The trafficking of the cellulose synthase complex in higher plants

Background

Cellulose is an important constituent of plant cell walls in a biological context, and is also a material commonly utilized by mankind in the pulp and paper, timber, textile and biofuel industries. The biosynthesis of cellulose in higher plants is a function of the cellulose synthase complex (CSC). The CSC, a large transmembrane complex containing multiple cellulose synthase proteins, is believed to be assembled in the Golgi apparatus, but is thought only to synthesize cellulose when it is localized at the plasma membrane, where CSCs synthesize and extrude cellulose directly into the plant cell wall. Therefore, the delivery and endocytosis of CSCs to and from the plasma membrane are important aspects for the regulation of cellulose biosynthesis.

Scope

Recent progress in the visualization of CSC dynamics in living plant cells has begun to reveal some of the routes and factors involved in CSC trafficking. This review highlights the most recent major findings related to CSC trafficking, provides novel perspectives on how CSC trafficking can influence the cell wall, and proposes potential avenues for future exploration.     

Confocal microscopy as a tool to reveal the tridimensional organization of intracellular lumens and intercellular cysts in a human colon adenocarcinoma cell line

Adenocarcinoma cells often form intracellular lumens and intercellular cysts. In order to study the structural relationships between these lumens and the apical domain of normal enterocytes, we have applied electron microscopy and confocal microscopy to a cloned cell line derived from the human colon adenocarcinoma cell line LoVo which express a high number of intracellular lumens and intercellular cysts. Microvilli reminiscent of those detected in the brush border of small intestinal cells are formed in the two types of compartments. By immunofluorescence, we found that a 135 kDa membrane glycoprotein characterized by a monoclonal Ab and normally associated with the brush-border of enterocytes is expressed at the surface of the intracellular lumens and intercellular cysts present in the adenocarcinoma cells. Comparison of fluorescence and reflection contrast micrographs obtained by confocal microscopy demonstrate the presence of spherical intracellular lumens in the juxtanuclear region of single cells, and of more complex shaped intercellular cysts located within clusters of cells. The later cells form junctional complexes limiting an apical plasma membrane domain in contact with the intercellular cyst. It is suggested that the intracellular lumens may represent the abortive form of an apical plasma membrane due to the lack of components required to establish epithelial cell contacts. As opposed to conventional fluorescence microscopy, confocal microscopy allows rapid inspection of the tridimensional organization of intracellular lumens and intercellular cysts even when they are located in cell multilayers.     

Effect of plasma membrane contacts between cells on uridine incorporation and RNA polymerase activity

The effect of lateral plasma membrane contacts between neighbouring cells on uridine incorporation and endogenous RNA polymerase activity has been studied in kidney epithelial cells growing on a solid substrate in culture. A gradual increase of intercellular membrane contacts was parallelled by a gradual decrease in the rate of uridine incorporation. In contrast, the endogenous RNA polymerase activity remained unaltered when cells established membrane contacts between each other. The results are interpreted as reflecting a decreased uptake of uridine through the plasma membrane rather than a decline in RNA synthesis in cells with intercellular membrane contacts.     

Potassium pyroantimonate and osmium-zinc iodide reactivity in the tubular epithelium of the opisthonephric kidney of the sea lamprey,Petromyzon marinus L

Pyroantimonate precipitate indicates that the epithelium of the proximal tubule is the only segment of the tubular nephron of the fresh water lamprey where large accumlations of cations are distributed. Unusually large amounts of reaction product are located within the lateral intercellular spaces and within vesicles closely associated with the plasma membrane at the lateral and basal surfaces. This technique suggests the continuity of these vesicles with the plasma membrane and alludes to the possibility of an endomembranous system of vesicles and the intercellular spaces as vehicles for ion transport. Lateral intercellular spaces of proximal tubules of lower vertebrates may play a different role in kidney function that their counterparts in higher vertebrates. Osmium-zinc iodide has a specificity for certain cells within the proximal, intermediate, and distal segments, but no structural differences are noted when these cells are compared to unstained cells. Smooth endoplasmic reticulum remains unstained in the distal segment but the stain has a strong affinity for elements of the Golgi apparatus, lysosomes, and the nuclear envelope of all cell types. This technique does not suggest a structural or functional similarity between cells of the distal segment and the chloride cells of the gills of teleosts.     

