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PubMed1991

Microinjection of the catalytic fragment of myosin light chain kinase into dividing cells: effects on mitosis and cytokinesis.

Myosin light chain kinase (MLCK) is thought to regulate the contractile activity in smooth and non-muscle cells, and may play an important role in controlling the reorganization of the actin-myosin cytoskeleton during cell division. To test this hypothesis we have microinjected the 61-kD catalytic fragment of MLCK into mitotic cells, and examined the effects of unregulated MLCK activity on cell division. The microinjection of active 61 kD causes both a significant delay in the transit time from nuclear envelope breakdown to anaphase onset, and an increase in motile surface activity during and after metaphase. Control experiments with intact MLCK or with inactive catalytic fragment suggest that these effects are specifically induced by the unregulated myosin light chain kinase activity. Immunofluorescence analysis suggests that delays in mitosis are coupled to disruptions of spindle structures, while increased surface motility may be related to changes in the organization of actin and myosin at the cell cortex. Most importantly, despite the expression of strong phenotypes, 61 kD-injected cells still form functional cleavage furrows that progress through cytokinesis at rates identical to those of control cells. Together, these results suggest that the activity of MLCK can affect mitosis and cortical activities, however additional control mechanisms are likely involved in the regulation of cytokinesis.

Actins↗

PubMed1992

Transport of microinjected alcohol oxidase from Pichia pastoris into vesicles in mammalian cells: involvement of the peroxisomal targeting signal.

This report describes the microinjection of a purified peroxisomal protein, alcohol oxidase, from Pichia pastoris into mammalian tissue culture cells and the subsequent transport of this protein into vesicular structures. Transport was into membrane-enclosed vesicles as judged by digitonin-permeabilization experiments. The transport was time and temperature dependent. Vesicles containing alcohol oxidase could be detected as long as 6 d after injection. Coinjection of synthetic peptides containing a consensus carboxyterminal tripeptide peroxisomal targeting signal resulted in abolition of alcohol oxidase transport into vesicles in all cell lines examined. Double-label experiments indicated that, although some of the alcohol oxidase was transported into vesicles that contained other peroxisomal proteins, the bulk of the alcohol oxidase did not appear to be transported to preexisting peroxisomes. While the inhibition of transport of alcohol oxidase by peptides containing the peroxisomal targeting signal suggests a competition for some limiting component of the machinery involved in the sorting of proteins into peroxisomes, the organelles into which the majority of the protein is targeted appear to be unusual and distinct from endogenous peroxisomes by several criteria. Microinjected alcohol oxidase was transported into vesicles in normal fibroblasts and also in cell lines derived from patients with Zellweger syndrome, which are unable to transport proteins containing the ser-lys-leu-COOH peroxisomal targeting signal into peroxisomes (Walton et al., 1992). The implications of this result for the mechanism of peroxisomal protein transport are discussed.

3T3 Cells↗

PubMed1993

Roles of kinesin and kinesin-like proteins in sea urchin embryonic cell division: evaluation using antibody microinjection.

Previous studies suggest that kinesin heavy chain (KHC) is associated with ER-derived membranes that accumulate in the mitotic apparatus in cells of early sea urchin embryos (Wright, B. D., J. H. Henson, K. P. Wedaman, P. J. Willy, J. N. Morand, and J. M. Scholey. 1991. J. Cell Biol. 113:817-833). Here, we report that the microinjection of KHC-specific antibodies into these cells has no effect on mitosis or ER membrane organization, even though one such antibody, SUK4, blocks kinesin-driven motility in vitro and in mammalian cells. Microinjected SUK4 was localized to early mitotic figures, suggesting that it is able to access kinesin in spindles. In contrast to KHC-specific antibodies, two antibodies that react with kinesin-like proteins (KLPs), namely CHO1 and HD, disrupted mitosis and prevented subsequent cell division. CHO1 is thought to exert this effect by blocking the activity of a 110-kD KLP. The relevant target of HD, which was raised against the KHC motor domain, is unknown; HD may disrupt mitosis by interfering with an essential spindle KLP but not with KHC itself, as preabsorption of HD with KHC did not alter its ability to block mitosis. These data indicate that some KLPs have essential mitotic functions in early sea urchin embryos but KHC itself does not.

