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Cells of any organism are consistently exposed to changes over time in their environment. The kinetics by which these changes occur are critical for the cellular response and fate decision. It is therefore important to control the temporal changes of extracellular stimuli precisely to understand biological mechanisms in a quantitative manner. Most current cell culture and biochemical studies focus on instant changes in the environment and therefore neglect the importance of kinetic environments. To address these shortcomings, we developed two experimental methodologies to precisely control the environment of single cells. These methodologies are compatible with standard biochemistry, molecular, cell and quantitative biology assays. We demonstrate applicability by obtaining time series and time point measurements in both live and fixed cells. We demonstrate the feasibility of the methodology in yeast and mammalian cell culture in combination with widely used assays such as flow cytometry, time-lapse microscopy and single-molecule RNA Fluorescent in-situ Hybridization (smFISH). Our experimental methodologies are easy to implement in most laboratory settings and allows the study of kinetic environments in a wide range of assays and different cell culture conditions.
IMPACT STATEMENT - Successful clinical tissue engineering requires functional fidelity of the cultured cell to its counterpart, but this has been elusive in renal tissue engineering. Typically, renal proximal tubule cells in culture have a flattened morphology and do not express key transporters essential to their function. In this article, we show for the first time that substrate mechanical properties dictate differentiation of cultured renal proximal tubule cells. Remarkably, this effect was only discernable after 4 weeks in culture, longer than usually reported for this cell type. These results demonstrate a new tunable parameter to optimize cell differentiation in renal tissue engineering.
OBJECTIVE - Endoglin, a transforming growth factor-β superfamily coreceptor, is predominantly expressed in endothelial cells and has essential roles in vascular development. However, whether endoglin is also expressed in vascular smooth muscle cells (VSMCs), especially in vivo, remains controversial. Furthermore, the roles of endoglin in VSMC biology remain largely unknown. Our objective was to examine the expression and determine the function of endoglin in VSMCs during angiogenesis.
APPROACH AND RESULTS - Here, we determine that endoglin is robustly expressed in VSMCs. Using CRISPR/CAS9 knockout and short hairpin RNA knockdown in the VSMC/endothelial coculture model system, we determine that endoglin in VSMCs, but not in endothelial cells, promotes VSMCs recruitment by the endothelial cells both in vitro and in vivo. Using an unbiased bioinformatics analysis of RNA sequencing data and further study, we determine that, mechanistically, endoglin mediates VSMC recruitment by promoting VSMC migration and spreading on endothelial cells via increasing integrin/FAK pathway signaling, whereas endoglin has minimal effects on VSMC adhesion to endothelial cells. In addition, we further determine that loss of endoglin in VSMCs inhibits VSMC recruitment in vivo.
CONCLUSIONS - These studies demonstrate that endoglin has an important role in VSMC recruitment and blood vessel maturation during angiogenesis and also provide novel insights into how discordant endoglin function in endothelial and VSMCs may regulate vascular maturation and angiogenesis.
© 2017 The Authors.
In familial pulmonary arterial hypertension (FPAH), the autosomal dominant disease-causing BMPR2 mutation is only 20% penetrant, suggesting that genetic variation provides modifiers that alleviate the disease. Here, we used comparison of induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from three families with unaffected mutation carriers (UMCs), FPAH patients, and gender-matched controls to investigate this variation. Our analysis identified features of UMC iPSC-ECs related to modifiers of BMPR2 signaling or to differentially expressed genes. FPAH-iPSC-ECs showed reduced adhesion, survival, migration, and angiogenesis compared to UMC-iPSC-ECs and control cells. The "rescued" phenotype of UMC cells was related to an increase in specific BMPR2 activators and/or a reduction in inhibitors, and the improved cell adhesion could be attributed to preservation of related signaling. The improved survival was related to increased BIRC3 and was independent of BMPR2. Our findings therefore highlight protective modifiers for FPAH that could help inform development of future treatment strategies.
Copyright © 2017 Elsevier Inc. All rights reserved.
