Maturation of Cell-Substratum Focal Adhesions Induced by Depolymerization of Microtubules is Mediated by Increased Cortical Tension

Dynamics of alterations of focal adhesions (FA) induced by a microtubule-depolymerizing drug, colcemid, was examined in several types of fibroblastic cells. Evolution of individual FA in cultured cells was monitored by interference-reflection microscopy (IRM); at the end of the monitoring period (3 hours) the cells were fixed and immunofluorescence microscopy of the same FA was performed with an antibody against vinculin. Control and colcemid-treated cells remained non-motile and did not show lamellipodial activity at the edges. During the incubation, formation of new FA or disappearance of pre-existing FA did not occur in either colcemid-treated or control cultures. However, FA in colcemid-treated cells significantly increased in size in the course of a 3 hour incubation. The growth of FA was centripetal and sometimes was accompanied by the fusion of several adjacent FA.

Immunofluorescence examination showed that colcemid-induced growth of FA was accompanied by accumulation of several proteins specific for these structures including vinculin, talin, paxillin and pp125FAK kinase. Immunoblotting with anti-vinculin antibody showed that incubation with colcemid considerably increased the amount of vinculin associated with the ventral membranes due to its partial redistribution from a soluble pool into the growing adhesions. A substantial increase in tyrosine phosphorylation of pp125FAK was also observed in colcemid-treated cells. In cells plated on elastic silicone rubber films, colcemid induced formation of wrinkles in the films and these wrinkles relaxed after treatment with cytochalasin D. These results confirm that microtubule depolymerization increases traction transmitted to the substratum by the actin cortex and shows that an increase in cortical tension accompanies maturation of FA.

Taken together, these data show that short-term incubation with colcemid does not affect the formation of initial FA. In contrast, microtubule depolymerization considerably stimulates the maturation FA, manifested by their centripetal growth. Maturation is proposed to be mediated by increased cortical tension, which is caused by microtubule depolymerization.     

Crossroads of integrins and cadherins in epithelia and stroma remodeling

Adhesion events mediated by cadherin and integrin adhesion receptors have fundamental roles in the maintenance of the physiological balance of epithelial tissues, and it is well established that perturbations in their normal functional activity and/or changes in their expression are associated with tumorigenesis. Over the last decades, increasing evidence of a dynamic collaborative interaction between these complexes through their shared interactions with cytoskeletal proteins and common signaling pathways has emerged not only as an important regulator of several aspects of epithelial cell behavior, but also as a coordinated adhesion module that senses and transmits signals from and to the epithelia surrounding microenvironment. The tight regulation of their crosstalk is particularly important during epithelial remodeling events that normally take place during morphogenesis and tissue repair, and when defective it leads to cell transformation and aggravated responses of the tumor microenvironment that contribute to tumorigenesis. In this review we highlight some of the interactions that regulate their crosstalk and how this could be implicated in regulating signals across epithelial tissues to sustain homeostasis.     

Crossroads of integrins and cadherins in epithelia and stroma remodeling

Adhesion events mediated by cadherin and integrin adhesion receptors have fundamental roles in the maintenance of the physiological balance of epithelial tissues, and it is well established that perturbations in their normal functional activity and/or changes in their expression are associated with tumorigenesis. Over the last decades, increasing evidence of a dynamic collaborative interaction between these complexes through their shared interactions with cytoskeletal proteins and common signaling pathways has emerged not only as an important regulator of several aspects of epithelial cell behavior, but also as a coordinated adhesion module that senses and transmits signals from and to the epithelia surrounding microenvironment. The tight regulation of their crosstalk is particularly important during epithelial remodeling events that normally take place during morphogenesis and tissue repair, and when defective it leads to cell transformation and aggravated responses of the tumor microenvironment that contribute to tumorigenesis. In this review we highlight some of the interactions that regulate their crosstalk and how this could be implicated in regulating signals across epithelial tissues to sustain homeostasis.     

Glycoprotein contributions to mammary gland and mammary tumor structure and function: roles of adherens junctions, ErbBs and membrane MUCs

Mammary function is dependent on its three-dimensional organization, which is established and maintained by cell adhesive junctions linked through the membrane to the cell cytoskeleton. These junctions serve not only as structural elements, but also function as initiators and integrators of cell signals. In this review we discuss three types of glycoproteins whose interactions impinge on the function of mammary cell-cell junctions, cadherins, ErbB receptor tyrosine kinases and membrane mucins, as a microcosm of events regulating mammary cell behaviors. Actions of these components are integrated by the critical signaling element beta-catenin. When functioning properly, these glycoproteins, beta-catenin and associated signaling pathways mesh into a highly structured program for development and function of the gland. However, disruption or dysfunction of these glycoproteins or the signaling elements can lead to disorganization of the epithelia and ultimately to neoplasia.     

