Confinement induces actin flow in a meiotic cytoplasm

In vivo, F-actin flows are observed at different cell life stages and participate in various developmental processes during asymmetric divisions in vertebrate oocytes, cell migration, or wound healing. Here, we show that confinement has a dramatic effect on F-actin spatiotemporal organization. We reconstitute in vitro the spontaneous generation of F-actin flow using Xenopus meiotic extracts artificially confined within a geometry mimicking the cell boundary. Perturbations of actin polymerization kinetics or F-actin nucleation sites strongly modify the network flow dynamics. A combination of quantitative image analysis and biochemical perturbations shows that both spatial localization of F-actin nucleators and actin turnover play a decisive role in generating flow. Interestingly, our in vitro assay recapitulates several symmetry-breaking processes observed in oocytes and early embryonic cells.     

The Ser/Thr protein kinase AfsK regulates polar growth and hyphal branching in the filamentous bacteria Streptomyces

In cells that exhibit apical growth, mechanisms that regulate cell polarity are crucial for determination of cellular shape and for the adaptation of growth to intrinsic and extrinsic cues. Broadly conserved pathways control cell polarity in eukaryotes, but less is known about polarly growing prokaryotes. An evolutionarily ancient form of apical growth is found in the filamentous bacteria Streptomyces, and is directed by a polarisome-like complex involving the essential protein DivIVA. We report here that this bacterial polarization machinery is regulated by a eukaryotic-type Ser/Thr protein kinase, AfsK, which localizes to hyphal tips and phosphorylates DivIVA. During normal growth, AfsK regulates hyphal branching by modulating branch-site selection and some aspect of the underlying polarisome-splitting mechanism that controls branching of Streptomyces hyphae. Further, AfsK is activated by signals generated by the arrest of cell wall synthesis and directly communicates this to the polarisome by hyperphosphorylating DivIVA. Induction of high levels of DivIVA phosphorylation by using a constitutively active mutant AfsK causes disassembly of apical polarisomes, followed by establishment of multiple hyphal branches elsewhere in the cell, revealing a profound impact of this kinase on growth polarity. The function of AfsK is reminiscent of the phoshorylation of polarity proteins and polarisome components by Ser/Thr protein kinases in eukaryotes.     

CDC42在HBx介导肝细胞恶性转化中作用的定量蛋白质组学研究

目的寻找在乙肝病毒 X 蛋白（HBx）诱发肝细胞恶性转化过程中 CDC42信号通路发挥作用的介导因子。方法利用基于质量亏损的准等重二甲基标记（pIDL）的定量蛋白质组学方法,检测 CDC42基因敲除前后HBx 转基因细胞蛋白质组的差异。筛选差异蛋白并对差异蛋白进行基因本体（gene ontology,GO）功能分析。结果与结论共定量到3409个蛋白,筛选出220个差异蛋白,通过 GO 分析发现与细胞骨架组织相关的 palladin、成蛋白样亚家族1（formin-like 1,FMNL1）和角蛋白-19均发生显著下调。该研究为 CDC42在 HBx 介导 Huh7细胞恶性转化的机制研究提供了候选基因和蛋白。     

Regulation of cytochrome P450 gene expression by ketamine: a review

Introduction: Although used as an anesthetic drug for decades, ketamine appears to have garnered renewed interest due to its potential therapeutic uses in pain therapy, neurology, and psychiatry. Ketamine undergoes extensive oxidative metabolism by cytochrome P450 (CYP) enzymes. Considerable efforts have been expended to elucidate the ketamine-induced regulation of CYP gene expression. The safety profile of chronic ketamine administration is still unclear. Understanding how ketamine regulates CYP gene expression is clinically meaningful.

Areas covered: In this article, the authors provide a brief review of clinical applications of ketamine and its metabolism by CYP enzymes. We discuss the effects of ketamine on the regulation of CYP gene expression, exploring aspects of cytoskeletal remodeling, mitochondrial functions, and calcium homeostasis.

