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Cardiovascular Research 2006 71(3):506-516; doi:10.1016/j.cardiores.2006.04.001
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Copyright © 2006, European Society of Cardiology

Microtubule-associated protein-4 (MAP-4) inhibits microtubule-dependent distribution of mRNA in isolated neonatal cardiocytes

Dimitri Scholza,c,*, Paul McDermotta,c, Maria Garnovskayab,c, Thomas N. Galliena,c, Stefan Huettelmaierd, Christina DeRienzoa,c and George Cooper, IVa,c

aGazes Cardiac Research Institute, Cardiology Division, Medical University of South Carolina, United States
bMedical and Research Services of the Ralph H. Johnson Veteran Affairs Medical Center, Nephrology Division, Medical University of South Carolina, United States
cDepartment of Veterans Affairs Medical Center, Charleston, SC 29401, United States
dDepartment of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, United States

* Corresponding author. Gazes Cardiac Research Institute, Medical University of South Carolina, 114 Doughty Street, Rm. 302 Charleston, SC 29403, United States. Tel.: +1 843 876 5067, fax: +1 843 876 5068. Email address: scholzd{at}musc.edu

Received 3 October 2005; revised 27 March 2006; accepted 9 April 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Appendix A. Supplementary data
 Acknowledgments
 References
 
Objectives Active mRNA distribution in the form of ribonucleoprotein particles moving along microtubules has been shown in several cell types, but not yet in cardiocytes. This study addresses two hypotheses: 1) a similar mRNA distribution mechanism operates in cardiocytes; 2) decoration of microtubules with microtubule-associated proteins compromises this distribution.

Methods To visualize ribonucleoproteins in cultured neonatal rat cardiocytes, they were transfected with vectors encoding zipcode binding protein-1 and Staufen fused with GFP. The velocity of microtubular transport and elongation were calculated on time-lapse confocal pictures.

Results: ZBP-1 and Staufen labeled particles co-localized with each other and with microtubules and moved along microtubules over a distance of 1–20 µm with a mean speed of 80 nm/s. The average speed decreased about 50% after decoration of microtubules by adenoviral microtubule-associated protein-4 (MAP-4). The elongation speed measured using the GFP-tagged end-binding protein-1 exceeded 200 nm/s and was not influenced by MAP-4.

Conclusions: We demonstrate for the first time ribonucleoprotein particles in cardiocytes, their microtubular-related movement, and its inhibition (but not of the microtubular elongation), by the MAP-4 decoration of microtubules.

KEYWORDS mRNA; Microtubule; Cardiocyte; Translocation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Appendix A. Supplementary data
 Acknowledgments
 References
 
Cardiocytes have a high rate of protein turnover, with proteins of both the myofilament and extramyofilament cytoskeleton being renewed to the extent of ~20% per day [1]. These processes are greatly enhanced during hypertrophy [2]. Thus, a consistent supply of translation-competent mRNA for these structural proteins to the polysomes is needed for normal cellular homeostasis, and it is essential for hypertrophic growth. However, several unique aspects of cardiocyte cytoarchitecture challenge this biosynthetic pathway. In terms of cardiocyte volume, myofibrils are 50–60%, mitochondria are 25–35%, nuclei are 3–5%, the T-system is 1–2%, and the sarcoplasmic reticulum is 1–3% [3–5]. "Free" cytoplasm (containing glycogen, ribosomes and the extramyofilament cytoskeleton) makes up only ~10%. This high concentration of organelles markedly limits the free diffusion of macromolecules within these large cells. Therefore, mRNAs for cardiocyte structural proteins must be actively delivered to their sites of translation rather than simply arriving via diffusion.

In 1993, James Wilhelm and Ron Vale [6] formulated a hypothesis according to which the transport of mRNAs, and not of the translated proteins, is responsible for the localization of cytoplasmic proteins. This model, slightly modified by Jansen [7], entails the binding of specific shuttling factors to the so-called "zip-code" sequence of nascent mRNA in the nucleus [8,9]. The term "zip code" as a definition for mRNA localization signals within the 3'UTR was proposed by Singer in 1993 [10] and since then has been broadly employed [7,10–12].

Zip code function is mediated by mRNA-binding proteins [7,13–18]. The best described and the most promising for the study of mRNA trafficking in cardiocytes are zipcode-binding protein-1 (ZBP-1) [10–12,19,20] and Staufen [21–28]. ZBP-1 was shown to participate in mRNA granule formation and is needed for mRNA–cytoskeleton attachment [19]. Further, ZBP-1 co-localizes with a β-actin transcription site [20]. Staufen, found also in skeletal muscle [26], was co-immunoprecipitated with the microtubule-based + end directed motor protein kinesin in Xenopus oocytes and was proposed to represent a link between specific mRNAs and the transportation machinery [29].

The active distribution of mRNA has been described in several cell types, including ascidian embryos [30], mosquito salivary glands [31], Drosophila oocytes [9,32], Xenopus oocytes [33], rat embryonic neurons [34,35], rat oligodendrocytes [8], and chicken embryo fibroblasts [19]. The organization of mRNA in ribo-nucleo-protein (RNP) granules in living cells and their movement along the cytoskeleton was shown for the first time in dendrites of mammalian neurons [36], then in oligodendrocytes [37] and the dendrites of rat hippocampal neurons [38]. RNP granules have been hypothesized to represent storage containers for mRNA under translational arrest, which could be poised for release to actively translated pools [35]. However, RNP granules and their active distribution have not been described in cardiocytes.

As one example of active intracellular distribution, microtubules serve as rails for the transport of vesicles, cytoplasmic particles [39,40], membrane receptors [41], and translation-competent mRNAs [6,42–44]. Active, microtubule-based mRNA transport has been described in Drosophila oocytes, yeast, neurons, oligodendrocytes, and fibroblasts [15,16,22,37,43–45].

