Melpomeni Platani

Cajal Body dynamics and association with chromatin are ATP-dependent

Cajal bodies (CBs) are nuclear organelles that contain factors required for splicing, ribosome biogenesis and transcription. Our previous analysis in living cells showed that CBs are dynamic structures. Here, we show that CB mobility is described by anomalous diffusion and that bodies alternate between association with chromatin and diffusion within the interchromatin space. CB mobility increases after ATP depletion and inhibition of transcription, suggesting that the association of CB and chromatin requires ATP and active transcription. This behaviour is fundamentally different from the ATP-dependent mobility observed for chromatin and suggests that a novel mechanism governs CB, and possibly other, nuclear body dynamics.

Direct membrane protein–DNA interactions required early in nuclear envelope assembly

Among the earliest events in postmitotic nuclear envelope (NE) assembly are the interactions between chromatin and the membranes that will fuse to form the NE. It has been proposed that interactions between integral NE proteins and chromatin proteins mediate initial membrane recruitment to chromatin. We show that several transmembrane NE proteins bind to DNA directly and that NE membrane proteins as a class are enriched in long, basic domains that potentially bind DNA. Membrane fractions that are essential for NE formation are shown to bind directly to protein-free DNA, and our data suggest that these interactions are critical for early steps in NE assembly.

Macromolecular mobility inside the cell nucleus

Abstract The highly organized interior of the living cell imposes major constraints on the dynamic properties of macromolecules and macromolecular complexes in both the cytoplasm and the nucleus. Although there are many examples of cytoplasmic molecular assemblies that can be propelled by molecular motors along microtubules and actin filaments, a view that had emerged during the past few years was that intranuclear mobility results largely from incessant Brownian motion. However, more recent studies now suggest that the movement of an increasing number of macromolecular entities inside the nucleus is controlled by a combination of passive diffusion and ATP-dependent processes.

Nup53 Is Required for Nuclear Envelope and Nuclear Pore Complex Assembly

Transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). These structures are composed of various subcomplexes of proteins that are each present in multiple copies and together establish the eightfold symmetry of the NPC. One evolutionarily conserved subcomplex of the NPC contains the nucleoporins Nup53 and Nup155. Using truncation analysis, we have defined regions of Nup53 that bind to neighboring nucleoporins as well as those domains that target Nup53 to the NPC in vivo. Using this information, we investigated the role of Nup53 in NE and NPC assembly using Xenopus egg extracts. We show that both events require Nup53. Importantly, the analysis of Nup53 fragments revealed that the assembly activity of Nup53 depleted extracts could be reconstituted using a region of Nup53 that binds specifically to its interacting partner Nup155. On the basis of these results, we propose that the formation of a Nup53-Nup155 complex plays a critical role in the processes of NPC and NE assembly.

In Vivo Imaging of Mammalian Cells: Image Acquisition and Analysis

Live cell imaging provides a powerful technique for the analysis of molecular dynamics within cells. Advances in imaging technology and probe design have established this approach as an important tool in modern biology. It is now possible to obtain commercial turnkey systems for digital imaging using a number of different imaging modalities. Nevertheless, it still requires considerable technical care and expertise to conduct a successful experiment. To perform a successful imaging experiment, it is important to maximize the signal-to-noise ratio (S:N) while minimizing damage to the cells. In this article, we focus on the use of fluorescence microscopy in live cell imaging, although most of the points discussed are relevant to any type of imaging. We describe many of the methods and considerations that are required for performing a successful imaging experiment in living cells. However, we do not provide a single recipe for success: The approach is much too empirical and depends on careful observation of the particular cells under investigation.

In Vivo Imaging of Mammalian Cells: Cell Engineering and Viability

Cold Spring Harb Protoc Jason R. Swedlow, Paul D. Andrews and Melpomeni Platani In Vivo Imaging of Mammalian Cells: Cell Engineering and Viability Service Email Alerting click here. Receive free email alerts when new articles cite this article Categories Subject Cold Spring Harbor Protocols. Browse articles on similar topics from (338 articles) Visualization, general (458 articles) Visualization (150 articles) Video Imaging / Time Lapse Imaging (226 articles) Live Cell Imaging (272 articles) Labeling for Imaging (235 articles) In Vivo Imaging (520 articles) Imaging/Microscopy, general (218 articles) Imaging for Neuroscience (192 articles) Imaging Development (200 articles) Fluorescent Proteins (347 articles) Fluorescence (565 articles) Developmental Biology (435 articles) Cell Imaging (241 articles) Cell Culture (989 articles) Cell Biology, general