The role of the nuclear envelope in cellular organization

Over the last years it has become evident that the nuclear envelope (NE) is more than a passive membrane barrier that separates the nucleus from the cytoplasm. The NE not only controls the trafficking of macromolecules between the nucleoplasm and the cytosol, but also provides anchoring sites for chromosomes and cytoskeleton to the nuclear periphery. Targeting of chromatin to the NE might actually be part of gene expression regulation in eukaryotes. Mutations in certain NE proteins are associated with a diversity of human diseases, including muscular dystrophy, neuropathy, lipodistrophy, torsion dystonia and the premature aging condition progeria. Despite the importance of the NE for cell division and differentiation, relatively little is known about its biogenesis and its role in human diseases. It is our goal to provide a comprehensive view of the NE and to discuss possible implications of NE-associated changes for gene expression, chromatin organization and signal transduction. Received 8 August 2005; received after revision 13 October 2005; accepted 13 October 2005     

Regulation of microRNA biogenesis and turnover by animals and their viruses

MicroRNAs (miRNAs) are a ubiquitous component of gene regulatory networks that modulate the precise amounts of proteins expressed in a cell. Despite their small size, miRNA genes contain various recognition elements that enable specificity in when, where and to what extent they are expressed. The importance of precise control of miRNA expression is underscored by functional studies in model organisms and by the association between miRNA mis-expression and disease. In the last decade, identification of the pathways by which miRNAs are produced, matured and turned-over has revealed many aspects of their biogenesis that are subject to regulation. Studies in viral systems have revealed a range of mechanisms by which viruses target these pathways through viral proteins or non-coding RNAs in order to regulate cellular gene expression. In parallel, a field of study has evolved around the activation and suppression of antiviral RNA interference (RNAi) by viruses. Virus encoded suppressors of RNAi can impact miRNA biogenesis in cases where miRNA and small interfering RNA pathways converge. Here we review the literature on the mechanisms by which miRNA biogenesis and turnover are regulated in animals and the diverse strategies that viruses use to subvert or inhibit these processes.     

Multiple roles for the actin cytoskeleton during regulated exocytosis

Regulated exocytosis is the main mechanism utilized by specialized secretory cells to deliver molecules to the cell surface by virtue of membranous containers (i.e., secretory vesicles). The process involves a series of highly coordinated and sequential steps, which include the biogenesis of the vesicles, their delivery to the cell periphery, their fusion with the plasma membrane, and the release of their content into the extracellular space. Each of these steps is regulated by the actin cytoskeleton. In this review, we summarize the current knowledge regarding the involvement of actin and its associated molecules during each of the exocytic steps in vertebrates, and suggest that the overall role of the actin cytoskeleton during regulated exocytosis is linked to the architecture and the physiology of the secretory cells under examination. Specifically, in neurons, neuroendocrine, endocrine, and hematopoietic cells, which contain small secretory vesicles that undergo rapid exocytosis (on the order of milliseconds), the actin cytoskeleton plays a role in pre-fusion events, where it acts primarily as a functional barrier and facilitates docking. In exocrine and other secretory cells, which contain large secretory vesicles that undergo slow exocytosis (seconds to minutes), the actin cytoskeleton plays a role in post-fusion events, where it regulates the dynamics of the fusion pore, facilitates the integration of the vesicles into the plasma membrane, provides structural support, and promotes the expulsion of large cargo molecules.     

Chromatin-remodeling complex specificity and embryonic vascular development

Vascular development is a dynamic process that relies on the coordinated expression of numerous genes, but the factors that regulate gene expression during blood vessel development are not well defined. ATP-dependent chromatin-remodeling complexes are gaining attention for their specific temporal and spatial effects on gene expression during vascular development. Genetic mutations in chromatin-remodeling complex subunits are revealing roles for the complexes in vascular signaling pathways at discrete developmental time points. Phenotypic analysis of these models at various stages of vascular development will continue to expand our understanding of how chromatin remodeling impacts new blood vessel growth. Such research could also provide novel therapeutic targets for the treatment of vascular pathologies.     

RNA processing and human disease

Gene expression involves multiple regulated steps leading from gene to active protein. Many of these steps involve some aspect of RNA processing. Diseases caused by mutations that directly affect RNA processing are relatively rare compared with mutations that disrupt protein function. The vast majority of diseases of RNA processing result from loss of function of a single gene due to mutations in cis-acting elements required for pre-messenger RNA (mRNA) splicing. However, a few diseases are caused by alterations in the trans-acting factors required for RNA processing and in the vast majority of cases it is the pre-mRNA splicing machinery that is affected. Clearly, alterations that disrupt splicing of pre-mRNAs from large numbers of genes would be lethal at the cellular level. A common theme among these diseases is that only subsets of genes are affected. This is consistent with an emerging view that different subsets of exons require different sets of cis-acting elements and trans-acting factors.     

