Effects of macromolecular crowding on intracellular diffusion from a single particle perspective

Compared to biochemical reactions taking place in relatively well-defined aqueous solutions in vitro, the corresponding reactions happening in vivo occur in extremely complex environments containing only 60–70% water by volume, with the remainder consisting of an undefined array of bio-molecules. In a biological setting, such extremely complex and volume-occupied solution environments are termed ‘crowded’. Through a range of intermolecular forces and pseudo-forces, this complex background environment may cause biochemical reactions to behave differently to their in vitro counterparts. In this review, we seek to highlight how the complex background environment of the cell can affect the diffusion of substances within it. Engaging the subject from the perspective of a single particle’s motion, we place the focus of our review on two areas: (1) experimental procedures for conducting single particle tracking experiments within cells along with methods for extracting information from these experiments; (2) theoretical factors affecting the translational diffusion of single molecules within crowded two-dimensional membrane and three-dimensional solution environments. We conclude by discussing a number of recent publications relating to intracellular diffusion in light of the reviewed material

Molecular sorting by stochastic resonance

To sort a targeted species from a mixture, we introduce a procedure that relies on the enhancement of its effective diffusion coefficient. We use the formation of a host–guest complex between α-cyclodextrin and a dye to evidence the dye dispersion when the medium is submitted to an oscillating field. In particular, we demonstrate that the effective diffusion coefficient of the dye may be increased far beyond its intrinsic value by tuning the driving field frequency in the stochastic resonance regime. We use this effect to selectively sort from a mixture a dye that is addressed by its rate constants for association with α-cyclodextrin

Intramolecular and intermolecular interactions of protein kinase B define its activation in vivo.

Protein kinase B (PKB/Akt) is a pivotal regulator of diverse metabolic, phenotypic, and antiapoptotic cellular controls and has been shown to be a key player in cancer progression. Here, using fluorescent reporters, we shown in cells that, contrary to in vitro analyses, 3-phosphoinositide-dependent protein kinase 1 (PDK1) is complexed to its substrate, PKB. The use of Förster resonance energy transfer detected by both frequency domain and two-photon time domain fluorescence lifetime imaging microscopy has lead to novel in vivo findings. The preactivation complex of PKB and PDK1 is maintained in an inactive state through a PKB intramolecular interaction between its pleckstrin homology (PH) and kinase domains, in a "PH-in" conformer. This domain-domain interaction prevents the PKB activation loop from being phosphorylated by PDK1. The interactive regions for this intramolecular PKB interaction were predicted through molecular modeling and tested through mutagenesis, supporting the derived model. Physiologically, agonist-induced phosphorylation of PKB by PDK1 occurs coincident to plasma membrane recruitment, and we further shown here that this process is associated with a conformational change in PKB at the membrane, producing a "PH-out" conformer and enabling PDK1 access the activation loop. The active, phosphorylated, "PH-out" conformer can dissociate from the membrane and retain this conformation to phosphorylate substrates distal to the membrane. These in vivo studies provide a new model for the mechanism of activation of PKB. This study takes a crucial widely studied regulator (physiology and pathology) and addresses the fundamental question of the dynamic in vivo behaviour of PKB with a detailed molecular mechanism. This has important implications not only in extending our understanding of this oncogenic protein kinase but also in opening up distinct opportunities for therapeutic intervention

Synthesis and Analysis of Novel Glycerolipids for the Treatment of Metabolic Syndrome Synthesis and Analysis of Novel Glycerolipids for the Treatment of Metabolic Syndrome

Tetradecylthioacetic acid (TTA) 1 is a peroxisome proliferator-activated receptor (PPAR) agonist found to improve insulin sensitivity, lower blood lipid levels, enhance fatty acid oxidation, and promote antiinflammation in vivo. In an attempt to enhance these properties, two key thioether fatty acid (Thefa) lipids, ditetradecylthioacetyl phosphatidylcholine 2 and tritetradecylthioacetyl glycerol 3, are synthesized and administered po to male Wistar rats at two different doses to study and compare metabolic outcomes relative to the administration of 1 alone after 6 days. Liposomal formulations of 1 and 2 are also prepared to evaluate acute metabolic responses (at 3 h) post iv injection. Across all metrics measured, 1-induced responses post po administration are in line with previous data. Responses induced from 3 are mostly equivalent to 1-induced responses. By contrast, 2-induced responses almost always outperform those of 1 and 3. Therefore, 2 may represent a new lead for the treatment of metabolic syndrome

Well-defined surface tungstenocarbyne complexes through the reaction of [W(=CtBu)(CH(2)tBu)(3)] with silica

The molecular complex [W(&3bond; CtBu)(CH(2)tBu)(3)], 1, reacts with SiO2-(700) to give as major species 2a, [(&3bond; SiO)W(CtBu)(CH(2)tBu)(2)], while a bisgrafted surface species 3a, [(&3bond; SiO)(2)W-(&3bond; CtBu)(CH(2)tBu)], is obtained on SiO2-(200). As in molecular organometallic chemistry, the alkylalkylidyne tautomeric form is favored. Despite these structural features, these surface organometallic complexes are very active olefin metathesis catalysts, as reported earlier, and it is very likely that the necessary metallocarbene intermediates are generated under the reaction conditions

Synthesis and analysis of novel glycerolipids for the treatment of metabolic syndrome

Tetradecylthioacetic acid (TTA)1 is a peroxisome proliferator-activated receptor (PPAR) agonist found to improve insulin sensitivity, lower blood lipid levels, enhance fatty acid oxidation, and promote anti-inflammation in vivo. In an attempt to enhance these properties, two key thioether fatty acid (Thefa) lipids, ditetradecylthioacetyl phosphatidylcholine 2 and two different doses to study and compare metabolic outcomes relative to the administration of 1 alone after 6 days. Liposomal formualtions of 1 and 2 are also prepared to evaluate responses post administration are in line with previous data. Responses induced from 3 are mostly equivalent to 1-induced responses. By contrast, 2-induced responses almost always outperform theose of 1 and 3. Therefore, 2 may represent a new lead for the treatment of metabolic syndrome