasmic shuttling by distinct molecular mechanisms

The observation that some antibodies can enter the nucleus after their microinjection into the cytoplasm established the principle of protein nucleocytoplasmic shuttling. Here, we introduce the concept of stationary antibodies for studying nuclear transport, particularly of native proteins. Contrary to the aforementioned translocating immunoglobulins, stationary antibodies do not cross the nuclear envelope. They are distinguished by their ability to trigger the nucleocytoplasmic redistribution of their antigen. What determines these apparently contradictory outcomes has not been explored. We studied a stationary STAT1 antibody and a translocating importin-beta antibody. The stationary phenotype resulted from the inhibition of carrier-independent transport. This was not due to crosslinking or precipitation of antigen, because the antigen-antibody complex remained highly mobile. Rather, decoration with stationary antibody precluded actual nuclear pore passage of antigen. In addition, both antibodies inhibited the carrier-dependent translocation via importin-alpha, but by diverse mechanisms. The translocating antibody blocked the association with importin-alpha, whereas the stationary antibody prevented the phosphorylation of its antigen, and thus functioned upstream of the importin-alpha binding step. We identified a stationary antibody to green-fluorescent protein (GFP) and probed the translocation of GFP fusions of STAT1, thyroid hormone receptor and histones, demonstrating general application of this approach. Our results provide an experimental rationale for the use of antibodies as unique tools for dissecting protein nuclear translocation. As the microinjection of stationary antibodies extends to analyses of native proteins, this method can complement and validate results obtained with fluorescent-labeled derivatives.

BCAR3 regulates EGF-induced DNA synthesis in normal human breast MCF-12A cells

BCAR3 (breast cancer anti-estrogen resistance 3) is a signal transducer containing an SH2 domain, a proline/serine-rich domain and a GDP-exchange factor homologous domain, whose role in signaling pathways is currently unclear. Furthermore, BCAR3 is implicated in anti-estrogen resistance of breast cancer cells. In the present study, we investigated the functional role of BCAR3 in a mitogenic signaling pathway of EGF in non-tumorigenic human breast epithelial MCF-12A cells. Microinjection of an anti-BCAR3 antibody, siRNAs targeting BCAR3 and an SH2 domain of BCAR3 inhibited EGF-induced DNA synthesis. Direct association of BCAR3 with activated EGF receptor and Cas was observed. Lastly, microinjection of a BCAR3 expression plasmid induced DNA synthesis. These findings suggest that the BCAR3 protein, through its SH2 domain, is involved in the signaling pathways of EGF leading to cell cycle progression, and that BCAR3 itself is part of a mitogenic signaling pathway.

The regulated exocytosis of enlargeosomes is mediated by a SNARE machinery that includes VAMP4

The mechanisms governing the fast, regulated exocytosis of enlargeosomes have been unknown, except for the participation of annexin-2 in a pre-fusion step. We investigated whether any SNAREs are involved. In PC12-27 cells, which are enlargeosome-rich, the expressed SNAREs exhibited various distributions (trans-Golgi network, scattered puncta, plasma membrane); however, only VAMP4 was colocalized in discrete puncta with the enlargeosome marker desmoyokin. The exocytosis of the organelle, revealed by capacitance increases and by surface appearance of desmoyokin, was largely inhibited by microinjection of anti-VAMP4, anti-syntaxin-6 and anti-SNAP23 antibodies, by incubation with botulinum toxin E, and by transfection of VAMP4 and syntaxin-6 siRNAs. Microinjection of the antibodies anti-VAMP7, anti-VAMP8 and anti-syntaxin-4, and transfection with the VAMP8 siRNA were ineffective. Inhibition of enlargeosome exocytosis by VAMP4 siRNA also occurred in a cell type that was competent for neurosecretion, SH-SY5Y. Moreover, in cells expressing a VAMP4-GFP construct, enlargeosome exocytosis and surface appearance of fluorescence occurred concomitantly, and many ensuing surface patches were co-labelled by GFP and desmoyokin. VAMP4, an R-SNARE that has never been shown to participate in regulated exocytoses, therefore appears to be harboured in the membrane of enlargeosomes and to be a member of the machinery mediating their regulated exocytosis. Syntaxin-6 and SNAP23 appear also to be needed for the process to occur; however, the mechanism of their participation, whether direct or indirect, remains undefined.

