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PubMed1999

The kinetoplast structure-specific endonuclease I is related to the 5' exo/endonuclease domain of bacterial DNA polymerase I and colocalizes with the kinetoplast topoisomerase II and DNA polymerase beta during replication.

The mitochondrial DNA (kinetoplast DNA) of the trypanosomatid Crithidia fasciculata has an unusual structure composed of minicircles and maxicircles topologically interlocked into a single network and organized in a disc-shaped structure at the base of the flagellum. We previously purified a structure-specific endonuclease (SSE1), based on its RNase H activity, that is enriched in isolated kinetoplasts. The endonuclease gene has now been cloned, sequenced, and found to be closely related to the 5' exonuclease domain of bacterial DNA polymerase I proteins. Although the protein does not contain a typical mitochondrial leader sequence, the enzyme is shown to colocalize with a type II DNA topoisomerase and a DNA polymerase beta at antipodal sites flanking the kinetoplast disc. Cell synchronization studies with an epitope-tagged construct show that the localization of the endonuclease to the antipodal sites varies in a cell cycle-dependent manner similar to that of the DNA polymerase beta [Johnson, C. E. & Englund, P. T. (1998) J. Cell Biol. 143, 911-919]. Immunofluorescent localization of SSE1 to the antipodal sites is only observed during kinetoplast replication. Together, these results suggest a point of control for kinetoplast DNA replication through the regulation of the availability of DNA replication proteins and a possible role for the antipodal sites in removal of RNA primers and the repair of gaps in newly replicated minicircles.

Amino Acid Sequence↗

PubMed2000

Calcium imaging demonstrates colocalization of calcium influx and extrusion in fly photoreceptors.

During illumination, Ca(2+) enters fly photoreceptor cells through light-activated channels that are located in the rhabdomere, the compartment specialized for phototransduction. From the rhabdomere, Ca(2+) diffuses into the cell body. We visualize this process by rapidly imaging the fluorescence in a cross section of a photoreceptor cell injected with a fluorescent Ca(2+) indicator in vivo. The free Ca(2+) concentration in the rhabdomere shows a very fast and large transient shortly after light onset. The free Ca(2+) concentration in the cell body rises more slowly and displays a much smaller transient. After approximately 400 ms of light stimulation, the Ca(2+) concentration in both compartments reaches a steady state, indicating that thereafter an amount of Ca(2+), equivalent to the amount of Ca(2+) flowing into the cell, is extruded. Quantitative analysis demonstrates that during the steady state, the free Ca(2+) concentration in the rhabdomere and throughout the cell body is the same. This shows that Ca(2+) extrusion takes place very close to the location of Ca(2+) influx, the rhabdomere, because otherwise gradients in the steady-state distribution of Ca(2+) should be measured. The close colocalization of Ca(2+) influx and Ca(2+) extrusion ensures that, after turning off the light, Ca(2+) removal from the rhabdomere is faster than from the cell body. This is functionally significant because it ensures rapid dark adaptation.

Animals↗

PubMed2000

Tid1/Rdh54 promotes colocalization of rad51 and dmc1 during meiotic recombination.

Two RecA homologs, Rad51 and Dmc1, assemble as cytologically visible complexes (foci) at the same sites on meiotic chromosomes. Time course analysis confirms that co-foci appear and disappear as the single predominant form. A large fraction of co-foci are eliminated in a red1 mutant, which is expected as a characteristic of the interhomolog-specific recombination pathway. Previous studies suggested that normal Dmc1 loading depends on Rad51. We show here that a mutation in TID1/RDH54, encoding a RAD54 homolog, reduces Rad51-Dmc1 colocalization relative to WT. A rad54 mutation, in contrast, has relatively little effect on RecA homolog foci except when strains also contain a tid1/rdh54 mutation. The role of Tid1/Rdh54 in coordinating RecA homolog assembly may be very direct, because Tid1/Rdh54 is known to physically bind both Dmc1 and Rad51. Also, Dmc1 foci appear early in a tid1/rdh54 mutant. Thus, Tid1 may normally act with Rad51 to promote ordered RecA homolog assembly by blocking Dmc1 until Rad51 is present. Finally, whereas double-staining foci predominate in WT nuclei, a subset of nuclei with expanded chromatin exhibit individual Rad51 and Dmc1 foci side-by-side, suggesting that a Rad51 homo-oligomer and a Dmc1 homo-oligomer assemble next to one another at the site of a single double-strand break (DSB) recombination intermediate.

