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Background: mRNA localization in somatic cells is an important mechanism for gene expression regulation. In fibroblasts, the protein ZBP1 associates with the sequence that localizes β-actin mRNA to the leading edge of fibroblasts, augmenting motility. β-actin mRNA localizes in a cytoskeleton-dependent manner, depending on intact actin and myosin ATP-hydrolysis, and is largely bound to the actin cytoskeleton. The ZBP1 protein contains four KH RNA binding domains and a classic RBD RNA binding domain. It also contains a putative nuclear import and export sequence, suggesting a nuclear phase in this protein''s function.Results: Using high-speed imaging, we show here the targeting of this RNA binding protein to β-actin pre-mRNA transcripts in the nuclei of living cells and measure the residence time of the RNA-protein complex before it leaves the transcription site. Then, the RNA-protein particle is exported to the cytoplasm, where it localizes at velocities of 0.6 μm/s by using actin filaments and/or microtubules. This RNA-ZBP1 complex is required for cytoplasmic localization in fibroblasts; mislocalizing the protein also mislocalizes the RNA, and expressing the protein in a ZBP1-deficient cell line induces β-actin mRNA localization.Conclusions: This work demonstrates that the RNA-protein association, essential for cytoplasmic localization, begins as soon as the RNA is transcribed. The ZBP1 then forms a ribonucleoprotein particle and moves in a myosin-dependent fashion by using the cytoskeleton for directional transport. [Copyright &y& Elsevier]

Summary: Limited chromosome mobility has been observed in mammalian interphase nuclei. Live imaging shows unidirectional and actin-dependent movement of HSP70 loci towards speckles upon heat shock, resulting in enhanced transcription. This adds further impetus to understanding compartmentalization of function in the nucleus. [Copyright &y& Elsevier]

RNA localization during development is required for proper sorting of developmental determinants. Direct visualization of endogenous transcribed RNA is now possible and should provide new insights into how this process occurs. [Copyright &y& Elsevier]

Summary: It has not been possible to view the transcriptional activity of a single gene within a living eukaryotic cell. It is therefore unclear how long and how frequently a gene is actively transcribed, how this is modulated during differentiation, and how transcriptional events are dynamically coordinated in cell populations. By means of an in vivo RNA detection technique , we have directly visualized transcription of an endogenous developmental gene. We found discrete “pulses” of gene activity that turn on and off at irregular intervals. Surprisingly, the length and height of these pulses were consistent throughout development. However, there was strong developmental variation in the proportion of cells recruited to the expressing pool. Cells were more likely to reexpress than to initiate new expression, indicating that we directly observe a transcriptional memory. In addition, we used a clustering algorithm to reveal synchronous transcription initiation in neighboring cells. This study represents the first direct visualization of transcriptional pulsing in eukaryotes. Discontinuity of transcription may allow greater flexibility in the gene-expression decisions of a cell. [ABSTRACT FROM AUTHOR]

Cell migration is crucial for many biological and pathological processes such as chemotaxis of immune cells, fibroblast migration during wound healing, and tumor cell invasion and metastasis. Cells migrate forward by extending membrane protrusions. The formation of these protrusions is driven by assembly of actin filaments at the leading edge . Neural Wiskott-Aldrich syndrome protein (N-WASP), a ubiquitous member of the WASP family, induces actin polymerization by activating Arp2/3 complex and is thought to regulate the formation of membrane protrusions . However, it is totally unclear how N-WASP activity is spatially and temporally regulated inside migrating cells. To detect and image sites of N-WASP activity during cell motility and invasion in carcinoma cells, we designed an N-WASP fluorescence resonance energy transfer (FRET) biosensor that distinguishes between the active and inactive conformations and mimics the function of endogenous N-WASP. Our data show that N-WASP is involved in lamellipodia extension, where it is activated at the leading edge, as well as in invadopodia formation of invasive carcinoma cells, where it is activated at the base. This is the first time that the activity of full-length N-WASP has been visualized in vivo, and this has lead to new insights for N-WASP function. [Copyright &y& Elsevier]

Cytoplasmic mRNA movements ultimately determine the spatial distribution of protein synthesis. Although some mRNAs are compartmentalized in cytoplasmic regions, most mRNAs, such as housekeeping mRNAs or the poly-adenylated mRNA population, are believed to be distributed throughout the cytoplasm . The general mechanism by which all mRNAs may move, and how this may be related to localization, is unknown. Here, we report a method to visualize single mRNA molecules in living mammalian cells, and we report that, regardless of any specific cytoplasmic distribution, individual mRNA molecules exhibit rapid and directional movements on microtubules. Importantly, the β-actin mRNA zipcode increased both the frequency and length of these movements, providing a common mechanistic basis for both localized and nonlocalized mRNAs. Disruption of the cytoskeleton with drugs showed that microtubules and microfilaments are involved in the types of mRNA movements we have observed, which included complete immobility and corralled and nonrestricted diffusion. Individual mRNA molecules switched frequently among these movements, suggesting that mRNAs undergo continuous cycles of anchoring, diffusion, and active transport. [Copyright &y& Elsevier]