The geometry of the cleavage furrow during mitosis is often asymmetric in vivo and plays a critical role in stem cell differentiation and the relative positioning of daughter cells during development. Early observations of adhesive cell lines revealed asymmetry in the shape of the cleavage furrow, where the bottom (i.e., substrate attached side) of the cleavage furrow ingressed less than the top (i.e., unattached side). This data suggested substrate attachment could be regulating furrow ingression. Here we report a population of mitotic focal adhesions (FAs) controls the symmetry of the cleavage furrow. In single HeLa cells, stronger adhesion to the substrate directed less ingression from the bottom of the cell through a pathway including paxillin, focal adhesion kinase (FAK) and vinculin. Cell-cell contacts also direct ingression of the cleavage furrow in coordination with FAs in epithelial cells-MDCK-within monolayers and polarized cysts. In addition, mitotic FAs established 3D orientation of the mitotic spindle and the relative positioning of mother and daughter centrosomes. Therefore, our data reveals mitotic FAs as a key link between mitotic cell shape and spindle orientation, and may have important implications in our understanding stem cell homeostasis and tumorigenesis.
Electrospun microfibers are attractive for the engineering of oriented tissues because they present instructive topographic and mechanical cues to cells. However, high-density microfiber networks are too cell-impermeable for most tissue applications. Alternatively, the distribution of sparse microfibers within a three-dimensional hydrogel could present instructive cues to guide cell organization while not inhibiting cell behavior. In this study, thin (∼5 fibers thick) layers of aligned microfibers (0.7 μm) were embedded within collagen hydrogels containing mesenchymal stem cells (MSCs), cultured for up to 14 days, and assayed for expression of ligament markers and imaged for cell organization. These microfibers were generated through the electrospinning of polycaprolactone (PCL), poly(ester-urethane) (PEUR), or a 75/25 PEUR/PCL blend to produce microfiber networks with elastic moduli of 31, 15, and 5.6 MPa, respectively. MSCs in composites containing 5.6 MPa fibers exhibited increased expression of the ligament marker scleraxis and the contractile phenotype marker α-smooth muscle actin versus the stiffer fiber composites. Additionally, cells within the 5.6 MPa microfiber composites were more oriented compared to cells within the 15 and 31 MPa microfiber composites. Together, these data indicate that the mechanical properties of microfiber/collagen composites can be tuned for the engineering of ligament and other target tissues. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1894-1901, 2016.
© 2016 Wiley Periodicals, Inc.
In vertebrate epithelia, p120-catenin (hereafter referred to as p120; also known as CTNND1) mediates E-cadherin stability and suppression of RhoA. Genetic ablation of p120 in various epithelial tissues typically causes striking alterations in tissue function and morphology. Although these effects could very well involve p120's activity towards Rho, ascertaining the impact of this relationship has been complicated by the fact that p120 is also required for cell-cell adhesion. Here, we have molecularly uncoupled p120's cadherin-stabilizing and RhoA-suppressing activites. Unexpectedly, removing p120's Rho-suppressing activity dramatically disrupted the integrity of the apical surface, irrespective of E-cadherin stability. The physical defect was tracked to excessive actomyosin contractility along the vertical axis of lateral membranes. Thus, we suggest that p120's distinct activities towards E-cadherin and Rho are molecularly and functionally coupled and this, in turn, enables the maintenance of cell shape in the larger context of an epithelial monolayer. Importantly, local suppression of contractility by cadherin-bound p120 appears to go beyond regulating cell shape, as loss of this activity also leads to major defects in epithelial lumenogenesis.
© 2016. Published by The Company of Biologists Ltd.
Leukocyte residence in lymphoid organs is controlled by a balance between retention and egress-promoting chemoattractants sensed by pertussis toxin (PTX)-sensitive Gαi protein-coupled receptors (GPCRs). Here, we use two-photon intravital microscopy to show that immature B cell retention within bone marrow (BM) was strictly dependent on amoeboid motility mediated by CXCR4 and CXCL12 and by α4β1 integrin-mediated adhesion to VCAM-1. However, B lineage cell egress from BM is independent of PTX-sensitive GPCR signaling. B lineage cells expressing PTX rapidly exited BM even though their motility within BM parenchyma was significantly reduced. Our experiments reveal that when immature B cells are near BM sinusoids their motility is reduced, their morphology is predominantly rounded, and cells reverse transmigrate across sinusoidal endothelium in a largely nonamoeboid manner. Immature B cell egress from BM was dependent on a twofold CXCR4 down-regulation that was antagonized by antigen-induced BCR signaling. This passive mode of cell egress from BM also contributes significantly to the export of other hematopoietic cells, including granulocytes, monocytes, and NK cells, and is reminiscent of erythrocyte egress.
