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PubMed1992

Microinjection of biotin-tubulin into anaphase cells induces transient elongation of kinetochore microtubules and reversal of chromosome-to-pole motion.

During prometaphase and metaphase of mitosis, tubulin subunit incorporation into kinetochore microtubules occurs proximal to the kinetochore, at the plus-ends of kinetochore microtubules. During anaphase, subunit loss from kinetochore fiber microtubules is also thought to occur mainly from microtubule plus-ends, proximal to the kinetochore. Thus, the kinetochore can mediate both subunit addition and loss while maintaining an attachment to kinetochore microtubules. To examine the relationship between chromosome motion and tubulin subunit assembly in anaphase, we have injected anaphase cells with biotin-labeled tubulin subunits. The pattern of biotin-tubulin incorporation was revealed using immunoelectron and confocal fluorescence microscopy of cells fixed after injection; chromosome motion was analyzed using video records of living injected cells. When anaphase cells are examined approximately 30 s after injection with biotin-tubulin, bright "tufts" of fluorescence are detected proximal to the kinetochores. Electron microscopic immunocytochemistry further reveals that these tufts of biotin-tubulin-containing microtubules are continuous with unlabeled kinetochore fiber microtubules. Biotin-tubulin incorporation proximal to the kinetochore in anaphase cells is detected after injection of 3-30 mg/ml biotin-tubulin, but not in cells injected with 0.3 mg/ml biotin-tubulin. At intermediate concentrations of biotin-tubulin (3-5 mg/ml), incorporation at the kinetochore can be detected within 15 s after injection; by approximately 1 min after injection discrete tufts of fluorescence are no longer detected, although some incorporation throughout the kinetochore fiber and into nonkinetochore microtubules is observed. At higher concentrations of injected biotin-tubulin (13 mg/ml), incorporation at the kinetochore is more extensive and occurs for longer periods of time than at intermediate concentrations. Incorporation of biotin-tubulin proximal to the kinetochore can be detected in cells injected during anaphase A, but not during anaphase B. Analysis of video records of microinjection experiments reveals that kinetochore proximal incorporation of biotin-tubulin is accompanied by a transient reversal of chromosome-to-pole motion. Chromosome motion is not altered after injection of 0.3 mg/ml biotin-tubulin or 5 mg/ml BSA. These results demonstrate that kinetochore microtubules in anaphase cells can elongate in response to the elevation of the tubulin concentration and that kinetochores retain the ability to mediate plus-end-dependent assembly of KMTs and plus-end-directed chromosome motion after anaphase onset.

Anaphase↗

PubMed1992

Export of mRNA from microinjected nuclei of Xenopus laevis oocytes.

Export of mRNA from the nucleus to the cytoplasm was studied in mature Xenopus laevis oocytes. In vitro transcribed, capped 32P-labeled mRNA was microinjected into nuclei, and its appearance in the cytoplasm measured by counting radioactivity or by RNA extraction and gel electrophoresis. Both for a 5.0-kb transferrin receptor mRNA and a 2.0-kb 4F2 antigen heavy chain mRNA we found saturable transport with an apparent Km of 3.6 x 10(8) molecules per oocyte nucleus. Under non-saturating conditions the half-time for mRNA export from the nucleus was approximately 2 min at 20 degrees C. At higher concentrations of injected mRNA this half-time was prolonged, and the maximal transport rate was reached at approximately 1.6 x 10(8) molecules/min. mRNA transport showed properties of an energy-dependent mechanism, since it was inhibited at 4 degrees C or by ATP depletion. Co-injection of the cap dinucleotide m7GpppG blocked the export effectively, suggesting a role for the cap in this process. The export was also inhibited by the pre-injection of wheat germ agglutinin. The effect of the lectin was specific and abolished by co-injection of N-acetylglucosamine. Finally, we found significant competitive inhibition in mRNA export by the presence of tRNA. Our results suggest that mRNA transport is a facilitated process which may share common steps with tRNA transport. Preliminary gel retardation experiments show that injected mRNA associates with endogenous nuclear proteins and suggest an exchange of some of the bound components during the transport to the cytoplasm.

Adenosine Triphosphate↗

PubMed1962

The intracellular distribution of ferritin following microinjection.

