Intracellular trafficking of hnRNP A2 in oligodendrocytes.

Heterogeneous ribonucleoprotein (hnRNP) A2 is a trans-acting factor that mediates intracellular trafficking of specific RNAs containing the A2 response element. HnRNP A2 is localized in the nucleus and also in granules in the perikaryon and processes in oligodendrocytes. The distribution of the cytoplasmic pool of hnRNP A2 is microtubule-dependent. HnRNP A2 is composed of two sequential RNA binding domains (RBDI and RBDII), a glycine-rich domain, and a nuclear import domain (M9). In order to analyze the roles of individual domains in determining the intracellular distribution of hnRNP A2, chimeric mRNAs encoding various domains fused with green fluorescent protein (GFP) were injected into oligodendrocytes, and the subcellular distribution of the GFP hybrid proteins was analyzed by fluorescence microscopy. Chimeric GFP proteins containing the M9 domain were localized to the nucleus. In the absence of the M9 domain, proteins containing the RBDII domain were preferentially concentrated in the distal processes of the cells. Localization of RBDII-containing proteins in the periphery was dependent on the presence of intact microtubules. These data suggest that the RBDII domain of hnRNP A2 targets hnRNP A2 to the periphery of the cell in a microtubule-dependent manner.

The balance of power in RNA trafficking.

In the past two years, several different RNA trafficking pathways have been characterized in oligodendrocytes; similar trafficking pathways have been discovered in neuronal and retroviral systems; co-assembly of multiple different RNAs into the same granules has been analyzed as a mechanism for coordinating gene expression; and a new hypothesis for RNA trafficking, based on the balance of power between kinesin and dynein in individual RNA granules, has been proposed.

Computational cell biologists snowed in at Cranwell.

Cell biology is being inundated by an avalanche of data from the genomics and proteomics enterprises. The complexity and sheer volume of information threaten to overwhelm the ability of traditional cell biologists to grasp its implications and develop experimentally testable hypotheses. For this reason, some have begun to explore computational approaches towards organizing complex data into quantitative models. This requires communication and collaboration between the biological science community and and the physical and mathematical sciences communities. A recent meeting [The First International Symposium on Computational Cell Biology, Cranwell Resort, Lenox, MA, USA; 4-6 March 2001. Organizers: J.H. Carson, A. Cowan, and L.M. Loew (www.nrcam.uchc.edu/conference).] made a first attempt to bring these two communities together. Three feet of new snow fell during the meeting, but the 125 attendees, an unusual mixture of cell biologists, computer scientists, mathematicians, physicists, and engineers, were having too much fun defining the new field of computational cell biology to notice that they were literally snowed in.

RNA trafficking in oligodendrocytes.

A2RE and hnRNP A2 have been identified as important cis/trans determinants for MBP RNA trafficking in oligodendrocytes. Since A2RE-like sequences are found in several different transported RNAs, and since hnRNP A2 is expressed in most cell types, this may represent a general RNA trafficking pathway shared by a variety of different RNAs in different cell types. In oligodendrocytes, A2RE/hnRNP A2 determinants are involved in at least four steps in the RNA trafficking pathway: (1) export from the nucleus to the cytoplasm, (2) granule assembly in the perikaryon, (3) transport along microtubules in the processes, and (4) translation activation in the myelin compartment. The components of the cellular machinery mediating each of these steps are known. How A2RE/hnRNP A2 determinants interact with these components to mediate RNA trafficking is being investigated by a combination of: biochemistry to analyze molecular interactions in vitro, imaging to visualize molecular interactions in living cells, and computational modeling to simulate molecular interactions in the Virtual Cell.

RNA trafficking signals in human immunodeficiency virus type 1.

Intracellular trafficking of retroviral RNAs is a potential mechanism to target viral gene expression to specific regions of infected cells. Here we show that the human immunodeficiency virus type 1 (HIV-1) genome contains two sequences similar to the hnRNP A2 response element (A2RE), a cis-acting RNA trafficking sequence that binds to the trans-acting trafficking factor, hnRNP A2, and mediates a specific RNA trafficking pathway characterized extensively in oligodendrocytes. The two HIV-1 sequences, designated A2RE-1, within the major homology region of the gag gene, and A2RE-2, in a region of overlap between the vpr and tat genes, both bind to hnRNP A2 in vitro and are necessary and sufficient for RNA transport in oligodendrocytes in vivo. A single base change (A8G) in either sequence reduces hnRNP A2 binding and, in the case of A2RE-2, inhibits RNA transport. A2RE-mediated RNA transport is microtubule and hnRNP A2 dependent. Differentially labelled gag and vpr RNAs, containing A2RE-1 and A2RE-2, respectively, coassemble into the same RNA trafficking granules and are cotransported to the periphery of the cell. tat RNA, although it contains A2RE-2, is not transported as efficiently as vpr RNA. An A2RE/hnRNP A2-mediated trafficking pathway for HIV RNA is proposed, and the role of RNA trafficking in targeting HIV gene expression is discussed.