Synthetic oligonucleotides as RNA mimetics: 2'-modified Rnas and N3'-->P5' phosphoramidates

Significant interest in synthetic DNA and RNA oligonucleotides and their analogues has marked the past two decades of research in chemistry and biochemistry. This attention was largely determined by the great potential of these compounds for various therapeutic applications such as antisense, antigene and ribozyme-based agents. Modified oligonucleotides have also become powerful molecular biological and biochemical research tools that allow fast and efficient regulation of gene expression and gene functions in vitro and in vivo. These applications in turn are based on the ability of the oligonucleotides to form highly sequence-specific complexes with nucleic acid targets of interest. This review summarizes recent advances in the design, synthesis, biochemical and structural properties of various RNA analogues. These comprise 3'-modified oligonucleotide N3'-->P5' phosphoramidates, analogues with modifications at the 2'-position of nucleoside sugar rings, or combinations of the two. Among the properties of the RNA minetics reviewed here are the thermal stability of their duplexes and triplexes, hydrolytic resistance to cellular nucleases and biological activity in in vitro and in vivo systems. In addition, key structural aspects of the complexes formed by the RNA analogues, including interaction with water molecules and ions, are analyzed and presented.     

Biomimetic actuators: where technology and cell biology merge

The structural and functional analysis of biological macromolecules has reached a level of resolution that allows mechanistic interpretations of molecular action, giving rise to the view of enzymes as molecular machines. This machine analogy is not merely metaphorical, as bio-analogous molecular machines actually are being used as motors in the fields of nanotechnology and robotics. As the borderline between molecular cell biology and technology blurs, developments in the engineering and material sciences become increasingly instructive sources of models and concepts for biologists. In this review, we provide a--necessarily selective--summary of recent progress in the usage of biological and biomimetic materials as actuators in artificial environments, focussing on motors built from DNA, classical cellular motor systems (tubulin/kinesin, actin/myosin), the rotary motor F1F0-ATPase and protein-based 'smart' materials.     

DNA damage repair and transcription

DNA mutations and aberrations are a problem for all forms of life. Eukaryotes specifically have developed ways of identifying and repairing various DNA mutations in a complex and refractory chromatin environment. The chromatin structure is much more than a packaging unit for DNA; it is dynamic. Cells utilize and manipulate chromatin for gene regulation, genome organization and maintenance of genome integrity. Once a DNA aberration has occurred, the various DNA repair machineries interact with chromatin proteins, such as the histone variant H2A.X, and chromatin remodeling machines of the SWI/SNF family to gain access and repair the lesion in a timely manner. Recent studies have thus begun to address the roles of chromatin proteins in DNA repair as well as to dissect the functions of DNA repair machinery in vitro on more physiological, nucleosomal templates.     

KIF1Bβ transports dendritically localized mRNPs in neurons and is recruited to synapses in an activity-dependent manner

KIF1Bβ is a kinesin-like, microtubule-based molecular motor protein involved in anterograde axonal vesicular transport in vertebrate and invertebrate neurons. Certain KIF1Bβ isoforms have been implicated in different forms of human neurodegenerative disease, with characterization of their functional integration and regulation in the context of synaptic signaling still ongoing. Here, we characterize human KIF1Bβ (isoform NM015074), whose expression we show to be developmentally regulated and elevated in cortical areas of the CNS (including the motor cortex), in the hippocampus, and in spinal motor neurons. KIF1Bβ localizes to the cell body, axon, and dendrites, overlapping with synaptic-vesicle and postsynaptic-density structures. Correspondingly, in purified cortical synaptoneurosomes, KIF1Bβ is enriched in both pre- and postsynaptic structures, forming detergent-resistant complexes. Interestingly, KIF1Bβ forms RNA–protein complexes, containing the dendritically localized Arc and Calmodulin mRNAs, proteins previously shown to be part of RNA transport granules such as Purα, FMRP and FXR2P, and motor protein KIF3A, as well as Calmodulin. The interaction between KIF1Bβ and Calmodulin is Ca⁺²-dependent and takes place through a domain mapped at the carboxy-terminal tail of the motor. Live imaging of cortical neurons reveals active movement by KIF1Bβ at dendritic processes, suggesting that it mediates the transport of dendritically localized mRNAs. Finally, we show that synaptic recruitment of KIF1Bβ is activity-dependent and increased by stimulation of metabotropic or ionotropic glutamate receptors. The activity-dependent synaptic recruitment of KIF1Bβ, its interaction with Ca²⁺ sensor Calmodulin, and its new role as a dendritic motor of ribonucleoprotein complexes provide a novel basis for understanding the concerted co-ordination of motor protein mobilization and synaptic signaling pathways.     

A llama-derived gelsolin single-domain antibody blocks gelsolin–G-actin interaction

RNA interference has tremendously advanced our understanding of gene function but recent reports have exposed undesirable side-effects. Recombinant Camelid single-domain antibodies (VHHs) provide an attractive means for studying protein function without affecting gene expression. We raised VHHs against gelsolin (GsnVHHs), a multifunctional actin-binding protein that controls cellular actin organization and migration. GsnVHH-induced delocalization of gelsolin to mitochondria or the nucleus in mammalian cells reveals distinct subpopulations including free gelsolin and actin-bound gelsolin complexes. GsnVHH 13 specifically recognizes Ca²⁺-activated gelsolin (K _d ~10 nM) while GsnVHH 11 binds gelsolin irrespective of Ca²⁺ (K _d ~5 nM) but completely blocks its interaction with G-actin. Both GsnVHHs trace gelsolin in membrane ruffles of EGF-stimulated MCF-7 cells and delay cell migration without affecting F-actin severing/capping or actin nucleation activities by gelsolin. We conclude that VHHs represent a potent way of blocking structural proteins and that actin nucleation by gelsolin is more complex than previously anticipated.