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The compartmentalization of protein synthesis: importance of cytoskeleton and role in mRNA targeting.

Following the synthesis of mRNA molecules in eukaryotic cells, the transcripts are processed in the nucleus and subsequently transported through the nuclear membrane into the cytoplasm before being sequestered into polysomes where the information contained in the RNA molecule is translated into an amino acid sequence. Recent evidence suggests that an association of mRNAs with the cytoskeleton might be important in targeting mechanisms and, furthermore, in the transport of mRNA from the nucleus to its correct location in the cytoplasm. Until recently, polysomes have been considered to exist in two classes, namely free or membrane-bound. There is now compelling evidence, however, that ribosomes, in addition to being associated with endoplasmic reticulum membranes, also are associated with components of the cytoskeleton. Thus, a large number of morphological and biochemical studies have shown that mRNA, polysomes and translational factors are associated with cytoskeletal structures. Although the actual nature and significance of the interaction between components of the translational apparatus and the cytoskeleton is not yet understood in detail, it would seem evident that such interactions are important in both the spatial organization and control of protein synthesis. Recent work has shown that a subcellular fraction, enriched in cytoskeletal components, contains polysomes and these (cytoskeletal-bound) polysomes have been shown to contain specific mRNA species. Thus, a population of cytoskeletal-bound polysomes may provide a specialized mechanism for the sorting, targeting and topographical segregation of mRNAs. In this review, current knowledge of the subcellular compartmentalization of mRNAs is discussed.

Animals↗

Influence of compartmental localization on the function of yeast NADP+-specific isocitrate dehydrogenases.

Three differentially compartmentalized isozymes of isocitrate dehydrogenase (mitochondrial IDP1, cytosolic IDP2, and peroxisomal IDP3) in the yeast Saccharomyces cerevisiae catalyze the NADP(+)-dependent oxidative decarboxylation of isocitrate to form alpha-ketoglutarate. These enzymes are highly homologous but exhibit some significant differences in physical and kinetic properties. To examine the impact of these differences on physiological function, we exchanged promoters and altered organellar targeting information to obtain expression of IDP2 and IDP3 in mitochondria and of IDP1 and IDP3 in the cytosol. Physiological function was assessed as complementation by mislocalized isozymes of defined growth defects of isocitrate dehydrogenase mutant strains. These studies revealed that the IDP isozymes are functionally interchangeable for glutamate synthesis, although mitochondrial localization has a positive impact on this function during fermentative growth. However, IDP2, whether located in mitochondria or in the cytosol, provided the highest level of defense against endogenous or exogenous oxidative stress.

Cell Compartmentation↗

Compartmentalization of NO signaling cascade in skeletal muscles.

Skeletal muscle functions regulated by NO are now firmly established. However, the literature on the compartmentalization of NO signaling in myocytes is highly controversial. To address this issue, we examined localization of enzymes engaged in L-arginine-NO-cGMP signaling in the rat quadriceps muscle. Employing immunocytochemical labeling complemented with tyramide signal amplification and electron microscopy, we found NO synthase expressed not only in the sarcolemma, but also along contractile fibers, in the sarcoplasmic reticulum and mitochondria. The expression pattern of NO synthase in myocytes showed striking parallels with the enzymes engaged in L-arginine-NO-cGMP signaling (arginase, phosphodiesterase, and soluble guanylyl cyclase). Our findings are indicative of an autocrine fashion of NO signaling in skeletal muscles at both cellular and subcellular levels, and challenge the notion that the NO generation is restricted to the sarcolemma.

Animals↗

Endoplasmic reticulum: one continuous network compartmentalized by extrinsic cues.

The endoplasmic reticulum (ER) is an extensive three-dimensional network that stretches from the inner nuclear envelope to the cell cortex with a single, continuous membrane and a single, continuous lumen. Yet the ER contains specialized regions that carry out unique functions. The question that immediately arises is how the ER can be compartmentalized if it is continuous, and the answer to this is that cellular landmarks with unique sub-cellular distributions impose non-uniformity on the ER from outside, creating structural and functional sub-domains of the ER.

