Organizing the genome: enhancers and insulators.

Enhancers can activate their target genes over large linear distances. Insulators can delimit the influence of an enhancer to an appropriate target. There are a number of intertwined mechanisms by which the regulatory functions of enhancers and insulators might be carried out at the level of the chromatin fiber. Recent evidence suggests that both enhancers and insulators participate in higher-order organization of chromatin in the nucleus and in localization of their regulated sequences to both subnuclear structures and compartments. Novel experimental approaches are helping to reveal the mechanisms underlying nuclear organization of developmentally regulated genes.     

Trafficking Guidance Receptors

Wiring of the brain relies initially on the correct outgrowth of axons to reach the appropriate target area for innervation. A large number of guidance receptors present in the plasma membrane of axonal growth cones and elsewhere on the neuron read and execute directional cues present in the extracellular environment of the navigating growth cone. The exact timing, levels, and localization of expression of the guidance receptors in the plasma membrane therefore determine the outcome of guidance decisions. Many guidance receptors are localized in exquisitely precise spatial and temporal patterns. The cellular mechanisms ensuring these localization patterns include spatially accurate sorting after synthesis in the secretory pathway, retrieval of inappropriately expressed receptors by endocytosis followed by degradation or recycling, and restriction of diffusion. This article will discuss the machinery and regulation underlying the restricted distribution of membrane receptors, focusing on the currently best-studied example, the L1 cell adhesion molecule. In addition to the long-range mechanisms ensuring appropriate localization, the same mechanisms can act locally to adjust levels and localization of receptors. These local mechanisms are regulated by ligand binding and subsequent activation of local signaling cascades. It is likely that the localization of all guidance receptors is regulated by a combination of sorting, retrieval, recycling and retention, similar to the ones we discuss here for L1.     

Flagellar protein localization mediated by a calcium-myristoyl/palmitoyl switch mechanism

The mechanisms by which proteins are targeted to flagella and cilia are poorly understood. We set out to determine the basis for the specific localization of a 24 kDa flagellar calcium-binding protein (FCaBP) expressed in all life cycle stages of Trypanosoma cruzi. Through the study of trypanosome transfectants expressing various FCaBP deletion mutants, we found that the N-terminal 24 amino acids of the protein are necessary and sufficient for flagellar localization. Subsequent experiments revealed that FCaBP is myristoylated and palmitoylated and, in fact, is one of very few proteins in the cell possessing these acyl modifications. Both fatty acids are required for flagellar localization, suggesting that FCaBP localization may be mediated through association with the flagellar plasma membrane. Indeed, FCaBP associates with the flagellar membrane in a calcium-dependent manner, reminiscent of the recoverin family of calcium-myristoyl switch proteins. Thus, FCaBP is a novel member of the calcium-acyl switch protein family and is the only member described to date that requires two fatty acid modifications for specific membrane association. Its unique localization mechanism is the first described for any flagellar protein. The existence of such a protein in this protozoan suggests that acylation and calcium switch mechanisms for regulated membrane association are conserved among eukaryotes.     

Fundamental features of chromatin structure

Which mechanisms regulate nuclear plasticity? Part of the answer to that question lies in understanding how genes are expressed and regulated in the context of chromatin structure. It is now clear that the genes are regulated in discrete and controlled stages, from packaging into chromatin to their localization within the nucleus. Whereas the genetic information provides the framework for the manufacture of all proteins necessary to create a living cell, chromatin structure controls how, where, and when the genetic information should be used. In this minireview, I summarize the main characteristics of chromatin structure and highlight some of the modifications usually associated with the regulation of gene expression.     

Silent glutamatergic synapses in the mammalian brain

Excitatory synaptic transmission in the mammalian brain is mediated primarily by alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors that are thought to be co-localized at individual synapses. However, recent electrophysiological and anatomical data suggest that the synaptic localization of AMPA and NMDA receptors may be independently regulated by neural activity. These data are reviewed here and the implications of these findings for the mechanisms underlying synaptic plasticity are discussed.     

Localization,anchoring and translational control of oskar,gurken, bicoid and nanos mRNA during Drosophila oogenesis

mRNA localization, and translation that is regulated spatially and temporally, are key mechanisms in the execution of polarized developmental programs. For over two decades, the Drosophila oocyte has served as a valuable model to study these mechanisms. Genetic and biochemical studies in flies have greatly contributed to the identification and understanding of factors that govern RNA localization and translational control. Embryonic axis formation is mediated through the subcellular localization and precise translational regulation of four key determinant mRNAs during oogenesis encoded by oskar, bicoid, gurken and nanos. In this review we aim to summarize recent insights into the mechanisms governing the asymmetric distribution and translation of these mRNAs.     