Animals↗

PubMed1976

"Cleavage" and cortical granule breakdown in Rana pipiens oocytes induced by direct microinjection of calcium.

Microinjection of approximately 0.3 mug of calcium into maturing oocytes of Rana pipiens after nuclear dissolution resulted in cleavage-like constrictions, cortical granule breakdown, and formation of a structure resembling a two-cell embryo. Mg2+, Na+, or K+ did not induce any of these reactions. Larger amounts of Ca2+-induced contraction over the entire surface of oocytes or eggs, but did not induce cleavage-like constrictions; smaller amounts of Ca2+ produced either a local cortical granule reaction of the formation of one large and one small "blastomere." Furrow formation was not observed during normally induced maturation until after germinal vesicle breakdown. The location of microinjected Ca2+ determined the orientation of the resulting furrow. Ca2+-induced cortical granule breakdown occurred in full-grown nonmaturing oocytes near the site of injection. Cortical granule breakdown also occurred in maturing oocytes (after germinal vesicle breakdown but before second meiotic metaphase), but only in the blastomere containing the infected Ca2+. As expected, in mature oocytes (at second meiotic metaphase) cortical granule breakdown occurred over the entire oocyte surface, including both blastomeres. The results indicate that furrow formation and cleavage-like constrictions may be directly influenced by Ca2+, and that functional contractile elements are present near all areas of the oocyte surface. Furthermore, Ca2+ injection initiates localized cortical granule breakdown in full-grown immature and maturing oocytes.

Animals↗

PubMed1983

Microinjection of fluorescent tubulin into dividing sea urchin cells.

To follow the dynamics of microtubule (MT) assembly and disassembly during mitosis in living cells, tubulin has been covalently modified with the fluorochrome 5-(4,6-dichlorotriazin-2-yl)aminofluorescein and microinjected into fertilized eggs of the sea urchin Lytechinus variegatus. The changing distribution of the fluorescent protein probe is visualized in a fluorescence microscope coupled to an image intensification video system. Cells that have been injected with fluorescent tubulin show fluorescent linear polymers that assemble very rapidly and radiate from the spindle poles, coincident with the position of the astral fibers. No fluorescent polymer is apparent in other areas of the cytoplasm. When fluorescent tubulin is injected near the completion of anaphase, little incorporation of fluorescent tubulin into polymer is apparent, suggesting that new polymerization does not occur past a critical point in anaphase. These results demonstrate that MT polymerization is very rapid in vivo and that the assembly is both temporally and spatially regulated within the injected cells. Furthermore, the microinjected tubulin is stable within the sea urchin cytoplasm for at least 1 h since it can be reutilized in successive daughter cell spindles. Control experiments indicate that the observed fluorescence is dependent on MT assembly. The fluorescence is greatly diminished upon treatment of the cells with cold or colchicine agents known to cause the depolymerization of assembled MT. In addition, cells injected with fluorescent bovine serum albumin or assembly-incompetent fluorescent tubulin do not exhibit fluorescence localized in the spindle but rather appear diffusely fluorescent throughout the cytoplasm.

Anaphase↗

PubMed1983

Microinjection of Ca++-calmodulin causes a localized depolymerization of microtubules.

The microinjection of calcium-saturated calmodulin into living fibroblasts causes the rapid disruption of microtubules and stress fibers in a sharply delimited region concentric with the injection site. This effect is specific to the calcium-bearing form of calmodulin; neither calcium-free calmodulin nor calcium ion at similar levels affects the cytoskeleton. If cells have previously been microinjected with calcium-free calmodulin, elevation of their intracellular calcium levels to 25 mM potentiates the disruption of microtubules throughout the cytoplasm. Approximately 400 mM free calcium is required to cause an equivalent disruption in uninjected cells. The level of calmodulin necessary to disrupt the full complement of cellular microtubules is found to be approximately in 2:1 molar ratio to tubulin dimer. These results indicate that calmodulin can be localized within the cytoplasm in a calcium-dependent manner and that it can act to regulate the calcium lability of microtubules at molar ratios that could be achieved locally within the cell. Our results are consistent with the hypothesis that calmodulin may be controlling microtubule polymerization equilibria in areas of high local concentration such as the mitotic spindle.