Avoiding a Traffic Jam

Proteins destined for secretion are delivered from the endoplasmic reticulum (ER) to the Golgi complex, which they must traverse en route to the plasma membrane. Noting that exit from the ER of procollagen-I is temperature-dependent, Pulvirenti et al . shifted the temperature of human skin fibroblasts from 40 o C to 32 o C to deliver a "pulse" of cargo to the Golgi and found that tyrosine phosphorylation in the Golgi region increased in parallel with the arrival and accumulation of procollagen-I after relief of ER export block. A combination of immunoblot and pharmacological analysis of cells homogenized during or after block indicated that trafficking was associated with the phosphorylation and activation of Src-family tyrosine kinases (SFKs). Immunofluorescence and immunoelectron microscopy confirmed localization of trafficking-induced phosphorylated SFK to the Golgi; trafficking did not, however, affect the total amount of Golgi-SFK. ER chaperones that accompany cargo proteins bind to the transmembrane KDEL receptor (KDEL-R) in the Golgi, triggering retrograde movement of KDEL-R and thereby recycling chaperones to the ER (see Asp and Nilsson), and the authors explored the role of KDEL-R in SFK activation in various cell types. Introduction of KDEL-R ligands (through endocytosis or transfection) stimulated phosphorylation of Golgi-associated SFKs, as did KDEL-R overexpression. In contrast, transfection of a KDEL-R dominant-negative mutant, microinjection of antibody directed against the KDEL-R cytosolic C-terminal tail, or depletion of KDEL-R with siRNA inhibited trafficking-induced activation of Golgi-localized SFKs. The KDEL-R C-terminal domain bound Src in a yeast two-hybrid assay; moreover, SFK inhibitors, depletion of Src with siRNA, and a Src dominant-negative mutant all inhibited intra-Golgi trafficking of a cargo protein. In contrast, a constitutively active Src mutant antagonized inhibition of trafficking by the KDEL-R dominant-negative. The authors thus conclude that KDEL-R binding by chaperones that accompany cargo initiates an SFK-mediated signaling cascade that stimulates Golgi trafficking. T. Pulvirenti, M. Giannotta, M. Capestrano, M. Capitani, A. Pisanu, R. S. Polishchuk, E. San Pietro, G. V. Beznoussenko, A. A. Mironov, G. Turacchio, V. W. Hsu, M. Sallese, A. Luini, A traffic-activated Golgi-based signalling circuit coordinates the secretory pathway. Nat. Cell Biol. 10 , 912-922 (2008). [PubMed] L. Asp, T. Nilsson, Golgi gets wired up. Nat. Cell Biol. 10 , 885-887 (2008). [PubMed]

Role of epsin 1 in synaptic vesicle endocytosis

Epsin has been suggested to act as an alternate adaptor in several endocytic pathways. Its role in synaptic vesicle recycling remains, however, unclear. Here, we examined the role of epsin in this process by using the lamprey reticulospinal synapse as a model system. We characterized a lamprey ortholog of epsin 1 and showed that it is accumulated at release sites at rest and also at clathrin-coated pits in the periactive zone during synaptic activity. Disruption of epsin interactions, by presynaptic microinjection of antibodies to either the epsin-N-terminal homology domain (ENTH) or the clathrin/AP2 binding region (CLAP), caused profound loss of vesicles in stimulated synapses. CLAP antibody-injected synapses displayed a massive accumulation of distorted coated structures, including coated vacuoles, whereas in synapses perturbed with ENTH antibodies, very few coated structures were found. In both cases coated pits on the plasma membrane showed a shift to early intermediates (shallow coated pits) and an increase in size. Moreover, in CLAP antibody-injected synapses flat clathrin-coated patches occurred on the plasma membrane. We conclude that epsin is involved in clathrin-mediated synaptic vesicle endocytosis. Our results support a model, based on in vitro studies, suggesting that epsin coordinates curvature generation with coat assembly and further indicating that epsin limits clathrin coat assembly to the size of newly formed vesicles. We propose that these functions of epsin 1 provide an additional mechanism for generation of uniformly sized synaptic vesicles.