Ferritin solutions were microinjected into the ground cytoplasm of intact amebae. At several time-intervals after injection the cells were fixed and the distribution of the protein in various organelles was studied with the aid of the electron microscope. Individual molecules of ferritin were found randomly dispersed throughout the ground cytoplasm and the ground nucleoplasm. Within the mitochondria, the ferritin was localized between the outer and inner membranes. Aggregates of ferritin were found in vacuoles, some of which could be identified as food vacuoles. The findings, which provide evidence for a rapid penetration of large molecules into the nucleus, the outer compartment of the mitochondria, and the digestive vacuoles, are discussed in relation to other reports on intracellular permeability.

Cell Nucleus↗

PubMed1993

Transport and localization of exogenous myelin basic protein mRNA microinjected into oligodendrocytes.

We have studied transport and localization of MBP mRNA in oligodendrocytes in culture by microinjecting labeled mRNA into living cells and analyzing the intracellular distribution of the injected RNA by confocal microscopy. Injected mRNA initially appears dispersed in the perikaryon. Within minutes, the RNA forms granules which, in the case of MBP mRNA, are transported down the processes to the periphery of the cell where the distribution again becomes dispersed. In situ hybridization shows that endogenous MBP mRNA in oligodendrocytes also appears as granules in the perikaryon and processes and dispersed in the peripheral membranes. The granules are not released by extraction with non-ionic detergent, indicating that they are associated with the cytoskeletal matrix. Three dimensional visualization indicates that MBP mRNA granules are often aligned in tracks along microtubules traversing the cytoplasm and processes. Several distinct patterns of granule movement are observed. Granules in the processes undergo sustained directional movement with a velocity of approximately 0.2 micron/s. Granules at branch points undergo oscillatory motion with a mean displacement of 0.1 micron/s. Granules in the periphery of the cell circulate randomly with a mean displacement of approximately 1 micron/s. The results are discussed in terms of a multi-step pathway for transport and localization of MBP mRNA in oligodendrocytes. This work represents the first characterization of intracellular movement of mRNA in living cells, and the first description of the role of RNA granules in transport and localization of mRNA in cells.

Animals↗

PubMed1996

Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes.

Mammalian polo-like kinase 1 (Plk1) is structurally related to the polo gene product of Drosophila melanogaster, Cdc5p of Saccharomyces cerevisiae, and plo1+ of Schizosaccharomyces pombe, a newly emerging family of serine-threonine kinases implicated in cell cycle regulation. Based on data obtained for its putative homologues in invertebrates and yeasts, human Plk1 is suspected to regulate some fundamental aspect(s) of mitosis, but no direct experimental evidence in support of this hypothesis has previously been reported. In this study, we have used a cell duplication, microinjection assay to investigate the in vivo function of Plk1 in both immortalized (HeLa) and nonimmortalized (Hs68) human cells. Injection of anti-Plk1 antibodies (Plk1+) at various stages of the cell cycle had no effect on the kinetics of DNA replication but severely impaired the ability of cells to divide. Analysis of Plk1(+)-injected, mitotically arrested HeLa cells by fluorescence microscopy revealed abnormal distributions of condensed chromatin and monoastral microtubule arrays that were nucleated from duplicated but unseparated centrosomes. Most strikingly, centrosomes in Plk1(+)-injected cells were drastically reduced in size, and the accumulation of both gamma-tubulin and MPM-2 immunoreactivity was impaired. These data indicate that Plk1 activity is necessary for the functional maturation of centrosomes in late G2/early prophase and, consequently, for the establishment of a bipolar spindle. Additional roles for Plk1 at later stages of mitosis are not excluded, although injection of Plk1+ after the completion of spindle formation did not interfere with cytokinesis. Injection of Plk1+ into nonimmortalized Hs68 cells produced qualitatively similar phenotypes, but the vast majority of the injected Hs68 cells arrested as single, mononucleated cells in G2. This latter observation hints at the existence, in nonimmortalized cells, of a centrosome-maturation checkpoint sensitive to the impairment of Plk1 function.

Antibodies↗

PubMed1998

Microinjection of antibody to Mad2 protein into mammalian cells in mitosis induces premature anaphase.

