Showing papers by "Worcester Foundation for Biomedical Research published in 1998"

The plurifunctional nucleolus

Abstract: The nucleolus of eukaryotic cells was first described in the early 19th century and was discovered in the 1960s to be the seat of ribosome synthesis. Although rRNA transcription, rRNA processing and ribosome assembly have been clearly established as major functions of the nucleolus, recent studies suggest that the nucleolus participates in many other aspects of gene expression as well. Thus, the nucleolus has been implicated in the processing or nuclear export of certain mRNAs. In addition, new results indicate that biosyntheses of signal recognition particle RNA and telomerase RNA involve a nucleolar stage and that the nucleolus is also involved in processing of U6 RNA, one of the spliceosomal small nuclear RNAs. Interestingly, these three nucleolus-associated small nuclear RNAs (signal recognition particle RNA, telomerase RNA and U6 RNA) are components of catalytic ribonucleoprotein machines. Finally, recent work has also suggested that some transfer RNA precursors are processed in the nucleolus. The nucleolus may have evolutionarily descended from a proto-eukaryotic minimal genome that was spatially linked to vicinal RNA processing and ribonucleoprotein assembly events involved in gene read-out. The nucleolus of today's eukaryotes, now surrounded by the chromatin of over 2 billion years of genome expansion, may still perform these ancient functions, in addition to ribosome biosynthesis. The plurifunctional nucleolus concept has a strong footing in contemporary data and adds a new perspective to our current picture of the spatial-functional design of the cell nucleus.

Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells

Abstract: Fluorescein-labeled oligodeoxynucleotides (oligos) were introduced into cultured rat myoblasts, and their molecular movements inside the nucleus were studied by fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP). FCS revealed that a large fraction of both intranuclear oligo(dT) (43%) and oligo(dA) (77%) moves rapidly with a diffusion coefficient of 4 × 10−7 cm2/s. Interestingly, this rate of intranuclear oligo movement is similar to their diffusion rates measured in aqueous solution. In addition, we detected a large fraction (45%) of the intranuclear oligo(dT), but not oligo(dA), diffusing at slower rates (≤1 × 10−7 cm2/s). The amount of this slower-moving oligo(dT) was greatly reduced if the oligo(dT) was prehybridized in solution with (unlabeled) oligo(dA) prior to introduction to cells, presumably because the oligo(dT) was then unavailable for subsequent hybridization to endogenous poly(A) RNA. The FCS-measured diffusion rate for much of the slower oligo(dT) population approximated the diffusion rate in aqueous solution of oligo(dT) hybridized to a large polyadenylated RNA (1.0 × 10−7 cm2/s). Moreover, this intranuclear movement rate falls within the range of calculated diffusion rates for an average-sized heterogeneous nuclear ribonucleoprotein particle in aqueous solution. A subfraction of oligo(dT) (15%) moved over 10-fold more slowly, suggesting it was bound to very large macromolecular complexes. Average diffusion coefficients obtained from FRAP experiments were in agreement with the FCS data. These results demonstrate that oligos can move about within the nucleus at rates comparable to those in aqueous solution and further suggest that this is true for large ribonucleoprotein complexes as well.

Isolation and Characterization of New Fission Yeast Cytokinesis Mutants

Abstract: Schizosaccharomyces pombe is an excellent organism in which to study cytokinesis as it divides by medial fission using an F-actin contractile ring. To enhance our understanding of the cell division process, a large genetic screen was carried out in which 17 genetic loci essential for cytokinesis were identified, 5 of which are novel. Mutants identifying three genes, rng3(+), rng4(+), and rng5(+), were defective in organizing an actin contractile ring. Four mutants defective in septum deposition, septum initiation defective (sid)1, sid2, sid3, and sid4, were also identified and characterized. Genetic analyses revealed that the sid mutants display strong negative interactions with the previously described septation mutants cdc7-24, cdc11-123, and cdc14-118. The rng5(+), sid2(+), and sid3(+) genes were cloned and shown to encode Myo2p (a myosin heavy chain), a protein kinase related to budding yeast Dbf2p, and Spg1p, a GTP binding protein that is a member of the ras superfamily of GTPases, respectively. The ability of Spg1p to promote septum formation from any point in the cell cycle depends on the activity of Sid4p. In addition, we have characterized a phenotype that has not been described previously in cytokinesis mutants, namely the failure to reorganize actin patches to the medial region of the cell in preparation for septum formation.