Perturbation of Syndapin/PACSIN Impairs Synaptic Vesicle Recycling Evoked by Intense Stimulation

Synaptic vesicle recycling has been proposed to depend on proteins which coordinate membrane and cytoskeletal dynamics. Here, we examine the role of the dynamin- and N-WASP (neural Wiskott-Aldrich syndrome protein)-binding protein syndapin/PACSIN at the lamprey reticulospinal synapse. We find that presynaptic microinjection of syndapin antibodies inhibits vesicle recycling evoked by intense (5 Hz or more), but not by light (0.2 Hz) stimulation. This contrasts with the inhibition at light stimulation induced by perturbation of amphiphysin (Shupliakov et al., 1997). Inhibition by syndapin antibodies was associated with massive accumulation of membranous cisternae and invaginations around release sites, but not of coated pits at the plasma membrane. Cisternae contained vesicle membrane, as shown by vesicle-associated membrane protein 2 (VAMP2)/synaptobrevin 2 immunolabeling. Similar effects were observed when syndapin was perturbed before onset of massive endocytosis induced by preceding intense stimulation. Selective perturbation of the Src homology 3 domain interactions of syndapin was sufficient to induce vesicle depletion and accumulation of cisternae. Our data show an involvement of syndapin in synaptic vesicle recycling evoked by intense stimulation. We propose that syndapin is required to stabilize the plasma membrane and/or facilitate bulk endocytosis at high release rates.

Targeted disruption of Aurora A causes abnormal mitotic spindle assembly, chromosome misalignment and embryonic lethality

Aurora A (also known as STK15/BTAK in humans), a putative oncoprotein naturally overexpressed in many human cancers, is a member of the conserved Aurora protein serine/threonine kinase family that is implicated in the regulation of G(2)-M phases of the cell cycle. In vitro studies utilizing antibody microinjection, siRNA silencing and small molecule inhibitors have indicated that Aurora A functions in early as well as late stages of mitosis. However, due to limitations in specificity of the techniques, exact functional roles of the kinase remain to be clearly elucidated. In order to identify the physiological functions in vivo, we have generated Aurora A null mouse embryos, which show severe defects at 3.5 d.p.c. (days post-coitus) morula/blastocyst stage and lethality before 8.5 d.p.c. Null embryos at 3.5 d.p.c. reveal growth retardation with cells in mitotic disarray manifesting disorganized spindle, misaligned and lagging chromosomes as well as micronucleated cells. These findings provide the first unequivocal genetic evidence for an essential physiological role of Aurora A in normal mitotic spindle assembly, chromosome alignment segregation and maintenance of viability in mammalian embryos.

Microinjection of recombinant O-GlcNAc transferase potentiates Xenopus oocytes M-phase entry

In order to understand the importance of the cytosolic and nuclear-specific O-linked N-acetylglucosaminylation ( O-GlcNAc) on cell cycle regulation, we recently reported that inhibition of O-GlcNAc transferase (OGT) delayed or blocked Xenopus laevis oocyte germinal vesicle breakdown (GVBD). Here, we show that increased levels of the long OGT isoform (ncOGT) accelerate X. laevis oocyte GVBD. A N-terminally truncated isoform (sOGT) with a similar in vitro catalytic activity towards a synthetic CKII-derived peptide had no effect, illustrating the important role played by the N-terminal tetratrico-peptide repeats. ncOGT microinjection in the oocytes increases both the speed and extent of O-GlcNAc addition, leads to a quicker activation of the MPF and MAPK pathways and finally results in a faster GVBD. Microinjection of anti-OGT antibodies leads to a delay of the GVBD kinetics. Our results hence demonstrate that OGT is a key molecule for the timely progression of the cell cycle.

Dynein-dependent Movement of Autophagosomes Mediates Efficient Encounters with Lysosomes

Autophagy is a membrane trafficking pathway that carries cytosolic components to the lysosome for degradation. During this process, the autophagosome, a double-membraned organelle, is generated de novo, sequesters cytoplasmic proteins and organelles, and delivers them to lysosomes. However, the mechanism by which autophagosomes are targeted to lysosomes has not been determined. Here, we observed the real-time behavior of microtubule-associated protein light chain 3 (LC3), which localizes to autophagosomes, and showed that autophagosomes move in a microtubule- and dynein-dynactin motor complex-dependent manner. After formation, autophagosomes show a rapid vectorial movement in the direction of the centrosome, where lysosomes are usually concentrated. Microinjection of antibodies against LC3 inhibited this movement; furthermore, using FRAP, we showed that anti-LC3 antibody injection caused a defect in targeting of autophagosomes to lysosomes. Collectively, our data demonstrate the functional significance of autophagosome movement that enables effective delivery from the cytosol to lysosomes.