In yeast, the Mad2 protein is required for the M phase arrest induced by microtubule inhibitors, but the protein is not essential under normal culture conditions. We tested whether the Mad2 protein participates in regulating the timing of anaphase onset in mammalian cells in the absence of microtubule drugs. When microinjected into living prophase or prometaphase PtK1 cells, anti-Mad2 antibody induced the onset of anaphase prematurely during prometaphase, before the chromosomes had assembled at the metaphase plate. Anti-Mad2 antibody-injected cells completed all aspects of anaphase including chromatid movement to the spindle poles and pole-pole separation. Identical results were obtained when primary human keratinocytes were injected with anti-Mad2 antibody. These studies suggest that Mad2 protein function is essential for the timing of anaphase onset in somatic cells at each mitosis. Thus, in mammalian somatic cells, the spindle checkpoint appears to be a component of the timing mechanism for normal mitosis, blocking anaphase onset until all chromosomes are aligned at the metaphase plate.

Anaphase↗

PubMed1998

Microinjection of anti-coilin antibodies affects the structure of coiled bodies.

The coiled body is a distinct subnuclear domain enriched in small nuclear ribonucleoprotein particles (snRNPs) involved in processing of pre-mRNA. Although the function of the coiled body is still unknown, current models propose that it may have a role in snRNP biogenesis, transport, or recycling. Here we describe that anti-coilin antibodies promote a specific disappearance of the coiled body in living human cells, thus providing a novel tool for the functional analysis of this structure. Monoclonal antibodies (mAbs) were raised against recombinant human coilin, the major structural protein of the coiled body. Four mAbs are shown to induce a progressive disappearance of coiled bodies within approximately 6 h after microinjection into the nucleus of HeLa cells. After their disappearance, coiled bodies are not seen to re-form, although injected cells remain viable for at least 3 d. Epitope mapping reveals that the mAbs recognize distinct amino acid motifs scattered along the complete coilin sequence. By 24 and 48 h after injection of antibodies that promote coiled body disappearance, splicing snRNPs are normally distributed in the nucleoplasm, the nucleolus remains unaffected, and the cell cycle progresses normally. Furthermore, cells devoid of coiled bodies for approximately 24 h maintain the ability to splice both adenoviral pre-mRNAs and transiently overexpressed human beta-globin transcripts. In conclusion, within the time range of this study, no major nuclear abnormalities are detected after coiled body disappearance.

Antibodies, Monoclonal↗

PubMed1975

Protein migration into nuclei. I. Frog oocyte nuclei in vivo accumulate microinjected histones, allow entry to small proteins, and exclude large proteins.

A technique is presented which enables one to measure the extent to which a protein enters and accumulates in the nucleus of the frog oocyte. In this method, the protein, labeled with 125-I, is microinjected into the oocyte. After incubation, the oocyte is manually enucleated and the radioactivity in the nucleus and cytoplasm is determined. Using this technique, proteins lighter than 20,000 daltons were found to enter the nucleus and completely equilibrate between the nucleus and cytoplasm within 24 h. The entry of proteins heavier than 69,000 daltons was severely hindered. Histones and histone fractions entered as quickly as other small proteins, but, in contrast to these proteins, they accumulated in the nucleus to different extents, depending on the total amount of histone injected into the oocyte and the identity of the histone. Evidence is presented that histone fractions compete with each other for accumulation in the nucleus.

Animals↗

PubMed1975

Nucleocytoplasmic movement of fluorescent tracers microinjected into living salivary gland cells.

The permeability of the nuclear envelop of a somatic cell, the C. thummi larval salivary gland cell, was studied by intracellular microinjection of fluorescent molecular tracers. As shown previously in oocytes (4,5,15,16), the envelop is permeable to a wide variety of materials, including molecules which are large enough to possess condiderable biological specificities and to play important roles in regulation of cellular activities. The envelop exhibits transport selectivity on the basis of size in the range of naturally occurring intracellular materials and it may thus perform important controlling functions in nucleocytoplasmic exchange. The nucleus to cytoplasm movement of in vivo ribonucleoprotein particulates in these synthetically active cells probably requires conformational changes in the particulates and/or the envelope pore complexes; morphological evidence exists for such processess in these cells (20).