A major impetus to this study was our finding that in pressure-overload cardiac hypertrophy, cardiocytes contain an increased amount of microtubules, heavily decorated with and stabilized by the predominant cardiac microtubule-associated protein, MAP-4 [46], which contributes to cell viscosity and contractility deficits [47–56]. Depolymerization of microtubules restores both the contractility of cardiocytes and cardiac function to normal [54,56–58]. However, the present study was based not on cardiocyte mechanics but on the question of whether the extensive MAP-4 decoration of a dense microtubule network that we find in pressure-overload cardiac hypertrophy might disrupt important cellular functions by interfering with microtubule-based intracellular transport of vesicles and cytoplasmic particles [6,39,40].

Thus, the aim of present study was to initially test in neonatal cardiocytes the hypothesis that cardiocyte mRNA transport is microtubule-dependent and that excessive MAP-4 decoration of microtubules compromises this transport function, thereby affecting processes important for the protein biosynthesis that is essential for the anabolic cellular response to hemodynamic overloads.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 5. Conclusions
 Appendix A. Supplementary data
 Acknowledgments
 References
 
2.1 Ethical statement
This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2 Animals and isolation of cells
Primary neonatal rat heart cell cultures were prepared from ventricular myocardium of 0- to 3-day-old rats as described [59]. Briefly, the cardiocytes were dissociated in (Ca2+–Mg2+)-free Hank's salt solution buffered with 30 mM HEPES, pH 7.4, in a Celstir apparatus (Wheaton Instruments) at 37 °C by the addition of trypsin (Cooper Biomedical Inc.), chemotrypsin, and elastase (Sigma) at concentrations of 2.4, 2.7 and 0.94 U/ml, respectively. After each of six successive 20-min incubations, the dissociated cells were mixed with minimum Eagle's medium (GIBCO) containing 10% newborn calf serum, centrifuged at 500 x g, and pooled. Cells were transfected by nucleofection (see below) and plated at a density of about 5 x 105 cells in 22 mm (170 µm thick-bottomed) gelatin-coated WillcoWells® culture dishes for live cell microscopy [28]. The majority of cells remained quiescent in vitro, and only a small percentage was beating at a low frequency spontaneously or under the laser beam; these cells were excluded from measurements.

2.3 Plasmids and transfection of cardiocytes
The vector encoding the fusion EYFP–Staufen protein [21–28] was constructed in the lab of Dr. M. Kiebler, Max-Planck-Institute for Developmental Biology, Tuebingen, Germany. The vector encoding the fusion EYFP–ZBP-1 and the ECFP–ZBP-1 proteins [10–12,19,20] were constructed in the lab of Dr. R. Singer, Albert Einstein College of Medicine, NY, USA. The vector encoding the fusion EGFP–EB-1 protein [60–62] was constructed in the lab of Dr. Y. Mimori-Kiyosue, Exploratory Research for Advanced Technology, Japan Science and Technology Corporation, Kyoto, Japan. The vector encoding the fusion EYFP–{alpha}-tubulin [63] protein was purchased from Clontech.

A NucleofectorTM kit designed for transfection of freshly isolated primary cardiocytes from newborn rats (Amaxa Biosystems, Giessen, Germany) was used according to the manufacturer's protocol. About 2 x 106 cells and 2–5 µg of plasmid per transfection (single plasmid or double) were used for each transfection. Up to 10% of cardiocytes were transfected.

2.4 Adenoviral infection
Adenoviruses expressing bacterial β-gal and human MAP-4 (Adβ-gal and AdMAP-4) were generated in our laboratory as described [64]. Neonatal rat cardiocytes were infected in serum-free medium [64] at a multiplicity of infection (MOI) of about 10 plaque forming units per cell, thus infecting about 90% of the cardiocytes as determined by immunofluorescence microscopy using either our polyclonal MAP-4 antibody or an X-gal reaction; there was no observable cytotoxicity.

Preliminary results have shown that MAP-4 appears mostly diffuse until day 3 after infection, after which it decorates the microtubules. Therefore, all experiments were conducted after day 3.

2.5 Immunofluorescent labeling
Neonatal heart cells were fixed in freshly prepared 4% formaldehyde for 10 min, permeabilized in 0.1% Triton X-100 and were covered with antibody against MAP-4 and then with the anti-rabbit antiserum tagged with Alexa Fluor® 488 (Molecular Probes, Eugene, OR). To generate the MAP-4 antibody, we made a bacterial expression construct using the 1–740 NH2-terminal residues of human MAP-4 [41]. Briefly, the recombinant protein, which had a hexahistidine tag inserted at the COOH terminus, was overexpressed in Escherichia coli, purified on a nickel–chelate affinity column, and submitted to SDS–PAGE. The purified protein band was excised, eluted from the gel, and sent to Lampire Biological Laboratories for preparation of a rabbit polyclonal MAP-4 antibody.

2.6 Microscopy and time lapse photography
A Zeiss LSM510META microscope with Ar-Laser (458, 477, 488 and 514 nm) 30 mW, Plan-Apochromat 63/1.4 objective and a chamber maintaining 37 °C and 5% CO2 was used. The minimal possible laser intensity was chosen to decrease the thermal damage to cells. Excitation wavelength and emission filters used for imaging fluorescence: CFP-458 nm/BP475-525, GFP-488 nm/LP505, YFP-514 nm/LP 530. CFP and YFP were visualized simultaneously on Figs. 2 and 4Go.


Figure 2
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  		Fig. 2 Co-localization of ZBP-1 (red) and Staufen (green) in the same granules. Four out of six visible ZBP-1 granules also contain Staufen. A small shift is caused by color aberration of the lens at the critical magnification. Neonatal rat cardiocytes were double-nucleofected with plasmids encoding the CFP–ZBP-1 and EYFP–Staufen. Scale bar=10 µm.

 

Figure 4
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  		Fig. 4 Movement of two ZBP-1–EYFP granules (red, arrows) along microtubules (green). Neonatal rat cardiocytes were nucleofected with two plasmids encoding: 1) a-Tubulin–EYFP (green) and 2) ZBP-1–ECFP (red). A–E represent sequential pictures with a time interval of 14 s. Scale bar=10 µm. A movie of this figure is available in the Appendix.

