Interphase HeLa Cells Research Articles

Mobility of tethering factor EEA1 on endosomes is decreased upon stimulation of EGF receptor endocytosis in HeLa cells

Tethering factor EEA1, mediating homotypic fusion of early endosomes, was shown to be localized in membrane-bound state both in serum-deprived and stimulated for EGF receptor endocytosis cells. However, it is not known whether dynamics behavior of EEA1 is affected by EGF stimulation. We investigated EEA1 cytosol-to-membrane exchange rate in interphase HeLa cells by FRAP analysis. The data obtained fitted two-states binding model, with the bulk of membrane-associated EEA1 protein represented by the mobile fraction both in serum-starved and EGF-stimulated cells. Fast recovery state had similar half-times in the two cases: about 1.6 s and 2.8 s, respectively. However, the recovery half-time of slowly cycled EEA1 fraction significantly increased in EGF-stimulated comparing to serum-starved cells (from 21 to 99 s). We suppose that the retardation of EEA1 fluorescence recovery upon EGF-stimulation may be due to the increase of activated Rab5 on endosomal membranes, the growth of the number of tethering events between EEA1-positive vesicles and their clustering.

MAP-kinase pathway inhibitors U0126 and PD98059 differentially affect organization of the tubulin cytoskeleton after stimulation of EGF receptor endocytosis

To confirm the hypothesis that EGF-stimulated MAP-kinase ERK1/2 is involved in regulation of functioning of the microtubule (MT) system, the influence of two widely used ERK1/2 inhibitors, U0126 and PD98059, on tubulin cytoskeleton organization in interphase HeLa cells during EGF receptor endocytosis has been investigated. It was found that addition of U0126 or PD98059 to cells that are not stimulated with EGF for 30 min had no influence on the radially organized MT system. However, in the case of U0126 addition before EGF endocytosis stimulation, 15 min after this stimulation, a decrease in MT number per cell was seen, which was followed by complete MT depolymerization for 60–90 min. If endocytosis was stimulated in the presence of PD98059, only a very small decrease in MT number, mainly from their minus ends, could be detected. At the same time, MT regions close to plasma membrane became stabilized, which was proven by increase in tubulin acetylation level. This was characteristic of all periods of the experiment. It was also found that inhibitors affected endocytosis dynamics of EGF-receptor complexes. Quantitative analysis has shown that, upon endocytosis stimulation in the presence of U0126, more endosomes were formed than in the case of control cells and their number did not change significantly during the experiment. All these endosomes were localized peripherally. The effect of PD98059 resulted in the formation of fewer endosomes than in control, but they exhibited very slow clusterization in the juxtanuclear area in spite of the presence of a few intact MTs. Both inhibitors decreased EGFR colocalization with the early endosomal marker EEA1, which indicates a delay in endosome fusion and maturation. The inhibitors were also shown to differently affect the phospho-ERK 1 and 2 forms: U0126 completely inhibited phospho-ERK1 and 2, while, in the presence of PD98059, the two ERK forms exhibited a sharp transient activation at 15 min, while only phospho-ERK2 could be detected after 60 min of endocytosis. In both cases, the dynamics of MAP-kinase activation was significantly different from that in control. Our results suggest that the EGF-stimulated MAP-kinase pathway plays a role in cytoskeleton regulation. At the same time, they exhibit nonequivalency of the two widely used inhibitors in relation to not only MAP-kinase activity, but also to such interdependent processes as changes in cytoskeleton organization and signaling receptor endocytosis.

Dissecting the Nanoscale Distributions and Functions of Microtubule-End-Binding Proteins EB1 and ch-TOG in Interphase HeLa Cells

Recently, the EB1 and XMAP215/TOG families of microtubule binding proteins have been demonstrated to bind autonomously to the growing plus ends of microtubules and regulate their behaviour in in vitro systems. However, their functional redundancy or difference in cells remains obscure. Here, we compared the nanoscale distributions of EB1 and ch-TOG along microtubules using high-resolution microscopy techniques, and also their roles in microtubule organisation in interphase HeLa cells. The ch-TOG accumulation sites protruded ∼100 nm from the EB1 comets. Overexpression experiments showed that ch-TOG and EB1 did not interfere with each other’s localisation, confirming that they recognise distinct regions at the ends of microtubules. While both EB1 and ch-TOG showed similar effects on microtubule plus end dynamics and additively increased microtubule dynamicity, only EB1 exhibited microtubule-cell cortex attachment activity. These observations indicate that EB1 and ch-TOG regulate microtubule organisation differently via distinct regions in the plus ends of microtubules.