Animals↗

PubMed1984

Microinjected fluorescent polystyrene beads exhibit saltatory motion in tissue culture cells.

Microinjected 0.26-micron fluorescent, carboxylated microspheres were found to display classical saltatory motion in tissue culture cells. The movement of a given particle was characterized by a discontinuous velocity distribution and was unaffected by the activity of adjacent particles. The microspheres were translocated at velocities of up to 4.7 micron/s and sometimes exhibited path lengths greater than 20 micron for a single saltation . The number of beads injected into a cell could range from a few to over 500 with no effect on the cell's ability to transport them. Neither covalent cross-linking nor preincubation of the polystyrene beads with various proteins inhibited the saltatory motion of the injected particles. The motion of the injected beads in cultured cells was reversibly inhibited by the microtubule poison nocodazole, under conditions in which actin-rich, nitrobenzoxadiazol - phallacidin -staining structures remain intact. Whole-cell high voltage electron microscopy of microinjected cells that were known to be moving the fluorescent microspheres revealed that the beads were embedded in the cytoplasmic matrix and did not appear to be membrane bound. The enhanced detectability of the fluorescent particles over endogenous organelles and the ability to modify the surfaces of the beads before injection may enable more detailed studies on the mechanism of saltatory particle motion.

Animals↗

PubMed2003

Evaluation of conditions for calpain inhibition in the rat spinal cord: effective postinjury inhibition with intraspinal MDL28170 microinjection.

Calpains (calcium-activated cysteine proteases) are strongly implicated in the secondary damage that follows contusion injury to the spinal cord. Calpains are activated within a few minutes following injury and their elevated activity persists for 24 h, thereby providing a reasonable window of opportunity for postinjury inhibition. Previous studies demonstrated decreased axonal damage and neurofilament proteolysis with postinjury intravenous administration of relatively low concentrations of the calpain inhibitors leupeptin, E-64-D, and calpeptin. We sought to determine if conditions under which calpain inhibitors were administered in previous studies resulted in effective calpain inhibition, and to identify conditions that result in significant calpain inhibition following spinal cord injury. Contusive spinal cord injury was produced in female Long-Evans rats using the NYU impactor at the 12.5-25-mm height setting. The results demonstrate that intravenous administration of 1 mg/kg E-64-D or 250 micro g/kg calpeptin does not inhibit total calpain activity in the rat spinal cord, measured using a BODIPY-FL labeled casein assay. Intravenous administration of MDL28170 (20 mg/kg) resulted in mild calpain inhibition and a modest decrease in the proteolysis of calpain substrates alpha-spectrin and MAP2. Intraspinal microinjection of 50 nmoles/19 micro g MDL28170, either 30 min prior to or 20 min following contusion injury, resulted in a more robust inhibition of total calpain activity and greater attenuation of alpha-spectrin breakdown and MAP2 proteolysis. The decreased proteolysis persisted 24 h postinjury. Together, the results demonstrate that direct microinjection of the calpain inhibitor MDL28170 is more effective than intravenous infusion in reducing calpain activity and decreasing the injury-induced proteolysis of calpain substrates.

Animals↗

PubMed1991

Permanent rescue of a non-Mendelian mutation of Paramecium by microinjection of specific DNA sequences.