Cell expansion in the epidermis: microtubules, cellulose orientation and wall loosening enzymes

Two models of isolated epidermis were used to demonstrate that the net orientation of cellulose microfibrils in the cell wall is related to mechanical properties of the tissue, and can be used as an indicator for wall anisotropy. In the developing plant epidermis, cells expand in one or two directions in the plane of the plant surface. In epidermis cells actively expanding in one direction (elongation), the orientation of cortical microtubules closely matches the net cellulose orientation. In epidermis cells expanding in two directions, the orientation of the parallel microtubules does not coincide with the net cellulose orientation in the adjacent cell wall. The orientation of cortical microtubules is thus not always a reliable indicator of wall characteristics. In both types of epidermis, a high rate of expansion correlates with a high activity of xyloglucan endotransglycosylase (XET), as determinedin situ. This high activity alone cannot explain unidirectional wall expansion.     

Cellular Dimorphism in the Maize Root Cortex: Involvement of Microtubules,Ethylene and Gibberellin in the Differentiation of Cellular Behaviour in Postmitotic Growth Zones

Detailed morphometric analysis of cell shapes and an immunofluorescent study of microtubules were carried out on primary roots of Zea mays L. Two types of cells were found to be formed within the postmitotic isodiametric growth (PIG) region of the root cortex that were differentially responsive to low level of exogenous ethylene. The innermost and central cell rows of the cortex were sensitive to ethylene treatment and showed a disturbed distribution of cortical microtubules (CMTs) as well as changed polarity of cell growth, whereas the 2–3 outermost cell rows were less sensitive in this respect. This suggests that post-mitotic cells of the inner cortex are specific targets for ethylene action. These properties of the inner cortex are compatible with its cells being involved in the formation of aerenchyma; they may also favour root growth in compacted soil. By contrast, the specific properties of the outer cortex indicate that this tissue domain is necessary for the gaseous impermeability and the mechanical strengthening of subjacent aerenchymatous cortex, especially in the mature region of the root. Ethylene affected neither the pattern of cortical cell expansion in the meristem nor the position of the PIG region with respect to the root tip. This contrasts with gibberellin-deficiency which affected these parameters in both parts of the cortex. These observations indicate a fundamental difference between the role of these two phytohormones in the morphogenesis and development of maize roots.     

膜蛋白的表达：在无细胞体系中实现功能性表达、折叠和组装

膜蛋白在人类和其他物种的生命活动中都起着至关重要的作用.在已完成测序的基因组中,膜蛋白占据30%.药物作用靶向位点、细胞之间的信号传递以及对外界环境的探测等功能,大多是通过生物膜上的特殊膜受体蛋白实现的.膜蛋白研究在工业、环境、国防、医学等领域具有重大意义.嗅觉受体蛋白是一类典型的膜蛋白,属于G蛋白偶联受体家族.嗅觉受体蛋白调控着生物的食物寻找、危险趋避和求偶行为.其主要分布于脊椎动物鼻腔和昆虫触角.嗅觉受体蛋白可以直接识别气味分子,将生物信号转化为电信号,最终传递至神经中枢,进而做出相关应答.膜蛋白的获取并不容易.天然组织中的膜蛋白含量太低不足以支撑学术研究.异源表达难以实现膜蛋白整合上膜,这给膜蛋白的结构和功能研究带来很大挑战.无细胞蛋白质合成系统是一个开放体系,且不依赖细胞活性,是体外表达蛋白质的有效方法.通过无细胞蛋白合成体系,在体外实现膜蛋白二聚体的自组装,将为膜蛋白研究带来全新突破.本文总结了用于无细胞表达的膜蛋白研究进展.     

Cell junction and cyclic AMP: II. Modulations of junctional membrane permeability,dependent on serum and cell density

Summary Junctional molecular transfer (as indexed by the number of cell interfaces transferring fluorescent-labelled molecules) and concentration of endogenous cAMP were determined in mammalian cells in culture at varying serum concentration and cell density. In several cell types, on stepping the serum concentration from 10% (the concentration to which the cells had been adapted) to zero, the junctional transfer rose (reversibly) within 48 hr, as the endogenous cAMP concentration rose. The junctional transfer was inversely related to serum concentration over a range, most steeply so the transfer of large and charged molecules. one cell type showed no junctional change in response to serum; it showed also no endogenous cAMP change. Junctional transfer varied inversely with cell density over the range of 0.7–7 (10⁴ cells/cm²) in 3T3 cells. In cultures seeded to various densities, or growing to various densities on their own, junctional transfer fell with rising density, and so did the endogenous cAMP concentration. Upon downstep from high density, junctional transfer rose over 24–48 hr. In B cells, junctional transfer was independent of cell density over the aforementioned range, and so was the endogenous cAMP concentration. These results, in conjunction with the effects of exogenous cAMP described in the preceding paper of this series, point to a cAMP-mediated junctional effect; a possible teleonomy for control of membrane junction is discussed.     