Gap junctions in the ovary of Drosophila melanogaster: localization of innexins 1, 2, 3 and 4 and evidence for intercellular communication via innexin-2 containing channels

BackgroundIn the Drosophila ovary, germ-line and soma cells are interconnected via gap junctions. The main gap-junction proteins in invertebrates are members of the innexin family. In order to reveal the role that innexins play in cell-cell communication during oogenesis, we investigated the localization of innexins 1, 2, 3 and 4 using immunohistochemistry, and analyzed follicle development following channel blockade.ResultsWe found innexin 1 predominantly localized to the baso-lateral domain of follicle cells, whereas innexin 2 is positioned apico-laterally as well as apically between follicle cells and germ-line cells. Innexin 3 was observed laterally in follicle cells and also in nurse cells, and innexin 4 was detected in the oolemma up to stage 8 and in nurse-cell membranes up to stage 12. In order to test whether innexins form channels suitable for intercellular communication, we microinjected innexin antibodies in combination with a fluorescent tracer into the oocyte of stage-10 follicles. We found that dye-coupling between oocyte and follicle cells was largely reduced by innexin-2 antibodies directed against the intracellular C-terminus as well as against the intracellular loop. Analyzing in vitro, between stages 10 and 14, the developmental capacities of follicles following microinjections of innexin-2 antibodies revealed defects in follicle-cell differentiation, nurse-cell regression, oocyte growth and choriogenesis.ConclusionOur results suggest that all analyzed innexins are involved in the formation of gap junctions in the ovary. While innexins 2 and 3 are colocalized between soma cells, innexins 2 and 4 are colocalized between soma and germ-line cells. Innexin 2 is participating in cell-cell communication via hemichannels residing in the oolemma. It is obvious that gap-junctional communication between germ-line and soma cells is essential for several processes during oogenesis.

A CRM1-mediated nuclear export signal governs cytoplasmic localization of BRCA2 and is essential for centrosomal localization of BRCA2

Germ-line mutations of the BRCA2 gene cause inherited susceptibility to breast and ovarian cancers. BRCA2 contains two nuclear localization signals, predominantly localizes in the nucleus and plays significant roles in DNA double-strand break repair. Recently, we reported that BRCA2 localizes to the centrosomes during the S and early M phases of the cell cycle. In this study, for the first time, we identified a functional nuclear export sequence (NES1; (1383)DLSDLTFLEVA(1393)) in BRCA2. The green fluorescent protein (GFP)-NES1 fusion protein was localized in the cytoplasm and could be blocked by the chromosomal region maintenance 1-specific export inhibitor leptomycin B. Mutation of a leucine residue in the NES1 motif to alanine (L1384A) resulted in both cytoplasmic and nuclear localization of the GFP-NES1 fusion protein and a nuclear accumulation of ectopic full-length BRCA2-FLAG. Moreover, treatment of cells with leptomycin B decreased centrosomal localization of BRCA2. Finally, by microinjection of an anti-BRCA2 antibody into the cytoplasm of HeLa S3 cells, we found that depletion of normal BRCA2 proteins in the cytoplasm leads to centrosome amplification and binucleated cells. Our results suggest that disruption of the NES function by genetic changes results in deregulation of BRCA2 export, which ultimately leads to centrosome disorder.

Dynamin 2 mediates fluid-phase micropinocytosis in epithelial cells

It is well-known that dynamin 2 (Dyn2) participates in clathrin- and caveolae-mediated endocytosis; however, the role of Dyn2 in coat-independent endocytic processes remains controversial. Here we demonstrate a role for specific spliced variants of Dyn2 in the micropinocytosis of fluid in epithelial cells, independent of coat-mediated endocytic pathways. A general inhibition of Dyn2 was first performed using either microinjection of anti-dynamin antibodies or Dyn2-siRNA treatment. Both of these methods resulted in reduced uptake of transferrin, a marker for clathrin-mediated endocytosis, and, under unstimulated conditions, reduced the uptake of the fluid-phase markers dextran and horseradish peroxidase (HRP). By contrast, cells treated similarly but stimulated with serum or EGF internalized substantial amounts of dextran or HRP, indicating that Dyn2 is not required for stimulated fluid uptake via macropinocytosis. We next tested whether a specific spliced variant might selectively affect fluid-phase endocytosis. Mutation of specific Dyn2 spliced variants resulted in a differential attenuation of transferrin and dextran internalization. Furthermore, the reduction in fluid uptake in Dyn2-siRNA-treated cells was only rescued upon re-expression of select spliced variants. These findings suggest that Dyn2 function is required for the coat-independent internalization of fluid through endocytic pathways distinct from macropinocytosis and, in addition, implicate different Dyn2 spliced variants in specific endocytic functions.