Animals↗

Compartmentalized PKA signaling events are required for synaptic tagging and capture during hippocampal late-phase long-term potentiation.

Synaptic plasticity, the activity-dependent change in the strength of neuronal connections, is a proposed cellular mechanism of memory storage that is critically regulated by protein kinases such as cAMP-dependent protein kinase (PKA). Despite the fact that a neuron contains thousands of synapses, the expression of synaptic plasticity can be specific to subsets of synapses. This is surprising because signal transduction pathways underlying synaptic plasticity involve diffusible second messenger molecules such as cAMP and diffusible proteins such as the catalytic subunit of PKA. One way in which this specificity can be achieved is by the localization of signal transduction molecules to specific subcellular domains. Spatial compartmentalization of PKA signaling is achieved via binding to A kinase-anchoring proteins (AKAPs). We report here that pharmacological inhibition of PKA anchoring impairs synaptically activated late-phase long-term potentiation (L-LTP) in hippocampal slices. In contrast, potentiation that is induced by the pharmacological activation of the cAMP/PKA pathway, which can potentially affect all synapses within the neuron, is not impaired by inhibition of PKA anchoring. These results suggest that PKA anchoring may be particularly important for events that occur at the synapse during the induction of L-LTP, such as synaptic tagging and capture. Indeed, our results demonstrate that blocking PKA anchoring impairs synaptic tagging and capture. Thus our data highlight the idea that PKA anchoring may allow for specific populations of synapses to change in synaptic strength in the face of plasticity-related transcription that is cell-wide.

Adaptor Proteins, Signal Transducing↗

Compartmentalization of prokaryotic DNA replication.

It becomes now apparent that prokaryotic DNA replication takes place at specific intracellular locations. Early studies indicated that chromosomal DNA replication, as well as plasmid and viral DNA replication, occurs in close association with the bacterial membrane. Moreover, over the last several years, it has been shown that some replication proteins and specific DNA sequences are localized to particular subcellular regions in bacteria, supporting the existence of replication compartments. Although the mechanisms underlying compartmentalization of prokaryotic DNA replication are largely unknown, the docking of replication factors to large organizing structures may be important for the assembly of active replication complexes. In this article, we review the current state of this subject in two bacterial species, Escherichia coli and Bacillus subtilis, focusing our attention in both chromosomal and extrachromosomal DNA replication. A comparison with eukaryotic systems is also presented.

Bacillus subtilis↗

Modelling the compartmentalization of splicing factors.

Splicing factor (SF) compartments, also known as speckles, are heterogeneously distributed compartments within the nucleus of eukaryotic cells that are enriched in pre-mRNA SFs. We derive a fourth-order aggregation-diffusion model that describes a possible mechanism underlying the organization of SFs into speckles. The model incorporates two hypotheses, namely (1) that self-organization of dephosphorylated SFs, modulated by a phosphorylation-dephosphorylation cycle, is responsible for the formation and disappearance of speckles, and (2) that an underlying nuclear structure plays a major role in the organization of SFs. A linear stability analysis about homogeneous steady-state solutions of the model reveals how the self-interaction among dephosphorylated SFs can result in the onset of spatial patterns. A detailed bifurcation analysis of the model describes how phosphorylation and dephosphorylation modulate the onset of the compartmentalization of SFs.

Animals↗

GnRH receptor signalling to ERK: kinetics and compartmentalization.

Many hormones, neurotransmitters and growth factors influence their target cells by activation of mitogen-activated protein kinase cascades. The consequences of such activation reflect not only the magnitude, but also the kinetics and cellular compartmentalization of kinase activity. Gonadotropin-releasing hormone (GnRH) receptors are seven-transmembrane receptors that have undergone a period of rapidly accelerated molecular evolution in which the advent of type I mammalian GnRH receptors has been associated with the loss of the carboxyl-terminal tail, a structure present in all other seven-transmembrane receptors. Here, we review spatiotemporal aspects of extracellular-signal-regulated kinase activation by gonadotropin-releasing hormone receptors, emphasizing how the absence or presence of the carboxyl-terminal tail dictates the receptors' ability to engage and signal via arrestins.

