Many signaling pathways regulate the function of the cellular cytoskeleton. Yet we know very little about the proteins involved in the cross-talk between the signaling and the cytoskeletal systems. Here we show that myosin II-B, an important cytoskeletal protein, resides in a complex with p21-activated kinase 1 (PAK1) and atypical protein kinase C (PKC) zeta (aPKC) and that the interaction between these proteins is EGF-dependent. We further show that PAK1 is involved in aPKC phosphorylation and that aPKC phosphorylates myosin II-B directly on a specific serine residue in an EGF-dependent manner. This latter phosphorylation is specific to isoform B of myosin II, and it leads to slower filament assembly of myosin II-B. Furthermore, a decrease in aPKC expression in the cells alters myosin II-B cellular organization. Our finding of a new signaling pathway involving PAK1, aPKC, and myosin II-B, which is implicated in myosin II-B filament assembly and cellular organization, provides an important link between the signaling system and cytoskeletal dynamics.
The organisation of the large volume of mammalian genomic DNA within cell nuclei requires mechanisms to regulate chromatin compaction involving the reversible formation of higher order structures. The compaction state of chromatin varies between interphase and mitosis and is also subject to rapid and reversible change upon ATP depletion/repletion. In this study we have investigated mechanisms that may be involved in promoting the hyper-condensation of chromatin when ATP levels are depleted by treating cells with sodium azide and 2-deoxyglucose. Chromatin conformation was analysed in both live and permeabilised HeLa cells using FLIM-FRET, high resolution fluorescence microscopy and by electron spectroscopic imaging microscopy. We show that chromatin compaction following ATP depletion is not caused by loss of transcription activity and that it can occur at a similar level in both interphase and mitotic cells. Analysis of both live and permeabilised HeLa cells shows that chromatin conformation within nuclei is strongly influenced by the levels of divalent cations, including calcium and magnesium. While ATP depletion results in an increase in the level of unbound calcium, chromatin condensation still occurs even in the presence of a calcium chelator. Chromatin compaction is shown to be strongly affected by small changes in the levels of polyamines, including spermine and spermidine. The data are consistent with a model in which the increased intracellular pool of polyamines and divalent cations, resulting from depletion of ATP, bind to DNA and contribute to the large scale hyper-compaction of chromatin by a charge neutralisation mechanism.
). † These authors contributed equally to this work. SUMMARYTargeted modification of the genome is an important genetic tool, which can be achieved via homologous, nonhomologous or site-specific recombination. Although numerous efforts have been made, such a tool does not exist for routine applications in plants. This work describes a simple and useful method for targeted mutagenesis or gene targeting, tailored to floral-dip transformation in Arabidopsis, by means of specific protein expression in the egg cell. Proteins stably or transiently expressed under the egg apparatus-specific enhancer (EASE) were successfully localized to the area of the egg cell. Moreover, a zinc-finger nuclease expressed under EASE induced targeted mutagenesis. Mutations obtained under EASE control corresponded to genetically independent events that took place specifically in the germline. In addition, RAD54 expression under EASE led to an approximately 10-fold increase in gene targeting efficiency, when compared with wild-type plants. EASE-controlled gene expression provides a method for the precise engineering of the Arabidopsis genome through temporally and spatially controlled protein expression. This system can be implemented as a useful method for basic research in Arabidopsis, as well as in the optimization of tools for targeted genetic modifications in crop plants.
Chromatin, once thought to serve only as a means to package DNA, is now recognized as a major regulator of gene activity. As a result of the wide range of methods used to describe the numerous levels of chromatin organization, the terminology that has emerged to describe these organizational states is often imprecise and sometimes misleading. In this review, we discuss our current understanding of chromatin architecture and propose terms to describe the various biochemical and structural states of chromatin.
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