Cell-to-cell signaling influences the fate of prostate cancer stem cells and their potential to generate more aggressive tumors

An increasing number of malignancies has been shown to be initiated and propelled by small subpopulations of cancer stem cells (CSC). However, whether tumor aggressiveness is driven by CSC and by what extent this property may be relevant within the tumor mass is still unsettled. To address this issue, we isolated a rare tumor cell population on the basis of its CD44(+)CD24(-) phenotype from the human androgen-independent prostate carcinoma cell line DU145 and established its CSC properties. The behavior of selected CSC was investigated with respect to the bulk DU145 cells. The injection of CSC in nude mice generated highly vascularized tumors infiltrating the adjacent tissues, showing high density of neuroendocrine cells and expressing low levels of E-cadherin and β-catenin as well as high levels of vimentin. On the contrary, when a comparable number of unsorted DU145 cells were injected the resulting tumors were less aggressive. To investigate the different features of tumors in vivo, the influence of differentiated tumor cells on CSC was examined in vitro by growing CSC in the absence or presence of conditioned medium from DU145 cells. CSC grown in permissive conditions differentiated into cell populations with features similar to those of cells held in aggressive tumors generated from CSC injection. Differently, conditioned medium induced CSC to differentiate into a cell phenotype comparable to cells of scarcely aggressive tumors originated from bulk DU145 cell injection. These findings show for the first time that CSC are able to generate differentiated cells expressing either highly or scarcely aggressive phenotype, thus influencing prostate cancer progression. The fate of CSC was determined by signals released from tumor environment. Moreover, using microarray analysis we selected some molecules which could be involved in this cell-to-cell signaling, hypothesizing their potential value for prognostic or therapeutic applications.     

Tumor Cells Positive and Negative for the Common Cancer Stem Cell Markers Are Capable of Initiating Tumor Growth and Generating Both Progenies

The cancer stem cell (CSC) model depicts that tumors are hierarchically organized and maintained by CSCs lying at the apex. CSCs have been “identified” in a variety of tumors through the tumor-forming assay, in which tumor cells distinguished by a certain cell surface marker (known as a CSC marker) were separately transplanted into immunodeficient mice. In such assays, tumor cells positive but not negative for the CSC marker (hereby defined as CSC⁺ and CSC⁻ cells, respectively) have the ability of tumor-forming and generating both progenies. However, here we show that CSC⁺ and CSC⁻ cells exhibit similar proliferation in the native states. Using a cell tracing method, we demonstrate that CSC⁻ cells exhibit similar tumorigenesis and proliferation as CSC⁺ cells when they were co-transplanted into immunodeficient mice. Through serial single-cell derived subline construction, we further demonstrated that CSC⁺ and CSC⁻ cells from CSC marker expressing tumors could invariably generate both progenies, and their characteristics are maintained among different generations irrespective of the origins (CSC⁺-derived or CSC⁻-derived). These findings demonstrate that tumorigenic cells cannot be distinguished by common CSC markers alone and we propose that cautions should be taken when using these markers independently to identify cancer stem cells due to the phenotypic plasticity of tumor cells.     

ICAM1 Is a Potential Cancer Stem Cell Marker of Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) accounts for about 90% of esophageal cancer diagnosed in Asian countries, with its incidence on the rise. Cancer stem cell (CSC; also known as tumor-initiating cells, TIC) is inherently resistant to cytotoxic chemotherapy and radiation and associates with poor prognosis and therapy failure. Targeting therapy against cancer stem cell has emerged as a potential therapeutic approach to develop effective regimens. However, the suitable CSC marker of ESCC for identification and targeting is still limited. In this study, we screened the novel CSC membrane protein markers using two distinct stemness characteristics of cancer cell lines by a comparative approach. After the validation of RT-PCR, qPCR and western blot analyses, intercellular adhesion molecule 1 (ICAM1) was identified as a potential CSC marker of ESCC. ICAM1 promotes cancer cell migration, invasion as well as increasing mesenchymal marker expression and attenuating epithelial marker expression. In addition, ICAM1 contributes to CSC properties, including sphere formation, drug resistance, and tumorigenesis in mouse xenotransplantation model. Based on the analysis of ICAM1-regulated proteins, we speculated that ICAM1 regulates CSC properties partly through an ICAM1-PTTG1IP-p53-DNMT1 pathway. Moreover, we observed that ICAM1 and CD44 could have a compensation effect on maintaining the stemness characteristics of ESCC, suggesting that the combination of multi-targeting therapies should be under serious consideration to acquire a more potent therapeutic effect on CSC of ESCC.     