Identification of two independent nucleosome-binding domains in the transcriptional co-activator SPBP

Transcriptional regulation requires co-ordinated action of transcription factors, co-activator complexes and general transcription factors to access specific loci in the dense chromatin structure. In the present study we demonstrate that the transcriptional co-regulator SPBP [stromelysin-1 PDGF (platelet-derived growth factor)-responsive element binding protein] contains two independent chromatin-binding domains, the SPBP-(1551-1666) region and the C-terminal extended PHD [ePHD/ADD (extended plant homeodomain/ATRX-DNMT3-DNMT3L)] domain. The region 1551-1666 is a novel core nucleosome-interaction domain located adjacent to the AT-hook motif in the DNA-binding domain. This novel nucleosome-binding region is critically important for proper localization of SPBP in the cell nucleus. The ePHD/ADD domain associates with nucleosomes in a histone tail-dependent manner, and has significant impact on the dynamic interaction between SPBP and chromatin. Furthermore, SPBP and its homologue RAI1 (retinoic-acid-inducible protein 1), are strongly enriched on chromatin in interphase HeLa cells, and both proteins display low nuclear mobility. RAI1 contains a region with homology to the novel nucleosome-binding region SPBP-(1551-1666) and an ePHD/ADD domain with ability to bind nucleosomes. These results indicate that the transcriptional co-regulator SPBP and its homologue RAI1 implicated in Smith-Magenis syndrome and Potocki-Lupski syndrome both belong to the expanding family of chromatin-binding proteins containing several domains involved in specific chromatin interactions.

Phosphorylation of STIM1 Underlies Suppression of Store-Operated Calcium Entry During Mitosis

When endoplasmic reticulum (ER) Ca2+ stores are depleted, Ca2+ influx via plasma membrane (PM) Ca2+ channels is activated by store-operated Ca2+ entry (SOCE). SOCE involves Orai1 Ca2+ influx channels and STIM1 ER Ca2+ sensors. ER Ca2+ depletion induces rearrangement of STIM1 from a diffuse localization throughout the ER membrane into punctate structures near the PM, where it activates Orai1 channels. Interestingly, SOCE is strongly suppressed during mitosis, the only known physiological situation in which SOCE is negatively regulated; however, the mechanisms that underlie SOCE suppression during mitosis are unknown. We found that both endogenous STIM1 and expressed eYFP-tagged STIM1 (eYFP-STIM1) immunoprecipitated from mitotic but not interphase HeLa and HEK293 cells were recognized by the phosphospecific MPM-2 antibody, suggesting mitosis-specific phosphorylation of STIM1. We also found that rearrangement of eYFP-STIM1 into near-PM puncta in response to ER Ca2+ depletion was suppressed during mitosis. We therefore hypothesized that STIM1 phosphorylation underlies prevention of STIM1 puncta formation and suppression of SOCE during mitosis. MPM-2 recognizes phospho-serine or threonine followed by proline, and human STIM1 contains 10 occurrences of S/T-P, all downstream of amino acid 482. eYFP-STIM1 truncated at amino acid 482 (482STOP) was not recognized by MPM-2 when immunoprecipitated from mitotic cells, suggesting lack of phosphorylation. In support of our hypothesis, non-phosphorylatable 482STOP co-expressed with Orai1 rearranged into near-PM puncta in response to ER Ca2+ depletion in mitotic cells, and also significantly rescued mitotic SOCE. A combination of mass spectrometry and site-directed mutagenesis identified S486 and S668 as mitosis-specific phosphorylated residues, and mutation of both to alanine also resulted in partial but significant rescue of SOCE in mitotic cells. Therefore, our data suggest that phosphorylation of S486 and S668 underlies suppression of SOCE during mitosis, although additional phosphorylation sites are likely involved.