Specificity of RNA Binding by CPEB: Requirement for RNA Recognition Motifs and a Novel Zinc Finger

Abstract: CPEB is an RNA binding protein that interacts with the maturation-type cytoplasmic polyadenylation element (CPE) (consensus UUUUUAU) to promote polyadenylation and translational activation of maternal mRNAs in Xenopus laevis. CPEB, which is conserved from mammals to invertebrates, is composed of three regions: an amino-terminal portion with no obvious functional motif, two RNA recognition motifs (RRMs), and a cysteine-histidine region that is reminiscent of a zinc finger. In this study, we investigated the physical properties of CPEB required for RNA binding. CPEB can interact with RNA as a monomer, and phosphorylation, which modifies the protein during oocyte maturation, has little effect on RNA binding. Deletion mutations of CPEB have been overexpressed in Escherichia coli and used in a series of RNA gel shift experiments. Although a full-length and a truncated CPEB that lacks 139 amino-terminal amino acids bind CPE-containing RNA avidly, proteins that have had either RRM deleted bind RNA much less efficiently. CPEB that has had the cysteine-histidine region deleted has no detectable capacity to bind RNA. Single alanine substitutions of specific cysteine or histidine residues within this region also abolish RNA binding, pointing to the importance of this highly conserved domain of the protein. Chelation of metal ions by 1,10-phenanthroline inhibits the ability of CPEB to bind RNA; however, RNA binding is restored if the reaction is supplemented with zinc. CPEB also binds other metals such as cobalt and cadmium, but these destroy RNA binding. These data indicate that the RRMs and a zinc finger region of CPEB are essential for RNA binding.

Localization of signal recognition particle RNA in the nucleolus of mammalian cells

Abstract: The signal recognition particle (SRP) of eukaryotic cells is a cytoplasmic ribonucleoprotein machine that arrests the translational elongation of nascent secretory and membrane proteins and facilitates their transport into the endoplasmic reticulum. The spatial pathway of SRP RNA processing and ribonucleoprotein assembly in the cell is not known. In the present investigation, microinjection of fluorescently tagged SRP RNA into the nucleus of mammalian cells was used to examine its intranuclear sites of localization. Microinjection of SRP RNA into the nuclei of normal rat kidney (NRK) epithelial cells maintained at 37°C on the microscope stage resulted in a very rapid initial localization in nucleoli, followed by a progressive decline of nucleolar signal and an increase of fluorescence at discrete sites in the cytoplasm. Nuclear microinjection of a molecule corresponding to a major portion of the Alu domain of SRP RNA revealed a pattern of rapid nucleolar localization followed by cytoplasmic appearance of signal that was similar to the results obtained with full-length SRP RNA. In contrast, a molecule corresponding to the S domain of SRP RNA did not display nucleolar localization to the extent observed with full-length SRP RNA. An SRP RNA molecule lacking helix 6 of the S domain displayed normal nucleolar localization, whereas one lacking helix 8 of the S domain did not. These results, obtained by direct, real-time observation of fluorescent RNA molecules inside the nucleus of living mammalian cells, suggest that the processing of SRP RNA or its ribonucleoprotein assembly into the SRP involves a nucleolar phase.