Animals↗

PubMed1981

Direct visualization of fluorescein-labeled microtubules in vitro and in microinjected fibroblasts.

Microtubule proteins and tubulin have been purified from brain and labeled with dichlorotriazinyl fluorescein (DTAF). This procedure compromises neither the polymerizability of the proteins nor their affinities for unlabeled proteins. Within 15 min after microinjection of either DTAF-microtubule proteins or DTAF-tubulin into cultured gerbil fibroma cells, there was an evolution of a fluorescent fibrillar pattern with a distribution similar to that of the microtubular network seen after staining with fluorescent antitubulin. These filaments were colchicine sensitive and could be seen to elongate with time. DTAF-labeled microtubule accessory proteins from brain were not incorporated into filaments and appeared to label autophagic vacuoles.

Animals↗

PubMed2002

A microinjected COL7A1-PAC vector restores synthesis of intact procollagen VII in a dystrophic epidermolysis bullosa keratinocyte cell line.

Dystrophic epidermolysis bullosa (DEB) comprises a family of inherited blistering skin disorders for which no corrective therapy currently exists. In the most severe form, the Hallopeau-Siemens subtype (RDEB-HS), the epidermal adhesion protein collagen VII is absent from the skin as a consequence of null mutations in the COL7A1 gene. In order to develop an ex vivo gene therapy approach for DEB, we aimed to restore expression of intact procollagen VII in RDEB-HS keratinocytes. The entire human COL7A1 locus in a P1-derived artificial chromosome (PAC) was transferred to RDEB-HS keratinocytes by microinjection, after which sustained biosynthesis and secretion of procollagen VII was detected for 1 year in vitro. Protein chemical analysis demonstrated that the chain composition, domain structure, N-glycosylation and protein folding of the newly produced procollagen VII were similar, if not identical, to its authentic counterpart, indicating that transgenic procollagen VII was structurally normal. These data demonstrate a "proof of principle" for genomic DNA vectors as a means of restoring collagen VII production in RDEB-HS skin and help develop future gene therapy protocols.

Chromosomes, Artificial, P1 Bacteriophage↗

PubMed2002

Comparison of microinjection (piezo-electric) and cell fusion for nuclear transfer success with different cell types in cattle.

Amongst the many variables that can determine success of cloning, the source of nuclei, the procedure used for nuclear transfer, and the activation of the reconstructed embryo are very important aspects. In this study, we have compared the two most common procedures for transferring nuclei to enucleated oocytes--cell fusion (CF) and piezoelectric microinjection (PEM) using different somatic cells--and we have investigated the effect of different activation procedures. Granulosa cells and fibroblasts were grown to confluency or in low serum to induce a quiescent state, while lymphocytes were thawed immediately prior to use. Enucleated oocytes were reconstructed either with CF or PME by 21-23 h postmaturation. For cell fusion, one pulse of 1 kVolt/cm for 30 microsec was used; for PEM, the cell membrane was broken by repeated pipetting and transferred in a 12% PVP solution to facilitate injection. Manipulated oocytes were activated with ionomycin and cycloheximide (CHX) or 6-DMAP (DMAP) and cultured in microdrops of SOF-BSA-AA. On day 7 (day 0: nuclear transfer), embryo development was evaluated and embryos were either transferred fresh or were frozen. More embryos were successfully reconstructed with PEM than CF, but a higher number of reconstructed embryos by CF developed to blastocyst at D + 7. In addition, in both systems more embryos were obtained after activation with DMAP than with CHX. The transfer of 141 embryos to recipients resulted in a pregnancy rate of 50%, and no differences were observed between the source of donor cell, the reconstruction methods, or the activation protocol. Six calves were delivered at term, and four survived. High pregnancy losses were observed throughout the gestation period.

Animals↗

PubMed1993

Rapid transcriptional assay for the expression of two distinct reporter genes by microinjection.