Genomic Models of Metastatic Cancer: Functional Analysis of Death-from-Cancer Signature Genes Reveals Aneuploid,Anoikis-Resistant,Metastasis-Enabling Phenotype with Altered Cell Cycle Control and Activated PcG Protein Chromatin Silencing Pathway

A recent discovery of death-from-cancer signature genes identifies potential markers predicting the high likelihood of treatment failure in cancer patients. This knowledge provides the opportunity to analyze in functional terms the therapy-resistant and metastasis-enabling phenotypes of cancer cells. Here we summarize the current data regarding the biological functions of genes comprising a death-from-cancer signature. This analysis predicts that cancer cells manifesting a stem cell-like expression profile of a death-from-cancer signature would exhibit the following features: a concomitantly increased expression of certain members of inhibitor of apoptosis protein (IAP) family (Survivin and XIAP); activation of mitotic spindle check point proteins (BUB1, BUB3, KNTC2, Mad2, PLK1, PLK4, STK6/Aurora A); and elevated levels of certain cell cycle control/marker proteins (CCNB1, CCNB2, CCND1, CCNA2, CDC2, CDC25, Ki67, USP22). Consequently, these cancer cells would acquire metastasis-enabling anoikis-resistance aneuploid phenotype with aberrant cell cycle control. A functionally complementary role of multiple cooperating oncogenic pathways and the essential role of Polycomb Group (PcG) protein chromatin silencing pathway in emergence of the stem cell cancer phenotype is highlighted.     

Presynaptic Control of the Synthesis and Release of Dopamine from Striatal Synaptosomes: A Comparison Between the Effects of 5-Hydroxytryptamine, Acetylcholine, and Glutamate

The effects of 5-HT and glutamate on dopamine synthesis and release by striatal synaptosomes were investigated and compared with the action of acetylcholine, which acts presynaptically on this system. 5-HT inhibited (28%) synthesis of [¹⁴C]dopamine from L-[U-¹⁴C]tyrosine, at 10-⁵M and above. This contrasts with the action of acetylcholine, which stimulated [¹⁴C]-dopamine synthesis by 24% at 10-⁴ M. Tissue levels of GABA were unaffected by either 5-HT or acetylcholine up to concentrations of 10-⁴ M. The inhibitory action of 5-HT (5 × 10^?5 M and 2 × 10^?4 M) on [¹⁹C]dopamine synthesis was completely abolished by methysergide (2 × 10^?6 M). Higher concentrations of methysergide (10^?4 M) or cyproheptadine (10^?5 M) inhibited [¹⁴C]dopamine synthesis by 28% and 25%, respectively, when added alone to synaptosomes. However, only methysergide prevented the further inhibition of synthesis caused by 5-HT. At concentrations of 2 × 10^?5 M and above, 5-HT stimulated [¹⁴C]dopamine release. This releasing action differed from that of acetylcholine, which occurred at lower concentrations (e.g., 10^?6 M). Methysergide (up to 10^?4 M) or cyproheptadine (2 × 10^?4 M) did not reduce the 5-HT (5 × 10^?5 M)-induced release of [¹⁴C]dopamine, but methysergide (10^?4 M) showed a potentiation (49%) of this increased release. The stimulatory effects of 5-HT (2 × 10^?5 M) and K⁺ (56 mM) on [¹⁴C]dopamine release were additive, indicating that two separate mechanisms were involved. However, when both agents were present the stimulatory effect of K⁺ (56 mM) on [¹⁴C]dopamine synthesis was not seen above the inhibitory effect of 5-HT. Glutamate (0.1-5 mM) did not affect [⁴C]dopamine release or its synthesis from L-[U-¹⁴C]tyrosine. It is concluded that 5-HT modulates the synthesis of dopamine in striatal nerve terminals through a presynaptic receptor mechanism, an action antagonised by methysergide. The releasing action of 5-HT apparently occurs through a separate mechanism which is also distinct from that involved in the response to K⁺ depolarisation.     