Expert opinion: Ketamine may inhibit CYP gene expression through inhibiting calcium signaling, decreasing ATP levels, producing excessive reactive oxygen species, and subsequently perturbing cytoskeletal dynamics. Further research is still needed to avoid possible ketamine–drug interactions during long-term use in the clinic.     

Microcystin‐LR induces protein phosphatase 2A alteration in a human liver cell line

Microcystin‐LR (MC‐LR) is a potent inhibitor of protein phosphatases 1 and 2A, and has potent hepatotoxicity and tumor promotion activity. Numerous studies on MC‐LR toxicity have been conducted in rat hepatocytes, but few studies of the effects of microcystins on human hepatocytes have been done. In this study, HL7702 cells (a human normal liver cell line) were incubated in MC‐LR for 24 h. The existence of MC‐LR in HL7702 cells was confirmed. Furthermore, PP2A activity and the alteration of PP2A subunits were assessed. The results show that PP2A activity decreased from the concentration of 1 μM MC‐LR, showing a concentration‐dependent decline, to about 34% at 10 μM MC‐LR. This activity undergone opposite change with alternations of phosphorylated Y307‐PP2A/C and PP2A/C subunit but showed same change with the alteration of the ratio of methylated L309‐PP2A/C to PP2A/C. B55α, a regulatory subunit of PP2A, was slightly increases in cells treated with the highest concentration of MC‐LR (10 μM), and colocalized increasedly with rearranged‐microtubules after 1 μM MC‐LR exposure. However, the proportion of early apoptotic cells did not show any change at various concentration of MC‐LR for 24 h. To our knowledge, this is the first report showing MC‐LR‐induced alteration of PP2A phosphatase in human cultured hepatocytes, and the mechanism of action seems to be similar as described before in vitro. The alteration of PP2A and microtubule seems to be the early event induced by MC‐LR exposure. © 2013 Wiley Periodicals, Inc. Environ Toxicol 29: 1236–1244, 2014.     

Intracellular distribution of differentially phosphorylated dual‐specificity tyrosine phosphorylation‐regulated kinase 1A (DYRK1A)

The gene encoding dual‐specificity tyrosine phosphorylation‐regulated kinase 1A (DYRK1A) is located within the Down syndrome (DS) critical region of chromosome 21. DYRK1A interacts with a plethora of substrates in the cytosol, cytoskeleton, and nucleus. Its overexpression is a contributing factor to the developmental alterations and age‐associated pathology observed in DS. We hypothesized that the intracellular distribution of DYRK1A and cell‐compartment‐specific functions are associated with DYRK1A posttranslational modifications. Fractionation showed that, in both human and mouse brain, almost 80% of DYRK1A was associated with the cytoskeleton, and the remaining DYRK1A was present in the cytosolic and nuclear fractions. Coimmunoprecipitation revealed that DYRK1A in the brain cytoskeleton fraction forms complexes with filamentous actin, neurofilaments, and tubulin. Two‐dimensional gel analysis of the fractions revealed DYRK1A with distinct isoelectric points: 5.5–6.5 in the nucleus, 7.2–8.2 in the cytoskeleton, and 8.7 in the cytosol. Phosphate‐affinity gel electrophoresis demonstrated several bands of DYRK1A with different mobility shifts for nuclear, cytoskeletal, and cytosolic DYRK1A, indicating modification by phosphorylation. Mass spectrometry analysis disclosed one phosphorylated site in the cytosolic DYRK1A and multiple phosphorylated residues in the cytoskeletal DYRK1A, including two not previously described. This study supports the hypothesis that intracellular distribution and compartment‐specific functions of DYRK1A may depend on its phosphorylation pattern. © 2013 Wiley Periodicals, Inc.     

