Differential nuclear import sets the timing of protein access to the embryonic genome.

The development of a fertilized egg to an embryo requires the proper temporal control of gene expression. During cell differentiation, timing is often controlled via cascades of transcription factors (TFs). However, in early development, transcription is often inactive, and many TF levels stay constant, suggesting that alternative mechanisms govern the observed rapid and ordered onset of gene expression.

Nucleoplasmin is a limiting component in the scaling of nuclear size with cytoplasmic volume.

How nuclear size is regulated relative to cell size is a fundamental cell biological question. Reductions in both cell and nuclear sizes during embryogenesis provide a robust scaling system to study mechanisms of nuclear size regulation. To test if the volume of embryonic cytoplasm is limiting for nuclear growth, we encapsulated gastrula-stage embryonic cytoplasm and nuclei in droplets of defined volume using microfluidics.

Induction of a Spindle-Assembly-Competent M Phase in Xenopus Egg Extracts.

Normal mitotic spindle assembly is a prerequisite for faithful chromosome segregation and unperturbed cell-cycle progression. Precise functioning of the spindle machinery relies on conserved architectural features, such as focused poles, chromosome alignment at the metaphase plate, and proper spindle length. These morphological requirements can be achieved only within a compositionally distinct cytoplasm that results from cell-cycle-dependent regulation of specific protein levels and specific post-translational modifications.

Tau-based fluorescent protein fusions to visualize microtubules.

The ability to visualize cytoskeletal proteins and their dynamics in living cells has been critically important in advancing our understanding of numerous cellular processes, including actin- and microtubule (MT)-dependent phenomena such as cell motility, cell division, and mitosis. Here, we describe a novel set of fluorescent protein (FP) fusions designed specifically to visualize MTs in living systems using fluorescence microscopy. Each fusion contains a FP module linked in frame to a modified phospho-deficient version of the MT-binding domain of Tau (mTMBD).

Changes in cytoplasmic volume are sufficient to drive spindle scaling.

The mitotic spindle must function in cell types that vary greatly in size, and its dimensions scale with the rapid, reductive cell divisions that accompany early stages of development. The mechanism responsible for this scaling is unclear, because uncoupling cell size from a developmental or cellular context has proven experimentally challenging. We combined microfluidic technology with Xenopus egg extracts to characterize spindle assembly within discrete, geometrically defined volumes of cytoplasm.

New nuclear partners for nucleosome assembly protein 1: unexpected associations.

Histone chaperones are important players in chromatin dynamics. They are instrumental in nucleosome assembly and disassembly and in histone variant exchange reactions that occur during DNA transactions. The molecular mechanisms of their action are not well understood and may involve interactions with various protein partners in the context of the nucleus.

The middle region of an HP1-binding protein, HP1-BP74, associates with linker DNA at the entry/exit site of nucleosomal DNA.

In higher eukaryotic cells, DNA molecules are present as chromatin fibers, complexes of DNA with various types of proteins; chromatin fibers are highly condensed in metaphase chromosomes during mitosis. Although the formation of the metaphase chromosome structure is essential for the equal segregation of replicated chromosomal DNA into the daughter cells, the mechanism involved in the organization of metaphase chromosomes is poorly understood. To identify proteins involved in the formation and/or maintenance of metaphase chromosomes, we examined proteins that dissociated from isolated human metaphase chromosomes by 0.

Nucleosome assembly depends on the torsion in the DNA molecule: a magnetic tweezers study.

We have used magnetic tweezers to study nucleosome assembly on topologically constrained DNA molecules. Assembly was achieved using chicken erythrocyte core histones and histone chaperone protein Nap1 under constant low force. We have observed only partial assembly when the DNA was topologically constrained and much more complete assembly on unconstrained (nicked) DNA tethers.

Simplified method for recombinant linker histone H1 purification.

We report a simplified alternative protocol for purification of recombinant linker histone H1 under non-denaturing conditions. This method takes advantage of the strong affinity of H1 to DNA and comprises nucleoprotein complex extraction from the lysate of bacterial cells overexpressing the protein, followed by two ion-exchange purification steps. The purity of the protein was at least 95%; the purified H1 was tested for nucleosome binding and was successfully fluorescently labeled for further studies.

Does BLM helicase unwind nucleosomal DNA?

RecQ helicases maintain chromosome stability by resolving several highly specific DNA structures. BLM, the protein mutated in Bloom's syndrome, is a member of the RecQ helicase family, and possesses both DNA-unwinding and strand-annealing activity. In this study, we have investigated the unwinding activity of BLM on nucleosomal DNA, the natural nuclear substrate for the enzyme.

H2A.Z and H3.3 histone variants affect nucleosome structure: biochemical and biophysical studies.

Histone variants play important roles in regulation of chromatin structure and function. To understand the structural role played by histone variants H2A.Z and H3.

Nucleosome dynamics as studied by single-pair fluorescence resonance energy transfer: a reevaluation.

Accessibility of nucleosomal DNA to protein factor binding is ensured by at least three mechanisms: post-synthetic modifications to the histones, chromatin remodeling, and spontaneous unwrapping of the DNA from the histone core. We have previously used single-pair fluorescence resonance energy transfer (spFRET) experiments to investigate long-range conformational fluctuations in nucleosomal DNA (Tomschik M, Zheng H, van Holde K, Zlatanova J, Leuba SH in Proc Natl Acad Sci USA 102(9):3278-3283, 2005). Recent work has drawn attention to a major artifact in such studies due to photoblinking of the acceptor fluorophore.

The linker-protein network: control of nucleosomal DNA accessibility.

Numerous studies have recently addressed the accessibility of nucleosomal DNA to protein factors. Two popular concepts - the histone code and chromatin remodeling - consider the nucleosome as a passive entity that 'waits' to be marked by histone modifications and is 'mobilized' by ATP-dependent remodelers. Here, we propose a holistic view of the nucleosome as an active, dynamic entity, the accessibility of which is controlled by binding of different linker proteins to the DNA entry/exit site.

Nap1: taking a closer look at a juggler protein of extraordinary skills.

The nucleosome assembly protein Nap1 is used extensively in the chromatin field to reconstitute nucleosomal templates for structural and functional studies. Beyond its role in facilitating experimental investigation of nucleosomes, the highly conserved Nap1 is one of the best-studied members of the histone chaperone group. Here we review its numerous functions, focusing mainly on its roles in assembly and disassembly of the nucleosome particle, and its interactions with chromatin remodeling factors.

Fast, long-range, reversible conformational fluctuations in nucleosomes revealed by single-pair fluorescence resonance energy transfer.

The nucleosome core particle, the basic repeated structure in chromatin fibers, consists of an octamer of eight core histone molecules, organized as dimers (H2A/H2B) and tetramers [(H3/H4)2] around which DNA wraps tightly in almost two left-handed turns. The nucleosome has to undergo certain conformational changes to allow processes that need access to the DNA template to occur. By single-pair fluorescence resonance energy transfer, we demonstrate fast, long-range, reversible conformational fluctuations in nucleosomes between two states: fully folded (closed), with the DNA wrapped around the histone core, or open, with the DNA significantly unraveled from the histone octamer.

Assembly of single chromatin fibers depends on the tension in the DNA molecule: magnetic tweezers study.

We have used magnetic tweezers to study in real time chaperone-mediated chromatin assembly/disassembly at the level of single chromatin fibers. We find a strong dependence of the rate of assembly on the exerted force, with strong inhibition of assembly at forces exceeding 10 pN. During assembly, and especially at higher forces, occasional abrupt increases in the length of the fiber were observed, giving a clear indication of reversibility of the assembly process.