Evidence for common machinery utilized by the early and late RNA localization pathways in Xenopus oocytes

In Xenopus, an early and a late pathway exist for the selective localization of RNAs to the vegetal cortex during oogenesis. Previous work has suggested that distinct cellular mechanisms mediate localization during these pathways. Here, we provide several independent lines of evidence supporting the existence of common machinery for RNA localization during the early and late pathways. Data from RNA microinjection assays show that early and late pathway RNAs compete for common localization factors in vivo, and that the same short RNA sequence motifs are required for localization during both pathways. In addition, quantitative filter binding assays demonstrate that the late localization factor Vg RBP/Vera binds specifically to several early pathway RNA localization elements. Finally, confocal imaging shows that early pathway RNAs associate with a perinuclear microtubule network that connects to the mitochondrial cloud of stage I oocytes suggesting that motor driven transport plays a role during the early pathway as it does during the late pathway. Taken together, our data indicate that common machinery functions during the early and late pathways. Thus, RNA localization to the vegetal cortex may be a regulated process such that differential interactions with basal factors determine when distinct RNAs are localized during oogenesis.     

Chronic activation of ERK and neurodegenerative diseases

The extracellular-signal regulated kinases 1/2 (ERK or ERKs) are involved in the regulation of important neuronal functions, including neuronal plasticity in normal and pathological conditions. We present findings that support the notion that the kinetics and localization of ERK are intrinsically linked, in that the duration of ERK activation dictates its subcellular compartmentalization and/or trafficking. The latter, in turn, dictates whether ERK-expressing cells would enter a program of cell death, survival or differentiation. We summarize experimental data showing that chronic activation of ERK plays a role in the mechanisms that trigger neurodegeneration. We also discuss how MKPs, members of the subclass of dual specificity phosphatases, might be the link between ERK kinetics and its subcellular localization.     

RNA localization in the cytoplasm

RNA localization in subcytoplasmic areas is a process known for more than twenty years, and more than a hundred RNAs have now been shown to be spatially regulated. In most cases, RNA localization is involved in cell polarity, either by reading spatial clues and translating them into a spatial regulation of gene expression, or more directly by controlling cytoskeletal polarity. In this review, the various functions of RNA localization will be presented, and by analyzing two examples, Ash1 mRNA in yeast and retroviral genomic RNAs in mammals, the reader will be taken step by step into the detailed mechanisms of this fascinating process.     

Regulation of ROCKII membrane localization through its C-terminus

RhoA activated kinases (ROCKs) are potent effectors of RhoA signaling for regulation of the cytoskeleton. ROCKs have been shown to be localized to several different subcellular locations, suggesting that its localization is context specific and regulated. However, the signaling mechanisms that control ROCK localization have not been clearly described. In this study we measured ROCKII localization following stimulation with the chemokine CXCL12 or adhesion to collagen 1. Strikingly, each of these extracellular signals targeted ROCKII to membrane protrusions. We further determined that both RhoA and PI3-kinase signaling are required for these stimuli to induce efficient membrane localization. Furthermore, we used a mutational approach to show that two separate domains predicted to respond to these localization signals, the Rho Binding Domain (RBD) and the Pleckstrin Homology domain (PH). Unexpectedly, we found that these two domains work synergistically to lead to membrane localization. This suggests a novel mechanism for controlling ROCKII localization at the membrane, in which the ROCKII C-terminus acts as a coincidence detector for spatial regulatory signals. In other words, efficient membrane targeting requires the ROCKII RBD to receive the RhoA signal and the PH domain to receive the phospholipid signal.     

Aurora B phosphorylates multiple sites on mitotic centromere-associated kinesin to spatially and temporally regulate its function

Chromosome congression and segregation require the proper attachment of microtubules to the two sister kinetochores. Disruption of either Aurora B kinase or the Kinesin-13 mitotic centromere-associated kinesin (MCAK) increases chromosome misalignment and missegregation due to improper kinetochore-microtubule attachments. MCAK localization and activity are regulated by Aurora B, but how Aurora B phosphorylation of MCAK affects spindle assembly is unclear. Here, we show that the binding of MCAK to chromosome arms is also regulated by Aurora B and that Aurora B-dependent chromosome arm and centromere localization is regulated by distinct two-site phosphoregulatory mechanisms. MCAK association with chromosome arms is promoted by phosphorylation of T95 on MCAK, whereas phosphorylation of S196 on MCAK promotes dissociation from the arms. Although targeting of MCAK to centromeres requires phosphorylation of S110 on MCAK, dephosphorylation of T95 on MCAK increases the binding of MCAK to centromeres. Our study reveals a new role for Aurora B, which is to prevent excess MCAK binding to chromatin to facilitate chromatin-nucleated spindle assembly. Our study also shows that the interplay between multiple phosphorylation sites of MCAK may be critical to temporally and spatially control MCAK function.     