Impaired mechanisms of leukocyte adhesion in vitro by the calcium channel antagonist mibefradil

Purpose. Enhanced adhesion to the vascular endothelium and excessive trafficking to extravascular locations can lead to serious tissue injury and destruction. Therefore, interfering with molecular mechanisms of leukocyte adhesion to the vascular endothelium is an important goal to block diseases like chronic inflammations and atherosclerosis. Methods. We studied the influence of the calcium antagonists mibefradil (T-type channel blocker), amlodipine and verapamil (both L-type channel blockers) on mechanisms related to leukocyte adhesion using isolated peripheral human blood leukocytes. Results. Mibefradil but not amlodipine and verapamil attenuated leukocyte adhesion in vitro. Regarding the mechanisms we found that mibefradil reduced the surface expression of 2 integrins and L-selectin. The immobilization of the 2 integrin subunit to the cytoskeleton that was inducible by receptor cross linking was impaired. Mibefradil was able to significantly inhibit the formyl-methionyl-leucyl-phenylalanine (fMLP) induced calcium rise, which suggests that mibefradil interfered with integrin signaling through blocking the intracellular calcium rise. SK&F 96365, a blocker of the capacitative calcium entry had no effect on cell adhesion and was less effective to influence integrin mediated mechanisms than mibefradil. Conclusion. Our data suggest that mibefradil or chemically related substances are promising to serve as potent drugs to prevent excessive adhesion of leukocytes.     

The role of microtubule movement in bidirectional organelle transport

We study the role of microtubule movement in bidirectional organelle transport in Drosophila S2 cells and show that EGFP-tagged peroxisomes in cells serve as sensitive probes of motor induced, noisy cytoskeletal motions. Multiple peroxisomes move in unison over large time windows and show correlations with microtubule tip positions, indicating rapid microtubule fluctuations in the longitudinal direction. We report the first high-resolution measurement of longitudinal microtubule fluctuations performed by tracing such pairs of co-moving peroxisomes. The resulting picture shows that motor-dependent longitudinal microtubule oscillations contribute significantly to cargo movement along microtubules. Thus, contrary to the conventional view, organelle transport cannot be described solely in terms of cargo movement along stationary microtubule tracks, but instead includes a strong contribution from the movement of the tracks.     

Heavy ion and X-ray irradiation alter the cytoskeleton and cytomechanics of cortical neurons

Heavy ion beams with high linear energy transfer exhibit more beneifcial physical and biological performance than conventional X-rays, thus improving the potential of this type of radiotherapy in the treatment of cancer. However, these two radiotherapy modalities both cause inevitable brain injury. The objective of this study was to evaluate the effects of heavy ion and X-ray irra-diation on the cytoskeleton and cytomechanical properties of rat cortical neurons, as well as to determine the potential mechanism of neuronal injury after irradiation. Cortical neurons from 30 new-born mice were irradiated with heavy ion beams at a single dose of 2 Gy and X-rays at a single dose of 4 Gy;subsequent evaluation of their effects were carried out at 24 hours after irradiation. An immunolfuorescence assay showed that after irradiation with both the heavy ion beam and X-rays, the number of primary neurons was signiifcantly decreased, and there was ev-idence of apoptosis. Radiation-induced neuronal injury was more apparent after X-irradiation. Under atomic force microscopy, the neuronal membrane appeared rough and neuronal rigidity had increased. These cell changes were more apparent following exposure to X-rays. Our ifnd-ings indicated that damage caused by heavy ion and X-ray irradiation resulted in the structural distortion and rearrangement of the cytoskeleton, and affected the cytomechanical properties of the cortical neurons. Moreover, this radiation injury to normal neurons was much severer after irradiation with X-rays than after heavy ion beam irradiation.     