© 2014 Beck et al.
Neurofibromin has been identified as a critical regulator of osteoblast differentiation. Osteoblast specific inactivation of neurofibromin in mice results in a high bone mass phenotype and hyperosteoidosis. Here, we show that inactivation of the Nf1 gene also impairs osteocyte development. We analyzed cortical bone tissue in two conditional mouse models, Nf1Prx1 and Nf1Col1, for morphological and molecular effects. Backscattered electron microscopy revealed significantly enlarged osteocyte lacunae in Nf1Prx1 and Nf1Col1 mice (level E2: ctrl=1.90±0.52%, Nf1Prx1=3.40±0.95%; ctrl 1.60±0.47%, Nf1Col1 2.46±0.91%). Moreover, the osteocyte lacunae appeared misshaped in Nf1Prx1 and Nf1Col1 mice as indicated by increased Feret ratios. Strongest osteocyte and dendritic network disorganization was observed in proximity of muscle attachment sites in Nf1Prx1 humeri. In contrast to control cells, Nf1Prx1 osteocytes contained abundant cytosolic vacuoles and accumulated immature organic matrix within the perilacunar space, a phenotype reminiscent of the hyperosteoidosis shown Nf1 deficient mice. Cortical bone lysates further revealed approx. twofold upregulated MAPK signalling in osteocytes of Nf1Prx1 mice. This was associated with transcriptional downregulation of collagens and genes involved in mechanical sensing in Nf1Prx1 and Nf1Col1 bone tissue. In contrast, matrix gla protein (MGP), phosphate regulating endopeptidase homolog, X-linked (PHEX), and genes involved in lipid metabolism were upregulated. In line with previously described hyperactivation of Nf1 deficient osteoblasts, systemic plasma levels of the bone formation markers osteocalcin (OCN) and procollagen typ I N-propeptide (PINP) were approx. twofold increased in Nf1Prx1 mice. Histochemical and molecular analysis ascertained that osteocytes in Nf1Prx1 cortical bone were viable and did not undergo apoptosis or autophagy. We conclude that loss of neurofibromin is not only critical for osteoblasts but also hinders normal osteocyte development. These findings expand the effect of neurofibromin onto yet another cell type where it is likely involved in the regulation of mechanical sensing, bone matrix composition and mechanical resistance of bone tissue.
Copyright © 2014 Elsevier Inc. All rights reserved.
Mechanical forces play a key role in a wide range of biological processes, from embryogenesis to cancer metastasis, and there is considerable interest in the intuitive question, "Can cellular forces be inferred from cell shapes?" Although several groups have posited affirmative answers to this stimulating question, nagging issues remained regarding equation structure, solution uniqueness and noise sensitivity. Here we show that the mechanical and mathematical factors behind these issues can be resolved by using curved cell edges rather than straight ones. We present a new package of force-inference equations and assessment tools and denote this new package CellFIT, the Cellular Force Inference Toolkit. In this approach, cells in an image are segmented and equilibrium equations are constructed for each triple junction based solely on edge tensions and the limiting angles at which edges approach each junction. The resulting system of tension equations is generally overdetermined. As a result, solutions can be obtained even when a modest number of edges need to be removed from the analysis due to short length, poor definition, image clarity or other factors. Solving these equations yields a set of relative edge tensions whose scaling must be determined from data external to the image. In cases where intracellular pressures are also of interest, Laplace equations are constructed to relate the edge tensions, curvatures and cellular pressure differences. That system is also generally overdetermined and its solution yields a set of pressures whose offset requires reference to the surrounding medium, an open wound, or information external to the image. We show that condition numbers, residual analyses and standard errors can provide confidence information about the inferred forces and pressures. Application of CellFIT to several live and fixed biological tissues reveals considerable force variability within a cell population, significant differences between populations and elevated tensions along heterotypic boundaries.