Antagonistic Roles of Neurofilament Subunits NF-H and NF-M Against NF-L in Shaping Dendritic Arborization in Spinal Motor Neurons

Abstract: Dendrites play important roles in neuronal function. However, the cellular mechanism for the growth and maintenance of dendritic arborization is unclear. Neurofilaments (NFs), a major component of the neuronal cytoskeleton, are composed of three polypeptide subunits, NF-H, NF-M, and NF-L, and are abundant in large dendritic trees. By overexpressing each of the three NF subunits in transgenic mice, we altered subunit composition and found that increasing NF-H and/or NF-M inhibited dendritic arborization, whereas increasing NF-L alleviated this inhibition. Examination of cytoskeletal organization revealed that increasing NF-H and/or NF-M caused NF aggregation and dissociation of the NF network from the microtubule (MT) network. Increasing NF-H or NF-H together with NF-M further reduced NFs from dendrites. However, these changes were reversed by elevating the level of NF-L with either NF-H or NF-M. Thus, NF-L antagonizes NF-H and NF-M in organizing the NF network and maintaining a lower ratio of NF-H and NF-M to NF-L is critical for the growth of complex dendritic trees in motor neurons.

Localization of Motor-Related Proteins and Associated Complexes to Active, but Not Inactive, Centromeres

Abstract: Multicentric chromosomes are often found in tumor cells and certain cell lines. How they are generated is not fully understood, though their stability suggests that they are non-functional during chromosome segregation. Growing evidence has implicated microtubule motor proteins in attachment of chromosomes to the mitotic spindle and in chromosome movement. To better understand the molecular basis for the inactivity of centromeres associated with secondary constrictions, we have tested these structures by immunofluorescence microscopy for the presence of motor complexes and associated proteins. We find strong immunoreactivity at the active, but not inactive, centromeres of prometaphase multicentric chromosomes using antibodies to the cytoplasmic dynein intermediate chains, three components of the dynactin complex (dynamitin, Arp1 and p150 Glued ), the kinesin-related proteins CENP-E and MCAK and the proposed structural and checkpoint proteins HZW10, CENP-F and Mad2p. These results offer new insight into the assembly and composition of both primary and secondary constrictions and provide a molecular basis for the apparent inactivity of the latter during chromosome segregation.

Indirect immunofluorescence microscopy in cultured cells.

Abstract: The technique of fluorescence immunolocalization has evolved steadily since its first application in the mid-1960s, incorporating innovations in probe chemistry, microscopy, and image detection. This chapter provides an overview of the current status of indirect immunofluorescence for those starting to use the method. It includes both general considerations from cell culture to image detection and several protocols that should serve as an entry point for this technique.

The role of the dynein stalk in cytoplasmic and flagellar motility

Abstract: We have recently identified a microtubule binding domain within the motor protein cytoplasmic dynein. This domain is situated at the end of a slender 10–12 nm projection which corresponds to the stalks previously observed extending from the heads of both axonemal and cytoplasmic dyneins. The stalks also correspond to the B-links observed to connect outer arm axonemal dyneins to the B-microtubules in flagella and constitute the microtubule attachment sites during dynein motility. The stalks contrast strikingly with the polymer attachment domains of the kinesins and myosins which are found on the surface of the motor head. The difference in dynein's structural design raises intriguing questions as to how the stalk functions in force production along microtubules. In this article, we attempt to integrate the myriad of biochemical and EM structural data that has been previously collected regarding dynein with recent molecular findings, in an effort to begin to understand the mechanism of dynein motility.

A 7-methylguanosine cap commits U3 and U8 small nuclear RNAs to the nucleolar localization pathway