Septin assemblies form by diffusion-driven annealing on membranes

Septins assemble into filaments and higher-order structures that act as scaffolds for diverse cell functions including cytokinesis, cell polarity, and membrane remodeling. Despite their conserved role in cell organization, little is known about how septin filaments elongate and are knitted together into higher-order assemblies. Using fluorescence correlation spectroscopy, we determined that cytosolic septins are in small complexes, suggesting that septin filaments are not formed in the cytosol. When the plasma membrane of live cells is monitored by total internal reflection fluorescence microscopy, we see that septin complexes of variable size diffuse in two dimensions. Diffusing septin complexes collide and make end-on associations to form elongated filaments and higher-order structures, an assembly process we call annealing. Septin assembly by annealing can be reconstituted in vitro on supported lipid bilayers with purified septin complexes. Using the reconstitution assay, we show that septin filaments are highly flexible, grow only from free filament ends, and do not exchange subunits in the middle of filaments. This work shows that annealing is a previously unidentified intrinsic property of septins in the presence of membranes and demonstrates that cells exploit this mechanism to build large septin assemblies.Septin filaments form rings, bars, and gauzes that serve as a scaffold at cell division sites; act to retract blebbed regions of membrane; and restrict diffusion between cell compartments (1–4). Septin function is required for cell division and viability in many eukaryotes whereas misregulation is associated with cancers and neurodegenerative disorders (5–8). Furthermore, septins mediate entry of both bacterial and fungal pathogens into host cells (9–11). In vivo, septin assembly is restricted both in time and in space through local activation of small GTPases such as Cdc42. Localized signaling leads to higher-order septin structures forming closely apposed to the plasma membrane at the plane of division, sites of polarity, and curved membranes (10, 12–14). Notably, eukaryotic cells of different geometries build higher-order septin assemblies of various shapes, sizes, and functions (4, 15, 16). Although septins are critical for spatial organization of cell plasma membranes, their assembly and disassembly dynamics are not understood (15).Electron microscopy (EM) studies of recombinant and immunoprecipitated Saccharomyces cerevisiae septins have shown that septins form nonpolar hetero-octameric rod-shaped complexes in high-salt buffers (>300 mM) and elongated filaments when dialyzed into low-salt buffers (<100 mM) (17, 18). Structural analyses of worm and mammalian septins have revealed that the heteromeric, rod-shaped complex is conserved (19–21). Thus, septin rods characterized to date contain two copies of each septin subunit assembled into a nonpolar, heteromeric complex (Fig. S1). Association of purified septin proteins with phosphoinositide-containing membrane monolayers placed on EM grids can promote the assembly of septin filaments in otherwise nonpermissive conditions such as high salt or the presence of mutant septins in the complex (22). A polybasic region in the N terminus of septin proteins as well as other surfaces of the septin protein have been proposed to be the basis for septin association with phosphoinositides; however, the functional role of membranes in filament formation is not yet known (22–24).Previous work has defined a possible starting state of assembly (the rod) and endpoint (filaments and gauzes); however, there is nothing known about how filaments elongate either in vivo or in vitro. Do septin filaments extend by stepwise addition of rods? Does addition occur from both ends of the filament? Can subunits be added in the middle and/or sides of a filament? Do septin filaments grow in the cytosol or on plasma membranes? Thus, it is still not known where filaments form in vivo, how filaments elongate, and how filaments are brought together to construct higher-order assemblies.The goal of this study was to identify the locations and mechanism of septin filament polymerization. Using fluorescence correlation spectroscopy (FCS), we observed that cytosolic septins are likely rods, not monomers or filaments. Using total internal reflection fluorescence (TIRF) microscopy, we found septins form short filaments on the plasma membrane and then long filaments and higher-order assemblies grow when filaments merge. We have called the process of short filaments coming together “annealing” as this has previously been described for F-actin in vitro (25). We reconstituted septin assembly, using purified proteins and supported lipid bilayers, and found that annealing is an intrinsic property of septins that occurs at very low protein concentrations in the presence of a supported phospholipid bilayer. Our results suggest that the plasma membrane concentrates septins and provides a platform for 2D diffusion that promotes polymerization.