Pix1 and Pix2 are novel WD40 microtubule-associated proteins that colocalize with mitochondria in Xenopus germ plasm and centrosomes in human cells

In many animals, the germ line develops from a distinct mitochondria-rich region of embryonic cytoplasm called the germ plasm. However, the protein composition of germ plasm and its formation remain poorly understood, except in Drosophila. Here, we show that Xpat, a recently identified protein component of Xenopus germ plasm, interacts via its C-terminal domain with a novel protein, xPix1. Xpat and xPix1 are co-expressed in ovaries, eggs and early embryos and colocalize to the mitochondrial cloud and germ plasm in stage I and stage VI oocytes, respectively. Although Xpat appears unique to Xenopus, Pix proteins, which contain an N-terminal WD40 domain and C-terminal coiled-coil, are widely conserved. In humans, two proteins, Pix1 and Pix2, are expressed at varying levels in different cancer cell lines. Importantly, as well as localizing to mitochondria, human Pix proteins localize to centrosomes and associate with microtubules in vitro and in vivo. Although, Pix proteins are stably expressed through the cell cycle, Pix2 concentrates on microtubule structures in mitosis and microinjection of Pix antibodies interferes with cell division. Based on these data, we propose that Pix1 and Pix2 are microtubule-associated adaptor proteins that likely contribute to a range of developmental and cell division processes.

Mammalian SEPT2 Is Required for Scaffolding Nonmuscle Myosin II and Its Kinases

Mammalian septin SEPT2 belongs to a conserved family of filamentous GTPases that are associated with actin stress fibers in interphase cells and the contractile ring in dividing cells. Although SEPT2 is essential for cytokinesis, its role in this process remains undefined. Here, we report that SEPT2 directly binds nonmuscle myosin II (myosin II), and this association is important for fully activating myosin II in interphase and dividing cells. Inhibition of the SEPT2-myosin II interaction in interphase cells results in loss of stress fibers, while in dividing cells this causes instability of the ingressed cleavage furrow and dissociation of the myosin II from the Rho-activated myosin kinases ROCK and citron kinase. We propose that SEPT2-containing filaments provide a molecular platform for myosin II and its kinases to ensure the full activation of myosin II that is necessary for the final stages of cytokinesis.

Maintenance of genomic methylation patterns during preimplantation development requires the somatic form of DNA methyltransferase 1

DNA methylation at cytosine residues in CpG dinucleotides is a component of epigenetic marks crucial to mammalian development. In preimplantation stage embryos, a large part of genomic DNA is extensively demethylated, whereas the methylation patterns are faithfully maintained in certain regions. To date, no enzymes responsible for the maintenance of DNA methylation during preimplantation development have been identified except for the oocyte form of DNA (cytosine-5)-methyltransferase 1 (Dnmt1o) at the 8-cell stage. Herein, we demonstrate that the somatic form of Dnmt1 (Dnmt1s) is present in association with chromatin in MII-stage oocytes as well as in the nucleus throughout preimplantation development. At the early one-cell stage, Dnmt1s is asymmetrically localized in the maternal pronuclei. Thereafter, Dnmt1s is recruited to the paternal genome during pronuclear maturation. During the first two cell cycles after fertilization, Dnmt1s is exported from the nucleus in the G2 phase in a CRM1/exportin-dependent manner. Antibody microinjection and small interfering RNA-mediated knock-down decreases methylated CpG dinucleotides in repetitive intracisternal A-type particle (IAP) sequences and the imprinted gene H19. These results indicate that Dnmt1s is responsible for the maintenance methylation of particular genomic regions whose methylation patterns must be faithfully maintained during preimplantation development.