The mutant Paramecium tetraurelia cell line d48 is unable to express the serotype A protein on its surface. Although the A gene is intact in the micronuclei of d48, the A gene copies in the macronucleus contain a large deletion eliminating virtually the entire coding sequence. Previous studies showed that microinjection of a plasmid containing the entire A gene into the macronucleus of d48 permanently restored A expression after autogamy. Together with other data, this result suggests that in wild type cells the A gene in the old macronucleus ensures the presence of a cytoplasmic factor that prevents A gene deletions at autogamy. In d48, where there are few, if any copies of the intact A gene in the old macronucleus, deletions occur during macronuclear formation. To elucidate the specific molecular mechanisms involved in this unusual phenomenon, we attempted to define the region(s) of the A gene necessary for rescuing d48. We show that microinjection of a 4.5-kb internal A gene fragment is sufficient for proper processing at autogamy and leads to permanent rescue of d48; i.e., the rescued strain is indistinguishable from wild type. Thus, rescue of d48 does not require upstream transcriptional control sequences, intact A mRNA or A serotype protein. We also show that various fragments of the A gene have the ability to rescue d48 to different extents, some being more efficient than others. We find no evidence to suggest that the A gene gives rise to a small stable RNA that might act as or encode a cytoplasmic factor. Molecular mechanisms that may be involved in the rescue of d48 are discussed.

Animals↗

PubMed2002

Predictive value of testicular histology in secretory azoospermic subgroups and clinical outcome after microinjection of fresh and frozen-thawed sperm and spermatids.

BACKGROUND: A retrospective study was carried out on 159 treatment cycles in 148 secretory azoospermic patients to determine whether histopathological secretory azoospermic subgroups were predictive for gamete retrieval, and to evaluate outcome of microinjection using fresh or frozen-thawed testicular sperm and spermatids. METHODS: Sperm and spermatids were recovered by open testicular biopsy and microinjected into oocytes. Fertilization and pregnancy rates were assessed. RESULTS: In hypoplasia, 97.7% of the 44 patients had late spermatids/sperm recovered. In maturation-arrest (MA; 47 patients), 31.9% had complete MA, and 68.1% incomplete MA due to a focus of early (36.2%) or late (31.9%) spermiogenesis. Gamete retrieval was achieved in 53.3, 41.2 and 93.3% of the cases respectively. In Sertoli cell-only syndrome (SCOS; 57 patients), 61.4% were complete SCOS, whereas incomplete SCOS cases showed one focus of MA (5.3%), or of early (29.8%) and late (3.5%) spermiogenesis. Only 29.8% of the patients had a successful gamete retrieval, 2.9% in complete and 77.3% in incomplete SCOS cases. In total, there were 87 ICSI, 39 elongated spermatid injection (ELSI) and 33 round spermatid injection (ROSI) treatment cycles, with mean values of fertilization rate of 71.4, 53.6 and 17%, and clinical pregnancy rates of 31.7, 26.3 and 0% respectively. CONCLUSIONS: Histopathological subgroups were positively correlated with successful gamete retrieval. No major outcome differences were observed between testicular sperm and elongated spermatids, either fresh or frozen-thawed. However, injection of intact round-spermatids showed very low rates of fertilization and no pregnancies.

Cell Size↗

PubMed1993

A method for the generation of YAC transgenic mice by pronuclear microinjection.

Yeast artificial chromosomes (YACs) represent the latest generation of vectors which have the great advantage of large insert size. The introduction of YACs into mammalian cells and organisms has become an important goal, since it offers the potential to study the control of large and complex transcription units and identify genes by complementation. Microinjection into the nucleus is the most direct and efficient way of delivering YAC DNA into cells, but requires the purification of the YAC from the remaining yeast chromosomes. Here we describe a detailed method for the isolation of pure, intact and highly concentrated YAC DNA. As a model system the murine tyrosinase gene was chosen and four YACs covering this locus were isolated. Introduction by homologous recombination in yeast of sequences permitting YAC amplification greatly facilitated the isolation of YAC DNA at high concentrations. YAC DNA stabilized in a salt and polyamine containing buffer did not compromise the survival of microinjected oocytes and was suitable for the generation of transgenic mice. Applications and benefits of this technique will be discussed.

Animals↗

PubMed1981

Microinjected Xenopus oocytes synthesize active human plasminogen activator.

