Modulation of N-cadherin junctions and their role as epicenters of differentiation-specific actin regulation in the developing lens

Extensive elongation of lens fiber cells is a central feature of lens morphogenesis. Our study investigates the role of N-cadherin junctions in this process in vivo. We investigate both the molecular players involved in N-cadherin junctional maturation and the subsequent function of these junctions as epicenters for the assembly of an actin cytoskeleton that drives morphogenesis. We present the first evidence of nascent cadherin junctions in vivo, and show that they are a prominent feature along lateral interfaces of undifferentiated lens epithelial cells. Maturation of these N-cadherin junctions, required for lens cell differentiation, preceded organization of a cortical actin cytoskeleton along the cells' lateral borders, but was linked to recruitment of α-catenin and dephosphorylation of N-cadherin-linked β-catenin. Biochemical analysis revealed differentiation-specific recruitment of actin regulators cortactin and Arp3 to maturing N-cadherin junctions of differentiating cells, linking N-cadherin junctional maturation with actin cytoskeletal assembly during fiber cell elongation. Blocking formation of mature N-cadherin junctions led to reduced association of α-catenin with N-cadherin, prevented organization of actin along lateral borders of differentiating lens fiber cells and blocked their elongation. These studies provide a molecular link between N-cadherin junctions and the organization of an actin cytoskeleton that governs lens fiber cell morphogenesis in vivo.     

Defining the role of mesenchymal stromal cells on the regulation of matrix metalloproteinases in skeletal muscle cells

Recent studies indicate that mesenchymal stromal cell (MSC) transplantation improves healing of injured and diseased skeletal muscle, although the mechanisms of benefit are poorly understood. In the present study, we investigated whether MSCs and/or their trophic factors were able to regulate matrix metalloproteinase (MMP) expression and activity in different cells of the muscle tissue. MSCs in co-culture with C2C12 cells or their conditioned medium (MSC-CM) up-regulated MMP-2 and MMP-9 expression and function in the myoblastic cells; these effects were concomitant with the down-regulation of the tissue inhibitor of metalloproteinases (TIMP)-1 and -2 and with increased cell motility. In the single muscle fiber experiments, MSC-CM administration increased MMP-2/9 expression in Pax-7⁺ satellite cells and stimulated their mobilization, differentiation and fusion. The anti-fibrotic properties of MSC-CM involved also the regulation of MMPs by skeletal fibroblasts and the inhibition of their differentiation into myofibroblasts. The treatment with SB-3CT, a potent MMP inhibitor, prevented in these cells, the decrease of α-smooth actin and type-I collagen expression induced by MSC-CM, suggesting that MSC-CM could attenuate the fibrogenic response through mechanisms mediated by MMPs. Our results indicate that growth factors and cytokines released by these cells may modulate the fibrotic response and improve the endogenous mechanisms of muscle repair/regeneration.     

WJSC 6th Anniversary Special Issues（2）:Mesenchymal stem cells Purinergic receptors and nucleotide processing ectoenzymes:Their roles in regulating mesenchymal stem cell functions

Human mesenchymal stem cells(MSCs)are a rare population of non-hematopoietic stem cells with multilineage potential,originally identified in the bone marrow.Due to the lack of a single specific marker,MSCs can be recognized and isolated by a series of features such as plastic adherence,a panel of surface markers,the clonogenic and the differentiation abilities.The recognized role of MSCs in the regulation of hemopoiesis,in cell-degeneration protection and in the homeostasis of mesodermal tissues through their differentiation properties,justifies the current interest in identifying the biochemical signals produced by MSCs and their active crosstalk in tissue environments.Only recently have extracellular nucleotides(eNTPs)and their metabolites been included among the molecular signals produced by MSCs.These molecules are active on both ionotropic and metabotropic receptors present in most cell types.MSCs possess a significant display of these receptors and of nucleotide processing ectoenzymes on their plasma membrane.Thus,from their niche,MSCs give a significant contribution to the complex signaling network of eNTPs and its derivatives.Recent studies have demonstrated the multifaceted aspects of eNTP metabolism and their signal transduction in MSCs and revealed important roles in specifying differentiation lineages and modulating MSC physiology and communication with other cells.This review discusses the roles of eNTPs,their receptors and ectoenzymes,and the relevance of the signaling network and MSC functions,and also focuses on the importance of this emerging area of interest for future MSC-based cell therapies.     