Animals↗

PubMed1995

Factor V is complexed with multimerin in resting platelet lysates and colocalizes with multimerin in platelet alpha-granules.

Factor V stored in platelets is an important source of factor Va for the prothrombinase complex. Investigations of potential platelet factor Va-binding proteins, using factor Va light chain affinity chromatography, identified a disulfide-linked multimeric protein with a reduced mobility of 155 kDa in the column eluate. Immunodepletion and immunoblotting indicated that this protein was multimerin. Multimerin specifically bound factors V and Va and the isolated factor Va light chain, but not the heavy chain of factor Va. Factor V stored in platelets, but not plasma factor V, was found to be complexed with multimerin. Multimerin immunodepletion of resting platelet lysates was associated with the removal of factor V and the loss of factor V coagulant activity. Immunoelectron microscopic studies colocalized factor V with multimerin in the alpha-granules of resting platelets. With thrombin-induced platelet activation, we observed dissociation of factor Va-multimerin complexes, multimerin-independent membrane binding of factor Va, and prothrombinase activity that was not inhibitable by multimerin antibodies. This study indicates that platelet factor V is stored as a complex with multimerin and suggests a possible role for multimerin as a carrier protein for factor V stored in platelets.

Animals↗

PubMed1996

Expression and insulin-regulated distribution of caveolin in skeletal muscle. Caveolin does not colocalize with GLUT4 in intracellular membranes.

Caveolin is believed to play an important role in sorting processes, vesicular trafficking, transmembrane signaling, and molecular transport across membranes. In this study we have evaluated the expression and distribution of caveolin in skeletal muscle and its interaction with GLUT4 glucose carriers. Caveolin was expressed to substantial levels in muscle and its expression was regulated in muscle; aging and high fat diet enhanced caveolin expression in skeletal muscle and inversely, myogenesis down-regulated caveolin in L6E9 cells. Under fasting conditions, most of caveolin was found in intracellular membranes and the caveolin present in the cell surface was found in both sarcolemma and T-tubules. Insulin administration led to a redistribution of caveolin from intracellular high density membrane fractions to intracellular lighter density fractions and to the cell surface; this pattern of insulin-induced redistribution was different to what was shown by GLUT4. These results suggests that caveolin is a component of an insulin-regulated machinery of vesicular transport in muscle. Quantitative immunoisolation of GLUT4 vesicles obtained from different intracellular GLUT4 populations revealed the absence of caveolin which substantiates the lack of colocalization of intracellular GLUT4 and caveolin. This indicates that caveolin is not involved in intracellular GLUT4 trafficking in skeletal muscle.

Animals↗

PubMed1996

Fibrillin-1 and fibulin-2 interact and are colocalized in some tissues.

Microfibrils 10-12 nm in diameter are found in elastic and non-elastic tissues with fibrillin as a major component. Little is known about the supramolecular structure of these microfibrils and the protein interactions it is based on. To identify protein binding ligands of fibrillin-1, we tested binding of recombinant fibrillin-1 peptides to different extracellular matrix proteins in solid phase assays. Among the proteins tested, only fibulin-2 showed significant binding to rF11, the N-terminal half of fibrillin-1, in a calcium-dependent manner. Surface plasmon resonance demonstrated high affinity binding with a Kd = 56 nM. With overlapping recombinant fibrillin-1 peptides, the binding site for fibulin-2 was narrowed down to the N terminus of fibrillin-1 (amino acid positions 45-450). Immunofluorescence in tissues demonstrated colocalization of fibrillin and fibulin-2 in skin, perichondrium, elastic intima of blood vessels, and kidney glomerulus. Fibulin-2 was not present in ocular ciliary zonules, tendon, and the connective tissue around kidney tubules and lung alveoli, which all contain fibrillin. Immunogold labeling of fibulin-2 on microfibrils in skin was found preferentially at the interface between microfibrils and the amorphous elastin core, suggesting that in vivo the interaction between fibrillin-1 and fibulin-2 is regulated by cellular expression and deposition as well as by protein-protein interactions.