Association Between a Low-Carbohydrate Diet,Glycemic Control,and Quality of Life in Australian Adults Living With Type 1 Diabetes: A Pilot Study

ObjectiveTo examine if there is an association between a low-carbohydrate diet (LCD), glycemic control, and quality of life (QoL) in Australian adults with type 1 diabetes.MethodsThis single-group, pre-post, mixed methods (quantitative and qualitative) study was conducted in an outpatient tertiary hospital. Eligible participants were those aged ≥18 years, with type 1 diabetes for ≥1 year, and using multiple daily insulin injections. Participants followed a 12-week individualized LCD (<100 g/d). Daily glucose levels were monitored using a continuous glucose monitor. Glycated hemoglobin (HbA1c) and QoL were measured preintervention and postintervention. A post-hoc exploratory regression analysis determined whether changes in carbohydrate intake was associated with changes in HbA1c and QoL. Qualitative data collected postintervention explored participants’ perceptions relating to a LCD, glycemic control, and QoL.ResultsParticipants (n = 22) completed the 12-week LCD intervention. An LCD provided a statistically, significant improvement in HbA1c 0.83% (95% CI 0.32%-1.33%), P = .003 but did not impact QoL: estimated change 1.14 units (95% CI: ?5.34 to 7.61); P = .72. The post-hoc exploratory regression analysis showed that participants with poorer baseline glycemic control were more likely to respond to an LCD resulting in significant reductions in HbA1c. Participant perceptions relating to the study variables were mixed.ConclusionsAn LCD (<100 g/d) is a potentially effective and safe strategy to improve glycemic control without negatively effecting QoL in Australian adults with type 1 diabetes.     

Biological Control of Thrips on Ornamental Crops: Interactions Between the Predatory Mite Neoseiulus cucumeris (Acari: Phytoseiidae) and Western Flower Thrips, Frankliniella occidentalis (Thysanoptera: Thripidae), on Cyclamen

Difficulties in controlling outbreaks of Western flower thrips, Frankliniella occidentalis, have obstructed the widespread adoption of biological control in many ornamental crops. The efficacy of the predatory mite, Neoseiulus cucumeris, in controlling F. occidentalis on two cultivars of cyclamen was tested in glasshouse experiments. The establishment and development of F. occidentalis populations was compared in three treatment introductions of N. cucumeris (50, 200 and 350 mites m -2 per week) and an untreated control. F. occidentalis were sampled in the flowers over eight weeks and counted into different life stages. No differences were observed between the two cultivars. All treatments with the predator resulted in a decline in numbers of F. occidentalis compared to the untreated control. Although the proportion of first instar F. occidentalis was similar in all treatments, the level of control varied with the number of N. cucumeris introduced. Lower populations of F. occidentalis, combined with a more rapid decline in their numbers, were observed at the 200 and 350 mites m -2 rates. Numbers of F. occidentalis remained low in the 350 N. cucumeris m -2 rate and the proportion of second instar F. occidentalis in the samples was consistently lower than in the other treatments. Trap counts of adult F. occidentalis were strongly correlated with the numbers of both adult and total F. occidentalis in flower samples. High inoculative releases of N. cucumeris early in the flowering cycle followed by frequent low introductions of predators should provide a strong basis for preventative control of F. occidentalis and other thrips species on cyclamen.     

Mutual Inhibition Kinetic Analysis of γ-Aminobutyric Acid, Taurine, and β-Alanine High-Affinity Transport into Neurons and Astrocytes: Evidence for Similarity Between the Taurine and β-Alanine Carriers in Both Cell Types

The transport kinetics of gamma-aminobutyric acid (GABA), taurine, and beta-alanine in addition to the mutual inhibition patterns of these compounds were investigated in cultures of neurons and astrocytes derived from mouse cerebral cortex. A high-affinity uptake system for each amino acid was demonstrated both in neurons (Km GABA = 24.9 +/- 1.7 microM; Km Tau = 20.0 +/- 3.3 microM; Km beta-Ala = 73.0 +/- 3.6 microM) and astrocytes (Km GABA = 31.4 +/- 2.9 microM, Km Tau = 24.7 +/- 1.3 microM; Km beta-Ala = 70.8 +/- 3.6 microM). The maximal uptake rates (Vmax) determined were such that, in neurons, Vmax GABA greater than Vmax beta-Ala = Vmax Tau, whereas in astrocytes, Vmax beta-Ala greater than Vmax Tau = Vmax GABA. Taurine was found to inhibit beta-alanine uptake into neurons and astrocytes in a competitive manner, with Ki values of 217 microM in neurons and 24 microM in astrocytes. beta-Alanine was shown to inhibit taurine uptake in neurons and astrocytes, also in a competitive manner, with Ki values of 72 microM in neurons and 71 microM in astrocytes. However, beta-alanine was found to be a weak noncompetitive inhibitor of neuronal and astrocytic GABA uptake, whereas in reverse experiments, GABA displayed weak noncompetitive inhibition of neuronal and astrocytic uptake of beta-alanine. Likewise, taurine was a weak noncompetitive inhibitor of GABA uptake in neurons and similarly, GABA was a weak noncompetitive inhibitor of taurine uptake into neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of membrane/lipid rafts with the cytoskeleton: Impact on signaling and function : Membrane/lipid rafts,mediators of cytoskeletal arrangement and cell signaling