Induction of synthesis of the protease plasminogen activator (PA) by hormones, oncogenic viruses and tumor promoters occurs at the transcription level. A novel bioassay for PA messenger RNA was developed to study the regulation of PA synthesis and the genetic elements involved in it. Poly(A)-containing RNA from HEp-3, a PA-rich tumor of human origin, was found to direct the synthesis of a new proteolytic activity when microinjected into Xenopus oocytes. Newly synthesized protease can be detected within a few hours after microinjection of minute quantities of unfractionated mRNA. The new enzymatic activity is indistinguishable from human PA: it is absolutely dependent on human plasminogen; it is neutralized by serum raised against urokinase, the human urinary PA; and it comigrates with urokinase and HEp-3 PA in gel electrophoresis, exhibiting a molecular weight of 60,000.

Animals↗

PubMed2005

Tracking of microinjected DNA in live cells reveals the intracellular behavior and elimination of extrachromosomal genetic material.

We here addressed the basic question, how does extrachromosomal DNA behave when it is placed in the nuclear or the cytoplasmic environment and how is it eliminated? To do this, we tracked microinjected DNA molecules in live cells. In the cytoplasm, the diffusion of microinjected DNA was inhibited in a size- and linearity-dependent manner, probably by the intermediate filament. This was followed by the rapid disappearance of the DNA fluorescent signal. In the nucleus, the diffusion was also dependent on the size of the molecule and was accompanied by the aggregation of the DNA. The aggregation may be due to a putative DNA-binding molecule, whose level is high during the G1 phase. Surprisingly, the injected DNA could move across the nuclear membrane and appeared in the cytoplasm, which suggests the presence of a transport system. The intracytoplasmic behavior and the elimination of such DNA was obviously different from the DNA that was directly injected at the cytoplasm. The DNA remaining in the nucleus appeared to be stable and persisted in the nucleus or, after cell division, in the cytoplasm, for more than one cell cycle. These findings provide a novel and basic understanding of the behavior and elimination of a wide variety of extrachromosomal genetic material.

Active Transport, Cell Nucleus↗

PubMed1996

Centrifugation of bovine oocytes for nuclear micromanipulation and sperm microinjection.

The reproductive biotechnologies of intracytoplasmic sperm microinjection, nuclear transfer and DNA microinjection require the visualization of cytoplasmic components and nuclei of oocytes and early embryonic cells. Bovine oocytes were matured and fertilized in vitro, and then centrifuged at the germinal vesicle, metaphase II and pronuclear stages and at syngamy. These (n = 536) were examined using light and transmission electron microscopy. The organelles stratified in five distinct zones in a consistent pattern in both oocytes and zygotes, though relative fractions changed in organelle composition after fertilization. These comprised a centripetal lipid zone, below which was a vesicular zone, then a supra-equatorial band of smooth endoplasmic reticulum (SER), a clear zone and a centrifugal mitochondrial zone. Cortical granules were located peripherally, single or clumped together, in the clear and mitochondrial zones. The nuclei were usually found associated with the SER or Golgi membranes, and chromatin was clumped at one pole within the nucleus. The maturation spindles were often located beneath the oolemma in all zones, while the first mitotic spindle was usually located in the clear zone. Some of the oocytes were activated by centrifugation and completed maturation.

Animals↗

PubMed1988

Interspecies differences in the stability of mammalian sperm nuclei assessed in vivo by sperm microinjection and in vitro by flow cytometry.

To assess the structural stability of mammalian sperm nuclei and make interspecies comparisons, we microinjected sperm nuclei from six different species into hamster oocytes and monitored the occurrence of sperm nuclear decondensation and male pronucleus formation. The time course of sperm decondensation varied considerably by species: human and mouse sperm nuclei decondensed within 15 to 30 min of injection, and chinchilla and hamster sperm nuclei did so within 45 to 60 min, but bull and rat sperm nuclei remained intact over this same period of time. Male pronuclei formed in oocytes injected with human, mouse, chinchilla, and hamster sperm nuclei, but rarely in oocytes injected with bull or rat sperm nuclei. However, when bull sperm nuclei were pretreated with dithiothreitol (DTT) in vitro to reduce protamine disulfide bonds prior to microinjection, they subsequently decondensed and formed pronuclei in the hamster ooplasm. Condensed rat spermatid nuclei, which lack disulfide bonds, behaved similarly. The same six species of sperm nuclei were induced to undergo decondensation in vitro by treatment with DTT and detergent, and the resulting changes in nuclear size were monitored by phase-contrast microscopy and flow cytometry. As occurred in the oocyte, human sperm nuclei decondensed the fastest in vitro, followed shortly by chinchilla, mouse, and hamster and, after a lag, by rat and bull sperm nuclei. Thus species differences in sperm nuclear stability exist and appear to be related to the extent and/or efficiency of disulfide bonding in the sperm nuclei, a feature that may, in turn, be determined by the type(s) of sperm nuclear protamine(s) present.