We have developed a technique in which gene expression from multiple reporter plasmids introduced by needle microinjection can be monitored simultaneously in individual cells by double-label indirect immunofluorescence. With constitutively active viral promoters, expression from lacZ or chloramphenicol acetyl transferase (CAT) reporter genes can be detected within as little as 30 min after injection. Expression from such strong promoters reaches a maximum level after about 2 hr. In place of the constitutive promoter, promoters containing different enhancer elements respond as expected to different stimuli, allowing for the comparison of two defined transcriptional control elements in living cells. Reporter expression can be analyzed temporally and can be compared to expression of endogenous genes. This technique is complementary to transfection and allows for the targeted analysis of expression in specific cells, for example, in a mixed cell population, and for the analysis of expression in cells that are available only in small numbers.

Animals↗

PubMed2002

Secretion and assembly of zona pellucida glycoproteins by growing mouse oocytes microinjected with epitope-tagged cDNAs for mZP2 and mZP3.

The zona pellucida (ZP) is a highly organized extracellular coat that surrounds all mammalian eggs. The mouse egg ZP is composed of three glycoproteins, called mZP1-3, that are synthesized, secreted, and assembled into a ZP exclusively by growing oocytes. Here, we microinjected epitope-tagged (Myc and Flag) cDNAs for mZP2 and mZP3 into the germinal vesicle (nucleus) of growing oocytes isolated from juvenile mice. Specific antibodies and laser scanning confocal microscopy were used to follow nascent, recombinant ZP glycoproteins in both permeabilized and nonpermeabilized oocytes. When such cDNAs were injected, epitope-tagged mZP2 (Myc-mZP2) and mZP3 (Flag-mZP3) were synthesized, packaged into large intracellular vesicles, and secreted by the vast majority of oocytes. Secreted glycoproteins were incorporated into only the innermost layer of the thickening ZP, and the amount of nascent glycoprotein in this region increased with increasing time of oocyte culture. Consistent with prior observations, the putative transmembrane domain at the C terminus of mZP2 and mZP3 was missing from nascent glycoprotein incorporated into the ZP. When the consensus furin cleavage site near the C terminus of mZP3 was mutated, such that it should not be cleaved by furin, secretion and assembly of mZP3 was reduced. On the other hand, mZP3 incorporated into the ZP lacked the transmembrane domain downstream of the mutated furin cleavage site, suggesting that some other protease(s) excised the domain. These results strongly suggest that nascent mZP2 and mZP3 are incorporated into only the innermost layer of the ZP and that excision of the C-terminal region of the glycoproteins is required for assembly into the oocyte ZP.

Animals↗

PubMed1999

Ultrastructural analysis of transcription and splicing in the cell nucleus after bromo-UTP microinjection.

In this study we demonstrate, at an ultrastructural level, the in situ distribution of heterogeneous nuclear RNA transcription sites after microinjection of 5-bromo-UTP (BrUTP) into the cytoplasm of living cells and subsequent postembedding immunoelectron microscopic visualization after different labeling periods. Moreover, immunocytochemical localization of several pre-mRNA transcription and processing factors has been carried out in the same cells. This high-resolution approach allowed us to reveal perichromatin regions as the most important sites of nucleoplasmic RNA transcription and the perichromatin fibrils (PFs) as in situ forms of nascent transcripts. Furthermore, we show that transcription takes place in a rather diffuse pattern, without notable local accumulation of transcription sites. RNA polymerase II, heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins, general transcription factor TFIIH, poly(A) polymerase, splicing factor SC-35, and Sm complex of small nuclear ribonucleoproteins (snRNPs) are associated with PFs. This strongly supports the idea that PFs are also sites of major pre-mRNA processing events. The absence of nascent transcripts, RNA polymerase II, poly(A) polymerase, and hnRNPs within the clusters of interchromatin granules rules out the possibility that this domain plays a role in pre-mRNA transcription and polyadenylation; however, interchromatin granule-associated zones contain RNA polymerase II, TFIIH, and Sm complex of snRNPs and, after longer periods of BrUTP incubation, also Br-labeled RNA. Their role in nuclear functions still remains enigmatic. In the nucleolus, transcription sites occur in the dense fibrillar component. Our fine structural results show that PFs represent the major nucleoplasmic structural domain involved in active pre-mRNA transcriptional and processing events.

Cell Line↗

PubMed2000

Selective and continuous elimination of mitochondria microinjected into mouse eggs from spermatids, but not from liver cells, occurs throughout embryogenesis.