Immunomodulation of mesenchymal stromal cells on regulatory T cells and its possible mechanism

Mesenchymal stromal cells (MSCs) and regulatory T cells (Tregs) have both garnered abundant interests from immunologists worldwide, as both MSCs and Tregs can be considered immunosuppressive in their own right. But a little attention has been paid to the impacts of MSCs on Tregs. To clarify the effects of MSCs on Tregs, we performed the coculture systems within MSCs and Tregs. We confirmed that MSC-exposed Tregs are capable of more immunosuppressive than Tregs without coculturing with MSCs. And this augmenting suppressive capacity was accompanied with an upregulation of programmed cell death 1 receptor (PD-1) on Tregs. Importantly, we found that cell viability of Tregs was excluded from the influences of MSCs. Finally, we showed that PD-1/B7-H1 interactions and IL-10 might be responsible for the enhanced suppressive capability of MSC-exposed Tregs. Further analysis revealed that PD-1/B7-H1 interactions were not responsible for the productions of IL-10 and TGF-β₁ in the MSC-Treg coculture systems; in contrast, IL-10 rather than TGF-β₁ played a role in the upregualtion of PD-1. Furthermore, this is the first explorative study to evaluate the immunomodulation of MSCs on the suppressive capacity of Tregs in MSC–Treg in vitro coculture setting.     

Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.     

Long helical filaments are not seen encircling cells in electron cryotomograms of rod-shaped bacteria

How rod-shaped bacteria form and maintain their shape is an important question in bacterial cell biology. Results from fluorescent light microscopy have led many to believe that the actin homolog MreB and a number of other proteins form long helical filaments along the inner membrane of the cell. Here we show using electron cryotomography of six different rod-shaped bacterial species, at macromolecular resolution, that no long (>80 nm) helical filaments exist near or along either surface of the inner membrane. We also use correlated cryo-fluorescent light microscopy (cryo-fLM) and electron cryo-tomography (ECT) to identify cytoplasmic bundles of MreB, showing that MreB filaments are detectable by ECT. In light of these results, the structure and function of MreB must be reconsidered: instead of acting as a large, rigid scaffold that localizes cell-wall synthetic machinery, moving MreB complexes may apply tension to growing peptidoglycan strands to ensure their orderly, linear insertion.     

Molecular evolution: Actin''s long lost relative found

The bacterial protein MreB has been identified as a prokaryotic homolog of the eukaryotic cytoskeletal protein actin. While we still know little about MreB's function, the structural similarities and differences between MreB and actin provide more insight into the remarkable properties of actin.     

Localization of cytoskeletal proteins in Pneumocystis carinii by immuno-electron microscopy

Pneumocystis carinii causes serious pulmonary infection in immunosuppressed patients. This study was undertaken to observe the cytoskeletal proteins of P. carinii by immuno-electron microscopy. P. carinii infection was experimentally induced by immunosuppression of Sprague-Dawley rats for seven weeks, and their lungs were used for the observations of this study. The gold particles localized actin, tropomyosin, and tubulin. The actin was irregularly scattered in the cytoplasm of the trophic forms but was much more concentrated in the inner space of the cell wall of the cystic forms called the inner electron-lucent layer. No significant amount of tropomyosin was observed in either trophic forms or cystic forms. The tubulin was distributed along the peripheral cytoplasm and filopodia of both the trophic and cystic forms rather than in the inner side of the cytoplasm. Particularly, in the cystic forms, the amount of tubulin was increased and located mainly in the inner electron-lucent layer of the cell wall where the actin was concentrated as well. The results of this study showed that the cell wall of P. carinii cystic forms is a structure whose inner side is rich in actin and tubulin. The location of the actin and tubulin in P. carinii suggests that the main role of these proteins is an involvement in the protection of cystic forms from the outside environment by maintaining rigidity of the cystic forms.     

Tetraspanins regulate the protrusive activities of cell membrane

Tetraspanins have gained increased attention due to their functional versatility. But the universal cellular mechanism that governs such versatility remains unknown. Herein we present the evidence that tetraspanins CD81 and CD82 regulate the formation and/or development of cell membrane protrusions. We analyzed the ultrastructure of the cells in which a tetraspanin is either overexpressed or ablated using transmission electron microscopy. The numbers of microvilli on the cell surface were counted, and the radii of microvillar tips and the lengths of microvilli were measured. We found that tetraspanin CD81 promotes the microvillus formation and/or extension while tetraspanin CD82 inhibits these events. In addition, CD81 enhances the outward bending of the plasma membrane while CD82 inhibits it. We also found that CD81 and CD82 proteins are localized at microvilli using immunofluorescence. CD82 regulates microvillus morphogenesis likely by altering the plasma membrane curvature and/or the cortical actin cytoskeletal organization. We predict that membrane protrusions embody a common morphological phenotype and cellular mechanism for, at least some if not all, tetraspanins. The differential effects of tetraspanins on microvilli likely lead to the functional diversification of tetraspanins and appear to correlate with their functional propensity.     

