Cell cycle progression is regulated by a wide variety of external factors, amongst them are growth factors and extracellular matrix factors. During the last decades evidence has been obtained that reactive oxygen species (ROS) may also play an important role in cell cycle progression. ROS may be generated by external and internal factors. In this overview we describe briefly the generation of ROS and their effects on processes that have been demonstrated to play an essential role in cell cycle progression, including such systems as signal transduction cascades, protein ubiquitination and degradation, and the cytoskeleton. These different effects of ROS influence cell cycle progression dependent upon the amount and duration of ROS exposure. Activation of growth factor stimulated signaling cascades by low levels of ROS result in increased cell cycle progression, or, in case of prolonged exposure, to a differentiation like growth arrest. From many studies it seems clear that the cyclin kinase inhibitor protein p21 plays a prominent role, leading to cell cycle arrest at higher but not directly lethal levels of ROS. Dependent upon the nature of p21 induction, the cell cycle arrest may be transient, coupled to repair processes, or permanent. At high concentrations of ROS all of the above processes are activated, in combination with enhanced damage to the building blocks of the cell, leading to apoptosis or even necrosis.

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Over the past two decades, it has become increasingly apparent that Alzheimer’s disease neuropathology is characterized by activated microglia (brain resident macrophages) as well as the classic features of amyloid plaques and neurofibrillary tangles. The intricacy of microglial biology has also become apparent, leading to a heightened research interest in this particular cell type. Over the years a number of different microglial cell culturing techniques have been developed to study either primary mammalian microglia, or immortalized cell lines. Each microglial system has advantages and disadvantages and should be selected for its appropriateness in a particular research context. This review summarizes several of the most common microglial cell culture systems currently being employed in Alzheimer’s research including primary microglia; BV2 and N9 retroviral immortalized microglia; human immortalized microglia (HMO6); and spontaneously immortalized rodent microglial lines (EOC lines and HAPI cells). Particularities of cell culture requirements and characteristics of microglial behavior, especially in response to applied inflammogen stimuli, are compared and discussed across these cell types.

Axon formation, the initial step in establishing neuronal polarity, critically depends on local microtubule reorganization and is characterized by the formation of parallel microtubule bundles. How uniform microtubule polarity is achieved during axonal development remains an outstanding question. Here, we show that the tripartite motif containing (TRIM) protein TRIM46 plays an instructive role in the initial polarization of neuronal cells. TRIM46 is specifically localized to the newly specified axon and, at later stages, partly overlaps with the axon initial segment (AIS). TRIM46 specifically forms closely spaced parallel microtubule bundles oriented with their plus-end out. Without TRIM46, all neurites have a dendrite-like mixed microtubule organization resulting in Tau missorting and altered cargo trafficking. By forming uniform microtubule bundles in the axon, TRIM46 is required for neuronal polarity and axon specification in vitro and in vivo. Thus, TRIM46 defines a unique axonal cytoskeletal compartment for regulating microtubule organization during neuronal development.

Decrypting the structure, function, and molecular interactions of complex molecular machines in their cellular context and at atomic resolution is of prime importance for understanding fundamental physiological processes. Nuclear magnetic resonance is a wellestablished imaging method that can visualize cellular entities at the micrometer scale and can be used to obtain 3D atomic structures under in vitro conditions. Here, we introduce a solid-state NMR approach that provides atomic level insights into cell-associated molecular components. By combining dedicated protein production and labeling schemes with tailored solid-state NMR pulse methods, we obtained structural information of a recombinant integral membrane protein and the major endogenous molecular components in a bacterial environment. Our approach permits studying entire cellular compartments as well as cell-associated proteins at the same time and at atomic resolution. cellular envelope | Escherichia coli | lipoprotein | PagL | magic angle spinning P hysiological processes rely on the concerted action of molecular entities in and across different cellular compartments. Whereas advancements in molecular imaging have provided unprecedented insights into the macromolecular organization in the subnanometer range (1), studying atomic structure and motion in situ has been challenging for structural biology. NMR has provided insight into cellular processes (2-4) and can determine entire 3D molecular structures inside living cells (5) provided that molecular entities tumble rapidly in a cellular setting. In principle, solid-state NMR (ssNMR) spectroscopy offers a complementary spectroscopic tool to monitor molecular structure and dynamics at atomic resolution in a complex setting (see ref. 6 for a recent review). Indeed, ssNMR has already been used to study individual molecular components in the context of natural bilayers (7,8), bacterial cell walls (9), and cellular organelles (10).Here, we introduce a general approach to investigate structure and dynamics of an arbitrary molecular target and its potential molecular partners in a cellular setting. Our studies focuses on the Gram-negative bacterial cell that is characterized by a molecularly complex but architecturally unique envelope, consisting of two lipid bilayers, the inner and outer membrane (IM, OM), separated by the periplasm containing the peptidoglycan (PG) layer (Fig. 1A). The IM is a phospholipid bilayer and harbors α-helical proteins, whereas the OM is asymmetrical and consists of phospholipids, lipopolysaccharides (LPS), lipoproteins, and β-barrelfold integral membrane proteins. LPS forms the outermost layer of the OM and protects the cell against harmful compounds from the environment. PG is a large macromolecule that gives the cell its shape and rigidity.Using uniformly 13 C, 15 N-labeled cellular preparations of Escherichia coli, we characterized the structure and dynamics of a recombinant integral membrane protein (PagL) and other major endogenous molecular components of the cell envelope in...