 
For Fig. 6, an Olympus IX71 microscope equipped with a mercury lamp as a light source, and filter sets for DAPI (Chroma, excitation S403/12, emission S457/50) and for Alexa488 (Chroma, excitation S490/20, emission D528/38) have been used.


Figure 6
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  		Fig. 6 Neonatal rat cardiocytes labeled with anti-MAP-4 antibodies (green) after AdMAP-4 infection (A) and without infection (B). Nuclei are stained with DAPI (red). Scale bar=20 µm.

 
Time-lapse pictures of about 100 frames gathered over 10 to 25 min without intervals between successive frames were scanned. Fluorescence-labeled particles moving straight in at least 3 frames in the row and over a distance more than 1 µm were considered to be moving actively and were measured using LSM 5 Image Browser software. Contracting or laser-damaged cells were excluded from the study. The movement speed was measured separately for ZBP-1, Staufen, and EB-1 labeled particles in control and in MAP-4 or β-gal infected cells for at least 60 particles per group. Results were analyzed statistically using Student's T-test.

2.7 Radioautography
Radioautography was conducted as described [65], applied here to neonatal cardiocytes. Briefly, cells were cultured on cover slips in 35 mm culture dishes. 3H-uridine (10 µCi/ml) was added to the culture medium for 1–6 h. After that, cells were fixed with formaldehyde, dried in alcohol and coated with undiluted Kodak NTB2 photographic emulsion at 42 °C. After 7 days of exposure at 4 °C, they were developed in Kodak D-19 developer as recommended by the manufacturer, counterstained with toluidine blue and mounted in Entellan.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 5. Conclusions
 Appendix A. Supplementary data
 Acknowledgments
 References
 
3.1 Assembly of RNPs
In order to visualize mRNA-related proteins in rat neonatal cardiocytes, we transfected freshly isolated cells with plasmids encoding fluorescently tagged RNP proteins Staufen and ZBP-1. The diffuse fluorescence became detectable in the cytoplasm as early as 3–4 h after transfection and persisted throughout the period of observation (7 days). This most likely corresponds to newly synthesized protein which is not yet bound to mRNA. Fluorescent granules, most likely representing RNP particles, appeared first in a perinuclear location (Fig. 1A) and then populated after 24 h the entire cytoplasm (Fig. 1B).


Figure 1
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  		Fig. 1 Distribution of Staufen–EYFP fusion protein 4 h (A) and 48 h (B) after nucleofection of neonatal rat cardiocytes. Light diffuse labeling over the entire cytoplasm corresponds to unbound protein; the nucleus appears as a dark oval. Granular labeling, most likely representing RNP particles, appears in the perinuclear region shortly after nucleofection (A) and later populates the entire cytoplasm (B). Scale bar=10 µm.

 
Double transfection of neonatal rat cardiocytes with plasmids encoding two different and differently labeled fusion proteins (Staufen–YFP and ZBP-1–CFP) revealed their co-localization in many instances (Fig. 2).

3.2 Movement of RNPs
Time-lapse pictures were analyzed to measure the speed of RNP movement. We found that in live cells the vast majority of labeled particles were oscillating in place, and only a small subpopulation moved straight over a distance more than 1 µm, and only rarely up to 10–20 µm (Fig. 3 and Fig. 3 movie in the Appendix). The measured speed of granules averaged 77.2±6.3 nm/s for ZBP-1 and 76.7±8.5 nm/s for Staufen (mean±S.E.M.). The close correspondence of the transport rates for these two types of proteins indicates that they may be transported by the same cellular motor and (combined with their co-localization) suggests indirectly that we may really be dealing with two proteins being transported as part of the same RNPs.


Figure 3
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  		Fig. 3 Linear movement of a single ZBP-1–EYFP granule (green, arrow) over a distance of about 20 µm. Scale bar=10 µm. A movie of this figure is available in the Appendix.

 
3.3 Co-localization of RNP and microtubules and movement along the microtubules
To test our hypothesis that microtubules serve as a railroad system for RNP movement, microscopic assessment of co-localization of microtubules and RNPs was undertaken. Cardiocytes double-transfected with the {alpha}-tubulin–EYFP and the ZBP-1–CFP demonstrated consistent co-localization: the RNP granules were localized close to the microtubules (Fig. 4). In addition, RNP granules moved along the microtubules from the nucleus towards the cell periphery, as shown by Fig. 4 and Fig. 4 movie in the Appendix.

3.4 Microtubule elongation is about 3-fold faster than RNP transport
Elongation of microtubules in live rat neonatal cardiocytes was measured using the GFP-labeled EB-1, which binds to the + end of growing microtubules (Fig. 5 and Fig. 5 movie in the Appendix). The mean speed of microtubule elongation was calculated to be 204±6 nm/s, or about three times as fast as the transport of the RNP particles (Fig. 7). Thus, the RNP transport kinetics that we have measured are not a function of the rate of microtubule polymerization-based elongation.


Figure 5
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  		Fig. 5 Elongation of microtubules. Neonatal rat cardiocytes were nucleofected with the plasmid encoding the microtubule+end-binding protein EGFP–EB-1. Scale bar=10 µm. A movie of this figure is available in the Appendix.

 

Figure 7
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  		Fig. 7 Average speed of labeled granules in live neonatal cardiocytes, nm/s, mean±S.E.M.

 
3.5 MAP-4 decoration slows down RNP transport but not microtubule elongation
MAP-4 decoration of microtubules at day 3 after the AdMAP-4 infection was documented in cardiocytes by immunofluorescence using antibodies against MAP-4 (Fig. 6). Non-infected cardiocytes demonstrated no detectable labeling whereas about 90% of infected cells were positively labeled, just as we demonstrated before [51]. In AdMAP-4-treated cells, the mean speed of the ZBP-1 and Staufen-labeled particles was reduced to about 50% of the control value (Fig. 7). On the other hand, β-gal infection did not cause significant changes.