Amino Acid Sequence↗

PubMed1999

HRad17 colocalizes with NHP2L1 in the nucleolus and redistributes after UV irradiation.

The rad17 gene of Schizosaccharomyces pombe plays an important role as a checkpoint protein following DNA damage and during DNA replication. The human homologue of S. pombe rad17, Hrad17, was recently identified, but its function has not yet been established. Using the yeast two-hybrid system, we determined that HRad17 can interact with a nucleolar protein, NHP2L1. This interaction was also demonstrated biochemically, in human cells. Immunofluorescence studies revealed that HRad17 and NHP2L1 colocalize to the nucleolus, and immunogold labeling further resolved the location of NHP2L1 to the dense fibrillar component of the nucleolus. Interestingly, the localization of HRad17 in the nucleolus was altered in response to UV irradiation. These results provide some insight into the DNA damage and replication checkpoint mechanisms of HRad17.

Amino Acid Sequence↗

PubMed2000

NBS1 and TRF1 colocalize at promyelocytic leukemia bodies during late S/G2 phases in immortalized telomerase-negative cells. Implication of NBS1 in alternative lengthening of telomeres.

Nijmegen breakage syndrome, a chromosomal instability disorder, is characterized in part by cellular hypersensitivity to ionizing radiation. The NBS1 gene product, p95 (NBS1 or nibrin) forms a complex with Rad50 and Mre11. Cells deficient in the formation of this complex are defective in DNA double-strand break repair, cell cycle checkpoint control, and telomere length maintenance. How the NBS1 complex is involved in telomere length maintenance remains unclear. Here we show that the C-terminal region of NBS1 interacts directly with a telomere repeat binding factor, TRF1, by both yeast two-hybrid and in vivo DNA-coimmunoprecipitation assays. NBS1 and Mre11 colocalize with TRF1 at promyelocytic leukemia (PML) nuclear bodies in immortalized telomerase-negative cell lines, but rarely in telomerase-positive cell lines. The translocation of NBS1 to PML bodies occurs specifically during late S to G(2) phases of the cell cycle and coincides with active DNA synthesis in these NBS1-containing PML bodies. These results suggest that NBS1 may be involved in alternative lengthening of telomeres in telomerase-negative immortalized cells.

Abnormalities, Multiple↗

PubMed2001

Human DNA polymerase epsilon colocalizes with proliferating cell nuclear antigen and DNA replication late, but not early, in S phase.

DNA polymerase epsilon (pol epsilon) has been implicated in DNA replication, DNA repair, and cell cycle control, but its precise roles are unclear. When the subcellular localization of human pol epsilon was examined by indirect immunofluorescence, pol epsilon appeared in discrete nuclear foci that colocalized with proliferating cell nuclear antigen (PCNA) foci and sites of DNA synthesis only late in S phase. Early in S phase, pol epsilon foci were adjacent to PCNA foci. In contrast to PCNA foci that were only present in S phase, pol epsilon foci were present throughout mitosis and the G(1) phase of cycling cells. It is hypothesized from these observations that pol epsilon and PCNA have separate but associated functions early in S phase and that pol epsilon participates with PCNA in DNA replication late in S phase.

Antimetabolites↗

PubMed2002

Human Herpesvirus 6 immediate-early 1 protein is a sumoylated nuclear phosphoprotein colocalizing with promyelocytic leukemia protein-associated nuclear bodies.