Exclusion of paternal mitochondria in fertilized mammalian eggs is very stringent and ensures strictly maternal mtDNA inheritance. In this study, to examine whether elimination was specific to sperm mitochondria, we microinjected spermatid or liver mitochondria into mouse embryos. Congenic B6-mt(spr) strain mice, which are different from C57BL/6J (B6) strain mice (Mus musculus domesticus) only in possessing M. spretus mtDNA, were used as mitochondrial donors. B6-mt(spr) mice and a quantitative PCR method enabled selective estimation of the amount of M. spretus mtDNA introduced even in the presence of host M. m. domesticus mtDNA and monitoring subsequent changes of its amount during embryogenesis. Results showed that M. spretus mtDNA in spermatid mitochondria was not eliminated by the blastocyst stage, probably due to the introduction of a larger amount of spermatid mtDNA than of sperm mtDNA into embryos on fertilization. However, spermatid-derived M. spretus mtDNA was eliminated by the time of birth, whereas liver-derived M. spretus mtDNA was still present in most newborn mice, even though its amount introduced was significantly less than that of spermatid mtDNA. These observations suggest that mitochondria from spermatids but not from liver have specific factors that ensure their selective elimination and resultant elimination of mtDNA in them, and that the occurrence of elimination is not limited to early stage embryos, but continues throughout embryogenesis.

Animals↗

PubMed1995

Interallelic complementation of beta-subunit defects in fibroblasts of patients with propionyl-CoA carboxylase deficiency microinjected with mutant cDNA constructs.

Propionic acidemia results from deficiency of propionyl-CoA carboxylase (PCC) activity. PCC is a biotin-dependent, mitochondrial enzyme composed of alpha- and beta-subunits (structure, alpha 4 beta 4), with the alpha-subunit containing the biotin ligand. About two-thirds of fibroblast lines from patients with mutations in the PCCB (beta-subunit) gene show interallelic complementation in cell fusion experiments (the pccB and pccC subgroups of the pccBC major group defining beta-subunit mutations, where pccB x pccC fusions show complementation). We previously identified the mutations in several pccB or pccC cell lines and suggested that point mutations or small, in-frame insertions or deletions were likely responsible for the complementation obtained between beta-subunit defects. To test this hypothesis, we have introduced five different mutations (three pccB and two pccC) that fit these criteria into a PCC beta-subunit cDNA plasmid expressed from a cytomegalovirus promoter. The cDNA plasmids were microinjected into mutant fibroblasts and the cells were assayed by radioautographic detection of 14C-propionate incorporation into cellular macromolecules. Four different mutations (Pro228Leu or dupKICK140 from pccB or delta IIe408 or Arg410Trp from pccC) complemented cells from complementation subgroups in a pattern congruent with the results obtained in cell fusion experiments. The fifth mutation, Arg536Asn, which was found both in a complementing pccB and a non-complementing pccBC cell line, failed to complement any of the mutant cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Metabolism, Inborn Errors↗

PubMedDate not supplied

Can we justify spermatid microinjection for severe male factor infertility?

During 1995 and 1996 the first spermatid pregnancies were announced with both round spermatid (ROSI) and elongated spermatid (ELSI) injections. These publications were flanked by live births from ROSI in a number of animal species, with resulting offspring appearing normal, healthy and fertile. However, the live births in humans heralded a scientific and ethical debate on the clinical use of this technology; and in a number of countries nationwide moratoria prohibiting spermatid microinjection were enjoined. Concerns surrounded the biological condition of spermatids and clinical implications of utilizing an immature spermatozoon for conception. Nevertheless, case reports and a few scientific studies on human spermatid conception have been published in recent years, and further polemic on testicular histopathology and prognosis has ensued. This paper reviews the current arguments on the clinical use of ROSI and ELSI, and evaluates the biology of the main contributory components of a spermatozoon to the subsequent embryo, namely the genetic material, the microtubular organizing complex and the putative oocyte activating factor. We also consider the relevant testicular histopathology and likely outcome in the context of the current birth rate from ROSI and ICSI. We conclude by considering the way forward for infertile men who require this technology to become genetic fathers, and whether the time is now appropriate to consider clinical trials.

Clinical Trials as Topic↗