In contrast, the mean speed of the EB-1-labeled particles in AdMAP-4-infected cells did not change (204±6 nm/s control vs. 227±10 nm/s AdMAP-4, n.s.).

3.6 Radioautographic dispersion of RNA
The incorporated radioactive 3H-uridine first localized in the nuclei, predominantly in the nucleoli (Fig. 8A). The nuclear and nucleolar labeling remained strong during 1–6 h (Fig. 8A–D) and up to 24 h (not shown), which reflects the steady high level of RNA synthesis. One hour after the onset of the experiment, labeling over the cytoplasm was minimal (Fig. 8A). Cells incorporating 3H-uridine for 2.5, 4 and 6 h demonstrated gradual dispersion of labeling over the entire cytoplasm. Since we could not follow individual silver grains or establish a cytoplasmic starting point, we roughly estimated that the wave front of labeled RNA moves from the nucleus to the cell periphery over a distance of 20–30 µm in 2.5 h to 6 h, which corresponds to a mean velocity of about 1–3.5 nm/s.


Figure 8
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  		Fig. 8 Radioautography of neonatal rat cardiocytes 1 h (A), 2.5 h (B), 4 h (C) and 6 h (D) after 3H-uridine incorporation. The labeling over the nucleus (mostly nucleoli) persists from A–D, indicating ongoing transcription. The labeling over the cytoplasm is barely detectable over the background in A and increases gradually in B–D, indicating ongoing cytoplasmic transport of nascent RNA. Scale bar=50 µm.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
5. Conclusions
 Appendix A. Supplementary data
 Acknowledgments
 References
 
This study provides the first experimental evidence for an active, microtubule-based mechanism of mRNA distribution in heart muscle cells. We demonstrated microscopically for the first time the existence of RNP particles in cardiocytes, their co-localization with microtubules, and their microtubule-related transport. We calculated the speed of this transport and its inhibition when microtubules are decorated by MAP-4. Finally, we found that in cardiocytes microtubule-based transport occurs independently from microtubule elongation, and that the latter is not influenced by MAP-4.

4.1 Nature of RNP particles in cardiocytes
In the present study, RNP particles ranged in size from about 30 to 100 nm. However, this is only an estimate, since we could not measure particle size precisely via fluorescence microscopy because of resolution limitations. Moreover, overexposed particles appear larger, and some studies have reported RNP particle sizes of up to 500 nm [34] or even 700 nm [66]. Electron microscopy studies [67] described nuclear RNP particles of 20 to 50 nm. However, electron microscopy data on RNP particles outside the nucleus [22] have described them as loose and amorphous particles associated with polyribosomes, about 200 nm in diameter. While Oleynikov and Singer described ZBP-1 shuttling between nucleus and cytoplasm in chicken embryo fibroblasts [20], we could not detect any nuclear ZBP-1 or Staufen labeling.

Pre-RNP exported through the nuclear pores to the cytoplasm [68,69] matures into RNP particle binding proteins, recently classified [70] into 1) RNA-binding proteins that associate with specific mRNAs such as Staufen [42], ZBP-1 [12], and Vera [71]; 2) motor proteins that associate with RNPs to transport them, such as dynein [45] and kinesin [72]; 3) adaptor proteins that interact with other elements of RNPs and are essential for their transport, such as Barentsz [27,73], She3p [74] and Miranda [75]; 4) cytoskeletal structures along which RNPs move, such as microtubules [42] and actin thin filaments [76]; and 5) specific repressors of protein synthesis that prevent translation before the target mRNA is properly localized, such as Bruno [77]. Obviously, after passing the nuclear pore bottleneck, tens or hundreds of particles may come together and bind additional regulatory and transport proteins and ribosomes [78]. They thereby grow about 10-fold in size (which corresponds to a 1000-fold gain in volume), bind to the microtubules, and move to their destination where the translation may finally begin [79].

4.2 Speed of RNP movement
Twenty years ago, Lasek et al. [80] differentiated fast (0.5–4 µm/s) axonal transport of vesicles from slow (10–50 nm/s) transport of cytoplasmic proteins based solely on radiolabeling, the only technique available at that time. The measurements for mRNA showed even slower movement than that for proteins [65].

Since fluorescence study of live cells after microinjection of labeled mRNA [37], labeling with mRNA-dyes [34] or transfection with fluorescent fusion proteins [23,81] became available, many investigators have followed intracellular movement directly and calculated the speed; the results are summarized in Table 1.


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  		Table 1 Literature data for the velocity of microtubule transport for RNP and other non-vesicle cytoplasmic particles

 
In the present study, we estimated an average speed of RNPs in the cytoplasm of about 80 nm/s, which corresponds closely to the literature data for RNP transport in neurons, oligodendocytes and fibroblasts [20,34,37,38,82].

In cells infected with AdMAP-4, the mean speed of RNP particles was significantly reduced. We excluded the influence of adenoviral infection by using Adβ-gal as a negative control and suggest that the reason for the speed reduction by MAP-4 could be the competition of the molecular motor with the MAP for binding sites on microtubules.

In contrast, the mean speed of microtubule elongation was almost 3-fold higher, very close to that described for HeLa cells [83], and it was MAP-4-independent. Thus, microtubule elongation and microtubule-dependent transport of RNPs are based on different molecular mechanisms.

At any time point the majority of particles do not exhibit net movement but instead oscillate in place. Similar behavior of RNP-labeled granules using GFP–ZBP-1 or GFP–Staufen has been reported in chicken embryo fibroblasts [20], chicken neurons [84], and rat hippocampal neurons [23]. This transport pattern could be explained by saltatory motion or by very short-term movement of intracellular cargos along microtubules [85]. Further, RNPs have been shown not to be bound to motor proteins or microtubules most of the time [86].