Immediate-early (IE) proteins are the first proteins expressed following viral entry and play a crucial role in the initiation of infection. We report the cloning and characterization of a full-length IE1 transcript and protein (IE1B) from human herpesvirus 6 (HHV-6) variant B. The IE1B transcript consists of five exons (3720 nucleotides), three of which are coding for the IE1 protein. The 1078-amino acid-long IE1B protein is 62% identical and 75% similar to the 941-amino acid IE1 from HHV-6 variant A. IE1B protein can be detected at 4 h post-infection (P.I.), and it is distributed as small intranuclear structures. The maximal number of IE1 bodies ( approximately 10-12/nucleus) is detected at 12 h P.I. after which the IE1 bodies condense into 1-3 larger entities by 24-48 h P.I. During infection the IE1B protein is phosphorylated on serine and threonine residues. IE1B undergoes further post-translational modification with its conjugation to the small ubiquitin-like modifier (SUMO-1) peptide. IE1B colocalizes with SUMO-1 and promyelocytic leukemia nuclear bodies during infection as well as in transfection experiments. Finally, IE1 from variant B is a weaker transactivator than IE1 from variant A, when assayed using heterologous promoters. Overall, the characterization of the HHV-6 IE1B protein presented highlights the similarity and divergence between IE1 from both variants and provides useful information pertaining to the early phase of infection.

Cell Nucleus↗

PubMed2002

Colocalization, physical, and functional interaction between Werner and Bloom syndrome proteins.

The RecQ helicase family comprises a conserved group of proteins implicated in several aspects of DNA metabolism. Three of the family members are defective in heritable diseases characterized by abnormal growth, premature aging, and predisposition to malignancies. These include the WRN and BLM gene products that are defective in Werner and Bloom syndromes, disorders which share many phenotypic and cellular characteristics including spontaneous genomic instability. Here, we report a physical and functional interaction between BLM and WRN. These proteins were coimmunoprecipitated from a nuclear matrix-solubilized fraction, and the purified recombinant proteins were shown to interact directly. Moreover, BLM and WRN colocalized to nuclear foci in three human cell lines. Two regions of WRN that mediate interaction with BLM were identified, and one of these was localized to the exonuclease domain of WRN. Functionally, BLM inhibited the exonuclease activity of WRN. This is the first demonstration of a physical and functional interaction between RecQ helicases. Our observation that RecQ family members interact provides new insights into the complex phenotypic manifestations resulting from the loss of these proteins.

Adenosine Triphosphatases↗

PubMed2004

Interaction, cooperative promoter modulation, and renal colocalization of GCMa and Pitx2.

The transcription factor GCMa is a member of a new small family of transcription factors with a conserved zinc-containing DNA-binding domain. All members of this transcription factor family play crucial roles as master regulators during development. GCMa is restricted to placenta during development and to kidney and thymus at postnatal stages. It is essential for the formation of the placental labyrinth and as a consequence for survival of the embryo from mid-embryogenesis onwards. Here, we identify Pitx transcription factors as GCMa-interacting proteins. We show that Pitx proteins interact via their conserved homeodomain with the DNA-binding domain of GCMa. As a consequence, Pitx proteins and GCMa exhibit cooperative DNA binding. Furthermore, Pitx proteins influence GCMa-dependent promoter activation in a cell-specific manner. One of the three Pitx paralogues in mice, Pitx2, is the predominant Pitx member present in the placenta and colocalizes on the cellular level with GCMa in the kidney. This is the first description of a regulatory cross-talk between a transcription factor of the GCM family and a homeodomain protein.

Animals↗

PubMed2004

Physical and functional interaction between Dorfin and Valosin-containing protein that are colocalized in ubiquitylated inclusions in neurodegenerative disorders.