Animals↗

Novel proteins in emulsions using in vitro compartmentalization.

IVC (in vitro compartmentalization) provides a complete cell-free approach for the production of novel targeted proteins. IVC uses aqueous droplets, which contain DNA and components for protein production, within water-in-oil emulsions. Recent advances in the composition and formation, as well as the detection, sorting and recovery, of the droplets enable the evolution of the encoded protein. Furthermore, IVC technology permits the step-wise addition of reagents into the droplets, making them suitable for high-throughput applications - where synthetic enzymes with substrate specificity are selected for catalytic activity, binding and regulation. In the broad field of in vitro display, developments such as the incorporation of unnatural amino acids and the production of cell toxic proteins expand the diverse spectrum of future applications for IVC.

Biomimetics↗

Compartmental inhibition of hepatic glutathione reductase activities by 1,3-bis(2-chloroethyl)-N-nitrosourea (BCNU) in Sprague-Dawley and Fischer-344 rats.

Administration of 1,3-bis(2-chloroethyl)-N-nitrosourea (BCNU) inhibits hepatic activities of glutathione reductase (GR) comparably in both adult male Fischer-344 (F344) and Sprague-Dawley (SD) rats in vivo. BCNU pretreatment greatly exacerbates the hepatic necrosis caused by diquat in F344 rats, but does not similarly potentiate liver injury in SD rats. The primary purpose of the present studies was to test the hypothesis that BCNU pretreatment would exhibit differences between the two strains in inhibition of GR activities in hepatic subcellular compartments that would correlate with the differing effects on diquat-induced hepatic necrosis. In the present studies, 16 h after administration of 80 mg/kg of BCNU, GR activities in the hepatic homogenates were 20-30% of activities in vehicle-treated controls. Neither the extents of inhibition nor the GR activities in hepatic cytosol, microsomes. mitochondria, or purified nuclei isolated by differential centrifugation were different between SD and F344 rats treated with BCNU. The results indicate that differences between SD and F344 rats in the effects of BCNU on susceptibilities to diquat-induced hepatic necrosis are not readily attributable to compartmentally selective inhibition of GR. In addition, hepatic O6-alkylguanine-DNA alkyltransferase (AGT, MGMT) levels were almost completely depleted in BCNU-treated rats of both strains, thus indicating that MGMT-dependent pathways are unlikely to be critical determinants of the effects of BCNU on this model of acute cell death mediated by reactive oxygen species in vivo.

Animals↗

Separate pathways of RNA recruitment lead to the compartmentalization of the zebrafish germ plasm.

The maternal RNAs vasa, dead end, nanos1, and daz-like all become localized to the peripheral ends of the first and second cleavage furrows, where they form part of the zebrafish germ plasm. We show that aggregates of a first class of germ plasm components, which include dead end, nanos1, and vasa RNAs, are initially present in a wide cortical band at the animal pole. Aggregates containing these three RNAs appear to be associated with f-actin, which during the first cell cycle undergoes a microtubule-dependent movement towards the periphery as well as circumferential alignment. These cytoskeletal rearrangements lead to the further aggregation of particles containing these RNAs and their concomitant recruitment to the forming furrow. Aggregates containing a second class of germ plasm RNA components, which include the transcript for daz-like, translocate along the plane of the cortex towards the animal pole, where they are recruited to the germ plasm. After recruitment to the furrow, these two classes of RNAs occupy overlapping yet distinct regions of the germ plasm, and this arrangement is maintained during the early cleavage stages. Our observations suggest that separate pathways of RNA recruitment facilitate the compartmentalization of the zebrafish germ plasm.

Actins↗

Intracellular compartmentalization of leukotriene synthesis: unexpected nuclear secrets.