Mutational analysis of a heterogeneous nuclear ribonucleoprotein A2 response element for RNA trafficking.

Cytoplasmic transport and localization of mRNA has been reported for a range of oocytes and somatic cells. The heterogeneous nuclear ribonucleoprotein (hnRNP) A2 response element (A2RE) is a 21-nucleotide segment of the myelin basic protein mRNA that is necessary and sufficient for cytoplasmic transport of this message in oligodendrocytes. The predominant A2RE-binding protein in rat brain has previously been identified as hnRNP A2. Here we report that an 11-nucleotide subsegment of the A2RE (A2RE11) was as effective as the full-length A2RE in binding hnRNP A2 and mediating transport of heterologous RNA in oligodendrocytes. Point mutations of the A2RE11 that eliminated binding to hnRNP A2 also markedly reduced the ability of these oligoribonucleotides to support RNA transport. Oligodendrocytes treated with antisense oligonucleotides directed against the translation start site of hnRNP A2 had reduced levels of this protein and disrupted transport of microinjected myelin basic protein RNA. Several A2RE-like sequences from localized neuronal RNAs also bound hnRNP A2 and promoted RNA transport in oligodendrocytes. These data demonstrate the specificity of A2RE recognition by hnRNP A2, provide direct evidence for the involvement of hnRNP A2 in cytoplasmic RNA transport, and suggest that this protein may interact with a wide variety of localized messages that possess A2RE-like sequences.

The cis-acting RNA trafficking signal from myelin basic protein mRNA and its cognate trans-acting ligand hnRNP A2 enhance cap-dependent translation.

The 21 nucleotide RNA trafficking signal (RTS), originally identified in myelin basic protein mRNA, but also found in a variety of other localized RNAs, is necessary and sufficient for transport of RNA along microtubules in oligodendrocytes. The RTS binds specifically to the RNA binding protein, hnRNP A2. Together, the RTS and hnRNP A2 comprise cis/trans determinants for several steps in the RNA trafficking pathway. Here we show that insertion of the RTS into green fluorescent protein (GFP) RNA enhances translation without affecting stability of microinjected RNA. In dicistronic RNA, the RTS enhances cap-dependent translation without affecting internal ribosome entry site (IRES)-dependent translation. The translation enhancer function of the RTS is position, copy number, and cell type independent, hnRNP A2 dependent, and saturable with increasing amounts of injected RNA. This represents one of the first specific translation enhancer elements identified in a mammalian system.

RNA on the road to myelin.

In oligodendrocytes some mRNAs are transported from the perikaryon to the distal processes and localized in the myelin compartment where they are translated. This review describes the cis-acting signals and trans-acting factors that mediate intracellular trafficking of myelin basic protein (MBP) RNA, the prototype for such mRNAs in myelinating glia.

Fluorescence quenching and dequenching analysis of RNA interactions in vitro and in vivo.

This paper describes the use of fluorescence quenching and dequenching to analyze molecular interactions of RNA in vitro and in vivo. Fluorescein-labeled ribonucleotide was incorporated into an RNA substrate by in vitro transcription. The fluorescence quantum yield of the intact RNA was reduced by intramolecular quenching. When the RNA was degraded by ribonuclease digestion, the quantum yield increased by approximately 50%, reflecting dequenching due to separation of proximate fluorophores. Dequenching was dependent on the concentration of enzyme and substrate and was inhibited by the ribonuclease inhibitor RNasin. Comparable rates of dequenching were observed with RNase A and RNase T1. Dequenching provides a sensitive, quantitative, and convenient assay for RNA degradation. When fluorescent RNA was microinjected into cells in culture the intracellular fluorescence declined gradually with time after injection reflecting "superquenching: due to intermolecular interactions between the injected RNA and intracellular components. Capped RNA exhibited greater superquenching than uncapped RNA. Superquenching provides a sensitive, quantitative, and specific assay with subcellular resolution for intermolecular interactions of RNA in vivo. When RNase was injected into the same cells, fluorescence increased by approximately 50%, indicating that fluorescence dequenching due to RNA degradation can be measured in vivo as well as in vitro.