Localization-dependent oskar protein accumulation; control after the initiation of translation

The appearance of Oskar protein occurs coincident with localization of oskar mRNA to the posterior pole of the Drosophila oocyte, and earlier accumulation of the protein is prevented by translational repression. We find that the nascent polypeptide-associated complex (NAC) is required for correct localization of oskar mRNA. The timing of the defects suggests that, if NAC acts directly via an interaction with nascent Oskar protein, oskar mRNA should be undergoing translation prior to its localization. Polysome analysis confirms that oskar mRNA is associated with polysomes even in the absence of localization of the mRNA or accumulation of Oskar protein. Thus, the mechanisms that prevent accumulation of Oskar protein until it can be secured at the posterior pole of the oocyte include regulated degradation or inhibition of translational elongation.     

Local protein synthesis during axon guidance and synaptic plasticity

mRNA localization and regulated translation take central roles in axon guidance and synaptic plasticity. By spatially restricting gene expression within neurons, local protein synthesis provides growth cones and synapses with the capacity to autonomously regulate their structure and function. Studies in a variety of systems have provided insight into the specific roles of local protein synthesis during axonal navigation and during synaptic plasticity, and have begun to delineate the mechanisms underlying mRNA localization and regulated translation. Several powerful new tools have recently been developed to visualize each of these processes.     

Distinct subcellular localization patterns contribute to functional specificity of the Cln2 and Cln3 cyclins of Saccharomyces cerevisiae

The G(1) cyclins of budding yeast drive cell cycle initiation by different mechanisms, but the molecular basis of their specificity is unknown. Here we test the hypothesis that the functional specificity of G(1) cyclins is due to differential subcellular localization. As shown by indirect immunofluorescence and biochemical fractionation, Cln3p localization appears to be primarily nuclear, with the most obvious accumulation of Cln3p to the nuclei of large budded cells. In contrast, Cln2p localizes to the cytoplasm. We were able to shift localization patterns of truncated Cln3p by the addition of nuclear localization and nuclear export signals, and we found that nuclear localization drives a Cln3p-like functional profile, while cytoplasmic localization leads to a partial shift to a Cln2p-like functional profile. Therefore, forcing Cln3p into a Cln2p-like cytoplasmic localization pattern partially alters the functional specificity of Cln3p toward that of Cln2p. These results suggest that there are CLN-dependent cytoplasmic and nuclear events important for cell cycle initiation. This is the first indication of a cytoplasmic function for a cyclin-dependent kinase. The data presented here support the idea that cyclin function is regulated at the level of subcellular localization and that subcellular localization contributes to the functional specificity of Cln2p and Cln3p.     

The role of regulated CFTR trafficking in epithelial secretion

The focus of this review is the regulated trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) in distal compartments of the protein secretory pathway and the question of how changes in CFTR cellular distribution may impact on the functions of polarized epithelial cells. We summarize data concerning the cellular localization and activity of CFTR and attempt to synthesize often conflicting results from functional studies of regulated endocytosis and exocytosis in CFTR-expressing cells. In some instances, findings that are inconsistent with regulated CFTR trafficking may result from the use of overexpression systems or nonphysiological experimental conditions. Nevertheless, judging from data on other transporters, an appropriate cellular context is necessary to support regulated CFTR trafficking, even in epithelial cells. The discovery that disease mutations can influence CFTR trafficking in distal secretory and recycling compartments provides support for the concept that regulated CFTR recycling contributes to normal epithelial function, including the control of apical CFTR channel density and epithelial protein secretion. Finally, we propose molecular mechanisms for regulated CFTR endocytosis and exocytosis that are based on CFTR interactions with other proteins, particularly those whose primary function is membrane trafficking. These models provide testable hypotheses that may lead to elucidation of CFTR trafficking mechanisms and permit their experimental manipulation in polarized epithelial cells.