New function of the proline rich domain in dynamin-2 to negatively regulate its interaction with microtubules in mammalian cells

Microtubule reorganization is necessary for many cellular functions such as cell migration, cell polarity and cell division. Dynamin was originally identified as a microtubule-binding protein. Previous limited digestion experiment revealed that C-terminal 100-amino acids proline rich domain (PRD) of dynamin is responsible for microtubule binding in vitro. However, as obvious localization of dynamin along microtubules is only observed at the spindle midzone during mitosis but not in interphase cells, it remains unclear how dynamin interacts with microtubules in vivo. Here, we report that GFP-dynamin-2-(1–786), a truncated mutant lacking a C-terminal portion of the PRD, localized along microtubules in interphase HeLa cells. GFP-dynamin-2-wild type (WT) and GFP-dynamin-2-(1–745), a construct that was further truncated to remove the entire PRD, localized in discrete punctate structures but not along microtubules. These data suggest that the N-terminal (residues 746–786) but not the entire PRD is necessary for the interaction of dynamin-2 with microtubules in the cell and that the C-terminus of PRD (787–870) negatively regulate this interaction. Microtubules in cells expressing GFP-dynamin-2-(1–786) were stabilized against exposure to cold. These results provide a first evidence for a regulated interaction of dynamin-2 with microtubules in cultured mammalian cells.

Correlation of Golgi localization of ZW10 and centrosomal accumulation of dynactin

ZW10 participates in the termination of the spindle checkpoint during mitosis by interacting with dynamitin, a subunit of the dynein accessory complex dynactin. We previously showed that ZW10 is attached to the endoplasmic reticulum through RINT-1 in interphase HeLa cells and involved in membrane transport between the endoplasmic reticulum and Golgi. Although a recent study demonstrated that ZW10 is localized in the Golgi in COS7 cells, the mechanism that regulates ZW10 localization remains unknown. In this study we showed a correlation between the Golgi localization of ZW10 and the centrosomal accumulation of dynactin. The amounts of ZW10 associated with dynactin were larger in cells where ZW10 was present in the Golgi than those where ZW10 was not in the Golgi. The targeting of ZW10 to the perinuclear Golgi region was found to depend on the perinuclear accumulation of dynactin, suggesting that dynactin regulates ZW10 localization.

Mitotic Phosphorylation of Dynamin-related GTPase Drp1 Participates in Mitochondrial Fission

Organelles are inherited to daughter cells beyond dynamic changes of the membrane structure during mitosis. Mitochondria are dynamic entities, frequently dividing and fusing with each other, during which dynamin-related GTPase Drp1 is required for the fission reaction. In this study, we analyzed mitochondrial dynamics in mitotic mammalian cells. Although mitochondria in interphase HeLa cells are long tubular network structures, they are fragmented in early mitotic phase, and the filamentous network structures are subsequently reformed in the daughter cells. In marked contrast, tubular mitochondrial structures are maintained during mitosis in Drp1 knockdown cells, indicating that the mitochondrial fragmentation in mitosis requires mitochondrial fission by Drp1. Drp1 was specifically phosphorylated in mitosis by Cdk1/cyclin B on Ser-585. Exogenous expression of unphosphorylated mutant Drp1S585A led to reduced mitotic mitochondrial fragmentation. These results suggest that phosphorylation of Drp1 on Ser-585 promotes mitochondrial fission in mitotic cells.

Dynamic nucleation of Golgi apparatus assembly from the endoplasmic reticulum in interphase hela cells.