Leukotrienes are important lipid mediators implicated in the regulation of various cellular processes and in disease states as well as homeostasis. Regulation of leukotriene biosynthesis is therefore of considerable interest. Although the levels of expression and catalytic activity of leukotriene-forming proteins have long been recognized as important determinants of leukotriene biosynthesis, it has recently become apparent that their intracellular compartmentalization also affects the integrated output of this biosynthetic pathway. In this minireview, we focus on the unexpected discovery that the nucleus is the key intracellular site for leukotriene biosynthesis and discuss the mechanisms that regulate protein localization and the potential implications of these findings.

Animals↗

Calcium compartmentation in cardiac tissue culture: the effects of extracellular sodium depletion.

The effect of extracellular sodium depletion upon cellular calcium distribution in myocardial tissue culture is studied with the use of sucrose or lithium chloride as substitutes for sodium. With sucrose substitution more than 50% of the increase in calcium at the cellular surface secondary to [Na]0 depletion is probably localized to a screening layer with the remainder bound to the sarcolemma of the cell. Calcium distribution (sucrose substituted) is studied under three different perfusion conditions: (1) In HEPES-buffered perfusate (pH = 7.1-7.35) the 113% gain in Ca at the cellular surface upon 33 mM [Na]0 perfusion is rapidly exchangeable, completely lanthanum (La) displaceable and more than 90% reversible. 5 X 10(-5) verapamil does not affect the response. (2) In 10 mM Pi (phosphate) solution at pH = 7.15, in which a slowly exchangeable mitochondrial Ca compartment is added, the gain in Ca after low [Na]0 is again rapidly exchangeable and 85% reversible that is, similar to the response in HEPES-buffered solution. (3) In 10 mM Pi solution at pH = 7.35 in which slow phase calcium uptake in 133 mM [Na]0 is increased, the initial rapid Ca response to low [Na]0 is similar to that in (1) and (2) but the slow phase uptake rate is further increased by 3.75 times. The major portion of this uptake is not reversible upon return to 133 mM [Na]0. The results distinguish two markedly different effects of [Na]0 depletion in tissue culture dependent upon the pre Na-depletion state of Ca compartmentation: (1) Addition of almost all the calcium to rapidly exchangeable sites at the cellular surface when exchange is limited to these sites and to mitochondria. (2) Further addition of calcium to more slowly exchangeable cellular site(s) when slow phase uptake is increased, by elevation of Pi at pH = 7.35, prior to low [Na]0 perfusion.

Animals↗

Muscle fiber type compartmentalization and expression of an immature myosin isoform in the sternocleidomastoid muscle of rabbits and primates.

The sternocleidomastoid muscle is located in the neck and is both a neck rotator and flexor. Cervical dystonia, a focal dystonia disorder, is characterized by forceful involuntary contraction of a group of neck muscles, usually including the sternocleidomastoid. Little is known about the fiber type composition, fiber type compartmentalization and innervation patterns in this muscle in rabbit and primates. Sternocleidomastoid muscles from rabbit and monkey were analyzed for muscle fiber type composition and number, muscle fiber cross-sectional area and patterns of innervation. The sternocleidomastoid muscle was composed of two distinct regions, or compartments, with different fiber type compositions: an outer or superficial region composed of mostly type 2 myofibers and an inner deep region composed of both type 2 and type 1 myofibers. Neonatal myosin heavy chain isoform was detected in approximately 25% of the myofibers in both regions of the muscle. Neuromuscular junctions were located in seven endplate bands approximately 1-3 cm apart throughout the length of the muscle. There is clear evidence of anatomical subdivisions within this muscle. Not only is there variation in fiber type composition between superficial and deep regions of the muscle, but unlike most other mature skeletal muscles, it continues to express neonatal myosin heavy chain isoform in the adult. The motor program for neck movements is extremely complex, and the histological complexity plays a role in allowing for a continuum of movements of the head and neck, from maintenance of posture to rapid head movements.

Adenosine Triphosphatases↗

Compartmentalization of steroidogen esis by the equine corpus luteum.