To approximate the rate of movement of RNA pools over a longer period of time, we used radiolabeling, taking into account that the measurement could be contaminated by rRNA movement but hoping that rRNA might be transported in the same RNP particles [22]. We roughly estimated the mean velocity of newly synthetisized RNA in cytoplasm to be about 1–3 nm/s or 20 to 80 times less than that measured by our fluorescence method, which probably means that at any given time only one out of 20 to 80 granules is moving. This result is consistent with our observation by fluorescence microscopy that at any given time only a small fraction of the particles show substantial vectorial movement.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
Appendix A. Supplementary data
 Acknowledgments
 References
 
We demonstrate here for the first time RNP particles in cardiocytes, their microtubule-related movement, and the inhibition of this movement, but not of microtubule elongation, by MAP-4 microtubule decoration.


   Appendix A. Supplementary data
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Appendix A. Supplementary data
Acknowledgments
 References
 
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cardiores.2006.04.001.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Appendix A. Supplementary data
 Acknowledgments
References
 
This study was supported by Program Project Grant HL-48788 from the National Heart, Lung, and Blood Institute and by a Merit Award from the Research Service of the Department of Veterans Affairs. We thank Dr. Michael Kiebler and Dr. Bernhard Goetze from Max Planck Institute for Developmental Biology, Tuebingen 72076, Germany and Dr. Yuko Mimori-Kiyosue from Tsukita Cell Axis Project, Exploratory Research for Advanced Technology, Japan Science and Technology Corporation, Kyoto Research Park, Shimogyo-ku, Kyoto 600-8813, Japan, for providing vectors and for the fruitful critical discussion and comments during the manuscript preparation.


   Notes
 
Time for primary review 23 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Appendix A. Supplementary data
 Acknowledgments
 References
 