RNA trafficking in myelinating cells.

In the past year, several key molecular components of the RNA trafficking pathway in myelinating cells have been identified: distinct cis-acting elements for RNA transport and localization have been characterized in myelin basic protein mRNA; hnRNP A2 has been identified as a trans-acting factor in oligodendrocytes that binds specifically to the RNA transport sequence; and microtubules and kinesin have been identified as cytoskeletal elements required for RNA transport in oligodendrocytes.

hnRNP A2 selectively binds the cytoplasmic transport sequence of myelin basic protein mRNA.

Segregation of mRNAs in the cytoplasm of polar cells has been demonstrated for proteins involved in Xenopus and Drosophila oogenesis, and for some proteins in somatic cells. It is assumed that vectorial transport of the messages is generally responsible for this localization. The mRNA encoding the basic protein of central nervous system myelin is selectively transported to the distal ends of the processes of oligodendrocytes, where it is anchored to the myelin membrane and translated. This transport is dependent on a 21-nucleotide cis-acting segment of the 3'-untranslated region (RTS). Proteins that bind to this cis-acting segment have now been isolated from extracts of rat brain. A group of six 35-42-kDa proteins bind to a 35-base oligoribonucleotide incorporating the RTS, but not to several oligoribonucleotides with the same composition but randomized sequences, thus establishing specificity for the base sequence in the RTS. The most abundant of these proteins has been identified, by Edman sequencing of tryptic peptides and mass spectroscopy, as heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a 36-kDa member of a family of proteins that are primarily, but not solely, intranuclear. This protein was most abundant in samples from rat brain and testis, with lower amounts in other tissues. It was separated from the other polypeptides by using reverse-phase HPLC and shown to retain preferential association with the RTS. In cultured oligodendrocytes, hnRNP A2 was demonstrated by confocal microscopy to be distributed throughout the nucleus, cell soma, and processes.

Transport and localization elements in myelin basic protein mRNA.

Myelin basic protein (MBP) mRNA is localized to myelin produced by oligodendrocytes of the central nervous system. MBP mRNA microinjected into oligodendrocytes in primary culture is assembled into granules in the perikaryon, transported along the processes, and localized to the myelin compartment. In this work, microinjection of various deleted and chimeric RNAs was used to delineate regions in MBP mRNA that are required for transport and localization in oligodendrocytes. The results indicate that transport requires a 21-nucleotide sequence, termed the RNA transport signal (RTS), in the 3' UTR of MBP mRNA. Homologous sequences are present in several other localized mRNAs, suggesting that the RTS represents a general transport signal in a variety of different cell types. Insertion of the RTS from MBP mRNA into nontransported mRNAs, causes the RNA to be transported to the oligodendrocyte processes. Localization of mRNA to the myelin compartment requires an additional element, termed the RNA localization region (RLR), contained between nucleotide 1,130 and 1, 473 in the 3' UTR of MBP mRNA. Computer analysis predicts that this region contains a stable secondary structure. If the coding region of the mRNA is deleted, the RLR is no longer required for localization, and the region between nucleotide 667 and 953, containing the RTS, is sufficient for both RNA transport and localization. Thus, localization of coding RNA is RLR dependent, and localization of noncoding RNA is RLR independent, suggesting that they are localized by different pathways.

A general computational framework for modeling cellular structure and function.

The "Virtual Cell" provides a general system for testing cell biological mechanisms and creates a framework for encapsulating the burgeoning knowledge base comprising the distribution and dynamics of intracellular biochemical processes. It approaches the problem by associating biochemical and electrophysiological data describing individual reactions with experimental microscopic image data describing their subcellular localizations. Individual processes are collected within a physical and computational infrastructure that accommodates any molecular mechanism expressible as rate equations or membrane fluxes. An illustration of the method is provided by a dynamic simulation of IP3-mediated Ca2+ release from endoplasmic reticulum in a neuronal cell. The results can be directly compared to experimental observations and provide insight into the role of experimentally inaccessible components of the overall mechanism.

Rearrangement and reactivation of the myelin basic protein locus in myelin-deficient (mld) mouse brain.