肿瘤干细胞靶向治疗研究进展

肿瘤干细胞是存在于肿瘤组织中的一小部分具有自我更新、多向分化和无限增殖能力的细胞,其控制着肿瘤的生长、分化,并且与 肿瘤的耐药性、转移以及复发密切相关。简述近年来国内外学者针对肿瘤干细胞的靶向治疗产品,包括抗体、多肽以及靶向药物的研究进展。 肿瘤干细胞的靶向治疗为临床肿瘤治疗提供了新的希望。     

Immunologic selection of simian virus 40 (SV40) T-antigen-negative tumor cells which arise by excision of early SV40 DNA.

A clonal line of highly oncogenic spontaneously transformed mouse cells (104C) was transformed in tissue culture by simian virus 40 (SV40) and subsequently recloned (106CSC). This 106CSC cell line expressed T antigen and transplantation antigen but was about 100 times less tumorigenic than the 104C parent. When 10(5) 106CSC cells were injected into immunocompetent syngeneic mice, tumors were produced. From such tumors, cell lines were established in culture, all of which were consistently negative for T antigen. We found previously by solution DNA hybridization methods that the tumor cells were depleted in the early region of SV40 DNA which codes for the T antigen. We postulated that this loss occurs through a DNA rearrangement of unknown mechanism in one or a few 106CSC cells and that the tumors are then produced from such a cell or cells, whereas all the T-antigen-positive 106CSC cells are rejected by immunologic means. In this investigation we showed by the DNA transfer method using appropriately selected SV40 DNA probes that indeed the tumor cell clone (130CSCT) we selected to investigate came from one 106CSC cell in which the T-antigen-coding SV40 DNA sequences (but not all the early SV40 DNA sequences) were lost by an excision and recombination mechanism. We also showed that the 130CSCT cells, which are highly tumorigenic, could again be transformed by SV40 and that the resulting T-antigen-positive cloned derivative cells became much less tumorigenic (approximately 10(5)-fold), apparently again because of immunologic recognition and rejection. Indeed, when 10(7) T-antigen-positive cloned cells were injected, all the T-antigen-positive cells were rejected and the tumor was produced again from one or more T-antigen-negative cells. Thus, a one-step in vivo transplantation experiment allowed a selection (for tumorigenicity and against the SV40 T antigen) of a mutant mammalian cell with a DNA deletion at a definable site.     

PATHS OF TRANSTUBULAR WATER FLOW IN ISOLATED RENAL COLLECTING TUBULES

The cells of perfused rabbit collecting tubules swell and the intercellular spaces widen during osmotic flow of water from lumen to bath induced by antidiuretic hormone (ADH). Ouabain had no influence on these changes. In the absence of net water flow intercellular width was unaffected when tubules were swollen in hypotonic external media. Therefore, during ADH-induced flow widening of intercellular spaces is not a consequence of osmotic swelling of a closed intercellular compartment containing trapped solutes, but rather is due to flow of solution through the channel. Direct evidence of intercellular flow was obtained. Nonperfused tubules swollen in hypotonic media were reimmersed in isotonic solution with resultant entry of water into intercellular spaces. The widened spaces gradually collapsed completely. Spaces enlarged in this manner could be emptied more rapidly by increasing the transtubular hydrostatic pressure difference. In electron micrographs a path of exit of sufficient width to accommodate the observed rate of fluid flow was seen at the base of the intercellular channel. It is concluded that the intercellular spaces communicate with the external extracellular fluid and that water, having entered the cells across the luminal plasma membrane in response in ADH, leaves the cells by osmosis across both the lateral and basilar surface membranes.     

Mechanisms Underlying the Increased Plasma ACTH Levels in Chronic Psychosocially Stressed Male Mice

Mice exposed to chronic subordinate colony housing (CSC, 19 days), an established paradigm for chronic psychosocial stress, show unaffected basal morning plasma corticosterone (CORT) concentrations, despite enlarged adrenal glands and an increased CORT response to an acute heterotypic stressor. In the present study we investigate the mechanisms underlying these phenomena at the level of the pituitary. We show that both basal and acute stressor-induced (forced swim (FS), 6 min) plasma adrenocorticotropic hormone (ACTH) concentrations, the number of total and corticotroph pituitary cells, and relative protein expression of pituitary mineralocorticoid receptor and FK506-binding protein 51 was increased in CSC compared with single-housed control (SHC) mice, while relative corticotropin releasing hormone (CRH) receptor 1 (CRH-R1) and glucocorticoid receptor protein expression was down-regulated. Relative pituitary pro-opiomelanocortin and arginine vasopressin (AVP) receptor 1b (AVPR-1b) protein expression, FS (6 min)-induced ACTH secretion in dexamethasone-blocked mice, and the number of AVP positive magnocellular and parvocellular neurons in the paraventricular hypothalamic nucleus (PVN) was unaffected following CSC. Taken together, the data of the present study indicate that 19 days of CSC result in pituitary hyperactivity, under both basal and acute heterotypic stress conditions. Although further studies have to assess this in detail, an increased number of pituitary corticotrophs together with unaffected relative pituitary AVPR-1b and decreased CRH-R1 protein expression following CSC suggests that pituitary hyperdrive is mediated by newly formed corticotrophs that are more sensitive to AVP than CRH. Moreover, our data indicate that changes in PVN AVP and negative feedback inhibition seem not to play a major role in pituitary hyperactivity following CSC.     