The plasma membrane in eukaryotic cells contains microdomains that are enriched in certain glycosphingolipids, gangliosides, and sterols (such as cholesterol) to form membrane/lipid rafts (MLR). These regions exist as caveolae, morphologically observable flask-like invaginations, or as a less easily detectable planar form. MLR are scaffolds for many molecular entities, including signaling receptors and ion channels that communicate extracellular stimuli to the intracellular milieu. Much evidence indicates that this organization and/or the clustering of MLR into more active signaling platforms depends upon interactions with and dynamic rearrangement of the cytoskeleton. Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to MLR and help regulate lateral diffusion of membrane proteins and lipids in response to extracellular events (e.g., receptor activation, shear stress, electrical conductance, and nutrient demand). MLR regulate cellular polarity, adherence to the extracellular matrix, signaling events (including ones that affect growth and migration), and are sites of cellular entry of certain pathogens, toxins and nanoparticles. The dynamic interaction between MLR and the underlying cytoskeleton thus regulates many facets of the function of eukaryotic cells and their adaptation to changing environments. Here, we review general features of MLR and caveolae and their role in several aspects of cellular function, including polarity of endothelial and epithelial cells, cell migration, mechanotransduction, lymphocyte activation, neuronal growth and signaling, and a variety of disease settings. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.     

Endometrial stromal cells and decidualized stromal cells: Origins,transformation and functions

Decidualization of endometrium, which is characterized by endometrial stromal cell (ESC) decidualization, vascular reconstruction, immune cell recruitment, and plentiful molecule production, is a crucial step for uterus to become receptive for embryo. When implantation takes place, ESCs surround and directly interact with embryo. Decidualized stromal cells (DSCs) are of great importance in endometrial decidualization, having a broad function in regulating immune activity and vascular remodeling of uterus. DSCs are shown to have a higher metabolic level and looser cytoskeleton than ESCs. What's the origin of ESCs and how ESCs successfully transform into DSCs had puzzled scientists in the last decades. Breakthrough had been achieved recently, and many studies had elucidated some of the characters and functions of DSCs. However, several questions still remain unclear. This paper reviews current understanding of where ESCs come from and how ESCs differentiate into DSCs, summarizes some characters and functions of DSCs, analyzes current studies and their limitations and points out research areas that need further investigation.     

Liquid–Liquid Phase Separation at the Plasma Membrane–Cytosol Interface: Common Players in Adhesion,Motility, and Synaptic Function

Networks of scaffold proteins and enzymes assemble at the interface between the cytosol and specific sites of the plasma membrane, where these networks guide distinct cellular functions. Some of these plasma membrane–associated platforms (PMAPs) include shared core components that are able to establish specific protein–protein interactions, to produce distinct supramolecular assemblies regulating dynamic processes as diverse as cell adhesion and motility, or the formation and function of neuronal synapses. How cells organize such dynamic networks is still an open question. In this review we introduce molecular networks assembling at the edge of migrating cells, and at pre– and postsynaptic sites, which share molecular players that can drive the assembly of biomolecular condensates. Very recent experimental evidence has highlighted the emerging role of some of these multidomain/scaffold proteins belonging to the GIT, liprin-α and ELKS/ERC families as drivers of liquid–liquid phase separation (LLPS). The data point to an important role of LLPS: (i) in the formation of PMAPs at the edge of migrating cells, where LLPS appears to be involved in promoting protrusion and the turnover of integrin–mediated adhesions, to allow forward cell translocation; (ii) in the assembly of the presynaptic active zone and of the postsynaptic density deputed to the release and reception of neurotransmitter signals, respectively. The recent results indicate that LLPS at cytosol–membrane interfaces is suitable not only for the regulation of active cellular processes, but also for the continuous spatial rearrangements of the molecular interactions involved in these dynamic processes.