Models of Golgi apparatus biogenesis and maintenance are focused on two possibilities: one is self-assembly from the endoplasmic reticulum, and the other is nucleation by a stable template. Here, we asked in three different experimental situations whether assembly of the Golgi apparatus might be dynamically nucleated. During microtubule depolymerization, the integral membrane protein p27 and the peripheral Golgi protein GM130, appeared in newly formed, scattered Golgi elements before three different Golgi apparatus cisternal enzymes, whereas GRASP55, a medial peripheral Golgi protein, showed, if anything, a tendency to accumulate in scattered Golgi elements later than a cisternal enzyme. During Golgi formation after brefeldin A washout, endoplasmic reticulum exit of Golgi resident enzymes could be completely separated from that of p27 and GM130. p27 and GM130 accumulation was onto newly organized perinuclear structures, not brefeldin A remnants, and preceded that of a cisternal enzyme. Reassembly was completely sensitive to guanosine 5'-diphosphate-restricted Sar1p. When cells were microinjected with Sar1pWT DNA to reverse a guanosine 5'-diphosphate-restricted Sar1p endoplasmic reticulum-exit block phenotype, GM130 and p27 collected perinuclearly with little to no exit of a cisternal enzyme from the endoplasmic reticulum. The overall data strongly indicate that the assembly of the Golgi apparatus can be nucleated dynamically by GM130/p27 associated structures. We define dynamic nucleation as the first step in a staged organelle assembly process in which new component association forms a microscopically visible structure onto which other components add later, e.g. Golgi cisternae.

Identification of Signal Sequences Determining the Specific Nucleolar Localization of Fibrillarin in HeLa Cells

Fibrillarin is one of the major nucleolar proteins and is involved in pre-rRNA maturation. Its three main regions are a glycine and arginine-rich (GAR) domain, an RNA-binding domain, and an alpha-helical region, which presumably has a methyltransferase activity. Yet the roles of these regions in nucleolus-specific localization of fibrillarin are still unclear. To elucidate this issue, a series of plasmids was constructed to express human fibrillarin mutants fused with the green fluorescent protein. Localization of the chimeric proteins was studied in interphase and mitotic HeLa cells after single transfection with the plasmids. Deletion or a mutation of any domain proved to alter the specific fibrillarin location coinciding with sites of pre-rRNA synthesis. The GAR domain and the first spacer together were sufficient for fibrillarin migration into the nucleolus. Fibrillarin mutants located within the interphase nucleolus did not differ in mitotic location from the wild-type fibrillarin.

Dynamic distribution of TTK in HeLa cells: insights from an ultrastructural study.

Entry into mitosis is driven by signaling cascades of mitotic kinases. Our recent studies show that TTK, a kinetochore-associated protein kinase, interacts with CENP-E, a mitotic kinesin located to corona fiber of kinetochore. Using immunoelectron microscopy, here we show that TTK is present at the nuclear pore adjacent complex of interphase HeLa cells. Upon nuclear envelope fragmentation, TTK targets to the outermost region of the developing kinetochores of monoorient chromosome as well as to spindle poles. After stable attachment, throughout chromosome congression, TTK is a constituent of the corona fibers, extending up to 90 nm away from the kinetochore outer plate. Upon metaphase alignment, TTK departs from the kinetochore and migrates toward the centrosomes. Taken together, this evidence strongly supports a model in which TTK functions in spindle checkpoint signaling cascades at both kinetochore and centrosome.

In vitro modulation of the interaction between HA95 and LAP2beta by cAMP signaling.

The nuclear envelope mediates key functions by interacting with chromatin. We recently reported an interaction between the chromatin- and nuclear matrix-associated protein HA95 and the inner nuclear membrane integral protein LAP2beta, implicated in initiation of DNA replication (Martins et al. (2003) J. Cell Biol. 160, 177-188). Here, we show that in vitro, interaction between HA95 and LAP2beta is modulated by cAMP signaling via PKA. Exposure of an anti-HA95 immune precipitate from interphase HeLa cells to a mitotic extract promotes ATP-dependent release of LAP2beta from the HA95 complex. This coincides with Ser and Thr phosphorylation of HA95 and LAP2beta. Inhibition of PKA with PKI abolishes phosphorylation of HA95 and dissociation of LAP2beta from HA95, although LAPbeta remains phosphorylated. Antagonizing cAMP signaling in mitotic extract also abolishes the release of LAP2beta from HA95; however, disrupting PKA anchoring to A-kinase anchoring proteins has no effect. Inhibition of CDK activity in the extract greatly reduces LAP2beta phosphorylation but does not prevent LAP2beta release from HA95. Inhibition of PKC, MAP kinase, or CaM kinase II does not affect mitotic extract-induced dissociation of LAP2beta from HA95. PKA phosphorylates HA95 but not LAP2beta in vitro and elicits a release of LAP2beta from HA95. CDK1 or PKC phosphorylates LAP2beta within the HA95 complex, but neither kinase induces LAP2beta release. Our results indicate that in vitro, the interaction between HA95 and LAP2beta is influenced by a PKA-mediated phosphorylation of HA95 rather than by CDK1- or PKC-mediated phosphorylation of LAP2beta. This suggests an additional level of regulation of a chromatin-nuclear envelope interaction in dividing cells.