Myelin-deficient (mld) is a complex mutation affecting the myelin basic protein (MBP) locus of the mouse. It consists of duplication and partial inversion of the MBP gene and results in a dysfunctional MBP locus. The mutant phenotype is reversed, both in vivo and in vitro, in approximately 5% of mld oligodendrocytes. One possible mechanism for the somatic reversion is recombination between homologous sequences of the duplicated gene copies to reconstitute a functional MBP locus. There are several possible recombination events that could reconstitute a functional MBP locus by DNA rearrangement. Two of these would result in reinversion and circularization of specific MBP gene sequences, respectively. In this work polymerase chain reaction analysis was used to detect both reinverted and circularized MBP gene sequences in mld mouse tissues, indicating that DNA rearrangement at the MBP locus does occur. Analysis of individually harvested cells showed that in revertant MBP-positive mld oligodendrocytes DNA rearrangement at the MBP locus was correlated with reactivation of the MBP gene. Fluctuation analysis showed that reactivation of the MBP locus is a stochastic event occurring with a frequency of approximately 1.4 x 10(-6) per cell per cell cycle during oligodendrocyte development. The frequency of rearrangement and reactivation of the MBP locus was comparable in double mutant (mld/mld, scid/scid) and single mutant (mld/mld, +scld/+scld) mice, indicating that the scid factor is not required for MBP gene reactivation in mld. The significance of DNA rearrangement in mammalian development is discussed.

Translocation of myelin basic protein mRNA in oligodendrocytes requires microtubules and kinesin.

Myelin basic protein (MBP) mRNA is localized to the myelin membranes of oligodendrocytes. When exogenous MBP mRNA is microinjected into oligodendrocytes in culture, it is transported along the processes and localized to the myelin compartment in a multistep intracellular RNA trafficking pathway. In the work described here, oligodendrocytes were treated with agents that affect the cytoskeleton including: nocodazole, to disrupt microtubules; taxol, to stabilize microtubules; cytochalasin, to disrupt microfilaments; and kinesin anti-sense oligonucleotide, to suppress kinesin expression. Digoxigenin-labeled MBP mRNA was microinjected into the treated cells and the extent of translocation of the microinjected RNA was determined by confocal microscopy. Nocodazole, taxol, and kinesin anti-sense oligonucleotide inhibited translocation of microinjected MBP mRNA, while cytochalasin B and kinesin sense oligonucleotide did not. These results indicate that translocation of MBP mRNA in oligodendrocytes requires intact microtubules and kinesin but does not require intact microfilaments. The results are discussed in relation to the current multistep model for intracellular RNA trafficking in oligodendrocytes.

Localization of histidyl-tRNA synthetase (Jo-1) in human laryngeal epithelial carcinoma cell line (HEp-2 cells).

The present study was designed to determine the subcellular localization of histidyl-tRNA synthetase (Jo-1) in human laryngeal epithelial carcinoma cell line (HEp-2 cells). Indirect immunofluorescence using commercial HEp-2 cells with human serum and human-affinity-purified anti-Jo-1 antibodies was performed using confocal microscopy. Anti-histidyl-tRNA-synthetase-positive sera showed distinct nuclear and cytoplasmic granular staining in HEp-2 cells. Affinity purified anti-Jo-1 produced an identical pattern to the whole serum, whereas the serum fraction that did not bind to the affinity column was negative by immunofluorescence on HEp-2 cells. Two commercial human anti-Jo-1-positive control sera and seven anti-Jo-1-positive sera from patients with myositis reproduced the nuclear and cytoplasmic granular pattern. We conclude that Jo-1 is present in cytoplasm and in intact nuclei from HEp-2 cells. The presence of tRNA synthetases in intact nuclei suggests that they have an unsuspected function in the nucleus.

Brain cytoplasmic and flagellar outer arm dyneins share a highly conserved Mr 8,000 light chain.

Sequence comparisons with the Mr 8,000 light chain from Chlamydomonas outer arm dynein revealed the presence of highly conserved homologues (up to 90% identity) in the expressed sequence tag data base (King, S. M. & Patel-King, R. S. (1995a) J. Biol. Chem. 270, 11445-11452). Several of these homologous sequences were derived from organisms and/or tissues that lack motile cilia/flagella, suggesting that these proteins may function in the cytoplasm. In Drosophila, lack of the homologous protein results in embryonic lethality (Dick, T., Ray, K., Salz, H. K. & Chia, W.(1996) Mol. Cell. Biol., 16, 1966-1977). Fractionation of mammalian brain homogenates reveals three distinct cytosolic pools of the homologous protein, one of which specifically copurifies with cytoplasmic dynein following both ATP-sensitive microtubule affinity/sucrose density gradient centrifugation and immunoprecipitation with a monoclonal antibody specific for the 74-kDa intermediate chain (IC74). Quantitative densitometry indicates that there is one copy of the Mr 8,000 polypeptide per IC74. Dual channel confocal immunofluorescent microscopy revealed that the Mr 8,000 protein is significantly colocalized with cytoplasmic dynein but not with kinesin in punctate structures (many of which are associated with microtubules) within mammalian oligodendrocytes. Thus, it appears that flagellar outer arm and brain cytoplasmic dyneins share a highly conserved light chain polypeptide that, at least in Drosophila, is essential for viability.