Overexpression of <Emphasis Type="Italic">PSP1</Emphasis> enhances growth of transgenic Arabidopsis plants under ambient air conditions

Cellulose biosynthesis is mediated by cellulose synthases (CesAs), which constitute into rosette-like cellulose synthase complexe (CSC) on the plasma membrane. Two types of CSCs in Arabidopsis are believed to be involved in cellulose synthesis in the primary cell wall and secondary cell walls, respectively. In this work, we found that the two type CSCs participated cellulose biosynthesis in differentiating xylem cells undergoing secondary cell wall thickening in Populus. During the cell wall thickening process, expression of one type CSC genes increased while expression of the other type CSC genes decreased. Suppression of different type CSC genes both affected the wall-thickening and disrupted the multilaminar structure of the secondary cell walls. When CesA7A was suppressed, crystalline cellulose content was reduced, which, however, showed an increase when CesA3D was suppressed. The CesA suppression also affected cellulose digestibility of the wood cell walls. The results suggest that two type CSCs are involved in coordinating the cellulose biosynthesis in formation of the multilaminar structure in Populus wood secondary cell walls.     

Corticosteroid side-chain isomerase in the circulatory system

Corticosteroid side-chain (CSC) isomerase catalyzes ketol-aldol interconversion of the corticosteroid side chain. The enzyme was present in the blood of mouse, rat, guinea pig, chicken, pig, horse, sheep, cow, and human. The patterns of substrate specificity, measuring 3H-1H exchange of 21-tritiated forms of 11-deoxycorticosterone, corticosterone, and cortisol, were species specific. Based on enzyme activity and immunostaining of mouse blood fractions, red blood cells had the most isomerase activity, plasma had less, and white blood cells had low but highly variable levels of enzyme. Purified mouse liver CSC isomerase was found to be adsorbed by red blood cells. The results suggest that circulating CSC isomerase is derived in part from tissue sources and is in part an intrinsic blood enzyme.     

The role of dynamin 3 in the testis

We report here that dynamin 3 in the testis is associated with structures termed tubulobulbar complexes that internalize intact intercellular junctions during sperm release and turnover of the blood-testis barrier. The protein lies adjacent to an actin-Arp2/3 network that cuffs the double plasma membrane tubular invagination at the core of each complex. To explore the possible relationship between dynamin 3 and nectin-based adhesion junctions, we transiently transfected DsRed-tagged dynamin 3 into MDCK cells stably transfected with eGFP-tagged nectin 2, one of the adhesion molecules known to be expressed in Sertoli cells at adhesion junctions. Cells transfected with the dynamin 3 construct had less uniformly distributed nectin 2 at intercellular contacts when compared to control cells expressing only nectin 2 or transfected with the DsRed plasmid alone. Significantly, tubular extensions positive for nectin 2 were visible projecting into the cells from regions of intercellular contact. Our findings are consistent with the conclusion that dynamin 3 is involved with tubulobulbar morphogenesis. Dynamin 3 also occurs in concentrated deposits around the capitulum and striated columns in the connecting piece of sperm tails suggesting that the protein in these cells may function to stabilize the base of the tail or serve as a reservoir for use during or after fertilization.     

Source and target cell specificities of a conditioned medium factor that increases choline acetyltransferase activity in cultured spinal cord cells

A macromolecular factor(s) in muscle conditioned medium (CM), when applied to spinal cord (SC) cells in culture, causes large increases in the activity of choline acetyltransferase (CAT), the enzyme which synthesizes the neurotransmitter acetylcholine. We have found apparent specificity of both species and cell type for the production, release, or action of this CAT stimulation component (CSC). Rat and mouse muscle CMs contained CSC which was active in mouse SC cells; chick muscle CM did not. In addition to muscle CM, the CM from cell cultures of mouse heart, liver, and kidney contained CSC. However, CM from secondary cultures of liver cells contained little if any CSC. These apparent specificities were not due to differences in the protein content of either the cells providing CM or of the CM itself. There was also apparent specificity of response to CSC among cholinergic cells in culture. Cultures of cells from only two of four regions of the mouse central nervous system, and from one of five neuronal cell lines tested, had increased CAT activity after treatment with muscle CM. The response in NG108-15 neuroblastoma-glioma hybrid cells was further characterized, and was used to develop a more convenient and rapid assay for CSC.     