Tropomyosin isoform 3 promotes the formation of filopodia by regulating the recruitment of actin-binding proteins to actin filaments

Tropomyosins are believed to function in part by stabilizing actin filaments. However, accumulating evidence suggests that fundamental differences in function exist between tropomyosin isoforms, which contributes to the formation of functionally distinct filament populations. We investigated the functions of the high-molecular-weight isoform Tm3 and examined the molecular properties of Tm3-containing actin filament populations. Overexpression of the Tm3 isoform specifically induced the formation of filopodia and changes in actin solubility. We observed alterations in actin-binding protein recruitment to filaments, co-incident with changes in expression levels, which can account for this functional outcome. Tm3-associated filaments recruit active actin depolymerizing factor and are bundled into filopodia by fascin, which is both up-regulated and preferentially associated with Tm3-containing filaments in the Tm3 overexpressing cells. This study provides further insight into the isoform-specific roles of different tropomyosin isoforms. We conclude that variation in the tropomyosin isoform composition of microfilaments provides a mechanism to generate functionally distinct filament populations.     

Cytoskeletal organization of human mesenchymal stem cells (MSC) changes during their osteogenic differentiation

Human MSCs have been studied to define the mechanisms involved in normal bone remodeling and the regulation of osteogenesis. During osteogenic differentiation, MSCs change from their characteristic fibroblast-like phenotype to near spherical shape. In this study, we analyzed the correlation between the organization of cytoskeleton of MSCs, changes in cell morphology, and the expression of specific markers (alkaline phosphatase activity and calcium deposition) of osteogenic differentiation. For osteoblastic differentiation, cells were cultured in a culture medium supplemented with 100 nM dexamethasone, 10 mM beta- glycerophosphate, and 50 microg/ml ascorbic acid. The organization of microfilaments and microtubules was examined by inmunofluorescence using Alexa fluor 594 phalloidin and anti alpha-tubulin monoclonal antibody. Cytochalasin D and nocodazole were used to alter reversibly the cytoskeleton dynamic. A remarkable change in cytoskeleton organization was observed in human MSCs during osteogenic differentiation. Actin cytoskeleton changed from a large number of thin, parallel microfilament bundles extending across the entire cytoplasm in undifferentiated MSCs to a few thick actin filament bundles located at the outermost periphery in differentiated cells. Under osteogenic culture conditions, a reversible reorganization of microfilaments induced by an initial treatment with cytochalasin D but not with nocodazole reduced the expression of differentiation markers, without affecting the final morphology of the cells. The results indicate that changes in the assembly and disassembly kinetics of microfilaments dynamic of actin network formation may be critical in supporting the osteogenic differentiation of human MSCs; also indicated that the organization of microtubules appears to have a regulatory role on the kinetic of this process.     

Correlation between proliferative activity and cellular thickness of human mesenchymal stem cells

A cell’s shape is known to be related to its proliferative activity. In particular, large and flat mammalian adult stem cells seem to show slow proliferation, however using quantitative analysis to prove the phenomenon is difficult. We measured the proliferation and cellular thickness of human mesenchymal stem cells (MSCs) by atomic force microscopy and found that MSCs with high proliferative activity were thick while those with low proliferative activity were thin, even though these MSCs were early passage cells. Further, low proliferative MSCs contained many senescence-associated β-galactosidase positive cells together with high senescence-associated gene expression. These findings suggest that the measurement of cellular thickness is useful for estimating the proliferative activity of human MSCs and is expected to be a practical tool for MSC applications in regenerative medicine.     

Oxidative stress induces senescence in human mesenchymal stem cells

Mesenchymal stem cells (MSCs) contribute to tissue repair in vivo and form an attractive cell source for tissue engineering. Their regenerative potential is impaired by cellular senescence. The effects of oxidative stress on MSCs are still unknown. Our studies were to investigate into the proliferation potential, cytological features and the telomere linked stress response system of MSCs, subject to acute or prolonged oxidant challenge with hydrogen peroxide. Telomere length was measured using the telomere restriction fragment assay, gene expression was determined by rtPCR. Sub-lethal doses of oxidative stress reduced proliferation rates and induced senescent-morphological features and senescence-associated β-galactosidase positivity. Prolonged low dose treatment with hydrogen peroxide had no effects on cell proliferation or morphology. Sub-lethal and prolonged low doses of oxidative stress considerably accelerated telomere attrition. Following acute oxidant insult p21 was up-regulated prior to returning to initial levels. TRF1 was significantly reduced, TRF2 showed a slight up-regulation. SIRT1 and XRCC5 were up-regulated after oxidant insult and expression levels increased in aging cells. Compared to fibroblasts and chondrocytes, MSCs showed an increased tolerance to oxidative stress regarding proliferation, telomere biology and gene expression with an impaired stress tolerance in aged cells.     