Immunological Analysis and Purification of Centromere Complex

This chapter describes the methods for isolating CENP-A chromatin complex from interphase HeLa cells. The method was originally developed for isolating CENP-A, CENP-B, and CENP-C chromatin complex and for analyzing its molecular structures. The methods for chromatin immunoprecipitation (ChIP) described consist of two steps; solubilization of bulk chromatin and immunoprecipitation of the CENP-A/B/C complex using anti-CENP-A antibody beads. These steps are used to analyze the protein components as well as the DNA. This method differs from analyses in the Saccharomyces cerevisiae or Schizosaccharomyces pombe system in following points: the amount of starting materials is increased as much as possible; a cross-linker like HCOOH is not used; bulk chromatin is solubilized not by sonication but only by micrococcal nuclease (MNase) digestion under the 0.3 M NaCl condition; and the antibodies used for immunoprecipitation are chemically linked to a solid support in order to repress IgG contamination to the least level during recovery of the proteins from the antibody beads.

Evidence that the entire Golgi apparatus cycles in interphase HeLa cells: sensitivity of Golgi matrix proteins to an ER exit block.

We tested whether the entire Golgi apparatus is a dynamic structure in interphase mammalian cells by assessing the response of 12 different Golgi region proteins to an endoplasmic reticulum (ER) exit block. The proteins chosen spanned the Golgi apparatus and included both Golgi glycosyltransferases and putative matrix proteins. Protein exit from ER was blocked either by microinjection of a GTP-restricted Sar1p mutant protein in the presence of a protein synthesis inhibitor, or by plasmid-encoded expression of the same dominant negative Sar1p. All Golgi region proteins examined lost juxtanuclear Golgi apparatus–like distribution as scored by conventional and confocal fluorescence microscopy in response to an ER exit block, albeit with a differential dependence on Sar1p concentration. Redistribution of GalNAcT2 was more sensitive to low Sar1pdn concentrations than giantin or GM130. Redistribution was most rapid for p27, COPI, and p115. Giantin, GM130, and GalNAcT2 relocated with approximately equal kinetics. Distinct ER accumulation could be demonstrated for all integral membrane proteins. ER-accumulated Golgi region proteins were functional. Photobleaching experiments indicated that Golgi-to-ER protein cycling occurred in the absence of any ER exit block. We conclude that the entire Golgi apparatus is a dynamic structure and suggest that most, if not all, Golgi region–integral membrane proteins cycle through ER in interphase cells.

The peptidyl-prolyl isomerase Pin1 interacts with hSpt5 phosphorylated by Cdk9

We identify and characterize several phosphorylated forms of the hSpt5 subunit of the DRB sensitivity-inducing factor (DSIF). A 175-kDa phosphorylated form of hSpt5 is bound to nuclei of interphase HeLa cells. This form is rapidly dephosphorylated when cultured cells are exposed to various drugs belonging to distinct chemical families. All these compounds are known to inhibit the protein kinase Cdk9, which phosphorylates in vitro hSpt5 and Rpb1, the largest subunit of RNA polymerase II. The efficiency to promote the dephosphorylation of both proteins matches their capacity to inhibit purified Cdk9 kinase, suggesting that Cdk9 is the major kinase phosphorylating hSpt5 and Rpb1 in vivo. We show that Cdk9 phosphorylates both the CTR1 and the CTR2 domains of recombinant hSpt5. These domains contain numerous serine-proline and threonine-proline residues similar to those found in the carboxyl-terminal domain (CTD) of Rpb1. The structural homology between hSpt5 CTRs and the Rpb1 CTD is further highlighted by the presence on both proteins of a phosphoepitope recognized by the monoclonal antibody CC-3. Of particular interest, the peptidyl-prolyl isomerase Pin1 interacts with Cdk9-phosphorylated hSpt5. Cdk9 dependent phosphorylation of Rpb1 and hSpt5 followed by Pin1 interaction might thus contribute to the regulation of transcription, pre-mRNA maturation, and the dynamics of these proteins in interphase and mitosis.