The presence of cytochrome P450C17 within equine follicles and corpora lutea (CL) was detected by immunostaining. Two different antibodies were used which had previously been shown by immunoblotting to cross-react with equine P450C17. Strong positive immunostaining was present in the theca-derived cells of the CL during the estrous cycle and pregnancy. In the CL from mares after Day 40 of pregnancy there were also occasional bands of positively stained cells which resembled the polyhedral-shaped theca cells seen in preovulatory follicles. The pattern of immunostaining suggested compartmentalization of steroidogenesis within the equine CL with small cells possessing the potential to produce androgen which could then be aromatized to estrogen by the large luteal cells.

Animals↗

Compartmentation of metabolism probed by [2-13C]alanine: improved 13C NMR sensitivity using a CryoProbe detects evidence of a glial metabolon.

The labelling of metabolites with the NMR active nucleus 13C allows not only metabolite enrichments to be monitored, but also the relative fluxes through competing pathways to be delineated. [2-13C, 15N]alanine was used as a metabolic probe to investigate compartmentation in superfused cerebral slices. Perchloric acid extracts of the tissue were investigated using 13C NMR spectroscopy. The spectra were obtained using a CryoProbe optimised for 13C detection (dual CryoProbe [13C, 1H]) in which the receiver and transmitter coils are cooled to approximately 20K to reduce contributions to noise in the signal obtained. Compared with conventional inverse geometry probe, the signal-to-noise ratio (S/N) was increased by approximately 17-fold using this device. A large proportion of alanine was initially metabolised over the first 20 min by glial cells, as indicated by the relative importance of the glial, only enzyme pyruvate carboxylase to the labelling pattern of glutamate, with the ratio of pyruvate carboxylase to pyruvate dehydrogenase derived glutamate being 0.25, and exported [2-13C, 15N]aspartate. Using the increased sensitivity of the CryoProbe, [2-13C, 15N]aspartate was also detected in the extracts of cerebral tissue. This metabolite could only have been derived via the pyruvate carboxylase pathway, and given the large export of the metabolite into the superfusion buffer suggests the occurrence of a "metabolon" arrangement of enzymes within glial cells.

Alanine↗

Compartmentalization of secretory proteins in pancreatic zymogen granules as revealed by immunolabeling on cryo-fixed and molecular distillation processed tissue.

Immunogold labeling of amylase obtained over zymogen granules of rat pancreatic acinar cells processed through cryofixation, molecular distillation drying and embedding in resins was found to be of high intensity and displayed a particular pattern. Indeed, it was concentrated in certain areas of the granules leaving others devoid of gold particles. This pattern of labeling reflects a strong compartmentalization of the secretory proteins within each granule. In order to assess this phenomenon, we have compared the intensities and the pattern of distribution of the labelings in tissues processed through: chemical fixation with embedding in various resins, cryo-ultramicrotomy and cryo-fixation followed by molecular distillation drying. Serials sections and double labeling experiments were performed for further evaluation of the results and for assessing artefactual displacement of proteins during tissue preparation. The results obtained indicate that the secretory proteins are indeed segregated within the granule which appears thus as a well organized structure. Cryo-fixation combined with molecular distillation appears thus to be superior in terms of preservation of protein antigenicity and retention of cellular components close to their living state.

Amylases↗

Compartmentalized vesicular traffic around the hair cell cuticular plate.

Through thin-section and freeze-fracture electron microscopy, we identify structural correlates of an intense vesicular traffic in a narrow band of cytoplasm around the cuticular plate of the bullfrog vestibular hair cells. Myriads of coated and uncoated vesicles associated with longitudinally oriented microtubules populate the narrow cytoplasmic region between the cuticular plate and the actin network of the apical junctional belt. If microtubules in the sensory hair cells, like those in axons, are pathways for organelle transport, then the characteristic distribution of microtubules around the cuticular plate represents transport pathways across the apical region of the hair cells. This compartmentalized membrane traffic system appears to support an intense vesicular release and uptake along a band of apical plasma membrane near the cell border. Functions of this transport system may include membrane recycling as well as exocytotic and endocytotic exchange between the hair cell cytoplasm and the endolymphatic compartment.

Acoustic Maculae↗