Abstract: U3 and U8 small nucleolar RNAs (snRNAs) participate in pre-rRNA processing Like the U1, U2, U4 and U5 major spliceosomal snRNAs, U3 and U8 RNAs are transcribed by RNA polymerase II and their initial 7-methylguanosine (m7G) 5' cap structures subsequently become converted to 2,2,7-trimethylguanosine However, unlike the polymerase II transcribed spliceosomal snRNAs, which are exported to the cytoplasm for cap hypermethylation, U3 and U8 RNAs undergo cap hypermethylation within the nucleus Human U3 and U8 RNAs with various cap structures were generated by in vitro transcription, fluorescently labeled and microinjected into nuclei of normal rat kidney (NRK) epithelial cells When U3 and U8 RNAs containing a m7G cap were microinjected they became extensively localized in nucleoli U3 and U8 RNAs containing alternative cap structures did not localize in nucleoli nor did U3 or U8 RNAs containing triphosphate 5'-termini The nucleolar localization of m7G-capped U3 RNA was competed by co-microinjection into the nucleus of a 100-fold molar excess of dinucleotide m7GpppG but not by a 100-fold excess of ApppG dinucleotide Although it was obviously not possible to assess formation of di- and trimethylguanosine caps on the microinjected U3 and U8 RNAs in these single cell experiments, these results indicate that the initial presence of a m7G cap on U3 and U8 RNAs, most likely together with internal sequence elements, commits these transcripts to the nucleolar localization pathway and point to diverse roles of the m7G cap in the intracellular traffic of various RNAs transcribed by RNA polymerase II

Mechanism of induction of rat hepatic CYP2B and 3A by the pesticide methoxychlor.

Abstract: Our earlier investigation demonstrated that methoxychlor treatment induced hepatic cytochrome P450 2B1/2B2 and 3A proteins in rats and enhanced their respective enzymatic activities. To determine the mechanism of the methoxychlor-mediated induction and whether methoxychlor acts as a frank inducer and not merely by causing stabilization of the cytochrome P450 proteins or of their mRNAs, we assessed the effect of methoxychlor treatment on the transcriptional rate and mRNA levels of these hemeproteins by nuclear run-on and northern blot analyses. Methoxychlor treatment markedly elevated the transcriptional rates of cytochrome P4502B1/2B2 and 3A. Also, higher levels of mRNA of these CYPs were observed. By contrast, mRNA levels of CYP1A1/1A2 were not affected. Using a nuclear run-on method that apparently measures specifically the transcription initiation rate of CYP2B1 and 2B2, we observed that the transcriptional rate of 2B2 was increased substantially more than that of CYP2B1. This differential induction of CYP2B2 versus 2B1 may account for the discrepancy between the levels of induced CYP2B protein and the much lower-than-expected CYP2B-mediated enzymatic activity observed in our earlier study.

Digital deconvolution of fluorescence images for biologists.

Abstract: Publisher Summary The performance of optical microscopes is limited both by the aperture of the lens that causes light from a point source to spread (or diffract) over a finite volume, and by the cross-contamination of light that originates from out of focus planes. To overcome these limitations, approaches have been developed both to improve the microscope design, as exemplified by confocal scanning microscopy, and to reverse mathematically the degrading effects of the conventional microscope. This latter approach is commonly referred to as “deconvolution,” because the degradation effects of the microscope can be described mathematically as the convolution of input signals by the point spread function of the optical system. This chapter introduces the basic rationale of these two methods in languages easily understood by biologists. It also provides details for the implementation of nearest- neighbor deconvolution using readily available hardware and software. A number of deconvolution algorithms have been tested for restoring fluorescence images. The most straightforward approach—three-dimensional (3-D) inverse filtering (Agard et al ., Holmes and Liu) attempts to reverse the effects of image degradation through direct calculations. The two methods currently in wide use are constrained iterative deconvolution and nearest-neighbor deconvolution.

Identification of the photocrosslinking sites in troponin-I with 4-maleimidobenzophenone labelled mutant troponin-Cs having single cysteines at positions 158 and 21

Abstract: Our previous studies have shown that 4-maleimidobenzophenone (BP-Mal) attached to troponin-C (TnC) mutants with single cysteines at positions 12, 57, 89 and 98 forms crosslinks to troponin-I (TnI), and the identified crosslinking regions indicate an antiparallel course of the two interacting polypeptide chains, in agreement with other studies using fragments of TnC and TnI. In this work we extended the mapping of the TnC–TnI interface by analysing photocrosslinking between TnI and BP-Mal labelled TnC mutants with single Cys residues at positions 21 (TnC21) and 158 (TnC158). We determined the sites of these photocrosslinks in TnI by progressive proteolysis of the crosslinked product, followed by N-terminal sequencing and mass spectrophotometric analyses. The results show that whereas TnC158 forms a specific crosslink with Met-21, TnC21 forms multiple crosslinks in the range of residues 96 to 134 of TnI. The results are discussed in light of the antiparallel model of the TnI–TnC complex and a structural model derived from low-angle X-ray and neutron scattering studies.