Phosphorylation and cell cycle-dependent regulation of Na+/H+ exchanger regulatory factor-1 by Cdc2 kinase.

Na(+)/H(+) exchanger regulatory factor (NHERF)-1 is a PDZ domain-containing adaptor protein known to bind to various receptors, channels, cytoskeletal elements, and cytoplasmic signaling proteins. We report here that the phosphorylation state of NHERF-1 is profoundly regulated by the cell cycle: NHERF-1 in HeLa cells is hyperphosphorylated in mitosis phase and much less phosphorylated at other points of the cell cycle. This mitosis phase-dependent phosphorylation of NHERF-1 could be blocked by roscovitine, consistent with phosphorylation by cyclin-dependent kinases. In vitro studies with purified NHERF-1 fusion proteins and purified kinases revealed that NHERF-1 was robustly phosphorylated by the cyclin-dependent kinase Cdc2. In contrast, the NHERF-1 relative NHERF-2 was not phosphorylated at all by Cdc2. NHERF-1 possesses two serines (Ser(279) and Ser(301)) that conform to the SPX(K/R) motif preferred for phosphorylation by Cdc2. Mutation of either of these serines reduced Cdc2-mediated phosphorylation of NHERF-1 in vitro, and mutation of both residues together completely abolished Cdc2-mediated phosphorylation. When the S279A/S301A NHERF-1 mutant was expressed in cells, it failed to exhibit the mitosis phase-dependent phosphorylation observed with wild-type NHERF-1. Mutation of both Ser(279) and Ser(301) to aspartate, to mimic Cdc2 phosphorylation of NHERF-1, resulted in a NHERF-1 mutant with a markedly impaired ability to oligomerize in vitro. Similarly, endogenous NHERF-1 from lysates of mitosis phase HeLa cells exhibited a markedly reduced ability to oligomerize relative to endogenous NHERF-1 from lysates of interphase HeLa cells. Mitosis phase NHERF-1 furthermore exhibited the ability to associate with Pin1, a WW domain-containing peptidylprolyl isomerase that does not detectably bind to NHERF-1 in interphase lysates. The association of NHERF-1 with Pin1 facilitated dephosphorylation of NHERF-1, as shown in experiments in which cellular Pin1 activity was blocked by the selective inhibitor juglone. These data reveal that cellular NHERF-1 is phosphorylated during mitosis phase by Cdc2 at Ser(279) and Ser(301) and that this phosphorylation regulates NHERF-1 oligomerization and association with Pin1.