Plakoglobin is a component of the filamentous subplasmalemmal coat of lens cells

The plasma membranes of the cells of the superficial layer of the eye lens and the lens fibres are in close intercellular contact, leaving an intermembrane space of approximately 20 nm or less throughout their entire length. This plasma membrane is underlaid by a filamentous, cytoplasmic web containing actin, proteins of the spectrin and band 4.1 families, alpha-actinin and vinculin. Using immunofluorescence microscopy and immunoblotting of gel electrophoretically separated proteins, we show that plakoglobin, the plaque protein common to desmosomal and nondesmosomal adhering junctions, is present in lens cells and is also a component of the subplasmalemmal coat of these cells. Plakoglobin also exists in the extended regions of intercellular contacts between cultured lenticular cells where it often colocalizes with vinculin but does not occur in other vinculin-rich plasma membrane regions such as the focal adhesions at the ventral cell surface. Plakoglobin associated with plasma membrane regions can also be identified in various other adhesive cultured cells, but it is not detected in cells and tissues that do not establish firm intercellular junctions such as erythrocytes, platelets, cultured myeloma cells and smooth muscle tissue. We conclude that plakoglobin occurs, at least in lens cells, throughout the entire subplasmalemmal coat, coexisting in this situation not only with vinculin but also with spectrin and 4.1 protein(s). This colocalization infers the presence of a distinct, complex type of membrane-skeleton assembly involving the actin filament-associated junctional plaque elements plakoglobin and vinculin together with actin-associated proteins of the spectrin and band 4.1 protein families.     

Stem cells as the root of pancreatic ductal adenocarcinoma

Emerging evidence suggests that stem cells play a crucial role not only in the generation and maintenance of different tissues, but also in the development and progression of malignancies. For the many solid cancers, it has now been shown that they harbor a distinct subpopulation of cancer cells that bear stem cell features and therefore, these cells are termed cancer stem cells (CSC) or tumor-propagating cells. CSC are exclusively tumorigenic and essential drivers for tumor progression and metastasis. Moreover, it has been shown that pancreatic ductal adenocarcinoma does not only contain one homogeneous population of CSC rather than diverse subpopulations that may have evolved during tumor progression. One of these populations is called migrating CSC and can be characterized by CXCR4 co-expression. Only these cells are capable of evading the primary tumor and traveling to distant sites such as the liver as the preferred site of metastatic spread. Clinically even more important, however, is the observation that CSC are highly resistant to chemo- and radiotherapy resulting in their relative enrichment during treatment and rapid relapse of disease. Many laboratories are now working on the further in-depth characterization of these cells, which may eventually allow for the identification of their Achilles heal and lead to novel treatment modalities for fighting this deadly disease.     

Revisiting the role of lysophosphatidic acid in stem cell biology

Stem cells possess unique biological characteristics such as the ability to self-renew and to undergo multilineage differentiation into specialized cells. Whereas embryonic stem cells (ESC) can differentiate into all cell types of the body, somatic stem cells (SSC) are a population of stem cells located in distinct niches throughout the body that differentiate into the specific cell types of the tissue in which they reside in. SSC function mainly to restore cells as part of normal tissue homeostasis or to replenish cells that are damaged due to injury. Cancer stem-like cells (CSC) are said to be analogous to SSC in this manner where tumor growth and progression as well as metastasis are fueled by a small population of CSC that reside within the corresponding tumor. Moreover, emerging evidence indicates that CSC are inherently resistant to chemo- and radiotherapy that are often the cause of cancer relapse. Hence, major research efforts have been directed at identifying CSC populations in different cancer types and understanding their biology. Many factors are thought to regulate and maintain cell stemness, including bioactive lysophospholipids such as lysophosphatidic acid (LPA). In this review, we discuss some of the newly discovered functions of LPA not only in the regulation of CSC but also normal SSC, the similarities in these regulatory functions, and how these discoveries can pave way to the development of novel therapies in cancer and regenerative medicine.