Innervation and Target Tissue Interactions Induce Rab-GDP Dissociation Inhibitor (GDI) Expression during Peripheral Synapse Formation in Developing Chick Ciliary Ganglion Neurons In Situ

Abstract: Regulated exocytosis of neurotransmitter from synaptic vesicles involves the function of a small GTP-binding protein, Rab3A. Rab-GDP dissociation inhibitor (GDI) is an important modulator of Rab function and subcellular distribution. We have characterized the respective roles of innervation and target tissue interactions in regulating GDI expression during synapse formation in chick ciliary ganglion (CG) neurons developing in situ. Here we report the first full-length chick GDI cDNA sequence. It is highly homologous to mammalian GDI isoforms and includes all of the sequence-conserved regions critical for Rab3A binding. This chick GDI mRNA is predominantly expressed in neurons as judged by Northern blot analysis of tissue distribution and by in situ hybridization of CG sections. Developmental increases in CG GDI mRNA levels occur in two phases as determined by reverse transcription (RT)-PCR and by Northern analysis of both normal-developing and input- or target tissue-deprived ganglia. The initial phase appears to be independent of cell-cell interactions. In contrast, the second, larger increase is induced by both presynaptic inputs and postganglionic target tissues but does not occur until target tissue innervation. Synaptic interaction with the target seems necessary for the regulatory response to both inputs and target tissues. GDI protein levels show similar changes. The developmentally delayed ability of inputs and targets to influence GDI levels differs from the regulation of neurotransmitter receptor expression in CG neurons. These results suggest that distinct extrinsic regulatory signals influence the expression of synapse-related components at the presynaptic axon terminal versus postsynaptic membrane in an individual neuron.

Spatial organization of olfactory receptor neurons on the antenna of the cabbage looper moth

Abstract: Electrophysiological responses to stimulation with behaviorally relevant compounds were recorded from receptor neurons within specialized classes of sensilla on the antennae of male cabbage looper moths. Receptor neurons were found that specifically respond to stimulation with Z-7,12:AC, Z-7,14:AC, Z-9,14:AC, and Z-7,12:OH. Specialized receptor neurons were not found for the three remaining pheromone blend components, 12:AC, Z-5,12:AC, or 11,12:AC. However, a new class of sensillum containing a pair of neurons insensitive to all of the cabbage looper pheromone components was encountered. In addition, spatial patterns of distribution along the flagellum for identified classes of sensilla are described. Sensilla containing Z-7,12:AC-sensitive neurons are preferentially located on the proximal half of the antennal flagellum. In addition to this distribution along the length of the antenna, a pattern across individual flagellar subsegments is described. Sensilla containing neurons sensitive to Z-9,14:AC were found exclusively on the lateral margins of individual flagellar subsegments.

Video basics: use of camera and monitor adjustments.

Abstract: Publisher Summary This chapter discusses the practical aspects of adjusting the video camera and the monitor to prevent the loss of specimen image gray-level information, a subject that is often treated in a most cursory fashion in the owners manuals for cameras and monitors. The problem one faces in coordinating camera and monitor adjustments is that there are too many interacting variables and no standard against which to evaluate the performance of the system. One can vary the intensity of the optical image on the photosensitive surface of the camera, camera settings, and monitor settings. All three appear interactive and can be used individually to seemingly optimize the quality of the image to some extent. When done in a haphazard fashion, empirical “knob twiddling” opens the real possibility of loosing specimen information, typically in the brightest and/or darkest portions of the image. The chapter also reviews, in a simplified fashion, a few aspects of the monochrome composite video signal, produced by tube-type cameras and conventional charge-coupled device (CCD) cameras.