Dorfin, a RING-IBR type ubiquitin ligase (E3), can ubiquitylate mutant superoxide dismutase 1, the causative gene of familial amyotrophic lateral sclerosis (ALS). Dorfin is located in ubiquitylated inclusions (UBIs) in various neurodegenerative disorders, such as ALS and Parkinson's disease (PD). Here we report that Valosin-containing protein (VCP) directly binds to Dorfin and that VCP ATPase activity profoundly contributes to the E3 activity of Dorfin. High through-put analysis using mass spectrometry identified VCP as a candidate of Dorfin-associated protein. Glycerol gradient centrifugation analysis showed that endogenous Dorfin consisted of a 400-600-kDa complex and was co-immunoprecipitated with endogenous VCP. In vitro experiments showed that Dorfin interacted directly with VCP through its C-terminal region. These two proteins were colocalized in aggresomes in HEK293 cells and UBIs in the affected neurons of ALS and PD. VCP(K524A), a dominant negative form of VCP, reduced the E3 activity of Dorfin against mutant superoxide dismutase 1, whereas it had no effect on the autoubiquitylation of Parkin. Our results indicate that VCPs functionally regulate Dorfin through direct interaction and that their functional interplay may be related to the process of UBI formation in neurodegenerative disorders, such as ALS or PD.

Adenosine Triphosphatases↗

PubMed2003

Nuclear staining for the small heat shock protein alphaB-crystallin colocalizes with splicing factor SC35.

AlphaB-Crystallin has for a long time been considered a specific eye lens protein. Later on it appeared that this protein belongs to the family of the small heat shock proteins and that it occurs also extra-lenticularly in many different cell types. AlphaB-Crystallin is mainly present in the cytoplasm, but there are some indications that it might have a function in the nucleus too. However, till now its presence in the nucleus is uncertain. We therefore compared the localization of alphaB-crystallin in nine cell lines cultured under normal conditions using four different antisera. All four antisera gave a diffuse staining for alphaB-crystallin in the cytoplasm, but one of the antibodies consistently showed nuclear staining in eight of the cell types, in the form of distinct speckles. These speckles are equally pronounced in the different cell types, whether or not cytoplasmic alphaB-crystallin is present. Preabsorption of the antiserum with alphaB-crystallin abolished the staining. Furthermore we demonstrate that if only minor amounts of alphaB-crystallin are present, the protein seems to be located exclusively in the nucleus. However, in case of higher amounts of protein, alphaB-crystallin is distributed between cytoplasm and nucleus. The nuclear alphaB-crystallin exists, like the cytoplasmic alphaB-crystallin, in non-phosphorylated and phosphorylated forms, is Triton-insoluble but can be extracted by 2 M NaCl. These data suggest that alphaB-crystallin might be bound to the nuclear matrix per se or to nuclear matrix proteins via other proteins. In agreement with other nuclear matrix proteins, nuclear alphaB-crystallin staining turns diffuse upon mitosis and leaves the chromosomes unstained. Double staining experiments revealed colocalization of alphaB-crystallin with the splicing factor SC35 in nuclear speckles, suggesting a role for alphaB-crystallin in splicing or protection of the splicing machinery.

3T3 Cells↗

PubMed2003

JC virus agnoprotein colocalizes with tubulin.

The human polyomavirus JC (JCV) encodes an agnoprotein that consists of 71 amino acid residues, with a molecular weight of approximately 8 kDa, from the late protein coding region. The agnoprotein of JCV shares 50% to 60% homology with those of simian virus 40 (SV40) and BK virus (BKV), and the carboxyl-terminal region of JCV agnoprotein is relatively unique. By using specific antibody to the carboxyl-terminal region of JCV agnoprotein, the authors have demonstrated that JCV agnoprotein expressed in the JCV-infected cells, where it localized predominantly in the perinuclear region of the cytoplasm, and colocalizes with the cellular cytoskeletal protein, tubulin. The results suggest that JCV agnoprotein may play a role in the stability of microtubules and the preservation of JCV infected cells via an interaction with tubulin.

Cytoplasm↗

PubMed1990

Spatial and temporal colocalization of the Golgi apparatus and microtubules rich in detyrosinated tubulin.