Animals↗

PubMed2002

Microinjection of an adenosine A1 agonist into the medial pontine reticular formation increases tail flick latency to thermal stimulation.

BACKGROUND: Both pain and the pharmacologic management of pain can cause the undesirable effect of sleep disruption. One goal of basic and clinical neuroscience is to facilitate rational drug development by identifying the brain regions and neurochemical modulators of sleep and pain. Adenosine is thought to be an endogenous sleep promoting substance and adenosinergic compounds can contribute to pain management. In the pontine brain stem adenosine promotes sleep but the effects of pontine adenosine on pain have not been studied. This study tested the hypothesis that an adenosine agonist would cause antinociception when microinjected into pontine reticular formation regions that regulate sleep. METHODS: The tail flick latency (TFL) test quantified the time in seconds for an animal to move its tail away from a thermal stimulus created by a beam of light. TFL measures were used to evaluate the antinociceptive effects of the adenosine A1 receptor agonist N6-p-sulfophenyladenosine (SPA). Pontine microinjection of SPA (0.1 microg/0.25 microl, 0.88 mm) was followed by TFL measures as a function of time after drug delivery and across the sleep-wake cycle. RESULTS: Compared with saline (control), pontine administration of the adenosine agonist significantly increased latency to tail withdrawal (P < 0.0001). The increase in antinociceptive behavior evoked by the adenosine agonist SPA was blocked by pretreatment with the adenosine A1 receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 0.75 ng/0.25 microl, 10 microm). CONCLUSIONS: These preclinical data encourage additional research on the cellular mechanisms by which adenosine in the pontine reticular formation contributes to the supraspinal modulation of pain.

Analgesics↗

PubMed1993

Analgesic effect of morphine, clonidine and serotonin microinjected into the PTN of rats.

The study was aimed to delineate the neurotransmitter receptors involved in pretectal analgesic mechanisms by direct microinjection of neurotransmitter agonists and antagonists through chronically implanted cannulae in the pretectal nucleus of rats. Morphine, clonidine and serotonin, at doses of 2.5 and 5.0 micrograms microinjected into the pretectal nucleus, produced a significant and prolonged analgesia as measured by the tail-flick test. The analgesia produced by morphine, clonidine and serotonin is significantly attenuated by pretreatment of the animals with naloxone (1 micrograms), yohimbine (5 micrograms) and methysergide (5-10 micrograms) respectively. The results indicate the possible involvement of opioid, adrenergic and serotonergic mechanisms in pretectal analgesia.

Analgesics↗

PubMed1994

Anxiolytic effect of nitric oxide synthase inhibitors microinjected into the dorsal central grey.

Nitric oxide synthase (NOS) accounts for most of the NADPH-diaphorase neuronal activity in the brain. NADPH-diaphorase-positive neurones have been localized at the dorso-lateral part of the periaqueductal grey (PAG), a region related to anxiety. Microinjections of the NOS inhibitors L-NAME (10-200 nmol 0.5 microliter-1) and L-NOARG (10-100 nmol) at this site induced anxiolytic-like effects in the elevated plus maze. These effects, however, occurred only at a limited range of doses and the dose-effect curve had a bell shape, higher doses of both compounds tending to be anxiogenic. The anxiolytic effect of L-NAME was antagonized by a previous microinjection of L-arginine (50 nmol 0.5 microliter-1). These results suggest that NO may play a role in PAG neurotransmission involved in anxiety.

Amino Acid Oxidoreductases↗