A motor goes mitotic and divorces the Golgi complex

Use of two-hybrid screening has previously shown that the Rab6-binding kinesin Rab6-KIFL is a partner of Rab6, a Golgi-associated GTPase implicated in intra-Golgi membrane traffic. Hill et al.1xThe Rab6-binding kinesin, Rab6-KIFL, is required for cytokinesis. Hill, E. et al. EMBO J. 2000; 19: 5711–5719Crossref | PubMedSee all References1 have now characterized the expression, localization and tentative function of human Rab6-KIFL. Rab6-KIFL accumulates in mitotic HeLa cells, where it localizes to central spindle microtubules and to the midbody during cytokinesis. Using an antiserum raised against the Rab6-binding domain of Rab6-KIFL, Hill et al. demonstrate that levels of the protein oscillate in a cell-cycle-controlled manner, mirroring the behavior of cyclin B2. This suggests that Rab6-KIFL is a kinesin found solely in cells during mitosis. Most interestingly, microinjection of antibodies against Rab6-KIFL blocks cytokinesis and results in a dramatic accumulation of binucleate cells. Furthermore, overexpression of Rab6-KIFL in HeLa cells causes cell death. Thus, Rab6-KIFL might be a microtubule motor with functions during formation of the cleavage furrow and cytokinesis.These new data on Rab6-KIFL create a conundrum as we had grown accustomed to think about this protein more as a Golgi-associated motor protein involved in vesicular traffic in interphase cells 2xInteraction of a Golgi-associated kinesin-like protein with Rab6. Echard, A. et al. Science. 1998; 279: 580–585Crossref | PubMed | Scopus (349)See all References2. Echard et al.2xInteraction of a Golgi-associated kinesin-like protein with Rab6. Echard, A. et al. Science. 1998; 279: 580–585Crossref | PubMed | Scopus (349)See all References2 had demonstrated a staining of the Golgi complex in interphase HeLa cells with an antiserum raised against the same region of (mouse) Rab6-KIFL as used by Hill et al. Intriguingly, any such Golgi staining pattern is absent with the new Rab6-KIFL antiserum raised by Hill et al. There are several possible explanations for this difference. The antiserum used by Echard et al. might contain a cross-reactivity against a Golgi-associated protein that masks the real localization of Rab6-KIFL. Indeed, Hill et al. show that their antiserum recognizes only the 100-kDa band of Rab6-KIFL, whereas the antiserum raised by Echard et al. additionally sees a band of 200 kDa. However, a much more interesting possibility could perhaps be that the two laboratories have characterized highly related but functionally distinct isoforms of Rab6-KIFL that perform functions in mitotic and interphase cells, respectively. Future experiments will show us whether Rab6-KIFL has divorced the Golgi complex or whether we might yet see a reconciliation.

A Monoclonal Antibody Raised Against an 86-kD Subunit of Human DNA Helicase II

Abstract Here we produced a monoclonal antibody (termed as mH2) which specifically recognized an 86-kD subunit of Human DNA helicase II (p86) in HeLa cells. Immunohistochemical analysis of p86 throughout the cell cycle showed that its localization was changed from the nuclei to the cytoplasm, when the cells entered into prophase, and p86 localized diffusely in the cytoplasm during mitosis. Double staining of interphase HeLa cells with mH2 and an antibody, which recognized a component of nuclear pore complex, showed that p86 did not localize at the nuclear envelop in interphase cells.

Coiled bodies preferentially associate with U4, U11, and U12 small nuclear RNA genes in interphase HeLa cells but not with U6 and U7 genes.

Coiled bodies (CBs) are nuclear organelles involved in the metabolism of small nuclear RNAs (snRNAs) and histone messages. Their structural morphology and molecular composition have been conserved from plants to animals. CBs preferentially and specifically associate with genes that encode U1, U2, and U3 snRNAs as well as the cell cycle-regulated histone loci. A common link among these previously identified CB-associated genes is that they are either clustered or tandemly repeated in the human genome. In an effort to identify additional loci that associate with CBs, we have isolated and mapped the chromosomal locations of genomic clones corresponding to bona fide U4, U6, U7, U11, and U12 snRNA loci. Unlike the clustered U1 and U2 genes, each of these loci encode a single gene, with the exception of the U4 clone, which contains two genes. We next examined the association of these snRNA genes with CBs and found that they colocalized less frequently than their multicopy counterparts. To differentiate a lower level of preferential association from random colocalization, we developed a theoretical model of random colocalization, which yielded expected values for chi2 tests against the experimental data. Certain single-copy snRNA genes (U4, U11, and U12) but not controls were found to significantly (p < 0.000001) associate with CBs. Recent evidence indicates that the interactions between CBs and genes are mediated by nascent transcripts. Taken together, these new results suggest that CB association may be substantially augmented by the increased transcriptional capacity of clustered genes. Possible functional roles for the observed interactions of CBs with snRNA genes are discussed.

Coiled Bodies Preferentially Associate with U4, U11, and U12 Small Nuclear RNA Genes in Interphase HeLa Cells but Not with U6 and U7 Genes

Coiled Bodies Preferentially Associate with U4, U11, and U12 Small Nuclear RNA Genes in Interphase HeLa Cells but Not with U6 and U7 Genes