The integrity and intracellular distribution of the Golgi apparatus appear to depend upon microtubules. We have found that the microtubules rich in detyrosinated tubulin are located preferentially in the vicinity of the Golgi. Cells were double-stained with antibodies specific for either tyrosinated or detyrosinated tubulin and an antibody to prolactin or wheat germ agglutinin (Golgi markers). Microtubules rich in detyrosinated tubulin showed a close codistribution with the Golgi in three different cultured cell lines GH3, BS-C-1, and AtT20. Disruption of microtubules with nocodazole in GH3 cells resulted in fragmentation and dispersal of the Golgi apparatus as reported previously. During recovery of the microtubules and the Golgi complex after removal of the nocodazole, there was a spatial and temporal colocalization of the Golgi apparatus and microtubules rich in detyrosinated tubulin. Our results suggest that a functional relationship may exist between the structure and organization of the Golgi complex and the detyrosination of alpha-tubulin in microtubules.

Animals↗

PubMed1990

BIK1, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin.

BIK1 function is required for nuclear fusion, chromosome disjunction, and nuclear segregation during mitosis. The BIK1 protein colocalizes with tubulin to the spindle pole body and mitotic spindle. Synthetic lethality observed in double mutant strains containing a mutation in the BIK1 gene and in the gene for alpha- or beta-tubulin is consistent with a physical interaction between BIK1 and tubulin. Furthermore, over- or underexpression of BIK1 causes aberrant microtubule assembly and function, bik1 null mutants are viable but contain very short or undetectable cytoplasmic microtubules. Spindle formation often occurs strictly within the mother cell, probably accounting for the many multinucleate and anucleate bik1 cells. Elevated levels of chromosome loss in bik1 cells are indicative of defective spindle function. Nuclear fusion is blocked in bik1 x bik1 zygotes, which have truncated cytoplasmic microtubules. Cells overexpressing BIK1 initially have abnormally short or nonexistent spindle microtubules and long cytoplasmic microtubules. Subsequently, cells lose all microtubule structures, coincident with the arrest of division. Based on these results, we propose that BIK1 is required stoichiometrically for the formation or stabilization of microtubules during mitosis and for spindle pole body fusion during conjugation.

Amino Acid Sequence↗

PubMed1991

Colocalization of synaptophysin with transferrin receptors: implications for synaptic vesicle biogenesis.

We have reported previously that the synaptic vesicle (SV) protein synaptophysin, when expressed in fibroblastic CHO cells, accumulates in a population of recycling microvesicles. Based on preliminary immunofluorescence observations, we had suggested that synaptophysin is targeted to the preexisting population of microvesicles that recycle transferrin (Johnston, P. A., P. L. Cameron, H. Stukenbrok, R. Jahn, P. De Camilli, and T. C. Südhof. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:2863-2872). In contrast to our results, another group reported that expression of synaptophysin in cells which normally do not express SV proteins results in the generation of a novel population of microvesicles (Leube, R. E., B. Wiedenmann, and W. W. Franke. 1989. Cell. 59:433-446). We report here a series of morphological and biochemical studies conclusively demonstrating that synaptophysin and transferrin receptors are indeed colocalized on the same vesicles in transfected CHO cells. These observations prompted us to investigate whether an overlap between the distribution of the two proteins also occurs in endocrine cell lines that endogenously express synaptophysin and other SV proteins. We have found that endocrine cell lines contain two pools of membranes positive for synaptophysin and other SV proteins. One of the two pools also contains transferrin receptors and migrates faster during velocity centrifugation. The other pool is devoid of transferrin receptors and corresponds to vesicles with the same sedimentation characteristics as SVs. These findings suggest that in transfected CHO cells and in endocrine cell lines, synaptophysin follows the same endocytic pathway as transferrin receptors but that in endocrine cells, at some point along this pathway, synaptophysin is sorted away from the recycling receptors into a specialized vesicle population. Finally, using immunofluorescent analyses, we found an overlap between the distribution of synaptophysin and transferrin receptors in the dendrites of hippocampal neurons in primary cultures before synapse formation. Axons were enriched in synaptophysin immunoreactivity but did not contain detectable levels of transferrin receptor immunoreactivity. These results suggest that SVs may have evolved from, as well as coexist with, a constitutively recycling vesicular organelle found in all cells.

Animals↗