  1. Simpson D., Decker M., Clark W., Decker R. Contractile activity and cell–cell contact regulate myofibrillar organization in cultured cardiac myocytes. J Cell Biol (1993) 123:323–336.[Abstract/Free Full Text]
  2. Imamura T., McDermott P.J., Kent R.L., Nagatsu M., Cooper G. IV, Carabello B.A. Acute changes in myosin heavy chain synthesis rate in pressure versus volume overload. Circ Res (1994) 75:418–425.[Abstract/Free Full Text]
  3. Page E., McCallister L.P. Quantitative electron microscopic description of heart muscle cells. Application to normal, hypertrophied and thyroxin-stimulated hearts. Am J Cardiol (1973) 31:172–181.[CrossRef][Web of Science][Medline]
  4. Anversa P., Loud A.V., Vitali-Mazza L. Morphometry and autoradiography of early hypertrophic changes in the ventricular myocardium of adult rat: an electron microscopic study. Lab Invest (1976) 35:475–483.[Web of Science][Medline]
  5. Rumyantsev P.P. Cardiomyocytes in the reproduction, differentiation and gegeneration. (1982) Leningrad: Nauka. [in Russian].
  6. Wilhelm J.E., Vale R.D. RNA on the move: the mRNA localization pathway. J Cell Biol (1993) 123:269–274.[Free Full Text]
  7. Jansen R. mRNA localization: message on the move. Nat Rev Mol Cell Biol (2001) 2:247–256.[CrossRef][Web of Science][Medline]
  8. Hoek K., Kidd G., Carson J.R.S. hnRNP A2 selectively binds the cytoplasmic transport sequence of myelin basic protein mRNA. Biochemistry (1998) 37:7021–7029.[CrossRef][Web of Science][Medline]
  9. Norvell A., Kelley R., Wehr K.T.S. Specific isoforms of squid, a Drosophila hnRNP, perform distinct roles in Gurken localization during oogenesis. Genes Dev (1999) 13:864–876.[Abstract/Free Full Text]
  10. Singer R. RNA zipcodes for cytoplasmic addresses. Curr Biol (1993) 3:719–721.[CrossRef][Web of Science][Medline]
  11. Oleynikov Y., Singer R.H. RNA localization: different zipcodes, same postman? Trends Cell Biol (1998) 8:381–383.[CrossRef][Web of Science][Medline]
  12. Ross A.F., Oleynikov Y., Kislauskis E.H., Taneja K.L., Singer R.H. Characterization of a β-actin mRNA zipcode-binding protein. Mol Cell Biol (1997) 17:2158–2165.[Abstract]
  13. Kislauskis E., Li Z., Singer R., Taneja K. Isoform-specific 3'-untranslated sequences sort {alpha}-cardiac and β-cytoplasmic actin messenger RNAs to different cytoplasmic compartments. J Cell Biol (1993) 123:165–172.[Abstract/Free Full Text]
  14. Bashirullah A., Cooperstock R., Lipshitz H. RNA localization in development. Annu Rev Biochem (1998) 67:335–394.[CrossRef][Web of Science][Medline]
  15. Bassell G., Oleynikov Y., Singer R. The travels of mRNAs through all cells large and small. FASEB (1999) 13:447–454.[Free Full Text]
  16. Farina K., Singer R. The nuclear connection in RNA transport and localization. Trends Cell Biol (2002) 12:466–472.[CrossRef][Web of Science][Medline]
  17. Roegiers F. Insights into mRNA transport in neurons. Proc Natl Acad Sci (2002) 100:1465–1466.[CrossRef][Web of Science]
  18. Jockusch B., Hüttelmaier S., Illenberger S. From the nucleus toward the cell periphery: a guided tour for mRNAs. News Physiol Sci (2003) 18:7–11.[Abstract/Free Full Text]
  19. Farina K., Hüttelmaier S., Musunuru K., Darnell R., Singer R. Two ZBP1 KH domains facilitate β-actin mRNA localization, granule formation, and cytoskeletal attachment. J Cell Biol (2003) 160:77–87.[Abstract/Free Full Text]
  20. Oleynikov Y., Singer R. Real-time visualization of ZBP1 association with. Curr Biol (2003) 13:199–207.[CrossRef][Web of Science][Medline]
  21. Wickham L., Duchaine T., Luo M., Nabi I.R., DesGroseillers L. Mammalian Staufen is a double-stranded-RNA- and tubulin-binding protein which localizes to the rough endoplasmic reticulum. Mol Cell Biol (1999) 19:2220–2230.[Abstract/Free Full Text]
  22. Kiebler M.A., Hemraj I., Verkade P., Kohrmann M., Fortes P., Marion R.M., et al. The mammalian Staufen protein localizes to the somatodendritic domain of cultured hippocampal neurons: implications for its involvement in mRNA transport. J Neurosci (1999) 19:288–297.[Abstract/Free Full Text]
  23. Kohrmann M., Luo M., Kaether C., DesGroseillers L., Dotti C.G., Kiebler M.A. Microtubule-dependent recruitment of Staufen–green fluorescent protein into large RNA-containing granules and subsequent dendritic transport in living hippocampal neurons. Mol Biol Cell (1999) 10:2945–2953.[Abstract/Free Full Text]
  24. Duchaine T.F., Hemraj I., Furic L., Deitinghoff A., Kiebler M.A., DesGroseillers L. Staufen2 isoforms localize to the somatodendritic domain of neurons and interact with different organelles. J Cell Sci (2002) 115:3285–3295.[Abstract/Free Full Text]
  25. Martin S., Leclerc V., Smith-Litiere K., D.S.J. The identification of novel genes required for Drosophila anteroposterior axis formation in a germline clone screen using GFP–Staufen. Development (2003) 130:4201–4215.[Abstract/Free Full Text]
  26. Belanger G., Stocksley M.A., Vandromme M., Schaeffer L., Furic L., DesGroseillers L., et al. Localization of the RNA-binding proteins Staufen1 and Staufen2 at the mammalian neuromuscular junction. J Neurochem (2003) 86:669–677.[CrossRef][Web of Science][Medline]
  27. Macchi P., Kroening S., Palacios I.M., Baldassa S., Grunewald B., Ambrosino C., et al. Barentsz, a new component of the Staufen-containing ribonucleoprotein particles in mammalian cells, interacts with Staufen in an RNA-dependent manner. J Neurosci (2003) 23:5778–5788.[Abstract/Free Full Text]
  28. Mallardo M., Deitinghoff A., Muller J., Goetze B., Macchi P., Peters C., et al. Isolation and characterization of Staufen-containing ribonucleoprotein particles from rat brain. Proc Natl Acad Sci U S A (2003) 100:2100–2105.[Abstract/Free Full Text]
  29. Yoon Y., Mowry K. Xenopus Staufen is a component of a ribonucleoprotein complex containing Vg1 RNA and kinesin. Development (2004) 131:3035–3045.[Abstract/Free Full Text]
  30. Jeffery W., Tomlinson C., Brodeur R. Localization of actin messenger RNA during early ascidian development. Dev Biol (1983) 99:408–417.[CrossRef][Web of Science][Medline]
  31. Mehlin H., Daneholt B., Skoglund U. Translocation of a specific premessenger ribonucleoprotein particle through the nuclear pore studied with electron microscope tomography. Cell (1992) 69:605–613.[CrossRef][Web of Science][Medline]
