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Regulation of gene expression is key determinant to cell structure and function. RNA localization, where specific mRNAs are transported to subcellular regions and then translated, is highly conserved in eukaryotes ranging from yeast to extremely specialized and polarized cells such as neurons. Messenger RNA and associated proteins (mRNP) move from the site of transcription in the nucleus to their final destination in the cytoplasm both passively through diffusion and actively via directed transport. Dysfunction of RNA localization, transport and translation machinery can lead to pathology. Single-molecule live-cell imaging techniques have revealed unique features of this journey with unprecedented resolution. In this review, we highlight key recent findings that have been made using these approaches and possible implications for spatial control of gene function. [ABSTRACT FROM AUTHOR]

Analyzing the expression of single genes in single cells appears minimalistic in comparison to gene expression studies based on more global approaches. However, stimulated by advances in imaging technologies, single-cell studies have become an essential tool in understanding the rules that govern gene expression. This quantitative view of single-cell gene expression is based on counting mRNAs in single cells, monitoring transcription in real time, and visualizing single proteins. Parallel advances in mathematical models based on stochastic, discrete descriptions of biochemical processes have provided crucial insights into the underlying cellular mechanisms that control expression. The view that has emerged is rooted in a probabilistic understanding of cellular processes that quantitatively explains both the mean and the variation observed in gene-expression patterns among single cells. Thus, the close coupling between imaging and mathematical theory has established single-cell analysis as an essential branch of systems biology. [Copyright &y& Elsevier]

Studies of the intracellular localization of mRNA have clearly demonstrated that certain subsets of mRNA are concentrated in discrete locations within the cytoplasm. Localization is one aspect of the post-transcriptional control of gene expression, and is intertwined with the translation and turnover of mRNA to achieve the goal of local protein production. Different mechanisms have been identified that enable localized mRNAs to target different subcellular compartments, and recent advances in understanding these pathways is reviewed here. [Copyright &y& Elsevier]

Understanding the different molecular mechanisms responsible for gene expression has been a central interest of molecular biologists for several decades. Transcription, the initial step of gene expression, consists of converting the genetic code into a dynamic messenger RNA that will specify a required cellular function following translocation to the cytoplasm and translation. We now possess an in-depth understanding of the mechanism and regulations of transcription. By contrast, an understanding of the dynamics of an individual gene''s expression in real time is just beginning to emerge following recent technological developments. [Copyright &y& Elsevier]

We all know that gene expression occurs within cells, yet we do not think of expression in terms of its fundamental unit – a single cell. Instead, we understand the expression of genes in terms of a cell population as all of our information comes from samples containing millions of cells. From a complex mixture of cells, we attempt to infer the probable state of an average cell in the population. In truth, what we obtain is an averaged cell, a contrivance for representing biological knowledge beyond the limits of detection. We never know the variation among the members of the population that our methods average into a mean. Recent technological advances allow the precise measurement of single-cell transcriptional states to study this variability more rigorously. How genes are expressed in the population is strikingly different to what we have assumed from extrapolating to an average cell. Does the average cell actually exist? As we discuss, it is becoming increasingly clear that it doesn''t. [Copyright &y& Elsevier]

Genes are transcribed in irregular pulses of activity termed transcriptional bursts. Cellular differentiation requires coordinated gene expression; however, it is unknown whether the burst fraction (i.e. the number of active phases of transcription) or size/intensity (the number of RNA molecules produced within a burst) changes during cell differentiation. In the ocular lens, the positions of lens fiber cells correlate precisely with their differentiation status, and the most advanced cells degrade their nuclei. Here, we examined the transcriptional parameters of the β-actin and lens differentiation–specific α-, β-, and γ-crystallin genes by RNA fluorescent in situ hybridization (FISH) in the lenses of embryonic day (E) E12.5, E14.5, and E16.5 mouse embryos and newborns. We found that cellular differentiation dramatically alters the burst fraction in synchronized waves across the lens fiber cell compartment with less dramatic changes in burst intensity. Surprisingly, we observed nascent transcription of multiple genes in nuclei just before nuclear destruction. Nuclear condensation was accompanied by transfer of nuclear proteins, including histone and nonhistone proteins, to the cytoplasm. Although lens-specific deletion of the chromatin remodeler SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (Smarca5/Snf2h) interfered with denucleation, persisting nuclei remained transcriptionally competent and exhibited changes in both burst intensity and fraction depending on the gene examined. Our results uncover the mechanisms of nascent transcriptional control during differentiation and chromatin remodeling, confirm the burst fraction as the major factor adjusting gene expression levels, and reveal transcriptional competence of fiber cell nuclei even as they approach disintegration. [ABSTRACT FROM AUTHOR]

Recent developments in reagent design can address problems in single cells that were not previously approachable. We have attempted to foresee what will become possible, and the sorts of biological problems that become tractable with these novel reagents. We have focused on the novel fluorescent proteins that allow convenient multiplexing, and provide for a time-dependent analysis of events in single cells. Methods for fluorescently labeling specific molecules, including endogenously expressed proteins and mRNA have progressed and are now commonly used in a variety of organisms. Finally, sensitive microscopic methods have become more routine practice. This article emphasizes that the time is right to coordinate these approaches for a new initiative on single cell imaging of biological molecules. [Copyright &y& Elsevier]

Abstract: The Karpas-620 human myeloma cell line (HMCL) expresses high levels of Cyclin D1 (CCND1), but has a der(8)t(8;11) and a der(14)t(8;14), and not a conventional t(11;14). Fluorescent in situ hybridization (FISH) and array comparative genomic hybridization (aCGH) studies suggest that der(14)t(11;14) from a primary translocation underwent a secondary translocation with chromosome 8 to generate der(8)t(8;[14];11) and der(14)t(8;[11];14). Both secondary derivatives share extensive identical sequences from chromosomes 8, 11, and 14, including MYC and the 3′ IgH enhancers. Der(14), with MYC located ∼700kb telomeric to the 3′IGH enhancer, expresses MYC. By contrast, der(8), with both CCND1 and MYC repositioned near a 3′IGH enhancer, expresses CCND1, which is telomeric of the enhancer, but not MYC, which is centromeric to the enhancer. The secondary translocation that dysregulated MYC resulted in extensive regions from both donor chromosomes being transmitted to both derivative chromosomes, suggesting a defect in DNA recombination or repair in the myeloma tumor cell. [Copyright &y& Elsevier]