CXC chemokine receptor 1 enhances the ability of human umbilical cord blood-derived mesenchymal stem cells to migrate toward gliomas

In this study, we showed that knocking-down interleukin-8 (IL-8) in glioma cells, or its receptor, CXC chemokine receptor 1 (CXCR1) in hUCB-MSCs reduced hUCB-MSC migration toward glioma cells in a Transwell chamber. In contrast, CXCR1-transfected hUCB-MSCs (CXCR1-MSCs) showed a superior capacity to migrate toward glioma cells in a Transwell chamber compared to primary hUCB-MSCs. Furthermore, these transfected cells also demonstrated the same ability to migrate toward tumors in mice bearing intracranial human gliomas as shown by histological and in vivo imaging analysis. Our findings indicate that overexpression of CXCR1 could be a useful tool for MSC-based gene therapy to achieve a sufficient quantity of therapeutic MSCs that are localized within tumors.     

Dopamine regulates mobilization of mesenchymal stem cells during wound angiogenesis

Angiogenesis is an important step in the complex biological and molecular events leading to successful healing of dermal wounds. Among the different cellular effectors of wound angiogenesis, the role of mesenchymal stem cells (MSCs) is of current interest due to their transdifferentiation and proangiogenic potentials. Skin is richly innervated by sympathetic nerves which secrete dopamine (DA) and we have recently shown that concentration of DA present in synaptic cleft can significantly inhibit wound tissue neovascularization. As recent reports indicate that MSCs by mobilizing into wound bed play an important role in promoting wound angiogenesis, we therefore investigated the effect of DA on the migration of MSCs in wound tissues. DA acted through its D(2) receptors present in the MSCs to inhibit their mobilization to the wound beds by suppressing Akt phosphorylation and actin polymerization. In contrast, this inhibitory effect of DA was reversed after treatment with specific DA D(2) receptor antagonist. Increased mobilization of MSCs was demonstrated in the wound site following blockade of DA D(2) receptor mediated actions, and this in turn was associated with significantly more angiogenesis in wound tissues. This study is of translational value and indicates use of DA D(2) receptor antagonists to stimulate mobilization of these stem cells for faster regeneration of damaged tissues.     

Differential mechanical response of mesenchymal stem cells and fibroblasts to tumor-secreted soluble factors

The progression of neoplastic malignancies is a complex process resulting not only from the accumulation of mutations within tumor cells, but also modulation of the tumor microenvironment. Recent advances have shown that the recruitment and subsequent heterotypic interactions of stromal cells--including fibroblasts and bone marrow-derived mesenchymal stem cells (MSCs)--are crucial for carcinogenesis. Though extensive work has been done analyzing the signals that recruit these cells, the governing mechanical properties have not been fully investigated. Here, we report that despite their initial similarities, MSCs respond not only faster but also more dramatically to pro-migratory tumor-secreted soluble factors. Utilizing multiple particle tracking microrheology to probe the cytoskeletal mechanical properties, we show that MSCs stiffen completely within one hour, three times faster than fibroblasts. In addition, unlike fibroblasts, MSCs exposed to tumor-secreted soluble factors display a functionally different phenotype characterized by morphological elongation, decreased actin stress fiber density, and decreased adhesion. Quantitative real-time PCR indicates these phenomena occur based on differential expression of small GTPases RhoA and Cdc42, but not Rac1. These findings demonstrate a fundamental difference in the recruitment of fibroblasts and MSCs.     

Actin nucleation: Nucleation-promoting factors are not all equal

Arp2/3 complex plays a key role in regulated actin polymerization. A recent study has revealed marked differences in the ability of two nucleation-promoting factors - N-WASP and Scar/WAVE1 - to activate the Arp2/3 complex. Further insights have come from determination of the Arp2/3 crystal structure.     

Gelsolin co-occurs with Lewy bodies in vivo and accelerates α-synuclein aggregation in vitro

Deposition of fibrillar α-synuclein as Lewy bodies is the neuropathological hallmark of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Apart from α-synuclein, these intraneuronal inclusions contain over 250 different proteins. The actin binding protein gelsolin, has previously been suggested to be part of the Lewy body, but its potential role in α-synuclein aggregation remains unknown. Here, we studied the association between gelsolin and α-synuclein in brain tissue from PD and DLB patients as well as in a cell model for α-synuclein aggregation. Moreover, the potential effect of gelsolin on α-synuclein fibrillization was also investigated. Our data demonstrate that gelsolin co-occured with α-synuclein in Lewy bodies from affected human brain as well as with Lewy body-like inclusions in α-synuclein over expressing cells. Furthermore, in the presence of calcium chloride, gelsolin was found to enhance the aggregation rate of α-synuclein in vitro. Moreover, no apparent structural differences could be observed between fibrils formed in the presence or absence of gelsolin. Further studies on gelsolin and other Lewy body associated proteins are warranted to learn more about their potential role in the α-synuclein aggregation process.