  32. Frigerio G., Burri M., Bopp D., Baumgartner S., Noll M. Structure of the segmentation gene paired and the Drosophila PRD gene set as part of a gene network. Cell (1986) 47:735–746.[CrossRef][Web of Science][Medline]
  33. Rebagliati M., Weeks D., Harvey R., Melton D. Identification and cloning of localized maternal RNAs from Xenopus eggs. Cell (1985) 42:769–777.[CrossRef][Web of Science][Medline]
  34. Knowles R., Sabry J., Martone M., Deerinck T.J., Ellisman M.H., Bassell G.J., et al. Translocation of RNA granules in living neurons. J Neurosci (1996) 16:7812–7820.[Abstract/Free Full Text]
  35. Krichevsky A., Kosik K. Neuronal RNA granules: a link between RNA localization and stimulation-dependent translation. Neuron (2001) 32:683–696.[CrossRef][Web of Science][Medline]
  36. Tiedge H., Fremeau R.J., Weinstock P., Arancio O., Brosius J. Dendritic location of neural BC1 RNA. Proc Natl Acad Sci U S A (1991) 88:2093–2097.[Abstract/Free Full Text]
  37. Ainger K., Avossa D., Morgan F., Hill S.J., Barry C., Barbarese E., et al. Transport and localization of exogenous myelin basic protein mRNA microinjected into oligodendrocytes. J Cell Biol (1993) 123:431–441.[Abstract/Free Full Text]
  38. Fredj N., Grange J., Sadoul R., Richard S., Goldberg Y., V.B. Depolarization-induced translocation of the RNA-binding protein Sam68 to the dendrites of hippocampal neurons. J Cell Sci (2004) 117:1079–1090.[Abstract/Free Full Text]
  39. Prahlad V., Helfand B.T., Langford G.M., Vale R.D., Goldman R.D. Fast transport of neurofilament protein along microtubules in squid axoplasm. J Cell Sci (2000) 113:3939–3946.[Abstract]
  40. Vale R.D. The molecular motor toolbox for intracellular transport. Cell (2003) 112:467–480.[CrossRef][Web of Science][Medline]
  41. Cheng G., Iijima Y., Ishibashi Y., Kuppuswamy D., Cooper G. IV. Inhibition of G protein-coupled receptor trafficking in neuroblastoma cells by MAP-4 decoration of microtubules. Am J Physiol Heart Circ Physiol (2002) 283:H2379–H2388.[Abstract/Free Full Text]
  42. Ferrandon D., Elphick L., Nüsslein-Volhard C., St Johnston D. Staufen protein associates with the 3'UTR of bicoid mRNA to form particles that move in a microtubule-dependent manner. Cell (1994) 79:1221–1232.[CrossRef][Web of Science][Medline]
  43. Carson J., Worboys K., Ainger K., Barbarese E. Translocation of myelin basic protein mRNA in oligodendrocytes requires microtubules and kinesin. Cell Motil Cytoskeleton (1997) 38:318–328.[CrossRef][Web of Science][Medline]
  44. Carson J., Kwon S., Barbarese E. RNA trafficking in myelinating cells. Curr Opin Neurobiol (1998) 8:607–612.[CrossRef][Web of Science][Medline]
  45. Schnorrer F., Bohmann K., Nüsslein-Volhard C. The molecular motor dynein is involved in targeting swallow and bicoid RNA to the anterior pole of Drosophila oocytes. Nat Cell Biol (2000) 2:185–190.[CrossRef][Web of Science][Medline]
  46. Takahashi M., Shiraishi H., Ishibashi Y., Blade K.L., McDermott P.J., Menick D.R., et al. Phenotypic consequences of β1-tubulin expression and MAP4 decoration of microtubules in adult cardiocytes. Am J Physiol Heart Circ Physiol (2003) 285:H2072–H2083.[Abstract/Free Full Text]
  47. Tsutsui H., Ishihara K., Cooper G. IV. Cytoskeletal role in the contractile dysfunction of hypertrophied myocardium. Science (1993) 260:682–687.[Abstract/Free Full Text]
  48. Tsutsui H., Tagawa H., Kent R.L., McCollam P.L., Ishihara K., Nagatsu M., et al. Role of microtubules in contractile dysfunction of hypertrophied cardiocytes. Circulation (1994) 90:533–555.[Abstract/Free Full Text]
  49. Tagawa H., Rozich J.D., Tsutsui H., Narishige T., Kuppuswamy D., Sato H., et al. Basis for increased microtubules in pressure-hypertrophied cardiocytes. Circulation (1996) 93:1230–1243.[Abstract/Free Full Text]
  50. Tagawa H., Wang N., Narishige T., Ingber D.E., Zile M.R., Cooper G. IV. Cytoskeletal mechanics in pressure-overload cardiac hypertrophy. Circ Res (1997) 80:281–289.[Abstract/Free Full Text]
  51. Sato H., Nagai T., Kuppuswamy D., Narishige T., Koide M., Menick D.R., et al. Microtubule stabilization in pressure overload cardiac hypertrophy. J Cell Biol (1997) 139:963–973.[Abstract/Free Full Text]
  52. Koide M., Nagatsu M., Zile M.R., Hamawaki M., Swindle M.M., Keech G., et al. Premorbid determinants of left ventricular dysfunction in a novel model of gradually induced pressure overload in the adult canine. Circulation (1997) 95:1601–1610.[Abstract/Free Full Text]
  53. Zile M.R., Richardson K., Cowles M.K., Buckley J.M., Koide M., Cowles B.A., et al. Constitutive properties of adult mammalian cardiac muscle cells. Circulation (1998) 98:567–579.[Abstract/Free Full Text]
  54. Zile M.R., Koide M., Sato H., Ishiguro Y., Conrad C.H., Buckley J.M., et al. Role of microtubules in the contractile dysfunction of hypertrophied myocardium. J Am Coll Cardiol (1999) 33:250–260.[Abstract/Free Full Text]
  55. Koide M., Hamawaki M., Narishige T., Sato H., Nemoto S., DeFreyte G., et al. Microtubule depolymerization normalizes in vivo myocardial contractile function in dogs with pressure-overload left ventricular hypertrophy. Circulation (2000) 102:1045–1052.[Abstract/Free Full Text]
  56. Schroder E.A., Tobita K., Tinney J.P., Foldes J.K., Keller B.B. Microtubule involvement in the adaptation to altered mechanical load in developing chick myocardium. Circ Res (2002) 91:353–359.[Abstract/Free Full Text]
  57. Cicogna A.C., Robinson K.G., Conrad C.H., Singh K., Squire R., Okoshi M.P., et al. Direct effects of colchicine on myocardial function: studies in hypertrophied and failing spontaneously hypertensive rats. Hypertension (1999) 33:60–65.[Abstract/Free Full Text]
  58. Roos K.P., Palmer R.E., Miller T.W. The role of microtubules in structural remodeling and the progression to heart failure. J Card Fail (2002) 8:S300–S310.[CrossRef][Web of Science][Medline]
  59. McDermott P.J., Morgan H.E. Contraction modulates the capacity for protein synthesis during growth of neonatal heart cells in culture. Circ Res (1989) 64:542–553.[Abstract/Free Full Text]
  60. Mimori-Kiyosue Y., Shiina N., Tsukita S. The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules. Curr Biol (2000) 10:865–868.[CrossRef][Web of Science][Medline]