Protein translation components are colocalized in granules in oligodendrocytes.

The intracellular distribution of various components of the protein translational machinery was visualized in mouse oligodendrocytes in culture using high resolution fluorescence in situ hybridization and immunofluorescence in conjunction with dual channel confocal laser scanning microscopy. Arginyl-tRNA synthetase, elongation factor 1a, ribosomal RNA, and myelin basic protein mRNA were all co-localized in granules in the processes, veins and membrane sheets of the cell. Colocalization was evaluated by dual channel cross correlation analysis to determine the correlation index (% colocalization) and correlation distance (granule radius), and by single granule ratiometric analysis to determine the distribution of the different components in individual granules. Most granules contained synthetase, elongation factor, ribosomal RNA and myelin basic protein mRNA. These results indicate that several different components of the protein synthetic machinery, including aminoacyl-tRNA synthetases, elongation factors, ribosomes and mRNAs, are colocalized in granules in oligodendrocytes. We propose that these granules are supramolecular complexes containing all of the necessary macromolecular components for protein translation and that they represent a heretofore undescribed subcellular organization of the protein synthetic machinery. This spatial organization may increase the efficiency of protein synthesis and may also provide a vehicle for transport and localization of specific mRNAs within the cell.

Three-dimensional organization of endoplasmic reticulum in the ventral photoreceptors of Limulus.

Living Limulus ventral photoreceptor cells were injected with long chain lipophilic carbocyanine fluorescent dyes to label the endoplasmic reticulum (ER). The purpose of this study was to examine the continuity, dynamic changes, and structure of the ER in the living cell, using laser scanning confocal microscopy and three-dimensional image reconstruction. In this highly polarized neuron, three lines of evidence indicate that the ER is a continuous network extending throughout both lobes of the cell. First, injection of DiO or DiI results in the labeling of ER throughout both lobes of the cell. Second, three-dimensional image reconstruction of the optical sections reveals a dispersed membrane meshwork which may be the structure that serves to interconnect the ER in the two lobes. Third, in cells fixed before dye injection, the pattern of labeling was similar to that in living cells, indicating that vesicle transport was not responsible for the spread of dye throughout the cell. The overall organization of the ER in the photoreceptor cell is relatively stable; however, the fine structure changes over time. This dynamic process appears to represent continual reorganization of the intracellular membranes in the cell. Three morphological types of ER were observed. The ER of the light-sensitive lobe, identified by coinjection of rhodamine-phalloidin to label the microvillar actin, is characterized by a concentration of stratiform membranes interconnected by thin tubular cross-bridges. The perinuclear ER is characterized by a tangle of convoluted tubules sometimes terminating in bulbous structures. Finally, there is a fine tubular reticulum dispersed throughout the cell.

Scanning concentration correlation spectroscopy using the confocal laser microscope.

Concentration correlation spectroscopy allows the assessment of molecular motions in complex systems. The technique generally monitors concentration fluctuations by means of some method such as the intensity of fluorescent molecules (fluorescence correlation spectroscopy). We describe here the use of scanning confocal laser microscopy to measure correlation functions in both space and time. This methodology offers two major advantages over conventional methods. First, collecting data from different regions of the sample significantly increases the signal-to-noise ratio. Second, molecular motions of colloidal gold can be analyzed by correlation methods with high temporal and spatial resolution. Using a MRC 600 laser scanning system, we collect data from an ensemble of 768 independent subvolumes and determine the space-time correlation function. We demonstrate the technique using two different types of samples, fluorescently labeled DNA molecules in solution and colloidal gold-tagged lipids in a planar bilayer. This approach, which we term "scanning concentration correlation spectroscopy," provides a straightforward means of performing high resolution correlation analysis of molecular motions with available instrumentation.