  61. Mimori-Kiyosue Y., Tsukita S. "Search-and-capture" of microtubules through plus-end-binding proteins (+TIPs). J Biochem (Tokyo) (2003) 134:321–326.[Abstract/Free Full Text]
  62. Piehl M., Tulu U.S., Wadsworth P., Cassimeris L. Centrosome maturation: measurement of microtubule nucleation throughout the cell cycle by using GFP-tagged EB1. Proc Natl Acad Sci U S A (2004) 101:1584–1588.[Abstract/Free Full Text]
  63. Kimble M., Kuzmiak C., McGovern K.N., de Hostos E.L. Microtubule organization and the effects of GFP–tubulin expression in Dictyostelium discoideum. Cell Motil Cytoskeleton (2000) 47:48–62.[CrossRef][Web of Science][Medline]
  64. Cheng G., Qiao F., Gallien T., Kuppuswamy D., Cooper G. IV. Inhibition of β-adrenergic receptor trafficking in adult cardiocytes by MAP4 decoration of microtubules. Am J Physiol Heart Circ Physiol (2005) 288:H1193–H1202.[Abstract/Free Full Text]
  65. Davis L., Banker G., Steward O. Selective dendritic transport of RNA in hippocampal neurons in culture. Nature (1987) 330:477–479.[CrossRef][Medline]
  66. Barbarese E., Koppel D., Deutscher M., Smith C.L., Ainger K., Morgan F., et al. Protein translation components are colocalized in granules in oligodendrocytes. J Cell Sci (1995) 108:2781–2790.[Abstract]
  67. Stevens B.J., Swift H. RNA transport from nucleus to cytoplasm in Chironomus salivary glands. J Cell Biol (1966) 31:55–77.[Abstract/Free Full Text]
  68. Daneholt B. Look at messenger RNP moving through the nuclear pore. Cell (1997) 88:585–588.[CrossRef][Web of Science][Medline]
  69. Levesque L., Guzik B., Guan T., Coyle J., Black B.E., Rekosh D., et al. RNA export mediated by tap involves NXT1-dependent interactions with the nuclear pore complex. J Biol Chem (2001) 276:44953–44962.[Abstract/Free Full Text]
  70. Villace P., Marion R.M., Ortin J. The composition of Staufen-containing RNA granules from human cells indicates their role in the regulated transport and translation of messenger RNAs. Nucleic Acids Res (2004) 32:2411–2420.[Abstract/Free Full Text]
  71. Deshler J., Highett M., Schnapp B. Localization of Xenopus Vg1 mRNA by Vera protein and the endoplasmic reticulum. Science (1997) 276:1128–1131.[Abstract/Free Full Text]
  72. Brendza R., Serbus L., Duffy J., Saxton W. A function for kinesin I in the posterior transport of oskar mRNA and Staufen protein. Science (2000) 289:2120–2122.[Abstract/Free Full Text]
  73. van Eeden F., Palacios I., Petronczki M., Weston M., St Johnston D. Barentsz is essential for the posterior localization of oskar mRNA and colocalizes with it to the posterior pole. J Cell Biol (2001) 154:511–523.[Abstract/Free Full Text]
  74. Takizawa P.A., Vale R.D. The myosin motor, Myo4p, binds Ash1 mRNA via the adapter protein, She3p. Proc Natl Acad Sci U S A (2000) 97:5273–5278.[Abstract/Free Full Text]
  75. Shen C., Knoblich J., Chan Y., Jiang M., Jan L., Jan Y. Miranda as a multidomain adapter linking apically localized Inscuteable and basally localized Staufen and Prospero during asymmetric cell division in Drosophila. Genes Dev (1998) 12:1837–1846.[Abstract/Free Full Text]
  76. Takizawa P.A., Sil A., Swedlow J.R., Herskowitz I., Vale R.D. Actin-dependent localization of an RNA encoding a cell-fate determinant in yeast. Nature (1997) 389:90–93.[CrossRef][Medline]
  77. Kim-Ha J., Kerr K., Macdonald P. Translational regulation of oskar mRNA by Bruno, an ovarian RNA-binding protein, is essential. Cell (1995) 81:403–412.[CrossRef][Web of Science][Medline]
  78. Brendel C., Rehbein M., Kreienkamp H., Buck F., Richter D., Kindler S. Characterization of Staufen 1 ribonucleoprotein complexes. Biochem J (2004) 384:239–246.[CrossRef][Web of Science][Medline]
  79. Huettelmaier S., Zenklusen D., Lederer M., Dictenberg J., Lorenz M., Meng X., et al. Spatial regulation of β-actin translation by Src-dependent phosphorylation of ZBP1. Nature (2005) 438:512–515.[CrossRef][Medline]
  80. Lasek R., Garner J., Brady S. Axonal transport of the cytoplasmic matrix. J Cell Biol (1984) 99:212s–221s.[Free Full Text]
  81. Chalfie M., Tu Y., Euskirchen G., Ward W., Prasher D. Green fluorescent protein as a marker for gene expression. Science (1994) 263:802–805.[Abstract/Free Full Text]
  82. Abaza A., Soleilhac J.M., Westendorf J., Piel M., Crevel I., Roux A., et al. M phase phosphoprotein 1 is a human plus-end-directed kinesin-related protein required for cytokinesis. J Biol Chem (2003) 278:27844–27852.[Abstract/Free Full Text]
  83. Perez F., Diamantopoulos G., Stalder R., Kreis T. CLIP-170 highlights growing microtubule ends in vivo. Cell (1999) 96:517–527.[CrossRef][Web of Science][Medline]
  84. Zhang H.L., Eom T., Oleynikov Y., Shenoy S.M., Liebelt D.A., Dictenberg J.B., et al. Neurotrophin-induced transport of a β-actin mRNP complex increases β-actin levels and stimulates growth cone motility. Neuron (2001) 31:261–275.[CrossRef][Web of Science][Medline]
  85. Sheetz M.P. Motor and cargo interactions. Eur J Biochem (1999) 262:19–25.[Web of Science][Medline]
  86. Kloc M., Etkin L. RNA localization mechanisms in oocytes. J Cell Sci (2005) 118:269–282.[Abstract/Free Full Text]
  87. Tiruchinapalli D.M., Oleynikov Y., Kelic S., Shenoy S.M., Hartley A., Stanton P.K., et al. Activity-dependent trafficking and dynamic localization of zipcode binding protein 1 and β-actin mRNA in dendrites and spines of hippocampal neurons. J Neurosci (2003) 23:3251–3261.[Abstract/Free Full Text]
  88. Yisraeli J., Sokol S., Melton D. A two-step model for the localization of maternal mRNA in Xenopus oocytes: involvement of microtubules and microfilaments in the translocation and anchoring of Vg1 mRNA. Development (1990) 108:289–298.[Abstract]
  89. Bertrand E., Chartrand P., Schaefer M., Shenoy S.M., Singer R.H., Long R.M. Localization of ASH1 mRNA particles in living yeast. Mol Cell (1998) 2:437–445.[CrossRef][Web of Science][Medline]
  90. Shav-Tal Y., Darzacq X., Shenoy S.M., Fusco D., Janicki S.M., Spector D.L., et al. Dynamics of single mRNPs in nuclei of living cells. Science (2004) 304:1797–1800.[Abstract/Free Full Text]
  91. Mundy D., Machleidt T., Ying Y., Anderson R., Bloom G. Dual control of caveolar membrane traffic by microtubules and the actin cytoskeleton. J Cell Sci (2002) 115:4327–4339.[Abstract/Free Full Text]

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